ISSN 0962-8436

volume 366

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In this Issue

Evolutionary developmental biology (evo-devo) and behaviour

Papers of a Theme issue compiled and edited by Rinaldo C. Bertossa

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Evolutionary developmental biology (evo-devo) and behaviour

Papers of a Theme issue compiled and edited by Rinaldo C. Bertossa

Contents

Introduction
Morphology and behaviour: functional links in development and evolution R. C. Bertossa 2056

Articles
Evo-devo and accounting for Darwins endless forms P. M. Brakeeld The levels of analysis revisited S. A. MacDougall-Shackleton Neural mechanisms underlying the evolvability of behaviour P. S. Katz Conservation of gene function in behaviour C. J. Reaume and M. B. Sokolowski Mapping behavioural evolution onto brain evolution: the strategic roles of conserved organization in individuals and species B. L. Finlay, F. Hinz and R. B. Darlington Evo-devo, deep homology and FoxP2: implications for the evolution of speech and language C. Scharff and J. Petri Evolution of time-keeping mechanisms: early emergence and adaptation to photoperiod R. A. Hut and D. G. M. Beersma Social molecular pathways and the evolution of bee societies G. Bloch and C. M. Grozinger A comparative analysis of neural taste processing in animals G. de Brito Sanchez and M. Giurfa 2069

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Phil. Trans. R. Soc. B (2011) 366, 20562068 doi:10.1098/rstb.2011.0035

Introduction

Morphology and behaviour: functional links in development and evolution

Rinaldo C. Bertossa*
Centre for Behaviour and Neurosciences & Centre for Ecological and Evolutionary Studies, University of Groningen, PO Box 11103, 9700 Groningen, The Netherlands Development and evolution of animal behaviour and morphology are frequently addressed independently, as reected in the dichotomy of disciplines dedicated to their study distinguishing object of study (morphology versus behaviour) and perspective (ultimate versus proximate). Although traits are known to develop and evolve semi-independently, they are matched together in development and evolution to produce a unique functional phenotype. Here I highlight similarities shared by both traits, such as the decisive role played by the environment for their ontogeny. Considering the widespread developmental and functional entanglement between both traits, many cases of adaptive evolution are better understood when proximate and ultimate explanations are integrated. A eld integrating these perspectives is evolutionary developmental biology (evo-devo), which studies the developmental basis of phenotypic diversity. Ultimate aspects in evo-devo studies which have mostly focused on morphological traitscould become more apparent when behaviour, the integrator of form and function, is integrated into the same framework of analysis. Integrating a trait such as behaviour at a different level in the biological hierarchy will help to better understand not only how behavioural diversity is produced, but also how levels are connected to produce functional phenotypes and how these evolve. A possible framework to accommodate and compare form and function at different levels of the biological hierarchy is outlined. At the end, some methodological issues are discussed. Keywords: evo-devo; evolutionary developmental biology; evolution of behaviour; emergence; levels of analysis; biological hierarchy

1. INTRODUCTION Behaviour has been studied for decades from both proximate (i.e. ontogenetic mechanisms and causes of actual behaviour) as well as ultimate perspectives (i.e. survival value and evolution) [1]. Is there a need for a new perspective? In recent years, fostered by unprecedented technical developments, we have noticed a new way to address (classic) questions that makes use of a more integrative approach and focuses on the system in addition to its component parts (e.g. [2,3]). On the other hand, different disciplines join forces to answer common questions. A clear trend is the integration of proximate and ultimate perspectives for a comprehensive understanding of phenotypes. But why combine development with evolution? A quick, off-the-shelf answer is: because nothing in biology makes sense except in the light of evolution [4]. The fact is that, here, this quotation is appropriate. In fact, explaining phenotype and its diversity is fundamentally a problem of explaining the evolution of phenotype development [5]. This goes beyond evolutionary biology. Medicine,

*rinaldo.bertossa@gmail.com One contribution of 10 to a Theme Issue Evolutionary developmental biology (evo-devo) and behaviour.

to name a glossy subject, has begun to realize the importance of evolution, not only in its large praxis [6,7] but also for addressing specic and burning issues such as cancer [8]. A discipline that is successfully integrating proximate with ultimate aspects of phenotype is evolutionary developmental biology, or evo-devo in short. Evodevo, as dened by one of its early exponents, is the study of how developmental processes evolve to produce new patterns of development, new developmental gene regulation, new morphologies, new life histories and new behavioural capabilities [9]. Evo-devo has recorded many important achievements [10] and has now a diversied research programme that ranges from comparative embryology and morphology, developmental genetics to theoretical approaches [11]. Although evo-devo has successfully succeeded in identifying developmental mechanisms underlying phenotype diversity (e.g. [12,13]), a more evident integration of proximate and ultimate explanations seems to be required [14,15]. The objective could be not less than a mechanistic theory for explaining phenotype development and evolution [16]. Since behaviour, considered frequently as integrator of form and function [16], plays an important role in adaptive evolution, it would be simplistic, and indeed potentially misleading,
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Introduction. Morphology and behaviour R. C. Bertossa to consider the evolution of form without taking into account its interactions with behaviour in the arena of selection [10]. Although notable efforts to consider development and evolution of organisms within their natural contextand highlight so the role of behaviourare being made (e.g. [17,18]), evo-devo studies have mostly focused on morphological aspects of the phenotype, leaving behaviour largely unaddressed [19]. The present issue is a rst, more direct attempt to address behaviour within an evo-devo framework. The rst obvious benet we can expect is to understand more about the developmental mechanisms underlying behaviour diversity. But there is another, complementary interest peculiar to addressing traits at different levels of biological organization, such are morphology and behaviour. Although organisms develop and evolve as functional unitary entities, they are composed of a hierarchy of levels (i.e. proteins, cells, organs and so on) [20]. To achieve the tness of the whole, the individual parts are matched. To understand how the levels are matched to each other and how variation at the genetic level translates into a functional phenotype (up to behaviour), it is crucial to understand the kind of relationships that connect levels. A hypothesis could be that different levels of biological organization may share principles of development and evolution and that, by looking at one level, one may discover principles that are relevant for others. Conversely, principles may be level-specic and focusing on only one level or even a priori assuming that principles obtained by studying one level can be applied tout court to other levels may be misleading. It is hence important to extend the evodevo paradigm to other levels of the biological hierarchy and include the whole functional phenotype [10]. In this introductory article, I am mainly concerned with showing that morphology and behaviour share many aspects, beginning with the importance that the environment plays for their development. Behaviour and morphology constitute in fact different qualities of the same unique phenotype, tightly linked in development and evolution. Since these qualities appear to be present at each level of the biological hierarchy, a unique framework of analysis encompassing levels seems already possible. Based on that assumption, I provide an example of comparison across levels and discuss some implications. I conclude with some critical considerations on methods used for discovering the genetic basis underlying behavioural diversity. At the end of the article, summaries of the contributions presented in this issue can be found.

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functional phenotype while analysing individual traits may help to better understand aspects of their development and evolution, as I discuss here. (a) Developmental aspects (i) When does development of behaviour begin? While a distinction may be made between a morphological and a behavioural trait, a clear separation between development of morphology and behaviour does not exist. When does behaviour, and so its development, begin during the lifetime of an animal? Development of behaviour may be considered as beginning when the organism interacts with the environment. But what is the environment? Is this the ambience external to the developing organism? In many classical examples, behavioural development starts already before a mature behaviour is used to interact with the external world [21]. For instance, highly specic responsiveness to the mallard maternal call at the species-typical repetition rate develops in the Peking duck embryo in advance of auditory experience with its own or sib vocalizations [22]. Similarly, correct wiring of the visual system in higher mammals occurs in utero long before vision, when waves of spontaneous neuronal activity are generated by ganglion cells in the retina, even before photoreceptors are present, and propagated across central synapses to target neurons in the lateral geniculate nucleus [23]. In these cases, development of behaviour starts during embryogenesis. Basically, neuronal circuits underlying behaviour develop long before the behaviour they support, and the stimulation of many of them is crucial for their development [24]. Thus, the distinction between the internal and the external environment in order to dene when development of behaviour begins is problematic. Further, if inuences internal to the developing organism can be considered part of behaviour development, which of these should be viewed as behaviour development and which not? The development of many neuronal circuits underlying behaviour does not require action potentials and synaptic transmission but is regulated by molecular cues as found on cell surfaces or diffusing in tissues, the same class of signals that regulate morphological development [25]. How should the reaction of a neuron (growth; changing neuronal activity) later involved in behaviour to an impinging stimulus be categorized? Morphological or behavioural development? In an extreme view, behaviour can be simply considered as a series of neuronal impulses within a specied neuronal circuit. In this case, development of behaviour would start when the rst neuron is formed. From the perspective of a developing neuronal circuit and neuronal impulses (and hence for behaviour), it does not matter whether a stimulus comes from outside or inside the developing organism or whether it has a chemical or neuronal origin: it will cause a change in developmental events. Thus, development of behaviour and morphology merges at a certain point into each other, but where? (ii) Development of both morphology and behaviour is sensitive to environmental inuences Changing perspective on what to consider environment helps us to recognize more similarities

2. DIFFERENT TRAITS, ONE PHENOTYPE In order to facilitate analyses, the phenotype is often fragmented in distinct traits and every trait analysed separately. This is reected also in the disciplines dedicated to the study of the different traits, such as developmental biology (i.e. morphology) and ethology (behaviour), as well as their perspectives, such as proximate (e.g. developmental genetics) and ultimate (e.g. behavioural ecology). However, although the independence of traits in development and evolution is a known phenomenon [5], organisms develop and evolve as one entity. Keeping an eye on the whole
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Introduction. Morphology and behaviour system in mice, for instance, requires sensory feedback from muscle contractions to adjust correct synaptic organization [30]. An extreme example illustrating this mutual inuence is provided by the so-called two-legged goat and discussed by West-Eberhard [5]. The goat, born without forelegs, adopted a semiupright posture and bipedal locomotion from the time of its birth. This behaviour caused morphological changes in bones, pelvic skeleton and musculature and thoracic skeleton not found in normal goats of the same age. Besides demonstrating the interplay between morphology and behaviour in development, this example reveals something else: development is contingent and accommodating. That is, if there is a developmental programme, this is highly sensitive to feedbacks from the environment. If these change, then developmental events can, to a certain degree, adapt. Changes in the environment can have a genetic origin (e.g. altered expression of a transcription factor during limb development) but also a non-genetic basis (e.g. paralysis of forelimbs owing, for example, to polio). It is clear that there was no programme telling a priori that bones and muscles should have developed in a certain way if some components had changed. There were no genes telling the limbs to develop differently or telling the animal to behave differently. The ability to adapt to changing developmental conditionsregardless of the cause of variation, whether genetic or environmentalis known as phenotypic accommodation [5]. But a changing environment, we have seen, is a constant of all development, not only of a pathological state. Indeed, many aspects of phenotype development simply emerge from interactions in time and space of underlying component parts and do not probably map one-to-one onto a genetic programme. For instance, if shear stress is a key player for the development of haematopoietic cells, how is this information encoded in the genome? Is there a gene or genetic programme dedicated to shear stress? For this reason, it has been argued that organisms are not reducible to the component parts [31,32] and several authors [5,3335] warn against seeing a strictly dened programme in the genome and considering genes as the sole source of developmental information. This realization has been made earlier in behavioural sciences. Probabilistic epigenesis for instance emphasizes the reciprocity of inuences within and between levels of an organisms developmental manifold (like genetic activity, neural activity as well as inuences of the environment external to the developing organism) and the ubiquity of gene environment interaction in the realization of all phenotypes [36,37]. Similarly, in neuroconstructivism, cognitive development is explained as emerging from the experience-dependent development of neural structures supporting mental representations [38]. Attempts to include the environment into a theory of development and evolution are being made in developmental systems theory (DST), whichnot surprisinglyis rooted primarily in developmental and behavioural psychology. For DST developmental information resides neither in the genes nor in the environment, but rather emerges from the interactions

shared by morphology and behaviour. Plasticity is the ability of an organism to react to an internal or external environmental input with a change in form, state, movement, or rate of activity [5]. Behaviour is commonly viewed as the plastic phenotype, one organismal property that displays high degrees of phenotypic plasticity [16]. Certainly, behaviour is highly, and reversibly, combinatorial and recombinatorial during a single life time [5], but this is a quality of a trait as it appears in a specic moment. And yet, also its development is considered as being particularly plastic, in which the continuous interaction with the environment is an intrinsic property. Experience and learning are concepts commonly associated with that. Morphological development, in contrast, is mostly believed to be less inuenced by environmental perturbations. But which environment are we talking about? If we shift the focus away from the whole organism and dene environment as everything different from the genome [5], we realize that the development of morphology is highly plastic and does not require fewer interactions with the environment than behaviour does. Morphological development relies on continuous interactions between developing parts of the body. For instance, shear stress produced during ow is necessary for the correct development of haematopoietic cells [26]. A classical example in morphogenesis is neural induction, when the mesoderm contacts and induces the overlying ectoderm to develop into neural tissue. Manipulations of developmental events reveal how crucial the environment is for a normal morphological development: if a small piece of tissue from around the dorsal rim of the blastopore of a developing amphibian embryo is transplanted to the ventral side of another embryo, the host responds by forming an additional embryonic axis containing a virtually complete central nervous system [27]. Interactions among developing tissues can certainly be viewed as a process of experience and learning, not less than what is known for development of behaviour. Not surprisingly, sensitive periodsphases in development during which a specic trait of an organism can be permanently altered by a particular experience or treatment, preceded and followed by a period of lower sensitivity to the same treatmentare readily described in behavioural [28] as well as morphological development [29]. When stressing the role of the environment in development and evolution [16], it is important to realize that the environment internal to a developing organism plays a role as important as the environment external to it. But basically, external/internal are relative concepts. What is external to the cell is internal to a tissue, which is internal to an organ and the organism. Even stimuli impinging on an organism may still be considered internal to a social group, a colony and so on. The best focus for an integral evo-devo is simply the whole functional phenotype, which may be dened, according to one denition, as all traits of an organism other than its genome [5]. (iii) Genes and environment Morphology and behaviour inuence each others development. Development of the withdrawal reex
Phil. Trans. R. Soc. B (2011)

Introduction. Morphology and behaviour R. C. Bertossa of disparate, dispersed developmental resources [39]. While genes play clearly an important role in development, they do not seem to be alone. But who plays which role is still debated [40]. An important question is thus understanding which the other developmental resources are, the role they play, and whether and how they could be integrated into the evo-devo framework of analysis. (b) Evolutionary considerations (i) Reciprocal inuences in evolution Evolutionary studies have preferred to focus either on morphology or on behaviour. However, if the phenotype develops as one entity, reciprocal inuences in evolution are expected, as described in several studies on adaptive evolution. McPeek [41] compared Damsely larvae of species inhabiting lakes in which predation was exerted by shes and of species from sh-less lakes, in which dragonies were the main predators. The latter are morphologically very similar to one another and differ greatly from sh-lake species. They are large and are active swimmers. All larvae of species from lakes with shes move very slowly and infrequently but species are morphologically very diverse. Since species in sh-less lakes are more closely related to species in lakes with shes, McPeek concluded that, following habitat shifts, selection pressures exerted by dragony predation apparently favoured swimming as an escape tactic, which mediated selection pressures onto morphologies used in swimming to increase swimming performance. Similar considerations may apply in other cases. Adaptive differences in the jaw morphology in cichlid shes are related to their feeding habits [42], and the same seems true for variations in beak morphology in Darwin nches [43]. In more extreme cases, such as the evolution of secondary male sexual ornaments, which in some species are driven by female choice [44], the function of morphological structures can only be understood in conjunction with behaviour. These are just a few examples but the potential complexity of morphological and behavioural interactions in the evolution of new adaptive types could be much greater than previously considered [45]. All elements of the phenotype inuence, directly (e.g. neurons) or indirectly (e.g. muscle size), patterns of behavioural adaptation. Katz [46] argues that the functional organization of the nervous system constrains but also promotes the evolvability of behaviour, and Chiel & Beer [47] remind that adaptive behaviour also depends on interactions among the nervous system, body and environment: sensory preprocessing and motor post-processing lter inputs to and outputs from the nervous system; coevolution and co-development of nervous system and periphery create matching and complementarity between them; body structure creates constraints and opportunities for neural control; and continuous feedback between nervous system, body and environment are essential for normal behaviour. (ii) Integration of proximate and ultimate explanations Purely functional considerations of behaviour seem sometimes to forget the historical dimension of the
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phenotype. Behavioural ecology has thrived on verifying theoretical predictions in natural populations [48]: which is the best behaviour to survive in a particular ecological context? But where does behavioural diversity in developmental terms come from? How comes this diversity to be there in evolutionary terms? [5]. As exemplied by the two-legged goat, new behaviour may be the result of phenotypic accommodation or of ancestral inheritance instead of selective pressure alone [49,50]. Moreover, the past evolutionary history constrains future evolutionary trajectories [51,52]: under the same selection pressure, different genomes will respond in different ways, whereas similar traits can be the result of different developmental paths [53,54]. Thus, functional considerations may be used to predict successful behavioural strategies such as particular cognitive abilities [55], but probably not to predict their genetic and developmental basis [5658]. Behaviour observed in an animal is the result of the interplay between genetic and developmental constraints as well as selection pressures [59]. A topical example to illustrate the above are behavioural syndromes, a suite of correlated behaviours reecting between-individual consistency in behaviour across multiple situations [60]. Their existence appears in conict with the expectation that organisms are adapted to every situation in an optimal fashion. Without knowing the genetic and developmental basis of behaviour, it is not possible to fully explain why behavioural syndromes are present in some species but not in others, or why behavioural syndromes display in some species correlations across domains (e.g. aggression in feeding as well as mating domain) whereas in others they show domain-specic correlations (e.g. in the mating domain, correlation between aggression towards females and males; no correlation between aggression in the mating versus feeding domain) [61]. Another conundrum is explaining the adaptive evolution of similar (or homologous) behaviours in different taxa (gure 1). Not only are common behaviours like aggression, sex, or learning and memory present across taxa [63], but also more specic ones, such as tool use [64] or web-spinning [65]. A key developmental question is: can similar behaviours in different taxa rest upon completely different neuronal and/or genetic mechanisms or do similar behaviours (independently of the taxon) rely (or even have to rely) on conserved neuronal and/or genetic mechanisms (gure 1)? Answering this question correctly also depends on clarifying what homologous traits are. Homology in biology refers to an historical continuity of traits. As aptly formulated by Wagner [66], intuitively, one would expect that the historical continuity of morphological characters is underpinned by the continuity of the genes that govern the development of these characters. However, things are not that simple: one of the most important results of the past 15 years of molecular developmental genetics is the realization that homologous characters can have different genetic and developmental bases [53,54,67]. These ndings have occasionally caused misunderstandings as to the correct interpretation of homologous characters [6870] and new ways to dene them are being continuously

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genetic mechanisms

Introduction. Morphology and behaviour

neuronal mechanisms behaviours

A B A B A

A B

2 3 4

A A B

Figure 1. Evolution of homologous behaviours. The evolution of similar behaviours across taxa, such as sex or aggression, represents a key developmental and evolutionary phenomenon for understanding genetic and developmental paths through which specic behaviours can be realized. Here, the question is whether similar behaviours can have a different neuronal basis (1), whether particular behaviours can only rest upon conserved neuronal (2) and genetic mechanisms (3) or whetheras in the construction of functionally similar morphological structures [62]similar behaviours can have the same genetic but different neuronal (i.e. developmental) basis (4).

discussed [66,7173]. The same kind of difculties are expected when discussing homology in behavioural traits and, especially, their underlying genetic and developmental bases [63]. If the above discussion reminds us of the importance to correctly [74] integrate proximate and ultimate aspects for an accurate understanding of behaviour [75], the latter example raises another, related problem: how are the levels in the biological hierarchy connected? 3. TOWARDS FORMULATING A FRAMEWORK OF ANALYSIS ACROSS LEVELS OF ORGANIZATION (a) Broadening the perspective Upto here, I have tried to highlight the importance of keeping an eye on the whole phenotype while studying individual traits. This helps us to recognize qualities shared by morphology and behaviour (e.g. the importance of the environment) and reciprocal inuences in development and evolution. Here I change perspective. I use broader denitions of form and behaviour to develop a possible framework to describe and compare traits at different levels of the biological hierarchy. (i) The form of behaviour One apparent quality of morphology is to have a dened structure, a form. As can be found in any English dictionary, form is dened as the spatial arrangement of something as distinct from its substance. In that respect, also behaviour has, like morphology, a form. Different from morphology, behaviour may be viewed as a dynamic (i.e. involving movement) spatial arrangement of e.g. body limbs, but this is nevertheless form. In this sense
Phil. Trans. R. Soc. B (2011)

and in analogy with a morphological trait, the form of a particular species-specic behavioural pattern (e.g. the courtship displays) remains within certain limits (i.e. is repeatable) and can be recognized as such despite some intra- and inter-individual variation in members of the same species. Additionally, this characteristic allows a particular behaviour to achieve a determined function (i.e. grooming behaviour, or a communication display). Frequently, a specic motoric pattern with specic parts of the body produces a reaction but not when the same motoric pattern is produced with other parts of the body (if possible at all) or if another motoric pattern is produced using the same body parts. For instance, a hand movement in a certain fashion signals hello! Another movement with the same hand may signify completely opposite intentions. This specicity is more evident in elementary behaviours involving motoric patterns such as feeding, swallowing, excreting (urinating and defecating), displacing in space (walking, ying), yawning, vomiting, grooming and so on. But also more complex behaviours, such as aggression, sex, fear, to even more elaborate functions commonly dened under the term cognition, such as decision making, may still be characterized by having a determined form. As known for morphological traits, motoric patterns in a species are so constant that they can be used to classify species and build phylogenies (e.g. [76,77]). The idea of form in behaviour is not new. Lorenz, for instance, when referring to Instinkthandlungen (instinctive acts), insisted on viewing and studying species-specic behaviours as organs [78].

(ii) The behaviour of form and the link to function Up to here, behaviour has been considered from its ethological perspective (i.e. as the behaviour of an animal). But behaviour has, of course, a broader denition, as found for example in Wikipedia: the actions or reactions of an object or organism, usually in relation to the environment. Behaviour so dened is not restricted to any specic level of the biological hierarchy. Protozoa, for instance, display complex actions and reactions, from escaping from a threat, mating and replicating to moving towards a food source and feeding [79]. These movements are not coordinated by a nervous system but are nevertheless classied as animal behaviour. Basically, morphological structures at every level of the hierarchy are regularly engaged in behaviour: organs, cells and enzymes act or react. This acting or reacting is strictly associated with function, meant here as the way in which a morphological trait operates to achieve function. The heart behaves in a dened way in order to function as a pump. Enzymes behave (i.e. function) in a very specic way in order to operate (e.g. catalyze reactions). A second element in the denition is that behaviour may or may not involve movement. Even structures that appear static may owe their form to passive behaviour, like for instance the structure of a bone, which is meant to maximize resilience when involved in a specic motoric behaviour, or colour patterns on the exoskeleton of an insect, meant to provoke a specic behaviour, like sexual attraction in a potential partner or warning to competitors or other insects, or even to avoid being seen by predators.

Introduction. Morphology and behaviour R. C. Bertossa Generally, morphological structures have a determined form in view of a specic way of operating. The way in which they operate, involving or not movement, because it is an action or reaction, can be considered behaviour. Put differently, the mean of functioning (the behaviour) of a morphological structure reveals its (form and) function. This is why behaviour is frequently seen as an integrator of function [16]. This is not to say that morphology does not have function on its own, but a more correct view is that function is achieved through morphology (i.e. structure) behaving (or used ) in a specic way.
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(iii) A framework for comparing levels of organization Considering the similarities between morphology and behaviour highlighted above and, especially, the form characterizing morphology as well as behavioural displays, a unique evo-devo programme may now be more within reach. A common programme would for instance try to understand developmental mechanisms underlying form at each level of the biological hierarchy and how these levels relate to each other. In order to integrate proximate and ultimate perspectives, the mechanistic theory for evo-devo envisioned by Laubichler [16] should hence also integrate levels of biological organization. With respect to mechanistic explanations, evo-devo has been mainly considering gene regulatory networks acting in development and changing through evolution (e.g. [80]). Yet, we have seen above that many features of the phenotype have emergent properties that would not map necessarily one-to-one with the genotype. To the reductionistic criticism Hamilton replies by noticing that mechanistic explanations are reductionisticin the sense that a mechanistic explanation works by decomposing systems (. . .) into their component parts and operationsbut they need not lead to gene-centrism. In particular, reductionistic explanations need not lead anyone to ignore the overall system in favour of the actions of one fundamental part of it [81]. That is, the properties of a system depend as much on the component parts as on their organization. By reuniting the qualities of form and behaviour outlined above, one may recognize that behaviour in its broader denitionis in fact the way in which parts composing a specic level of the hierarchy function, their organization. The parts so organized (i.e. behaving in a certain way) acquire and have a specic form and so a function. This perspective applies to any level of the biological hierarchy (gure 2). Parts at the behavioural level may be body parts that, organized (i.e. moving, behaving) in a specic way, achieve a certain function; parts at a lower level may be amino acids in a protein that, organized in a determined way (i.e. having a specic three-dimensional structure) achieve a specic function. In this way, some of the difculties encountered above, as for example establishing when, during development, to consider the beginning of behaviour development, would disappear. Because biological systems are inherently functional, they are always composed of behaviour and form at the same time. Form and behaviour describe together the specic way in
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Figure 2. Organization of levels in the biological hierarchy. The organization of elements at each level may be similar across the biological hierarchy. Because elements or parts (indicated by the numbers) behave or interact (indicated by arrows connecting the parts) in a specic way, they acquire (in the case of dynamic interactions between the parts) or have (in the case of static interactions) a form with a dened function. (a) A level may be a protein, composed of amino acids having a specic spatial organization or, (b) in a behavioural display with an arm, a determined coordination in time and space of parts of the arm (hand, forearm). A key question is understanding how the levels are connected (vertical double arrow with question mark).

which parts at a certain level of the biological hierarchy are organized and function. As soon as this organization changes in time or space, development begins. A mechanistic theory for development and evolution would thus have to understand (i) which are the parts composing each level in the biological hierarchy, (ii) how these parts are organized in order to produce each level, (iii) how the levels are connected, and (iv) how the organization in levels evolves and affects evolutionary trajectories in order to produce phenotypic diversity. (b) Evo-devo on behaviour: comparing levels If developmental mechanisms are similarly organized through the levels of the biological hierarchy, then conserved principles may be expected across them. An approach to understand this is applying the available evo-devo framework to what we already know about development and evolution of behaviour (see e.g. [82]). Here, I briey illustrate an example of how this approach could look like and generate new specic questions that can be experimentally addressed.

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Introduction. Morphology and behaviour to behaviour are probably involved in other, nonbehavioural functions [105]. The genes needed for the construction of the central nervous system (e.g. Hox, Otx, Pax, Wnt), for instance, are used for the construction of other organs as well [106]. This applies also for many genes (e.g. Pax6) involved in the development of central pattern generators (CPGs), neuronal circuits underlying many rhythmic behaviours like breathing and walking [107]. This could suggest that also diversity in behaviour may be produced mainly through changes in the use rather than the coding sequence of genes, a hypothesis supported already by few data. For example, an important species-specic difference in afliative behaviour in voles is caused by changes in the regulatory region of the vasopressin V1a receptor [108]. The fact that in humans, retained non-coding sequences underwent accelerated evolution compared with other primates [109], and genes specically expressed in the brain display a lower degree of amino acid divergence than other genes [110], lent further support for such a mechanism. Finally, since FoxP2 is expressed in vocal learner and non-vocal learner species, its involvement in vocal learning has also been attributed to differences in expression [92]. With regard to neuronal mechanisms, since homologous neurons and neuronal circuits can be found in species that appear and act very differently, it has been suggested that functional differences could arise by respecication of common circuits [19,96]. Different authors have observed that rather subtle changes in the nervous system (e.g. change in neuromodulation) can cause important changes in behaviour [56,96]. For instance, the evolution of language in humans entailed modications of pre-existing neuronal circuits [111] and the switch between aggression and courtship in Drosophila relies on the neuromodulatory action of octopamine on male-specic neuronal circuits [112]. The rst impression when considering these data is that both at the genetic as well as the neuronal level the way for producing phenotypic diversity may be similar. That is, changes in the use (rather than the nature) of conserved elements at one phenotypic level (genes, neuronal circuits) seem responsible for changes at higher levels (morphology, behaviour). The data presented above are certainly not enough to make a conclusive assessment, but the idea may still serve as a working hypothesis. Basic questions related to this are: How do changes at the higher levels needed for producing new behaviour correlate with changes at the genetic level? Is it all about teaching old genes (or neuronal circuits) new tricksto use an expression familiar to evo-devo practitioners [113] or are other changes (i.e. the coding sequence of genes) also or even more important for producing new behaviour? Are the genetic and developmental changes responsible for behaviour variation within a species the same as those causing behaviour differences between species? These few considerations already raise excitement: the conservation of principles governing development and evolution of phenotypes across levels of phenotypic complexity (i.e. morphology, behaviour) could be more than a hypothesis.

(i) The conservation of developmental elements across species An ongoing discovery in evo-devo studies is that many genes and gene regulatory networks underlying morphology are evolutionarily conserved and inform the evolutionary process [83]. Some are used for similar tasks in distant taxa, such as genetic cascades underlying eye [62] or heart [84] formation, or for the specication of proximaldistal limb patterning [85]. Others are deployed multiple times during development for the construction of completely different structures [86,87]. Is there conservation in the elements (genes, neurons) underlying behaviour? Some behaviours, like aggression [88] or learning [89], seem to rely on molecular mechanisms that are conserved between vertebrates and invertebrates [63] (see also [63] for a broader discussion). This might be explained by the fact that a centralized nervous system was already present in the common ancestor of bilateria, as supported by genetic and developmental data [90,91]. These data suggest that even more molecular mechanisms underlying homologous behaviours could be shared by vertebrates and invertebrates. It may hence not be surprising to nd conservation within more related groups. FoxP2, for instance, even though it is also expressed in non-vocal learner species, is necessary for the elaboration of sensory-motor information underlying vocalization from crocodiles to humans [92,93]. Conservation seems also to be widespread in the neuronal mechanisms underlying behaviour. Neuronal structures responsible for acquisition and processing of odorant and taste information display a striking degree of similarity between invertebrates and vertebrates [59,94]. Since specic neuronal circuits can support different behaviours [95], conservation across taxa may not be surprising and could also explain the observation that entire circuits may be retained even across taxa that display different behaviours [96]. Even if limited, these data suggest that conservation might not be specic to a level of the biological hierarchy (e.g. genes, proteins) but might be common also at other levels (e.g. neuronal circuits). Still, many questions remain open. If elements are conserved, how is context-specicity during development realized? That is, how are conserved elements used to produce different developmental outcomes? Do conserved gene regulatory networks underlie particular behaviours as they do for particular morphological structures [83]? If elements (i.e. proteins, neuronal circuits) are conserved, how is behaviour diversity produced?

(ii) Developmental mechanisms underlying phenotypic novelty The fact that many proteins and protein cascades are conserved across taxa but are deployed for the construction of different morphologies suggested that their differential use (rather than their composition) could lie at the basis of the observed diversity. Since differential gene use is obtained through changes in regulatory regions, this came to be considered by some as a major mechanism behind the evolution of phenotypic novelty [97,98]. Although still debated [99,100], this conclusion is supported by studies on the evolution of morphological and physiological traits [101104]. Genes contributing
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Introduction. Morphology and behaviour R. C. Bertossa 4. THE SEARCH FOR GENETIC CHANGES UNDERLYING BEHAVIOURAL DIVERSITY: SOME CONSIDERATIONS (a) The limits of comparative genomics How to nd changes in the genome that are relevant for behavioural diversity? Comparisons between the genomes of distinct species may reveal associations between new genes and novel phenotypes [114]. In general, however, as already known for new morphological structures, new behaviours are probably the product of conserved genes rather than new, species-specic ones. For instance, the development of CPGs relies on genes involved in the development of many other structures [107]. For the same reason, it will be difcult to infer function from the evolutionary conservation of the DNA sequence [115], both regulatory and coding, unless the compared species are closely related and the involvement of some genes in behaviour is known at least in one species. In the case of new morphology, relevant genes can be inferred from their expression in the new structure they build (e.g. [43]). As to behaviour, it will be difcult to associate genes with a particular behaviour without knowing which specic neuronal elements the behaviour needs, which is rarely the case. Additionally, the fact that the same neuronal structures can support many behaviours [95] renders the analysis even more daunting. If new behaviour is the result of small changes in regulatory regions of conserved genes, it will be difcult to spot the relevant ones: small changes in the genome are frequently the product of drift and nonselective mutations. Between humans and mice, for instance, many regulatory elements are not conserved, and many conserved elements in regulatory regions appear not to have a regulatory function [116]. Ideally, changes in behaviour should be unambiguously associated with changes at the genetic level. This is only possible for experiments carried out in the same genetic background, as in mutagenesis screens and selection experiments.

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behavioural mutants in high-throughput format [120,121], or microarrays and next-generation sequencing methods to spot changes in gene expression as a consequence of the induced mutation (see below). However, understanding the primary function of a wild-type allele by looking at the effects of its mutated version could be misleading. In fact, if phenotypic accommodation is extensive, the mutant phenotype we observe would be caused primarily by the reaction of the developing system to the mutation, which has less to do with the primary function of the gene. For instance, if the two-legged goat phenotype was caused by a mutation, what is the primary function of the mutated gene? Causing the goat to walk normally? Or should we not better say that we have discovered the gene (i.e. the mutant allele) responsible for bipedal posture? Heritable behavioural variation in a population can be screened directly or used to select lines that behave differently. Differences in behaviour can then be correlated with genetic changes through microarrays [118], QTL analyses [122] or, nowadays, next-generation sequencing methods [123]. Although selection is applied on behavioural differences observed within and not between species, selection experiments can identify genes associated with a particular behaviour [118] and reveal genetic correlations underlying a particular behaviour, but alsosince development and evolution of behaviour and morphology inuence each otherunderlying particular associations between behaviour and morphology in adaptive evolution.

(b) Mutagenesis screens and selection experiments If diversity in behaviour would be caused by a change in the regulatory region of a gene, a small but relevant mutation in a regulatory element should affect only one or few functions (e.g. in behaviour) of that gene. Mutagenesis screens for behaviour have been crucial to identify genes underlying important behaviours in Drosophila [117], but this method seems nowadays not very popular. This may be partly due to the belief that behaviour is genetically more complex than morphology, because of, for example, more widespread pleiotropy. However, there are no reasons for assuming that genes affecting behaviour would display more pleiotropy than genes underlying complex morphology (compare for instance [118,119]). Another reason could be that we are possibly not used to reducing complex behaviours into dened and quantiable behavioural patterns as we easily do with complex morphology. New technologies can be combined with mutagenesis screens to overcome these problems, as for instance the use of software for screening
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5. CONCLUSIONS I have argued for the importance of keeping an eye on the whole phenotype while analysing individual traits. This helps for instance to recognize that key developmental elements are shared by morphology and behaviour. Behaviour is not more plastic than morphology. It is only dynamic and can be repeatedly used. Environment is a key element of development of both traits and should nd a place in the framework of analysis of evo-devo. The tight entanglement of morphology and behaviour in development and evolution suggests that an integration of proximate and ultimate perspectives could offer a better understanding of present phenotypes. The functional phenotype, made of morphology and behaviour alike, develops and evolves as one entity. Therefore, there is only one evo-devo that includes the whole functional phenotype [10]. Since behaviour can describe the way of interacting of elements at any level of the biological hierarchy and form is the specic waydynamic or staticin which these elements are organized, morphology and animal behaviour could be reunited within the same framework of analysis. This framework may be applied beyond the organism level, such as to social groups, to understand whether principles valid within organisms can be used to describe development and evolution beyond the organism level [81,124]. Behaviour hence is truly the missing partner: not only animal behaviour at the organism level, needed for a better interpretation of adaptive evolution but, more generally, behaviour as the way in

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Introduction. Morphology and behaviour behaviour can be addressed independently using different levels of investigation, including the DNA sequence, the genes position in a genetic pathway, spatial temporal tissue expression and neuronal circuit. Several examples are used to illustrate these points, including circadian rhythms, learning and memory, food-related behaviours and sleep. They also discuss how qualitative and quantitative comparisons of the behavioural phenotype, its function and the importance of the environmental and social context should be used in cross-species comparisons. In mammals, the statistics of allometric variation in brains within species (swine, mink, and multiple strains of laboratory mice) closely resemble phylogenetic brain variation over all mammals. Barbara Finlay, Flora Hinz and Richard Darlington [58] argue that, rst, this pattern reveals the selection of developmental parameters for brain organization robust to environmental and niche variation at the individual level, and, second, that only a restricted set of computational models of the brain can be consistent with an adaptive function for conserved patterns of variation at within- and betweenspecies scales. Language in animals evolved via modications of existing molecular and morphological hardware. Connstance Scharff & Jana Petri [93] focus on one protein, FoxP2, which is required for language in humans, for song learning in birds, and for motor skill learning in mice. They argue that it is a good candidate to study deep homologies of molecular toolkits and neural circuits relevant for the emergence of language. They also critically examine the (lack of) evidence for some of the claims about the uniqueness of human language. Circadian systems are ubiquitous, implying an essential role for daily timing in evolution. Roleof Hut & Domien Beersma [125] suggest that energy storage was crucial for the earliest evolution of circadian clock systems in primitive photosynthesizing life forms. The modication of ATP storage/release capacity of KaiC proteins through KaiA suggests a daylength adaptation mechanism that may explain the global expansion of cyanobacteria. In general, different models for photoperiodic adaptation predict different selection pressures on the circadian period. Hut & Beersma [125] therefore conclude that establishing latitudinal clines in circadian tau for various species is crucial to reveal circadian photoperiod adaptation mechanisms and how these may constrain responses to latitudinal expansion and global warming. Bee societies have fascinated humans for centuries because of their complexity, and led to many theories about the evolution of cooperative and altruistic behaviours. Guy Bloch & Christina Grozinger [124] review recent genomic studies on the development and evolution of social behaviour in bees and propose a model of social pathways, modules consisting in the detection, elaboration and output of signals involved in social behaviour. The authors then use the model to illustrate how the hormonal system and plasticity in the circadian clockwork have been modied during and contributed to the evolution of bee societies. Taste, the sense that distinguishes between chemical compounds and the sensations they produce based on contact with chemoreceptors, allows discriminating edible from non-edible items and is, therefore, crucial

which elements at any level of the hierarchy need to be organized in order to obtain a specic functional form. The question now is understanding which are the key players at every level, how they interact and how levels are connected. Perhaps, once having claried this, understanding the role of genesi.e. whether they are the major players in development and evolutionwill emerge as a natural consequence.

6. WHAT THE ARTICLES SAY While studies on behaviour development and evolution have been carried out for many years, the treatment of behaviour within an evo-devo paradigm is only beginning, as reected by the contributions presented in the present issue. The rst articles [10,46,63,74] set in motion (or refresh) important discussions on general aspects of development and evolution pertaining to behaviour. These are then more specically addressed in the studies discussed in the second series of articles [58,93,94,124,125]. For all those not familiar with evo-devo, the article by Paul Brakeeld [10] is a very useful introduction. He reviews some major themes and achievements of evo-devo, illustrating the scope of the eld to reveal how the processes of development can contribute to explaining patterns of evolutionary diversication in animal form. In addition to stressing the exciting potential of extending the approach of evo-devo to patterns of behavioural diversication, Brakeeld [10] reasons that to consider the evolution of form without taking into account its interactions with behaviour (. . .) is simplistic and potentially misleading. The author concludes by agreeing with some other workers in the eld that understanding more about evolvabilitythe capacity of a developmental system to evolveshould be a key step towards answering many of the open questions in evo-devo. Studies of the evolution and the development of behaviour take place at the ultimate and proximate levels of analyses, respectively. There has been much debate in past decades about how to (or how not to) integrate studies across these levels. Scott MacDougallShackleton [74] reviews different uses of the term levels of analysis and highlights how studies of function and mechanism can be integrated, an approach epitomized by evolutionary developmental biology. Although genetic differences can affect behaviour, genes ultimately act through the nervous system. Paul Katz [46] maintains that the functional organization of the nervous system constrains but also promotes the evolvability of behaviour. He reviews evidence showing how neural mechanisms such as activity-dependent plasticity and neuromodulation allow basal neural circuits to be employed for different behaviours, as observed in sensory processing, motor output and even in complex social behaviour. Katz concludes that the qualities of the nervous system that enable it to produce complex behaviour may also play a role in the evolvability of species-typical behaviour. Christopher Reaume and Marla Sokolowski [63] discuss how gene function, as a hierarchical biological phenomenon, relates to behavioural homology across species. They suggest that gene function homology in
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Introduction. Morphology and behaviour R. C. Bertossa for survival. Gabriela De Brito Sanchez & Martin Giurfa [94] discuss the principles of taste coding in the animal brain, from insects to mammals. They argue that an essential task for a neuroscience of taste is to determine the connectivity of taste-processing circuits in the central nervous system, and suggest that, beyond labelled-line or across-bre pattern coding, taste representations are conserved across species and seem to relate to the hedonic value of the tastant (e.g. palatable versus non-palatable).

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I thank D. Beersma, L. Beukeboom, P.-H. Clergeot, A. Kamping, S. Mitchell, M. Olmedo, S. Paolucci, T. Schwander, S. Verhulst, C. Vermeulen, L. van de Zande and three anonymous reviewers for useful discussions and comments on previous versions of the manuscript. I thank all contributors in this Special Issue for their tenaciousness in coping with my and reviewers comments and apologize with all those whose contributions could not be included in this issue. I wish to thank the following people at Philosophical Transactions of the Royal Society: Joseph James for his invitation to guest-edit the present issue; Claire Rawlinson and Joanna Bolesworth for their patience with many changes along the way. R.C.B. received support with a grant from the Swiss National Science Foundation (SNF) for prospective researchers and an ALW grant (nr. 817.02.020) from the Netherlands Organization for Scientic Research (NWO).

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Phil. Trans. R. Soc. B (2011) 366, 20692075 doi:10.1098/rstb.2011.0007

Review

Evo-devo and accounting for Darwins endless forms

Paul M. Brakeeld1,2,*
1

Department of Zoology, University Museum of Zoology Cambridge, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK 2 Institute of Biology, Leiden University, The Netherlands

Evo-devo has led to dramatic advances in our understanding of how the processes of development can contribute to explaining patterns of evolutionary diversication that underlie the endless forms of animal life on the Earth. This is increasingly the case not only for the origins of evolutionary novelties that permit new functions and open up new adaptive zones, but also for the processes of evolutionary tinkering that occur within the subsequent radiations of related species. Evo-devo has time and again yielded spectacular examples of Darwins notions of common ancestry and of descent with modication. It has also shown that the evolution of endless forms is more about the evolution of the regulatory machinery of ancient genes than the origin and elaboration of new genes. Evolvability, especially with respect to the capacity of a developmental system to evolve and to generate the variation in form for natural selection to screen, has become a pivotal focus of evo-devo. As a consequence, a balancing of the concept of endless forms in morphospace with a greater awareness of the potential for developmental constraints and bias is becoming more general. The prospect of parallel horizons opening up for the evolution of behaviour is exciting; in particular, does Sean Carrolls phrase referring to old genes learning new tricks in the evolution of endless forms apply equally as well to patterns of diversity and disparity in behavioural trait-space? Keywords: evo-devo; evolvability; developmental bias; morphology; endless forms

The model-T Ford was not the invention but the conveyor belt that built it. (from the lm Seabiscuit)

1. SOME ACHIEVEMENTS OF EVO-DEVO With respect to Darwins endless forms, modern evolutionary developmental biology, or evo-devo, has demonstrated beyond any doubt the truth of his principles of common ancestry and of descent with modication. A half-century ago, few would have considered it likely that diversication of a single ancient and highly conserved tool kit of signalling genetic pathways had provided the basis for the evolution of morphology throughout the animal kingdom, or that the set of developmental genes in a y would have so much in common with that of our own species. Research is revealing how, to employ Sean Carrolls [1] now famous phraseology, the evolution of form is largely about teaching old genes new tricks. Carroll [2] has emphasized how the multiple functions and contexts of these genes in development (both spatially and temporally) constrains evolutionary change to the regulatory machinery of the genes rather than to the proteins themselves (the demonstration by Halder et al. [3] of the ability of the mouse Pax-6 protein to induce ommatidium formation in Drosophila, just like the Drosophila Pax*pb499@cam.ac.uk One contribution of 10 to a Theme Issue Evolutionary developmental biology (evo-devo) and behaviour.

6 (eyeless) protein itself, retains its power to impress). Many of these developmental genes code for transcription factors that regulate the activity of other genes, often via interactions with other transcription factors and proteins. Typically they regulate the expression of numerous target genes in complex genetic regulatory networks and, thus, lead to the activity of cascades of genes in cell differentiation and development. There are of course demonstrations of protein change in morphological evolution. Some, but not all [4,5], of such examples involve proteins with more specialized roles in development such as in pigmentation or in the ear (e.g. [6,7]). While clearly there are also important examples of taxonomically restricted genes (e.g. [8]), some of which contribute to development, the conserved tool kit of signalling pathways is involved in so much of what happens in morphogenesis throughout the animal kingdom. Perhaps, in hindsight, there is little reason to nd such general properties of the diversication of form especially surprising or unexpected. Evolutionary biologists are used to the notion that the process of evolution is highly opportunistic; if a particular feature or property is available, evolution will exploit its potential and run with it, somewhere and somehow. Micro-RNAs and the growing evidence of their important role in gene regulation provide a good example of this. It seems then, rather logical that tinkering with, and the reuse of, existing systems will tend to happen much more often than inventing and designing new systems, de novo, for the evolution of form. The
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Review. Evo-devo and Darwins endless forms behavioural traits will inevitably also provide a critical input to the evo-devo of form. In turn, the whole eld will be able to edge towards explaining the coordinated evolution of the complex functional phenotypes that are screened for their performance by natural selection. Consider, for example, a species-specic scent brush on the wings of a male buttery that produces a certain sex pheromone dispersed in a particular phase of a courtship display which is then responded to by specialized cells on the antenna of a potential female partner to elicit a chain of events and behaviour leading to successful sperm transfer [10]; to understand a single stepsay the evo-devo of the scent brushis to understand very little of the whole functional sequence, let alone the underlying process of its evolution and its diversication among species. The latter will only become possible when the approach of the evo-devo for form has been extended successfully to the complete functional phenotype. Here, I will draw attention to some of the ongoing successes of evo-devo from the perspective of an evolutionary biologist interested in how adaptive morphological traits arise and are elaborated upon in the processes of evolutionary diversication.

extensive pleiotropy of such existing genetic systems in their developmental context will then lead to bias in the mechanisms by which evolutionary change tends to occur. Although evolution will happen in different ways, evo-devo has led to the recognition that certain types of genetic change predominate, and adaptive hotspots are likely to occur in the genome with respect to the diversication of form [9]. The details of the types of change in genetic regulation that have been associated with the origins of evolutionary novelties involving morphology may, however, tend to involve different ways of modifying the networks from those that dominate the subsequent tinkering process in character transformation [9]. It is in any case comparatively easy to begin to make generalizations now we know much about the principles of how genes coordinate the development of complex morphologies. Such general principles for the evolution and genetics of diversication of behavioural traits are yet to progress as far but will surely be an exciting eld in years to come, and one likely to yield different trends from those for form.

