The purpose of this forum is to introduce notable papers and books published by you and other persons. The work can be new or old, but it should be of wide interest and high quality. A brief comment on the significance of the work should be attached. The current categories of the subjects are (1) adaptation, (2) behavioral evolution, (3) dosage compensation, (4) evo-devo, (5) gene evolution, (6) genomic evolution, (7) molecular phylogeny, (8) natural selection, (9) phenotypic evolution, (10) sensory receptors, (11) sex chromosomes, (12) sex determination, (13) speciation, (14) symbiosis and evolution, and (15) horizontal gene transfer. However, new categories can be added if necessary. Emphasis will be given on the biological work rather than on the mathematical. Any person may post a paper by sending it to one of the editors listed below. We also welcome your comments on posted work, but we moderate all the comments to control spam. This forum is primarily for scientific discussion and to construct a database for good molecular evolution papers.

Tuesday, May 29, 2012

How Can There Be Orphan Genes?

Contributed by: Ken Weiss
Earlier discussions about genome evolution in this and other blogs coincided with my being reminded of a Trends in Genetics paper in 2009 by Khalturin et al. (1) on the subject of ‘orphan’ genes, and there have been two recent papers on this topic, Tautz et al. (2) and Ranz & Parsch (3) that seem worthy of comment here. Orphan genes are individual genes or small gene families that are sequestered within specific taxonomic groups, but have no known related genes outside the group.  The term is of course a misnomer, and could be highly misleading, because unless you’re a Creationist the DNA that’s a gene today had to be in some genome somewhere ancestrally.  But in what form and how did it arise? 
An important generalization at the core of modern molecular evolution is that evolution occurs by duplication events. The idea is that genes have from early days been so structured that they cannot arise just by random mutation of single nucleotides.  But duplication is clearly only part of the story: since every gene needs to be regulated, and regulatory elements are shorter, more fluid in number and location than the genes they regulate, they can arise more easily by mutation alone than whole genes can.  Thus even in modern theory a combination of duplication and ‘ordinary’ mutation is responsible for genome evolution.  But if genes themselves (and/or their exons) arise by duplication, that creates a family of related sequences.  So how can there be orphan genes, without a trace of relatives?
The above authors point out that in every taxonomic group so far studied up to 20% or even more of its genes have no recognizable homologues in other species.  Or, more properly, they are ‘Taxonomically Restricted Genes’ (TRG’s) with perhaps a small gene family that is, however, found only within a specific taxonomic clade.  Given the idea that genomes evolve by duplication, the prevalence of orphans needs some explanation, and these authors basically provide three.
First, the authors argue that the existing evidence suggests that the orphan genes fulfill some restricted taxon-specific adaptive needs (e.g., specific functional cell-types in cnidarians).  If that is the case, the relatives in collateral taxa must have lost their function, their trace erased by mutation or deletion not opposed by selection.  Khalturin et al. (1) suggest that “once a certain evolutionary time has elapsed” sequence similarity to the ancestral gene will be erased. 
Mutational erasure is clearly possible over time.  Marshall et al. (4) tried to quantify the idea in 1994, concluding that after about 10 million years, genes mutated into pseudogenhood could no longer be revived by mutation (but retain enough sequence to still be recognized as pseudogenes).
For this to be the case, we have to assume the ‘parental’ gene(s), that must have existed if genes at the time of the taxonomic split had previously been important when the new species branched off, but then were later removed by drift or selection from the descendants of the parental clade, while serving some strong, or new adaptive function in the new clade. Presumably the parental taxa didn’t include a large gene family related to the orphan, because if they did a large gene family would have been serving one—or many—important functions at the time and there would likely still be at least some of them around today. 
We know that gene families can persist in widespread branches of life, without sequence easily recognized by BLAST or other homology searches, based on work by Kazz Kawasaki (5) in my group for unusual kinds of proteins, such as the disordered proteins like those involved in biomineralization, in which the protein 3D structure is not as important for function as its ion-binding capacity.  At least one of the genes in the SCPP gene family (Amelogenin, responsible for capturing Ca+ ions in forming dental enamel crystals) was considered an orphan gene, until we identified its relatives (6).
