Major Grant Proposal, March 2011

Effect of elevational clines on local climate adaptation in D. melanogaster

Felicia King

PROPOSAL SUMMARY. The study of adaptation is at the heart of evolutionary biology. Nevertheless, after
over a century of rigorous empirical and theoretical developments, there still lacks a comprehensive view of the
mode and tempo of adaptive evolution.
The migration of the fruit fly species Drosophila melanogaster from tropical Africa to more temperate
climates is an excellent system for the study of adaptation. D. melanogaster has only recently colonized
temperate climates, yet displays a well characterized set of traits and behaviors that promote survival under the
stresses of winter.
Here I propose to research the adaptation of D. melanogaster to temperate climates by investigating
climate-adaptive traits and loci that are known to vary latitudinally along the East Coast of North America to
determine whether or not similar variation is present along elevational transects from the Central Valley up into
the Sierra Nevada through one growing season (Mar-Nov). I hypothesize that “Northernly” winter-adaptive
genes and phenotypes will vary linearly with elevation and through the season, being most prevalent at high
elevation and early in the growing season. Investigating whether or not such patterns exist will significantly
contribute to understanding the mode and tempo of adaptation in D. melanogaster and will fill fundamental gaps
in our knowledge of the ecology of the species.
The project presented in this proposal will form the foundation of my Honors Thesis in Ecology and
Evolutionary Biology at Stanford University.

A. OBJECTIVES. I propose to investigate adaptation of D. melanogaster to temperate climates in Central

California by looking at phenotypic and genetic adaptive variation (i) across elevation gradients, (ii) through the
growing season, or (iii) both.
Objective 1. Collect flies along elevation gradient through a complete growing season. Along with other
members of the Petrov Lab, I am working to establish collection sites within the Central Valley and Sierra
Nevada. Once per month, I will collect D. melanogaster from populations at five localities. Each locality will
be at a different elevation, associated with different average length growing and freeze-free seasons. The
collections will take place over an entire growing season, with a total of eight monthly collections, March-
November 2011. These collections across both space and time will provide the raw genetic material that will be

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subject to analysis in Objectives 2 and 3.
Objective 2. Assay phenotypes associated with temperate adaptation. I will assay descendants of the field
caught D. melanogaster to identify climate-adaptive phenotypic characteristics present within the populations.
To test my hypothesis that winter-adaptive phenotypes will be more prevalent in high elevations and early
season collections, I will measure known climate-adaptive traits including diapause incidence, thorax length,
development time, starvation resistance, cold tolerance, and fat content.
Once phenotypic frequencies are determined within each temporal sample, I will assess the extent to
which they vary across the elevational gradient. This cross-clinal analysis will be done for each temporal sample
individually to analyze climate-adaptive trait frequency variation across presumed elevational clines. This
course of study will make apparent any systematic phenotypic difference between clinal and/or temporal samples
that may imply the presence of selection.
Objective 3. Assay frequencies of known alleles underlying climate adaptation. I will perform pooled
sequencing of the collected D. melanogaster at loci linked with known winter-adaptive traits. Candidate loci
include: couch potato (cpo), a
major locus in determining
diapause phenotype in D.
melanogaster (Schmidt et al.
2008); timeless (tim), a
crucial circadian clock gene
that effects the incidence of
diapause (Tauber et al. 2007);
UDP-glucose pyrophosphorylase gene (UGPase) and Alcohol dehydrogenase (Adh), both important metabolic
enzymes (Aquardo et al. 1986; Sezgin et al. 2004); and Insulin-like Receptor gene (InR), which mediates the
determination of life history traits in D. melanogaster (Paaby et al. 2010). Using pyrosequencing DNA
sequencing, I will assay for allele frequency at cpo, InR, UDPase, Adh, and tim. I will use these data to test the
seasonal phase cline hypothesis that predicts allele frequency clines by changes in selection coefficients through
the growing season.