2. EVOLUTION OF FORM AND ON TO THE EVOLUTION OF BEHAVIOUR Development is central to the evolution of form because it translates genotypes into morphologies. Mutations and genetic variation are the raw material for evolution, but the developmental processes of morphogenesis build the variation in form that is screened by natural selection to yield organisms adapted to their environment. Animal groups with spectacular differences in body plan, not to mention all the products of evolutionary tinkering with such designs, have used the same basic machinery to yield disparity in form. The eld of evo-devo is unravelling the puzzle of how the ancient tool kit has been employed via the evolution of complex regulatory machinery to build such an astonishing variety of animals with such disparate morphologies. It is this architecture of development which when coupled with the processes of natural selection and evolution accounts for the awe-inspiring range of morphologies exhibited by biodiversity on the Earth. Biology is indeed edging ever closer to a detailed understanding of how the processes of development have become elaborated upon and modied in the evolution of Charles Darwins endless forms most beautiful [1]. Are the prospects equally exciting for behaviour? Charles Darwin was the rst scientist to think carefully about behaviour, just as morphology, as a product of natural selection and the process of adaptation. Thus, it is timely to consider the achievements and prospects of evo-devo from the perspective of form, before handing over to researchers on the evolution of behaviour at least some of the challenge of uncovering the role of developmental processes in translating genotype into phenotype. Not only will progress with the development of behaviour be important in its own right, but morphologies almost always interface within the arena of natural selection by interacting with behaviour and life histories. Thus, advances in understanding the evolution and development of
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3. SOME GOALS OF EVO-DEVO Evo-devo has led to a rich diversity in the themes of research and theory being pursued within the eld [9,11]. Put succinctly, it has been concerned with examining the evolution of developmental pathways, and with discovering how the processes of development contribute to the evolution of form. The eld has examined how changes in developmental pathways account for the evolution of disparity in form across distantly related taxa with highly divergent body plans and, increasingly, also among closely related species. This work is exploring the extent to which the same sorts of pathways and changes are involved at different phylogenetic levels. The connections between patterns at the macroevolutionary level and microevolutionary processes observed within populations on an experimental time scale remain essentially unmade [12]. However, advances that will help to resolve these connections are being made through work on subtle differences in phenotype such as occur for bristle patterns or wingspotting in dipteran ies [13 16], and the studies of evolutionary novelties involved in major, novel structures such as limbs, segments, eyes and ns [17 19]. Among the systems currently under study with exciting potential for integrating the microand macro-levels of evolution in form are vertebrate teeth [20,21], and the pelvic ns and armour of stickleback sh [22]. The results of research in evo-devo are also beginning to tackle whether the way in which development works could inuence the sets of morphologies that evolve within morphospace. Dramatic examples of parallel evolution in form, such as in the species ocks of haplochromine cichlid shes of the African Rift Valley lakes [21,23], could be inuenced by biases in the capacity of the relevant developmental pathways to generate variation in certain axes of

Review. Evo-devo and Darwins endless forms P. M. Brakeeld morphospace relative to others. These cichlids appear to show a small number of distinct trophic morphologies (not to mention behaviours and life histories), which may reect more or less discrete clusters of species, each with a different way of making a living. Developmental biases could also have played a role in the evolution of the different morphologies of teeth along the jaw of a mammal [20,21], or the appendages of different segments along the body of a crustacean [24,25]. Again, the way in which development works and the evolvability of teeth or appendages could help to explain the different functional morphologies that haveor indeed have notevolved from some ancestral jaw or body axis carrying repeated copies, each of similar form. The evolution of individuality in the form of the serially repeated eyespots along the margin of a buttery wing involves comparable issues [26 28]. Similar types of questions can also be posed with respect to the evolution of disparity in behaviour. Thirty years or so ago, development in an evolutionary context was essentially a black box positioned between genotype and form; development mapped form onto genotype but was largely unknown, at least from the genetical perspective. Thanks to the extraordinary advances of developmental and molecular genetics, the contents of the black box have become visible. What have we learnt and how is this likely to transfer to other components of the phenotype, especially behaviour? The concepts that have owed from opening up this box for morphogenesis may ultimately help with the challenge of understanding the evolution of the whole functional phenotype where physiology and other processes must play more prominent and integrative roles. Evolution of form is highly opportunistic. The machinery of tool kit genes, master control genes and signalling pathways is used time and again both within ontogeny from the zygote to the adult organism, and in very different structures, tissues and organs. It is the evolution and reorganization of the developmental gene regulatory networks for this machinery that appears to underlie much of the evolution of morphology [2,19,29]. To almost any student of biology from the 1970s or earlier, the great discoveries of developmental biology over recent decades are truly inspiring revelations. The next quarter of a century will clearly reveal more of how the genotype to phenotype map works for the whole functional phenotype, and will demonstrate the extent to which the evolution of form is special with its own largely private mechanisms and machinery. How do the principles being observed for the evo-devo of form apply to behaviours and life histories, and do the patterns of change in the genes which regulate such traits parallel those for form? Development and evolution are clearly deeply intertwined, and as Hall [30] has emphasized, it makes no sense to study them in isolation. Any evolutionary change in form must involve some modication in development. Also, any absence of change in form over a very long period of time (morphological stasis) begs the question of why in terms of development some pattern of dynamic evolution has not occurred
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[12]. Developmental and behavioural plasticity in response to environmental conditions are additional processes likely to play a crucial role in shaping the evolution of form, both in terms of the phenomena of phylogenetic constraints or inertia [31] and of facilitating patterns of diversication, perhaps involving genetic accommodation [32,33]. The study of such processes will contribute to a blossoming of the extension of evo-devo becoming known as eco-evo-devo [34], in which both plasticity and the environment are a focus of attention.

4. EVO-DEVO AS A DISCIPLINE A variety of concepts have become important focal points for discussion during the era of modern evodevo in an attempt to integrate the two elds, and tie down the goals of evo-devo. Modularity, developmental integration, developmental constraints, emergence and evolvability together with many related ideas have all been targets for much discussion [11]. Such discussions have not always been based on rich sets of empirical data, but this is changing as more experimental and analytical work is performed using an increasing range of systems. Comparative embryology has enjoyed a long and rich history, and is clearly ancestral to modern evodevo in terms of many of the research problems (e.g. [35]). Developmental constraints and the role of heterochrony were a core problem of comparative evolutionary embryology. The association between evolution in form and the timing of modication of development in ontogeny remains unclear, especially with respect to evolutionary novelties. Associations of this type will also prove fascinating to unravel for behavioural traits. The success of exploring the molecular genetics of embryogenesis in combination with the broad eld of developmental genetics is at the core of evo-devo. Developmental biologists have revealed how the processes of embryogenesis and morphogenesis are regulated by genes and signalling pathways. Much of the early effort in integrating evolution and development examined how differences in body plan are organized and specied [17,36,37]. Major body features such as segments, limbs and eyes were compared in a small number of model organisms with highly disparate morphologies. These studies discovered differences, as well as many remarkable similarities, in the developmental mechanisms employed by different phyla. They demonstrated that the signalling pathways used by animal cells in all phyla are ancient and highly conserved. The complete pathways are homologous across the metazoan from arthropods to vertebrates; they are also surprisingly few in number. The association of the Pax-6 gene with the development of the eyes of atworms, arthropods and vertebrates is a particularly dramatic example. An important focus in this work has been to examine how modications of developmental processes can lead to the production of the new morphological features of evolutionary novelties [18,38,39]. Similarly, the sorts of modications that are involved when organs are lost are being

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Review. Evo-devo and Darwins endless forms ecospace-limiting constraints do tend to be more inuential than any developmental constraints for the evolution of form (e.g. [54]), and given time biases arising through the properties of evolvability tend to be of comparatively minor consequence. However, time to adapt is not always available for a population confronted by an environmental challenge, especially in todays rapidly changing world. More research is clearly needed that seeks to explore in an integrative manner the intrinsic effects resulting from the generation of variation in the functional phenotype and the extrinsic effects resulting from the process of sieving that variation by natural selection. Similar considerations can also be applied to the origin of evolutionary novelties [55]: are these rare because of developmental constraints or is their appearance primarily driven by natural selection? Eberhard [56] argues that his surveys of male secondary sexual traits in sepsid ies support a retarding effect of developmental constraints, although his conclusion has been questioned [55]. Again, evo-devo is making advances here in our knowledge of the development of such structures [57,58], and it is to be hoped that this work will help to resolve the role of developmental processes in the evolution of the observed patterns in morphospace. This case study also illustrates again the intricate interactions between form and behaviour since these novel structures in male sepsid ies can only be functional and inuence mating success when coupled with the appropriate behaviours and signals [56,59]. The origin of evolutionary novelties is one aspect of evolvability; another is the role of standing genetic variation and mutational input in generating developmental exibility. However evolvability is denedand this is to some degree dependent on the perspective of the researcherit is crucial to discover the extent to which both genetics and development inuences the generation of variation in the phenotype. Evo-devo, at least for the forms of organisms, must in its broadest sense be capable of helping to reveal the importance of differences in evolvability for the observed patterns of occupancy in morphospace, as well as for making predictions about the likely effects of environmental change. Again, it is simplistic, and indeed potentially misleading, to consider the evolution of form without taking into account its interactions with behaviour in the arena of selection. The evolvability of buttery eyespot patterns in different directions of morphospace can be explored experimentally by articial selection and the responses analysed in terms of both genetical and developmental mechanisms [27], but ultimately how such phenotypes will evolve will depend on their interactions with behaviour and ecology [28]. An integration of experimental work on key traits using model species with a more comparative phylogenetic approach across species in eco- and trait-space is needed to progress further. A system can be considered to be evolvable if it can be modied through genetic change in a way that could potentially enhance survival and reproduction. Some traits may be more evolvable than others, and evolvability may depend on the direction of change

increasingly revealed, for example, in research on blind cave sh [40 42]. The eld of evo-devo is also becoming increasingly concerned with the differences in form among closely related species [43]. Waddington [44] considered how development could inuence the expression of phenotypic variation. Wim Scharloo and other researchers galvanized by the Edinburgh school of developmental and quantitative genetics were forerunners of current work on the developmental basis of more subtle differences in morphology. Examples in insects include the cuticular bristle patterns of ies [13,15], the wing spots of ies [14,16], the horns in dung beetles [45,46] and the eyespots along the margins of a buttery wing [26,27]. An early gathering of researchers [47] from both the developmental and the evolutionary elds established an important milestone in dening a developmental constraint as a bias on the production of various phenotypes caused by the structure, character, composition or dynamics of the developmental system. The eld remains a long way from any complete description and analysis of such bias and its effects on the occupancy of morphospace [48], but progress is being made [49]. Hopefully, future work will unravel more effectively the genetical from the developmental properties.

5. EVOLVABILITY AS A FOCUS FOR THE EVOLUTION OF FORM Evo-devo seeks to answer a variety of different questions but many of these have their roots in the concept of evolvability. Hendrikse et al. [50] have recently argued that the proper focus for evo-devo is evolvability [51]. Such a pivotal focus is indeed logical and certainly encapsulates the reason why so many evolutionary biologists have become enthused by the eld. Does knowledge of how forms are built help evolutionary biologists to account for the directions and rates of evolution in morphospace? Some even consider that the results of this endeavour may lead to a reassessment of the modern synthesis [2,11,52]. Knowledge about the properties of evolvability must be at the heart of explanations of patterns in evolution, especially perhaps with respect to how radiations of species explore morphospace [28]. From a developmental perspective, evolvability is perhaps best dened as the capacity of a developmental system to evolve, which is primarily a function of its ability to generate variation in form. The efcacy of natural selection depends on the availability of appropriate variation in the phenotype. This is in turn not only dependent on genetic variation, but how this is translated to yield variation in the phenotype; that is, in the case of form, on development and morphogenesis. In essence, if evolvability with respect to the ability of development to generate the fuel for natural selection to screen for functionally effective phenotypes differs among potential directions of change, the paths that evolution follows can be expected to be biased. The extent to which natural selection is all-powerful in different organisms remains unclear whether one is considering morphological, behavioural or life-history traits (e.g. [12,49,53]). Perhaps
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Review. Evo-devo and Darwins endless forms P. M. Brakeeld in phenotype. The relationship between evolvability and different forms of robustness, for example mutational robustness, is controversial (but see [60]). Thus, mutational robustness implies that the system produces little phenotypic variation in response to genetic variation, which would be expected to hamper the response to natural selection. On the other hand, robustness is expected to facilitate the accumulation of cryptic genetic variation which could foster such responses under certain conditions of environmental perturbation and some form of failure in the mechanisms of robustness. In particular, this could be an important process when considering access to new adaptive peaks or a region of high tness surrounded by a valley of low tness. From a developmental perspective, it will be exciting to be able to differentiate more clearly between these types of effects. Detailed experimental work to untangle such processes is progressing with proteins (e.g. [61]), RNA viruses (e.g. [62]) and bacteria (e.g. [63]), but has hardly begun for the development of morphological traits in higher organisms. Perhaps in the future, it will also be possible to extend considerations of how the evolution of behaviour depends on evolvability and robustness in development. Developmental bias [47] and genetical channelling [64] can in theory arise from patterns of shared developmental pathways or genetic covariance among traits. Distinguishing between such terms as the most appropriate label for a difference in patterns of response to natural selection on morphological traits will always be challenging. Because the processes of establishment of behavioural traits will involve a broader set of underlying mechanisms, one which will include development, this is likely to be even more challenging for such traits. However, in principle, sufcient information at different levels of the process of translation from genetic variation to expression of the trait itself should enable a proper distinction to be made. In any case, such distinctions may not be very informative except to illustrate that any one-sided or reductionist approach, one which, for example, considers only genetic variation, is unlikely to capture the underlying processes sufciently to be able to predict fully responses to natural selection and environmental change.

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and then elaborated upon in subsequent adaptive radiations in morphospace facilitated by the structure remains an exciting challenge for the future [19]. The roles of the processes of developmental plasticity and epigenetics, and of so-called cryptic variation, are also elds that are clearly important to understand more fully. It will also be fascinating to determine how readily generalizations for the evolution of form can be extended to behavioural traits, and to the evolution of interactions between form, physiology and behaviour that underlie any coordinated, functional phenotype. One of the clearest trends in evo-devo has been the increasing contribution made by research on a range of emerging model organisms [67,68]. Functional tests of candidate genes and genetic variants, especially using RNAi, are also becoming widely available for these organisms. These efforts have been coupled with an ever-widening variety of types of morphologies under investigation, and with an extending interest from the opening up of developmental genetics through to studies of function, performance and natural selection on the same traits. This reects one of the achievements of the eld in expanding its horizons beyond the deepest nodes in phylogenetic evolution towards understanding the contribution of development at all levels of biological organization, throughout ontogeny, and from evolutionary novelties to the effects of evolutionary tinkering with form. The era of modern evo-devo was ushered in by developmental biologists concerned with probing the differences in developmental processes underlying the major differences in body plan among the small number of model organisms, but this has been increasingly transformed to yield a much broader perspective. The same healthy trends can be predicted for the research community working on the development of behaviour.
I thank Rinaldo Bertossa and Gunter Wagner for their helpful comments on this article, and the ERC for funding support.

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Phil. Trans. R. Soc. B (2011) 366, 20762085 doi:10.1098/rstb.2010.0363

Review

The levels of analysis revisited

Scott A. MacDougall-Shackleton*
Advanced Facility for Avian Research, Departments of Psychology and Biology, University of Western Ontario, London, Ontario, Canada N6A 5C2 The term levels of analysis has been used in several ways: to distinguish between ultimate and proximate levels, to categorize different kinds of research questions and to differentiate levels of reductionism. Because questions regarding ultimate function and proximate mechanisms are logically distinct, I suggest that distinguishing between these two levels is the best use of the term. Integrating across levels in research has potential risks, but many benets. Consideration at one level can help generate novel hypotheses at the other, dene categories of behaviour and set criteria that must be addressed. Taking an adaptationist stance thus strengthens research on proximate mechanisms. Similarly, it is critical for researchers studying adaptation and function to have detailed knowledge of proximate mechanisms that may constrain or modulate evolutionary processes. Despite the benets of integrating across ultimate and proximate levels, failure to clearly identify levels of analysis, and whether or not hypotheses are exclusive alternatives, can create false debates. Such non-alternative hypotheses may occur between or within levels, and are not limited to integrative approaches. In this review, I survey different uses of the term levels of analysis and the benets of integration, and highlight examples of false debate within and between levels. The best integrative biology reciprocally uses ultimate and proximate hypotheses to generate a more complete understanding of behaviour. Keywords: integration; proximate; ultimate; levels of analysis

1. INTRODUCTION Behaviour is studied from a variety of perspectives, ranging from social sciences, to behavioural ecology and animal behaviour, to cellular and molecular neuroscience. One of the greatest challenges of integrative biology is to synthesize ideas and information across such approaches to seek a more complete understanding. Integrative biologists are fortunate in that there is a rich and deep theoretical framework that can be used to aid such synthesis across different approaches. This theoretical framework is, of course, the grand unifying theory of all biology: evolution by natural selection. The challenge of integration thus includes the challenge of considering evolution and adaptation when exploring the mechanisms of behaviour, and of considering mechanisms of behaviour when exploring evolution. Taking an evolutionary or adaptationist approach when studying mechanisms and developmental processes of behaviour has become increasingly popular in elds including behavioural ecology and evolutionary psychology, but has been extensively criticized (e.g. [14]). In this review, I explore the integration of adaptive function and causal mechanism. Central to this issue is seeking clarity about levels of analysis and levels of reductionism, and explicitly considering whether or not potentially

competing hypotheses are, or are not, mutually exclusive. First, I review varied concepts of the levels of analysis and attempts to synthesize or integrate between them. Second, I review several ways in which considering the ultimate function of behaviour can aid in exploring the mechanisms of behaviour. Finally, I make the point that clarity about whether or not competing hypotheses are mutually exclusive is required to eliminate false debate between and within levels of analysis. The examples I use below are biased to include studies on songbirds, in part because that is my own eld of research, but also because birds are studied extensively from different perspectives across a range of disciplines and provide an excellent case study.

*smacdou2@uwo.ca One contribution of 10 to a Theme Issue Evolutionary developmental biology (evo-devo) and behaviour.

2. CAUSE AND FUNCTION (a) Levels of analysis Baker [5] distinguished between ultimate and proximate factors that regulate the timing of reproduction. Ultimate factors are those variables that determine offspring survival and thus reproductive success (e.g. food availability) and proximate factors are those variables that organisms actually use to time reproduction (e.g. the annual change in photoperiod). Mayr [6] rened and extended the use of these terms to highlight two kinds of questions biologists can ask about a phenomenon: what is the proximate causation? (causes derived within the life of the organism) versus what is the ultimate causation? (causes derived over evolutionary time). These two kinds of questions
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Table 1. Three uses of the term levels of analysis. I suggest that the distinction between ultimate and proximate levels is the best use of this term, though the other uses are now engrained in the literature. levels of analysis ultimate/proximate Mayr [6]: four questions Sherman [8] following Tinbergen [9]: evolutionary history adaptive function development physiological mechanisms ?cognitive/ psychological ecological/ evolutionary organismal physiological cellular molecular levels of reductionism

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ultimate (evolution and adaptation) proximate (mechanisms)

have become generally accepted as two levels of analysis. The proximate level (how questions) deals with mechanistic causes such as genetic, neural, hormonal, or cognitive/behavioural processes, whereas the ultimate level (why questions) deals with adaptive and evolutionary aspects. Ultimate causes are causes only in the sense of an Aristotelian Final Cause and thus the ultimate and proximate levels have been summarized as function and cause, respectively [7]. In addition to the distinction between ultimate and proximate levels, the term levels of analysis has been used in other ways (table 1). A common example of this in animal behaviour is Tinbergens [9] four questions of evolutionary history, adaptive function, causation and development. Tinbergens framework is, justiably, adopted in every major textbook in animal behaviour, and answers to each of these four kinds of questions are required for a full understanding of behaviour. Some authors, however, have equated these questions with the levels of analysis, and even proposed a fth level for cognitive or psychological mechanisms (e.g. [8]). The term levels of analysis is additionally sometimes used to refer to levels of reductionism, such as behavioural, physiological, neural or molecular genetic levels of inquiry. I would argue that, given the distinct differences between mechanistic causes and adaptive functionsthat is given they are distinct logical categories [10,11]Mayrs division of ultimate and proximate levels is the most appropriate use of the term. Table 1 illustrates that the term levels of analysis has been used in very different ways. However, it is important to note that the reference to levels need not imply a hierarchy or ranking of importance. Ultimate and proximate explanations are different explanations, butdespite claims by somethese two types of questions are not hierarchically related, and research at one level is not inherently superior to research at the other level [10]. Apart from providing theoretical scaffolding by which to organize different approaches to the study of behaviour, the levels of analysis are important in that they clarify non-overlapping explanations and
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hypotheses [8]. For example, the statement that male birds sing in spring to attract mates is not mutually exclusive with the statement that male birds sing in spring due to increased levels of circulating testosterone. Of course, both of these hypotheses could be correct, or both could be incorrect. They are explanations at different levels of analysis; current adaptive function (ultimate) and physiological mechanism (proximate). Indeed, much fruitless debate could be avoided in some elds, such as evolutionary psychology, if opponents would acknowledge that both adaptive function and current socio-economic conditions can explain variation in human behaviour and that hypotheses at these two levels are not directly competitive. This issue is explored further in 2b below. One of the key issues in debates centred on levels of analysis is whether information from one level of analysis can inform research at the other. Sherman [8] notes that non-overlapping explanations occur even within the proximate or ultimate levels of analysis. For example, hypotheses regarding the current adaptive utility (trait X is adaptive because of Y ) and evolutionary history (trait X arose from a non-adaptive precursor) may be non-competing. I argue that mutually compatible explanatory hypotheses extend beyond traditional considerations of levels of analysis and do not only exist on different levels of analysis. An example of this is illustrated by the different hypotheses proposed to explain variation in immediate-early gene (IEG) expression in the auditory forebrain of songbirds (below). Whether or not two hypotheses are mutually exclusive is critical to preventing false debate either across, or within, levels of analysis.

(b) Non-alternative hypotheses: immediate-early gene expression in songbird brains Measuring the expression of IEGs in the brain has become a standard method for assessing which neurons are repeatedly depolarized during exposure to a stimulus. For example, expression of the gene c-fos is increased in the medial preoptic area and the medial and cortical amygdala of mother rats following exposure to pups [12]. In songbirds, increased expression of the IEG ZENK (hereafter Zenk response) occurs in auditory forebrain regions including the caudomedial mesopallium (CMM) and caudomedial nidopallium (NCM) following exposure to conspecic vocalizations. CMM and NCM exhibit greater Zenk response following playback of conspecic song as opposed to other stimuli [13]. The importance of these brain regions for auditory processing of song has been corroborated by electrophysiology [14] and a lesion study [15]. The amount of Zenk response in the auditory forebrain has been shown to correlate well with variation in the nature of the song stimuli. For example, the Zenk response in the NCM is positively related to the complexity of playback songs in budgerigars, Melopsittacus undulatus [16] and the bout length of playback songs in European starlings, Sturnus vulgaris [17]. Moreover, in white-crowned sparrows (Zonotrichia leucophrys), the Zenk response is higher in birds exposed to preferred local dialect songs, and

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Review. Levels of analysis the absence of attention if, for example, the birds were asleep (see [21]). The important point is that these two hypotheses are on the same level of analysis (proximate) and address the same category of question (neural and cognitive mechanisms) but are still nonalternative explanatory hypotheses (sensu [8]). Both of these hypotheses could be correct in that increased Zenk could both reect attentional processes and the locus of song memory; the two hypotheses are not mutually exclusive. Of course, future data could also prove both hypotheses incorrect. This example highlights the importance of formulating clear hypotheses and determining whether or not different hypotheses are truly alternatives. Hypotheses at different levels of analysis are explicitly nonalternatives, but so too are many hypotheses within the same level. Confusion over falsely competing hypotheses is not limited to hypotheses at different levels of analysis. Some integrative and adaptationist approaches have been criticized for confusing levels of analysis (discussed in [1,8]). However, this problem is not limited to elds that attempt to integrate between proximate and ultimate levels, and occurs within elds that are exploring the same mechanisms in the same research domain. Careful attention needs to be paid to determine whether both hypotheses are mutually exclusive of each other or not, regardless of whether one is a causal and one a functional hypothesis, or both are functional hypotheses. The example of IEG expression in the auditory forebrain highlights another point. Both of the above hypotheses can, and have, been criticized on the grounds that neither of them explains anything. Indeed, over the years, I have received several anonymous reviews pointing out that because it is unclear what is occurring at the cellular and molecular level, these hypotheses are void of mechanism. This category of comments reects a failure to distinguish levels of reductionism (sometimes referred to as levels of analysis, table 1), and an assumption that analyses at lower levels of reductionism are inherently superior. Dennett [27] refers to reductionism that values lower levels and ignores complexities and theory at higher levels as greedy reductionism. I refer to the view that research at lower levels of reductionism is inherently superior or more scientic as reductionist snobbery. These views value physics over chemistry, and chemistry over biology. Greedy reductionism and reductionist snobbery are also present within biology, for example, when authors restrict the use of the work mechanism to refer to cellular and molecular mechanisms and not behavioural or cognitive mechanisms, or explicitly value proximate explanations over ultimate [28]. Mechanisms, broadly speaking, include causal processes at the proximate level of analysis. These can include molecular, cellular or system level processes. The interaction of different organisms, different brain regions, different neurons, or different proteins can all be proximate mechanisms. We do not fully understand how auditory processing in the songbird auditory forebrain results in increased expression of Zenk, nor do we know the downstream target genes in these brain regions and how their regulation affects activity in these brain areas. However, incomplete understanding at one level of reductionism

correlates with the number of sexual displays emitted during song playback [18]. Thus, in a range of species, the Zenk response in auditory regions appears related to the biological salience of the song playback. What accounts for this correlation between the nature of the song playback and the ensuing Zenk expression? One explanation is that increased activation of neurons in NCM and/or CMM reects the auditory memories of songs learnt early in life. In support of this, the Zenk response to song playback is related to the accuracy of song learning in zebra nches (Taeniopygia guttata) [19,20]. Moreover, the Zenk response is higher in NCM and CMM following the playback of tutor song (compared with novel song) in young zebra nches in the process of song learning [21]. This differential Zenk response was absent in HVC (not an acronym, sometimes referred to as high vocal centre), a brain region critical for the production of learned song. Thus, growing evidence links the Zenk response in these caudal pallial regions with the memory of learned song that guides song production [22]. A different hypothesis used to explain variation in the Zenk response is that variation in IEG expression in the songbird auditory forebrain reects attention mechanisms. Kruse et al. [23] found that changing the playback context increases Zenk expression in response to the same song. Merely switching the speaker location resulted in an increased Zenk response compared with the song playing from the same location. Similarly, male song sparrows (Melospiza melodia) show increased Zenk response in NCM when they are exposed to novel territorial songs as compared to when they are exposed to songs they had previously heard [24]. In female house nches (Carpodacus mexicanus) and song sparrows, there was no differential Zenk expression in response to playback of tutor versus non-tutor songs; however, in both species, Zenk expression was higher in isolate-reared females than in tutored females, consistent with the idea that the isolates were more attentive to songs when compared with tutored birds [25,26]. Combined, the results of the studies above are consistent with the idea that increased Zenk expression is related to increased perceptual processing via some form of attentional gating mechanism. We thus have at least two testable hypotheses to explain variation in Zenk expression in the auditory forebrain. Hypothesis 1 is that increased Zenk response occurs because NCM and CMM provide the neural substrate for song memories. This hypothesis may or may not be correct for a number of reasons. For example, the locus of song memories may be elsewhere and the patterns of Zenk activation explained by other factors. Second, there may be no single locus of the song engram, and different aspects of song memories may be distributed over a range of perceptual and motor regions including NCM, CMM and the song-control regions such as HVC. Differentiating among these requires further experiments. Hypothesis 2 is that increased Zenk response reects increased perceptual processing owing to an attentional gating mechanism. Similarly, this hypothesis may or may not be correct. The patterns described above are consistent with birds exhibiting variation in attention to song playback, but could result even in
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Review. Levels of analysis S. A. MacDougall-Shackleton does not negate hypothesis testing at another level. Studies of cellular and molecular mechanisms, physiological mechanisms, developmental processes, and indeed cognitive and behavioural mechanisms are all at the proximate level of analysis.

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(c) Cause and function: integrating across ultimate and proximate levels of analysis Because explanations at different levels of analysis are not mutually exclusive alternatives, they cannot directly compete with each other. Indeed, there is almost unanimous consensus that we need explanations at both ultimate and proximate levels for a complete understanding of behaviour [2]. Some argue for boundaries between levels of analysis in order to eliminate confusion between them (e.g. [29]). Hogan [11] further points out that cause and function are logically distinct categories, and rejects Mayrs terminology of ultimate causation. Because adaptations are the outcome of selection, it is teleological to argue that adaptive function can, in any sense, be an ultimate cause of behaviour. More to the point, Hogan [11] argues that function can tell us nothing about proximate causation. Similarly, Bolhuis [2] argues that, at most, functional considerations can provide only clues as to potential mechanisms. Despite the clear distinction between proximate and ultimate levels of analysis, however, approaches that integrate across these levels are thriving. Behavioural ecology, traditionally focused on ultimate questions, has become more and more focused on proximate mechanisms including immunology, endocrinology, neurobiology and development. Such approaches use information from the proximate level of analysis to help formulate and revise hypotheses at the ultimate level. Indeed, many argue that a failure to integrate across levels is likely to lead to erroneous interpretations [30], and that data and interpretations drawn from one level can inform data and interpretations at the other. McNamara & Houston [31] argue that it is necessary to integrate mechanisms when exploring the evolution of behaviour. Not all agree that integration across levels of analysis is a useful research strategy, however (e.g. [1]), and suggest that instead research at the proximate level should focus on naturally occurring behaviours, but without presuppositions as concerning the evolutionary or functional signicance of them. I disagree. Taking an adaptationist stanceassuming that neural and cognitive mechanisms have function, and have been shaped by natural selectiondoes more than providing clues as to their mechanisms. Assuming adaptive function or design (the intentional or adaptationist stance) is an efcient and sensible way to gure out how something works [27,32]. In reverse engineering something, an adaptationist stance (assuming design) constrains and directs lines of inquiry from vast to those best suited to the data [27,32]. The discovery of how the heart acts as a pump, for example, was no doubt aided by the assumption that the heart has a (adaptive) function: that of, well, a pump. Similarly, the study of mechanisms of behaviour can benet
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from the adaptationist stance. A research programme aimed at understanding mechanisms of behaviour that truly and entirely ignores function becomes a Brownian random walk at the proximate level. It is important to note, however, that an adaptationist stance does not imply Panglossian hyper-adaptationism (sensu [33]). Assuming an adaptive function as a way to develop and test hypotheses is not synonymous with accepting such hypotheses despite evidence to the contrary. Particular neural or behavioural mechanisms cannot be automatically assumed, just because a behaviour has a particular adaptive function. An integrative approach uses considerations of function and evolutionary history to generate hypotheses as to proximate mechanisms, and then proceeds to test, and possibly to reject, these hypotheses [34]. Adaptationist integrative biologists use functional considerations to interpret data and generate hypotheses. Sherry [35,36] highlights several ways that a consideration of adaptive function can aid in the study of mechanisms. First, function can provide guidance in developing hypothesis at the proximate level (3a). Second, adaptive function can dene the categories of behaviour that are further explored at the proximate level (3b). Third, consideration of function can set criteria that proximate explanations must satisfy (3c). In addition to these benets, an integrative approach has at least one other major strength. Not only does a consideration of adaptive function help guide research and interpretation of proximate mechanisms, but data and interpretations of proximate mechanisms often reciprocally guide and rene functional and evolutionary hypotheses (3d).

3. THE ADAPTATIONIST INTEGRATIVE APPROACH (a) Generating hypotheses The power of the adaptationist stance to generate novel hypotheses is a major strength of an adaptationist integrative approach, such as neuroecology or behavioural ecology. Sherry [35] documents several examples including olfactory learning in rodents resulting in disassortative mating at the major histocompatibility complex [37] and the use of hippocampus-dependent spatial memory to retrieve cached food by chickadees [38,39]. The rst of these examples arose from evolutionary theory on inclusive tness [40], which raises the proximate question: how do animals recognize their relatives? The second example arose from a functional consideration that food-storing birds increase overwinter survival through caching, which raises the question: how do birds retrieve cached food? A third example is sex ratio manipulation. Following long-standing theory that sex ratios should be maintained at 1 : 1 [41], Trivers & Willard [42] predicted that parents should be able to adjust offspring sex ratio depending on their condition. This differential sex-allocation theory has been supported by numerous studies, and there is good evidence that this can even apply to primary sex ratios in vertebrates. For example, collared ycatcher (Ficedula albicollis) females appear able to adjust the sex ratio of eggs ovulated in response to the phenotype of their mate [43].

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Review. Levels of analysis zebra nches. As may be predicted, HVC lesions did not disrupt song preferences in female zebra nches, but CMM lesions did [15]. Earlier work on marsh wrens (Cistothorus palustris) similarly suggested that HVC might not be the primary brain region underlying song perception and song memories. Males of the western subspecies sing more complex songs and have a larger HVC than males of the eastern subspecies [50]. However, HVC size differences between these subspecies do not reect song-learning experience [51], nor are there differences between the subspecies in HVC size in females [52]. These studies took an adaptationist, integrative approach and assessed whether variation in HVC size might reect differences in song learning or song perception. The results did not support this idea, and the search for the neural substrates of song perception continued. If birds are adapted to process species-specic songs differently from other sounds, they should possess brain regions that receive auditory input and respond specically to song more strongly than other sounds. This is exactly what was found with the discovery of the connectivity of CMM and NCM to other auditory regions and the IEG response of these regions to song playback [13,53,54]. As noted in 2b, above, there is growing evidence that these regions are critical for song perception in many species, and may also function as the neural basis of song memories. It is important to note, however, that these neural regions important for specialized auditory processing of song, and which may also serve in storing auditory memories of song, were discovered by considering clues suggested by adaptive function, not by considering the behaviour in the absence of presuppositions concerning their evolutionary or functional signicance. It is too early to entirely rule out a role for the song-control brain regions as important for song perception and memories, as correlations and lesion data from at least canaries (Serinus canaria) do support this [55 58]. Further work is clearly required to better understand how the song-control system and specialized auditory regions such as CMM and NCM coordinate song production and learning, song memories and song perception. These efforts will benet from comparative approaches that test whether neural differences reect behavioural differences among taxa.

This raises the novel proximate question: how do birds adjust the primary sex ratio of their eggs? Recent evidence suggests that hormone levels of the mother may play a role. For example, high progesterone during meiosis can bias sex ratios towards overproduction of females [44]. Other hormones may affect primary sex ratios as well, and research is ongoing (reviewed in [45]). Much study remains to work out the molecular mechanisms of this remarkable process. However, it is unlikely that any of these research groups would have undertaken the detailed and expensive mechanistic studies to explore how birds manipulate primary sex ratio without previous evidence that birds do indeed manipulate primary sex ratio in the wild. These eld observations were in turn motivated by predictions from sex-allocation theory that assumed adaptive function. Consideration of adaptive function provides motivation and direction to explore novel proximate mechanisms. Of course, consideration of adaptive function sometimes generates proximate hypotheses that are not supported, or are not good ideas to begin with. For example, one might predict that since, in many songbird species, males learn to sing and females do not, males might have been selected to be better auditory learners than females. As noted by Bolhuis & Macphail [1], there is no evidence for this hypothesis. Of course, one might also predict the opposite. Since, in many species, females are the primary receivers of song, might they be superior to males in auditory perception or auditory learning? Evidence suggests that this may be the case in some species. Although data are scant [46], some evidence indicates that female Bengalese nches (Lonchuria striata) are more sensitive to changes in song stimuli than males, as assessed by changes in heart rate [47]. Similarly, female redwinged blackbirds (Agelaius phoeniceus) appear to be more perceptive of songs than are males [48]. Whether or not a hypothesis about proximate mechanisms that was derived from evolutionary predictions is supported by the data, or was a good idea to begin with, is unrelated to the fact the hypothesis was based on ideas from the ultimate level of analysis. As one further example of the importance of using information on adaptive function to explore mechanism I return to the issue of which regions in the songbird brain underlie song perception and auditory song memory. Much of the early research on the neural bases of song perception and auditory memories focused on the song-control system, brain regions demonstrated to be important for song production and acquisition [22]. Current evidence suggests that this may not be the case, and instead good candidates for the regions important for perception and song memories include auditory regions, such as CMM and NCM. Considering function, however, directly inuenced the discovery that these areas are important for song perception. For example, the consideration that song functions in female attraction led to the hypothesis that, because female zebra nches have a tiny HVC compared with males [49], it would be unlikely that the song region HVC is important for song perception. That is, if HVC is critical for song perception, it should not be so small in female
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(b) Dening categories Besides generating novel hypotheses, adaptive function denes the categories of behaviour that we study. Function denes elds. Indeed, physiological systems are often named for what they do: circulatory system, immune system, digestive system. Though not commonly thought of in this way, these elds reect the functional organization of physiology, shaped through natural selection in response to the competition for survival and reproduction. The function of the digestive system, though sometimes considered in the absence of evolution, is indeed an adaptive function. Individuals better able to extract required energy and nutrients from their food will outcompete those less able to do so.

Review. Levels of analysis S. A. MacDougall-Shackleton In behaviour, considering adaptive function similarly denes clusters of behaviour that are studied together: communication, foraging, kin recognition. Moreover, considering adaptive function can help resolve categorization of behaviours in ways that may assist understanding of mechanistic causes. For example, maternal aggression in rodents is phenotypically similar to other forms of aggression, but its endocrine regulation involves changes in hormones associated with pregnancy and maternal behaviour, such as oestrogen and progesterone [59]. Thus, considering the function of maternal behaviour can assist in the exploration of mechanism. That function denes categories of behaviour that are studied together is more or less self-evident. However, these categories can also guide the studies of proximate mechanism. For example, pre-existing neuroendocrine mechanisms can be co-opted through evolution to control different behaviours with similar function [60]. The gonadal steroids (e.g. testosterone, oestradiol) that regulate gamete production later evolved to control sexual behaviour. The peptide hormone prolactin triggers brooding and crop milk production in male ring doves (Streptopilia risoria; [61]) and is associated with paternal behaviour in California mice (Peromyscus californicus [62]). In this case, the common adaptive function (paternal care) may be underpinned by similar proximate mechanisms (prolactin). However, it is critical to also note that very different mechanisms can give rise to similar outcomes. For example, evolution can result in changes in the hormone-dependent behaviours that may not be initially predicted by ecological or evolutionary perspectives. Testosterone may be critical for the expression of sexual behaviour in one species, such as laboratory rats [63], but not in another, such as red-sided garter snakes (Thamnophis sirtalis [64]). Similarly, work in evolutionary developmental biology has shown gene expression underlying traits with the same adaptive function may, or may not, be the same. For example, eyes in mammals and insects appear to have evolved independently, but their development is inuenced by the regulatory gene Pax6 in both taxa [65]. Such deeply homologous traits that have different morphology may develop through expression of the same genes [66]. In this case, the adaptive function (vision) may be implemented by different non-homologous morphological structures (eyes) in different taxa, that are, in turn, dependent on developmental expression of (deeply) homologous regulatory genes. Adaptive function denes categories of behaviour. The mechanisms within these categories may or may not be similar across taxa. Thus, functional categories may or may not reect categories of mechanisms. However, considering adaptive function provides a useful framework to explore mechanisms within a category to determine where they differ and where they are the same.

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are to have any explanatory power [35]. Part of the reason for this involves design constraints [27]. The number of shapes that can be used to create a useful wheel are limited. Thus, hypotheses about how an object can be moved using rollers are limited in scope. Similarly, there are only so many ways that an object can be spatially located by sound, or that the earths magnetic eld can be used for navigation [35]. Hypotheses regarding the neural mechanisms of sound localization by vertebrates must deal with inter-aural time, intensity and phase differences. These are the mechanistic bases by which the problem can be solved. Hypotheses regarding the sensory and neural mechanisms of magnetic compasses must deal with polarity, strength and inclination of the earths magnetic eld. These are the kinds of information that could theoretically be used by a magnetic compass. It is the assumption that natural selection has designed sensory systems with a function that allows us to dene the problems that neurosensory systems must solve [35]. Thus, consideration of the adaptive function of the system (e.g. sound localization) sets criteria that any potential mechanistic hypotheses must satisfy. Although the adaptive function of behaviour sets criteria that must be met, the discovery of proximate mechanisms may take unexpected turns. Factors that might be expected to be important based on design criteria may turn out to not be. For example, the regulation of seasonal reproduction must rely on environmental cues that change seasonally which can affect internal timing mechanisms. Photoperiod is one such environmental cue, and it might be predicted that declining day-length would act as a proximate signal to terminate reproduction in spring/summer breeders. However, in many species of songbirds, the declining length of daylight in late summer has nothing to do with the termination of seasonal reproduction. Rather, exposure to long days in early spring, by mechanisms not fully understood, results in the eventual onset of photorefractoriness and termination of reproduction [67,68]. Similarly, design considerations constrain the types of cues that could be used by a magnetic compass, and strong evidence indicated early on that migratory birds use inclination [69]. However, the more recent discovery that cryptochromes may be the molecules that underlie birds light-dependent magnetic compass did not simply result from consideration of function, and depended on the input from physics theory [70]. Thus, considering adaptive function can set design criteria and inform exploration of proximate mechanisms, but is clearly not sufcient in and of itself for the discovery of such mechanisms.

(c) Setting criteria Considering function can also set criteria that hypotheses about proximate mechanisms must satisfy if they
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(d) Proximate ultimate reciprocity There is at least one other benet of considering adaptive function when exploring proximate mechanisms. This occurs when data on proximate mechanisms are then used to rene and direct hypotheses at the ultimate level of analysis. Such reciprocity between levels of analysis can be the hallmark of the best integrative biology. Here, I review several examples where

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Review. Levels of analysis simple correlation. Sex differences in HVC are affected by phylogeny, and exhibit a persistent male bias [83]. Indeed, a recent study of a species where females sing more than males still found a malebiased HVC size [84]. In another species, where males and females have identical songs, males have a larger HVC, but females HVC has much higher expression of synapse-related genes [85]. Such studies raise many further questions at both proximate and ultimate levels. What was the ancestral condition; have sex differences increased or decreased over evolutionary time? In species where females sing equally to males, but have a smaller HVC, how do they accomplish this? Do they use alternative neural mechanisms than males to learn and produce song? Do they incur any developmental trade-offs by using such mechanisms? Understanding of the evolution of the sex differences in the song-control system will require better understanding of the development of these differences. Although initially considered a model for the organizational effects of gonadal steroids, currently these sex differences are thought to emerge from sex differences in the genome [86]. Further work is required to determine both the evolution and mechanisms of sex differences in the song-control regions of the brain, but this will be aided by consideration of both ultimate and proximate levels of analysis. Finally, studies of proximate mechanisms can inuence studies at the ultimate level by highlighting constraints that need consideration. Animals are not innitely exible in their capacity to respond to the environment. A recent review has called for a systematic study of the evolution of mechanisms (evo-mecho), with a focus on simple mechanisms that work well in diverse environments as opposed to complex mechanisms that work well in simple environments [31]. It is unclear to what extent this call will be heeded, but it is important to note that there is growing consensus that studies of behaviour at the ultimate level of analysis cannot ignore proximate mechanisms.

consideration of mechanisms has guided the generation of hypotheses regarding evolution and function. Fluctuating asymmetry provides an example where a proximate mechanism resulted in a large number of functional hypotheses. Fluctuating asymmetries are random deviations from perfect symmetry in bilaterally symmetric organisms, and individuals with greater asymmetry are presumed to have lower ability to cope with environmental stressors during development (often referred to as developmental stability [71]). Consideration of mechanisms of development resulted in wide interest in behavioural ecology in the functional signicance of uctuating asymmetry in visual signals in a range of taxa [72,73]. In many species, symmetric individuals are preferred mates when compared with more asymmetric individuals [74]. In turn, considering the function of preferences for symmetry generated novel proximate hypotheses. At the ultimate level, individuals may be selected to prefer symmetrical mates because such mates may pass on heritable traits that allow stable development. Thus, perhaps symmetrical features are signals that convey information about an individuals developmental stability to potential mates. This ultimate hypothesis then begs the proximate question: can individuals perceptually detect uctuating asymmetry? In a series of studies, Swaddle [75] and Swaddle et al. [76] have shown that the ability of birds to detect asymmetry in visual features is limited. Birds can detect asymmetries in visual displays, but not the small differences such as observed in asymmetrical morphological traits. These new data on proximate mechanism must now be accounted for and lead to the revision of ultimate hypotheses. If asymmetry is not detected, how can it be a signal? Perhaps asymmetry is correlated with other traits that are detectable and do act as signals. Studies of uctuating asymmetry have had their share of methodological and theoretical challenges [73,77]; however, the reciprocal ow of data and interpretations have moved the eld forward. A second example of proximate and ultimate reciprocity involves sex differences in song-control regions of the songbird brain. Shortly after the discovery of the song-control system, Nottebohm & Arnold [49] found that brain regions such as HVC exhibited one of the largest anatomical neural sex differences observed in vertebrates. In zebra nches, females cannot sing, and male HVC is an order of magnitude larger than that in females. In canaries, however, where females can sometimes sing (but less than males), the sex difference was smaller. This proximate observation led Brenowitz & Arnold [78] to test the ultimate hypothesis that perhaps sex differences in the brain co-evolved with sex differences in behaviour. In a study of tropical wrens where males and females sing equally, they found no sex differences in the size of HVC. Since that time, the story has become more complex, and the interplay between proximate mechanism and function and evolutionary history has continued. In some species where females sing equal to males, sex differences in HVC are still observed [79 81]. However, comparative analyses do demonstrate a correlation between sex differences in behaviour and in the brain [82], but it is not a
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4. CONCLUSION In this review, I have highlighted different uses of the term levels of analysis, and explored the benets of using an integrative approach that uses an understanding of adaptive function to guide exploration of proximate mechanisms. This endeavour of integration is not without risk, however. It is critical to thoroughly evaluate whether or not two hypotheses are mutually exclusive. By denition, hypotheses at the ultimate and proximate levels of analysis are not mutually exclusive, but it is often ignored that presumably competing hypotheses within a level of analysis, or at different levels of reductionism, may also be non-exclusive. That is, two hypotheses may be non-exclusive even if they are at exactly the same level of analysis and level of reductionism. Much false debate could be avoided by careful consideration of whether or not alternative hypotheses are or are not mutually exclusive alternatives. Because ultimate and proximate levels do not directly compete, however, does not mean they should have nothing to do with each other. An

Review. Levels of analysis S. A. MacDougall-Shackleton adaptationist, integrative approach uses knowledge of one level to guide research at the other. Explanations at these two levels must at least be concordant, even if they do not directly compete. Failure to consider ultimate hypotheses when conducting research on proximate mechanism can result in greedy reductionism. Similarly, researchers at the ultimate level need to be careful to not underestimate the complexities of proximate mechanisms of the behaviours under their study. Integrating across levels of analysis is tricky business. Researchers often have primary expertise and training in one level or the other. They must thus tread carefully when in each others domain. A common charge is that evolutionary ecologists have too simple a view of proximate mechanisms, or that cellular and molecular biologists have too simple a view of behaviour and ecology. Indeed, I suspect that many of the critiques of integrative approaches stem from researchers in one eld failing to address or appreciate complexities in another. Integration across levels of analysis thus requires detailed knowledge of other research domains, and is thus often best conducted via collaborations. A complete understanding of behaviour, or any biological phenomenon, requires explanations of both cause and function. Achieving these explanations is best done in an integrative fashion. Consideration of function can guide research into mechanisms in many ways. Similarly, research into adaptive function can be stimulated and guided by a detailed appreciation of mechanisms. Although there is risk of confusion, careful consideration of one level of analysis can benet research at the other.
I thank David Sherry and Beth MacDougall-Shackleton for many discussions on this topic. Rinaldo Bertossa, Tom Smulders and anonymous reviewers provided helpful comments on the manuscript. My research is funded by NSERC Canada.

