The Origin of Plastids

Plastids are core components of photosynthesis in plants and algae. Scientists are currently
debating the events leading to the appearance of plastids in eukaryotic cells.

Organelles, called plastids, are the main sites of photosynthesis in eukaryotic cells.
Chloroplasts, as well as any other pigment containing cytoplasmic organelles that enables
the harvesting and conversion of light and carbon dioxide into food and energy, are
plastids. Found mainly in eukaryotic cells, plastids can be grouped into two distinctive
types depending on their membrane structure: primary plastids and secondary plastids.
Primary plastids are found in most algae and plants, and secondary, more-complex plastids
are typically found in plankton, such as diatoms and dinoflagellates. Exploring the origin of
plastids is an exciting field of research because it enhances our understanding of the basis
of photosynthesis in green plants, our primary food source on planet Earth.

Primary Plastids and Endosymbiosis

Where did plastids originate? Their origin is explained by endosymbiosis, the act of a
unicellular heterotrophic protist engulfing a free-living photosynthetic cyanobacterium and
retaining it, instead of digesting it in the food vacuole (Margulis 1970; McFadden 2001;
Kutschera & Niklas 2005). The captured cell (the endosymbiont) was then reduced to a
functional organelle bound by two membranes, and was transmitted vertically to
subsequent generations. This unlikely set of events established the ancestral lineages of the
eukaryote supergroup "Plantae" (Cavalier-Smith 1998; Rodriguez-Expeleta et al. 2005;
Weber, Linka, & Bhattacharya 2006), which includes many photosynthetic algae and land
plants.

The idea of endosymbiosis was first proposed by Konstantin Mereschkowski, a prominent

Russian biologist, in 1905. He coined the term "symbiogenesis" when he observed the
symbiotic relationship between fungi and algae (Mereschkowski 1905). The term
"endosymbiosis" has a Greek origin (endo, meaning "within"; syn, meaning "with"; and
biosis, meaning "living"), and it refers to the phenomenon of an organism living within
another organism. In 1923, American biologist Ivan Wallin expanded on this theory when
he explained the origin of mitochondria in eukaryotes (Wallin 1923). However, not until
the 1960s did Lynn Margulis, as a young faculty member at Boston University, substantiate
the endosymbiotic hypothesis. Based on cytological, biochemical, and paleontological
evidence, she proposed that endosymbiosis was the means by which mitochondria and
plastids originated in eukaryotes (Sagan 1967; Margulis 1970). In those days, the research
community viewed her unconventional idea with much skepticism, but her work was
eventually published in 1967 (Sagan 1967) after being rejected by fifteen scientific
journals! Today, endosymbiosis is a widely accepted hypothesis to explain the origin of
intracellular organelles.

Besides these original and bold ideas, what else have we learned? Since 1990 we have seen
rapid advancement in techniques in molecular biology and bioinformatics. Using molecular
phylogenetic approaches, numerous comparative studies have demonstrated the
cyanobacterial origin of genes encoded in the Plantae plastid and provide evidence for gene
transfer from the endosymbiont genome to the "host" nucleus (Bhattacharya & Medlin
1995; Delwiche 1999; Moreira, Le Guyader, & Phillippe 2000; McFadden 2001; Palmer
2003; Bhattacharya, Yoon, & Hackett 2004; Rodriguez-Ezpeleta et al. 2005; Reyes-Prieto,
Weber, & Bhattacharya 2007). These studies complement several independent lines of
evidence based on protein transport and the biochemistry of plastids (McFadden 2001;
Matsuzaki 2004; Weber, Linka, & Bhattacharya 2006; Reyes-Prieto & Bhattacharya 2007).
The establishment of primary plastids in eukaryotes is estimated to have occurred 1.5
billion years ago (Hedges 2004; Yoon et al. 2004; Blair, Shah, & Hedges 2005), but dating
such an ancient event based on molecular data remains controversial due to the limited
support provided by the fossil records (Douzer et al. 2004).

Whereas endosymbiosis involving a cyanobacterium explains the establishment of primary

plastids in Plantae, the story is more convoluted in other photosynthetic eukaryotes, which
harbor secondary plastids with more complex structures. The plastids found in Paulinella
chromatophora (a filose amoeba) are an exception to the rule. These organisms are derived
from a far more recent cyanobacterial primary endosymbiosis that occurred about 60
million years ago (Bhattacharya, Helmchen, & Melkonian 1995; Marin, Nowack, &
Meklonian 2005; Yoon et al. 2006). This plastid traces its origin to a cyanobacterial donor
of the Prochlorococcus-Synechococcus type (Yoon et al. 2006). The closely related
Paulinella ovalis, although lacking a plastid, is an active predator of cyanobacteria that are
commonly localized within food vacuoles (Johnson, Hargraves, & Sieburth 2005).
Therefore, the cyanobacterium-derived plastid in the photosynthetic P. chromatophora
provides an independent example of the phagotrophic origin of a primary plastid.