Second, the orphan-paper authors acknowledge that BLAST searching and our genome data bases are imperfect, so that  relatives of some of these orphans may be eventually identified.  However, it seems unlikely that that will account for all of them, and there should be some gene-age consequences both for the adaptive function (something new in the clade, for example) and time for the ancestral genes to be erased.  Some evidence cited by Tautz (2) is ambiguous in regard to the estimated age and functions of orphans, so the picture is not wholly clear.  Is it more plausible that in so many cases the homologies just haven’t been recognized for some reason, including incomplete genomic data currently available?
Three, the explanation that is most interesting is that orphan genes really are orphans in the sense of having arisen de novo, without being copies of functional genes.  Tautz (2) and Ranz (3) both suggest that regulatory sequences might arise near to DNA that has enough of the structure of genes (start, stop, and splice sequences, proper coding exons, polyA addition sites) to be transcribed as well as translated, and serve some function that over-rode any possible toxicity a new protein might have in the cell.  Regulatory sequences usually involve many different TF-binding elements, so may have been put in these places by translocation events of such elements from other genes.  Or in examples cited, the new gene may be in an exon of an existing (and hence already regulated) gene.
Tautz (2) provide a step-by-step scenario for de novo gene creation.  These authors recognize the stretched plausibility of such ideas, given the seemingly miniscule probability that functional genes—with strongly advantageous effects—could arise this way.  This is certainly a challenge to Ohno!
One possibility mentioned is that the recent discovery that much or most of DNA, including non-coding DNA, is transcribed into RNA.  The cell obviously tolerates this RNA litter, which could make it more likely that occasionally such an RNA has translatable properties.  Of course, one might suggest that any such RNA sequences are actually the unrecognized fragmentary trash of long-dead genes.  If de novo creation were to happen often, most of the time selection would perhaps remove it.  But over millions of years maybe it happens enough.
Could it be that the history of discovery has misled us to become Ohno-ized?  We discovered interrupted genes, which led to theories of gene origin by exon duplication (and many genes have repeat exons with high duplication/deletion properties).  Then we discovered gene families, and this led to the obvious conclusion that duplication was ‘the’ mode of genome evolution.  We excepted enhancer evolution which can easily come and go by normal point mutation.  But this led to the discovery of the generality of gene families and focused attention on them, and the networks in which they participate, and the related but diverging functions they fill.
Could it be that instead, new genes often really do arise by de novo mechanisms, and disappear by deletion before they generate large gene families?  If they are old enough, they and their paralogs would be less taxonomically restricted than if they are recent.  After all if you had a phylogenetic dart board and randomly through darts, most would hit on some branch, not at the very top: their descendants would be ‘taxonomically restricted’.  So it’s the lack of collateral relatives rather than the taxonomic restriction that seems most curious to me.
Other authors have suggested that human orphan genes are often expressed in the brain, but that seems to me to be yet another kind of forced human exceptionalism, because most genes old or new are expressed in the brain.   Likewise, Khalturin (1) propose that taxon-specific genes “drive morphological specification,” as part of “rewiring” of the networks of regulatory genes.  But isn’t this always the case?  Except in some special circumstances, traits are usually affected by many interacting genes.  They seem to evolve by gradually diverging functions emerging from selection acting (again gradually) on the diversity made possible by the redundancy generated by gene duplication.  It seems rather unlikely that a newcomer of basically random structure could participate in such a network (or be properly expressed in a relevant tissue context) to experience strong positive selection.
Orphan genes may be simply be lucky genes in complex systems that happened to survive for us to observe them--different contributing genes, for different reasons including drift, surviving in different taxa.  The 20% of such genes that are orphans may just be the normal passengers on the train of duplication and deletion. 
If de novo evolution is common, or more common than we thought relative to gene duplication, we may have to revisit the strong evidence for the evolution of gene evolution by exon duplication and the proliferation of ancient gene families.  Have we missed something?
This could be a startling realization.  I’m sure many Mol. Evol. readers will know more about this than I do, and I’d like to see what you think.