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25 Hernandez, A. M. & MacDougall-Shackleton, S. A. 2004 Effects of early song experience on song preferences and song control and auditory brain regions in female house nches (Carpodacus mexicanus). J. Neurobiol. 59, 247 258. (doi:10.1002/neu.10312) 26 Hernandez, A. M., Phillmore, L. S. & MacDougallShackleton, S. A. 2008 Effects of learning on song preferences and Zenk expression in female songbirds. Behav. Process. 77, 278 284. (doi:10.1016/j.beproc. 2007.11.001) 27 Dennett, D. C. 1995 Darwins dangerous idea. New York, NY: Simon & Schuster. 28 Sherry, D. F. 2004 Asking how? versus asking why?. Cerebrum 6, 8998. 29 Reeve, H. K. & Sherman, P. W. 1993 Adaptation and the goals of evolutionary research. Q. Rev. Biol. 68, 1 32. (doi:10.1111/j.1469-185X.1993.tb00731.x) 30 Ryan, M. J. 2005 The evolution of behaviour, and integrating it towards a complete and correct understanding of behavioural biology. Anim. Biol. 55, 419 439. (doi:10. 1163/157075605774841012) 31 McNamara, J. M. & Houston, A. I. 2009 Integrating function and mechanism. Trends Ecol. Evol. 24, 670 675. (doi:10.1016/j.tree.2009.05.011) 32 Dennett, D. C. 1987 The intentional stance. Cambridge, MA: MIT Press. 33 Gould, S. J. & Lewontin, R. C. 1979 The spandrels of San Marco and the Panglossian paradigm: a critique of the adaptationist programme. Proc. R. Soc. Lond. B 205, 581 598. (doi:10.1098/rspb.1979.0086) 34 MacDougall-Shackleton, S. A. & Ball, G. F. 2002 Revising hypotheses does not indicate a awed approach: reply to Bolhuis and Macphail. Trends Cogn. Sci. 6, 68 69. (doi:10.1016/S1364-6613(00)01847-7) 35 Sherry, D. F. 2005 Do ideas about function help in the study of causation? Anim. Biol. 55, 441 456. (doi:10. 1163/157075605774840950) 36 Sherry, D. F. 2006 Neuroecology. Annu. Rev. Psychol. 57, 167197. (doi:10.1146/annurev.psych.56.091103.070324) 37 Penn, D. J. 2002 The scent of genetic compatibility: sexual selection and the major histocompatibility complex. Ethology 108, 1 21. (doi:10.1046/j.1439-0310. 2002.00768.x) 38 Hampton, R. R. & Shettleworth, S. J. 1996 Hippocampal lesions impair memory for location but not color in passerine birds. Behav. Neurosci. 110, 831 835. (doi:10.1037/0735-7044.110.4.831) 39 Sherry, D. F. & Vaccarino, A. L. 1989 Hippocampus and memory for food caches in black-capped chickadees. Behav. Neurosci. 103, 308 318. (doi:10.1037/07357044.103.2.308) 40 Hamilton, W. D. 1964 The genetical evolution of social behaviour. J. Theoret. Biol. 7, 1 52. (doi:10.1016/00225193(64)90038-4) 41 Fisher, R. A. 1930 The genetical theory of natural selection. Oxford, UK: Clarendon. 42 Trivers, R. L. & Willard, D. E. 1973 Natural selection of parental ability to vary the sex ratio of offspring. Science 179, 9092. (doi:10.1126/science.179.4068.90) 43 Ellegren, H., Gustafsson, L. & Sheldon, B. C. 1996 Sex ratio adjustment in relation to paternal attractiveness in a wild bird population. Proc. Natl Acad. Sci. USA 93, 11 72311 728. (doi:10.1073/pnas.93.21.11723) 44 Correa, S. M., Adkins-Regan, E. & Johnson, P. A. 2005 High progesterone during avian meiosis biases sex ratios toward females. Biol. Lett. 1, 215 218. (doi:10.1098/ rsbl.2004.0283) 45 Alonso-Alvarez, C. 2006 Manipulation of primary sexratio: an updated review. Avian Poultry Biol. Rev. 17, 120. (doi:10.3184/147020606783437930)
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castrated male rats. J. Comp. Physiol. Psychol. 42, 433453. (doi:10.1037/h0059086) Camazine, B., Gartska, W., Tokarz, R. & Crews, D. 1980 Effects of castration and androgen replacement on male courtship behavior in the red-sided garter snake (Thamnophis sirtalis). Horm. Behav. 14, 358 372. (doi:10.1016/ 0018-506X(80)90025-2) Kozmik, Z. 2005 Pax genes in eye development and evolution. Curr. Opin. Genet. Dev. 15, 430 438. (doi:10. 1016/j.gde.2005.05.001) Shubin, N., Tabin, C. & Carroll, S. 2009 Deep homology and the origins of evolutionary novelty. Nature 457, 818 823. (doi:10.1038/nature07891) Dawson, A., King, V. M., Bentley, G. E. & Ball, G. F. 2001 Photoperiodic control of seasonality in birds. J. Biol. Rhythms 16, 365380. (doi:10.1177/074873001129002079) Nicholls, T. J., Goldsmith, A. R. & Dawson, A. 1988 Photorefractoriness in birds and comparison with mammals. Physiol. Rev. 68, 133176. Wiltschko, W. & Wiltschko, R. 1972 Magnetic compass of European robins. Science 176, 62 64. (doi:10.1126/ science.176.4030.62) Liedvogel, M. & Mouritsen, H. 2010 Cryptochromes: a potential magnetoreceptor: what do we know and what do we want to know? J. R. Soc. Interface 7, S147 S162. (doi:10.1098/rsif.2009.0411.focus) Parsons, P. A. 1992 Fluctuating asymmetry: a biological monitor of environmental and genomic stress. Heredity 68, 361364. Mller, A. P. & Swaddle, J. P. 1998 Asymmetry, developmental stability, and evolution. Oxford, UK: Oxford University Press. Swaddle, J. P. 2003 Fluctuating asymmetry, animal behavior, and evolution. Adv. Study Behav. 32, 169205. (doi:10.1016/S0065-3454(03)01004-0) Mller, A. P. & Thornhill, R. 1998 Bilateral symmetry and sexual selection: a meta-analysis. Am. Nat. 151, 174 192. (doi:10.1086/286110) Swaddle, J. P. 1999 Limits to length asymmetry detection in starlings: implications for biological signalling. Proc. R. Soc. Lond. B 266, 12991303. (doi:10.1098/ rspb.1999.0778) Swaddle, J. P., Ruff, D. A., Page, L. C., Frame, A. M. & Long, V. A. 2008 A test of receiver perceptual performance: European starlings ability to detect asymmetry in

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Phil. Trans. R. Soc. B (2011) 366, 20862099 doi:10.1098/rstb.2010.0336

Review

Neural mechanisms underlying the evolvability of behaviour

Paul S. Katz*
Neuroscience Institute, Georgia State University, PO Box 5030, Atlanta, GA 30302, USA The complexity of nervous systems alters the evolvability of behaviour. Complex nervous systems are phylogenetically constrained; nevertheless particular species-specic behaviours have repeatedly evolved, suggesting a predisposition towards those behaviours. Independently evolved behaviours in animals that share a common neural architecture are generally produced by homologous neural structures, homologous neural pathways and even in the case of some invertebrates, homologous identied neurons. Such parallel evolution has been documented in the chromatic sensitivity of visual systems, motor behaviours and complex social behaviours such as pair-bonding. The appearance of homoplasious behaviours produced by homologous neural substrates suggests that there might be features of these nervous systems that favoured the repeated evolution of particular behaviours. Neuromodulation may be one such feature because it allows anatomically dened neural circuitry to be re-purposed. The developmental, genetic and physiological mechanisms that contribute to nervous system complexity may also bias the evolution of behaviour, thereby affecting the evolvability of species-specic behaviour. Keywords: homoplasy; neuromodulation; convergent evolution; homologous neurons; evolutionary development; neural circuits

1. INTRODUCTION The eld of evolutionary development (evo-devo) seeks to explain phylogenetic differences in the form or function of organisms in terms of developmental and genetic processes [13]. This has been particularly successful in clarifying the origins of species differences in morphology that can be directly observed. Applying the principles of evo-devo to behaviour is more complicated because behaviour is produced by the nervous system interacting with the body and the environment. Therefore, mechanistic explanations for phylogenetic differences in behaviour must explain how species differences in behaviour are created by nervous systems that are derived from a common ancestor, i.e. what developmental and genetic processes led to the neural mechanisms underlying behaviours seen in the various species? The nervous systems in major animal phyla, such as vertebrates, arthropods, molluscs and annelids, contain a greater variety of cell types than any other organ in the body. Furthermore, these cells (neurons) form highly specic synaptic interconnections and exhibit temporally dynamic neural activity. Given the complex nature of the nervous system, one might wonder how it would be possible to evolve adaptive behaviour at all; any alteration of a complex system would be likely to produce deleterious results. In fact, large reorganizations or structural transformations

*pkatz@gsu.edu One contribution of 10 to a Theme Issue Evolutionary developmental biology (evo-devo) and behaviour.

have been rare, indicating that the nervous system and behaviour are to a large extent phylogenetically constrained. Paradoxically, the structure and dynamics of complex nervous systems may facilitate the evolution of particular behaviours, which appear repeatedly in different species within a lineage. The mechanisms underlying the development of nervous system complexity include rules that enable novel structures to be incorporated. Furthermore, neural dynamics allow the generation of multiple activity patterns. Thus, precisely because it is complex, the nervous system exhibits features that allow for and even promote the evolution of certain behaviours. Here, I will argue that such features can be said to affect the evolvability of behaviour, where evolvability is dened as the capacity of a lineage to evolve [4]. It has been asserted that assessing evolvability is critical for a mechanistic understanding of evolutionary phenomena [5,6]. This has been discussed from a genetic point of view [4,7,8], but not often from a macroscopic view. There has been disagreement on whether evolvability itself is a trait that can be selected for because evolvability never benets the tness of the individual, it acts at the level of species selection [9 13]. In this article, it will be argued that evolvability of behaviour (both positive and negative) can arise directly from the development and physiology of nervous systems. Thus, regardless of whether it has been selected for, evolvability of behaviour can arise as a secondary consequence of having a complex nervous system.
This journal is q 2011 The Royal Society

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Review. Neural basis of behavioural evolvability 2. NERVOUS SYSTEMS ARE PHYLOGENETICALLY CONSTRAINED In many respects, the gross structures of nervous systems have been strongly conserved within phyla. This has allowed neuroscientists to extrapolate the functions of homologous brain regions across species within a taxon. For example, work on rodent hippocampus informs our understanding of primate hippocampus. Clearly, the shapes and relative sizes of neural structures vary [14,15]; yet, all Gnathostomata (Vertebrata) have forebrain, midbrain and hindbrain and the cerebellum always has Purkinje cells that project to deep cerebellar nuclei [16]. Still, there are differences in the details across taxa. For example, in mammals, the cerebellum has a highly conserved pattern of transverse stripes that subdivide transverse zones, which can be revealed by gene or protein-expression patterns. Microchiropteran bats as a group, however, have an altered expression pattern of these markers, which may have functional signicance for echolocation [17]. Gross organizational characteristics of the brain map very well onto phylogenetic trees for mammals [18] as well as insects [19], suggesting that gross morphological characters exhibit phylogenetic constraints and do not account for species-level differences in behaviour. In insects, major areas and pathways are recognizable across members of a taxon such as Diptera [19]. These brain regions are also recognizable across major taxa such as between insects and Crustacea [20] and possibly even across phyla [21 23]. This is not to say that major changes in organization have not occurred; certainly, there are important differences in structure that correspond to functional divergence in the visual system and mushroom body of insects [24 26]. In contrast to gross morphology, there are species differences in microcircuitry that are not readily apparent in the overall connectivity in dipteran nervous systems [27,28]. The extent to which neural circuitry changed during evolution to produce species-specic behaviour in closely related species is still an open question. It might seem that the complexity of the nervous system would be a constraint on the evolution of behaviour [29]; random changes to a complex system would more probably have a negative impact on system function than be adaptive. This would result in the retention of ancestral neural traits and pathways, thereby decreasing the evolvability of the nervous system [30]. However, the very developmental mechanisms that allow the nervous system to be so complex might also enable it to accept novel inputs. For example, much of the wiring of the vertebrate nervous system self-assembles using simple developmental rules. One such rule is that neurons that tend to be active at the same time will form synapses with each other. Such activity-dependent sorting rules enable the nervous system to develop in a coherent manner even in the presence of a novel set of inputs such as occurs either experimentally or evolutionarily [31 34]. Thus, although these developmental rules play a role in setting up the nervous system, they also might enable the evolution of novel sensory systems.
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3. EVOLVABILITY OF SENSORY RECEPTION Behavioural responses to conspecics, predators or food sources depend upon sensory transduction. Species differences in the range or qualities of sensory stimuli that can be transduced could account for species-specic behaviour. For example, in butteries, the evolution of the ability to detect short-wavelength light is thought to have driven the evolution of blue wing coloration. Moreover, sexual dimorphisms in long-wavelength opsins may have evolved for conspecic recognition [35]. Similarly, it has been suggested that the evolution of trichromatic vision in primates was an adaptation that provided selective advantage for nding food sources [36 39]. There have been recurrent evolutionary gains and losses of chromatic sensitivity in vertebrates as well as in arthropods [40,41]. Changes in wavelength sensitivity have occurred several times through duplication of photopigment genes that allowed diversication of opsins and subsequent amino acid substitutions that shifted their absorbance spectra [4244]. In insects, long-wavelength photopigments arose independently in various lepidoptera (gure 1a) and hymenoptera clades [45,46]. The same amino acid substitutions repeatedly occurred in the transition to long-wavelength opsin in both groups (gure 1b) [44]. Models of the proteins function show that sites of parallel amino acid substitutions are close to the chromophore [47]. Mutagenesis of this site in bovine opsin alters spectral sensitivity [48]. There may be a limited number of protein congurations that generate long-wavelength sensitivity. Selection may therefore have repeatedly arrived at one of the available congurations that imparts long-wavelength sensitivity. Such structural determinants can even be seen across phyla; independently evolved shortwavelength opsins in both butteries and primates show similar substitutions in binding sites for 11-cisretinal that produce a shift in absorbance towards blue [49]. These results provide an example of the twin faces of evolvability; inherent in the nature of the photopigment is the ability to shift the absorbance to long or short wavelengths, but only certain critical amino acid substitutions will sufce. Thus, the same substitutions are observed to have occurred repeatedly over the course of evolution.

4. EVOLVABILITY OF SENSORY CIRCUITS Evolution of additional opsins seems straightforward; however, for those opsins to have an effect on behaviour, the neural circuits would need to respond adaptively. For instance, for trichromatic vision to evolve from dichromatic vision, not only would there need to be an additional photopigment, it would also have to be segregated into different photoreceptors and those photoreceptors would need to form the proper synaptic connections. In the mammalian retina, this presumably would involve the formation of antagonistic centre-surround receptive eld properties in retinal ganglion cells. Experiments in mice suggest that developmental rules and plasticity are sufcient to allow neural circuits to form trichromatic circuits when presented with additional photopigments. Transgenic mice that expressed modied human photopigments showed a

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(a)

Apodemia mormo LWRh2 (600) Lycaena rubidus (568) Apodemia mormo LWRh1 (505) Pieris rapae (563) Papilio xuthus PxRh3 (575) Papilio xuthus PxRh1 (530) Papilio xuthus PxRh2 (515) Manduca sexta 10#
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Adelpha bredowi Agraulis vanillae Amathusia phidippus LWRh1 Anartia jatrophae Apodemia mormo LWRh1 Apodemia mormo LWRh2 Papilio xuthus PxRh1 Papilio xuthus PxRh2 Papilio xuthus PxRh3

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(b)

IT

Figure 1. Parallel evolution of long-wavelength photopigments in butteries and moths. (a) Dendrogram showing parallel evolution of opsins that absorb long-wavelength light. The numbers in parentheses after the species names represent the maximum absorbance wavelength (in nanometres). Both Apodemia mormo (LWRh2) and Papilio xuthus (PxRh3) are signicantly redshifted with respect to other pigments. The shift occurred independently as intermediate pigments show absorbance at shorter wavelengths. (b) Alignment of partial coding sequences for lepidopteran opsins. Identical amino acid substitutions occurred in positions 10, 23 and 29 of transmembrane domain no. 1 (TM1) for Apodemia mormo (LWRh2) and Papilio xuthus (PxRh3) (adapted from Frentiu et al. [44]).

remarkable segregation of those opsin genes to different photoreceptors [50]. Subsequent work suggested that there is a developmental mechanism that causes mutually exclusive expression of opsin genes in cones [51]. This leads to chromatic sensitivity in retinal ganglion cells [52]. Adult mice with additional photopigments exhibit a novel ability to discriminate colour [53]. Thus, retinal circuits have mechanisms that seem to fortuitously allow them to process input from additional photopigments, thereby facilitating the evolution of trichromatic from dichromatic vision. Having a single receptor gene expressed per sensory cell has been thought to be important for the circuits to encode differences in the responses to each of the receptor types. It was asserted that there may be developmental mechanisms present in many sensory systems across phyla, which allow just a single receptor type to be expressed in a primary sensory neuron [54]. Such a pattern could be produced by a process of negative feedback [55]. These mechanisms may be in use for certain olfactory systems as well as visual systems [56]. Although such mechanisms would play a role in normal development, they also provide a means for the evolution of sensory neural circuits that respond to different qualities of the stimuli simply through the process of gene duplication and subsequent sequence divergence. Such simple developmental rules allow a complex system to evolve in a coherent manner and retain its functionality. Perhaps even more remarkable is the recent observation that the adult nervous system can adapt
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to the introduction of novel photopigments. In adult squirrel monkeys that were red green-decient dichromats, viral addition of another opsin allowed the monkeys to distinguish red and green [57]. In this case, the opsin was not introduced early in development and did not segregate to different photoreceptors. Instead, it was expressed unevenly, so that some photoreceptors expressed a single native opsin and some expressed both the native opsin and the exogenous opsin. Over the course of a few weeks, the monkeys were able to use the information provided by the exogenous opsin for behavioural tasks, revealing that even in the absence of developmental mechanisms, neural circuits exhibit exibility to novel inputs. It was previously shown in colour-decient humans that visual experience can modify the perception of colour in adults [58]. This suggests that there is ongoing plasticity that plays a role in the adjustment of neural circuits. Thus, neural circuit plasticity can facilitate the incorporation of novel changes to the transduction apparatus and thereby provide a mechanism for the evolution of novel sensory input.

5. MOTOR SYSTEM EVOLUTION Although sensory systems exhibit properties that clearly affect their evolvability, the question has arisen as to whether species differences in motor behaviours are caused by central circuitry at all. It has been argued that species differences in feeding behaviours in

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Review. Neural basis of behavioural evolvability sh and amphibians arise not from differences in the outow of the nervous system but from divergence in the organization of the musculo-skeletal system that the nervous system controls [59 62]. Others have noted though that quantication of the motor output is difcult in the absence of an understanding of the neural circuitry [63]. Nonetheless, clear examples of species differences in the motor patterns underlying feeding behaviour have been observed in reptiles [64], sh [65] and insects [66]. However, it is important to recognize the potential role that sensory feedback plays in shaping motor output [67], making it even more difcult to assess the extent to which the underlying motor output has changed. Still, important species differences in motor behaviour are caused by differences in motor output. Locomotor behaviour can be species-specic and species differences in locomotion are not always caused by differences in external anatomy. For example, white-tailed deer gallop when alarmed, whereas mule deer stott, i.e. spring up into the air with all four legs leaving the ground simultaneously [68]. Furthermore, the behaviours are genetically determined; hybrids of these two species produce a somewhat intermediate behaviour when startled. Similarly, hybrids between two species of crickets produce calling songs that are distinct from each of the parental lines in the temporal pattern of syllables [69]. The challenge of determining the neural basis of species-specic motor behaviour is complicated by the fact that behaviour is caused by the dynamic interactions of many neurons, making it difcult to understand the neural basis of behaviour at the cellular level even within a single species. For example, although it has been studied intensively for more than a century [70,71], there is still no agreement on the cellular basis for spinal-generated locomotion [7274]. Thus, in order to understand how motor behaviours evolved, one would need to study nervous systems with clear species differences that are accessible to analysis.

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example, homologous neurons have been found to serve similar roles in the feeding circuitry of gastropod molluscs [79,80]. Similarly, in the well-studied stomatogastric ganglion of decapod crustaceans, the AB neuron is the rhythmic pacemaker for the pyloric central pattern generator (CPG) in the stomatogastric ganglion of lobsters, crabs and spiny lobsters [81]. Despite the strong phylogenetic conservation, there are examples where homologous neurons have changed function. One dramatic example is in the evolution of a novel means of swimming by sand crabs [82]. Over the course of evolution, muscles have been modied in size and orientation and the articulation of the exoskeleton has been altered to allow these animals to swim with their tails up, using their tail fans as ippers [83]. Along with the transformation in morphology, there is the evolutionary emergence of stretch receptors that do not exist in other crustacean lineages [84]. Phylogenetic analysis suggests that these novel primary sensory neurons are actually homologous to motor neurons in other species [85]. In other words, neurons have had their functions converted from motor to sensory, an extraordinary transformation.

6. IDENTIFIED HOMOLOGOUS NEURONS AID IN THE STUDY OF MOTOR SYSTEMS The difculties in studying the neural basis of motor behaviour are lessened in some invertebrate nervous systems, in which neural circuits are composed of individually identiable neurons [7577]. This allows the activity and synaptic connectivity of particular neurons to be directly related to the behaviour of the animal. Furthermore, just as individual neurons can be uniquely recognized within a species based on a set of characteristics, homologous neurons can be recognized across species based on those same characteristics [77,78], allowing the neural function to be compared across species and related to neural properties and connectivity. As with gross morphological features, there is substantial phylogenetic conservation of function of homologous neurons across species within a taxon. So many examples of conservation of function exist that, in the absence of evidence to the contrary, a basic assumption when studying related species is that homologous neurons play similar roles. For
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7. BIOPHYSICAL PROPERTIES AND NEUROMODULATION IN MULTI-FUNCTIONAL CIRCUITS Work on neural circuits composed of identied neurons has shown that the dynamics of neural circuitry is dependent upon the biophysical properties of the neurons and synapses within the circuits. Therefore, species-specic motor behaviour could, in principle, arise from small differences in the expression of ion channels or other proteins. In this way, the same anatomically dened circuit might exist in different species, but produce different patterns of neural activity. If small changes in biophysical properties underlie species-specic behaviour, then there might be an almost innite exibility to the possible behaviours that a neural circuit can produce. However, recent evidence, particularly from the stomatogastric nervous system in crustaceans, suggests that the output of neural circuits is actually impervious to small changes in properties. In fact, there is evidence to suggest that similar behavioural outputs can be produced by neuronal circuits composed of neurons with different ion channel compositions and different synaptic strengths [8690]. The overall output of the network may be maintained through homeostatic mechanisms allowing different combinations of ion channels and synapses to achieve a similar set point of activity [9193]. Thus, if species-specic motor behaviours were caused by differences in the biophysical properties of the neurons and synapses, it would probably involve a suite of biophysical differences rather than one or two small differences. This could be good news for studying the evolution of neural circuits because it means that species differences that underlie behaviour are likely to be substantial, rather than subtle. On the other hand, the intra-species variability may make electrophysiological properties poor candidates for characters used in phylogenetic analysis. The biophysical properties of neurons and synapses within an animal are altered by neuromodulatory

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P. S. Katz Review. Neural basis of behavioural evolvability Pleurobranchaea evolved the dorsal ventral swim behaviour independently [108]. Thus, the underlying structure of the ancestral nervous system that contained the ancestral C2/A1 and DSI/As1 3 cells seems to have been predisposed to the evolution of the neural circuitry to produce dorsal ventral exions.

inputs to circuits [9496]. Species-specic behaviour might arise from differences in the activity of these neuromodulatory inputs or in the responses to them [81,97,98]. In the stomatogastric nervous systems of crustaceans, there are species differences in the presence of neurotransmitters and in the effects of neuromodulatory substances [99101]. In aplysiid molluscs, the effect of serotonin on sensory neuron excitability and synaptic strength varies in a phylogenetic manner and may underlie species differences in behavioural sensitization [102104]. Species differences in neuromodulatory actions also underlie differences in the swimming behaviours of frog embryos [105,106]. Thus, species-specic behaviour could arise from differences in neuromodulation of cellular and synaptic properties, allowing anatomically dened circuitry to be re-specied for another pattern of activity. Therefore, one might expect to see similar circuitry in closely related animals producing different patterns of activity.

8. SIMILAR BEHAVIOURS HAVE INDEPENDENTLY EVOLVED USING HOMOLOGOUS NEURONS The test of whether evolvability is affected by the structure of the nervous system is whether animals independently evolved analogous behaviour using the same structures. This has been examined at the level of individual neurons that produce the swimming behaviour of nudipleura molluscs [107,108]. Based on a phylogenetic analysis, it appears that homologous neurons have independently come to play similar roles in the swimming behaviours of two species: Tritonia diomedea and Pleurobranchaea californica. Both species swim by repeatedly exing their bodies in the dorsal and ventral directions. In Tritonia, the dorsal swim interneuron (DSI) and interneuron C2 re bursts of action potentials during the dorsal phase of this behaviour (gure 2a) and are part of the CPG circuit that underlies swimming [110,111] (gure 2b). Pleurobranchaea contains neurons called As13 and A1 that are homologous to the Tritonia DSI-AC and C2 based on anatomical and physiological criteria [109,112]. The neural circuitry for Pleurobranchaea swimming resembles the swim CPG circuit in Tritonia (gure 2c). As13 and A1 are rhythmically active during the swim motor pattern in a manner that strongly resembles the neural activity in Tritonia (gure 2d). Thus, homologous neurons play similar roles in the production of swimming behaviour in these two species. Tritonia and Pleurobranchaea belong to a clade called Nudipleura [113 115]. Most Nudipleura species do not swim as Tritonia and Pleurobranchaea do, i.e. using dorsal/ventral body exions. There are many species that swim with side-to-side or lateral exions and still others that do not exhibit any swimming behaviour. Plotting these traits on the phylogenetic tree of Nudipleura (gure 3) reveals that one or all three of these behaviours must have arisen independently several times. It is most probable that swimming was lost several times. If dorsal ventral exion swimming is ancestral, then lateral exion swimming must have arisen independently at least three times. A more plausible hypothesis given the phylogenetic distribution of swimming behaviours is that Tritonia and
Phil. Trans. R. Soc. B (2011)

9. SPECIES-SPECIFIC BEHAVIOURS ARE PRODUCED FROM NERVOUS SYSTEMS COMPRISING HOMOLOGOUS NEURONS Owing to phylogenetic constraints, animals that evolved divergent behaviours would nonetheless have homologous structures. Nudibranchs that do not swim like Tritonia or Pleurobranchaea also have homologues of the swim CPG neurons [119]. For example, Melibe leonina is more closely related to Tritonia than Pleurobranchaea, but swims by exing its body from side to side instead of dorsally and ventrally [120,121] (gure 3). This behaviour differs in several other fundamental ways from the Tritonia swim, including the duration of the episodes and the stimuli that will elicit the response. Different sets of neurons control swimming in Melibe and Tritonia [108,122]. Nonetheless, Melibe contains a homologue of the Tritonia DSI called CeSP [119], which is not rhythmically active during side-to-side swimming [107] (gure 2e). Thus, these homologous neurons play very different roles in the generation of swimming behaviour. Even though the DSI homologue is not part of the swim CPG in Melibe, it still affects the swimming behaviour in a way that sheds light on how these different behaviours may have evolved. In all of the Nudipleura, the DSI homologues use the neurotransmitter serotonin. In Tritonia, DSI uses serotonin to modulate the strength of synapses of other neurons in the swim circuit [123 126]. Serotonergic neuromodulation was found to be necessary for producing the swimming behaviour; serotonin receptor antagonists block the swim motor pattern [127]. Also, serotonin [127] or DSI stimulation [128] is sufcient to trigger swimming. In Melibe, CeSP is also serotonergic, but unlike in Tritonia, neither the DSI homologue nor serotonin is necessary for swimming [107]. However, serotonin can still modulate the swim motor programme in Melibe. Thus, the function of homologous serotonergic neurons differs in species with different behaviours; in the dorsal ventral exion swimmer, Tritonia, serotonergic neurons play an intrinsic neuromodulatory role, whereas in the lateral exion swimmer, Melibe, they are extrinsic to the CPG, but modulate its activity. Most nudibranch species do not swim at all; however, homologues of the DSI have been identied in 10 different genera, including three non-nudibranch opisthobranchs [109,119,129 131]. What are homologues of the so-called dorsal swim interneuron doing in species that do not swim? One answer to that the question is that the DSI in Tritonia is multi-functional; in addition to being part of the swim CPG, it also accelerates crawling when the animal is not swimming [132]. This function is mediated, in part, by specic synaptic connections to efferent neurons. The synaptic connections between the DSI homologues and the efferent neurons are also observed in each of the

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(a) DSI C2 nerve stim. (d) 5s (c) 20 mV
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CeSP Si1 5s Figure 2. Homologous identied neurons in sea slugs have divergent or similar roles in behaviour. (a) Tritonia diomedea swims by exing its body in the dorsal and ventral directions as shown in the diagram to the left. Simultaneous intracellular microelectrode recordings from DSI and C2, two neurons in the central pattern generator (CPG) for the swimming behaviour, display rhythmic bursts of action potentials after a body wall nerve is electrically stimulated (nerve stim.). This comprises the swim motor pattern. (b) The swim CPG in Tritonia contains three neuronal types: DSI, C2 and VSI. There are three DSIs: DSI-A, DSI-B, DSI-C. They are being grouped together for simplicity. The triangles represent excitatory synapses, the circles represent inhibitory synapses and multicomponent synapses are presented by combinations of the two. (c) The swim CPG in Pleurobranchaea has many similarities to that in Tritonia. As13 are homologous to the DSIs in Tritonia. There is an As4 that is in the same cell cluster and is in the swim CPG, but is not homologous to the DSIs. Its homologue exists in Tritonia, but the function of this neuron has not been determined in Tritonia. The IVS neuron has not been identied, but its synaptic actions, which can be inferred from recordings of the other neurons, are similar to those of the Tritonia VSI. A1 (which is homologous to C2 in Tritonia) is strongly electrically coupled to neuron A10 and so both are represented together. Homologues of A3 and A10 have not been identied in Tritonia. (d) Pleurobranchaea californica swims with dorsal ventral body exions. Intracellular recordings show that the As2,3 neurons and the A1 neuron both exhibit bursting behaviour during the swim motor pattern (adapted from [109], American Physiological Society, with permission). (e) Melibe leonina swims by exing its body from side-to-side. Intracellular recordings from CeSP (which is homologous to the DSI in Tritonia) and swim interneuron 1 (Si1) show that the CeSP neuron is not rhythmically active during the swim motor pattern. 20 mV

species where it was examined [119,133]. Furthermore, the DSI homologues also play a role in modulating feeding in Pleurobranchaea [133] and Aplysia [129]. Thus, homologous neurons can maintain one function while taking on additional functions. This suggests that the multi-functionality of neurons allows them to be re-purposed in different species without interfering with their other functions.

10. PARALLEL AND CONVERGENT EVOLUTION OF COMPLEX MOTOR BEHAVIOURS Homoplasy, such as the independent evolution of swimming in Tritonia and Pleurobranchaea, provides an opportunity to test how neural circuits evolved to produce species-specic motor behaviour [134]. There are many examples of independent evolution of locomotor behaviour. For instance, anatomical and fossil evidence suggests that knuckle-walking in apes evolved independently more than once [135] as did the pacing gait in Camelids [136]. Stick insects
Phil. Trans. R. Soc. B (2011)

re-evolved ight several times [137]. If two species independently evolved a particular behaviour, then the neural mechanisms for those behaviours can be examined to determine if the same mechanisms underlie the behaviour. If the mechanisms arise from nonhomologous components, then this is said to be convergent evolution. However, if the behaviours arise from independent evolution using homologous neural structures, then this would be parallel evolution. The presence of parallel evolution is an indication of the extent to which the nervous system provides a pathway for evolution and thus affects evolvability. Parallel evolution is common for animals that share homologous brain structures. For instance, different clades of monkeys independently evolved dexterity using expansion of similar brain areas [138]. Parallel evolution of brain areas in response to similar environmental pressures has occurred among different clades of shrews [139] and also between marsupials and placental mammals [140]. Reduction of the pretectal area occurred independently in two clades of eel, showing

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Tritonia (DV) Tochuina (N) Melibe (L) Dendronotus (L) Lomanotus (L) Fiona (N) Phestilla (N) Hermissenda (L) Flabellina tiophina (N) Flabellina iodinea (L) Armina (N) Aphelodoris (DV) Archidoris (N) Hexabranchus (DV) Plocamopherus (L) Triopha (N) Pleurobranchaea (DV) Berthatella (N)

Figure 3. Phylogeny of the Nudipleura based on both anatomical and molecular data [114,116118]. The phylogenetic tree shows selected genera and their swimming behaviours. DV (green), dorsalventral exion; L (blue), lateral exion; N (red), non-swimming.

pre-adapted for electrolocation and for jamming avoidance responses [151]. This is another way to say that the nervous system affected the evolvability of these behaviours by providing a substrate on which selection could readily act. However, the independent evolution of electrolocation behaviour also provides a clear example of convergent evolution, where non-homologous structures have come to have analogous properties. In particular, distinct areas of the brains of Mormyriformes and Gymnotiformes are responsible for some of the processing of the electrosensory signal; in the South American sh, timing and amplitude comparisons are made in the midbrain, whereas in the African sh, similar computations are carried out in a medullary structure [151,153]. Thus, both convergent and parallel evolutions have played roles in the independent evolution of electrolocation in South American and African electric sh. It would be of interest to determine if there are additional examples of similar computations being carried out in non-homologous brain regions.

that secondary loss of structures can also be a route for evolutionary change [141]. Vocal learning by birds is another example where homologous brain regions have come to assume similar functions in independently evolved behaviour. Although many species of birds produce vocalizations, only songbirds, hummingbirds and parrots learn their vocalizations. Given the phylogenetic relationships of these birds, it is likely that vocal learning evolved independently at least twice and possibly three times [142144]. Recent data suggest that the same anterior forebrain areas are used for learning the song in these species. These areas may be generally involved in motor learning. Therefore, the independent evolution of this sensory-motor behaviour may occur through the reuse of homologous brain areas, i.e. parallel evolution of neuronal circuits. Thus, the structure and organization of nervous systems may have provided unique avenues for evolution and biased the direction of evolutionary change, thus affecting the evolvability of behaviour. The independent evolution of active electrolocation behaviour of weakly electric sh involves both convergent and parallel evolution. The African Mormyriformes and the South American Gymnotiformes independently evolved electrosensory systems consisting of sensory structures (tuberous and ampullary electroreceptors), a motor structure (the electric organ) and neural circuitry to process the information [145153]. There are many similarities in the behaviours of sh in these two groups. Both groups have species with pulse-like electric organ discharges as well as species with wave-like discharges. Having wave-like discharges requires a jamming avoidance behaviour, which also evolved independently in the two clades [151]. Many of the similarities arose through parallel evolution. For example, both groups of sh have independently come to express the same sodium channel genes in their electric organs and these sodium channels have independently evolved similar changes in the gating region of the protein [152,154]. It has been said that the nervous system was
Phil. Trans. R. Soc. B (2011)

11. EVOLVABILITY OF NEURAL CIRCUITS UNDERLYING SOCIAL BEHAVIOUR Nervous system evolvability may play a signicant role in shaping the evolution of social behaviour. A classic example of this comes from work on male parental care and pair-bonding, which has been correlated with the expression of arginine-vasopressin 1a (V1a) receptors in particular brain areas in voles [155159]. Monogamous species such as the prairie vole (Microtus ochrogaster) exhibit high levels of V1a receptor expression in the ventral pallidum, an area that receives input from the nucleus accumbens. Non-monogamous species such as the meadow vole (Microtus pennsylvanicus) exhibit lower levels of expression in this area (gure 4a). It was shown that this receptor distribution was not just correlative, but causal; increasing the expression of V1a receptors in the ventral pallidum of the meadow vole brain, using viral vector gene transfer, transformed the behaviour of the meadow vole on a partner preference task to be more like that of a prairie vole [161]. This suggests that the underlying neural circuitry was already in place to allow the pair-bonding behaviour to occur if the receptor is expressed in this brain area (gure 4b). Pair-bonding is relatively rare in mammals, occurring in only 3 5% of species. Yet in several species that have independently evolved pair-bonding, the monogamous species exhibit a higher level of V1a receptor expression in the ventral pallidum than closely related non-monogamous species (gure 4a) [162]. This has been observed in other rodents such as the California mouse (Peromyscus californicus) as well as in a primate, the common marmoset (Callithrix jacchus) [162]. The association of V1a receptor expression in the ventral pallidum with monogamous behaviour is still another example of parallel evolution where homologous brain regions have independently undergone the same change to produce analogous behaviours. The parallel evolution of V1a receptor expression in the ventral pallidum suggests a mechanism for the

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Figure 4. (a) Comparison of vasopressin 1a (V1a) receptor distribution in the brains of six mammalian species. The species in the left column display monogamous behaviour and those in the right column are non-monogamous. The arrows in monogamous species point to the high level of expression in the ventral pallidum (VP). The boxes show the lack of staining in this region in non-monogamous species. Images provided by Larry Young. (b) A schematic of the reward circuitry, which is common to rodents. Dopamine (green) from the ventral tegmental area (VTA) is released in the prefrontal cortex (PFC) and the nucleus accumbens (NAcc). The NAcc also receives excitation from the periaqueductal grey (PAG) and nucleus tractus solitarus (NTS), which are activated during sex. The NAcc projects to the ventral pallidum (VP), which is the major output relay that helps reinforce motor behaviour. The medial amygdala (MeA), which gets input from the olfactory bulb (OB), projects bres to the VP that contain vasopressin (magenta). Differences in the level of V1a receptor expression in VP can modulate the reinforcement of mate-related odours (based upon Young & Wang [159] and Young et al. [160]).

evolution of male afliative behaviour that is inherent in the structure of the brain. The ventral pallidum is part of the reward circuitry [163]. Similar circuitry is found in most mammals (gure 4b). Expressing the appropriate receptors along this pathway can cause particular behaviours to be reinforced. The mechanism for directing the expression of V1a receptors may not be consistent across species. In the vole species, the expression differences can be accounted for by a region upstream of the gene for the V1A receptor; prairie voles have long tandem repeats in this area, whereas meadow voles have shorter repeats [164 166]. Although this gene is polymorphic in primates, the length of the tandem repeat does not appear to correlate with social structure [162,167,168]. This demonstrates the importance of considering the effect of the gene relative to the nervous system; a similar genetic alteration does not necessarily lead to an equivalent phenotype in a different neural environment.
Phil. Trans. R. Soc. B (2011)

12. SUMMARY A mechanistic understanding of the evolution of behaviour must take into account the bias that neural structures impose on the evolvability of particular behaviours. The nervous system is quite complex. Phylogenetic and developmental constraints presumably prevent large differences in nervous system structure from arising in closely related species. In spite of this, clear species differences in behaviour exist. Rather than impeding the evolution of behaviour, the developmental, physiological and genetic processes that allow the nervous system to be so complex may also bias the evolution of behaviour towards particular outcomes. This results in independent evolution of behaviour through parallel changes in the nervous system. Parallel evolution suggests that certain nervous system properties are easily achieved and thus can be selected for repeatedly. The nervous system, therefore, plays a role in the evolvability of behaviour by constraining the potential behaviours that can

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anonymous reviewers for their feedback. The authors research is funded by NSF ISO-0814411 and NSF IIS-0827418.

evolve while facilitating the evolution of particular behaviours. The end result is that the structure and physiology of the nervous system helps direct the evolution of behaviour down certain paths that recur over evolutionary time. The appearance of parallel changes to homologous structures is not the result of random chance or wild coincidence; it indicates a predisposition of the system towards that outcome. Modelling studies have shown that at the molecular level, the probability of parallel nucleotide substitutions under natural selection (i.e. homoplasious nucleotide substitutions that result in equivalent amino acid substitutions) is twice as high as neutral changes [169]. By analogy, at a macroscopic level, there might be certain developmental changes that have a higher probability of yielding functional outputs based on factors such as the constraints imposed by pre-existing neural pathways. This creates the paradox that although the complexity of the nervous system constrains evolution, it also may guide it. For a complex nervous system to develop and function in a coherent manner, it must be regulated by developmental and homeostatic rules. Homeostatic rules compensate for changes in the environment or in the activity of the brain area. These very rules play a role in the ability of the nervous system to compensate for changes in the periphery of the body, such as the appearance of novel photopigments. Such developmental plasticity assists the evolution of species-specic sensory processing. Motor networks are capable of producing exible patterns of activity through the actions of neuromodulatory inputs. Evidence suggests that different species express different behaviours using the same circuit components (such as Tritonia and Melibe). The conservation of the circuitry might allow behaviours to re-appear in other species (such as Tritonia and Pleurobranchaea). Mechanisms that allow for a exible motor output within a species might also contribute to phylogenetic exibility. Complex social behaviours, such as pair-bonding, could independently arise through the exploitation of basic reward circuitry that is conserved in all mammals. Once again, the change is not through gross alterations in connectivity, but rather in the expression pattern of G protein-coupled receptors (in this case V1a receptors). These receptors are likely to change the dynamics of activity in the reward circuitry and thereby change the behaviour. It points again to the importance of neuromodulation of neural circuits in shaping the evolution of behaviour. The presence of parallel evolution of behaviour through recurrent changes to neural circuits suggests that the nervous system affects the evolvability of behaviour by facilitating certain changes or conversely, by limiting the range of possible functional states. Thus, the evolvability itself, while not necessarily being selected for, results as a natural consequence of having a complex nervous system.
The author wishes to thank the editor for the invitation to contribute to this edition, members of his laboratory for helpful comments on the manuscript, as well as three
Phil. Trans. R. Soc. B (2011)

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Sci. USA 103, 36753680. (doi:10.1073/pnas. 0600160103) Hammock, E. A. 2007 Gene regulation as a modulator of social preference in voles. Adv. Genet. 59, 107 127. (doi:10.1016/S0065-2660(07)59004-8) Lim, M. M. & Young, L. J. 2006 Neuropeptidergic regulation of afliative behavior and social bonding in animals. Horm. Behav. 50, 506 517. (doi:10.1016/j. yhbeh.2006.06.028) Nair, H. P. & Young, L. J. 2006 Vasopressin and pairbond formation: genes to brain to behavior. Physiology (Bethesda) 21, 146 152. Lim, M. M., Hammock, E. A. & Young, L. J. 2004 The role of vasopressin in the genetic and neural regulation of monogamy. J. Neuroendocrinol. 16, 325 332. (doi:10. 1111/j.0953-8194.2004.01162.x) Young, L. J. & Wang, Z. 2004 The neurobiology of pair bonding. Nat. Neurosci. 7, 10481054. (doi:10. 1038/nn1327) Young, L. J., Murphy Young, A. Z. & Hammock, E. A. 2005 Anatomy and neurochemistry of the pair bond. J. Comp. Neurol. 493, 51 57. (doi:10.1002/cne.20771) Lim, M. M., Wang, Z., Olazabal, D. E., Ren, X., Terwilliger, E. F. & Young, L. J. 2004 Enhanced partner preference in a promiscuous species by manipulating the expression of a single gene. Nature 429, 754757. (doi:10.1038/nature02539) Young, L. J. 1999 Oxytocin and vasopressin receptors and species-typical social behaviors. Horm. Behav. 36, 212221. (doi:10.1006/hbeh.1999.1548)

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Phil. Trans. R. Soc. B (2011) 366, 21002110 doi:10.1098/rstb.2011.0028

Review

Conservation of gene function in behaviour

Christopher J. Reaume and Marla B. Sokolowski*
Department of Biology, University of Toronto, Mississauga, Ontario, Canada, L5L 1C6 Behaviour genetic research has shown that a given gene or gene pathway can inuence categorically similar behaviours in different species. Questions about the conservation of gene function in behaviour are increasingly tractable. This is owing to the surge of DNA and omics data, bioinformatic tools, as well as advances in technologies for behavioural phenotyping. Here, we discuss how gene function, as a hierarchical biological phenomenon, can be used to examine behavioural homology across species. The question can be addressed independently using different levels of investigation including the DNA sequence, the genes position in a genetic pathway, spatialtemporal tissue expression and neural circuitry. Selected examples from the literature are used to illustrate this point. We will also discuss how qualitative and quantitative comparisons of the behavioural phenotype, its function and the importance of environmental and social context should be used in cross-species comparisons. We conclude that (i) there are homologous behaviours, (ii) they are hard to dene and (iii) neurogenetics and genomics investigations should help in this endeavour. Keywords: gene function; behaviour; conservation; homology; genetics; genomics

1. INTRODUCTION It is well known that gene sequences are conserved even between distantly related species. For example, genes in the nematode Caenorhabditis elegans or the y Drosophila melanogaster exhibit sequence similarity to versions of human genes. But does such DNA sequence similarity reect a functionally conserved role for the genes in question? The answer is yes for developmental genes such as hox genes that specify anterior posterior morphology in organisms from ies to mammals suggesting that hox genes had this function in a common ancestor of arthropods and chordates [1]. Here, we ask if evolutionary developmental (evo-devo) approaches can be extended to behavioural phenotypes that exhibit extensive plasticity and are subject to real-time interaction with the environment.

2. HOMOLOGY AND THE CONSERVATION OF BEHAVIOURAL TRAITS A phenotype is homologous when two (or more) species share a common ancestor that exhibits the phenotype. Distinguishing between evolutionary conservation and convergence is challenging [2,3]. Descriptions of homology have traditionally been the purview of embryologists, anatomists and systematists. More recently, evo-devo biologists have addressed morphological homology and the role that gene function plays in specifying conserved phenotypes across species. Molecular phylogenies have been used as important baseline data for tests of homology and
* Author for correspondence (marla.sokolowski@utoronto.ca). One contribution of 10 to a Theme Issue Evolutionary developmental biology (evo-devo) and behaviour.

morphology [4]. Identifying homologous behavioural phenotypes is challenging because (i) behaviour exhibits plasticity in response to the environment and (ii) behaviour can show homology at one level of biological organization (e.g. gene pathway), but not at another (e.g. neural circuitry; see below) [5]. Homologous behaviours are hard to dene. In developmental biology, researchers consider the modular nature of an organisms body plan (e.g. a limb or an organ) in order to relate morphological features to phenomena at the cellular level to patterns of gene expression [6]. Similar to this is the concept of endophenotypes, where complex behaviours are constructed from simpler components or modules [79]. Endophenotypes and their behavioural components are useful for approaching investigations of homology in behaviour. For example, courtship behaviour in D. melanogaster comprises component behaviours, including orientation, tapping, wing extension and song [10]. Examining behavioural components is not only important for tractability and interspecic comparisons of behaviours but can also help identify species-specic modications in complex behavioural phenotypes that might not have been apparent. How can behavioural phenotypes be compared in species with drastically different morphology and natural history? We can start with similar categories of behaviour that are shared across species such as feeding, mating, parental care, aggression, learning, memory, circadian rhythms and sleep. Each category can be quantied using descriptions of the behaviours performed (e.g. for aggression: kick, lunge, punch, bite) as well as information about the frequency, duration and sequencing of behaviours. A signicant challenge is to develop and standardize informative and unbiased assays of behaviour across species using a comparative approach [11]. Part of this challenge is
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Figure 1. Schematic diagram of the hierarchical nature of gene function in behaviour. Included are a number of factors that can be examined for homology in inuencing behaviour through gene function. Terms coloured in red indicate factors that are intrinsic to the organism that are involved in gene function in behaviour while those that are coloured blue are extrinsic to the organism. Epistasis and epigenetic processes are not shown in the gure; however, gene interaction networks and heritable changes in chromosome methylation and histone modication patterns that underlie behaviours also might be conserved across species.

to design paradigms that are relevant to the natural histories of each species but at the same time can be compared functionally across species [12]. This is more straightforward for some categories of behaviour than others (e.g. characteristics of rest/wake cycles compared with courtship behaviour). Considerations of the social context of behaviours should be incorporated into analyses as this information is inseparable from the phenotypes. Finally, the extent of plasticity of the behaviour is also important as it tells us whether and how much the phenotype varies across environments. These latter considerations introduce considerable analytical difculties when comparing divergent species.