Secondary Plastids and the Current Scientific Debate

Figure 1: The concept of endosymbiosis

The chromalveolate hypothesis can explain some endosymbiotic events in dinoflagellates.
In comparison to Plantae and P. chromatophora, the origin of plastids in other
photosynthetic eukaryotes is more complicated. These organisms possess secondary
plastids, which have one or two additional membranes surrounding the existing two
membranes of primary plastids. However, an endosymbiosis event involving a
cyanobacterium cannot explain the origin of three — and four — membrane-bound
plastids. Instead, these extra membranes likely formed due to secondary endosymbiosis,
when an existing Plantae cell containing a primary plastid was engulfed and reduced to a
plastid. There is, however, disagreement among scientists about the number of secondary
endosymbioses in eukaryotes. In general, there are opposing viewpoints regarding their
establishment, namely, the chromalveolate hypothesis (Cavalier-Smith 1999, 2003; Keeling
2009) and the independent acquisition hypothesis (also known as serial eukaryote-
eukaryote endosymbiosis, or the serial EEE) (Archibald 2009; Bodyl, Stiller, &
Mackiewicz 2009; Baurain et al. 2010) (Figure 1).

What is the chromalveolate hypothesis? This controversial idea proposes that a free-living
red algal cell was captured and retained by a nonphotosynthetic heterotrophic protist soon
after the evolutionary split of red and green algae (i.e., secondary endosymbiosis), giving
rise to the pigmented ancestor of the supergroup "Chromalveolata" (Cavalier-Smith 1999).
The red algal endosymbiont was retained in a variety of chromalveolate lineages such as
cryptophytes, haptophytes, stramenopiles, and dinoflagellates. This hypothesis is based on
numerous phylogenetic studies that support the red algal origin of the plastid in most
chromalveolates and of most plastid-localized proteins that are encoded in the nucleus of
these taxa (Fast et al. 2001; Harper & Keeling 2003; Bhattacharya, Yoon, & Hackett 2004;
Li et al. 2006; Nosenko et al. 2006). Although independent horizontal gene transfer events
could explain this observation, the more reasonable explanation is that a secondary
endosymbiosis event in red alga involved multiple gene transfers.

What other changes have occurred in organisms containing secondary plastids? With the
exception of the cryptophytes, which retain a remnant of the red algal cell (Greenwood
1974), other chromalveolates do not have the nucleus of the red algal endosymbiont, which
means that the genes necessary to ensure a fully functional plastid have been transferred to
the host nucleus (Douglas & Penny 2001; Douglas et al. 2001). In addition, for a subgroup
of dinoflagellates that contain the peridinin pigment, including species that cause "red tide"
in the ocean (e.g., Alexandrium), the plastid genomes are highly reduced in size due to
substantial gene transfer from the endosymbiont to the host nucleus (Hackett 2004).

To further complicate matters, besides tracing their origin not only to red algae as expected,
plastid-targeted proteins in many dinoflagellates traced also include proteins derived from
other unicellular eukaryotes such as excavates and other chromalveolates (Ishida & Green
2002; Hackett 2004; Yoon et al. 2005; Nosenko 2006), suggesting additional (tertiary)
endosymbioses in their evolutionary histories. The recent discovery of a substantial number
of red and green algal-derived genes in diatoms (Moustafa et al. 2009) supports the idea of
an additional secondary endosymbiotic event with green alga during the early evolution of
chromalveolates.

If the chromalveolate hypothesis is true, then why are not all chromalveolates (ciliates and
apicomplexans) photosynthetic? Under the assumption of the chromalveolate hypothesis,
the lack of plastids in ciliates is explained by subsequent loss of the captured algal cell or
by genes of the endosymbiont that were acquired during serial endosymbiosis (Reyes-
Prieto, Moustafa, & Bhattacharya 2008). Interestingly, the parasitic Apicomplexans (e.g.,
Toxoplasma), although nonphotosynthetic, possess unique organelles called the apicoplast
(also known as the nonphotosynthetic "plastids") that share similar features with secondary
plastids (Maréchal & Cesbron-Delauw 2001; Ralph et al. 2004). The apicoplasts may have
shared common origins with secondary plastids of the closely related dinoflagellates, but
subsequently lost their photosynthetic capability, likely due to the transition to obligate
parasitism (Funes et al. 2002; Waller & McFadden 2005).