B. BACKGROUND AND SIGNIFICANCE.

B1. Adaptation to temperate environments. D. melanogaster is a tropical fruit fly that in the wake of human

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migration over the last 10,000 years has colonized much of the world (David and Capy 1988). As these flies
expanded their range into novel environments, they experienced new environmental pressures that likely resulted
in adaptation. In particular, D. melanogaster have colonized more temperate climates and have evolved in
response to the principal manifestation of temperate climates, cold winters. There are multiple lines of evidence
that support the idea that D. melanogaster has adapted to the harsh winters of temperate climates.
One strong line of evidence comes from the study of clinal variation in allele frequencies. Populations
of D. melanogaster along latitudinal gradients in several continents show robust clinal changes in allele
frequencies at a number of loci (Flowers et al. 2007; Sezgin at al. 2004). Although these clines could, in
principle, have been produced solely by stochastic, demographic processes associated with the migration of D.
melanogaster, a number of observations argue strongly against this purely demographic model and in favor of
climate adaptation: Clines in allele frequency at many loci are repeatable across several countries (Gonzalez et
al. 2010; Knibb 1988; Turner et al. 2008). Single-nucleotide polymorphisms (SNPs) close to clinally varying
loci generally do not show concordant shifts as would be expected from a purely demographic model, but instead
show panmixia, implying an adaptive model (Berry et al. 1993; Gockel et al. 2001). A recent study of a set of
putatively climate-adaptive transposable elements (TEs) found these TEs do vary in a clinal pattern in North
America and Australia, while a set of putatively neutral TEs does not (Gonzalez et al. 2010). Lastly, some of the
observed clines have shifted over the last 20 years in a pattern predicted by anthropogenic climate change
(Umina 2005). Pulling from this evidence, it is very unlikely that demography alone has produced all or even
most of the clines in allele frequencies; it consequently seems likely that natural selection has played a role in
generating this clinal variation.
Two specific models describing how natural selection could generate clinal variation have been
proposed. The classic model by Haldane (1948), often referenced as the “migration-selection balance” (MiSB)
model, posits that clines are consequence of the joint effects of migration between neighboring demes and
selection coefficients that vary linearly along the cline or vary strongly at the ends of the cline. Alternatively,
clines could be explained as a byproduct of selection coefficients that vary temporally over the course of a
season (Rhomberg et al. 1989). In this scenario, referred to as the “seasonal phase cline” (SPC), one allele is
favored in the winter, but suffers a fitness disadvantage in the summer. By this logic, the winter-favored allele
will be present in relatively high frequencies at the beginning of spring, but will decrease through the summer
months in proportion to the length of the growing season.
It has been proposed that due to the short growing season at higher latitudes, the winter-favored allele

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will not decrease in frequency as substantially in populations closer to the Equator (Rohmberg et al. 1989).
And, in a complementary fashion, due to shorter, milder winters felt by the populations at lower latitudes, the
winter-favored allele will not rise to the same high frequency it does at higher latitudes. By this logic, a similar
scenario plays out across elevational clines: the short growing season at high elevations and the shorter, milder
winters at low elevations predict analogous allelic frequency patterns.
The seasonal phase cline model predicts that estimates of allele frequency should vary through the
growing season in a predictable way, whereas the MiSB model predicts that allele frequencies will be relatively
constant through time once populations reach equilibrium. Thus, by assessing the dynamic change in allele
frequency through time, I will be able to test these alternative cline generating hypotheses. If alleles present at
higher frequency at high elevation are also at high frequency in the early part of the growing season and decrease
in frequency as the season progresses, then I will conclude that these clines are maintained by the SPC
mechanism.
B2. Diapause syndrome: an important phenotypic adaptation and example of seasonal phase cline. The
classic temperate climate adaptation in invertebrates is the expression of a diapause syndrome (Tauber et al.
1986) that is cued by low temperatures and reduced photoperiod. Reproductive diapause was first described in
D. melanogaster by Saunders et al. (1989) and has been further characterized both phenotypically (Saunders
1990; Saunders et al. 1989; Schmidt et al. 2006; Schmidt et al. 2008) and genetically (Schmidt et al. 2005;
Schmidt et al. 2005; Schmidt et al. 2008; Tauber et al. 2007; Williams et al. 1993;Williams et al. 2006). It has
been shown that diapause incidence varies predictably with latitude (Schmidt et al. 2005) and season (Schmidt et
al. 2006); these dynamics reflect fitness tradeoffs between high and low diapause propensity genotypes in
different environments. The genetic variance for diapause has pleiotropic effects on other life history traits
including lifespane, age-specific mortality rates, fecundity profiles, stress tolerance, lipid content, and
development time (Schmidt et al. 2005).
In Europe and Eastern North America the aptness of flies to enter diapause is greater at higher latitudes –
Schmidt et al. (2006) reported about 60% difference between Florida and Maine. Similarly, flies captured in the
spring and early summer are more likely capable of entering diapause than those captured later in the fall –
Schmidt et al. (2006) observed ̴40-50% difference between May and September for a Pennsylvania population.
The presence of this phenotypic variation through space and time suggests that the ability to enter diapause
comes at a cost. In fact, while flies able to enter diapause are better suited to survive winter, they are less
productive compared to flies unable to enter diapause during summer months due to increased development time