3. CONSERVATION OF GENE FUNCTION IN BEHAVIOUR Information from neurogenetics and genomics helps determine which behaviours are homologous. Investigations of gene function homology in behaviour can be approached through interspecic comparisons of the various components that affect the behavioural phenotype in question [5,13,14]. The implicated genes, their sequence variation and the relevant signalling pathways and tissues (cells, organs, circuits) are all informative (see case studies below). In this sense, we dene gene function in behaviour as a hierarchical phenomenon that includes not only sequence identity and transcriptional events but also the position and role of the gene product in a signalling pathway that acts in dened cells and circuits in the expression of
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the behavioural phenotype (gure 1). Evo-devo studies have found that homologous morphological phenotypes may result from genetic and developmental mechanisms that are not themselves necessarily homologous [3,5]. This suggests that questions related to gene function homology should focus on a single hierarchical level of gene function (as it relates to behaviour) at a time. This is discussed further below. We mentioned above the need for comparable and unbiased behavioural tests and well-known phylogenies for the species in question (see [12] for further discussion). Examples of other pertinent investigations include (i) genome or gene sequencing to test for sequence homology, (ii) transcriptional studies to assess gene expression, (iii) functional studies examining posttranslational and signalling pathways, (iv) comparative histological studies examining developmental and physiological aspects of the tissues and structures involved in gene function and execution of the behaviour in question, and (v) comparative studies examining geneenvironment interactions of the gene(s) and behaviour(s) under investigation. This list is not exhaustive but provides several perspectives from which a genes function in behaviour may be investigated. We suggest that the question of homology needs to be addressed and considered at each relevant level of gene function (cell, circuit, neural substrate, transcription, translation, signalling pathway, behaviour) independently (see case studies below). Conservation of a genes function can occur at the levels of molecular pathways, plasticity or gene environment interactions, neural circuitry, developmental

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Review. Gene function conservation in behaviour involved in serotonin re-uptake. Studies of this gene in non-human primates and in human populations have identied a promoter-linked polymorphic region that interacts with early experience to affect behaviours in the young and adults [23]. The long and short alleles result from a 43 bp insertion/deletion in the promoter region of the 5-HTT gene. In humans, the short allele has approximately three times less in vitro basal transcription of 5-HTT mRNA when compared with the long allele [24]. The allelic variants are differently associated with depression and other related behaviours when an individual has a history of adversity early in life [22]. In general, the short allele is thought to confer risk to early adversity while the long allele confers protection (but this is not always the case [25]). Although rats do not have the longshort polymorphism in their 5-HTT promoter region, polymorphisms in the rat 5-HTT gene have also been found to interact with early experience to affect similar behaviours to those reported in rhesus monkeys and humans [26].

functions and through its pleiotropy (gure 1). What is meant here by conservation of a genes pleiotropic function is when a gene affects the same suite of behaviours in two different species, suggesting shared pleiotropic functions of the gene in these species. Some scientists interested in genes and behaviour use the candidate gene approach to facilitate the identication of genes involved in the behaviours of a variety of species [15]. In this respect, candidate genes are those that are dened in one organism (often in well-dened genetic models like D. melanogaster and Mus musculus) and then investigated for similar effects in organisms without a genetic tool-box. Mutations in the genes of the former group of organisms are available or can be generated along with transgenic animals that can be used to increase or decrease expression of a gene and to target the expression of that gene in time and space. Numerous behaviours are studied in this way including courtship and mating, circadian rhythms, sleep, learning and memory, aggression, maternal behaviour and food-related behaviours [16,17]. The importance of olfaction, audition, taste, touch and other stimuli to these behaviours is also under investigation. In some cases, the neural substrates important to these behaviours have been identied and manipulated. Many genes that inuence these behaviours have been discovered using analyses of genetic mutants. Natural genetic variants in behaviours have also been studied and genes that affect normal individual differences in behaviour have been uncovered (e.g. foraging in Drosophila [18]; npr1 in C. elegans [19,20]; vasopressin receptor in mice and voles [21]). An important challenge to identifying homology of gene function in behaviour is that well-resolved phylogenies are lacking for many species, making it difcult to test alternative hypotheses [12]. Below, we examine several well-known and extensively studied examples of gene function that inuence behaviour across species. It is difcult to decipher what denition of homology remains once a system-based approach to gene function in behaviour is adopted. The case studies presented below show that there is no singular approach to assess homology, because examinations at different levels of the system can lead to different conclusions. This is not an exhaustive review of all pertinent examples but a selection of useful studies for illustrative purposes.

4. CASE STUDIES IN THE CONSERVATION OF GENE FUNCTION IN BEHAVIOUR (a) Gene-by-environment interactions and the serotonin transporter gene Behaviour as a phenotype is highly responsive to the environment. This plasticity makes it particularly challenging for studies of homology. Despite the plasticity that emerges from the abiotic and biotic factors experienced by organisms during development and adulthood, it is still possible to nd common patterns in their responses to environments. One way to investigate this is to use genetic variation to ask whether different genotypes differ in their sensitivity to the environment. One of the best examples of gene-by-environment interactions that apply across species involves allelic variation in the serotonin transporter gene (5-HTT) and its interaction with early experience [22]. 5-HTT encodes for a protein
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(b) Gene pathways and the biological clock Genetic analyses of the functions of biological clocks help us to understand the conservation of molecular pathways in behaviour. The molecular pathways involved in circadian phenotypes, such as sleep/wake cycles, have been well-described in diverse organisms. Additionally, as mentioned previously, the behavioural outputs of the clock (e.g. activity, sleep/wake cycles) are relatively easily compared between divergent species (gure 2). First, we provide some background on the biological clock in Drosophila, where the genetic underpinnings of the clock were rst discovered [28,29]. The period (per) gene, which affects circadian rhythm in D. melanogaster, has been used as a candidate gene to examine per homologues involved in other insects [28] and mammals [30]; per was the rst gene discovered to affect circadian behaviour [31]. Three mutations called long (per l), short (per s) and arrhythmic (per 0) alter eclosion rhythms and circadian patterns of locomotor activity. A second clock gene called timeless (tim) affects circadian rhythmicity and per expression [32 34]. Genes per and tim are transcriptionally regulated in a cyclic manner. Transcripts of both genes are present early in the day but the highest levels are found late in the day and at the beginning of the night [35 37]. PER and TIM proteins accumulate during the night and form a heterodimer that moves into the nucleus to bind to transcription factors Clock (CLK) and Cycle (CYC). This prevents CLK and CYC binding to the per and tim promoters which results in the transcriptional repression of per and tim. In early morning hours, TIM and PER degrade and allow for the rise in tim and per transcripts. This negative effect of PER and TIM on their own transcription creates the negative feedback loop that has been the central theme of clocks in many species [38]. Natural variants in per and tim are also known [39] and provide fertile ground for exploring gene function homology in behaviour. In fact, detailed comparisons among a number of different insect orders have already commenced [40]. There is general agreement that the function of per in biological rhythms is conserved across a broad range of species

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Figure 2. Actograms of behavioural rhythms in (a) humans and (b) rodents. Activity is measured as an episode of sleep (dark line) in the human and wheel running (dark line) in the rodent. Episodes of behaviour are shown relative to midnight with dark lines. Consecutive days are plotted top to bottom for each organism. The upper panels show behaviour rhythms of individuals maintained on light : dark cycles. The middle panels show a human with a sleep disorder called advanced sleep phase syndrome (ASPS) and a hamster with a mutation in the double-time gene which affects the biological clock. In the bottom panel the individuals are kept in constant darkness without any exposure to temporal cues. In both cases, the human with the sleep disorder and the mouse with a mutation in the period gene Per2 show drift (from the normal 24 h period) in their rhythms. This drift is indicative of shortened periods. This gure illustrates the similarities in the circadian phenotypes of humans and rodents (gure adapted from [27]).

from insects to humans; however, tims role in the biological clock is not. Gene and genome duplication events have produced four paralogues of per genes in mammals known as mPer1 to mPer4 [41]. The mPer1 and mPer2 genes appear to have a functionally similar role in the signalling pathways of ies and mammals. Circadian genes are thought to function in a number of human disorders [42]. Figure 3 shows the molecular pathways involved in clock function in the y and the mouse. What appears to be conserved is the structure of the clock molecular mechanism but not necessarily each particular gene or gene product. (See also a discussion of relationship of the cyanobacterial clock to eukaryote clocks [43]). While some genes play the same roles in insects and mammals (e.g. period), others do not (timeless), and still others are found in some but not all species. Nevertheless, similar to well-known examples in evodevo, we can conclude that the structure of the molecular mechanism underlying clocks is similar in both groups. The genes involved are highly conserved at the DNA level, and some genes function in the same way and position in the clock molecular mechanism. Qualitative comparisons of gure 3 suggest that the raw material from the y clock may have been tinkered with, in an evolutionary sense, to build the mammalian clocks. If we were to ask whether there is conservation of a specic genes function in behaviour, we would conclude that this is true for some genes and not others. For example, one could ask when and why the function of the timeless gene has changed over the course of vertebrate evolution. Or how genomic evolution has allowed for the potential conservation of the structure of clock molecular mechanisms while the body plan and organs in which it acts
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have changed dramatically. In general, it is valuable to focus on divergences and convergences found between species at any level of organization as these cases will be informative from an evolutionary perspective and may suggest novel hypotheses. To summarize this example, circadian rhythms are found in organisms from bacteria to humans, and these seem to be controlled by a clock mechanism. Interestingly, while there is broad overlap between the genes involved, they are not always the same. The evo-devo approach for circadian behaviour works well, and candidate gene approaches allowed us to make signicant headway in understanding the genetic basis of circadian behaviour. However, the precise details of clock function require the study of clock mechanisms in specic organisms.

(c) Regulation of the foraging gene across species Even when sequence homology is found, as is required for the candidate gene approach, the details of gene function may differ at any or all hierarchical levels. The candidate gene approach [14] has been successfully used for studies of the D. melanogaster foraging (for) gene, investigated in C. elegans, Apis mellifera and the ants Pogonomyrmex barbatus and Pheidole pallidula (see below). The for gene encodes a cGMPdependent protein kinase (PKG) in D. melanogaster and affects a large array of behaviours including food-related behaviours, responses to stress and learning and memory [44]. Foraging behaviour in insects has been a major focus of research on the behavioural effects of PKG. In nature, larval and adult ies behave as rovers or sitters [45]. Well-fed rovers exhibit signicantly more locomotion in the presence of food than

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Figure 3. The molecular mechanism of the (a) Drosophila and (b) mammalian clocks. The genes that show sequence homology are shown in the same colour, those that are not homologous are shown in shades of grey. Proteins that form dimerization partners are presented as a single complex (gure adapted from [41]).

sitters. When food is patchy, rovers have a greater tendency to leave a patch in search of new food patches compared with sitters [15]. Food deprived rover larvae behave as sitters and exhibit concomitant reductions in their PKG enzyme activity resembling well-fed sitters [46,47]. In addition, well-fed rover larvae have lower food intake than sitters. Adult rovers and sitters show exible responses to food deprivation as reected in behavioural, metabolomic and microarray data [48,49]. As mentioned above, the for gene also plays a role in food-related behaviours in C. elegans, D. melanogaster, A. mellifera, P barbatus . and P pallidula [50 53]. However, the particular . functions of for and the signalling pathways involved must differ between species in signicant ways. In D. melanogaster and A. mellifera, high levels of for result in rover and forager ies and bees, respectively. In C. elegans and P pallidula, high levels of for result . in dweller (not roamer) worms, and forager (not defender) ants, respectively. Furthermore, fors contribution to the plasticity of behaviour differs between these species. In D. melanogaster, both chronic food deprivation and short-term acute deprivation changes for expression and behaviour [46,47]. In A. mellifera and P barbatus, for is involved in long-term plastic .
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changes in behaviour tied to maturation (temporal polyphenism). In P pallidula, for is involved in a . worker ants rapid switch from defending the nest to foraging. Finally, only in P pallidula has a difference . in the spatial localization of FOR protein been reported. The brains of defender worker ants have ve more cells expressing FOR than do the brains of the forager worker ants [53]. Together, these differences suggest that in a very broad sense, for is involved in the plasticity of food-related behaviours, but the actual time scale and mechanisms involved in the various species probably differ. It seems clear, however, that for modulates many phenotypes. It is not known whether for affects the same suite of phenotypes in other species and, if so, whether the multiple functions of a given gene (i.e. its pleiotropy) could be useful for between species comparisons. Further research should shed light on these issues. The for gene example shows that a given gene or set of genes can contribute to phenotypic plasticity in behaviour across a diverse range of species [54]. This suggests, as in the biological clock example above, that the gene gene interactions may also show conservation. The gene for and genes in the insulin signalling pathways are involved in both rover/sitter foraging

Review. Gene function conservation in behaviour [48] and the transition from nurse to forager in A. mellifera where the behavioural phenotypes and patterns of gene expression are nutritionally regulated.

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(d) Genetic analyses of learning and memory Another eld of research, learning and memory, helps shed light on the conservation of gene function in behaviours across divergent species. The many types of learning and memory will not be discussed here nor will their relevance for the species life histories. There are many paradigms for testing learning and memory, both non-associative and associative, that are informative for interspecic comparisons. One non-associative paradigm is habituation distinguished by decreases in a response to a stimulus following repeated stimulation. Another is sensitization, an increase in response to a neutral stimulus following exposure to a stronger stimulus. Habituation and sensitization are observed in species as diverse as C. elegans [55], D. melanogaster [56], Rattus norvegicus [57] and humans [58]. Associative learning has also been observed in many species [59,60]. Associative learning paradigms can be separated into two categories: (i) operant and (ii) classical (Pavlovian) conditioning [60]. In operant conditioning, an animal acts on its environment to establish associations between unrelated stimuli. The concept can easily be demonstrated by imagining an operant conditioning chamber (Skinner box). In one of its most basic forms, the operant conditioning chamber contains a lever that an animal learns to press in order to acquire a food reward. Classical conditioning usually involves the pairing of two stimuli. There is a reexive stimulus that elicits a response (e.g. food reward, electric shock) and a conditioned stimulus (e.g. an odour). The animal learns to associate the conditioned stimulus with the reexive stimulus resulting in a conditioned response. These and other forms of non-associative and associative learning can be used to address questions of homology of gene function. However, some forms will be more tractable than others. For example, in operant conditioning, there is less control over the timing, duration and strength of the stimuli when compared with classical conditioning. Another consideration is the divergence of the organisms in question. Simpler learning paradigms such as habituation and sensitization are more suitable for addressing questions about the conservation of gene function. Similarities in the behavioural properties of classical conditioning are found in a wide array of species from molluscs to insects, sh, rodents and humans. Many of the genetic pathways underlying memory formation are shared in these species [61,62]. For example, the cAMP-dependent signalling pathway has been implicated in memory formation in Aplysia [63], honey bees [64], ies [65] and rodents [66] as well as other species. More specically, CREB (cAMP responsive element binding protein) is thought to be necessary for the formation of protein synthesis-dependent long-term memory in ies and rodents [67 70]. The importance of the cAMP signalling pathway for learning and memory is well established [71]. Some recent
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investigations in Drosophila and mice show that PKG (called cGKI in mammals) also functions in learning and memory. In the mouse, PKG plays a role in fear conditioning in the amygdala [72]. In Drosophila, PKG is encoded by for with the rover and sitter natural variants described above. for is the y homologue of mammalian cGK1 and is known to affect Pavlovian associative olfactory aversive learning [73,74], appetitive learning [46] and operant visual learning [75]. Many of the genes and signalling pathways associated with memory formation are conserved across a wide range of species. Drosophila genes involved in cAMP signalling, ras/MAP kinase signalling, Staufen RNA binding protein, and genes involved in human neurocognitive disorders all play a role in memory formation [61,62]. Furthermore, structural elements of the neural circuitry underlying associative learning are postulated to be homologous in insects and mammals [61]. Imaging analyses have advanced to the point where a common origin between the annelid mushroom bodies and the vertebrate pallium has been suggested [76]. Technological advancements in functional neuroanatomy and real-time imaging improve along with our understanding of the cellular mechanisms underlying learning and memory, and will facilitate further interspecic studies of learning and memory [71].

(e) Sleep Sleep is another good model to examine homology of gene function in behaviour [7780]. A number of behavioural criteria can be used to compare sleep between species [81]. They were designed to distinguish sleep from other states of quiescence and include: (i) a quiescent period, (ii) a reduction in the response to external stimuli (increased arousal threshold), (iii) increased rest after prolonged waking, and (iv) reversibility. These criteria led to the discovery of sleep states in a number of non-mammalian species including C. elegans [82], Leucophea maderae [83], A. mellifera [84], D. melanogster [85,86] and Danio rerio [87]. A cAMP CREB pathway has been implicated in sleep, providing examples of various components of hierarchical gene function homology in ies, worms and mammals [7880]. In Drosophila, mutants with increased cAMP levels have reduced sleep, while mutants with reduced cAMP levels have increased sleep [88]. Additionally, manipulations of CREB activity demonstrated its role in wakefulness. Further studies showed mice lacking either one or two CREB isoforms exhibited reduced wakefulness [89]. In worms, the mutants pde-4 (reduced-function cyclic nucleotide phosphodiesterase) and acy-1 (gain-of-function adenelyte cyclase) result in increased cAMP levels, and show increase sensory responsiveness during lethargus [82]. These data suggest that the role of cAMP signalling in sleep behaviour may be homologous at multiple levels in very diverse organisms. Interestingly, the cGMP pathway, involving PKG, has been implicated in sleep-like behaviour. Raizen et al. [82] showed that PKG regulates sleep-like behaviours in ies and worms ( for and egl-4, respectively). The authors compared gain- and loss-of-function

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Review. Gene function conservation in behaviour variation in social behaviour investigated in 13 species of primates [94]. It is difcult to determine whether the similarities in the behavioural functions of the vasopressin molecule and its receptors across distantly related species are due to homology. A category of behaviour that includes social and reproductive behaviours may be too broad to be used to address questions of homology. This suggests that the level of behavioural analysis (e.g. endophenotypes, behavioural components) that is chosen for investigations of homology can affect the conclusions. When vasopressin is investigated as a class of molecules, a great deal of conservation of the neural expression of these genes is found [93]. DNA sequence homologues of this neuropeptide found in animals from hydra to vertebrates existed hundreds of millions of years ago. Examination of tissue-specic expression patterns shows that in mammals, vasopressin is found in the hypothalamic brain regions and then in the pituitary where it travels to affect the brain and is also released into the periphery. Interestingly, genetic homologues of vasopressin are found in related neurosecretory structures in the brains of other organisms such as worms and sh. So at the tissue level, this neuropeptide is found in functionally related regions and tissue types in mammals, sh and worms. One way to ask whether the function of a gene has been conserved is to perform transgenic experiments between species. Transgenic experiments, using another neuropeptide called oxytocin, were performed to ask whether genes expressed in evolutionarily conserved neural tissue exhibit similar functions across species. Indeed transgenic rats carrying a blowsh oxytocin homologue were able to express oxytocin in neurons of the rat hypothalamus; this suggested consistency between the regulatory features of the blowsh and rat genes [95]. Additionally, the sh gene in transgenic rats exhibited normal physiological functions. While the behaviours investigated are highly species-specic, the levels of DNA sequence, tissue-specic expression, regulation and physiological function appear to be conserved in these neuropeptides. This example demonstrates that different levels of investigation can provide different insights into questions of gene function homology across diverse species.

egl-4 mutants and demonstrated that PKG is associated with the extent of behavioural quiescence as well as its time-dependence. They then used rover and sitter y lines to ask whether the behavioural effect of PKG on sleep is evolutionarily conserved. They found that rovers with higher PKG activity slept more than the sitters. These preliminary data suggest that PKG activity is positively associated with the amount of sleep that an animal displays in both species and appears to be conserved. In mice, a conditional knockout of mammalian PKG called cGKI results in increased sleep fragmentation, exaggerated delta rebound following deprivation and reduced rapid-eye movement sleep [90]. The cAMP and cGMP signalling pathways are important to sleep in diverse animals. (f) Dopamine and reward A given gene, or set of genes, may play a role in development and/or functioning of the neural circuitry of a behaviour phenotype. If this neural circuitry shows some conservation between species, then this circuitry can be investigated for conservation of behavioural function. Dopamine signalling regulates a variety of complex behaviours in a wide range of organisms [91]. The dopamine system is of interest because it functions in reward which is intimately linked with many behaviours such as feeding, mothering, sex, learning and addictive behaviours. Dopamine neurons express dopamine pathway genes whose products are involved in dopamine synthesis and transport in most organisms. All dopamine neurons share a small number of genes that code for enzymes and transporters important for the synthesis, packaging and re-uptake of dopamine. How these genes are regulated in diverse species is poorly understood. Flames & Hobert [91] recently found that the function of a dopamine cis regulatory motif called DA is conserved (and interchangeable) in C. elegans and M. musculus. These and other ndings will open the door towards understanding the evolution of structures and neural circuits in animal brains [92]. (g) Neuropeptides and social behaviour As discussed, questions about the conservation of behavioural phenotypes across distantly related species are difcult to answer. Donaldson & Young [93] discuss how vasopressin and its receptors play a role in the modulation of social and reproductive behaviours, a broad class of behaviours found in many organisms. However, the actual effects of this neuropeptide on components of these behaviours are highly speciesspecic. In closely related vole species, species-specic differences in the social bonding result from differences in the expression of the arginine vasopressin V1a receptor (V1aR). Monogamous prairie vole males have a higher number of V1a receptors than polygamous meadow vole males [21]. These voles differ in sequence variation at the 50 region of this gene. Genetic polymorphisms in this gene have also been associated with variation in sociobehavioral traits in humans, including autism spectrum disorders. However, the evolution of the 50 region of this vasopressin receptor gene did not directly contribute to
Phil. Trans. R. Soc. B (2011)

(h) Epigenetics, genomic responses to early adversity A relatively new area of research that can potentially be scrutinized for gene function conservation in behaviour is epigenetics. For instance, specic environmental factors that alter methylation patterns or histone modication of conserved genes that affect behaviour might be homologous across diverse taxa. Meaney, Szyf and colleagues have characterized an epigenetic mechanism which results in maternal programming that has been shown to affect stress responses in rats [9698]. This response is mediated by glucocorticoid receptors in the hippocampus. Maternal licking and grooming provided by the mothers during the rst week of life change the levels of RNA expression of the glucocorticoid receptors. High licking and grooming mothers produce offspring with higher levels of

Review. Gene function conservation in behaviour glucocorticoid receptor mRNA, while low licking and grooming mothers produce offspring with lower levels of this RNA. Individuals that received higher maternal stimulation showed less behavioural and neuroendocrine reactivity to stressful stimuli. These changes in gene activity in response to stress are controlled by patterns of methylation that dene an epigenetic response to mothering. The glucocorticoid receptor is affected by patterns of DNA methylation within the promotor region of the gene. In the case of low licking and grooming mothers, the offsprings promoter of the glucocorticoid receptor is methylated resulting in a decrease in the expression of this gene. This does not occur in offspring of high licking and grooming mothers. The patterns of methylation are maintained into later stages of life. There is some evidence that the stress response-related effects of maternal licking and grooming are passed on to female offspring. The female offspring mother their offspring according to how they themselves were reared (with high or low licking and grooming). The transmission of this maternal behaviour across generations is related to methylation of the oestrogen receptor gene passed from mother to daughter [98]. In a related human study, McGowan et al. [99] showed that methylation patterns and RNA expression levels of the glucocorticoid receptor in the hippocampus of the brains of suicide victims were altered when the victims had a history of abuse. This suggested that analogous to low licking and grooming in mice, early adversity in humans causes a downregulation of the glucocorticoid receptor gene. Thus, this gene-by-early environment adversity interaction is found in rats and humans.

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conserved phenotypes. Evo-devo has already used genome-wide linkage mapping and transcriptional proling for interspecic comparisons. Such genome-wide comparisons can be measured dynamically and in parallel between multiple species of interest. Combined with information on nervous system function, neural circuitry and plasticity, we will be able to compare and contrast molecular pathways and their neural substrates for well-dened behavioural phenotypes across species in different environments. This system-level approach will provide data to address issues of conservation of the molecular and neural pathways that underlie specic behavioural variation. Rapid developments should soon make it possible to link levels of organization on a genome and nervous system-wide scale, making it possible to address issues of conservation across all levels of organization in more quantitative ways.
We thank Dr James Burns, Dr Ken Dawson-Scully and Dr Scott Douglas for fruitful discussions realting to the topics discussed in the article, Dr Laurence Packer for useful comments on an early version of the manuscript and Bianco Marco for help with preparation of the manuscript. We also especially thank Rinaldo Bertossa and three anonymous reviewers for critical readings and very useful suggestions that improved the article. This work was supported by Natural Sciences and Engineering Research Council of Canada (NSERC) grants to Marla B. Sokolowski and Christopher J. Reaume.

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5. SUMMARY Early on, the eld of behaviour genetics focused on organisms that could be genetically manipulated. However, this limited the breadth of species and behaviours that could be studied using a genetic approach. Advances in DNA sequencing and the omics sciences as well as the use of candidate genes has allowed for a broader focus that includes new model species studied from ecological and neurobiological perspectives. Indeed, it has been argued that an understanding of how genes evolve to affect phenotypes should include a comparative approach, and should consider many species and collaborations between evolutionary and molecular biologists [100,101]. While relatively little is known about how evolutionary processes shape intra- and interspecic variation in behavioural phenotypes and the genes that underlie them [7,8], one intriguing theme is that genes which show homology at the level of DNA sequence appear to inuence similar categories of behaviours across taxa. What can functional genomics, systems biology and the plethora of data provided [102] tell us about conservation of a genes function in behaviour? As the sophistication of genome databases rapidly improve, we will learn more about molecular pathways involved in specic brain functions and how these pathways translate to species-specic differences in behaviour. By making comparisons across genomes, we can better understand how sequence variation, genetic architecture and expression patterns associate with
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78 Zimmerman, J. E., Naidoo, N., Raizen, D. M. & Pack, A. I. 2008 Conservation of sleep: insights from non-mammalian model systems. Trend. Neurosci. 31, 371 376. (doi:10.1016/j.tins.2008.05.001) 79 Harbison, S. T., Mackay, T. F. C. & Anholt, R. R. H. 2009 Understanding the neurogenetics of sleep: progress from Drosophila. Trends Genet. 25, 262 269. (doi:10.1016/j.tig.2009.04.003) 80 Crocker, A. & Sehgal, A. 2010 Genetic analysis of sleep. Genes Dev. 24, 12201235. (doi:10.1101/gad.1913110) 81 Hendricks, J. C. 2000 The need for a simple animal model to understand sleep. Progr. Neurobiol. 61, 339 351. (doi:10.1016/S0301-0082(99)00048-9) 82 Raizen, D. M., Zimmerman, J. E., Maycock, M. H., Ta, U. D., You, Y.-J., Sundaram, M. V. & Pack, A. I. 2008 Lethargus is a Caenorhabditis elegans sleeplike state. Nature 451, 569 572. (doi:10.1038/nature06535) 83 Tobler, I. 1983 Effect of forced locomotion on the restactivity cycle of the cockroach. Behav. Brain Res. 8, 351 360. (doi:10.1016/0166-4328(83)90180-8) 84 Sauer, S., Herrmann, E. & Kaiser, W. 2004 Sleep deprivation in honey bees. J. Sleep Res. 13, 145 152. (doi:10. 1111/j.1365-2869.2004.00393.x) 85 Hendricks, J. C., Sumei, L., Kume, K., Yin, J. C.-P., Zhaohai, Y. & Sehgal, A. 2003 Gender dimorphism in the role of cycle (BMAL1) in rest, rest regulation, and longevity in Drosophila melanogaster. J. Biol. Rhythms 18, 12 25. (doi:10.1177/0748730402239673) 86 Shaw, P. J., Cirelli, C., Greenspan, R. J. & Tononi, G. 2000 Correlates of sleep and waking in Drosophila melanogaster. Science 287, 18341837. (doi:10.1126/science. 287.5459.1834) 87 Yokogawa, T., Marin, W., Faraco, J., Pezeron, G., Applebaum, L., Zhang, J., Rosa, F., Mourrain, P. & Mignot, E. 2007 Characterization of sleep in zebrash and insomnia in hypocretin receptor mutants. PLoS Biol. 5, 23792397. (doi:10.1371/journal.pbio. 0050277) 88 Hendricks, J. C., Sehgal, A. & Pack, A. I. 2001 A noncircadian role for cAMP signaling and CREB activity in Drosophila rest homeostasis. Nat. Neurosci. 4, 11081115. (doi:10.1038/nn743) 89 Graves, L. A., Hellman, K., Veasey, S., Blendy, J. A., Pack, A. & Abel, T. 2003 Genetic evidence for a role of CREB in sustained cortical arousal. J. Neurophys. 90, 1152 1159. (doi:10.1152/jn.00882.2002) 90 Langmesser, S., Franken, P., Feil, S., Emmenegger, Y., Albrecht, U. & Feil, R. 2009 cGMP-dependent protein kinase type I is implicated in the regulation of the timing and quality of sleep and wakefulness. PLoS ONE 4, e4238. (doi:10.1371/journal.pone.0004238)

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Phil. Trans. R. Soc. B (2011) 366, 21112123 doi:10.1098/rstb.2010.0344

Review

Mapping behavioural evolution onto brain evolution: the strategic roles of conserved organization in individuals and species
Barbara L. Finlay1,2,*, Flora Hinz1,2, and Richard B. Darlington1,2
1

Department of Psychology, and 2Department of Neurobiology and Behavior, Cornell University, Ithaca, NY 14853, USA

The pattern of individual variation in brain component structure in pigs, minks and laboratory mice is very similar to variation across species in the same components, at a reduced scale. This conserved pattern of allometric scaling resembles robotic architectures designed to be robust to changes in computing power and task demands, and may reect the mechanism by which both growing and evolving brains defend basic sensory, motor and homeostatic functions at multiple scales. Conserved scaling rules also have implications for species-specic sensory and social communication systems, motor competencies and cognitive abilities. The role of relative changes in neuron number in the central nervous system in producing species-specic behaviour is thus highly constrained, while changes in the sensory and motor periphery, and in motivational and attentional systems increase in probability as the principal loci producing important changes in functional neuroanatomy between species. By their nature, these loci require renewed attention to development and life history in the initial organization and production of species-specic behavioural abilities. Keywords: brain evolution; allometry; domestication; individual variation; neocortex

1. INTRODUCTION: FRAMEWORKS TO RELATE NATURAL VARIATION IN BEHAVIOUR, BRAIN STRUCTURE AND GENES Individual variation is the foundation of natural selection, and the idea of inheritance of adaptive characteristics is fundamentally a simple one. Yet, understanding individual behavioural variability and its inheritance is one of the most complex tasks facing current researchers. A good theory of the organization of every level of analysis from behaviour to gene is necessary: individual animals in populations, the computational structure of the brain, the mechanisms of development of the organism and the translation from genome to organism. How do we begin to break into the complex chain from gene to brain to behaviour to population? For example, a fairly recent review of the evolutionary biology of animal cognition gives an ambitious catalogue of variations in behaviour ranging from very specic differences in sensory systems to very complex changes in communication systems and cognition, some with known tness consequences [1]. The associated brain phenotypes range from photoreceptor opsins, to neurotransmitters and
* Author for correspondence (blf2@cornell.edu). Present address: Howard Hughes Medical Institute and Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA. Electronic supplementary material is available at http://dx.doi.org/ 10.1098/rstb.2010.0344 or via http://rstb.royalsocietypublishing.org. One contribution of 10 to a Theme Issue Evolutionary developmental biology (evo-devo) and behaviour.

to whole brain organization and size, and the species range from humans to Drosophila, but in no case is the explanatory chain ever complete. For perfectly understandable reasons, researchers appreciate the complexity of the level of analysis of their primary area of expertise, and underestimate the complexity of other levels, producing predictable and recurring errors in explanations that attempt to bridge levels. In the following paper, we will present some new data about individual variation in brain parts and relate it to phylogenetic variability. Relating the size of brain parts to species-typical adaptive behaviours has been the subject of studies of brain allometry for many years [2 6]. The nature of individual variation in the size of brain parts offered to selection informs this work directly. For this journal issue, however, we will place these results in the context of three links in the behaviour-to-gene chain, where new information has appeared. First, while the behavioural signicance of changes in absolute and relative brain size in phylogeny has been the subject of analysis and speculation for many years, causal or even correlational links between relative brain size and tness had never been demonstrated. Newer studies now allow a better understanding of what behavioural advantage an increase in relative brain size permits animals in natural contexts. Second, new work in computer science in robotics, the design of machines that must function in the real world, are beginning to yield insights on what kinds of computational architectures are robust to change, damage and growth. Finally, developmental neurobiology increasingly demonstrates the importance
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Review. Individual variability in brains This produces better colour acuity in the green brown regions of colour space, with many plausible adaptive advantages [1518]. Startlingly, the identical modication may be knocked in to the retinas of mice or dichromatic monkeys, who can in short order learn to make behavioural use of the new colour information, with no other genetic changes in their nervous systems [19,20]. The lessons to be learned here are important, as they tend to recur over and over in brain behaviour mapping questions. First, researchers equated prominent adaptive features of the behavioural environment (such as a coloured fruit) with features of the eye and brain (such as opsin best frequency, a particular cell type or a brain nucleus) far too quickly: the categories of the world are not the categories of the brain. Second, when investigators were interested in speciestypical specializations in sensorimotor capacities, such as ability to identify a particular fruit, they tended to ignore the complex abilities in scene recognition, navigation and locomotion upon which such specializations ultimately depended. These complex, species-general abilities may dominate species-specic specializations in any currently measurable form of neural commitment like neural volume or energetic expenditure. Finally, the perceptual, cognitive and motor systems of any extant vertebrate are the end result of continual compromise, having proved competent to deal with both species-typical and species-general problems.

of epigenetic and developmental factors in aligning initially unspecied brain structures with their particular physical and social environments.

(a) Some insights from the evolution of colour vision Of all the work in evolution of perceptual and motor systems, vertebrate colour vision comes closest to having a full description at every level, including genetic, developmental, physiological, computational and ecological. The history of this investigation can inform current work on other aspects of brain and behavioural evolution directly. Vertebrate colour vision contrasts the output of two to ve very broadly tuned photoreceptors that cover the visible spectrum to code the reectance characteristics of objects and environments with relative independence from the spectrum of light illuminating them. A typical mammal has two photopigments for diurnal vision (opsins), and most anthropoid primates have three [79]. The opsin molecule, composed of amino acids, is a rough cylinder to which a short-chain molecule, 11-cis retinal, is attached, like a tab on a cola can. Light absorbed by the 11-cis retinal changes its conformation to all-trans, initiating the chain of phototransduction. Of the several 100 amino acids comprising the opsin molecule (using the redgreen opsin as a typical example), only a small fraction are placed in the opsin cylinder such that an amino acid substitution will change the best wavelength for reconformation of the 11-cis retinal molecule [10]. The probability of this class of mutations with direct functional consequences is reasonably well understood in terms of the ongoing jitter in base-pair substitution that the genome continuously undergoes. Overall, the steps from gene to opsin to phototransduction are exceptionally well worked out. From basic receptor photosensitivity to adaptive function in the real world, that is, to perception and behaviour, the path to understanding has been rockier. What does a changed best frequency of a photopigment signify for behavioural adaptation? A rst guess was that there might be a direct relationship between the most sensitive frequency of the photoreceptors and a specic aspect of the adaptive environment. Perhaps the best frequencies of photoreceptors evolved to facilitate foraging or conspecic recognitionthat is, detecting yellow preferentially to nd bananas, or discriminating the redness of faces (e.g. [11]). However, no case of direct matching of best opsin frequency and any particular feature of the environment have ever been established as a basis for species-typical preferences (not negating the fact of species-typical preferences, only where in the brain they may be situated!). Instead, the opsins appear to have been selected to adequately cover the range of available light in each species typical environment. In the case of signalling, the signaller (whether fruit or conspecic) typically evolves to be optimally detectable, while the receiver remains generic [12,13]. In the special case of primate trichromacy, a single amino acid change in the original opsin may produce two slightly different opsin forms whose output can be compared in the central nervous system [14].
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(b) Evolution shapes development New work in evo-devo adds a further dimension to our understanding of behavioural variability. The central researchers and theorists in evo-devo argue persuasively that the developmental programmes of existing creatures are as much a product of evolution as their mature phenotypes, and that these developmental programmes come to have the features of evolvability and robustness. Considering the various aspects of evolvability, which have been discussed at length elsewhere [21 24], the one of central interest here is facilitated variation. Readers interested in a discussion of basic issues in brain development, including organization of the body plan, neural proliferation and segmentation, and activity-dependent organization of the central nervous systems are directed to any one of these sources. Consider an evolutionary case of unfacilitated variation. Suppose we know that an animal would enjoy greater reproductive success if it had longer forelimbs, but we also know that there are no coordinating mechanisms between the developmental programmes producing its various organ systems. Production of longer forelimbs in this animal would require simultaneous random changes in its unlinked programmes for bone growth, vascular supply, muscle volume, muscle attachments, the length of other attached bones, the neural programme executing movement and so on. While gradual accretion of such changes is certainly not impossible, in principle, no such unlikely lineups of random events are needed to produce differences in relative and absolute amounts of limb growth in existing animals. A mutation changing only

Review. Individual variability in brains the rate or duration of bone production can be seen in adult limb morphology because of the epigenetic programmes controlling somatic growth in which bone growth is embedded. In the colour vision example, a random change in the coding of a single amino acid in a much larger opsin molecule can directly change its best wavelength. If the neural system supporting wavelength selectivity required new committed processors from retina to brain (banana detectors; command neurons for banana grasping), such changes at the photoreceptor level would rarely have consequences other than blindness. If the brain mechanism that looks at retinal information is a generalized wavelength comparator, however, as seems to be the case, prepared to analyse a new contrast and learn its relationship to environmental structure, the mutation can succeed, as has recently been demonstrated [19,20]. On the other hand, nonsense genetic changes unsupported by epigenetic embedding or robust neural programmes will likely be invisible or be deleterious. Normal development, via evolution, acts as a lter of genetic mutations to promote meaningful variability that it has previously and successfully experienced, and opsin variations appear to be in that class.

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existence proofs that the links from tness as measured in natural settings to genetic mechanisms can be made, when we understand the structure of individual and species variability in features with demonstrated tness relationships. 2. CONSERVATION AND VARIATION IN BRAIN SIZE AND BEHAVIOUR, IN AN EVO-DEVO CONTEXT (a) Relative brain size can be directly linked to aspects of tness The largest possible focus for brain behaviour relationships, the whole brain, is a surprisingly informative place to begin. Pervasive increases in relative brain size with respect to body size (encephalization) in multiple lineages over evolutionary time and its association with behavioural complexity (without any direct measure of tness) were rst described systematically by Jerison [2]. More recently, the same simple feature of relative brain size (at the species level) has been shown to have direct links to tness, in innovative studies of multiple species of both birds and mammals in natural ecology [32]. These tness indicators include annual mortality rate, probability of an individual species survival when introduced into a new niche, and behavioural innovation in diet. These ecological indicators can be further correlated with several measures of learning exibility in laboratory contexts in a smaller number of species. Although entirely different in emphasis, similar studies actually exist for individual differences in humans: individual variations in absolute brain size have a small, but statistically signicant link to intelligence quotient (e.g. [33]), and thus to multiple measures of socioeconomic status, if not tness. The relative sizes and developmental pattern of enlargement of the parts of the brain most disproportionately large in the largest individual brains, the frontal and parietal cortex, vary directly with individual differences in cognitive exibility [34]. (b) Allometric variability in brains, between species To make the links from behavioural exibility to brain size to initial genetic specication of brain size, via development, we have empirical work at every step. Phylogenetic variation between mammalian species brains is well described [3,35]. Developmental alterations associated with species variations in brain size and sensory systems are beginning to be understood [36 40]. A growing body of knowledge exists on those genes or gene loci involved in brain size regulation including studies in mouse models [41 44], in development [45 48] and in phylogeny [49]. (c) Theories of brain function underlying the rst hypotheses of brain evolution The history of our understanding the relationship of complex behavioural functions to phylogenetic brain variation is one of bootstrapping back and forth from the most general structure function mappings to progressively more sophisticated versions, in much the same way that the understanding of the evolution of colour vision changed over time. Here, we must digress

(c) From individual variation to species variation in morphology Links of natural phenotypic variation to evolutionary dimensions of tness, speciation and genotype have been made in non-behavioural contexts. Linking within-species individual phenotypic variability to patterns of speciation has been evaluated in wide-ranging natural contexts and phyla, the most well-known instances involving readily observable aspects of morphology, for example, plant ecology [25], skeletal alterations in three-spine sticklebacks [26] and the beaks of Darwins nches [27]. In a particularly relevant study, Schluter [28] related details of stickleback morphological individual variation in skeletal structure to speciation. For a founder species, the largest set of co-varying features was termed Gmax, the phylogenetic path of least resistance. For closely related species, it was predicted that this factor should be highly represented, as it is the corpus of accessible variation on which selection can easily act. For distantly related species, it was expected that the variation observed would diverge systematically. Instead, Gmax remained as high in the distant taxa as in the immediately related ones. Developmental constraint was offered to account for these results, operating over both short and long time spans. Alternatively, however, the co-varying dimensions could represent the operation of conserved developmental programmes, facilitating variability along the dimensions associated with viable outcomes in the past and ltering out others. In natural instances of variation and speciation, going to the second step of linking phenotypic changes directly to genotypic changes has been attempted in only a few contexts, such as bird plumage [29], the explosive radiation of the Lake Malawi cichlids [30] and, recently, adaptive specialization of mouse fur colour to the environment [31]. These studies are
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Review. Individual variability in brains not link readily to relative size of anatomically dened chunks of the brain in mammals (for example, equating motor skill and cerebellar volume), this does not signify that species-specic adaptations do not exist, or that they are not important. It simply suggests that species-typical specializations must arise through other mechanisms. Some plausible, demonstrated mechanisms producing species diversity are: (i) the sensory or motor periphery imposing its order on the central nervous system, (ii) changed distributions of neuromodulators and receptors altering social motivation and attention, and (iii) dynamic reallocation of neural tissue to the most active channels by instruction through the sensory channels and motivational preferences of each animal. We have nally set the stage for looking at the relationship between phylogenetic variability in brain structure and scaling, and individual differences in brain organization.

somewhat to consider contrasting theories of brain function in a general evolutionary context. As mentioned, Jerison [2] rst proposed a relationship between encephalization, the possession of a relatively large brain with respect to body size, and behavioural complexity. He also hypothesized the strategic allocation of neural volume according to niche, which is the principle of proper mass. Particularly, he argued more volume came to be allocated to neocortex in highly visual, diurnal primates and more to olfactory bulb and the limbic system in early nocturnal insectivores. This axis of variation in relative olfactory bulb and limbic brain size proves to be a recurring principal component of vertebrate brain variability, not only of primates [37,50,51], whose signicance we will explore later. In parallel with Jerisons description, the growth of neuroethology on the one hand, with its attention to species-specic adaptations, and the early discoveries of localization or modularization of mature neural function in cognitive neuroscience led the eld to concentrate on the relationship of lifestyle and niche to specic brain parts. The basic hypothesis appeared that brains are collections of special-purpose devices or modules that can be the objects of selection, a point of view that retains strong adherents in evolutionary psychology (e.g. [52]), and some elds of cognitive neuroscience [53]. It is often assumed without question in literature about brain evolution [3,5]. To compare species, the immediate corollary of the collection of devices hypothesis was that the relative size of a brain part should reect the importance or complexity of the behaviour dependent upon it, proper mass. When investigated widely across species and brain structures, relating non-shared residual variation in the relative size of brain parts to niche or behavioural specializations typically met with only modest success, capturing statistically signicant but small amounts of variation (e.g. [3,5]). Many reasonable predictions based on the idea of proper mass fail to nd any relationship of relative size and utility at all, for example whether there is a correlation between a mammals nocturnality or diurnality and the volume of its visual cortex [54]. Any behavioural or tness advantage directly owing to change in the relative size of a brain part has yet to be demonstrated. Several apparent exceptions to this generality occur, which we will introduce now and discuss fully later in terms of the component structure of brain variability. Dunbar [4] posited that the relative size of the neocortex in primates, compared with the rest of the brain, was related to species complexity in social structure, the social brain hypothesis, though more recent analyses target whole brain size rather than cortex alone [55]. Species and individual variation in the hippocampus also stands out in its consistent relationship to requirements of memory in foraging and navigation, often dependent on experience [5658]. The hippocampus associates with the olfactory bulb and olfactory cortex and dissociates from neocortex scaling across mammalian species [35], and is part of the neocortex/limbic contrast originally laid out by Jerison [2]. Simply because species differences in sensory capabilities, motor adaptations and social structure do
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3. PHYLOGENETIC VARIATIONS IN BRAIN STRUCTURE Three features predominate in mammalian brain scaling: high intercorrelation of structure volumes, distinct allometric scaling for each structure and relative independence of the olfactory limbic system from the rest of the brain. At this point, we can consider nine major taxonomic groups [35], using the brain divisions employed in the original analyses of Stephan & Manolescu [59]. The rst two principal components of volume variation account for 99.01 per cent of the total volume variation in this set of 160 mammals. The rst factor is highly loaded on the cortex, cerebellum, diencephalon and mesencephalon, accounting for 96.47 per cent of the total variance (gure 1a, black bars). The second principal component loads most highly on the olfactory bulb, and next on olfactory cortex, hippocampus, septum and subicular cortices, accounting for 2.61 per cent of the variance (gure 1a, white bars). In addition, a third component relates body size independently to the sizes of the medulla and cerebellumthis factor is small, but signicant, and has been reported in other kinds of analyses (e.g. [60]). Finally, each brain division has a distinct allometry, with each brain subdivision increasing at a different slope as brain volume increases overall. In particular, the neocortex and cerebellum scale with absolute brain volume at high slopes so that very large brains become disproportionately composed of these two structures. The regularity of allometric brain relationships across mammals is echoed in a corresponding conservation of patterns of neurogenesis [36]. In a pattern that we termed late equals large, the latest-generated neuronal populations in a conserved order of mammalian development are the ones that show hyperallometry. Extending the duration of development to produce a larger brain has the greatest effect on the relative numbers of cells in precursor pools generating cells for the longest duration, such as those producing the cortex or cerebellum. Furthermore, the duration of neurogenesis maps onto an axis in the conserved embryonic brain plan in vertebrates such that the medially located, basal cell groups in brain

Review. Individual variability in brains

(a) olf. bulb olf. cortex hippocamp septum subic. cortex medulla striatum mesenceph dienceph cerebellum neocortex olf. bulb olf. cortex hippocamp septum subic. cortex medulla striatum mesenceph dienceph cerebellum neocortex (e) olf. bulb olf. cortex hippocamp septum subic. cortex medulla striatum mesenceph dienceph cerebellum neocortex (d) (c) olf. bulb olf. cortex hippocamp septum subic. cortex medulla striatum mesenceph dienceph cerebellum neocortex

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(b) olf. bulb olf. cortex hippocamp septum subic. cortex medulla striatum mesenceph dienceph cerebellum neocortex 0 0.2 0.4 0.6 0.8 % variance explained 1.0

% variance explained

Figure 1. In all graphs, the percentage variance in each structure described by the rst principal component (PC1) is graphed by the black bars, and the second principal component (PC2) by the white bars, the total percentage variance differing in each case. (a) Phylogenetic variability, based on a sample of 131 species of bats, primates and insectivores. PC1 accounts in total for 96.47 and PC2, 2.61%. (b) Individual variability, Composite includes 47 individuals whose scores were entered as deviations from cell means so as to exclude species and sex differences, where the cells were six male wild mink, six female wild mink, six male domestic mink, six female domestic mink, six wild pigs of unknown sex, six domestic pigs of unknown sex and 11 mouse strains. PC1 accounts for 72.48% of the variance, and PC2 for 7.9%. For the individual species, (c) pig, (d ) mink and (e) mouse, data are plotted so that their overall pattern might be examined, but no statistical claims about factor loadings on individual structures are made at the individual species level.

segments (segments here are spinal cord segments, rhombomeres and prosomeres) cease precursor generation early. Laterally located, alar groups stop last, or in the case of hippocampus and olfactory bulb continue into adulthood [38]. Taxa included in the phylogenetic analysis of brain variability are very diverse, including species of megaand microbats, shrews, armadillos, polar bears, llamas, humans and manatees, with brain sizes ranging over 20 000-fold [35]. Within any particular species, brain sizes will only range over a tiny fraction of this amount, but as phylogenetic variability must arise from the heritable components of individual variability, it is entirely reasonable to ask what aspects of phylogenetic variability in brains are mirrored in individual variability. One excellent source of brain volume measurements of multiple individual members of single species exists, measured in a way comparable with the original Stephan
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dataset and the Reep extended dataset. Dieter Kruska measured a variety of individual animals to study the effects of domestication. He compared a sample of six wild boars with six domestic swine (brain sizes range from 92 to 204 g, ratio 2.21 [61]) and 12 adult wild mink brains with 12 adult ranch mink brains (brain sizes range from 7.2 to 10.4 g, ratio 1.44 [62]; the electronic supplementary material, table S1). He compared a number of other domestic and wild species with fewer individuals (reviewed and discussed in [63 65]). In the present analysis, our interest was not domestication per se, but the availability of measurements of a number of individuals of the same species with the bonus of the added variation produced by domestication. In addition, morphometric analyses of a wide variety of mouse brains used for genetic analyses are now available from an online database ([66]; Box 1 includes individual strain descriptions). In this

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Box 1. Mouse strain details. In general, most strains are wild-derived inbred strains with, at most, inducible non-neural diseases. Only WSB/Ei and CAST/Ei are referred to as wild strains. CAST/Eiis referred to as a wild strain (not inbred) and is often used as a control line. In a study characterizing behavioural phenotypes, CAST/Ei always came out somewhere between inbred strains, with no signicant behavioural phenotype evident from the approximately 13 tests described [67,68]. CASA/Rkwild-derived inbred strain, no abnormal phenotype. Molf/Eiwild-derived inbred strain, which has no abnormal behavioural phenotype but is extremely susceptible to infection with Salmonella typhimurium [69]. SWR/Jwild-derived inbred strain, which is susceptible to chemically induced colorectal cancer, but responds well to the anti-tumour drug lentinan [70,71]. The SWR/J strain also exhibits more extensive corneal clouding after UV exposure than other inbred strains do, and control SWR/J mice exhibits a low activity variant phenotype for the major ocular aldehyde dehydrogenase (ALDH) AHD-4, and decreased levels of soluble protein in corneal extracts. Theory: ALDH assists the cornea in protecting the eye against ultraviolet radiation-induced tissue damage [72]. WSB/Eiwild strain very rarely used. SM/Jnon-diabetic [73], small inbred strain. In the mouse the naturally occurring inbred strain SM/J presents with a number of phenotypic abnormalities that have been attributed to reduced neuraminidase activity. SM/J mice were originally characterized by their altered sialylation of several lysosomal glycoproteins. This defect was linked to a single gene, neu-1, on chromosome 17, which was mapped by linkage analysis to the H-2 locus. In addition, these mice have an altered immune response that has also been coupled to a deciency of the Neu-1 neuraminidase. Here, we report the identication in SM/J mice of a single amino acid substitution (L209I) in the Neu-1 protein that is responsible for the partial deciency of lysosomal neuraminidase. [74]. Also, Compared with other inbred strains, SM/J mice have both abnormally high responses to B cell mitogens and hyper NK cell and K cell activity. [75]. RIIIS/Jinbred strainhighly susceptible to collagen-induced arthritis [76] and produce low antibody responses to several polysaccharide Ag of bacterial origin. [77]. SJL/Jinbred strainnot much information, no abnormal phenotype described. Molc/Rkinbred straina light bellied-agouti. PL/Jinbred strain with susceptibility to skin diseasePsoriasis is a frequently occurring inammatory skin disease characterized by thickened erythematous skin that is covered with silvery scales. It is a complex genetic disease with both heritable and environmental factors contributing to onset and severity. The CD18 hypomorphic PL/J mouse reveals reduced expression of the common chain of b2 integrins (CD11/CD18) and spontaneously develops a skin disease that closely resembles human psoriasis. In contrast, CD18 hypomorphic C57BL/6J mice do not demonstrate this phenotype. [78]. Thr/ftwild-derived inbred strainno abnormal phenotype was described.

case, single individual examples of 11 different strains were chosen (ranging from 0.30 to 0.53 g, ratio 1.8), using the neuroanatomic delineations identical to those described in Reep et al. [35]. These individuals were examined for the same principal component structure examined previously, considering as covariates species, sex and domestication, as described in Reep et al. [35] and Finlay & Darlington [36] (brain measurements, table 1).