Alternative Explanations for the Origin of Secondary

Plastids
Is there an alternative explanation for the origin of secondary plastids? Indeed, the
independent acquisition hypothesis suggests that the origin of secondary plastids in
different groups of chromalveolates (e.g., haptophytes, cryptophytes, and stramenopiles)
results from independent, serial endosymbioses involving unicellular eukaryotes, not
necessarily red algae (Archibald 2009; Bodyl, Stiller, & Mackiewicz 2009; Baurain et al.
2010). Experimental evidence for this hypothesis is not as strong as that for the
chromalveolate hypothesis, however. In fact, many recent analyses are tailored to disprove
the chromalveolate hypothesis using selective data sampling (Baurain et al. 2010).

What is the advantage of the independent acquisition hypothesis? We know that over
evolutionary time plastid-lacking chromalveolates are likely to lose the plastid and nuclear-
encoded plastid targeted proteins derived from the red algal endosymbiont. In evolutionary
biology, we assume that the least complex (most parsimonious) explanation for an
observation is more plausible. The independent acquisition hypothesis avoids a convoluted
explanation of organelle and gene losses to explain the sporadic plastid distribution
observed today in nonphotosynthetic chromalveolates. In the extreme case, some scientists
who support this hypothesis claim that the fundamental grouping of the chromalveolates is
itself inaccurate. Given the paucity of empirical data, we need to understand this complex
chain of plastid acquisition events much better.

What about the origin of plastids in other eukaryotes? There are other photosynthetic
eukaryotes that are not members of Plantae or Chromalveolata, such as the photosynthetic
excavates (e.g., Euglena) and the chlorarachniophyte amoebae (Rhizaria), but the plastid
origins of these organisms are less well-studied. Nonetheless a number of phylogenetic
analyses show that these organisms have acquired their plastids in more recent instances of
secondary endosymbiosis, during which a green alga was independently captured by their
common ancestors (Rogers et al. 2007).

Summary
There is no simple way to explain the gain and loss of plastids in all eukaryotes. The origin
of primary plastids via endosymbiosis involving a cyanobacterium is well-established, but
the origin of secondary plastids is still controversial. However, the chromalveolate
hypothesis (and the secondary endosymbiosis involving a red alga) is the best-supported
hypothesis to date based on numerous empirical studies. In addition, subsequent tertiary
endosymbioses involving other free-living eukaryotes explain plastid origins in other
eukaryote lineages. Additional studies and biochemical validation (where possible) are
needed to better test existing hypotheses about the evolutionary origins of plastids in
eukaryotes.

Mitochondria arose through a fateful endosymbiosis more than 1.45 billion years ago.
Many mitochondria make ATP without the help of oxygen.

What variety is there in mitochondria? Mitochondria occur in various forms across various
eukaryotic groups, yet considerations on the origin of mitochondria sometimes neglect this
understanding. Four main mitochondrial types can be distinguished on the basis of
functional criteria concerning how or whether ATP is produced. These functional types do
not correspond to natural groups, because they occur in an interleaved manner across the
tree of eukaryotic life. Instead they correspond to ecological specializations.

Mitochondria: A Ubiquitous and Diverse Family of

Organelles

The mitochondria typical of mammalian cells respire O2 during the process of pyruvate
breakdown and ATP synthesis, generating water and carbon dioxide as end products. The
Krebs cycle and the electron transport chain in the inner mitochondrial membrane enable
the cell to generate about 36 moles (mol) of ATP per mole of glucose, with the help of O2–
respiring mitochondria. Such typical mitochondria also occur in plants and various groups
of unicellular eukaryotes (protists) that, like mammals, are dependent on oxygen and
specialized to life in oxic environments.

In contrast, the mitochondria of many invertebrates (worms like Fasciola hepatica and
mollusks like Mytilus edulis being well–studied cases) do not use O2 as the terminal
acceptor during prolonged phases of the life cycle. These mitochondria allow the
anaerobically growing cell to glean about 5 mol of ATP per mole of glucose, as opposed to
about 36 with O2. The typical excreted end products are carbon dioxide, acetate, propionate,
and succinate, which are generated mostly through the rearrangement of Krebs cycle
reactions and the help of the mitochondrial electron transport chain. These organelles are
commonly called anaerobic mitochondria.