1.  Khalturin, K, Hemmrich, G, Fraune, S, Augustin, R, and Bosch, TCG. More than just orphans: are taxonomically-restricted genes important in evolution?Trends in Genetics 25(9): 404-413, 2009.
2.  Tautz, D, and Domazet-Loso, T.  The evolutionary origin of orphan genes. Nature Reviews Genetics 12(10):692-702, 2011.
3.  Ranz, J, and Parsch, J.  Newly evolved genes: Moving from comparative genomics to functional studies in model systems.  BioEssays 34: 477-83, 2012.
4.  Marshall, CR, Raff, EC, and Raff, RA.  Dollo’s law and the death and resurrection of genes.  PNAS 91(25): 12283-7, 1994.
5.  Kawasaki, K, Buchanan, AV, and Weiss, KM. Biomineralization in humans: making the hard choices in life.  Ann. Rev Genet,43: 119-142, 2009.

Monday, May 21, 2012

Rodents with No Y Chromosome and No Sry Gene

In mammalian species sex is determined by the X and Y chromosomes. Individuals with two X chromosomes (XX) become female, and those with one X chromosome and one Y chromosome (XY) will be male. This occurs because the Y chromosome carries an Sry gene, which is a transcription factor and triggers the developmental pathway for testis formation. In the absence of the Sry gene, the developmental pathway for ovary formation is chosen as a default option.

Fig. 1. Evolution of sex chromosomes, SRY, and CBX2 in the genus Tokudaia. a Evolutionary events inferred from the present study (red) and previous studies (black) are shown in the phylogeny, together with the geographical distribution of Tokudaia species. Ma: million years ago. b Adult female of Okinawa spiny rat.  c Sub-adult male (2).

Fig. 2. Karyotypes of Amami and Tokunoshima spiny rats (3).

However, there are exceptional species in rodents. Two species of the genus Tokudaia living in small islands called Amami Oshima (T. osimensis) and Tokunoshima (T. tokunoshimensis) near Okinawa, Japan, have neither the Y chromosome nor the Sry gene (Fig. 1). Furthermore, both males and females of these species have only one X chromosome, so that their sex chromosome type is XO in both sexes. In addition, the number of autosomal chromosomes is different between the two species. T. osimensis (Amami spiny rat) has 24 autosomal chromosomes, whereas T. tokunoshimensis (Tokunoshima spiny rat) has 44 autosomal chromosomes (Fig. 2). The genus Tokudaia have three species, and the remaining one (T. muenninki) lives in Okinawa Island. This Okinawa spiny rat has the normal sex chromosome type XX/XY, and the number of autosomal chromosomes is 42.
Molecular phylogenetic analysis has suggested that Okinawa spiny rats diverged from the other two Tokudaia species about 2.5 million years ago and Amami and Tokunoshima spiny rats diverged about one million years ago. Therefore, the chromosome number has changed rapidly in these species by means of fusion, fission, inversion, deletion, translocation, etc. In fact, there is evidence that the chromosomes of Okinawa spiny rat have also experienced structural changes frequently. However, these chromosomal changes are not surprising because chromosomes can change quite often in small populations.
The surprising finding here is that the Sry gene has been lost and the male and the female are determined by the male and the female X chromosomes, respectively. Therefore, the gene contents of the male and the female X chromosomes must be different. In a recent paper Kuroiwa et al. (1) studied this problem by examining the copy number and chromosomal location of 10 genes (Artx, Cbx2, Dmrt1, Fgf9, NroB1, Nr5A1, Rspo1, Sox9, Wnt4, Wnt1) that are concerned with the differentiation of gonads into testis or ovary. The reason why they studied these genes is that duplicates of these genes are often used as signal proteins for sex determination in other organisms such as birds, frogs, and medaka fish. They then found that there are multiple copies of Cbx2 genes in the Tokudaia species and that there are two or more copies of Cbx2 genes in males than in females in both Amami and Tokunoshima spiny rats. Because the Cbx2 gene is known to repress ovarian development in mice and humans, they concluded that a larger number of Cbx2 genes in males is probably responsible for testis development. However, this hypothesis has not been confirmed by isolating and characterizing the Cbx2 genes.
There are two more rodent species which have the XO/XO sex chromosome type in both males and females. They are mole voles (4), and their effective population size again appears to be small. At present, the evolutionary change of the sex determination system in mammals is believed to be rare, but this may not be the case if we examine the sex chromosomes in species of small population size.
In reptiles, amphibians, and fish the sex determination system is known to change rapidly in the evolutionary process. Particularly in reptiles, there are the genetic sex determination (GSD) including both the XY and ZW systems and the temperature dependent sex determination (TSD), and these systems are interchangeable in the evolutionary process (5). It is also known that one species of frog, Rana rugosa, contains geographical races with the XY and ZW systems, and the XY ↔ ZW change appears to have occurred recently by an inversion event in the sex chromosomes (6).
In vertebrates Dmrt genes are believed to be responsible for imitating the developmental pathway for the formation of male and female phenotypes, but various signal proteins that trigger the function of Dmrt genes are used in different species (7). Dmrt-triggering genes are apparently subject to a rapid evolutionary change. How this evolutionary change occurs is not well understood. 