4. PRINCIPAL COMPONENT STRUCTURE AND ALLOMETRIC SCALING IN INDIVIDUAL BRAIN VARIABILITY (a) First principal component The factor loadings for phylogenetic variability already described are shown in gure 1a, and for individual variability in gure 1b. In each case, the per cent factor loading on each brain structure within the total variance of each component is plotted. The rst principal component (PC1; black bars) accounts for much more variance (phylogenetic variability, 96%; individual variability, 72%) than the second component (white bars; 3 and 8%, respectively). The principal component analysis is also broken down by individual species in gure 1c e. Although the number of individuals is too small for statistical comparison between species, these graphs are included so that the relationship of individual species data to massed data can be examined. Comparing the
Phil. Trans. R. Soc. B (2011)

individual analysis with the phylogenetic analysis, the PC1 loads on a similar range of brain parts. Though the total amount of variance explained is less than in the phylogenetic analysis, 72.48 per cent is remarkable considering the 20 000-fold range in the absolute brain sizes of the phylogenetic dataset versus the approximately twofold range of within-species variability. The similarity of the structure of the variance is the more striking in that this dataset includes the peculiar effects of directed selection for different aspects of domestication, and various indeterminate effects of laboratory rearing on the mouse strains, and not natural selection. (b) Second and third components The second principal component of variation (gure 1; white bars) loads most strongly on the olfactory bulb in both cross-species and individual cases. This component contributes more highly to total variance in the individual than in the phylogenetic comparison, 7.9 per cent versus 3 per cent, explaining 86 per cent of the residual variance not accounted for by the rst factor. In each of the individual species, (i.e. pig, mink and mouse), the second principal component loads most highly on olfactory bulb though loading on other brain subcomponents varies widely. We further examined the relationship of body size to brain components, because a relationship of body mass to medulla and mesencephalon, partialing out brain size, has been noted previously. Because

Review. Individual variability in brains

35.9 37.5 58.8 45.3 230.5 4.4 6.8 27.9 18.5 20.8 111.9 40.2 Table 1. Volumes of brain structures for eleven mouse strains. Denitions of the inclusions of each structure, which together comprise the entire brain, can be found in Reep et al. [35]. PL/J

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CAST/Ei 433a

34.6 28.5 54.9 35.64 224.21 4.85 7.2 27.3 22.36 21.2 97.7 43.6

SJL/J

individual body weight was not available for the mice, a regression analysis controlling for the effects of species, sex and domestication was done on the remaining animals. Body weight correlated highest with medulla (r 0.198) and second highest with mesencephalon (r 0.1143). Since the correlation coefcient for medulla is 74 per cent higher than even the second highest value for the mesencephalon, we can be reasonably condent that individual variability also echoes cross-species variability, in both cases, a small effect.

MOLC/Rk

38.6 32 59.7 42.5 218.8 4.9 6.4 21.9 25.9 26.8 95.8 37.1

(c) Mice follow the pattern, but some mouse strains are outliers An interesting feature to the pattern of variability for the rst and second principal components is plotted in gure 2. For each individual pig, mink and mouse, a point is plotted for their value of PC1 and PC2. Of the four individual animals lying outside the 90 per cent condence circle, three are commonly used strains of laboratory mice. Two are aberrant on PC1, one loading abnormally high (WSB/ei) and one abnormally low (MOLF/ei). This means that for WSB/ei, the major brain parts associated with the PC1 (cortex, cerebellum and so forth) are unusually invariant with respect to each other (two standard deviations from the mean), while MOLF/ei is unusually variable. The third mouse strain, abnormally high on PC2[MOLC/ei], has large and highly correlated limbic system components, ve deviations from the mean. What feature of the ordinary domestication of minks and pigs retains the factorial structure in brain variation resembling phylogenetic variability, which appears to be lost in part in the form of selection exerted on individual mouse strains, is not clear. This observation suggests that unusual variation in brain organization should be considered as a factor in the research use of these strains. A recent magnetic resonance imaging study of recombinant inbred individuals of two strains of mice for the purposes of identifying genetic inuences on brain volumes and neuron numbers used different subdivisions of brain from those employed here, but retrieves a generally similar structure, though the range in brain sizes is reduced still further [44]. The PC1 in both strains loads highest on cortex, and the third highest on olfactory bulb; the second principal component loads highest on midbrain and medulla, reversing the order of the second and third components we observed. Earlier work of this same group [41] strongly linked overall control of brain neuron number to gene regions related to transcription factors and to overall somatic growth, consistent with the idea of the PC1 of the brain region related to duration or rate of development linking the proliferation of all brain regions. Another study of control of neuron number and size of the olfactory bulb noted that the bulb size (unlike the brain) was highly variable, was related to sex and experience, and also changed in volume late in maturation [79]. Four different genetic loci from the one controlling whole brain size were related to olfactory bulb variation.

RIIIS/J

Molf/Ei

CASA/RK

mouse strains

CAST/Ei

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structure volume (mm3) mes di cer med tel sept schiz striat obulb hippo neo palaeo

32.23 34.75 51.87 41.8 208.469 4.28 6.04 23.42 20.14 25.43 88.37 40.789

28.19 32.58 54.19 43.24 218.98 3.76 8.15 24.12 17.86 21.3 103.69 40.1

25.38 25.24 45.47 36.09 163.48 2.53 6.99 20.9 16.1 14.7 77.78 24.48

32.88 29.51 49.69 43.91 230.29 3.89 8.08 28.34 20.34 20.83 103.3 45.51

SWR/J

34.51 46.45 61.06 39.54 251.218 8.628 9.67 29.4 14.09 31.14 114.86 43.43

WSB/Ei

48.8 50.07 80.86 55.7 295.57 6.4 11.9 40.4 32.8 30.9 122.1 51.07

Thr/ft

41.5 47.28 67.1 47.8 253.5 6.4 11.4 28.3 27.6 26.1 104.8 48.9

SM/J

39.06 42.61 77.69 55.88 255.1 6.7 8 32.6 23.4 26.8 110.3 47.3

47.2 34.2 53.1 33.1 235.1 5.9 11.5 25.1 23.7 33.2 93.5 42.2

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0.6 ratio of wild/domestic cortex volumes, ln 0.5 0.4 0.3 0.2 0.1 0 boar/pig

3 principal component 2

guanaco/alpaca and llama wild/domestic ferret

wild/lab rat

wild/domestic sheep gerbil

wild/domestic mink 0 0.1 0.2 0.3 0.4 ratio of wild/domestic brain volumes, ln 0.5

3 2 0 2 principal component 1

Figure 2. Values of PC1 and PC2, expressed as a z-score for each individual animal for the population of 47 individuals. Three of the mice and one pig fall outside the 90% delimiter (dashed line); these mouse strains are WSB/ei, MOLC/ei and MOLf/ei. Mouse strains may be intrinsically more variable because of greater genetic difference than the minks and pigs, whose lineages are unknown, but whatever the cause, their deviation from the phylogenetic and the other individuals is pronounced. Diamonds, minks; open circles, pigs; lled circles, mice.

Figure 3. Comparison of the ratio of cortex to total brain volume in seven wild/domestic comparisons gathered by Kruska, compared with equal proportionality (dashed line) or to cortex hyperallometry determined phylogenetically (solid line). In ve of the seven comparisons, cortex volume is reduced more than overall brain volume.

(d) Cortex hyperallometry Finally, the hyperallometry of the cortex can be examined in this dataset by comparison of the various domesticated and wild animals studied by Kruska [62,64,65]. We conrm his observations of the relative greater reduction of the cortex compared with whole brain volume. We are able to further describe the relative reduction in cortex size of the domesticated species compared with our cross-species database (gure 3). In general, the effect of domestication is a decrease in relative brain size to body size or encephalization, ranging from a 26 per cent reduction from the wild guanaco to the domesticated alpaca or llama to equality between the wild and domesticated minks; no pair has the reverse relationship. The solid line plotted in gure 3 is the regression line of slope of 1.3 previously computed for the phylogenetic relationship of neocortex volume to the total brain volume [35], not the regression line for these data. For visual comparison, the dashed line plots the slope of 1.0, the line that would be observed if the cortex decreased isometrically with the total brain volume. Five of the seven comparisons fall on or above equal proportionality. So, as domesticated species decrease in relative brain volume, they decrease disproportionately in neocortex volume, in accord with the phylogenetic pattern. (e) Species differences and principal component 2 Both across and within mammalian taxa, the volume of the neocortex (the structure loading highest on PC1) versus the volume of PC2 limbic structures
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(olfactory bulb and cortex, hippocampus, amygdala and septum), both compared with the rest of the brain, are negatively correlated [35]. Various multivariate analyses of the initial Stephan dataset (e.g. [5,36,50] and others) capture this same contrast. The negative correlation of the volume of limbic structures and the cortex contributes to a large number of studies showing mosaic, part-by-part, or systemby-system covariation (e.g. [80,81]). The olfactory bulb and hippocampus have the further unusual feature of continued neurogenesis into adulthood in mammals, which is one clear substrate for their linkage to sex and experiential factors [79]. In the very large number of studies linking hippocampus size to niche and foraging strategy, in mammals and in birds, across and within species, the whole brain or some adjacent structure is typically used for calibration of hippocampus volume, so it is unclear if the hippocampus varies independently from the rest of PC2 in all cases [56,57]; see reviews in [58,82,83]. Of the various contrasts that have been made in the studies of hippocampus, avian versus mammalian, foraging range and the presence of hording, seasonality of foraging and gender, it would be interesting to see if some contrasts claim the entire set of principal component 2 structures, and if some are specic to the hippocampus. In any case, the two dominant components of mammalian brain variation, which we have recently shown also characterize chondrichthyans (sharks and rays; [51]), are an enduring hot spot for species-typical variation.

5. A COMPUTATIONAL CONTEXT FOR BRAIN VARIATION The close resemblance of phylogenetic variation to individual variation was quite surprising, given the orders of magnitude differentiating the variation of phylogenetic and individual samples. Reiterating, we

Review. Individual variability in brains nd consistent covariation in the relative size of brain parts with respect to each other. We nd the same dissociation, the independent variation of olfactory bulb with respect to the rest of the brain. Finally, we nd the predicted disproportionate reduction of the neocortex with respect to the total brain volume, in those domesticated species that have regressed in total brain volume. The similarity is the more impressive in that domestication and laboratory animal membership should minimally exert atypical genetic pressure on the individuals of these species. Independent of genetics and any kind of selection, domestication itself should have large effects on rearing conditions, nutrition and general experience of individual animals likely to confound an analysis of brain variability, not enhance it. Of course, the seeds of phylogenetic variability must be found in individual variability, but it is surprising that the pattern of phylogenetic variability should conform in such detail. The morphological evolution of stickleback species, where individual variation matched species variation, over both short and long phylogenetic differences, closely resembles these results [28].

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(a) Brain architectures that scale gracefully The hyperallometry of cortex (and cerebellum), which allocates the prots of extended brain production preferentially to multi-functional brain components, may be useful both at the individual and species level. We can take some hints from current computing research, where it is of obvious practical use to develop computing structures in which more elements can be added as necessary, or where an architecture is desired which can lose components and remain at least partially functional (as in warfare). These scalable computer architectures, or subsumption architectures, allow for the addition and subtraction of components gracefully without interference in fundamental operations [84,85]. One basic insight arising from this literature is that locating new computational circuits directly in command lines executing central functions impairs processing speed and prohibits scaling, but locating new computational power as ancillary loops modifying basic functions improves speed and enables scaling. Restating, if more computing power is located between sensory input and motor output, it slows the entire device and makes it vulnerable to damage at any point. If, however, a model brain is produced beside the basic command lines but able to intercept and modify commands as they are made, a more robust architecture with more computing power and no loss in speed is the result. This, of course, is a good description of the computational position of most of the cortex and cerebellum. Since this computational claim is a property about brain scaling in general, and not taxonomic levels of brain organization, it should thus hold true for variations in brain growth, adult variations in brain size and species level. To draw an analogy with our original colour vision example, while it might be extremely useful for a monkey to rapidly recognize bananas, placing a banana-recognition device in the retina may never be computationally feasible if the design requirements of
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the whole brain are considered. Testing this hypothesis about the evolvability of particular types of circuit organization will eventually require examination of circuits known to range in size in diverse vertebrate and invertebrate groups, and comparison of their properties. Considering the partial dissociation of the olfactory bulb and limbic structures, we speculate that the pattern of variation at the individual level may be either or both the source and product of evolution. Evolution must certainly serve as a lter against deleterious variations, but might pass on neutral ones. We speculate that the independent variation of olfactory bulb from the rest of the brain may be not so much selection for olfactory variability, but rather selection for tighter coupling of the other sensory systems that must share thalamic projections and neocortical representations. Modelling an independent enlargement of a single dened module in a neural net consumed disproportionate amounts of the neural resources of the other modules sharing the same net resources as its excessive input output requirements propagated through the net [86]. Sequestering the variation of the single mammalian brain part that can vary its size by generation of neurons throughout life may aid this computational requirement.

6. TWO LARGE CLASSES OF BEHAVIOURAL VARIATION? Returning to our initial discussion of brain variability and its behavioural correlates, we suggest there are two very different kinds of change typical in brain evolution. The rst is the general increase in computational power and exibility afforded by a large brain, which can be measured in the very most general ways: surviving longer by avoiding predation, exploiting new food sources and learning new strategies to nd shelter, attract mates and protect offspring [32]. These classes of behaviour are only incidentally niche- and modality dependent, and would be ill served by strong modular commitments. Variation in the relative size of cortex among humans has a small but signicant relationship to general intelligence. As yet, no such relationship between brain size and general behavioural capacity has been shown at the individual level for any other species; the work by Lefebvre et al. [32] compares species, but the relationship should hold for individuals as well. Overall, for general problems, the demands of general-purpose architectures should predominate, and should be seen both in individual and species variations, the kind we describe here. On the other hand, species-typical behaviours of every possible sort have evolved. On the sensory side, we have everything from variants of colour vision, to whole new sensory systems like electroreception in mammals, to sensorimotor combinations like echolocation. On the motor side, we have crawling, ying and competitive gymnastics. Every variation of social preference and aggression exists, generating from individual preferences lifelong monogamy, cooperative predation, solitary individuals and herds. The data here do not bear directly on the mechanisms of these changes. However, evolutionary changes must occur

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Review. Individual variability in brains production systematically varies may be essential to a useful brain architecture, one that scales gracefully and is robust to damage. Note also that most of the mechanisms that transform initial, generic brain architecture into a species-specic instantiation are developmental or epigenetic mechanisms, which take key changes, often in the sensory and motor periphery, and amplify their effects throughout the nervous system. We return to our visual system example. Rather than producing a more visual primate with trichromatic colour vision and fovea by adding partsgenerating more cells in the fovea, a new set of photoreceptors, more stages of processing, a larger-than-expected lateral geniculate, larger primary visual cortex, genetically specied colour vision modules and so forthalterations are made within the overall scaling architecture [102,103]. Nevertheless, the restructuring and processing changes are profound. To produce the high visual acuity of the primate fovea, the retina is differentially stretched, a topological rather than additive change, to compact cells in the fovea and spread out the cells of the periphery, conserving the expected number of cells in the retina. Consequently, processing resources of the cortex are concentrated automatically on the central few degrees of the visual eld. A new opsin is expressed, but adds no total photoreceptors to the retina, subdividing the initial set. The new chromatic information is then analysed by the generalized comparator mechanism for wavelength already present in the animal [20]. More interest and attention to visual features, perhaps mediated by subcortical changes in motivation, will cause activity-dependent processes to allocate more and more brain space to visual information. Aspects of modularity may emerge in cortex, for example, for colour processing, or face areas from the same Hebbian re together, wire together processes. Some of these changes arise from the immediate epigenetic effects of connecting up the nervous system, but many more depend on later interactions with the environment, guided by the animals attentional and motivational preferences. The study of brain evolution and special behavioural adaptations may essentially become the study of guided brain development.
Supported by NSF DBI0848612 to B. Finlay. We thank Christine Charvet for her help with the production of this manuscript, and also the three anonymous reviewers for their clarifying suggestions.

largely within the numeric connes established by general brain scaling. Specializations in the sensory periphery impose their structure on the brain, causing the representation of the whole body surface to disproportionately represent the specialization, from the palpating nose of the star-nosed mole, to hands and tongues, to whiskers and tusks. Many such cases of redirection of generic brain structure are well described (e.g. [8790]). These allocations may develop in infancy, or may dynamically relocate as required, as in the reallocation of the visual cortex for Braille reading even in the late blind [91]. A second source of rich variability is just beginning to be investigated, the churning of the nonapepetide modulator and receptor distribution in basal forebrain and midbrain networks, where small changes in expression patterns give rise to large changes in the motivational and attentional structure of individual animals, likely to result in the emergence of new social organization in populations [92,93]. The third source of evolutionary behavioural variation is intrinsically developmental: over and over again, although the cortex comes equipped with initially highly specied input connectivity, it has been shown that computational space is allocated in cortex on the basis of activity, from micro- to macro-scales (reviewed in [94]). Since the source of our information on this has often come from developmental accidents of deafness or blindness [91,95,96] or deliberate experimental subtractions [97], the plasticity observed is often seated in the context of recovery from pathology. Dynamic reallocation of function according to relative activity, however, both during development and at adulthood (e.g. [98]) is probably the normal state of affairs, and should be studied more systematically in non-manipulated brain, and as an emergent property in species adaptations. The massive re-use of brain tissue implied by imaging studies, showing the same tissue lighting up repeatedly in diverse contexts and tasks (e.g. [99,100]), is probably no artefact of experimental design or poor task distinctions, but a basic operating principle of a general purpose device put to use in multiple adaptive contexts.

7. EVO-DEVO, THE BRAIN AND BEHAVIOUR We can now return to highlight how some features of brain scaling may be examples of the facilitated variability and evolvability that have been much discussed in the evo-devo literature. In general, in the mammals discussed here, simple duration of brain growth is tightly linked to brain size [101]. Changes in the duration of growth (of genetic, but possibly environmental origin as well) do not produce isometric changes in all brain parts, but allocate volume changes preferentially to the structures produced over the longest embryonic duration. In neural development, the basal-to-alar axis of repeating brain segments (the embryonic medial-to-lateral direction) is the axis in which increasing duration of neurogenesis is roughly represented [35,38]. While this conserved axis could be argued to be a developmental constraint on brain structure, we have presented arguments here that a brain axis in which duration of neuron
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Phil. Trans. R. Soc. B (2011) 366, 21242140 doi:10.1098/rstb.2011.0001

Review

Evo-devo, deep homology and FoxP2: implications for the evolution of speech and language
Constance Scharff * and Jana Petri
Department of Animal Behavior, Freie Universitat Berlin, Takustr. 6, 14195 Berlin, Germany The evolution of novel morphological features, such as feathers, involves the modication of developmental processes regulated by gene networks. The fact that genetic novelty operates within developmental constraints is the central tenet of the evo-devo conceptual framework. It is supported by ndings that certain molecular regulatory pathways act in a similar manner in the development of morphological adaptations, which are not directly related by common ancestry but evolved convergently. The Pax6 gene, important for vision in molluscs, insects and vertebrates, and Hox genes, important for tetrapod limbs and sh ns, exemplify this deep homology. Recently, evo-devo has expanded to the molecular analysis of behavioural traits, including social behaviour, learning and memory. Here, we apply this approach to the evolution of human language. Human speech is a form of auditory-guided, learned vocal motor behaviour that also evolved in certain species of birds, bats and ocean mammals. Genes relevant for language, including the transcription factor FOXP2, have been identied. We review evidence that FoxP2 and its regulatory gene network shapes neural plasticity in cortico-basal ganglia circuits underlying the sensory-guided motor learning in animal models. The emerging picture can help us understand how complex cognitive traits can descend with modication. Keywords: basal ganglia; bird; vocal learning; avian; song learning; zebra nch

1. INTRODUCTION The purpose of this paper is two fold. On the one hand, we will review recent contributions from the eld of molecular genetics and neurobiology to understanding acoustic communication in humans and animals and we will place those ndings into a larger evolutionary developmental framework bearing on the evolution of language. On the other hand, we would like to critically re-examine the evidence for the claims that certain features of language are unique to humans and absent from all animals. The inclusion of concepts from developmental biology to evolutionary theory has led to the eld of evo-devo [1]. Others, including DArcy Thompson [2], Alan Turing [3] and John Maynard Smith [4], had previously entertained the notion that the structure, composition and dynamics of the developmental system pose limits on the phenotypic variability. During the last decade, experimental data from many systems have illustrated how developmental principles and constraints actually contribute to morphological novelty. Drawing on molecular and genetic methods, developmental biologists have uncovered conserved molecular networks that shape the morphology of different species [5]. How these molecular pathways change during the course of evolution and thereby contribute

* Author for correspondence (scharff@zedat.fu-berlin.de). One contribution of 10 to a Theme Issue Evolutionary developmental biology (evo-devo) and behaviour.

to morphological adaptations is a central theme in the current evo-devo research [68]. General concepts are emerging that may not only apply to the evolution of form, but also extend to the evolution of behaviour ([9]; see also [1012]). Language and music are behaviours that constitute a fascinating evolutionary puzzle. Animals are usually considered to have neither language nor music, so how, when and why did these traits evolve in the human lineage? Did they evolve gradually [13] or through a sudden change [14]; were they driven by natural [15] or sexual [16] or relaxed [17] selection; or did they emerge via other processes [18]? Academics throughout the documented history of human scholarship have written on the subject. To quote Noam Chomsky There are libraries of books and articles about evolution of languagein rather striking contrast to the literature, say, on the evolution of the communication system of bees [14, p. 14] and many different scenarios of how language evolved have been proposed [19 28]. However, as one eminent contemporary scholar in the eld, Tecumseh Fitch, succinctly put it . . . discussion of the evolution of language often involve more speculation than data. . . [29, p. 258]. Given this skewed relationship between the amount of empirical data and the number of publications, it may not be surprising that there has been a shift in appraisal concerning the potential contribution of animal studies to the subject of human language evolution, from initially very little [30] to recently a lot more [13,28,31]. Where one stands on this issue
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Review. Evo-devo and FoxP2 depends on ones view of the uniquely human aspects of language. Which aspects of language are unique to humans are still a matter of debate [32 34], but that some aspects of language are uniquely human and absent from all animals communication is not questioned in the literature. This is actually a bit surprising, given that the communication systems in many animals have hardly been exhaustively studied. We will return to this question below. What is clear though is that human language must have emerged through qualitative and quantitative modications of morphology that existed in our primate ancestors, e.g. changes in the size of brain regions, alterations of the strength of neural connections, creation of new neural pathways, and morphological alterations of the peripheral sound production and sound perception machinery. Among the molecular mechanisms that are known to shape such changes are heterotypy (altered gene products), heterochrony (altered timing of gene expression), heterotopy (altered spatial gene expression) and heterometry (altered amounts of expression). How these changes come about in evolution is a topic of lively debate and investigations. An evo-devo framework and the concept of deep homology [35,36] increasingly permeates the thinking of biologists of any eld, but may be less familiar to linguists, cognitive scientists, psychologists and philosophers who are interested in the evolution of language. We propose that, in considering the evolution of human language, an evo-devo approach can provide a useful theoretical framework to study genetic modules that are necessary components of language. Specically, we argue that the FoxP2 transcription factor and the regulatory molecular network that it interacts with may be part of a molecular toolkit that is essential for sensory-guided motor learning in cortico-striatal and cortico-cerebellar circuits in humans, mice and songbirds and maybe even invertebrates. The nomenclature for Forkhead (Fox) genes follows Kaestner et al. [37]. Essentially, the spelling is: human, FOXP2; mouse, Foxp2; and all other species, FoxP2. As per convention, genes and mRNA are italicized, proteins not.

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2. LANGUAGE, SPEECH, ANIMAL COMMUNICATION AND VOCAL LEARNING: SOME DEFINITIONS When discussing the evolution of language in the context of animal vocalizations, it is necessary to dene some terms. Human communication can be verbal, using words or non-verbal, such as cries, sighs, laughter and gestures. The latter may have been the substrate for the development of so-called protolanguage [38]. Spoken language expresses facts, thoughts and feelings orally using specic sounds that are created via a precisely coordinated motor programme involving jaw and orofacial musculature as well as the muscles in larynx, neck, chest and abdomen. This motor aspect of language is called speech. Non-verbal and verbal vocalizations occur in many behavioural contexts, voiced in a communicative context but also when alone (inner voice). Animals also communicate in many different contexts, including mate attraction, territorial defence, group cohesion, foraging or parent offspring
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interactions [39,40]. In addition, animals may vocalize outside of any obvious communicative context, as is the case when male zebra nches sing undirected song, which often occurs while birds are alone [41]. Interestingly, talking to oneself is one of the traits that has been stated to be human unique (e.g. It seems likely that these private soliloquizing, praying or talking to oneself are uniquely human activities that evolved on top of prior purely social communicative form of language) [42]. In fact, not only songbirds sing when they are alone, parrots and chimps that have been taught to imitate human words, vocally or as signs, also do not limit their use to a communicative context [43,44]. A complementary approach to organizing animal vocalizations by behavioural context is to sort them by bioacoustic features. Calls are mostly short utterances, often with variable temporal sequences that occur usually in specic circumstances such as begging calls, alarm calls, contact calls, food calls or distress calls. Songs are typically longer utterances with more stereotypically ordered temporal sequences, frequently associated with territorial defence or reproduction, including pair cohesion and mate attraction. Songs occurring in the aforementioned contexts have been described, for instance, for birds [45], mice [46,47], bats [48 50], whales [51,52] and gibbons [53 55]. Learning of human language and birdsong is often used synonymously with the terms vocal learning or production learning, which refer to the change in sound signal as a result of experience [56]. For instance, the rst uttered words emerge from non-verbal infant babbling as a result of infants experience with adult language. This type of learning has only been documented in a few species, among them all songbird species studied [45,57], various species of parrots [45,58,59], Anna hummingbirds [60], sack-winged bats [61], a harbour seal [62] and two elephants [63]. In contrast, many animals can associate an existing sound signal with a new context (referred to as contextual or auditory learning) [56]. For instance, a young vervet monkey can learn to associate hearing a particular alarm call with the presence of a particular predator (comprehension learning) and it can even learn to produce a particular innately specied call in a particular predator context, as a result of observing conspecics (usage learning) [64,65]. Contextual auditory learning is common in animals, whereas vocal production learning is not. Both language and birdsong are best learned during a period of opportunity also called sensitive period during early development [66].

3. DECONSTRUCTING LANGUAGE INTO BIOLOGICALLY TRACTABLE UNITS In broadest terms, language can be divided into a conceptual intentional system that deals with thoughts and meaning, and a sensorimotor system that deals with the acoustic analysis of speech sounds and their production [32]. Of course, this is an oversimplication and those two systems feed back on each other, the same way the brain inuences behaviour and behaviour inuences the brain.

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C. Scharff & J. Petri Review. Evo-devo and FoxP2 In animals with large repertoires, the potential limitation of combinatorial coding space is even less of an issue. However, bird song researchers have compared the number of distinct sound elements in the songs of different species with the number of combinations in which they occur. In contrast to the situation with language, in all investigated bird species there are more song elements in the repertoire than combinations of those elements [74], which argues at rst sight against the existence of duality of patterning. In addition, in many bird species, strings of song elements that occur together usually do so in a hierarchically structured, mostly unidirectional way, again, in contrast to language. This is also the case for instance for free-tailed bat song [49]. However, if one considers the size of the combinatorial unit not to be the smallest song element (also called a note, a syllable or an element in bird song literature) but the next larger unit of song, e.g. a string of ordered elements occurring together (called song type, or motif or phrase in the literature), the situation is different. Song types in some species with large repertoires occur in long, non-random and non-unidirectional arranged sequences during song bouts that last from minutes to many hours [75 78]. At this organizational level, there is much more room for complex sequential rules that could be rich enough to carry semantic information. In fact, song type (or motif) order in many birds and some bats [49] can be much more dynamic than note order within song types. If animals were indeed using such sequences in a combinatorial way to convey semantic content (hypothetically speaking), then animals would have to integrate information conveyed over longer song sequences, which requires sufcient auditory memory capacity. Working memory for auditory sequences in at least one songbird, the starling, is in the same range as that of humans [79], and starlings may use similar strategies as humans to store and retrieve serially ordered auditory communication signals [80]. In addition, for items other than song, birds are known to have large memory capacities; for instance food-caching birds can memorize the location of hundreds of stored food items over a period of several days [81]. In addition, female birds of various species visit 10 or more mating partners before settling on one. In the well-documented case of satin bowerbirds, this search can take weeks during which females must remember features of those potential mates [82]. Thus, in principle, birds can hold many items in memory over varying periods of time. In human language, grammatical rules structure the plethora of combinatorial possibilities. For instance, an English speaker knows that a sentence starting with if will be followed by a then (implicit or explicitly stated), but the number of words in between can vary. The causality, however, will be completely clear. Likewise, the difference between the meaning of Anna laughed at Ben and Ben laughed at Anna being coded by syntax is a feature that is generally assumed not to exist in animal vocalizations. There is no known parallel in animal communication, but to our knowledge, the idea that combinatorial signalling carries semantic information at the level beyond song syllable combinations has not been formally explored.

Which features of language can be analysed in animal models? In the 1960s, some linguists considered human language and animal communication to be so categorically different that they were essentially incomparable [30]. Hockett postulated a continuum of complexity among animal communication systems, including human language [67]. Those positions still mark the two ends of the spectrum, but concomitant with the emergence of biolinguistics as a research eld [68 70], the abyss between the two camps is slowly being bridged [14,32,33]. The present review is a contribution to this biolinguistic perspective. Reviewing the literature on the link between FOXP2 and language in people and its role in birdsong and other animal models will lead us to propose, in 4, that this transcription factor and its associated molecular network may constitute one of the constraints that channel evolutionary patterns towards similar outcomes, e.g. learned vocal communication in diverse taxa. But before reviewing the evidence, we will spend a moment challenging the assertion in most if not all writings on the subject that animal studies can solely contribute to the sensorimotor speech aspects of language, whereas syntax and particularly semantics, the language domains that allow the externalization of the conceptual intentional system, do not exist in animal communication. Since animals are increasingly recognized as having concepts and intentions [71 73], we think it is useful to re-examine the reasons why animals are assumed not to communicate about those. Below, we will therefore review different aspects of language and which of those are known to exist in animals (3(a)) and lay out the advantages of a comparative approach to language evolution (3(b)).

(a) Conceptional intentional processes in animal communication? Hockett [67] proposed that human language is characterized by the sum of a set of design features, some of which he recognized as present in animals. Among those were auditory properties of sound, and the fact that communication systems have senders and receivers. Others he assumed to be exclusive to language. We will examine the evidence for their unique status one by one. Traditional transmission, e.g. the social learning of language, Hockett considered to be uniquely human. However, as mentioned above, a number of animals are also capable of socially mediated vocal imitation, a fact already recognized by Aristotle [19]. Duality of patterning refers to the ability to create from a nite set of sounds an innite set of words and from these an innite set of sentences. This is also regarded to be unique to human language. Two arguments against duality of patterning in animal vocal communication have been put forward. If an animal has a small sound repertoire, there are only a limited number of combinations possible, even though it is also clear that theoretically even very small (and not necessarily imitatively learned) repertoires can code large amounts of information (e.g. binary computer code, or 4-letter DNA alphabet).
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Review. Evo-devo and FoxP2 It would require analysing whether particular strings of song types sung in particular sequences correlate with different situations or behaviours. These are questions that are hard, but not impossible, to address experimentally. Productivity, the ability to create new utterances by combining existing utterances, Hockett also considered to be uniquely human. For example, enter can be combined into entertain or enterprise, meaning different things. Adding an im to patient reverses the meaning. To our knowledge, few studies have addressed whether different combinations of calls or song elements could have different meaning. Where it has been investigated, for instance in Campbells monkeys, it was found that they can combine calls in such a way that indeed the meaning can change [83]. Semanticity, e.g. the fact that language is about things and can express arbitrary thoughts is usually considered to be exclusive to humans. Two quotes exemplify this . . . the elements of birdsong or whale or gibbon song are not put together by the animals in such a way that the whole song conveys some complex message assembled from the meaning of the parts [42] or If there are nonhuman species with openended semantics, they are remarkably clever at hiding these abilities from generations of dedicated ethologists [84]. In fact, generations of dedicated ethologists have not addressed the meaning of parts of song but are instead operating under the assumption that animals communicate mainly about ghting or irting in a non-semantic way. What experiments have addressed song semanticity? The fact that a playback of a mere song fragment can be sufcient to elicit an agonistic response from a territory holder may not be any more indicative of song syntax and semantics than the fact that the mere you stupid? can be sufcient to start a brawl at a bar. Besides, bees communicate about food [85], many animals use calls that refer to different predators [86,87] and grey parrots can learn to use sounds to refer to objects [44]. Some great apes can also learn to use signs or sounds to refer to objects [88,89]. None of these examples capture all aspects of semanticity in the human language, but they indicate that animals are in principle able to use arbitrary vocal or other gestures associated with meaning. Whether they do this as extensively as humans do, what subjects other than food and predators they might communicate about, and what role syntax plays should be the subject of (difcult but not impossible) investigations. The absence of evidence should not be taken for evidence of absence. Displacement, the ability to refer to absent events, things or concepts is another design feature considered to be restricted to humans [90]. Again, what is the evidence for communication about future events not occurring in animals? What experiments have refuted this? Clearly, negative evidence is never completely satisfying to rule out the existence of something, but we think those experiments havent even been done yet. In fact, data from Nicky Clayton et al. are consistent with the possibility that birds in the corvid family act with an eye to future events, an interpretation that is controversially discussed [91 93]. If indeed
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animals are aware of the past and the future, they could in principle also communicate about them. In sum, we feel that there is insufcient evidence to conclude that vocalizations that externalize concepts, facts and intentions are necessarily an exclusively human domain. Needless to say, animal thoughts may differ in fundamental ways from ours, which makes empirical research on this topic challenging. In conclusion, we do not argue against the fact that the sum of Hocketts design features characterizes human language. We also do not expect animals to have a communication system that mirrors human language in all aspects. However, if all of Hocketts design features existed in some version in animals, it would help to shift the debate about the evolution of language from the categorical (language has unique attributes that do not exist in animals) to the graded (different attributes of languages exist in principle in other species, to varying degrees and with potentially different consequences). Adopting the latter standpoint is bound to lead to new experimental impulses and less conjecture [94 96].

(b) The comparative approach Deconstructing the complexity of language is a rst step towards the study of brain and gene networks involved in these constituents in diverse non-human species. When molecular biologists rst unravelled the mechanics of transcription and translation in bacteria, many scientists were sceptical about whether the emerging principles were applicable to eukaryotes as well. Even though the existence of general biological principles at this level is no longer surprising, the ndings that the Pax6 transcription factor and its target genes play a central role in the formation of eyes across the entire phylogenetic tree was considered astonishing news, because the morphology of eyes in different taxa had clearly evolved in different ways and not from a common ancestor eye [97]. As more evidence for conserved molecular toolkits emerges, for instance, for learning and memory in ies, slugs and mice [98], one wonders whether conserved molecular networks may also apply to learned vocal behaviour? Birds offer a great opportunity to address this question. There are hundreds of different species of songbirds, in addition to two other orders of birds, hummingbirds [60] and parrots [58], that are known to learn their vocalizations by imitation. In contrast, there is only one human species, speaking more than 4000 languages. Comparing languages within our own species, the domain of linguists, has provided data that are suggestive of both common principles, shared by all languages, and specic parameters for each [30]. Even though the behavioural strategies and neural systems mediating song in birds also have common principles, parameters differ across species [45]. We should not forget that there is usually much more genetic variety from species to species than within one. Talking about birds in general when comparing song to language can by denition only capture the common principles of song, and ignores the potentially interesting variations in parameters, some of which may be more comparable with language than others.

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C. Scharff & J. Petri Review. Evo-devo and FoxP2 activator of downstream genes [112,113]. A C2H2 type zinc nger may be relevant to DNA and protein interactions and a leucine zipper domain is required for the homodimerization and heterodimerization with two other FoxP family membersFoxP1 and FoxP4. To act as a transcriptional repressor, dimerization of FoxP2 is essential, at least in reporter-gene cell culture assays [114,115]. FoxP2 also contains a glutaminerepeat region within the N-terminal part of the protein akin to those occurring in poly-Q repeat disorders, such as Huntingtons disease and spinocerebellar ataxia [116], but in the case of FOXP2, the CAG repeat has not been linked to the speech pathology. As the name transcription factor implies, FoxP2 regulates the transcription of other genes. Which genes those are is an interesting question for two reasons. On the one hand, identication of these socalled downstream targets can help to pinpoint the cellular functions regulated by FoxP2 in a particular species. On the other hand, comparing and contrasting FoxP2 targets in non-human animals with those in humans could provide important cues for unravelling how, during the course of evolution, potential functional changes occurred that might have contributed to the emergence of speech and language. Recently, direct neural targets of FOXP2 were identied in a human neuronal cell line [113] and in various human embryonic tissues [117]. Both studies used chromatin-immunoprecipitation with antibodies that recognize FOXP2 in combination with human promoter microarrays (ChIP-chip). The main set of candidate genes from these studies are proposed to play a role in neurodevelopment and neurotransmission, for example neurite outgrowth and synaptic plasticity, predicting a possible disturbance of these pathways involved in the speech and language disorder of patients with FOXP2 mutations [113,117]. FOXP2 ChIP-chip of human foetal brain tissue at 16 20 weeks of gestation revealed 84 specic target genes in the basal ganglia (BG) and 83 specic targets in the inferior frontal cortex. These sets differed markedly from those in human lung. The identity of the majority of genes suggests specic neural functions of FOXP2 in regulatory networks for cell communication and signal transduction. This points towards a prenatal FOXP2 function during central nervous system (CNS) development. Of the 285 proposed targets, 14 are predicted to be also under positive selection in humans. In a study comparing gene expression in chimps, rhesus macaques and humans, Caceres et al. [118] found that of the differentially expressed genes, around 90 per cent were expressed at higher levels in the human brain, but did not differ in liver and heart. Of the FOXP2 target genes that Spiteri et al. [117] identied, 47 genes also belonged to the set that was expressed differentially in human and chimpanzee brains [117]. Among those were genes relevant for CNS development and neural transmission, suggesting a potential role of some FOXP2 target genes in human-specic traits. Konopka et al. [119] explored whether the human form of FOXP2 may have different functions than the chimpanzee version of FoxP2 by expressing chimpanzee or human

Returning to genes and molecules, looking for bird song principles and parameters may provide inroads into previously unrecognized structural determinants. These in turn could reect common molecular deep homologies, as well as associated cellular and neural substrates. The same comparative approach is possible in mammals, for instance by tapping into the species diversity of bats with different social vocalizations [48 50,99]. A comparative approach is already very successfully being employed to study common genetic mechanisms in the evolution of social behaviour in invertebrates [100] and vertebrates [101]. 4. FOXP2: HYPE AND HOPE FOXP2 has captured the imagination of scientists and laymen alike because it was the rst gene causally related to a fairly specic speech and language phenotype, called developmental verbal dyspraxia (DVD; alternatively called childhood apraxia of speech, CAS, American Speech-Language-Hearing Association, 2007) [102]. DVDs core symptoms include inaccurate and incomplete pronunciation of words, difculties in repeating multi-syllable nonsense words and impaired receptive speech [103 105]. Notably, FOXP2 belongs to a group of genes for which multiple studies have found clear evidence for positive selection in the hominin lineage [106,107]. The link between the transcription factor FOXP2 and language was rst recognized in the large KE family spanning three generations. About half of the members of this family suffer from an autosomal dominant speech and language disorder, which was shown to be due to a heterozygous point mutation in FOXP2, inherited by all the affected, but none of the unaffected individuals [102]. Similar speech and language phenotypes exist in unrelated individuals with different FOXP2 mutations [108]. The original hype in the popular press that touted the gene as THE language gene was replaced by a more differentiated picture about its role in language. This is based on ndings from in vitro and in vivo studies, including animal studies, which have made considerable progress in addressing the molecular and neural function of FoxP2 in different species [109,110]. We will review the evidence for the relevance of FoxP2 for neural development and for neural and behavioural plasticity in postnatal life. The hope is that with increasing information about the molecular regulatory networks that FoxP2 participates in, and with more information about its function in different animal vertebrate and invertebrate model systems, we may not only learn whether FoxP2 is indeed another case of deep homology [84,111], but it may illuminate how speech and language work mechanistically and how they evolved. (a) Gene structure, molecular upstream regulators and target genes FoxP2 is a member of the winged helix transcription factor family, characterized by a highly conserved Forkhead (Fox) domain that binds to distinct DNA sequences in its target genes regulatory regions. It can act both as a transcriptional repressor as well as an
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Review. Evo-devo and FoxP2 FoxP2 genes in human neuronal cell lines. Subsequent microarray analysis showed that gene expression levels of 116 genes differed quantitatively. As this set of genes included genes active in pathways and tissues relevant for speech and language, the authors speculated that the human version of FOXP2 might have contributed to the evolution of this trait. The differentially regulated sets are enriched for genes involved in transcription, cell cell signalling, protein and cell regulation. The authors nd key players of brain development and function, e.g. DLX5 and SYT4, among the highly connected genes when applying network analysis and observe other relevant candidates that seem to be co-regulated with some of the differentially expressed putative targets [119]. One of the downstream targets of FOXP2, CNTNAP2, recently received special attention. Particular single nucleotide polymorphisms are associated with core decits of children with specic language impairment and also coincide with language delays in autistic children [120]. In songbirds, CNTNAP2 is differentially expressed in some song control nuclei, but whether FoxP2 regulates CNTNAP2 in songbirds has not yet been addressed [121]. These ndings are encouraging in the light of potential deep homologies between human speech and bird song. To understand the role of FOXP2 for cellular and behavioural function and how this might have changed during the course of evolution, one also needs to identify how the transcription of FoxP2 is regulated. In fact, Carroll has argued that changes in gene regulation via non-coding sequences, including transcriptional cis-regulatory elements, the untranslated regions of messenger RNAs and RNA-splicing signals are a more important force than coding-sequence evolution in the morphological and behavioural evolution of hominins [122]. Two putative upstream transcriptional regulators of FoxP2 have been described, one in zebrash (Danio rerio) [123] and one in zebra nch (Taenigpygia guttata) [124]. Lef1 is a transcription factor that is activated via the Wnt/b-catenin signalling pathway. Its decreased expression or inhibition during the development of the zebra sh brain leads to decreased FoxP2 expression in particular brain areas. Furthermore, enhancer sites within the FoxP2 genomic DNA sequence show several Lef1 binding sites and two of these enhancers bind Lef1 in chromatin immunoprecipitation analysis in zebra sh. In juvenile zebra nches, administration of a cannabinoid agonist increases FoxP2 expression in the striatum, persisting into adulthood. Whether the inuence of cannabinoid signalling on FoxP2 expression in brain regions relevant for learning and practising song is due to direct interactions needs to be further studied. To recap, many potential FOXP2 target genes have been identied, including CNTNAP2 which also has been linked to language impairments [120]. Which target genes operate in which neurons remains to be discovered and this will narrow down hypotheses about the function of FOXP2 in different tissue contexts. The discovery that hetero- and homodimerization are an essential feature of the FoxP protein family offers the opportunity for combinatorial ne-control of gene expression in a cell-type specic manner.
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(b) FoxP2 expression studies during brain development and in postnatal brains There are two general, but not mutually exclusive, possibilities about how FOXP2 affects language and speech in humans. The protein could be involved in the formation of speech-circuits or it could affect the process of speech learning, the perception and/or the production itself. Surveying the existing evidence, both seem likely (tables 1 and 2). FOXP2 expression is already present in human foetal brains at sites that develop into those structures that show morphological and functional abnormalities in patients with FOXP2 mutations [125]. This is consistent with FOXP2 playing a role in establishing speech relevant circuits prenatally. The expression patterns in human foetal brains are highly similar to those in mice of a comparable embryonic stage. FoxP2 is also expressed during embryonic development in homologous brain regions of monkeys, various rodent species, different bird species, frogs and sh [112,126 128,136,140 144]. These ndings stress that FoxP2 is not exclusive to humans but likely to be relevant for the development of homologous brain regions in many vertebrates. A developmental role is underscored by the fact that homozygous Foxp2 mouse mutants (see below) develop cerebellar anomalies [130 133]. Continued expression of FoxP2 in postnatal and adult brains of different species suggests functions beyond developmental patterning. Foxp2 and its close homologue Foxp1 are expressed in different subpopulations of projection neurons in the cerebral cortex of young mice, and might therefore serve different functions during establishing and shaping of distinct cortical circuits in early postnatal stages [129]. In adult mice, Foxp2 continues to be expressed in layer 6 of the cortex, the striatum, dorsal thalamus, cerebellar deep nuclei and Purkinje cells, and the inferior olive in the medulla [126,128]. Except for the cortical expression, this pattern is also characteristic for a number of bird species and crocodiles [136]. In the striatum of mice and songbirds, FoxP2 is expressed by medium spiny neurons, co-localizing with DARPP32 and co-expressing dopamine D1 receptors [136,147]. Within the mammalian striatum, the striosomal and matrix compartments express neurochemical markers differentially and are characterized by distinct projection patterns [151]. In macaque, FoxP2 expression is enriched in the striosomal compartment [141]. Most attention so far has focused on the signicance of FoxP2 in cortico-striatal and cortico-cerebellar networks because of their clear connection to speech. Focusing on other regions that express FoxP2 supports potential common cellular and behavioural roles. For instance, the inferior olive strongly expresses FoxP2 in all species studied, and is, like the striatum, important for the timing of motor behaviour [152,153]. In addition, the midbrain auditory inferior colliculus nucleus and its avian equivalent Mld, and the auditory thalamic medial geniculate nucleus (MGN) and its avian equivalent nucleus ovoidalis express FoxP2 [127,128,136]. Although the neural FoxP2 expression patterns are overall strikingly conserved among vertebrate species, some interesting heterochronic changes exist. For