Figure 1: Enzymes and pathways found in various manifestations of mitochondria

Proteins sharing more sequence similarity to eubacterial than to archaebacterial
homologues are shaded blue; those with converse similarity pattern are shaded red; those
whose presence is based only on biochemical evidence are shaded grey; those lacking
clearly homologous counterparts in prokaryotes are shaded green. (A) Schematic summary
of salient biochemical functions in mitochondria, including some anaerobic forms.
(B)Schematic summary of salient biochemical functions in hydrogenosomes. (C) Schematic
summary of available findings for mitosomes and Eukaryotic evolution, changes and
challenges. Nature 440, 623–630 (2006)
Mitochondria of yet another kind yield even less ATP per molecule of glucose. These are
mitochondria of several distantly related unicellular eukaryotes (protists) that lack an
electron transport chain altogether. They synthesize ATP from pyruvate breakdown via
simple fermentations that typically involve the production of molecular hydrogen as a
major metabolic end product. These mitochondria are called hydrogenosomes and allow the
cell to gain about 4 mol of ATP per mole of glucose. Hydrogenosomes were discovered in
1973 in trichomonads, a group of unicellular eukaryotes. They were later found in
chytridiomycete fungi that inhabit the rumen of cattle, as well as some ciliates, and they
continue to be found in other groups. The enzymes of hydrogenosomes are not unique to
these anaerobes. They are found also in the mitochondria, the cytosol, or even the plastids
of other eukaryotes (Figure 1).

A fourth category of eukaryotes possesses small, inconspicuous mitochondria that are not
involved in ATP synthesis at all. These eukaryotes synthesize their ATP in the cytosol with
the help of enzymes that are otherwise typically found in hydrogenosomes. They obtain 2-4
mol of ATP per mole of glucose. Their typical end products are carbon dioxide, acetate,
and ethanol, and their mitochondria are called mitosomes. Mitosomes were discovered in
the human intestinal parasite Entamoeba histolytica in 1999, and were subsequently found
in many additional eukaryotes, including Giardia lamblia in 2003.

Knowledge about these different forms of mitochondria comes from decades of

biochemical and physiological investigations of eukaryotic anaerobes, many of which are
important pathogens or parasites of humans and livestock. Well into the 1990s it was
widely thought that several anaerobic eukaryotes, such as Giardia lamblia, lack
mitochondria altogether and had never possessed them in the evolutionary past. Newer
work, however, has shown that mitochondria are just as defining and ubiquitous among
eukaryotes as is the nucleus itself. That realization has had considerable impact on current
views about the origin of mitochondria.

The Endosymbiotic Origin of Mitochondria

There are currently two main, competing theories about the origin of mitochondria. They
differ with regard to their assumptions concerning the nature of the host, the physiological
capabilities of the mitochondrial endosymbiont, and the kinds of ecological interactions that
led to physical association of the two partners at the onset of symbiosis.

Figure 2: Models for eukaryote origins that are, in principle, testable with genome data
(A-D) Models that propose the origin of a nucleus-bearing but amitochondriate cell first,
followed by the acquisition of mitochondria in a eukaryotic host. (E-G) Models that
propose the origin of mitochondria in a prokaryotic host, followed by the acquisition of
eukaryotic-specific features. The relevant microbial players in each model are labelled.
Archaebacterial and eubacterial lipid membranes are indicated in red and blue, respectively.

The traditional view posits that the host that acquired the mitochondrion was an anaerobic
nucleus-bearing cell, a full-fledged eukaryote that was able to engulf the mitochondrion
actively via phagocytosis (Figure 2). This view is linked to the ideas that the mitochondrial
endosymbiont was an obligate aerobe, perhaps similar in physiology and lifestyle to
modern Rickettsia species; and that the initial benefit of the symbiosis might have been the
endosymbiont's ability to detoxify oxygen for the anaerobe host. Because this theory
presumes the host to have been a eukaryote already, it does not directly account for the
ubiquity of mitochondria. That is, it entails a corollary assumption (an add–on to the theory
that brings it into agreement with available observations) that all descendants of the initial
host lineage, except the one that acquired mitochondria, went extinct. The oxygen
detoxification aspect is problematic, because the forms of oxygen that are toxic to
anaerobes are reactive oxygen species (ROS) like the superoxide radical, O2-. In eukaryotes,
ROS are produced in mitochondria because of the interaction of O2 with the mitochondrial
electron transport chain. In that sense, mitochondria do not solve the ROS problem but
rather create it; hence, protection from O2 is an unlikely symbiotic benefit. This traditional
view also does not directly account for anaerobic mitochondria or hydrogenosomes, and
additional corollaries must be tacked on to explain why anaerobically functioning
mitochondria are found in so many different lineages and how they arose from oxygen-
dependent forebears.