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and reduced fecundity (Schmidt et al. 2005; Schmidt et al. 2005). Therefore, diapausing individuals suffer from
reduced relative fitness during the summer and their frequency declines over the season. Several generations
later, when winter sets in, the diapausing individuals are again more fit than their non-diapausing cohorts and
increase in frequency.
The ability to enter diapause has been linked to polymorphisms in components of the circadian clock
(tim; Tauber et al. 2007) and neural signaling (cpo; Schmidt et al. 2008) pathways. As part of this project I will
be assaying for the presence of the diapause phenotype and linked polymorphism frequencies to test my
hypothesis that Northernly alleles of the East Coast are present at higher frequencies in populations from higher
elevations and early in the growing season.
B3. History of D. melanogaster populations in the United States. D. melanogaster is well known as a model
organism is biology, but there is a surprising gap of information regarding its natural history. The species is a
known native of tropical Africa that colonized the New World alongside humans within the last several hundred
years. In the Eastern United States, populations exist across a relatively continuous environmental gradient from
Florida to Maine. For a tropical insect, temperate environments represent new habitats and the winter stresses
associated with them novel pressures; therefore, survivorship over the winter season may present a significant
challenge to these populations. (Schmidt et al. 2005)
Little is known about Drosophila populations in the Western United States. The project proposed here
will assess distribution, prevalence, and levels of genetic and phenotypic variability in western D. melanogaster
populations. Further, up to this point no one has investigated North American elevational clines in D.
melanogaster. Therefore, this project will help fill fundamental gaps in basic knowledge about the ecology of
this species.

C. METHODS.
C1. Objective 1: Field collection and establishment of
isofemale lines. I will collect flies at 5 field sites spread
along an elevational gradient from the Central Valley up
into the foothills of the Sierra Nevada range.
Collections will be made at orchards, fruit patches, and
vineyards spread along this gradient. At these locales,
adult D. melanogaster inhabit fermenting cracks and

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crevices in fruit; I will collect adults from these positions by aspiration or sweep netting. Once back in the lab,
individual females will be identified to the species level and used to establish “isofemale lines” for further
phenotypic and genetic analysis. I intend to collect ̴100 isofemale lines per locale, per month.
C2. Objective 2: Phenotypic Analysis. I will use the lab-reared F2 offspring of field caught females for
phenotypic analysis (the use of F2s removes potential confounding, maternal effects imposed by field caught
mothers). For each isofemale line, I will measure egg-adult development time; survival of eggs, pupae, and
adults to extreme heat and cold shock, and diapause incidence for 50 individuals per line. I will use standard,
well-developed phenotypic assays (Jensen et al. 2004; Schmidt et al. 2005) to measure these traits. To measure
triglyceride content, I will construct 5 subpools of 10 individuals per line and measure triglyceride content using
the Infinity Triglyceride Quantification Kit from ThermoScientific. For each phenotype, I will use linear models
to test if time measured as degree days since Dec. 1, location measured as altitude, or their interaction
significantly affect each phenotype.

Fig 3: (below) Phenotypic traits I will measure and citation of established methodology I will follow.

Trait Assayed individuals per Methodology

isofemale line, taken in F1

Egg-adult development time 50 Schmidt et al. 2005

Survival to extreme heat shock of eggs, 50 Jensen et al. 2004
pupae, and adults
Survival to extreme cold shock of eggs, 50 Jensen et al. 2004
pupae, and adults
Diapause incidence 50 Schmidt et al. 2005eastern united states
Triglyceride content 50 Infinity Triglyceride Quantification Kit

C3. Objective 3: Pyrosequencing. I will use pyrosequencing to estimate allele frequency at 5 genes known to
vary in a clinal fashion or known to strongly affect the phenotypes I will measure in Objective 2.
Pyrosequencing is a highly validated and efficient way to assess allele frequency in pooled samples (Cruber et al.
2002; Lavebratt et al. 2006). Allele frequency estimation by pyrosequencing works by measuring relative
heights of nucleotide peaks in a sequencing run; these heights are used to determine the proportion of DNA
molecules that contain each allele. To perform pyrosequencing, I will pool one individual per isofemale line per
locale per time point (100 individuals per sample) and extract its DNA. I will then use PCR to amplify a 100 bp
region surrounding the adaptive SNPs in each gene. These amplicon pools will be sent to the Stanford Protein