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Table 1. Developmental role of FoxP2 in vertebrate species. Many of the observations listed cannot clearly distinguish between only developmental or developmental and adult online effects and are listed here only when observations were made during the development of the organism. evidence developmental verbal dyspraxia, impairment of speech and language learning expression in foetal brain downstream target genes in foetal brains expression sites in mammalian brains Foxp1 and Foxp2 expression in different subpopulations of cortical neurons homozygous Foxp2 mutants: developmental delay, motor impairments, die shortly after birth heterozygous Foxp2 mutants: subtle brain abnormalities reduced number of ultrasonic isolation calls of pups from mutant mice ultrasonic isolation calls with different bioacoustic properties of pups with partially humanized FOXP2 mouse mutants with differences in motor skill learning and synaptic plasticity medium spiny neuron properties differ in mice with partially humanized FOXP2 expression in MGN elevated FoxP2 expression in Area X during the sensorimotor learning phase FoxP2 expression in proliferating neurons in post-hatch songbird brains declining expression of FoxP2 in Area X with maturation knockdown in Area X during the song learning phase results in incomplete and impaired song imitation homologous sites of foetal brain expression inferred function speech and language learning embryonic brain development central nervous system development cortico-striatal and olivo-cerebellar circuits differential shaping of distinct neural circuits essential for normal development essential for normal brain development ultrasonic mouse vocalizations in pups ultrasonic mouse vocalizations in pups motor skill learning and synaptic plasticity synaptic plasticity regulation by auditory activity song learning song learning song learning essential for song learning species investigated human mouse, rat and human human mouse, rat and human mouse mouse references [102,108] [125 127] [117] [125 128] [129] [130 133]

mouse mouse mouse

[130,133] [130,133] [134]

mouse mouse mouse zebra nch zebra nch zebra nch zebra nch

[132] [134] [135] [136] [137] [138] [139]

neural development not limited to circuits for vocalizations

monkeys, rodents, birds, frogs, sh

[112,126 128,136, 140144]

Table 2. Post-organizational role of FoxP2 in vertebrate species. Many of the observations listed cannot clearly distinguish between effects that result from adult, online decits or effects that manifested during the development and are listed here only, when observations were made during adulthood. species investigated human human mouse and rat mouse and songbirds mouse mouse monkeys zebra nch

evidence speech and language decit in adults perceive rhythmic differences less well mammalian brain expression pattern strong striatal medium spiny neuron expression Foxp2 missense mutation: alterations in auditory brainstem responses medium spiny neuron properties differ in mice with partially humanized FOXP2 decreased FoxP2 brain expression decreased expression of FoxP2 in Area X after singing
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inferred function receptive and expressive language perception of rhythm maintenance of cortico-striatal and olivo-cerebellar circuitries sensorimotor integration auditory brain stem processing synaptic plasticity ? online regulation of song in adults

references [103,145] [146] [126,128] [136,137,147] [148] [134] [141] [149,150]

Review. Evo-devo and FoxP2 instance, FoxP2 expression dramatically declines in monkey brains postnatally and disappears rst from the putamen and later from the caudate nucleus of the BG, whereas expression persists in homologous parts of the rat brain. Neural FoxP2 expression in mammals commences only after their last mitotic division, whereas in songbirds some dividing neuroblasts in the ventricular zone already express FoxP2 and this expression is maintained into adulthood, also differently than in adult mice, where the neurogenic zones do not express Foxp2 [137]. Postnatally, songbird FoxP2 expression is dynamically regulated in a BG structure called Area X. During development, songbirds acquire their species-specic and individual-specic song by imitating the sounds of adult conspecics. To achieve this, Area X is required [154,155]. Once song is stably learned, Area X continues to be relevant for online monitoring of song [154 156] and without Area X, normal song production deteriorates [157]. Juvenile male zebra nches consistently express 10 20% more FoxP2 mRNA within Area X than in the surrounding striatum during their song-learning phase. This change in FoxP2 expression is not related to immediately prior singing activity, because birds in this study had not sung before sacrice [136]. Other regions involved in controlling the learning and production of song show very low FoxP2 expression [127,136]. A further correlation between song plasticity and levels of FoxP2 expression exists in another species of songbirds, canaries. During the breeding season, male canaries sing highly regular and stereotyped song and FoxP2 expression in Area X is low. After the breeding season, song becomes variable and new syllables are incorporated, and concomitantly FoxP2 in Area X is upregulated [136]. As in juvenile zebra nches, canaries did not sing prior to sacrice, so the different FoxP2 mRNA levels cannot be explained by a direct link to singing activity. However, such an acute link between singing and FoxP2 expression has also been reported in zebra nches. When adult male zebra nches do not sing or sing undirected song (not directed towards another bird), FoxP2 mRNA is signicantly higher in Area X than in the surrounding striatum [149]. In contrast, when adult birds sing female-directed courtship song [149], FoxP2 mRNA levels are lower than in the surrounding striatum. In contrast to mRNA levels, after 2 h of either directed or undirected singing, FoxP2 protein levels in Area X (normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH)) are lower than in a non-singing group [150]. The interpretation of the difference between mRNA and protein levels could be owing to interesting regulatory mechanisms, but is currently difcult to evaluate, because the variability of the protein data within the groups is quite high and it is unclear whether GAPDH is suitable for normalization in avian Area X [158]. In addition, the most consistent and strongest difference in FoxP2 expression is an increase that occurs in non-singing birds during the rst 2 h of daylight, before they start singing [150]. During the late phase of song development, 75 days after hatching, FoxP2 mRNA levels, similar to brains of adult birds, decrease after 2 h of undirected singing in Area X of hearing as well as in deafened birds. Both
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constitutive and singing-induced changes in FoxP2 levels in Area X might thus be tied to song plasticity during development as well as in adulthood [136,138]. Whether FoxP2 functions cellularly as a positive or negative regulator awaits further study. Interestingly, 75 dayold birds, whose song imitation is already quite good, show a higher song and song sequence variability after 2 h of singing than after 2 h of silence [159]. Curiously, in 5057 day-old birds, the ongoing improvement of the juvenile birds ability to imitate the adult song of a tutor increases during each day, but decreases slightly over night before it improves again the next day [160]. Whether the singing-induced variability noted by Miller et al. [159] relates to the daily improvement of imitation noted by Deregnaucourt [160] will be interesting to pursue. Several other genes are specically regulated at the mRNA level in zebra nch Area X by undirected singing behaviour [161]. For example, two histone family members (H2AFX; H3f3B), two heat-shock proteins (Hsp25; Hsp90a) and calcyclin-binding protein (Cacybp) are upregulated, whereas ARHGEF9 [161] is downregulated. Mutations of the latter gene are involved in human mental retardation and sensory hyperarousal [162,163]. Interestingly, FoxP2 is also expressed in the song circuitry in species of two other avian orders, hummingbirds and parakeets, both vocal learners. Given that these three orders are not linked by an immediate common ancestor [164], it seems parsimonious that songbirds, parrots and hummingbirds evolved the neural circuitry for vocal learning independently. The alternative hypothesis, that a common ancestor to all extant birds possessed this trait that was subsequently lost in non-vocal learners (to varying degrees), is however also possible [165]. Considering both scenarios, one should bear in mind that only a few species have unequivocally been shown not to exhibit vocal learning [166] and there may be so far unrecognized intermediate phenotypes between accurate imitative production learning and usage learning [56]. If so, the existence of neural structures similar to those in hummingbirds, parakeets and songbirds should be checked in those species, as well as FoxP2 expression patterns. Together, these kinds of experiments are necessary to determine how universal deep molecular homologies relating to the neurobiology and the behaviour of learned motor behaviours really are. In mice, activity-driven Foxp2 expression has also recently been noted in the MGN, the principal auditory relay nucleus of the mouse thalamus. Foxp2 is strongly expressed after white noise stimulation of young mice [135]. Whether activity-driven FoxP2 expression plays a role in expression differences, such as the developmental downregulation in monkey striatum should be taken into account in future expression studies. In insects, only one FoxP family member exists [167,168], indicating that the four FoxP paralogues in vertebrates arose via gene duplication. The functional role of FoxP in Drosophila is just starting to be investigated [169] and together with the information about its target genes may provide rich insights into deeper levels of molecular homologies for similar

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upstream of FoxP2 downstream of FoxP2 FoxP2 FoxP2 FoxP2 FoxP2 FoxP2 auditory gene C gene B motor gene A developmental/ post-organizational cognition

species-specific conditions spatial conservation, temporal differences cortical subcortical peripheral Figure 1. Schematic depiction of the levels at which differential regulation of FoxP2 and its target genes can affect cognitive, motor and peripheral functions in different organs and species. FoxP2 is controlled by a so far mostly unknown set of upstream regulators. It regulates a large set of target genes, which are known for only a very limited number of species, cell types and developmental stages. FoxP2 acts in cortical, subcortical and peripheral areas of different vertebrate species. The future challenge is to discover how the similarities and differences in the amount, space and time of FoxP2 expression in different species is regulated, and how this in turn affects its downstream targets and eventually their behaviour.

cellular and behavioural functions. In honeybees (Apis mellifera), AmFoxP mRNA is expressed at higher levels in the brains of worker bees around 4 days after eclosion, when bees become foragers, searching for food and communicating information to their hive members via dancing. Unfortunately, whether or not AmFoxP is also increasing in drones that leave the hive, but do not dance, is not known. Division of labour or caste does not seem to have any inuence on AmFoxP expression levels in the bee brain. One day after eclosion, foragers express AmFoxP in cell bodies clustered in the optic, protocerebral and dorsal lobes. The latter is suggested to play a role in the processing of mechanosensory information, but without knowledge of the projection patterns of the AmFoxP expression neurons, it is currently difcult to make predictions about their function [168]. In summary, heterochrony of FoxP2 expression in different bird species exists with respect to development, season and circadian rhythm and heterometry occurs as a result of acute singing activity and social context. How these are regulated is one of the challenges for the future (gure 1). Altered amounts of expression during song learning in birds and as a result of auditory stimulation in mice are compatible with a role of FoxP2 in auditory processing, vocal motor behaviour or the integration of both. Evoked brainstem responses after auditory stimulation in mouse mutants (see below) further support this idea.

(c) Functional analysis of Foxp2 in mutant mice Foxp2 mouse models include knock-out mice [130,131,133], mice that carry the aetiological
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mutations that mirror those of the KE family or other patients [132] and mice in which the murine Foxp2 gene has been humanized by including the two human-specic amino acids [134]. Heterozygote mice carrying a Foxp2 null-allele or the aetiological mutation show a reduction of Foxp2 protein by 50 per cent and thus mirror the haploinsufciency of patients with FOXP2 mutations [102]. Homozygous FOXP2 mutations in humans have not been reported, presumably because they are lethal. Likewise, mice carrying two alleles with aetiological or loss-offunction Foxp2 mutations die a few weeks after birth [130,131,133]. In some studies, these heterozygous mice have subtle abnormalities in brain morphology as well as mild developmental delays [130,133], while other investigations reported that heterozygotes are macroscopically normal without such abnormalities [131,132]. Differences in these observations may relate to modier effects in the genetic background, given that some of these investigations did not perform back-crossing of the mutant mice [109]. Altered auditory brainstem processing was observed in mutant mice (Foxp2-R552H) that carry the equivalent of the human aetiological R553H point mutation in the forkhead domain, but not in mice with a Foxp2 mutation that mirrors the human aetiological R328X mutation that truncates the protein before the known functional DNA binding and protein interaction domains [148]. These results may reect preserved dimerization capacity of the R553H, but not the R328X mutation. These two mutant proteins also differ in their intracellular localization when expressed transiently in human neuronal-like cell lines [170]. How much the interactions with other

Review. Evo-devo and FoxP2 Foxp proteins contribute to these effects is not known, but both questions are accessible to further studies. Analysis of vocalizations in Foxp2 mutant mice has so far been limited to pup vocalizations, which are thought to be innate. A reduced Foxp2 dosage does not prevent pups from producing audible calls, ultrasounds and clicks [132] even though KO and R552H-KE mutants tested under less stressful conditions did not produce any ultrasonic isolation calls [130,133]. Heterozygous mouse models, either carrying the Foxp2-R552H missense or the Foxp2-S321X nonsense mutation, produce all sound types and do not show any signicant differences in the temporal domain that are not routinely observed for different mouse strains [171]. Whether adult ultrasound mouse courtship song [46] and its neural substrates differ in Foxp2 mutants and wild-type mice is not known, nor is the answer to the question of whether mouse song is learned via imitation-like speech and birdsong. Four muroid species with different levels of complex vocal output do not show any species-specic Foxp2 expression patterns that can be related to their different abilities to vocalize [172], but the latter study presents interesting incidences of heterotopic expression of Foxp2 in some parts of cortex and amygdala in the different murine species studied. In the absence of more specic information about mouse vocalization pathways and a potential role of Foxp2 in those, it is interesting that Groszer et al. [132] describe signicant behavioural and physiological differences of R552H heterozygote mice when compared with littermates. Mutants with reduced Foxp2 dosage perform worse on the tilted voluntary running-wheel system and on accelerated rotarods, consistent with impaired motor skill learning and affected frontostriatal and/or frontocerebellar circuitry. They also show impaired long-term depression (a form of altered synaptic plasticity) at glutamatergic synapses of the dorsolateral striatum. Cerebellar synaptic plasticity also subtly differs, although the circuitry was found to be grossly normal. Consistent with a role of FoxP2 in the olivo-cerebellar [173,174] and cortico-striatal system [153] are also ndings that affected KE family members perceive rhythmic differences in stimuli less well and imitate rhythms less accurately than control subjects [146]. The bottom line of the studies in mice is that reduced levels of Foxp2 protein, equivalent to the amounts of cellular protein in patients carrying heterozygote human FOXP2 mutations, affect neuronal plasticity in the striatum and motor learning but have less of an effect on vocalizations.

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(d) Functional analysis of FoxP2 in songbirds Birdsong, just like human speech and language, has to be learned by integrating auditory input with vocal motor output through practice. Searching for a causal relationship between FoxP2 and learned complex vocalizations, FoxP2 was knocked-down using lentivirally mediated RNA-interference during the sensorimotor learning phase in the striatal BG nucleus Area X of male zebra nches [139]. Birds with reduced FoxP2 levels in Area X did not learn a complete copy of their tutors song, omitting several of the tutors
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syllables. Furthermore, they copied the imitated syllables less accurately than did the control birds. In contrast to their control siblings that expressed normal FoxP2 levels, they also sang song more variably from rendition to rendition [139]. This production variability also characterizes people with FOXP2 mutations [145]. The relative contribution of sensory, motor or sensorimotor integration to the impairments is difcult to dissect unambiguously with the current animal models. However, a number of ndings suggests that the decits resulting from FoxP2 knockdown are not restricted to motor performance; imitation success differs signicantly, already during the learning phase, between FoxP2 knockdown and control zebra nches and then plateaus early in the knockdown birds, whereas variability of song element production (syllables) continues to increase in knockdown nches when it no longer does in controls [139]. A more denite dissection of a sensory role for FoxP2 in the BG would require conditional knockdown of FoxP2 during the purely auditory learning phase in bird species with a sensory memorization phase that precedes motor practice by months, such as canaries, reactivating FoxP2 expression during the time when vocal motor practice ensues. Do these ndings allow us to make any predictions about the role of FoxP2 at the neural level? The anterior forebrain pathway of song learning birds echoes the mammalian cortico-BG-thalamo-cortical loops, but important differences exist. Like the striatum in mammals, striatal Area X in songbirds receives cortical glutamatergic afferents that synapse onto spiny neurons with histochemical and electrophysiological features strongly resembling those of mammalian medium spiny neurons. The songbird cortical input to the spiny neurons of Area X is also modulated presynaptically by midbrain dopaminergic input. However, Area X also contains aspiny, tonically active, fast-ring GABAergic neurons similar to mammalian pallidal neurons [175]. Recording from Area X in singing birds, Goldberg et al. [176] recently identied two types of these neurons, differing in connectivity and ring pattern, in a very similar way as do the two different pallidal neuron types in primates. Importantly, Area X within the songbird striatum has slightly different connectivity patterns than the surrounding striatum [177]. These differences could reect the small evolutionary modications postulated for new traits, such as avian vocal learning. What are the cellular consequences of knocking down FoxP2 in Area X spiny neurons? Schulz et al. [178] found that spiny neurons with knocked-down FoxP2 levels had fewer spines than control-injected neurons. This effect was even more pronounced when neurons received the knockdown before differentiation, i.e. as neuroblasts in the ventricular zone, where adult neurogenesis takes place. To sum up, using gene manipulation in striatal Area X of young zebra nches during their song-learning period caused song impairments that phenotypically echo aspects of developmental verbal dyspraxia in humans. Like patients with FOXP2 mutations, birds with reduced levels of FoxP2 do not develop their full articulatory potential and produce their smaller set of

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C. Scharff & J. Petri Review. Evo-devo and FoxP2 evolution of language. However, recent data show that Neanderthals already possessed the human-like FOXP2 version, even though their lineage diverged from the one leading to modern man approximately 300 000400 000 years ago, before language was so far thought to have arisen [182]. Alternatively, there may have been gene ow between Neanderthals and humans or contamination of the Neanderthal samples with modern human DNA [183]. A nal interesting, but currently not understood, nding concerns bats, which show a much greater sequence divergence of FoxP2 than all other vertebrates studied [184]. While it is tempting to speculate that this diversity might be related to echolocation or different vocal behaviours that bats exhibit [61,185,186], comparing FoxP2 sequences in other animals that learn their vocalizations and those that do not did not turn up sequence variants that segregate with vocal learning [187,188]. One of the great challenges of characterizing FoxP2s function and its evolutionary contribution to vocal learning is to understand the dynamics of the genes expression in different species. There are a number of questions that need to be answered. (i) How do non-coding DNA sequence changes affect where and when FoxP2 is expressed in different species; (ii) how do coding changes affect the structure of the protein and its interaction with other proteins and the DNA; (iii) what is the role of the differential expression of proteins that interact with FoxP2; (iv) how does FoxP2 expression respond to internal and external signals? All of the factors could be important sources of evolutionary change. Songbirds are a fruitful model system to explore heterochrony, since agerelated differences, seasonal changes and differences co-occurring with singing style exist in Area X [127,136,138,149]. It will be interesting to follow those studies in different species of songbirds that vary in the timing of song learning, who sing during behavioural contexts for which FoxP2 expression has not been tested yet, and who show differences in adult song plasticity. In addition, more efforts should be directed at identifying the genomic loci regulating temporal expression differences of FoxP2.

vocal elements more variably than is typical. The fact that FoxP2 knockdown in adult Area X affects the structural plasticity of dendritic spines nicely complements the data from the mouse models demonstrating altered synaptic plasticity [132,134,179].

(e) Evolution of FoxP2 The evolution of FoxP2 is enigmatic. On the one hand, the DNA sequences for most of the species studied differ by less than 10 per cent. This, together with the similar brain expression patterns in vertebrates, suggests that the protein fulls an evolutionarily ancient and important role, not limited to humans. On the other hand, there is clear evidence that the human FOXP2 gene was subject to a selective sweep during hominin evolution [106,180], as was a subset of putative FOXP2 target genes [117]. These ndings, together with the genes role in language, have led to the proposal that the human FOXP2 gene is one of the things that helped us become human. What, then, is the nature of the differences between the non-human and human FOXP2 genes? Only two amino acid changes distinguish the protein sequence of chimps and humans, not counting the extra glutamine within the polyglutamine repeat in the chimpanzee FoxP2. Neither of the two amino acids lies within the characterized protein functional domains, so their molecular function is unclear. Do these differences make any contribution to the fact that human speak and chimpanzees do not? Testing this idea, Enard et al. [134] introduced the two human-specic amino acids into the murine Foxp2 locus. Interestingly, mice pups that carry a partially humanized version of FOXP2 (because only the coding changes were introduced) had isolation calls that differed bioacoustically from those of control mice. In addition, the mice carrying the partially humanized version of FOXP2 showed less exploratory behaviour, altered synaptic plasticity of the striatal medium spiny neurons, lower dopamine levels in ve brain regions including the frontal cortex and the caudate-putamen, and had longer dendrites in culture [134]. In a follow-up study, the humanized version of FOXP2 introduced into mice was shown to specically affect the cortico-BG circuits, but not the cortico-cerebellar circuits [179]. These ndings are consistent with a different function of the human FOXP2 from the wild-type mouse version, but it is unclear whether the chimpanzee FoxP2 would also behave differently in a mouse background, and if so, how. The fact that one of the human-specic amino acids also occurs in carnivores questions the uniqueness of the link between the two human-specic amino acids and the human language capacity. More importantly, data from a recent analysis of genomic regions up- and downstream of the region coding for the two human-specic amino acids raise the possibility that the selective sweep was in fact not associated with the two human-specic amino acid substitutions [181]. Furthermore, the original estimate for the emergence of the human-specic FOXP2 sequence was dated around 260 000 years ago and therefore concomitant with the emergence of cultural artefacts that are thought to be related to the
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5. CONCLUSIONS Changes in the regulatory regions of genes can alter the timing, the amount or the place of gene expression in the course of evolution [6]. Likewise, changes in the protein-coding sequence can bring about altered gene products, leading to different functions [7]. In turn, both can result in changes to neural circuitry, as amply attested by differences in neuroanatomy among different species. Whether FoxP2 played a role in bringing about circuit changes that facilitated the emergence of human language is not clear. Cell lineage analyses and studies of neural microcircuitry in the mouse carrying the humanized FOXP2 allele could be a step in the right direction, but as a complex behaviour like language is bound to be a polygenic trait, other genes that presumably need to act together with FOXP2 might not be present or active in this mouse model.

Review. Evo-devo and FoxP2 Alternatively, one could imagine that other genes brought about the circuit changes required for vocal learning. Subsequently, FoxP2, which already functioned in the precursor circuits, then either acquired new importance because it operated in a new environment, or the gene also changed and altered its function. In the case of songbirds, song nuclei are embedded in regions that are active during stereotyped motor behaviours like hopping and walking [189]. In this evolutionary scenario, the expression of FoxP2 in Area X of the striatum thus became useful for sensory motor integration or precise timing of vocal gestures as supposed to other motor learning tasks in adjacent non-vocal circuitry cells. During the evolution of vocal learners, once the striatum got connected to other regions necessary for vocal learning to occur, FOXP2 mutated in humans to become human specic and this might have affected neural transmission. This would be a two-hit scenario of FOXP2s role in language evolution, circuit changes predating gene function changes. From the postnatal studies in mouse and bird, it is clear that FoxP2 plays a role in neural plasticity of certain circuits. As homozygous mutations are lethal in mice, one can assume that Foxp2 also plays a role in development. Whether this is true for brain circuits that are relevant for vocal learning in humans and birds is not clear. To reiterate, both human speech and some animal vocalizations, such as the song of many bird species, are a form of auditory-guided vocal motor learning. Research into the underlying neural and genetic substrates of vocal learning in humans and in animal models is starting to reveal similarities and differences. Whether vocal learning is really the hallmark of a selected few species and presents a case of parallel evolution, or whether it is an ancient trait that exists to some degree in many more species that have not been stringently tested should be further explored. Each species exhibits adaptations that are a mix of traits it shares with others and some that may be unique. Depending on ones point of view about the position of humans in the universe, one nds the aspects we share with animals or those that set us apart more appealing for study. While we are certainly limited by our own species-specic cognitive biases, maintaining as agnostic a stance about what animals can and cannot do, until supported by experimental evidence, will be useful to unravel whatever precursors of language exist in our various animal relatives.
We thank Christopher K. Thompson, Anna Zychlinsky, Roland L. Knorr and particularly the two reviewers for very helpful comments and edit suggestions. Supported by funds from the SFB 665.

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Apis mellifera. Insect Mol. Biol. 17, 5360. (doi:10.1111/ j.1365-2583.2008.00775.x) Brembs, B. P., Pauly, D., Schade, R., Mendoza, E., Puger, H.-J., Scharff, C. & Tzars, T. 2010 The Droso phila FoxP gene is necessary for operant self-learning: implications for the evolutionary origin of language. In Society for neuroscience. San Diego, CA: Society for Neuroscience. Vernes, S. C., Nicod, J., Elahi, F. M., Coventry, J. A., Kenny, N., Coupe, A. M., Bird, L. E., Davies, K. E. & Fisher, S. E. 2006 Functional genetic analysis of mutations implicated in a human speech and language disorder. Hum. Mol. Genet. 15, 31543167. (doi:10. 1093/hmg/ddl392) Gaub, S., Groszer, M., Fisher, S. E. & Ehret, G. 2010 The structure of innate vocalizations in Foxp2-decient mouse pups. Genes Brain Behav. 9, 390 401. (doi:10. 1111/j.1601-183X.2010.00570.x) Campbell, P., Reep, R. L., Stoll, M. L., Ophir, A. G. & Phelps, S. M. 2009 Conservation and diversity of Foxp2 expression in muroid rodents: functional implications. J. Comp. Neurol. 512, 84100. (doi:10.1002/ cne.21881) Welsh, J. P., Lang, E. J., Suglhara, I. & Llinas, R. 1995 Dynamic organization of motor control within the olivocerebellar system. Nature 374, 453457. (doi:10. 1038/374453a0) Yarom, Y. & Cohen, D. 2002 The olivocerebellar system as a generator of temporal patterns. Ann. NY Acad. Sci. 978, 122 134. (doi:10.1111/j.1749-6632. 2002.tb07561.x) Farries, M. A., Meitzen, J. & Perkel, D. J. 2005 Electrophysiological properties of neurons in the basal ganglia of the domestic chick: conservation and divergence in the evolution of the avian basal ganglia. J. Neurophysiol. 94, 454 467. (doi:10.1152/jn.00539.2004) Goldberg, J. H., Adler, A., Bergman, H. & Fee, M. S. 2010 Singing-related neural activity distinguishes two putative pallidal cell types in the songbird basal ganglia: comparison to the primate internal and external pallidal segments. J. Neurosci. 30, 70887098. (doi:10.1523/ JNEUROSCI.0168-10.2010) Person, A. L., Gale, S. D., Farries, M. A. & Perkel, D. J. 2008 Organization of the songbird basal ganglia, including area X. J. Comp. Neurol. 508, 840 866. (doi:10.1002/cne.21699)

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Phil. Trans. R. Soc. B (2011) 366, 21412154 doi:10.1098/rstb.2010.0409

Research

Evolution of time-keeping mechanisms: early emergence and adaptation to photoperiod

R. A. Hut* and D. G. M. Beersma
University of Groningen, Chronobiology Research Unit, Life Science building, Nijenborgh 7, 9747AG Groningen, The Netherlands Virtually all species have developed cellular oscillations and mechanisms that synchronize these cellular oscillations to environmental cycles. Such environmental cycles in biotic (e.g. food availability and predation risk) or abiotic (e.g. temperature and light) factors may occur on a daily, annual or tidal time scale. Internal timing mechanisms may facilitate behavioural or physiological adaptation to such changes in environmental conditions. These timing mechanisms commonly involve an internal molecular oscillator (a clock) that is synchronized (entrained) to the environmental cycle by receptor mechanisms responding to relevant environmental signals (Zeitgeber, i.e. German for time-giver). To understand the evolution of such timing mechanisms, we have to understand the mechanisms leading to selective advantage. Although major advances have been made in our understanding of the physiological and molecular mechanisms driving internal cycles (proximate questions), studies identifying mechanisms of natural selection on clock systems (ultimate questions) are rather limited. Here, we discuss the selective advantage of a circadian system and how its adaptation to day length variation may have a functional role in optimizing seasonal timing. We discuss various cases where selective advantages of circadian timing mechanisms have been shown and cases where temporarily loss of circadian timing may cause selective advantage. We suggest an explanation for why a circadian timing system has emerged in primitive life forms like cyanobacteria and we evaluate a possible molecular mechanism that enabled these bacteria to adapt to seasonal variation in day length. We further discuss how the role of the circadian system in photoperiodic time measurement may explain differential selection pressures on circadian period when species are exposed to changing climatic conditions (e.g. global warming) or when they expand their geographical range to different latitudes or altitudes. Keywords: circadian system; photoperiodism; cyanobacteria; seasonal adaptation; suprachiasmatic nucleus; chronobiology

1. EMPIRICAL EVIDENCE FOR A SELECTIVE ADVANTAGE OF CIRCADIAN RHYTHMS AROUND 24 h As a result of combined evolutionary pressures, almost all species have developed circadian timing systems that show persistent oscillations in molecular processes, even in constant conditions. Commonly, the observed periods are close to 24 h, hence the term circadian. Among the species that developed circadian rhythms are cyanobacteria (Synechococcus elongatus), one of the earliest and most primitive organisms known. In itself, this observation suggests that during the early development of life on Earth, rhythms with periods close to the light dark cycle already generated an evolutionary advantage. The hypothesis of optimization of circadian period was tested in competition

* Author for correspondence (r.a.hut@rug.nl). One contribution of 10 to a Theme Issue Evolutionary developmental biology (evo-devo) and behaviour.

experiments under light dark cycles of different periods with several S. elongatus mutants, each with different intrinsic circadian periods [1,2]. In these experiments, the reproductive success has been quantied of the various strains under different experimental circumstances. It was found that the strain that had a period of its timing system closest to the applied light dark cycle survived best. Under rhythmic conditions, rhythmic strains outcompeted arrhythmic strains. Under constant light conditions, there were no systematic differences in reproductive success, neither between rhythmic and arrhythmic strains nor between rhythmic strains with different periods. So there is clear adaptive value to the period of the circadian timing system. This may have led to the selection of the KaiA, KaiB and KaiC molecules that are known to play a prominent role in the circadian system of S. elongatus. In a world with large daily uctuation in energy supply, it is likely that all cells, including individual cells in complex organisms, have to perform a similar
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Evolution of time-keeping mechanisms or under continuous light conditions. Tomatoes grow faster under a 24 h light dark cycle than under shorter or longer cycles or even in continuous light [12 14]. But this is not universal. Arabidopsis grows faster in continuous light [15]. Yet, even if a species grows faster in a different environment, this does not exclude the possibility that it is optimally adjusted to the environmental circumstances where it lives. Furthermore, growth rate may not be related to tness and it remains therefore important to test directly the selective advantage of circadian rhythms in other species through competition experiments [9].

task of harvesting and storing energy when it is available and using up their stores when external energy supplies are insufcient. Yet, in complex organisms, the pathways of energy supply may be many, and the supply may come from various sources and at different times, depending on the specic tasks of the cells and their embedding in the organism. Therefore, in other species, the molecular processes on which the oscillations are based may be very different from cyanobacteria, and may even have evolved independently [3]. Yet, each cell will separately sense uctuations in energy supply and will adjust its own temporal pattern in response to those uctuations. In this way, complex organisms might be composed of cells that are rhythmic with a 24 h period but with different timing between the cells, each depending on their specic environments. This is indeed what is observed: many cell types of complex organisms, like mammals, show intrinsic circadian oscillation, but the phases of these oscillations differ between cell types [4]. Yet, in most complex organisms investigated (except plants), it has been observed that a central pacemaker has evolved; a structure that apparently has the specic task to keep track of environmental time and provide that time information to the rest of the body. Other cells use that time information as a reference to inuence their own behaviour, albeit in different ways for different cells. In mammals, this central pacemaker is located in the suprachiasmatic nucleus (SCN) of the hypothalamus [5,6]. Transplantations of SCN tissue between mutant hamsters with different periods of their locomotor activity in constant conditions have proved the circadian pacemaker function of the SCN beyond any doubt [7]. The SCN sets the pace of downstream processes. Since circadian pacemakers have evolved, it seems obvious that they provide tness value to the species. Yet, experiments to demonstrate the tness value of having a circadian pacemaker are almost absent. One important exception is an experiment in chipmunks by DeCoursey et al. [8]. She compared individuals having an intact timing system with individuals in which the timing system was shut down by lesioning of the SCN. SCN-lesioned animals and sham-operated animals were reintroduced in nature and their survival was monitored over time. It turned out that SCN-lesioned as well as sham-lesioned animals initially had a hard time, possibly owing to the fact that their territories were taken over by other animals during the few days that were needed for the surgery. After two weeks the survival curves stabilized, but the SCN-lesioned animals persisted to show higher mortality. This suggests specic tness value of having an intact circadian pacemaker. It is true that this study focused on survival and not on pure tness consequences [9]. Yet, it is clear that most animals of the SCN-lesioned group did not survive long enough to be able to produce and raise a litter, and therefore it seems to be justied for this specic study to extrapolate the results on survival to tness. Several other studies have been performed to test whether a species is optimally adjusted to an environment with 24 h cyclicity against other periods. Fruities [10] and blowies [11] live longer under a 24 h light dark cycle than under various other periods
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2. EARLY EVOLUTION OF CIRCADIAN TIMING MECHANISMS: ESCAPE FROM LIGHT OR ENERGY STORAGE? In the early development of life on our planet, the emerging photosynthesizing organisms that were exposed to sunlight faced several major problems owing to the alternation between day and night. They had to have access to energy throughout the day and night, and they had to protect themselves against damage by ultraviolet (UV) light [16], which was not ltered by the Earths atmosphere around 3 4 billion years ago when early life emerged [17]. In those cases where the usable form of energy (e.g. sunlight) was not constantly available, organisms had to harvest energy when available and store it for later use. For organisms that relied on photosynthesis, the solutions to these problems must have led almost automatically to rhythmic behaviour at the molecular level. During the day, light had to be absorbed for the formation of ATP through photosynthesis. Part of this ATP is generally used for primary cell functions such as division and growth, whereas another part somehow had to be stored for usage during the subsequent night when the energy supply through photosynthesis was lacking. Simultaneously, the organisms DNA had to be protected against UV radiation during the day. If this protection required mechanisms that interfered with transcription, those mechanisms should have been reversed during the night. Thus, night and day required different fundamental molecular processes. For cyanobacteria, which are among the most ancient photosynthesizing organisms on Earth, it has recently been proposed that their circadian timing system might have evolved to protect DNA from UV radiation [18,19]. This conjecture explains that the KaiC protein, as a key player in the evolution of timing systems, appeared about 3.5 billion years ago [17]. In these early organisms, KaiC forms hexamers in the presence of ATP during the light phase of the day. These hexamers seem to be involved in the compaction process of the DNA. Simons argues that this compaction protects against UV damage. During the night the hexamers fall apart, thereby allowing unfolding of the DNA. This conjecture is largely a specication of the escape from light hypothesis, put forward by Pittendrigh [20] and later studied by Hastings and co-workers [21,22]; for a review see [16]. Simons proposes that KaiB and KaiA, the other key players in the molecular circadian oscillator of the cyanobacterium S. elongatus, emerged much later in evolution,

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dephosphorylated KaiC Figure 1. Postulated temporal patterns of phosphorylation and dephosphorylation of KaiC in (a) summer and (b) winter. The phosphorylation phase is hypothesized to be stimulated by KaiA, starting with dephosphorylated KaiC hexamers at dawn and reaching fully phosphorylated hexamers at dusk.

KaiB for the purpose of signalling the end of the day, and KaiA to turn the system into a self-sustained oscillation, enabling cyanobacteria to anticipate the daynight cycle rather than just responding to the presence of light. A recent major breakthrough in the development of our knowledge on circadian regulation is the observation by Kondos group that DNA transcription is not required for obtaining molecular circadian oscillations in the Kai-system. They have demonstrated that it is sufcient for circadian oscillations in KaiC phosphorylation level to emerge in a test tube, when ATP is added to the proteins KaiA, KaiB and KaiC [23]. The resulting mixture shows oscillations in concentration of the various molecular complexes with large amplitudes and with a period close to 24 h. The resulting period is only marginally dependent on environmental temperature. In addition, measured circadian periods in vitro depended on specic mutations in the Kai proteins, which paralleled periods found in vivo. The apparent post-translational oscillation in the test tube suggests a major role for a similar oscillation in the cells, albeit possibly modied and shaped by transcriptional regulation [19,24]. Mechanistically, the idea is that KaiC occurs in its monomeric form at the beginning of the day. Simultaneously, ATP is produced through photosynthesis. Six KaiC monomers form a hexamer for which they use 12 ATP molecules, not as energy supply but as building blocks [25]. Apart from using ATP as structural elements in the hexamer complex, additional ATP is also used to phosphorylate KaiC. Each KaiC molecule has two phosphorylation sites and KaiA promotes their subsequent phosphorylation in the course of the light phase. Phosphorylation creates additional hydrogen bonds between the six monomers and tightens the hexamer [26,27]. With increasing phosphorylation of the hexamers at the end of the day, KaiB is more likely to attach to the molecular complex. The KaiABC complex is likely to undergo a conformational change, after which phosphorylation is more difcult, and dephosphorylation occurs during the night [28]. Upon dephosphorylation, the hexamers more easily disintegrate. During the night, the amount of KaiC decreases [29]. Upon complete
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dephosphorylation, the conformational state changes back to the original form, and the remaining KaiC molecules will be phosphorylated again during the next day. Simons [18] suggested that a major function of the circadian oscillation is to regulate compaction and decompaction of the DNA, in order to avoid UVinduced DNA damage [24]. Records of DNA compaction and decompaction [29] clearly show that in modern cyanobacteria compaction occurs at night, not during the day. Of course, the current timing of the DNA compaction cycle may be due to a more recent adaptation to the altered atmosphere. The phase of the cycle may have been different 3.5 billion years ago. Apart from DNA protection, however, we suggest that another evolutionary function of the phosphorylation dephosphorylation cycle of KaiC in cyanobacteria was to store ATP during the day and supply ATP at night. We suggest that ATP storage was the primary reason to develop circadian oscillations. In short (gure 1), we propose that hexamerization of KaiC during the photoperiod served to lock in ATP at a time during evolution when we think that other mechanisms for ATP storage (starch and sugar) had not yet evolved. Since ATP was generated by light exposure, the storage should occur in daytime. At night, in the absence of ATP production, the cells released ATP from the store. This release had to be controlled in order to provide the cells with an even ow of ATP. Phosphorylation has that effect. At the beginning of the night, phosphorylation of KaiC is maximal leading to a tight locking in of ATP. As a consequence, a relatively small fraction of the large number of available hexamers dissociates at the beginning of the night. At the end of the night, a relatively larger fraction of the smaller number of remaining hexamers dissociates. This results in a more or less even ow of ATP. Obviously, if the function of this mechanism indeed is to provide ATP at night, the ATP should become available for, say, housekeeping mechanisms, not for rephosphorylating the just dephosphorylated sites of the KaiC molecules. This may be the reason that the

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two phosphorylations and dephosphorylation per day per KaiC molecule occur in a specic order. Owing to this, rephosphorylation can only occur after complete dephosphorylation [30]. (a) KaiB: a signal of the night phase? KaiB probably emerged between 2.3 and 3.5 billion years ago [17], much earlier than KaiA. In modern S. elongatus, KaiB binds to hexamers in a specic phosphorylation status, which seems to occur roughly half way through the dark phase (gure 1; [31]). This behaviour is very suitable to signal a specic phase of the cycle in order to trigger relevant processes at that time [18,19]. This signal might ultimately be used to block cell division at night when energy supplies might be limiting to support new cells. Blocking of cell division in S. elongatus was indeed found to be under circadian control. Exposure to constant light revealed a steady increase in population size, except for short intervals of about 3 h that appeared at about 24 h intervals [3234]. The data show that the 3 h intervals without cell division occur at the very end of the subjective day. Under normal 12 h light : 12 h dark conditions, this leads to the situation that cell division stops 3 h before dusk. During the night, cells do not divide either, but this might be a direct response to the absence of light. At dawn, cell division does not immediately reappear. Apparently, the relevant processes need some time to resume. It might be that KaiB evolved to (indirectly) suppress cell division at the end of the day, at a time when no direct environmental signals are available to trigger this response. This signalling pathway can be considered a clock function: the mechanism is supposed to tell time to the organism through an endogenous mechanism, at a time when direct environmental signals are not available. (b) KaiA: an adaptation to photoperiod variation? About 1 billion years ago, KaiA appeared [17]. KaiA is known to accelerate KaiC phosphorylation (gure 2; [26,27]). While gene transcription of KaiB and KaiC depends on the same promoter, activation of the KaiA gene is controlled by a different promoter [35], reviewed in Williams [29]. We suggest several possible evolutionary advantages for the development of KaiA. In vitro, KaiA has been demonstrated to increase phosphorylation by a factor of approximately 2.5 in comparison with phosphorylation in the absence of KaiA [26,27]. If this also applies to the in vivo situation, more hexamers may reach the fully phosphorylated state in the presence of KaiA, which will change the timing of the KaiB signal to block cell division. This leads to a functional interpretation of the evolution of KaiA. We propose that KaiA serves to modulate the rate of phosphorylation of KaiC in order to meet the uctuating demands of changing photoperiod across the seasons (gure 2). Hence, the development of KaiA might have provided S. elongatus and similar cyanobacteria with a selective advantage by generating the capacity to invade areas at different latitudes. This hypothesis can be tested experimentally,
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KaiB detects KaiC phosphorylation state KaiA enhances KaiC phosphorylation rate

time of day (h) Figure 2. Proposed role of the Kai proteins in evolutionary adaptation to photoperiod. Both in short and in long days, the number of phosphorylated KaiC hexamers decreases at about the same constant low rate. In short days this process lasts longer, so more hexamers must be formed in the day, requiring more KaiC production per hour than in long days and more phosphorylation. Since phosphorylation of KaiC must be complete at the end of the shorter day, KaiA is expected to be present at an even higher concentration in short days. If KaiB signals the dephosphorylation status of the hexamer complexes, this will happen during the night, both in short and in long days.

by competition experiments in which different KaiA mutants compete with each other under different photoperiods.

3. ADJUSTMENT TO CHANGING DAY LENGTH AS A SELECTION FORCE FOR THE CIRCADIAN SYSTEM Although it is likely that circadian timing systems originally evolved on the basis of daily changes in the environment, there is another important function of the circadian timing system, and that is the regulation of annual rhythms in physiology and behaviour [36]. The duration of photoperiod can indicate the time of year, which relates to environmental changes in temperature and food availability. Timing of migration, moult, hibernation and, most importantly, reproduction are behaviours that are adapted to annual changes in the environment to maximize survival and offspring production at temperate climatic zones. Because these behaviours directly relate to tness, it can be expected that selection pressures for optimal annual timing are strong. Therefore, evolution will have shaped the readouts from the circadian clock to optimize annual timing. Theoretically, photoperiod can be assessed without a circadian oscillator being present. Organisms that are exposed to the full light phase of the day could have developed systems by which they directly measure photoperiod. However, the evidence so far indicates that all organisms (plants, insects, mammals and birds) that respond to photoperiod changes use a circadian timing mechanism to do so (see [37] for

Evolution of time-keeping mechanisms detailed overview). Circadian inuence in photoperiodism does not exclude mechanisms of direct responses to day length. This is best exemplied by melatonin secretion in the mammalian pineal. The duration of melatonin secretion during the night is critical in driving downstream photoperiod responses, but melatonin secretion itself is both driven by the circadian system and directly suppressed by light. The most parsimonious explanation for the ubiquitous involvement of a circadian oscillator in photoperiodic responses is that such timing mechanisms were present early in evolution and started to form an internal representation of the day night cycle. This internal representation now formed an adequate buffering against day-to-day uctuations in light conditions, yielding a much more reliable seasonal signalling.

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(a) Latitudinal clines in circadian parameters Seasonal modulation of the environment increases at latitudes closer to the poles. The most reliable signal to indicate the time of year is photoperiod, but the relationship between photoperiod and time of year depends on latitude [38]. The amplitude of the annual environmental temperature rhythm also increases with latitude [39]. Species that occur along a wide range of latitudes, therefore, have to adapt to the combined annual changes of temperature and photoperiod in order to optimize annual timing of reproduction. In some species, this has apparently led to latitudinal differences in characteristics of the circadian timing system. Together, these correlative observations between latitude and circadian characteristics form a strong indication that photoperiodic response mechanisms generate a major selection pressure for the circadian system, which, in itself, indicates also a crucial role for the circadian system in annual timing. One example was found in Arabidopsis. This model plant shows a latitudinal cline in free-running period of its circadian timing system [40], with the period being longer at higher latitudes. A similar positive correlation between circadian period and latitude was found in Drosophila auraria [41], and Drosophila ananassae [42], while Lankinen & Forsman [43] found a negative correlation between free-running period and latitude in Drosophila littoralis. Other examples of latitudinal clines can be found in the molecular makeup of the circadian system such as polymorphisms in clock genes. Latitudinal clines in circadian clock gene expression levels or clock gene polymorphisms were found in birds [44,45], sh [46] and insects [47 50]. Extreme situations occur in polar regions, where light levels are more or less constant for long periods of time. It has been demonstrated that reindeer (Rangifer tarandus) show daily modulation of behaviour only in spring and in autumn. During continuous darkness in winter and continuous light in summer, they show no 24 h modulation of their rest activity pattern [51,52] or plasma melatonin levels [53,54]. Apparently, the lack of 24 h rhythmicity in the environment at some parts of the year leads this species to a complete or partial loss of the circadian system at the molecular level [54]. The selective advantage of losing circadian organization under
Phil. Trans. R. Soc. B (2011)

these conditions might be to optimize food intake and ruminant digestion efciency by a more frequent ultradian organization of feeding [52]. Similar temporal loss of daily organization in an arctic species has also been found in the Svalbard ptarmigan (Lagopus mutus hyperboreus; [55]), but the results in both species do not mean that all species in arctic regions lose circadian organization during the arctic summer or winter. Arctic ground squirrels (Spermophilus parryi, a hind gut fermenter), for instance, maintain a 24 h activity rest cycle during arctic mid-summer under continuous light conditions well above the polar circle [56,57]. Taken together, the reindeer and ptarmigan data seem to suggest that a circadian system is only advantageous when the environment has a strong (continuous) 24 h rhythm. These conclusions are fully in line with the selection experiments in cyanobacteria [1,2], but do not explain why arctic ground squirrels maintain circadian organization during the arctic summer. One possible explanation may reside in the strong circannual organization of obligate seasonal hibernation in ground squirrels. Correct timing of hibernation onset and offset is essential to ground squirrels, and a continuous functionality of the circadian (and melatonin) system throughout the summer may therefore be essential to this species. No matter the specic evolutionary advantages of the disappearance of circadian rhythmicity in locomotor activity of reindeer in summer and winter, the data do demonstrate an inuence of photoperiod on melatonin [53,54], indicating that the crucial role for melatonin in photoperiodism is preserved. While much of the physiology underlying annual changes in reproduction or moult in mammals are regulated in other areas (like the pituitary and its pars tuberalis [58]), it is commonly assumed that the SCN (at least in mammals outside the arctic region) is involved in the detection of annual changes in photoperiod, and that this serves as a trigger to induce these behaviours. This notion is based on lesion studies in hamsters, showing that photoperiod-induced gonadal regression requires an intact pathway from SCN to the pineal gland [5865]. The duration of melatonin production in the pineal gland is the signal that regulates gonadal growth or regression [58,6668], and this melatonin signal is shaped by the SCN through the detection of photoperiod [69].

(b) The evolutionary importance of annual timing and the role of the suprachiasmatic nucleus Many important events that may have direct tness consequences are regulated on an annual basis, including reproduction, migration and hibernation. Deviations from optimal seasonal timing will have strong repercussions for survival and reproductive output. Thus, an accurate annual timing mechanism must have been heavily selected for through evolution. In seasonal organisms living in temperate zones, the adaptive signicance of the circadian system may lie in its day length signalling capacity rather than in its daily timing function.

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PVN IML and SCG

photoperiod pineal retina SCN daily melatonin rhythm entrainment of annual rhythms (hypothalamus, pituitary)

generation of daily rhythms (behaviour, physiology)

Figure 3. The dual role of the SCN in daily and annual rhythms as illustrated by its input and output pathways. Arrow connection ends indicate stimulatory pathways, at connection ends indicate inhibitory pathways. SCN, suprachiasmatic nucleus; PVN, paraventricular nucleus; IML, intermediolateral column of the spinal cord; SCG, superior cervical ganglion.