2. Murata C, Yamada F, Kawauchi N, Matsuda Y, and Kuroiwa A. 2012. The Y chromosome of the Okinawa spiny rat, Tokudaia muenninki, was rescued through fusion with an autosome. Chromosome Res 20:111-125.
4. Bagheri-Fam S, Sreenivasan R, Bernard P, Knower KC, Sekido R et al. 2012. Sox9 gene regulation and the loss of the XY/XX sex-determining mechanism in the mole vole Ellobius lutescens. Chromosome Res 20:191-199.
5. Sarre SD, Ezaz T, and Georges A. 2011. Transitions between sex-determining systems in reptiles and amphibians. Annu Rev Genomics Hum Genet 12:391-406.
6. Miura I, Ohtani H, and Ogata M. 2012. Independent degeneration of W and Y sex chromosomes in frog Rana rugosa. Chromosome Res 20:47-55.

Monday, May 14, 2012

Loss of Sweet Taste Genes in Carnivores

Contributed by: Naoko Takezaki

It is generally believed that most mammals perceive five basic taste qualities: sweet, umami (tasty), bitter, salty, and sour. The receptors for sweet, umami and bitter tastes are G protein-coupled receptors (GPCRs). Sweet taste is mediated largely by a heteromer of two closely related Tas1r (type 1 taste receptor) family GPCRs: Tas1r2 and Tas1r3. Tas1r1, another member of the Tas1r family, in combination with Tas1r3, forms an umami taste receptor. Tas1r receptors are class C GPCRs. Unlike sweet and umami tastes, bitter taste is mediated by Tas2r family GPCRs, which belong to class A GPCRs and are structurally unrelated to Tas1r family receptors. The genes encoding Tas2r receptors, the Tas2r genes, differ substantially in gene number and primary sequences among species, most likely reflecting the likelihood that these genes are required for detecting toxic or harmful substances in a species’ ecological niche.
Sweet taste Tas1r2 genes are pseudogenized in domestic cats which show indifference to sweet taste. Similarly, in other members (e.g., tiger and cheetah) of the cat family Tas1r2 genes are pseudogenized. In a recent study Jiang et al. (1) sequenced all exons of Tas1r2 genes from several carnivore species including obligate carnivores (e.g., domestic and wild cats), relatively omnivorous species (e.g., bears), and strict herbivores (e.g., giant panda). They found that loss-of-function mutations occurred in different positions of exons of Tas1r2 genes of obligate carnivores (Fig. 1), which are scattered on the phylogenetic tree (Fig. 2). This result indicates that pseudogenization of Tas1r2 genes resulting in loss of sweet taste occurred independently in several carnivore lineages in relatively short periods of time.