An alternative theory posits that the host that acquired the mitochondrion was a prokaryote,
an archaebacterium outright. This view is linked to the idea that the ancestral
mitochondrion was a metabolically versatile, facultative anaerobe (able to live with or
without oxygen), perhaps similar in physiology and lifestyle to modern Rhodobacteriales.
The initial benefit of the symbiosis could have been the production of H2 by the
endosymbiont as a source of energy and electrons for the archaebacterial host, which is
posited to have been H2 dependent. This kind of physiological interaction (H2 transfer or
anaerobic syntrophy) is commonly observed in modern microbial communities. The
mechanism by which the endosymbiont came to reside within the host is unspecified in this
view, but in some known examples in nature prokaryotes live as endosymbionts within
other prokaryotes. In this view, various aerobic and anaerobic forms of mitochondria are
seen as independent, lineage-specific ecological specializations, all stemming from a
facultatively anaerobic ancestral state. Because it posits that eukaryotes evolved from the
mitochondrial endosymbiosis in a prokaryotic host, this theory directly accounts for the
ubiquity of mitochondria among all eukaryotic lineages.

Eukaryotes are genetic chimeras. They possess genes that they inherited vertically from
their archaebacterially related host. Genes for cytosolic ribosomes in eukaryotes, for
example, reflect that origin. But eukaryotes also possess genes that they inherited vertically
from the endosymbiont - for example, mitochondrially encoded genes for mitochondrial
ribosomes. But even the largest mitochondrial genomes possess only about sixty protein-
coding genes, while typical mitochondria harbor up to a thousand proteins or more that are
encoded in the nucleus. During the course of mitochondrial genesis, many genes were
transferred from the genome of the mitochondrial endosymbiont to the genome of the host.
This kind of endosymbiotic gene transfer is nothing unusual; endosymbiosis very often
entails gene transfers from the endosymbiont to the host. It happened during the origin of
plastids too, and it is still ongoing in our own genome: Mitochondrial DNA constantly
escapes from the organelle and becomes integrated as copies into nuclear DNA. The vast
majority of mitochondrial proteins are encoded by nuclear genes, and many of these are
endosymbiotic acquisitions from the mitochondrial ancestor.

When and How Often Did Mitochondria Arise?

Figure 3
The oldest undisputedly eukaryotic microfossils go back 1.45 billion years in the fossil
record. Given the coincidence of mitochondria with the eukaryotic state, this can also be
seen as a minimum age for mitochondria and a rough best-guess starting date for eukaryotic
evolution. According to newer geochemical views, this date of origin corresponds to a
protracted phase in Earth history when the oceans were mostly anoxic — from 1.8 billion
years ago until about 580 million years ago — because of the workings of marine, H2S-
producing bacteria. Eukaryotes thus arose and diversified in an environment where anoxia
was commonplace. Accordingly it is hardly surprising that many independent eukaryotic
lineages have preserved anaerobic energy-producing pathways in their mitochondria
(Figure 3).

Like eukaryotes themselves, mitochondria appear to have arisen only once in all of
evolution. The best evidence for the single origin of mitochondria comes from a conserved
set of clearly homologous and commonly inherited genes preserved in the mitochondrial
DNA across all known eukaryotic groups. In the case of hydrogenosomes (which usually
lack DNA) and mitosomes (which so far always lack DNA), the strongest evidence for their
common ancestry with mitochondria is twofold. First, aspects and components of the
mitochondrial protein import process are conserved in hydrogenosomes and mitosomes,
arguing strongly for common ancestry with mitochondria. Second, all known lineages of
eukaryotes that possess hydrogenosomes or mitosomes branch as sisters to mitochondrion-
bearing lineages.

Summary

Mitochondria arose once in evolution, and their origin entailed an endosymbiosis

accompanied by gene transfers from the endosymbiont to the host. Anaerobic mitochondria
pose a puzzle for traditional views on mitochondrial origins but fit nicely in newer theories
on mitochondrial evolution that were formulated specifically to take the common ancestry
of mitochondria and hydrogenosomes into account. The presence of mitochondria in the
eukaryote common ancestor continues to change the way we look at eukaryote origins, with
endosymbiosis playing a more central role in considerations on the matter now than it did
twenty years ago. The integral part that mitochondria play in many aspects of eukaryote
biology might well reflect their role in the origin of eukaryotes themselves.