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and Nucleic Acid facility pyroseqencing service. To accurately estimate allele frequency from DNA pools, I
need to calibrate pyrosequencing reactions with pools of DNA with known 50:50 allele frequencies. To this end,
I will use the Drosophila Genetic Resource Panel, which is a population of isofemale lines from Raleigh, NC
each with individually resequenced genomes. I will identify strains that bear each allele, cross them to make
heterozygotes, PCR amplify the regions of interest and send them to the pyrosequencer along with my samples.
Similar to the analysis described in C2, I will use (generalized) linear models to test for changes in allele
frequency through time and space.

Fig 4: (below) Genotypic markers I will assay for and citation of established methodology I will follow.

Marker Assayed individuals per Methodology

isofemale line, taken in F2

couch potato (cpo) 50 Schmidt et al. 2008

timeless (tim) 50 Bradshaw and Holzapfel 2007; Tauber et
al. 2007
UDP-glucose pyrophosphorylase gene 50 Aquardo et al. 1986; Sezgin et al. 2004
(UGPase)
Insulin-like Receptor gene (InR) 50 Paaby et al. 2010
Alchol dehydrogenase (Adh) 50 Aquardo et al. 1986; Sezgin et al. 2004

D. RESOURCES AND PREPARATION. My great resource is the Petrov Lab. I joined the lab early Fall
Quarter and have been mentored by a postdoctoral fellow within the lab, Alan Bergland, whose research aims to
integrate the role of ecological variation with evolutionary processes through an understanding of the genetics,
physiology, and morphology that regulate life histories. Since the fall, I have met with Prof. Petrov quarterly
and Dr. Bergland weekly to discuss a course of investigation that has evolved into this proposed study. The lab
community brings together individuals from a wide variety of interests and expertise including genotypic and
phenotypic adaptation, purifying selection, and patterns of mutational processes. The lab group is an invaluable
resource and my interactions with them have helped hone my research questions and methodology.
Previous experience in the lab and the field have prepared me for undertaking this project. The summer
before entering Stanford I worked as a lab assistant at UCSC, gaining time-intensive experience with lab
protocol, DNA extraction, and qPCR. As a member of the Petrov Lab I've familiarized myself with the layout
and capacities of the lab, done DNA extractions, run PCR, and completed lab safety training. Previous field
experience includes over 3,000 hours work as a field assistant catching and processing frogs in nearby Elkhorn

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Slough, Monterey Bay, CA. As a senior in high school, I also designed and implemented a field-based project
studying trematode (a class of parasitic flat worms with cryptic life cycles) prevalence in fresh water snail
communities of California's Central Coast that involved collecting and dissecting several thousand snails from
over 20 pond sites.
Last quarter I took Ecology, which covered clinal adaptation and emphasized experimental methodology
in a field setting, and Molecular Biology, which explored the mechanisms of molecular evolution, focusing on
historical experimental methodology, and discussed DNA sequencing technologies. Next quarter I will take
Fundamentals of Molecular Evolution to become more familiar with key molecular evolutionary processes from
protein and DNA sequences.
This project will culminate in an Honors Thesis, required for my concentration in Ecology and
Evolutionary Biology within the Biology major. I plan to continue and expand on this project for the next two
years, ultimately producing a paper for publication. I also plan to present the results of this summer's work next
Fall at the Bay Area Population Genomics IV Conference and at the 2012 Evolution Society Meeting.

E. TIMELINE AND WORKFLOW CHART.

Tasks Week Week Week Week Week Week Week Week Week Week
1 2 3 4 6 7 8 9 10 11

Collections, identifying isofemale lines Temp Temp Temp

Sample Sample Sample
1 2 3

Extracting DNA from some progeny and set up new

generation
Pyrosequencing

Starting to assay for the F2 for phenotypes

Fig 5: (above) A magnified view of the first 11 weeks of the summer season field and lab work schedule that includes the
collection of 3 temporal samples and processing of 2. The five week cycle shown here repeats every month, with each new
field collection.

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 Tasks M A M J J A S O N D J F M A M J
2011 2012
Submit
proposal

Obtain
supplies and
prepare
Sampling
and
fieldwork
Pheno-
typing

Pyro-
sequencing

Data
analysis

Write paper

Fig 6: A visual depiction of the complete project timeline.

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