Fig. 1. Widespread pseudogenization of the sweet-taste receptor gene Tas1r2. The above diagram shows the positions of ORF-disrupting mutations in Tas1r2 from selected species of carnivores. The functional dog Tas1r2 gene structure is shown as a reference. The positions where ORF-disrupting mutations occurred are marked with a red asterisk (*).

Fig. 2. Evolutionary tree of Tas1r2 genes from 18 species of carnivores. Species with a pseudogenized Tas1r2 are marked with a diamond (red and gray depict species characterized in this study and the previous study, respectively).

Furthermore, Jiang et al. showed that in sea lion (Carnivora) and dolphin (Cetacea) which swallow whole food and have a reduced number of taste buds on tongue Tas1r1 and Tas1r3 genes are pseudogenized. They also failed to find intact Tas2r genes that encode bitter taste receptors in the dolphin genome. This result indicates the loss of umami taste as well as the loss of sweet taste in sea lion and dolphin and suggests the loss of bitter taste in the latter. Jiang et al. present the view that the loss of taste receptor genes is a consequence of dietary specialization in mammals (1).
            However, Zhao and Zhang (2) pointed out that (i) although Tas1r2 genes are pseudogenized only in obligate carnivores in Jiang et al. (1), ferret and Canadian otter, which are also obligate carnivores, still possess intact Tas1r2 genes and (ii) although three pinnipeds share the common ancestry of obligate meat-eating, none of the null mutations in Tas1r2 genes are shared by these species. Moreover, a previous study of Zhao and Zhang’s group (3) showed that (iii) Tas1r2 genes are pseudogenized in vampire bats which feed solely on blood containing carbohydrate and (iv) Tas1r2 genes are missing in herbivorous horse and omnivorous pig as well as in all available birds’ genome sequences irrespective of their diet. Tas1r1 genes that encode umami taste receptors are pseudogenized in all bats regardless of their diet (fruit, insect, blood) (4). In Zhao and Zhang’s view the relationship between the diet of species and the loss of function of taste receptor genes is quite complex and sometimes inconsistent.
Jiang et al. (5) responded to Zhao and Zhang (2) stating that (i) prefect correspondence between the exclusive meat-eating diet and the loss of taste receptor genes should not be expected because the loss of sweet taste receptor genes occurs stochastically and therefore is a time-dependent and ongoing process after diet switch, (ii) whether the common ancestor depended solely on meat-eating diet is not known, (iii) vampire bats unlikely require sweet taste because of the very small amount of carbohydrate in blood, and (iv) some studies showed the presence of Tas1r2 genes in horse and pig and these species show preference of sweet taste.
Although Jiang et al. (5) responded to Zhao and Zhang’s (2) questions, some questions remain unanswered. For example, why are no sweet taste receptor genes found in birds regardless of their diets?
Although the two groups of authors have different opinions on the relationship of the diet and loss of taste receptor genes, both of them recognize a need for better understanding of physiological mechanisms of taste and taste receptors to understand the relationship between diet and gene loss (1, 2).

The abstract of Jiang et al.’s paper (1) is as follows.