Most body cells show circadian rhythms, which in mammals are coordinated by a central hypothalamic pacemaker, the SCN [4]. To synchronize the bodys internal physiology and behaviour to the external daily environmental light cycle, the SCN is facing two tasks: daily synchronization to the light dark cycle, and adaptation to changing day length over the year (gure 3). The rst task drives daily timing (e.g. sleep and activity), the second drives a nocturnal pineal melatonin signal that acts as an input signal to annual timing systems driving seasonal responses in physiology and behaviour (gure 3; [70]). Interactions between both functions are illustrated by annual cycles in daily rhythms under (semi-) natural conditions [38,51,52,7173]. The heterogeneous structure of the SCN and its differentiation between species and sexes [7480] offers the fascinating possibility that the SCN carries differentiated neuroanatomical substrates for daily and annual timing. Annual variation in melatonin signal duration has been shown to be a critical input signal for the annual timing mechanism [81,82] that involves the pituitary and its pars tuburalis (PT), which is densely packed with melatonin receptors [8389]. Pineal melatonin release is driven by the SCN via the paraventricular nucleus (PVN), superior cervical ganglion (SCG) and intermediolateral column of the spinal cord (IML) pathway [58,69]. Pharmacological infusions at the PVN have shown the involvement of nocturnal glutamate excitatory signalling and diurnal GABA (g-aminobutyric acid) inhibitory signalling to PVN neurons that both shape the melatonin signal to signal the length of the night [6165]. Selection on annual timing can take place at different levels in the melatonin signalling cascade (gure 3), but most importantly also in the melatonin recipient areas like the PT. Good advances are currently being made in elucidating the molecular mechanism of melatonin signalling in the PT. Although these mechanisms also involve clock genes like Cry1 and Per1 [83,90,91], there are other genes involved that might be subject to selection on annual timing. For this paper, we will limit ourselves to discussing selection on day length measurement on the level of the circadian system.
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Recent ndings about the characteristics of pacemaker cells within the SCN of several mammalian species may lead to a better understanding of how the SCN might be capable of measuring photoperiod. The issue here is that cells within the SCN appear to show substantial differences with respect to the timing of their activity within the day [92]. While most cells are electrically active during the day, some cells (as deduced from electrical discharge and period gene expression rhythms) seem to be linked to dawn and others are linked to dusk [93]. The phase distribution of SCN cells seems to be organized along the rostro-caudal gradient, where morning cells are dominating the caudal region of the SCN and evening cells are dominating the mid-rostral region of the SCN [94 97]. A compression of photoperiod brings those cell groups closer together in time, while an increase in photoperiod moves them apart [93,98 100]. In this way, the duration of SCN activity becomes related to photoperiod, but so far it is unknown whether those SCN neurons that spread their internal phase relationship under long photoperiods are also involved in shaping the melatonin signal. Furthermore, it is tempting to match this rostro-caudal phase gradient in the SCN to the previously proposed morning evening model of photoperiod adaptation as proposed by Pittendrigh & Daan [101], but it remains to be established whether the circadian properties of rostral and caudal SCN cells follow the predictions specied for the morning and evening oscillator [101,102].

(c) Selection pressures on intrinsic circadian period: arguments from annual timing In virtually all circadian systems studied so far, environmental light dark cycles tune the intrinsic period of the system (t) to the external period of 24 h. In other words, the organism is synchronized to the outside world owing to the interaction of a response to external light and the organisms intrinsic period. Organisms with t close to 24 h have a selective advantage over conspecics that deviate considerably from 24 h (see 1). However, t measured under continuous darkness may vary between individuals of the

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Figure 4. Oscillator speed affects photoperiodic time measurement: differences between external (a) and internal (b) coincidence timings. (a) In an external coincidence timing model, longer circadian periods (slower speed) will generate larger phase angle differences with morning light (grey triangle) owing to a lagging phase angle of the circadian oscillator relative to the light dark cycle. Short circadian periods (faster speed) will generate smaller phase angle differences with morning light owing to the leading phase angle of the circadian oscillator relative to the light dark cycle. Hence, internal representation of day length will be longer in slower pacemakers (the reverse being true when evening light induces the photoperiodic response). (b) In an internal coincidence timing model, the circadian oscillator is composed of (at least) two oscillators, a morning (light grey curve) and an evening (dark grey curve) oscillator. The phase angle difference of these two oscillators determines the internal photoperiod representation. Intrinsic period of the combined system will generate leading and lagging properties of both oscillators causing a compression of the phase angle difference when intrinsic period deviates from 24 h. Hence, internal representation of day length will be longer when pacemaker period approaches 24 h.

same species, and even more so between species. In addition, intrinsic t within a species was found to vary with latitude (see 3(a)). These arguments indicate that t was subject to species and environmentspecic selection pressures, not tuning t simply to 24 h, but values slightly away from 24 h to match to specic needs of the organism and maximize tness through higher survival or reproductive output. There has been surprisingly little debate on what those selection pressures on intrinsic circadian period could be and how these selection mechanisms might work. Here, we will make an attempt to predict qualitative changes in selection pressures with varying environmental conditions, by considering two distinct hypotheses of the role of the circadian system in annual timing: the internal and external coincidence timing models. (d) Leading and lagging circadian systems: consequences for timing Before zooming in on relationships between circadian and circannual timing, it is important to understand the consequences of differences between the period, T, of the external Zeitgeber, and the endogenous period, t, of the organism. If T . t, the overt period
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of the organism has to lengthen in order to become equal to T. Since the external signal has the effect of slowing down the internal oscillation, the phase of the internal oscillation will be earlier than the phase of the entraining signal. In other words, the internal oscillation will lead relative to the Zeitgeber. By contrast, if T , t, the internal oscillation will lag relative to the Zeitgeber (gure 4a). As a consequence, under real-life 24 h conditions, the intrinsic period of the oscillator determines to some extent the time of day at which certain events of the internal cycle occur [103]. Correlations between circadian period deviations from 24 h and stability of the circadian rhythm [104] may be important for the organism. Additionally, deviation between the external period and the internal circadian period reduces tness in bacterial competition experiments [2], and reduces longevity in mammals [105], both conrming the circadian resonance hypothesis. Such properties of circadian systems may have direct tness consequences that eventually optimize circadian period, but evidence discussed below suggests that circadian period may also be shaped through its effect on day length measurement and subsequent annual responses in physiology, behaviour and reproduction.

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internal photoperiod
(a) (b)

Photoperiodic responses are characterized by a sharp transition between short- and long-day responses, indicating strong selection for optimal annual timing. For instance, photoperiodic control of testes growth in hamsters can distinguish between a 1 h change in photoperiod: regressed testes are switched to developing testes in a 12.5 h photoperiod but not in an 11.5 h photoperiod [106]. The involvement of the circadian system in this photoperiodic regulation of testis size is indicated by the observation that hamsters with a short circadian period (owing to the tau mutation) show regressed testes at dark durations of 9 h or longer, whereas their wild-type counterparts need at least 11 h darkness per day to regress their testes [107,108]. In addition, selection experiments in Peromyscus leucopus for short-day response of the reproductive system showed a difference in circadian period between responsive and non-responsive mice [109], although subsequent experiments in continuous darkness might indicate that photoresponsiveness in these strains is also determined by other factors. Two different models have been postulated to describe the role of the circadian system in photoperiodic time measurement: external coincidence timing [110,111] and internal coincidence timing [101,102,112]. External coincidence timing proposes that the circadian system acts as a single oscillator with a specic phase angle to the onset (or offset) of the light phase of the day. When day length increases, the additional light will eventually coincide with a certain sensitive phase of the oscillator and thereby trigger a long-day response (gure 4a). Internal coincidence timing proposes the existence of (at least) two oscillators, one entraining to dawn and the other one to dusk. When day length increases, the phase angle difference between these two oscillators will also increase, which will eventually trigger a long-day response (gure 4b). Both external and internal coincidence timing models assume an internal photoperiod representation, generated by the circadian system in response to the external day length. The two photoperiodic models appear to predict very different effects of external photoperiod on intrinsic photoperiod representation when variation in circadian period, and subsequent leading or lagging phase angles, is considered. In the external coincidence timing model, consider the case of a sensitive phase in the morning: if light hits the system at this phase, the long-day photoperiod response is triggered. If an individual has a longer intrinsic circadian period (a more lagging phase angle), the sensitive phase will occur at a later time of the day (gure 4a, the reverse being true for external coincidence with evening light). Hence, light will hit the sensitive phase at an earlier day in the season. Phrased differently, long-day photoperiod responses will occur at shorter external photoperiods in animals with longer t (gure 5a), given that all other things are being equal. Longer circadian periods will, therefore, lengthen the internal photoperiod representation (gure 5b, grey line). Conversely, when evening light induces a photoperiod response, lengthening of circadian period will shorten internal photoperiod (gure 5b, dashed line).
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internal photoperiod

(c)

(t (t > 24 2 h 4h ) (t <2 ) 4h )

(d)

24 h) (t (t > 2 <2 4 4 h h) )

(t

external photoperiod

24 23 25 circadian period, t (h)

Figure 5. External (a,b) and internal (c,d ) coincidence timing models lead to different predictions for directional selection pressure on circadian period. As follows from gure 4, the relationship between internal and external photoperiods will be differentially affected by circadian period when external (a) or internal (c) coincidence timing is assumed. Plotting internal photoperiod (for a given external photoperiod) against circadian period (t) yields different curves for external (b) and internal (d) coincidence timing. For external coincidence timing, morning light would generate an inverse relationship between t and internal photoperiod (b, continuous curve) compare with evening light (b, dashed curve). The curves in (b) and (d) enable predictions on how selection pressure would act on t when an earlier long-day response is favoured during spring (e.g. at lower latitudes or warmer climatic conditions) for external (b) coincidence and internal (d) coincidence timing. For this example, the directional selection pressures on t are indicated for both models (black arrows).

In the internal coincidence timing model, both the morning and evening oscillators will tend to lead when intrinsic period shortens and both oscillators will tend to lag when intrinsic period lengthens. Only when intrinsic period approaches 24 h, both oscillators will have no tendency to lead or lag and as a result their phase angle difference will be maximal when t approaches 24 h (gures 4b and 5c). When internal coincidence timing is assumed, the internal representation of any given external photoperiod is expected to be maximal when t 24 h (gure 5d). Once the inuence of intrinsic circadian period on the internal representation of photoperiod is known (gure 5b,d ), we can try to establish how selection would act upon intrinsic circadian period when, for instance, a changing selection pressure for earlier reproduction is present. There are at least two environmental conditions where it is relatively easy to see how environmental conditions would select for earlier reproduction in spring. First, we can expect a latitudinal cline in the relationship between spring temperatures and photoperiod. In general, a certain long-day photoperiod (longer than 12 h) corresponds to lower temperatures at higher latitudes for two reasons: (i) because it occurs

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Table 1. Properties of latitudinal clines in circadian period. Latitudinal clines in circadian period are summarized for Arabidopsis thaliana and several Drosophila species. The latitudinal range indicates the minimal and maximal latitudes from which the samples were taken; t range indicates the outcome of the regression of t against latitude over the latitudinal range that was sampled. The slope and signicance of the regression of t against latitude is given, as well as whether the regression crossed the 24 h limit over the latitudinal range sampled. No dataset was corrected for other geographical parameters like altitude or distance from large open water areas. Genus name abbreviation: A, Arabidopsis; D, Drosophila; asterisk (*) indicates European data only. species A. thaliana D. subobscura D. littoralis D. ananassae D. auraria D. melanogaster D. melanogaster development state seedling pupae pupae adult adult adult adult latitude range 5 65 28.2 62.9 41.6 69 6 36 3443 33.6 52.2 39.5 52.2

t range
23.724.9 24.725.9 20.723.4 22.225.4 22.424.4 23.623.8 23.923.6

regression slope 2 2 2 n.s. signicant signicant signicant signicant n.s. signicant

t range crosses 24 h?
yes no no yes yes no no

reference [40] [120] [43] [42] [41] [121] [121]*

earlier in spring closer to the poles, and (ii) because the Sun projects less heat on the Earth surface because it radiates from a position closer to the horizon at higher latitudes. For plants, invertebrates and poikilothermic vertebrates, development depends on ambient temperature and at higher latitudes reproduction and growth will commence later in spring. These species, but also the predators depending on them, will face a selection pressure favouring later breeding than their conspecics living closer to the equator. As a result, latitude-dependent selection pressure on timing of breeding may result in latitudinal clines in circadian clock gene polymorphism frequencies or expression levels, as indicated by studies in birds [44,45], sh [46] and insects [47 50]. Second, global warming generates a new relationship between photoperiod and ambient temperature. Without debating the cause of global warming, climate data show that a certain latitudinal location faces increasing ambient temperatures during the last 50 100 years, while the annual cycle in photoperiod duration remains constant. This yields a changing relationship between photoperiod and temperature [113,114]. Warm spring temperatures that allow for developmental growth or reproduction will occur at shorter photoperiods than before. This suggests that animals breeding in temperate zones could breed earlier for energetic reasons, but also because the growth season for plants and invertebrate prey species starts earlier [115 117]. Indeed, selection pressure for earlier reproduction attributed to global warming was found in long-term population records for temperate zone birds [118,119]. When selection pressure for earlier reproduction increases, chronobiology would predict an effect on circadian period, given the importance of the circadian system in photoperiodic time measurement. Both models for photoperiodic time measurement (external versus internal coincidence) yield different predictions when earlier reproduction in spring is favoured (gure 5b,d ). In such situations, external coincidence timing would select for circadian periods that may deviate from 24 h (increasing t when morning light induces the photoperiod response; decreasing t when evening light induces the photoperiod response), while internal coincidence timing would specically
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select for t closer to 24 h. Inherent to this line of reasoning is that the external coincidence timing model would, in principle, allow for a continuous increase or decrease of critical day length by changing circadian period. Internal coincidence timing would have a clear limitation in that maximal internal photoperiod representation is obtained when circadian period equals 24 h. Deviations from 24 h, in any direction, would always yield shortening of internal photoperiod representation. When selection pressures would call for even longer internal photoperiod representation, additional modifying response parameters would have to be recruited. Internal coincidence timing, therefore, seems to be the more complex solution, because it inherently carries its limitations in regulatory possibilities. For this reasoning, it seems likely that natural selection may have favoured external coincidence timing over internal coincidence timing, but we cannot exclude that a combination of both mechanisms can exist, even within a species. Neither can we exclude that in certain species, selection pressure acted on mechanisms downstream from the melatonin signal, leaving the circadian system untouched but yielding similar effects on annual timing.

(e) Data from latitudinal clines t external, but not internal coincidence timing When a population moves up towards higher latitudes, selection pressure would change to later reproduction. As a consequence, t can be predicted to move away from 24 h under the assumption of internal coincidence timing (gure 5d), but under the assumption of external coincidence timing t will continuously decline (photo induction phase in the subjective morning), or increase (photo induction phase in the subjective evening; gure 5b). Hence, both models will predict a latitudinal cline in circadian t, but only the external coincidence timing model predicts that this cline will cross the 24 h limit if the measured range of latitudes is large enough. This difference was tested using published data in insects (table 1). The data in table 1 indicate that, both in Arabidopsis and Drosophila, latitude can cause selection pressure that changes circadian period from above to below

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Evolution of time-keeping mechanisms system in photoperiodic responses. We suggest that these latitudinal clines can actually be used to distinguish between two leading hypotheses in circadian photoperiod regulation, of internal and external coincidence timings. Both models yield different predictions on the effect of changing selection pressures on circadian period when photoperiodic responses change with different latitudes. Using this line of reasoning enables us to determine which photoperiodic mechanism may be present in a specic species, when latitudinal clines of its circadian period are established. Data obtained in Drosophila seem to indicate that this species uses an external coincidence model since the latitudinal cline of circadian period crosses 24 h, while internal coincidence would predict a limit around 24 h. To obtain insight in mechanisms of photoperiodism in other taxa, it will be valuable to obtain information on latitudinal clines of circadian period in, for instance, mammals, birds and sh. The qualitative model presented here using latitudinal clines of circadian period will be equally relevant to predict what the directional selection pressures on circadian period may be when temperate zone species are exposed to changing climatic conditions (e.g. global warming) that shift their optimal timing of breeding and other annual events in physiology and behaviour.
We thank B. Helm, M. Menaker, S. Daan, M. Merrow, M. Maas, M. Simons, M. Sturre and the referees for helpful discussions and comments on the manuscript. Part of this work was developed during the 2008 Lorenz Center workshop Keeping track of the Seasons organized by M. Visser, A. Dawson and B. Helm. This work was supported by the European Commission 6th Framework Project EUCLOCK (No. 018741).

24 h or vice versa, as predicted by an external coincidence model (gure 5b). In these data, there is no indication that selection on circadian period pushes against a limit around 24 h, as can be expected when the system follows an internal coincidence model (gure 5d). It would be very valuable to obtain similar datasets in mammals and birds, but such data are presently not known to us.

4. CONCLUSIONS The everlasting alternation between light and darkness and the concomitant oscillation in environmental temperature provides systematic variation in the environment to many organisms on Earth. Those organisms have adjusted their physiology to those environmental changes to prot optimally from the daily changes. The underlying machinery is, however, very different between cyanobacteria and complex animals. In animals, the rhythms of individual cells and tissues are controlled by a master pacemaker that evolved because of the apparent benet of a conductor that regulates the phasing of the activities of all other cells in the organism. This line of reasoning seems rather straightforward: small evolutionary steps are required to reach more complex circadian adaptation known today and each step has tness advantage of its own. Available data seem to support quite a few of those steps. Yet, the proof of tness advantages of having a pacemaker is meagre and mostly indirect. The advantage for early cyanobacteria to be able to anticipate daily uctuations of the environment through oscillations in KaiC hexamers might also have created a new problem: adaptation to a specic light dark cycle would have caused limitations on expanding to geographical regions at different latitudes. We suggest that KaiA might have emerged to enable this system to adjust to different durations of day and night, thereby allowing cyanobacteria to expand their geographical range to different latitudes. A testable prediction that follows from this hypothesis is that KaiA would be upregulated under short photoperiods and downregulated under long photoperiods (gure 2). Specic experiments of Kai expression under different photoperiods would not only teach us more about the function of circadian regulation in photoperiodic adaptation in cyanobacteria, but it would also shed light from a different angle on the evolution of the earliest circadian clock system known. The proposed early evolutionary force of photoperiod adjustment in the earliest organisms that were equipped with a circadian system indicates that circadian clocks and photoperiodic adjustment must have been intricately connected from the start. Indeed, in higher plants, insects and mammals, the involvement of the circadian system in photoperiodic responses has been conrmed by various experiments, albeit that its precise mechanism remains to be elucidated. Another line of reasoning originates from accumulating evidence that latitudinal clines were found in circadian period in various species. This suggests that photoperiod-dependent selection pressures on circadian period exist, and that circadian period plays a critical role in the involvement of the circadian
Phil. Trans. R. Soc. B (2011)

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Phil. Trans. R. Soc. B (2011) 366, 21552170 doi:10.1098/rstb.2010.0346

Review

Social molecular pathways and the evolution of bee societies

Guy Bloch1,* and Christina M. Grozinger2
Department of Ecology, Evolution and Behavior, The Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem, Israel 2 Department of Entomology, Center for Pollinator Research, Center for Chemical Ecology, Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, PA, USA Bees provide an excellent model with which to study the neuronal and molecular modications associated with the evolution of sociality because relatively closely related species differ profoundly in social behaviour, from solitary to highly social. The recent development of powerful genomic tools and resources has set the stage for studying the social behaviour of bees in molecular terms. We review ground plan and genetic toolkit models which hypothesize that discrete pathways or sets of genes that regulate fundamental behavioural and physiological processes in solitary species have been co-opted to regulate complex social behaviours in social species. We further develop these models and propose that these conserved pathways and genes may be incorporated into social pathways, which consist of relatively independent modules involved in social signal detection, integration and processing within the nervous and endocrine systems, and subsequent behavioural outputs. Modications within modules or in their connections result in the evolution of novel behavioural patterns. We describe how the evolution of pheromonal regulation of social pathways may lead to the expression of behaviour under new social contexts, and review plasticity in circadian rhythms as an example for a social pathway with a modular structure. Keywords: social behaviour; evolution; gene network; bee; communication; circadian rhythm
1

1. INTRODUCTION Insect societies represent one of the major transitions in evolution [1] and have attracted the attention of biologists for centuries. Darwin [2] noted that insect societies provide some of the most difcult challenges to the theory of natural selection operating on individuals. In his words: Can we consider the sting of the wasp or of the bee as perfect, which, when used against many attacking animals, cannot be withdrawn, owing to the backward serratures, and so inevitably causes the death of the insect by tearing out its viscera? ([2], ch. VI, Difculties to the theory, p. 202). He addressed this and other difculties posed by the biology of social insects by suggesting that natural selection acts also at the group (colony) level: We can see how useful their production may have been to a social community of insects, on the same principle that the division of labour is useful to civilised man (p. 242). Since Darwins time, there has been much debate about the ultimate causes underlying the evolution of social behaviour, resulting in the development of insightful theories such as kin and multi-level selection (e.g. [1,3 5]). Because phylogenetic studies indicate that the solitary lifestyle is the

* Author for correspondence (bloch@vms.huji.ac.il). One contribution of 10 to a Theme Issue Evolutionary developmental biology (evo-devo) and behaviour.

ancestral state, it is assumed that social animals evolved from solitary ancestors [6]. In order to fully understand how natural selection may have been operating to produce this transition, it is necessary to elucidate the proximate mechanisms by which the behaviours of a solitary animal were modied to produce complex social behaviours, such as those underlying division of labour, colony defence and nest construction. With the development of new genomic tools and resources, it is now possible to employ a comparative approach for understanding the molecular basis of social behaviour within species, and how these behaviours evolved from solitary ancestors [7,8]. A similar approach has been particularly powerful in the eld of developmental biology, in which it was shown that reorganization of a core set of regulatory genes has led to a stunning diversity of structural forms [9]. The sociobiological evo-devo approach attempts to characterize suites of genes and molecular pathways and test their association with social behaviour. Thus far, most comparative genomics studies have focused only on species with well-developed genomic resources (such as the honeybee and Drosophila), but with the increasing accessibility of genomic information, more relevant comparisons between taxonomically related solitary and social species will become feasible. While it is possible that novel social behaviours evolved de novo as the result of the appearance of
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Box 1. Terms used in the manuscript (in alphabetical order). Alloethism Task specialization by different members of a colony of social insects that is related to variation in body size. Caste A group of individuals displaying a distinct morphological, physiological and behavioural phenotype that specializes on a particular task or set of tasks in a polymorphic social species. Circadian rhythm A repeating cycle with a period of approximately 24 h. Circadian rhythms are generated by endogenous processes but can be entrained/ adjusted by external cues. Developmental modules Modules that are involved in the developmental processes generating the form and function of the structures and networks necessary for the execution of a phenotype, including a social behaviour. Division of labour Specialization of individuals within a social group on specic tasks (see also polyethism and alloethism). Eusociality A form of social organization in which groups of conspecics display cooperative care of young, overlapping generations and reproductive division of labour. Functional modules Modules associated with dened and different functions. For example, in a social pathway, the detection of social signals, the processing of the information conveyed by the social signals or the regulation of exocrine functions. Genetic toolkit Set of genes with specialized and conserved molecular function (e.g. transcription factors) that is involved in the regulation of similar phenotypes across a wide variety of species and can acquire new or additional functions during evolution to regulate novel physiological or behavioural phenotypes. Gonadotropic Acting on or stimulating the reproductive organs; in social insects, this refers primarily to ovary activation. Ground plan Molecular pathways regulating fundamental physiological processes that acquire new or additional functions during evolution to regulate novel physiological or behavioural phenotypes. Microarray Genomic technology in which thousands of probes, corresponding to DNA with specic nucleotide sequences, are spatially distributed in an organized array. DNA, RNA or cDNA from a sample can be hybridized to the microarray to measure the relative abundance of these nucleotide sequences in the sample. Expression microarrays thus allow analysis of expression levels of thousands, potentially all, of genes in the genome. Module A set of components (e.g. genes, proteins, cells) that are strongly connected and have relatively weak connectivity to other components or modules. Modules could be subject to divergent evolutionary forces and therefore evolve relatively independently (see also physiological modules, functional modules and developmental modules). Pheromone Chemical signal that stimulates a stereotyped response in another individual of the same species. Responses are typically instinctive and not learned. Physiological modules Modules that are involved in the regulation of cellular, neuronal and endocrine processes necessary for the execution of a phenotype, including a social behaviour. Polyethism Task specialization by different members of a colony of social insects; tasks can be segregated among individuals by age (age polyethism) or morphological differences (caste polyethism). Polyphenism The ability of a single genome to produce two or more alternative phenotypes (e.g. morphologies) within a single population in response to environmental cues such as photoperiod, temperature, nutrition or social signals. Primer pheromone Chemical signal that elicits a long-term change in physiology and/or behaviour. Quantitative trait locus (QTL) A DNA region (which may contain several genes) that is statistically associated with a specic quantitative phenotypic trait. Releaser pheromone Chemical signal that triggers a rapid behavioural response (of the order of seconds or minutes). Social pathway The molecular, physiological and cellular components involved in detecting a social signal, processing the information and orchestrating an appropriate behavioural output. Each step of the process may be regulated by a relatively independent module. At the molecular level, the social pathway may encompass genetic toolkits or ground plans.

new genes, it seems more likely that discrete pathways that regulate specic behaviours in solitary species have been co-opted to regulate complex social behaviours in social species; this theory underlies the development of various ground plan and genetic toolkit models for social evolution, which have received support from several empirical studies (box 1, see below for further discussion). We further propose that in order to fully understand the evolution of sociality, it is necessary to characterize complete pathways that are involved in social behaviour (hereafter abbreviated as social pathways). We hypothesize that social pathways are modular, and consist of the detection of social signals, the processing of the information conveyed by these signals in the peripheral and the central nervous system and subsequent behavioural outputs (see below). Modications within modules or in the way modules interact with each other can lead to changes in behaviour and in the context under which it is expressed, and thus can give rise to the evolution of new forms of social behaviour. These models will not only help structure future studies of the evolution of social behaviour in bees,
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but may also serve as a framework for other behavioural systems in which animals respond to environmental stimuli or signals from other individuals (e.g. courtship and mating, aggression, predator prey interactions).

2. INSECT SOCIETIES Social insects, and bees in particular, provide an excellent model system with which to study the evolution of sociality because there is a broad range of taxonomically related species that encompass all levels of sociality, from species with essentially solitary lives all the way to eusocial species with colonies of thousands of highly specialized individuals (gure 1). Furthermore, there are clear effects of genes and social environment on both the social behaviour of individuals and the social organization of the group. Therefore, insect societies provide an excellent model system with which to dissect the effects of nature and nurture on complex behaviour [10 15]. One of the hallmarks of insect societies is a profound bias in reproduction. Some individuals in the

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Figure 1. Bees show diverse levels of social organization, from solitary to highly eusocial. (a) A solitary bee (Eucera palestina) at the entrance to her nest (photo: Abraham Hefetz). (b) Bumble-bee (B. terrestris) colony with a size-based division of labour. The photo shows several relatively small bees that are caring for the brood (photo: Guy Bloch). (c) A honeybee colony with an open queen cell (centre of photo). The rearing of new gynes is regulated by a pheromone blend secreted from the mandibular glands of the colonys queen (photo: Bernardo Nino).

colony (typically the queens) are extremely fecund, whereas others (the workers) are sterile or have relatively low reproductive potential. In primitively social species, adult females compete for reproductive dominance; subordinate females typically do not reproduce but rather perform brood care, nest maintenance or foraging activities. In more advanced social systems, females can develop into either queens or workers via alternative developmental processes (known as caste differentiation) that are initiated during pre-adult stages. In species with relatively small-sized colonies, regulation appears to occur mainly through physical aggression, while in larger colonies, regulation is commonly via pheromones. However, differential nutrition, genetic variation and environmental factors have been also implicated in caste determination, mostly in studies with ants [6,16]. A second organization principle is a division of labour among workers specializing in different activities (e.g. brood care, guarding the nest entrance and foraging for food). In bees, division of labour is most commonly related to age (age polyethism) or size (alloethism). Age-related division of labour has been best studied in honeybees (reviewed in [17,18]). In this system, young workers typically specialize in brood care (nursing) and other in-hive activities, while older bees perform activities outside the nest such as guarding and foraging. Size-based division of labour is common in bumblebees, and is characterized by profound morphological polymorphism with up to ninefold differences in body size [16]. Large individuals are more likely to perform foraging activities, and may start foraging as early as at their rst day as adults, while small bees tend to perform in-nest activities. Recent studies demonstrated that large workers are better suited for foraging activities because they have more sensitive sensory detection systems and stronger circadian rhythms [1921]. These functional differences between small nurses and large foragers are probably determined by pre-adult developmental processes, which are reminiscent of the caste polymorphism division of labour found in many species of ants [22]. Both reproductive and worker division of labour require the development of elaborated communication systems between the individuals in a colony. Information can be conveyed by distinct chemical, vibratory, visual or
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auditory signals, and can involve the simultaneous use of several modalities [6]. Social bees, particularly those with larger colony sizes such as honeybees, use a broad array of pheromones to organize the activities of the individuals in a colony. Pheromones are chemical signals that convey information between individuals within a species, and can trigger immediate short-term behavioural changes (releaser pheromones) or long-term changes in physiology followed by behavioural changes (primer pheromones). Pheromones are used to regulate both reproductive and worker division of labour in many systems, as well as other colony activities such as nest defence.

3. GROUND PLANS AND TOOLKITS Genes associated with social behaviour in bees have been identied by three main complementary approaches, which have been applied most broadly in honeybees. Candidate gene approaches (using orthologues of genes found in more genetically tractable systems, such as Drosophila melanogaster) have resulted in the characterization and manipulation of key genes, such as the foraging ( for) and vitellogenin (Vg) genes that are relatively conserved across species. Genomic approaches, such as quantitative trait locus (QTL) mapping, microarrays, differential display and proteomics, have identied large sets of genes that are associated with social behaviour, but there is typically little information on their molecular function in bees. Finally, genetic and molecular analyses of honeybee populations differing in their social behavioureither owing to natural variation or articial selectionhave identied genes or genetic loci associated with reproduction and foraging preference [23 25]. One of the important insights emerging from these molecular studies is that honeybee social behaviour may have evolved from modications in conserved molecular pathways regulating the physiology and behaviour of solitary species. These conserved pathways are commonly referred to as ground plans [26 28] or genetic toolkits [8]. There are excellent reviews on the social behaviours that are associated with molecular modications in conserved gene networks regulating reproduction, longevity, food gathering

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G. Bloch & C. M. Grozinger Review. Social pathways and bee societies division of labour in honeybees. These studies showed that JH, which functions as a major gonadotrophic signal in solitary insects, regulates worker behavioural maturation but not oogenesis in the adult honeybee (reviewed in [32,43,44]). In solitary insects, JH typically upregulates the fat body biosynthesis and accumulation in developing oocytes of the conserved yolk protein vitellogenin (Vg) [45,46]. JH activates Vg biosynthesis in honeybee pupae [47,48], but in adult bees, JH suppresses Vg gene activity [49] and Vg suppresses JH titres [29]. RNA interference-mediated downregulation of Vg caused bees to forage earlier in life [50]. These ndings suggest that a network involving JH and Vg was co-opted to regulate worker task (division of labour) in the adult honeybee. Since JH also appears to regulate adult oogenesis in other bee species, including bumblebees, the modication in this network in this bee lineage may have occurred after the divergence of bumble-bees and honeybees at about 6070 Ma and may not be a general mechanism for regulating division of labour in bees [32]. It is not yet known whether JH is more strongly associated with reproduction or division of labour in stingless bees, the other major group of highly eusocial bees [51]. However, in ants and wasps, there is also a similar variability in which JH is associated with reproduction in some species and with age polyethism in others [32]. Recent evidence suggests that JH regulatory pathways are involved in other forms of morphological polyphenism in solitary insects [52] and thus JH-regulated molecular pathways may represent a common target for the evolution of polyphenisms in insects. In addition to their role in regulating age polyethism in honeybees, reproductive pathways may have also been co-opted to regulate foraging preference in honeybees. Articial selection of lines of honeybees for high versus low levels of pollen hoarding revealed that high pollen-hoarding lines also had higher proportions of pollen foragers and an earlier transition to foraging, indicating that the processes regulating the transition to foraging and forager specialization are linked [53]. There is also a suite of correlated differences in sensory physiology and locomotion, which is also seen in typical unselected (wild-type) colonies in which pollen foragers are similar to bees from the selected high pollen-hoarding line, and nectar foragers are similar to bees from colonies selected for low pollen hoarding (reviewed in [29,30]). Both workers from high pollen-hoarding colonies and pollen foragers from unselected colonies have ovaries with more ovarioles and higher titres of Vg [54]. This association of foraging behaviour/preference and reproductive physiology is consistent with the reproductive ground plan hypothesis, which states that task specialization evolved through modication in molecular pathways regulating the reproductive cycle of solitary insects [26,42]. However, it will be important to study additional social species and to demonstrate that these traits are indeed correlated in solitary bee species. QTL mapping of the high and low selected pollenhoarding lines identied four major QTLs that have complex pleiotropic and epistatic interactions, and

and diapause in solitary insects [8,14,24,29,30]. We therefore review these lines of research briey below. The molecular basis for social foraging behaviour has been well-characterized, and has led to important insights into the evolution of this pathway. This system is also a good example of a social pathway (4), in that it appears that similar behaviours (foraging) are triggered by different cues in solitary versus social species. In solitary species that do not engage in brood care, foraging is required for personal consumption and triggered by hunger signals. In solitary species that engage in brood care, foraging is also used for offspring provisioning and presumably stimulated by cues from the brood. In honeybees, social foraging is regulated by the colonys nutrition needs (which includes signalling via pheromones produced by the brood [31]), and is carried out by forager bees who do not engage in brood care or reproduction. The ontogenic transition from nest activities to foraging is associated with modications in key physiological processes including changes in haemolymph titres of juvenile hormone ( JH), brain levels of the neuromodulator octopamine and metabolic traits such as lipid levels [17,32,33]. Similarly, there are genetic and physiological differences between foragers that specialize on nectar versus pollen, including differences in levels of JH and ovariole size [24,29]. One of the key questions in the evolution of division of labour in honeybees is whether the molecular factors that regulate social foraging are associated with food-gathering behaviour in solitary species. Candidate gene studies have revealed that some of the genes that are differentially expressed between nurse and forager honeybees are functionally conserved in solitary insects. For example, the foraging ( for) gene, which encodes protein kinase G and is upregulated in foragers, is associated with foraging behaviour in Drosophila, Caenorhabditis elegans, ants and mammals (reviewed in [34]). Additional genes involved in feeding-related pathways in solitary insects have been linked to social foraging, including malvolio [35], insulin-signalling genes [36] and carbohydrate metabolism genes [37]. Peptides that are involved in regulating food intake in solitary insects are also involved in social foraging in honeybees, but interestingly are not linked to ingestion [38]. In addition to these candidate gene studies, microarray comparisons of the brain expression patterns of nurses and foragers revealed signicant differences in expression levels of more than 3000 genes, many of which are associated with cell signalling, neuronal development and metabolism [39]. Importantly, large subsets of these genes are regulated by factors known to regulate the transition from nest activities to foraging, such as pheromones or hormone levels (e.g. [40,41]). Thus, these genes may be involved in regulating the transition to foragingrather than being differentially expressed as a result of foraging behaviourbut their function in food-seeking behaviour in solitary species is largely uncharacterized. Pathways regulating reproductive physiology appear to be another target for social evolution [27,28,42]. Some of the rst data leading to this idea were obtained in studies on the endocrine regulation of
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Review. Social pathways and bee societies account for much of the observed behavioural and physiological variation. Mapping these QTLs to the honeybee genome revealed that they encompassed a disproportionately high number of orthologues to genes involved in reproduction and insulin signalling in Drosophila, as well as the for gene and the receptor for tyramine, a biogenic amine that may play a role in regulating sensory responsiveness (reviewed in [24,30]). Two candidate genes, PDK1 and HR46, have subsequently been associated to ovarian development [55]. In solitary insects, the insulin/insulin-like signalling (IIS) pathway acts upstream of JH and ecdysteroid-mediated regulation of sensory tuning and reproductive physiology [56], and thus, the IIS pathway may play an important role in this system. Additional studies are necessary to test the expression pattern and function of each of the genes in these QTLs in relation to division of labour and reproduction. The hypothesis that ovarian physiology regulates division of labour was recently challenged, since no differences in task specialization were found between strains of honeybees that vary in reproduction capacity [57]. These ndings suggest that the links between ovariole number and foraging behaviour are not always detected or present. Moreover, in solitary bees that provision their brood, the networks regulating foraging and reproduction may need to operate simultaneously (and not separately at different phases of a reproductive cycle) because actively nesting females are foraging. Thus, further investigations on the generality of this model in highly social bees, as well as additional information on the physiology and molecular biology of reproduction in solitary bees, are needed to better understand the relationship between ovarian physiology and foraging behaviour. An additional hallmark of the division of labour in insect societies is the care of siblings (not offspring) by workers, which led to the theory that sib-care behaviour evolved from maternal behaviour [27,42], and thus these two processes may be regulated by similar pathways [28,58]. Studies with the primitively social paper wasp Polistes metricus support this hypothesis. Female paper wasps show several natural behavioural states that differ in the combination of brood care and reproductive behaviours. Foundresses and queens are reproductively active whereas gynes and workers are not. The pattern of brain gene expression in workers was more similar to that in foundresses, which show maternal care, than to that in queens and gynes, which do not. Genes of the IIS pathway were among the differentially regulated genes, consistent with the hypothesis that this pathway played a critical role in the evolution of eusociality in the hymenoptera [59]. Similarly, in honeybees, microarray analysis of the brains of queens, sterile workers and workers with activated ovaries revealed that genes associated with caste/reproductive differences signicantly overlapped with genes associated with nursing behaviour [60]. In many social insects, queens are not only highly fecund but also long lived. Workers, on the other hand, are typically sterile and relatively short lived. This relationship differs from most animals in which there is a trade-off between reproduction and longevity
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[61,62]. The IIS pathway is involved in the integration of ageing and fertility in vertebrates and invertebrates [63]. Recent studies support the notion that Vg and insulin signalling are also involved in the regulation of longevity in the honeybee [64,65], and queens and workers differ signicantly in the expression of genes associated with metabolism, oxidative pathways and immunity [60]. Corona et al. [64] hypothesized that insulin signalling plays a prominent role in the regulation of longevity in honeybees as in other insects, but the traditional relationship between nutrition and IIS is reversed in that high nutritional status inhibits the secretion of insulin-like peptide. Thus, well-fed queens have lower IIS signalling compared with workers.

4. SOCIAL MOLECULAR PATHWAYS The ndings reviewed above revealed a surprising degree of conservation between the molecular pathways regulating behaviour in solitary insects and social behaviour in social insects. But how are these pathways re-organized to produce social versus solitary behaviour? Social behaviour is elicited in response to stimuli from other individuals, and thus these conserved ground plan pathways must be in some way connected to the detection of social signals. We suggest that these ancient molecular pathways are encompassed by social pathways, in which there is a directional ow of information. The pathway starts with the detection of a social signal, subsequent processing and integration of the information conveyed by the signal, and ultimately resulting in a behavioural output (or the inhibition of a behaviour normally performed in a solitary species under certain contexts). We suggest that social pathways commonly include several relatively independent modules, which display strong connectivity within each module and relatively weak connectivity among modules, thereby enabling functional exibility. These modules could be under different selection pressures and thus serve as the building blocks for evolution [66 68]. How are the modules of a social pathway organized? At the higher, functional level, a social pathway would consist of a sensory module (e.g. pheromone receptor neurons) that detects the relevant social signal(s), sensory integration modules that process the information conveyed by the social signal (e.g. glomeruli in the antennal lobes), higher integration modules such as neuronal circuits and endocrine systems (e.g. a neuronal network in the mushroom bodies) and motor-controlling modules that regulate the execution of a socially relevant behaviour (e.g. brood care). These high-level functional modules contain nested lower level modules, including physiological and developmental modules [66 68]. Physiological modules (such as JH signalling pathways) regulate cellular, neuronal and endocrine processes, while developmental modules (such as pathways regulating worker body size or caste differentiation) regulate the form and function of the structures and networks necessary for the execution of social behaviour. Importantly, low-level modules are those in which genes (or proteins) interact to form gene networks and signalling pathways. These

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social signal b

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behavioural act Figure 2. A schematic of a social pathway. From a functional perspective, a social pathway includes sensory modules (S), sensory integration modules (SI), higher integration modules (I) and modules regulating effecter mechanisms such as motor control or exocrine glands (O). Each of these functional modules consisted of nested molecular modules (insert, note that specic genes can be involved in more than a single module). The molecular modules can be developmental or physiological (see text and box 1 for details). Light grey circles outline functional modules, arrows depict the ow of information, small circles within the insert depict individual genes (or proteins) and lines connecting the small circles depict activation (diamond end) or inhibition (line end) of gene (or protein) activity. The dotted line depicts a social signal directly activating integration modules, bypassing sensory integration (see text for details). a, a point in which two pathways converge; b, a point of bifurcation.

genes and gene networks may be conserved, and thus can be compared between species or individuals that differ in their social behaviour. It should be noted that regulatory gene networks and signalling pathways can be involved in several functional modules in the same way that hox genes and Delta Notch pathways are repeatedly used for the development of diverse morphological structures [66]. The term social pathway does not imply a single unidirectional route. Rather it may include bifurcations or points in which several pathways converge. For example, several sensory modules can converge into a single sensoryprocessing module, and a high-integration module can affect several downstream motor-controlling modules (gure 2). A similar complexity in connectivity can also occur among protein and gene network modules. What are the molecular mechanisms by which the connections between modules in a social pathway may be reorganized? New genes may have evolved (e.g. by gene duplication) that regulate or connect conserved modules; this seems likely in the case of sensory modules for olfactory signals, since there can be considerable diversity of olfactory receptor sequences even among related species [69]. Alternatively, expression patterns of existing genes may be altered by changes in protein structure/function or cis-regulatory regions, which
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allow these genes to be expressed in novel contexts allowing for the reorganization of connections between modules [7072]. Epigenetic regulation, which occurs without any changes in DNA sequence, can also alter expression patterns of a gene (or suite of genes) involved in regulating a particular module or forming connections between modules. Epigenetic regulation may be caused by modications to DNA or chromatin (the DNAprotein complex in chromosomes) structure, which alter the transcriptional potential of certain genes or chromosomal regions [73]. There is evidence that epigenetic regulation associated with DNA methylation is involved in caste determination, one of the key processes in the evolution of advanced insect societies [74,75]. DNA methylation has been shown to be involved in integrating social signals and result in long-term behavioural changes in rodents [76], but its function in regulating behaviour in social insects remains to be fully characterized [77]. The social pathway may be a useful conceptual framework for studying how an ancient ground plan pathway was modied to give rise to diverse forms of social behaviours. Comparative analyses of social pathways can be used for identifying parts of the pathways that diverge between species differing in their social behaviour. An effective study of social pathways therefore requires the availability of molecular and

Review. Social pathways and bee societies genomics resources for related insects differing in their social behaviour. The evolution of a certain social behaviour can be associated with modications in the interactions among modules or by changes within modules that change their function. One of the ways in which such a change can occur is by expressing a behaviour in a new (social) context [27,42]. From a social pathway perspective, this means that a sensory module that detects a social signal evolved to be linked to an ancient ground plan pathway. For example, social brood care could have evolved because a sensory module that detects brood signals has been connected to the ancient modular pathway that is associated with food seeking or processing in solitary insects, but the remainder of the pathways may remain mostly intact. Indeed, studies in honeybees and Drosophila have suggested that the for gene underlies food-related locomotion in both species, but it is regulated by factors associated with the division of labour in honeybees [78]. Similarly, the biogenic amine octopamine is implicated in feeding and food-related activity in several solitary insects [79,80] and in social foraging in honeybees [81]. Social pathways can be also modied such that a similar social signal evolved to regulate different social behaviours. We recently used the social pathway framework in a comparative study on the social behaviour of bumble-bees (Bombus terrestris) and honeybees (Apis mellifera). In honeybees, queen pheromone inhibits worker reproduction and slows down behavioural maturation of the worker bees; this latter process is probably modulated by pheromone-mediated regulation of JH levels [82,83]. In the bumble-bees, the presence of the queenwhich is communicated by behavioural and possibly chemical signals [84,85]inhibits worker reproduction via decreased JH levels, but was suggested to have no effect on division of labour [86,87]. In both species, the presence of the queen is associated with a decrease in JH biosynthesis rates [88,89], and therefore requires a pathway linking the sensory system detecting the queen signal with the regulation of the corpora allata, the endocrine glands that synthesize and release JH (gure 3). JH has pleiotropic effects and the pathways downstream of JH are predicted to at least partially diverge in order to accommodate the differential inuences on division of labour and reproduction in adult workers of the two species. We hypothesized that by comparing gene expression patterns across the two species, it should be possible to identify genes that are sensitive to the social environment versus genes that are directly linked to the downstream behavioural changes. We examined the expression patterns of a tranppel-homologue 1 (Kr-h1), to test scription factor, Kru this model. Kr-h1 expression is downregulated in honeybee workers upon exposure to queen pheromone, and upregulated in foragers [40,90]. In the bumblebee, Kr-h1 levels were similarly downregulated by the presence of the queen or the dominant worker. Kr-h1 levels were also associated with JH-mediated changes in reproductive state, but elevated levels were not associated with foraging behaviour [91]. Thus, it is possible that Kr-h1 is a part of a JH signalling pathway that in social bees is linked to social regulation, but was differentially co-opted during social evolution of
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Figure 3. A simplied scheme for a social pathway associated with juvenile hormone ( JH)-mediated regulation of social behaviour. The social inhibition of JH biosynthesis by the corpora allata is similar in bumble-bees and honeybees, but the inuence of JH on worker behaviour differs between the two species. Kruppel homologue 1 (Kr-h1) is a transcription factor that appears to be involved in the conserved part of the pathway. The arrows depict the ow of information, dotted arrows are hypothetical and the white boxes depict modules in which gene expression or activity can be studied.

bumble-bees and honeybees (gure 3). However, owing to the large pleiotropic effects of JH on insect physiology, further studies are necessary to determine whether the mechanisms by which JH and Kr-h1 interact are similar in the two species.