Mammalian sweet taste is primarily mediated by the type 1 taste receptor Tas1r2/Tas1r3, whereas Tas1r1/Tas1r3 act as the principal umami taste receptor. Bitter taste is mediated by a different group of G protein-coupled receptors, the Tas2rs, numbering 3 to ~ 66, depending on the species. We showed previously that the behavioral indifference of cats toward sweet-tasting compounds can be explained by the pseudogenization of the Tas1r2 gene, which encodes the Tas1r2 receptor. To examine the generality of this finding, we sequenced the entire coding region of Tas1r2 from 12 species in the order Carnivora. Seven of these nonfeline species, all of which are exclusive meat eaters, also have independently pseudogenized Tas1r2 caused by ORF-disrupting mutations. Fittingly, the purifying selection pressure is markedly relaxed in these species with a pseudogenized Tas1r2. In behavioral tests, the Asian otter (defective Tas1r2) showed no preference for sweet compounds, but the spectacled bear (intact Tas1r2) did. In addition to the inactivation of Tas1r2, we found that sea lion Tas1r1 and Tas1r3 are also pseudogenized, consistent with their unique feeding behavior, which entails swallowing food whole without chewing. The extensive loss of Tas1r receptor function is not restricted to the sea lion: the bottlenose dolphin, which evolved independently from the sea lion but displays similar feeding behavior, also has all three Tas1rs inactivated, and may also lack functional bitter receptors. These data provide strong support for the view that loss of taste receptor function in mammals is widespread and directly related to feeding specializations.


1. Jiang P, Josue J, Li X, Galser D, Li W, Brand JG, Margolskee RF, Reed DR, Beauchamp GK. 2012. Major taste loss in carnivorous mammals. Proc. Natl. Acad. Sci. USA. 109:4956-4961.
2. Zhao H, Zhang J. 2012. Mismatches between feeding ecology and taste receptor evolution: An inconvenient truth. Proc. Natl. Acad. Sci.USA, doi: 10.1073/pnas.1205205109.
3. Zhao H, Zhou Y, Pinto CM, Charles-Dominique P, Galindo-Gonzalez J, Zhang S, Zhang J. 2010. Evolution of the sweet taste receptor gene Tas1r2 in bats. Mol. Biol. Evol. 27; 2642-2650.
4. Zhao H, Xu D, Zhang S, Zhang J. 2012. Genomic and genetic evidence for the loss of umami taste in bats. Genome Biol. Evol. 4:73-79.
5. Jiang P, Josue J, Li X, Galser D, Li W, Brand JG, Margolskee RF, Reed DR, Beauchamp GK. 2012. Reply to Zhao and Zhnag: Loss of taste receptor function in mammals is directly related to feeding specializations. Proc. Natl. Acad. Sci. USA, doi: 10.1073/pnas.1205581109.

Wednesday, May 9, 2012

Endosymbiosis and Photosynthetic Animals

Contributed by: Hong Ma

            Endosymbiosis is thought to be crucial for the early evolution of eukaryotic cells, including the origins of the mitochondrion and the chloroplast, which is essential for photosynthesis. Photosynthesis is generally associated with plants, algae, and bacteria such as cyanobacteria, making these organisms the major primary producers. It is widely accepted that photosynthesis originated in bacteria and that chloroplasts in eukaryotic photosynthetic organisms were derived via ancient endosymbiosis after the capture of a cyanobacterium by an early eukaryote (Fig.1).  This primary endosymbiosis resulted in the ancestor of red algae and green algae. One branch of green algae then evolved into the green plants on land, such as mosses, ferns, and seed plants. The cyanobacterium taken in by the eukaryotic host cell evolved into the organelle chloroplast, which has a greatly reduced genome, whereas most of the original cyanobacterial genes were either lost or transferred into the host nuclear genome. For example, many of the proteins for photosynthesis are now encoded by nuclear genes.
After the origins of red and green algae, secondary symbiotic events (Fig.1) following the capture of one of the red or green algae by other eukaryotes generated a number of highly divergent algae, such as brown algae; however, the origins and histories of many of these algae and other organisms are still uncertain. In these algae, chloroplasts have one or two additional membranes compared with those in red or green algae (and the land plant descendents of green algae), with partial or complete loss of the nuclear genome of the captured red/green algal cells. Because both primary and secondary endosymbioses were both very ancient events, and there are few, if any, intermediate cases, the origin and history of these processes are not well understood.