5. PHEROMONE-REGULATED SOCIAL PATHWAYS The studies reviewed above suggest that the molecular pathways involved in key physiological processes in solitary insects have been modied in social insects, such that they are activated by social signals and therefore expressed in a novel social context. In order for a molecular pathway to be expressed in a social context, it needs to be linked to sensory systems associated with social communication. Communication can occur via multiple modalities, but pheromonal communication is particularly widespread and sophisticated in insects (reviewed in [92,93]). The evolution of social pathways regulated by pheromonal communication

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G. Bloch & C. M. Grozinger Review. Social pathways and bee societies directly altering activity of a neuronal network rather than acting via receptors and processing in the olfactory system. Thus, based on the social pathways model, HVA may have evolved to bypass the sensory detection system and act directly on neuroprocessing pathways (gure 2, see also [106]). In order to understand how pheromone-regulated social pathways may be modulated, it is necessary to rst characterize the molecular responses to the pheromonal signal. Pheromones may cause changes in endocrine or neuroendocrine signalling, which affect downstream physiological processes regulating behaviour. Several studies have demonstrated that exposure of honeybee workers to pheromones causes changes in brain gene expression that are associated with downstream changes in behaviour [40,107,108]. For example, exposure of young honeybee workers to QMP causes signicant changes in global brain gene expression patterns. These changes are behaviourally relevant: upregulated expression of genes that are upregulated in the brains of nurse bees (relative to foragers), and downregulated expression of genes that are upregulated in the brains of forager bees. These expression changes are consistent with behavioural and physiological data demonstrating that QMP delays the transition from nursing to foraging activities [83]. There is evidence that the organization of pheromone-regulated social pathways in honeybees may be modied by social context and behavioural/physiological state. For example, unlike nurse bees, forager bees are not behaviourally or molecularly responsive to QMP, despite the fact that antennal responses to QMP are similar [90,109,110]. Worker bees in social groups will respond behaviourally and physiologically to alarm pheromone, but isolated bees do not, though antennal responses remain intact [111,112]. Thus, the sensory module of the social pathway is functional, but the modules downstream that are involved in the processing or behavioural output are disabled or disconnected. Studies with brood pheromone (BP) suggest that the connections between the modules of this social pathway may be labile. BP is produced by the developing larvae, and has different effects on nurses and foragers. BP delays the transition from nursing to foraging in young nurse bees [113], but it stimulates pollen-foraging behaviour in forager bees [31,114]. Exposure of young bees to BP shifts brain gene expression patterns to be more nurse-like, while exposure of older bees produces a more forager-like brain expression pattern [107]. Thus, BP has age- or task-dependent effects on both behaviour and gene expression. These results suggest that the sensory module is intact, while the connections to the downstream processing or behavioural modules have been modied. The above studies highlight the fact that pheromone-regulated social pathways can be modied by both social context and behavioural state. However, social pathways may also be the target of selective pressures, which result in population- or even species-level differences in the molecular response to social cues. For example, exposure of young honeybees to alarm pheromone (which stimulates defensive

requires the development of multiple pheromone (exocrine) glands, a sensory system that can decode and process multiple pheromonal signals, and the linkage of this sensory system, via processing modules, to downstream behavioural modules that regulate reproduction, brood care, nest construction, defence and/ or foraging. Pheromone detection can lead to neurophysiological changes that result in the production of a specic behaviour, or changes in sensory thresholds that result in altered behaviour under different contexts. Below we discuss the putative modules of pheromonally regulated social pathways, and highlight examples in honeybees in which the modules or the connections between the modules of these pathways appear to have been altered to produce a different behavioural response. There can be considerable variation in the production of pheromonal signals, which can affect the meaning of the chemical signal. For example, the reproductive state of the honeybee queen is associated with variation in the pheromones she produces; queens that have a higher reproductive potential appear to signal this to the workers [94,95]. Modulation of pheromone biosynthetic pathways is also associated with caste differentiation in honeybees. Queens produce a specic pheromone blend in their mandibular glands (ve components of which are termed queen mandibular pheromone, or QMP). Modications in these biosynthetic pathways, presumably via modulation of gene expression, result in the production of a different blend in workers. Furthermore, queenless and reproductive workers can produce queen-like pheromone blends, demonstrating that these changes in biosynthetic pathways are plastic and regulated by the social environment [96 98]. A pheromone-regulated social pathway requires the detection of the chemical signal by receptors in the peripheral chemosensory systems. Binding of the pheromones to specic odorant receptors results in the activation of receptor neurons, which subsequently modulates downstream neural networks. These neurophysiological changes in the central nervous system are thought to result in downstream behavioural or physiological changes (reviewed in [69,99]). Sequencing of the honeybee genome has identied numerous olfactory receptors [100], most of which do not have any known function, though one (AmOR11) was recently demonstrated to detect 9-ODA, the major component of queen pheromone [101]. There is also some evidence that pheromones can interact directly with neuronal networks in the central brain (dotted line in gure 2). In Drosophila, male accessory gland proteins that are transferred to the female during copulation pass from the reproductive tract into the circulation system and affect female behaviour, perhaps by interacting with receptors in the central nervous system (reviewed in [102]). In honeybees, one of the components of QMP, homovanillyl alcohol (HVA), has a similar structure to the biogenic amine dopamine and appears to interact directly with and activate dopamine receptors in the worker bee brain [103105]. Since worker bees do lick and consume queen pheromone as part of a behavioural response to the pheromone, it appears that this component may be
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Review. Social pathways and bee societies behaviour) has large-scale effects on brain gene expression that largely overlap with expression differences between Africanized and European bees, two races that are characterized by differences in aggression and response to alarm pheromone [108]. Thus, genotypic differences in behaviour may be associated with changes in expression patterns of modules associated with pheromone response. In re ants, interactions between queens and workers differ between two genetic morphs, and this may also be related to changes in social pathways. Fire ants exhibit a natural social polymorphism with monogynous (single queen) or polygynous (multiple queen) colonies. This polymorphism in social organization is associated with allelic variation in the gene encoding putative olfactory binding protein general protein-9 (Gp-9). Homozygous workers with the BB allele will only accept a single BB queen, while heterozygous workers will accept multiple Bb queens. This strong association between a putative odorant binding protein and a social phenotype is consistent with the hypothesis that changes in a pheromonal communication system affect social organization (reviewed in [115]), perhaps at the level of the sensory detection module. However, Gp-9 allelic variation is also associated with a suite of queen-related behavioural and morphological traits, and thus may result from the combined effect of several genes linked to the Gp-9 locus, and not from the Gp-9 gene alone [106]. In summary, the organization of pheromone communication systems is inherently modular, encompassing sensory detection, neural processing and downstream endocrine and behavioural changes. Within honeybees, there is evidence for plasticity in pheromone production and responses to pheromones. Responses to pheromones can be modulated by physiological, genotypic and social factors, and, in the examples highlighted above, appear to be due to changes in the processing or behavioural output modules. With the identication of genes associated with pheromone-mediated behavioural changes in honeybees, it now becomes feasible to bridge into other systems and determine whether these pathways are conserved. Queen regulation of worker reproduction is common across social insect species, making this an ideal system for studying the conservation or divergence of different modules in social pathways.

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6. TASK-RELATED PLASTICITY IN THE CIRCADIAN CLOCKWORK The circadian clock is an internal system that organizes the temporal physiology and behaviour of animals [116]. Socially regulated plasticity in circadian rhythms provides a good model for a social pathway because the circadian clock is one of the systems in which the relationships between genes and behaviour are best understood, and the molecular clockwork is a well-dened module. In addition, the circadian clock appears to play important roles in the temporal organization of insect societies, and thus was probably inuenced by the evolution of social behaviour. For example, the circadian clock is involved in social synchronization of the behaviour of individual bees, in
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plasticity in circadian rhythms of queens that is associated with their reproductive status and in task related plasticity in circadian rhythms of workers that appears to contribute to the division of labour and overall colony efciency (reviewed in [117]). Task-related plasticity in circadian rhythms was rst discovered in the honeybee, where division of labour relates to age. Young bees (less than two weeks of age) typically perform activities inside the constantly dark and homeostatically regulated hive with no circadian rhythms. Foragers, which are typically older (more than three weeks of age), have strong circadian rhythms with elevated activity during the day in which they forage for nectar and pollen, while at night they sleep inside the hive [118 121]. Differences in the exposure of nest bees and foragers to environmental factors such as light and temperature cannot account for this plasticity in circadian rhythms; the rhythms are strongly linked to the behavioural state of the bee. Nurses are active around-the-clock even when they experience a light dark illumination regime that is similar to that of the foragers [121,122]. In addition, an ontogeny of circadian rhythms similar to that seen in the hive is also evident in young bees that are housed individually or in small broodless groups in constant laboratory environment, consistent with the premise that the ontogeny of circadian rhythms is not regulated by changes in ambient illumination or temperature [123,124]. The social context has profound inuence on circadian rhythmicity in bees. Foragers switch back to activity with no circadian rhythms when induced to revert to brood care activity [125,126]; nurse bees switch to activity with robust circadian rhythms shortly after removal from the hive [122,127]. Given the association between brood care and plasticity in circadian rhythms, it is not surprising that the brood is the most potent social factor inuencing plasticity in the clockwork. Young honeybees that are conned to a broodless piece of comb or develop on empty combs in small cages do show robust circadian rhythms in clock gene expression, whereas full-sister bees of a similar age developing on a comb containing brood are active around the clock with attenuated molecular oscillations [127]. The molecular module generating circadian rhythms in animals is well characterized; it consists of interlocked autoregulatory transcriptional/translational feedback loops with positive and negative elements [128]. This organizational principle of the circadian clock is manifested in an approximately 24 h cycle in both mRNA and protein abundance of a group of canonical clock genes, and provides the means to assess the molecular clockwork by measuring clock gene expression over the course of a day. The molecular clockwork in the honeybee appears to be in some characteristics more similar to that of mammals than to Drosophila. The honeybee genome does not encode orthologues for two of the canonical clock genes in Drosophila, Timeless (Tim1), and the Drosophila-type Cryptochrome (Cry-d, also known as Cry1), but it does encode the mammalian-type Cryptochrome (Cry-m, also known as Cry2), which is not encoded by the Drosophila genome [122,129]. Taskrelated plasticity in circadian rhythms is associated

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brood signals sensory organ (receptors) integration and processing of sensory information

with plasticity in the molecular clockwork. Brain mRNA abundance of Per, Cry-m, Tim2 and Cycle show robust oscillations in foragers but not in nurses. Social manipulations uncoupling age and task indicated the temporal pattern of clock gene expression is linked more strongly to task than to age [122,126,130]. Taken together these studies suggest that the circadian system in social insects was coopted to organize individual behaviour such that it ts with the social environment. Comparative studies support this hypothesis: there is a similar task-related plasticity in behavioural and molecular rhythms in the bumble-bee B. terrestris in which division of labour is based primarily on size rather than age as in honeybees [21,131], and in ants in which age-related division of labour evolved independently of that in honeybees [132,133]. In the bumble-bees, large workers, that are more likely to perform foraging activity, emerge from the pupae with stronger circadian rhythms [21], and more cells expressing the putative clock neuropeptide pigmentdispersing factor (PDF) [134]. This latter nding is different from the honeybee in which there are a similar number of PDF immunoreactive cells in nurses and foragers [135]. Thus, in bumble-bees with a size-based division of labour, developmental factors that control body size also appear to inuence the structure of the circadian system. The plasticity in circadian rhythms is hypothesized to improve the division of labour and colony efciency because around-theclock activity by nurses facilitates continuous brood care, whereas foraging behaviour relies on the circadian clock for timing visits to owers and for time-compensated sun-compass navigation. This suggests that the evolution of sociality was associated with an increased plasticity in an integrative module, the circadian system, and with linking this module to sensory modules detecting signals from the brood. Little is known about the molecular pathways linking the clock to sensory modules and to downstream output pathways (gure 4). But how did the clock of the worker evolve for this plasticity? In animals, including humans, imposing around-the-clock activity is typically associated with increased pathologies and deterioration in performance [116]. As reviewed above, it is commonly assumed that nursing behaviour in workers evolved from maternal behaviour in the ancestors of social insects. This hypothesis predicts an association between reproductive state and circadian rhythms in bees with developed maternal care. There is no information on the activity rhythms of solitary mother bees, but there is evidence suggesting that female cockroaches with active ovaries show attenuated circadian rhythms [136], suggesting an interaction between reproductive physiology and the circadian system in this species. The association of maternal behaviour, reproductive state and circadian rhythms was investigated in the bumble-bee B. terrestris in which the mother queen cares for the rst developing batch of brood. This study shows that queens with brood are active around the clock, but queens of similar age for which the brood was removed switched to activity with strong circadian rhythms [137]. Queens with
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clock input pathways brood circadian network

motor controlling centre brood care behaviour

other behaviours Figure 4. A simplied scheme for a social pathway associated with task-related plasticity in circadian rhythms. Nurse bees that interact with the brood are active around the clock with no circadian rhythms in behaviour or brain clock gene expression. Nurse bees that do not interact with the brood switch to activity with strong behavioural and molecular circadian rhythms. The brood signal and the sensory system detecting and conveying this information to the circadian clock network are yet unknown. It is not known whether social signals inuence the circadian system via dedicated clock input pathways or indirectly by changing motor activity or arousal state which then act on the circadian system. Other details as in gure 3.

only eggs were also active with no circadian rhythms, making it difcult to determine whether around-theclock activity is associated with brood care behaviour, reproductive physiology or both. In ants [138 140], and honeybees [141,142], egg-laying queens that do little, if any, maternal care are also active around the clock, suggesting an association of circadian rhythmicity with reproductive physiology, but not with brood care in these species. Thus, pathways that regulate reproductive physiology, maternal behaviour or both may be involved in regulating plasticity in circadian rhythms. Little is known about pathways linking reproductive physiology and the circadian system. Manipulations of JH levels in honeybee workers or cockroaches did not affect their behavioural rhythms or the pattern of brain clock gene expression [136,143,144]. The involvement of insulin signalling has not yet been explored in bees but there is some evidence that these two systems interact. In Drosophila, elevated insulin signalling and FOXO mutations increase the susceptibility of the circadian clock to oxidative stress. FOXO mutations also advanced ageing-related attenuation of circadian rhythms [145]. In addition, nutrition and metabolism, which are regulated by insulin signalling, have diverse

Review. Social pathways and bee societies inuences on circadian rhythms (reviewed in [146]). The IIS pathway is also a good candidate to inuence size-related variation in the circadian system of bumble-bees because it plays a major role in the regulation of cell number and body size [147,148]. A better characterization of the environmental and neuroendocrine regulation of the circadian system in social and solitary bees is necessary for understanding the social pathways underlying plasticity in circadian rhythms and their relationships with pathways regulating reproduction and nutrition.

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7. CONCLUDING REMARKS The recent development of genomic and molecular tools has revolutionized the study of insect sociobiology. Thus far, many of the studies that we reviewed suggest that modications in core pathways, such as insulin signalling, the circadian system, reproductive physiology and hormone signalling, play key roles in the evolution of social behaviour. However, how these conserved molecular pathways are incorporated into a social pathwayand especially how they have been linked to social communication systems remains to be elucidated. One obvious limitation of our current state of knowledge is that most of the characterized modications were inferred from comparisons between the highly social honeybee and the solitary fruity D. melanogaster. Thus, it is difcult to separate changes that are genuinely associated with social evolution from those related to phylogenetic variation between bees and ies, given that these lineages are estimated to have diverged over 300 Ma [149]. With the anticipated sequencing of the genomes of several bee species in the near future (G. Robinson 2010, personal communication), and the increasing accessibility of genomic technologies, it will become possible to conduct more informative comparative genomics analyses involving closely related solitary and eusocial species. Key bee species for socio-comparative genomics include halictid bees, a family of bees showing all levels of sociality, and Euglossine (orchid) bees, bumble-bees and stingless bees, which are closely related to honeybees. Another key challenge is to determine whether there are specic elements or modules of social pathways that serve as the main targets of social evolution. For example, it is possible that core pathways remain intact and highly conserved from solitary to eusocial species, while sensory modules regulating detection of a social signal are modied such that the behaviour is expressed in a social context. Are there specic hot spots that are a common target for the modications associated with social evolution? It is also important to determine whether social pathways are indeed modular, and whether these modules are subject to different selection pressures. Alternatively, selection may operate by targeting highly pleiotropic genes that may act as prime movers for evolutionary changes. For example, many of the studies that we reviewed above suggest that modications in insulinsignalling pathways were important in the evolution of social behaviour in bees (e.g. longevity, reproduction and feeding behaviour). Does this emphasis of
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IIS pathways represent the current state of knowledge and research focus, or did the evolution of sociality in bees indeed involve only a few modications in key pathways, such as insulin signalling, which act upstream of diverse processes involved in social behaviours? In order to answer these questions, it will be necessary to develop genomic resources in a range of species, from solitary to social, and to characterize the genes and pathways regulating similar behavioural processes. With the recent genomic sequencing of Nasonia [150], ants [151], transcriptome sequencing of P metricus [59] and Polistes dominulus (Toth & . Grozinger 2011, unpublished data) and ongoing sequencing projects for a number of other bee species, it will soon be possible to address these questions.
We would like to thank Abraham Hefetz and members of the Bloch and Grozinger laboratories for helpful discussions and critical reading of the manuscript. We thank two anonymous reviewers for their useful comments on an earlier version of this manuscript. The Bloch laboratory has been supported by grants for the Israel Science Foundation (ISF), the USIsrael Bi-National Science Foundation (BSF), the National Institute for Psychobiology in Israel (NIPI) and the German Israeli Foundation (GIF). The Grozinger laboratory has been supported by funding from the National Science Foundation (NSF), US Department of Agriculture (USDA) and BSF.

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Phil. Trans. R. Soc. B (2011) 366, 21712180 doi:10.1098/rstb.2010.0327

Review

A comparative analysis of neural taste processing in animals

Gabriela de Brito Sanchez1,2,* and Martin Giurfa1,2
1 2

Centre de Recherches sur la Cognition Animale, Universite de Toulouse, 31062 Toulouse Cedex 9, France Centre National de la Recherche Scientique (CNRS), Centre de Recherches sur la Cognition Animale, 31062 Toulouse Cedex 9, France

Understanding taste processing in the nervous system is a fundamental challenge of modern neuroscience. Recent research on the neural bases of taste coding in invertebrates and vertebrates allows discussion of whether labelled-line or across-bre pattern encoding applies to taste perception. While the former posits that each gustatory receptor responds to one stimulus or a very limited range of stimuli and sends a direct line to the central nervous system to communicate taste information, the latter postulates that each gustatory receptor responds to a wider range of stimuli so that the entire population of taste-responsive neurons participates in the taste code. Tastes are represented in the brain of the fruity and of the rat by spatial patterns of neural activity containing both distinct and overlapping regions, which are in accord with both labelled-line and acrossbre pattern processing of taste, respectively. In both animal models, taste representations seem to relate to the hedonic value of the tastant (e.g. palatable versus non-palatable). Thus, although the labelled-line hypothesis can account for peripheral taste processing, central processing remains either unknown or differs from a pure labelled-line coding. The essential task for a neuroscience of taste is, therefore, to determine the connectivity of taste-processing circuits in central nervous systems. Such connectivity may determine coding strategies that differ signicantly from both the labelled-line and the across-bre pattern models. Keywords: taste; gustation; labelled-line; across-bre pattern; invertebrates; vertebrates

1. INTRODUCTION: BASIC MODELS OF TASTE ENCODING A concept that has guided much taste research is the notion that taste is organized in basic categories that correspond to neuron types narrowly tuned to a single stimulus quality [1,2]. This idea is the basis of the labelled-line theory, which posits that each gustatory receptor neuron is highly specic, responds to one stimulus or a very limited range of stimuli and sends a direct line to the central nervous system to communicate information about this (or those) particular taste(s) (gure 1). An alternative view is proposed by the across-bre pattern theory [3,4] in which individual gustatory receptor neurons are not exclusively labelled for a particular sensation but cooperate with other gustatory receptor neurons in the ensemble to provide a ngerprint or neural pattern for the taste. In this case, each gustatory receptor neuron is less specic and responds to a wider range of stimuli; the entire population of taste-responsive neurons participates in the taste code (gure 1). Labelled-line processing has the advantage of providing very precise knowledge about a limited number of tastes because each separate channel is dedicated to one

* Author for correspondence (debrito@cict.fr). One contribution of 10 to a Theme Issue Evolutionary developmental biology (evo-devo) and behaviour.

taste. On the other hand, it cannot code, given the natural constraints of neural systems (e.g. number of neurons), all possible tastes in the environment. Labelled-line processing is therefore a good system for detecting and recognizing a given stimulus with a crucial biological value for the animal, but not for general taste coding. Conversely, the combinatorial across-bre processing can code a much higher number of tastes with the same number of gustatory receptor neurons. Individual receptor neurons have broad, overlapping response patterns (i.e. they are broadly tuned) so that an individual bre is non-specic, but collectively, the pattern of activity across multiple receptor neurons is unique for a given stimulus. These two opposing views are well represented by Scott [5] and Yarmolinsky et al. [6], who defend labelled-line coding, and by Smith & St John [7], who defend across-bre pattern coding. It seems to us that considering important novel ndings on neural processing of taste both in vertebrates and invertebrates can enlighten this debate. In these considerations, a critical question is how perceptual taste sensations arise in the central nervous system [8], or what are the neural mechanisms allowing a given label to be assigned to a taste. In colour vision, for instance, explaining colour sensations on the pure basis of photoreceptor excitations is obviously senseless, as we know that the basis for colour sensations resides in the existence of colour opponent neurons [911], which impose a subtractive interaction
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taste A 3 4 taste B 2 3

gustatory receptor neurons

sense in the case of taste perception. In this framework, we will focus on peripheral (gustatory receptor neuronlevel) and central (central nervous system-level) taste encoding to analyse the strategies of taste encoding across various insect and mammalian species.

molecular receptive range

(b)
gustatory receptor neurons 1

taste A 2 3 4

taste B 2 3

molecular receptive range

Figure 1. Schematic of two theories of taste coding. A simplied gustatory system (without lateral connections) is presented, with ve different gustatory receptor neurons. (a) Labelled-line: each molecular receptor has a very limited molecular receptive range, i.e. it is activated by a single (or very few) taste(s). Two different tastes, A and B, are each detected by only one molecular receptor, which activates only one gustatory receptor neuron (in black). Differentiation between A and B does not need further processing, but only ve different tastes can be thus coded. (b) Acrossbre pattern: each molecular receptor has a much broader molecular receptive range, i.e. it can be activated by a range of different tastes. The ve different molecular receptors have differentbut broadreceptive ranges. In our example, taste A will activate several gustatory receptor neurons, although with different intensities depending on the receptor neuron. Receptor neuron 2 will be highly activated by taste A, but only slightly by taste B. Receptor neuron 3 shows the opposite response prole. Among the other gustatory receptor neurons, some will be equally activated by the two tastes (receptor 4), others will show a contrasted response (i.e. responding to A but not to B; gustatory receptor neuron 1), while others will not be activated at all by either taste (gustatory receptor neuron 5). This system allows the ne coding of many tastes, but differentiation among tastes needs additional downstream processing as the representation of each taste is contained in the combination of activations of the different neuronal units.

upon photoreceptor inputs and which are present both in vertebrates and invertebrates capable of colour vision [11,12]. Thus, it is not the receptor signal which is relevant, but the kind of interaction that is imposed upon such receptor input in the neural networks that process tastes upstream of the receptor level. The critical question is, therefore, which kind of processing (i.e. which kind of receptor interaction) is imposed to information coming from taste receptors at the central level. Answering this question may allow deciding whether or not the labelled line or the across-bre pattern hypothesis makes
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2. PERIPHERAL TASTE ENCODING (a) The case of insects The fruity Drosophila melanogaster is one of the organisms for which much information has been gained in the last years concerning the neural basis of taste [13,14]. For this insect, the notion of basic tastes prevails, based on the characterization of molecular gustatory receptors. Sixty-eight gustatory receptors (DmGrs, where DM stands for Drosophila melanogaster and Grs for the molecular taste receptors) encoded by 60 genes through alternative splicing have been identied in the fruity [1517]. These encode putative heptahelical 7-transmembrane proteins but it is not clear whether the resulting gustatory receptors signal through G-proteindependent second-messenger cascades or operate as ligand-gated ion channels. Recently, DmX, a gustatory receptor of the fruity tuned to detect a natural toxic molecule, L-canavanine, has been explicitly identied as a G-protein-coupled receptor [18]. Interestingly, this DmX receptor has partially diverged in its ligandbinding pocket from the metabotropic glutamate receptor family and is not related to the Gr family. The expression of the DmX receptor is required in bittersensitive gustatory receptor neurons, where it triggers the premature retraction of the proboscis, thus leading to the end of food searching and food aversion. Another interesting class of receptors has been recently discovered in the fruity, the ionotropic receptors (IRs) [19], which are expressed in appendages where gustatory receptor neurons, but also olfactory receptor neurons, are located. These receptors constitute a family of ionotropic glutamate receptors (iGluRs) which do not belong to the well-described kainate, aamino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or N-methyl-D-aspartate (NMDA) receptor classes of iGluRs, and which have divergent ligand-binding domains that lack their characteristic glutamateinteracting residues. IRs are expressed in a combinatorial fashion in sensory neurons that respond to many distinct odours but do not express either insect odorant receptors or gustatory receptors. It has been proposed that IRs constitute a novel family of chemosensory receptors [19], which would be involved in fast-odour signalling [20]. However, their role in gustation cannot be excluded, in particular, because iGluRs are involved in peripheral chemosensing of amino acids in bacteria [21]. Some of the fruitys gustatory receptors (Grs) have been linked to specic gustatory stimuli. For instance, Gr5a has been associated with sweet taste as it responds specically to trehalose and is expressed in most sugar-responsive gustatory receptor neurons [22 25]. Similarly, Gr64a is involved in the detection of other sugars including sucrose, glucose and maltose [26,27]. Gr66a, on the other hand, has been associated with bitter taste as it responds to caffeine and its mutation eliminates caffeine-avoidance behaviour [25,28]. Similar results (inability to respond to caffeine

Review. Taste processing in animals and to theophyline) were obtained upon mutations in Gr93a, which is co-expressed with Gr66a [29]. Flies also possess a taste for carbonated water. A population of neurons was identied which detects CO2 in water and mediates taste-acceptance behaviour [30]. Using neurogenetic methods available in Drosophila, it has been possible to determine that gustatory receptor neurons expressing Gr5a respond to a broad spectrum of sweet substances, while gustatory receptor neurons expressing Gr66a respond to a broad spectrum of bitter substances [25]. A genetically encoded calcium sensor, G-CaMP (see gure 2 legend), was expressed in gustatory receptor neurons of the fruity, whose activity was then measured upon stimulation of the proboscis by application of a wide-pore pipette lled with taste solutions. Thus, taste-induced activation of gustatory receptor neurons, which results in calcium increase, could be visualized in terms of uorescence changes. Besides trehalose, receptor neurons expressing Gr5a also respond to arabinose, fructose, galactose, glucose, maltose and sucrose (gure 2, green bars) while receptor neurons expressing Gr66a responds to caffeine but also to aristolochic acid, berberine, azadirachtin, limonin, lobeline, papaverine, quinine, quassin and denatonium benzoate (gure 2, red bars). Furthermore, other gustatory receptor neurons expressing different Grs (Gr32a and Gr47a) exhibit practically the same prole of responses to a variety of bitter substances as Gr66a receptor neurons. Given the structural differences between these compounds and the fact that receptor neurons with different Grs exhibit similar response proles to a broad spectrum of bitter substances, the idea that taste receptor neurons in the y are specically tuned to single molecules is doubtful. This may be owing to the fact that Gr5a cells and Gr66a receptor neurons coexpress other molecular receptors whose tuning may differ from Gr5a and Gr66a molecular receptors. For instance, the Gr5a molecular receptor, reported as a trehalose receptor [22,24], is coexpressed with another molecular receptor, Gr64f, which is broadly required for the detection of most sugars. Gr64f may also be coexpressed with Gr64a, which appears to be tuned to detect other sugars, such as sucrose, glucose and maltose [27]. Thus, combinations of Gr5a/Gr64f and Gr64a/Gr64f may enhance the spectrum of responsiveness to sugars of a single gustatory receptor neuron [31]. In speaking about labelled lines, what counts is not the specicity of a molecular receptor but the specicity of the message conveyed by a gustatory receptor neuron to the higher order gustatory centres in the central nervous system. In the periphery of the fruity, therefore, tastant detection seems to be organized according to labelled lines segregated, not in terms of single molecule specialization, but in terms of the hedonic value of the tastants (e.g. aversive versus appetitive). Gustatory receptor neurons tuned to respond to bitter substances mediate aversive behaviours, while gustatory receptor neurons tuned to respond to sweet substances mediate appetitive behaviours [25]. If the mammalian molecular receptor for capsaicin, TrpV1, is expressed in Gr66a receptor neurons, ies exhibit aversion towards
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capsaicin, while expressing it in Gr5a receptor neurons results in preference for capsaicin. Similar tendencies are found if an olfactory molecular receptor is expressed in these cells: the odorant becomes attractive if its molecular receptor is expressed in Gr5a receptor neurons, while it is rejected if its molecular receptor is expressed in Gr66a receptor neurons [32]. This organization may not be shared by all insects as different lifestyles may lead to dramatic modications of the gustatory repertoire. In the case of the honeybee, for instance, experiments performed with harnessed bees in the laboratory could not nd clear evidence for bitter taste perception until now [33]. Electrophysiological as well as behavioural analyses performed on several gustatory appendages have shown that bees in the laboratory are quite insensitive to bitter substances [34] and even consume important quantities of them despite their high concentration, toxicity and resulting mortality [35]. However, when tested in free-ight conditions, bees exhibit avoidance of highly concentrated bitter substances thus suggesting that gustatory thresholds may dramatically vary with the experimental situation and the possibility of expressing avoidance in an overt way [36]. Interestingly, the honeybee presents only 10 gustatory receptor genes, a nding that has been interpreted as an indication of a limited gustatory world [37]. None of these receptors share homologies with the bitter-tuned gustatory receptor gene Gr66a of the fruity. Although the ligands of the honeybee gustatory receptor neurons have not yet been identied, the dramatic difference existing between bees and ies in gustatory receptor genes underlines the necessity of comparative studies on insect gustation. Clearly, focusing on a single species, even if it allows using state-of-the art techniques for molecular receptor characterization at different levels, will not allow understanding per se the logic of taste perception. What happens if one follows the projections of gustatory receptor neurons to central taste-processing organs in the fruity? Marella et al. [25] traced the projections of gustatory receptor neurons to the suboesophageal ganglion (SOG), the rst relay in the central nervous system for the processing of taste information in insects. They found that different gustatory receptor neurons show segregated projections in the SOG, with neurons expressing Gr5a projecting lateral and anterior to projections of neurons expressing Gr66a. Marella et al. [25] concluded that there is a spatial activity map of different taste modalities in the y brain that corresponds to the anatomical projections of Gr5a and Gr66a receptor neurons. Imaging taste responses in the SOG by expression of an exogenous ligand-gated ion channel showed that activation of Gr5a receptor neurons drives acceptance, while that of Gr66a receptor neurons drive rejection behaviour [25]. Although the spatial segregation of projections of receptor neurons seems to support the labelled-line hypothesis, this is not necessarily the case: such a spatial segregation refers to the receptor neuron level but not to secondorder neurons.

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G. de Brito Sanchez & M. Giurfa Review. Taste processing in animals while expressing the same molecular receptor in gustatory receptor neurons responding to bitter substances results in aversion towards spiradoline [47].

(b) The case of mammals Although insects and mammals diverged from a common ancestor in the Cambrian, 550 Ma, common principles can be identied between these phyla in the organization of their gustatory system [6]. Studies on taste detection in rats have yielded the notion that tastant quality is mediated by labelled lines dened by distinct and strictly segregated populations of taste receptor cells. Such cells are endowed with either heteromeric G-coupled receptors assembled by combinatorial arrangement of T1R1, T1R2 and T1R3 subunits or T2R receptors [6]. T1R3 combines with T1R2 (T1R2 3) to form a sweet taste receptor that responds to all classes of sweet tastants. Thus, cells expressing T1R2 3 are the sweet-sensing receptor cells [38]. On the other hand, T1R1 and T1R3 molecular receptors combine to form a broadly tuned amino acid taste receptor at the basis of umami sensations. Thus, cells expressing T1R1 3 are umami-sensing cells. Bitter taste is mediated by T2R molecular receptors [38]. T2R genes are selectively expressed in subsets of gustatory receptor cells distinct from those containing sweet and umami receptors. A large number of T2Rs have been shown to function as bitter taste receptors in heterologous expression assays [38] and several have distinctive polymorphisms that are associated with signicant variations in sensitivity to selective bitter tastants in mice [39], chimpanzees [40] and humans [41]. Salt is detected by various mechanisms, one of which is mediated by the sodium channel ENaC [42], while the membrane-tethered carbonic anhydrase CA IV is required for carbonated taste [43]. Sour tastes are mediated by a member of the transient receptor potential ion-channel family, PKD2L1 [44]. This molecular receptor is selectively expressed in a population of gustatory receptor cells distinct from those mediating sweet, umami and bitter tastes. Genetic ablation of PKD2L1-expressing cells produced animals that lost sour taste [44]. These results indicate that the PKD2L1 ion channel is the candidate mechanism involved in the sour taste detection. Variations in the peripheral coding of these basic tastessweet, umami, bitter, salty and sourare present in some mammal species. Cats, for instance, carry a naturally occurring deletion in their T1R2 gene, which explains why Felidae do not respond to sweets [45]. In fact, the specicity of the sweet receptors differs among carnivore species that vary substantially in dietary habits. In the case of cats and dogs, for instance, difference in dietary habits is reected in the different functional states of the T1R2 molecular receptor for sweet taste. Dogs are omnivores and have a functional T1R2 receptor gene, whereas cats are obligate carnivores and do not have such a gene [45]. As in Drosophila, gustatory receptor neurons tuned to respond to bitter substances mediate aversive behaviours, while gustatory receptor neurons tuned to respond to sweet substances mediate appetitive behaviours. For instance, spiradoline is a synthetic opiate that is tasteless to mice; however, expressing a spiradoline molecular receptor in gustatory receptor neurons responding to sweet substances results in attraction to spiradoline [46]
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3. CENTRAL TASTE ENCODING: HEDONIC VALUE OF TASTANTS AS BASIC ENCODING CRITERION? In general, what remains unclear, except in a few cases (see below), is the kind of neural interaction that is imposed upon taste receptor input at the central level. Whether or not taste neurons are embedded in interactive ensembles (i.e. whether the information conveyed by taste neurons is processed by networks of interconnected neurons in which a single neuron rarely encodes taste characteristics) remains a discussed question in most animals apart from a few exceptions discussed below. Do neuronal responses to taste follow the same organizational principle as gustatory receptor neurons in the central nervous system? In mammals, many central gustatory neurons exhibit a strong response to a specic tastant but are also able to respond to other tastants. Both narrowly and broadly tuned gustatory neurons can be found at every stage of the mammalian central gustatory pathways, which suggests that labelled-line and across-bre pattern coexist as coding mechanisms [48]. Recent studies performed on central processing of taste in the rat have provided a clearer picture in which an organized form of acrossbre pattern seems to be present [49]. In mammals, the process of encoding chemical information into taste perception extends from the receptor level to the primary gustatory cortex and to other multimodal areas [17,50]. Imaging the gustatory cortex upon gustatory stimulation in rats showed that the four different tastes tested (salty, sour, sweet and bitter) are represented by specic spatial patterns containing both distinct and overlapping regions, which can account for both labelled-line and across-bre pattern processing of taste, respectively (gure 3). Quantifying the overlap between different taste representations allowed the emergence of two groups of stimuli, related to what can be dened as the appetitive (or hedonic) value of the stimulus itself. Higher overlap values were found between NaCl and sucrose, associated with good nutrients, or between quinine and citric acid, associated with noxious substances. This may suggest a possible additional level of spatial organization representing a tastes hedonic value: a pattern for attractive and a pattern for aversive stimuli [49]. In associated behavioural tests, it was shown that it was possible to associate the positive or negative hedonic value of a given taste with the cortical patterns elicited by each taste in the imaging experiments [49]. These imaging results suggest that the hedonic value of the tastes (palatable versus non-palatable) corresponds to a neural classication principle, far beyond a pure labelled-line strategy. Thus, these imaging studies [49] indicate that an across-bre pattern, evinced through overlapping of activation areas between tastes (see yellow areas in gure 3c), coexists with specic maps for each taste category in the gustatory cortex. Thus, while taste-specic maps would allow

Review. Taste processing in animals

%F/F 10 arabinose fructose sugars galactose glucose maltose sucrose trehalose aristolochic acid azadirachtin berberine caffeine denatonium bitter limonin lobeline papaverine quassin quinine KCl 1 M NaCl 10 mM NaCl misc lysine threonine valine H2O *** *** *** **** * *** *** *** *** * *** ** *** ** * ** *

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20

30

*** *** *** ***

***

Figure 2. Gr5a cells respond to a broad spectrum of sugars and Gr66a cells respond to a broad spectrum of bitter substances in the fruity Drosophila melanogaster. G-CaMP, a circularly permutated green-uorescent protein (GFP) linked to calmodulin (CaM) and a CaM-binding peptide, was expressed in taste neurons. Upon neural activation, CaM binds the peptide in the presence of calcium and promotes GFP uorescence, with uorescence increasing as a function of calcium concentration. Fluorescence changes (%DF/F ), which indicate changes in neural activity, are shown for Gr5a cells (green) and Gr66a cells (red) to 23 substances. Gr5a responses were compared with the Gr5a water response, and Gr66a with the Gr66a water response (Students t-test, ***p , 0.005, **p , 0.01, *p , 0.05). Ten brains were monitored for each stimulus/genotype. Error bars are s.e.m. Adapted from Marella et al. [25].

differentiation of tastes within a hedonic category, their grouping in a common region would label them with a hedonic value. Again, the kind of neural interaction leading to the emergence of such hedonic maps remains unclear. Even more unclear are the neural interactions underlying the experience-dependent reorganization of such taste maps in the rat gustatory cortex. Associating a sweet taste with a visceral malaise leads to a plastic rearrangement of its cortical representation, becoming more similar to a bitter and unpleasant taste representation [51]. Thus, an internal state of malaise induces a plastic reshaping in the gustatory cortex, which is translated into a behavioural shift of the stimulus hedonic value [51]. This cortical reshaping considerably challenges the labelled-line hypothesis as it is difcult to reconcile with a rigid linear transmission of taste information from the periphery to the central nervous system.
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It rather supports the notion that taste information is subjected to considerable processing that reshapes the original message in its way to higher order centres. Are taste-encoding principles similar in insects and mammals at the central level? In the larva of Drosophila, neurons expressing the gene hugin, which arborize in the SOG, are closely related with gustatory receptor neurons expressing Gr66a [52] and may act as second-order neurons for these receptor neurons [53]. While silencing Gr66a results in ies accepting bitter substances (see above), eliminating hugin lowers the threshold for initiating feeding. In other words, blocking hugin neurons would not inuence the choice of food but rather the decision to begin feeding on a certain food. Which kind of interaction exists between projection of neurons expressing Gr66a and hugin neurons in the SOG remains to be determined. Thus, characterizing the computation

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(a) sucrose quinine

10 4 2 0 NaCl citric acid

2 DR/R

(b)

NaCl

sucrose

citric acid

quinine

max

0 Rat # (c) NaCl Suc NaCl CA NaCl Quin

Suc CA

Suc Quin

CA Quin

500 m Figure 3. Different tastants induce different activation patterns in the rat gustatory cortex. In vivo intrinsic optical imaging of the rat gustatory cortex. Following gustatory stimulation of the rats oral cavity, intrinsic signals from the blood vessel pattern of the gustatory cortex are recorded by illuminating it with a 546 nm lter. These intrinsic signals are associated with an initial increase in the concentration of deoxyhaemoglobin. (a) Different examples of activation maps induced by sucrose and quinine stimulation (upper row) and NaCl and citric acid (lower row). While sucrose (dashed yellow line) and quinine (dashed red line) induce patterns with low overlapping (upper left panel), NaCl and citric acid exhibited more overlapping (lower left panel). Twenty-eight presentations of each stimulus are averaged. (b) Population maps for the four basic taste modalities, NaCl (n 18), sucrose (n 15), citric acid (n 8) and quinine (n 8). Imaging was done on a total of 27 animals, testing at least two tastants chosen randomly among the four. Colour scale max represents 60% of total number of animals. (c) Comparison of activated areas between tastants (Suc, sucrose; CA, citric acid; Quin, quinine). The yellow areas represent overlapping regions. Single stimulus surfaces are derived from (b) considering pixels responding with a consistency index of 20%. Adapted from Accolla et al. [49], by generous courtesy of A. Carleton.

processes performed by second-order neurons on receptor input is a priority that needs to be addressed to determine whether beyond peripheral taste detection, an across-bre pattern model or a spike timing model (in which the precise pattern of action potentials that communicate taste quality) accounts for central taste coding in insects. In that sense, electrophysiological studies performed in the desert locus Schistocerca migratoria [54,55] provide fundamental information as they report how tastes detected by gustatory receptor neurons on the hind legs are encoded by a population of interneurons of the methathoracic ganglion (MG). Previous studies on eshies Sarcophaga bullata [56]
Phil. Trans. R. Soc. B (2011)

and the locust Locusta migratoria [57] have reported single-neuron recordings performed either at the level of the SOG (eshies) or the MG (locust) but they did not perform a systematic testing of a broad spectrum of tastants so that the gustatory response proles of these neurons was unclear. On the contrary, Rogers & Newland [55] focused on spiking interneurons located in the midline of the MG and analysed their responses upon stimulation of gustatory receptor neurons of the locust hind leg with various tastants. These gustatory receptor neurons send their afferents to the MG and contact the spiking interneurons of the midline of the MG [54]. These interneurons responded differently to various tastants

Review. Taste processing in animals such as NaCl, water, sucrose and nicotine hydrogen tartrate (NHT), thus showing that there is convergence of a large number of taste qualities onto the same interneurons [55]. Furthermore, the response durations of these interneurons are a function of chemical identity and concentration. Thus, at rst sight, spiking interneurons of the MG are broadly tuned to different chemical stimuli in a manner consistent with across-bre pattern coding. However, contrary to assumptions of the across-bre pattern theory, spiking interneurons of the locust MG showed all the same response prole instead of exhibiting broad but different, overlapping response proles (gure 1). Indeed, the seven interneurons recorded responded highly to the deterrent substances NHT and NaCl at a high concentration (250 mM), while showing a low response to attractive sucrose and water. Rogers & Newland [55] afrm that this response prole is inconsistent with a system that is concerned with establishing chemical identity because, for instance, water could not be distinguished from sucrose or NHT from NaCl. They propose instead that the duration of response to different chemicals provides a direct measure of aversiveness because the relative size of the neuronal response of spiking local interneurons and motor neurons correlates strongly with behavioural withdrawal responses. One could, in fact, push the argument further and state that what spiking interneurons in the MG of the locus encode is the hedonic value of the tastants perceived. Firstly, although all neurons recorded in the midline of the MG showed the same response prole, the number of neurons recorded upon stimulation with NaCl, water, sucrose and NHT was low so that further recordings could show different proles. Secondly, neurons in other regions of the MG may also show coincident response proles but different from those of the MG midline, responding more, for instance, to appetitive rather than to aversive substances. In any case, the idea of having a central hedonic coding is present in the work of Rogers & Newland [55] who stated that local circuits in the MG mediate motor responses that differentiate between acceptable and unacceptable, and a neural representation of this appears fully apparent at an early synaptic stage of chemosensory integration. Intracellular recordings from central gustatory neurons in the SOG of the moth Heliothis virescens combined with uorescent staining have recently revealed a large diversity of neurons responding with varying tuning breadth to sucrose, quinine, water and mechanosensory stimuli applied to the antennae, proboscis and right tarsus [58]. The integration of information across stimuli and appendages contradicts a simple labelled-line mechanism in the central nervous system for coding identity and location of taste stimuli. Instead, responses recorded suggest a population coding mechanism in which information is represented by distinct activity patterns in partly overlapping populations of SOG neurons [58]. With just one appetitive (sucrose 1 M) and one aversive stimulus (quinine hydrochloride 0.1 M) tested, it is difcult to determine whether or not a spatial form of hedonic coding can be found in the SOG of the moth
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H. virescens [58]. Further studies should analyse whether the moths SOG presents a hedonic spatial, organization.

4. A COMPARATIVE ANALYSIS OF TASTE PERCEPTION The fact that hedonics seems to be a guiding principle in the organization of the gustatory system raises the question of whether neural architectures allowing such encoding originate from a common ancestor or whether they represent cases of parallel evolution. So far, answering these questions is difcult given the lack of enough data allowing across-species comparisons. In that sense, even if model organisms such as the fruity and the rat paved the way of gustatory research, we should keep in mind that they offer a rather narrow view on the multiplicity of neural solutions that may underlie the neural processing of tastes. In that sense, focusing, either for commodity reasons or for pressures imposed by current scientic policies, on a couple of model species may be misleading as it might lead to loss of the evolutionary perspective necessary for interpreting the logics of a sensory system. In insects, for instance, the honeybee seems to differ dramatically from the fruity in its gustatory repertoire [33,37]. As mentioned above, the bee possesses fewer gustatory receptors (10 versus 68 in the fruity; [37]). Is it because bees have a lifestyle in which sucroseassociated taste of nectars is over-dimensioned so that other taste modalities have been lost? This hypothesis seems too simplistic: bees collect pollen and vegetal resins and these tasks could provide an adaptive framework for perceiving bitter substances and amino acids. Thus, the study of gustatory coding and perception should be enlarged to other species in order to answer questions on particularities and commonalities of functional principles and underlying neural architectures. What is nevertheless interesting is that, despite our limited dataset, insects and mammals seem to use a common principle for coding tastes at the central level: the segregation of tastes in terms of their hedonic value. This may be associated with the fact that organisms with different evolutionary histories are nevertheless confronted with the same problem: distinguishing palatable from non-palatable food items. This distinction is crucial for individual survival as non-palatable items are usually associated with toxicity and mortality. In that sense, taste encoding would follow a basic principle of the nervous system that is present across phyla, which is the capacity to encode in an unambiguous way the positive (appetitive) and negative (aversive) experiences. Dedicated neural systems exist to this end both in insects and mammals. In insects, for instance, appetitive reinforcements are signalled through the activity of octopaminergic neurons [5962] while aversive reinforcements are signalled through dopaminergic neurons [6166]. In mammals, dopaminergic activity underlies appetitive reward systems and dopamine neurons are said to encode the prediction error of rewarding outcomes [6771] but no specic punishment neurotransmitter system is

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G. de Brito Sanchez & M. Giurfa Review. Taste processing in animals provide the necessary framework for a complete understanding of the functioning of taste networks. Gustatory responses in the taste cortex of rats are dynamic. Indeed, chronic recordings in active, tasting rats uncovered slower dynamics but also oscillations [75] in the gustatory cortex, a region rife with inhibitory cross-talk based on GABAergic transmission [76]. These oscillations, however, are apparent only when rats are not engaged in taste processing. These and other results show that the emergence of taste sensations at the central level is complex and rather distant from a labelled-line coding strategy. In the case of insects, no study has determined so far how taste is encoded by higher order neurons at the level of the SOG. Single-cell recording combined with multi-electrode and imaging recording methods may allow progress in our understanding of central taste processing. We conclude that the labelled-line hypothesis can eventually account for peripheral processing of taste as sensory neurons can be tuned to basic tastes, but we argue that it is critical to unravel the neural processing of tastes occurring at the central level focusing on interactive neural population coding.
We thank four anonymous reviewers and Frederic Marion-Poll for helpful comments on previous versions of the manuscript, and Rinaldo Bertossa for careful editing of our work. We thank Alan Carleton for kindly providing gure 3 and the French National Research Agency (ANR; Project INSAVEL), the University Paul Sabatier (Project APIGENE) and the CNRS for support.

known. A critical question is if and how these reinforcement systems, positive and negative, interactwhen presentwith gustatory pathways to instruct gustatory processing circuits about the hedonic value of tastes. Such interaction, if any, would support the idea that the hedonics could grow out (at least in part) of the message conveyed by reinforcement neurons upon taste perception. In other words, a concomitant activation of a negative reinforcement neuron upon taste perception would result in a negative labelling of that taste. In the olfactory system of honeybees, this labelling has been clearly demonstrated. Any odour paired with the articial activation (through intracellular current injection via an electrode) of an octopaminergic, reinforcement neuron called VUMmx1 (ventral unpaired median neuron of the maxillar neuromere 1) acquires a positive value and is therefore learned as an appetitive stimulus ([72], see [73] for review). In the fruity, similar results have been found for aversive learning: optophysiological [65] or temperature-dependent [66] activation of a specic subset of dopaminergic neurons upon odour stimulation leads to the formation of odour-aversive memories and therefore to the avoidance of the odour that was learned as an aversive stimulus. Interactions between the gustatory pathways and reinforcement pathways have not been explored so far. Do these two pathways converge already at the peripheral level to facilitate fast hedonic classication of taste, which would be important for survival? Are they confused so that neurons tuned to respond to aversive tastes release a negative-reinforcement neurotransmitter to further signal the negative experience? Do these two systems share similar topologies at the central level? If yes, are there several classes of reinforcement (e.g. dopaminergic) neurons, among which particular classes would be dedicated to the gustatory system? Answering these and other questions, i.e. understanding how and where in the brain interactions between gustatory and reinforcement processing do occur, is therefore a research programme in which across-species comparisons should play a fundamental role.

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5. CONCLUSION The labelled-line design of the gustatory periphery remains uncontested, both for insects and mammals, as convincingly illustrated by the recent genetic and physiological data. Whether the labelled-line principle is carried on or not to higher taste centres remains unknown. On the one hand, it can be argued that a labelled-line organization of higher pathways cannot be ruled out a priori, given that basic taste qualities such as sweet, sour, bitter or salty are not only recognized by different receptor neurons, but represent clearly distinct perceptual entities, at least in humans. On the other hand, the facts that taste is encoded in broad hedonic categories and that even the hedonic value of a taste may change as a result of experience argue against the validity of the labelledline hypothesis when it comes to the central processing of taste. The study of interactive population coding in central areas related to taste processing has still a long way to go [74]. Characterizing such interactions will
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27 July 2011 volume 366

. number 1574 . pages 20552180

Evolutionary developmental biology (evo-devo) and behaviour

Papers of a Theme issue compiled and edited by Rinaldo C. Bertossa
Introduction
Morphology and behaviour: functional links in development and evolution R. C. Bertossa 2056

Articles
Evo-devo and accounting for Darwins endless forms P. M. Brakefield The levels of analysis revisited S. A. MacDougall-Shackleton Neural mechanisms underlying the evolvability of behaviour P. S. Katz Conservation of gene function in behaviour C. J. Reaume & M. B. Sokolowski Mapping behavioural evolution onto brain evolution: the strategic roles of conserved organization in individuals and species B. L. Finlay, F. Hinz & R. B. Darlington Evo-devo, deep homology and FoxP2: implications for the evolution of speech and language C. Scharff & J. Petri Evolution of time-keeping mechanisms: early emergence and adaptation to photoperiod R. A. Hut & D. G. M. Beersma Social molecular pathways and the evolution of bee societies G. Bloch & C. M. Grozinger A comparative analysis of neural taste processing in animals G. de Brito Sanchez & M. Giurfa 2069 2076 2086 2100

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