Fig.1. Diagrams illustrating primary and secondary endosymbiosis. Top: a eukaryotic cell (brown with flagella) takes up a cyanobacterium (blue) and then evolved into red and green algae, as well as glaucophytes (golden algae). Bottom, red algae and green algae are again taken up in secondary endosymbiotic events, one of which gave rise to Vaucheria.

Animals derive nutrients from foods that are ultimately produced by photosynthetic organisms. Lesser known are the amazing symbiotic relationships between members of diverse groups of invertebrates and single-cell photosynthetic algae or bacteria. Such symbiosis is found in sponges, corals, and some giant clams, and provides fixed carbon to the animals, whereas the photosynthetic algae or cyanobacteria also benefit from this relationship in the form of protection and nutrients from the animal partners, such as nitrogen and other minerals. In these mutually beneficial relationships, as much as 90% of the energy needed by the animals can be from the photosynthetic symbionts.
            In the primary and secondary endosynbiotic relationships for plants, algae and other organisms, the ancestral free-living photosynthetic cells had evolved into an organelle, the chloroplast, that depends on the host nuclear genes for biogenesis and function. In addition, chloroplast (or plastid) is maintained throughout the life cycle of plants or algae. In contrast, the relationships between the animal hosts and their photosynthetic symbionts are less intimate, with the symbionts still retaining their cellular structure and complete genomes. Furthermore, the symbiotic relationship is established de novo during animal development. Therefore, the relationships between animals and their symbionts are fundamentally different from those in the primary and secondary endosymbiotic relationships between the plant and algal hosts and their chloroplasts. It is thus difficult to draw inference from the animal – algal symbiotic relationships regarding the evolutionary history of the primary and secondary endosymbioses of the chloroplast.
            Recently, Rumpho et al. (1) described an unusual relationship between a sea slug (Elysia chlorotica) and an alga (Vaucheria litorea). Elysia chlorotica is a green marine animal commonly known as eastern emerald elysia and is found in Eastern US coastal marshes.  It is usually 20-30 mm long, but can grow to as long as 60 mm (Fig. 2). Its food at the juvenile stage is the intertidal Vaucheria litorea, a yellow-green alga and member of the Heterokonts, a diverse group that also includes brown algae. Before feeding on V. litorea, the sea slug has a brown color with red spots and feeding on V. litorea is necessary for the green color.

Fig. 2. Elysia chlorotica, showing highly branched digestive system and green color.

Using an artificial sea-water culture system, Rumpho and coworkers observed that metamorphosis of E. chlorotica from larva to juvenile depends on the presence of V. litorea, and newly formed juvenile starts to feed on the algae immediately. As the sea slug feeds on V. litorea, it breaks the unicellular filamentous alga, and sucks the contents, accumulating chloroplasts in its highly branched digestive system distributed throughout its body. The chloroplasts are taken up by and stored within the cells of the digestive system of the animal in a precess called “kleptoplasty” (Fig. 3), making the body green. The feeding continues until the number of chloroplasts accumulates to a sufficient level, then the sea slug can live as a photosynthetic organism without eating for several months. This ability to use photosynthesis as the sole energy source has endowed the name “solar-powered” sea slug. If, however, the sea slug is removed from the algae within a few days of the start of feeding, then there are insufficient chloroplasts and development arrests.

Fig.3. Kleptoplasty, by which cells of the digestive systems of the sea slug take up chloroplasts from V. litorea, and maintain the function of the chloroplasts.

            Although the V. litorea chloroplasts have four outer membranes, characteristic of such secondary endosymbiotic chloroplasts of heterokonts, the chloroplasts retained in the sea slug seem to lack the two outer membranes. In addition, the V. litorea nuclei or mitochondria were not detected in the sea slug cells, suggesting that only the chloroplasts (without their outer two membranes) are taken up by the cells of the host digestive tract. In other words, the algal nuclei that normally contain the needed gene functions to maintain chloroplast functions appear to be absent in the sea slug cells.
            Therefore, this sea slug –alga relationship is similar to other animal-algal symbiotic relationships in having to establish it de dovo during development, and unlike the endosymbiotic relationship between plants/algae and chloroplasts. However, the E. chlorotica - V. litorea symbiosis is distinct from other animal-algae relationship because the V. litorea chloroplasts are taken up by the E. chlorotica digestive cells, and V. litorea cells are not maintained in the animal. It is striking that during the time when the sea slug is not feeding, even for months, chloroplasts in its digestive cells are functional. One of consequences of the photosynthesis is damage to chloroplast proteins; thus new protein synthesis is needed for the maintenance of chloroplast function. Therefore, the sea slug should be able to provide needed protein activities to repair photo-oxidative damage to chloroplast proteins.
            An attractive hypothesis that explains the ability of E. chlorotica to maintain chloroplast function is that, through repeated uptake of V. litoria cellular content, crucial genes for chloroplast functions have been transferred to the E. chlorotica nuclear genome. Indeed, several lines of evidence support gene transfer from V. litoria to E. chlorotica: de novo protein synthesis of a nuclear-encoded light-harvesting protein, synthesis of the nuclear-encoded PRK protein and light-induced expression of the prk gene in algal-starved sea slugs, and detection of sequences highly similar to V. litoria nuclear genes encoding chloroplast proteins. However, recently a partial transcriptome analysis has failed to identify cDNA sequences that are similar to algal nuclear genes. Nevertheless, the absence of supporting evidence for gene transfer is not evidence for the absence of gene transfer. It is possible that such transferred genes are not expressed highly enough to be detected in the partial transcriptome analysis. Alternatively, the chloroplast functions might be supported via novel mechanisms. Therefore, more extensive and definitive evidence for gene transfer is needed to support the above hypothesis.
            Regardless of the exact mechanisms, E. chlorotica clearly can take up functional chloroplasts intracellularly and maintain their functions for extended periods of several months. These features make this relationship more advanced than other animal-algal symbiotic relationships with intact cellular symbionts and closer to endosymbiotic relationships present in algae. It will be instructive to understand the mechanistic interaction between E. chlorotica and V. litoria and more will be uncovered as much more extensive sequencing efforts are underway. It is possible that in the future, when the soma-germline barrier can be broken for the chloroplast, and when the symbiont chloroplasts can replicate in the sea slug, the relationship will then have evolved into true endosymbiosis.   
1. Rumpho ME, Pelletreau KN, Moustafa A, Bhattacharya D. 2011. The making of a photosynthetic animal. J. Exp. Biol. 214, 303-311.

Wednesday, May 2, 2012

Epigenetics and Speciation

Contributed by: Masafumi Nozawa

Epigenetic variation has recently been recognized as an additional layer of genetic changes. After the discovery of epigenetic variation, many researchers have studied its importance in adaptation and evolution. However, the role of epigenetic variation in speciation remains largely unexplored.
Durand et al. (1) recently showed that natural epigenetic variation in Arabidopsis thaliana contributes to the cause of genetic incompatibility responsible for post-zygotic reproductive isolation. When they crossed the Columbia-0 (col) strain with the Shahdara (sha) strain, some F2 hybrids showed reduced seed production by 80-90%. Linkage disequilibrium analysis identified two genomic regions, K4 and K5, that are responsible for the incompatibility. Further fine mapping revealed that K5 contains gene AtFOLT1, which encodes a folate transporter, and K4 contains a duplicate locus, AtFOLT2, only in sha. AtFOLT1 is expressed in col but not in sha, whereas AtFOLT2 exists only in sha. Interestingly, F2 hybrids become incompatible only when there is no AtFOLT transcript. Therefore, the lack of AtFOLT transcripts is responsible for the incompatibility (Fig. 1).

Fig. 1. Mechanism of the allelic incompatibility. In col, the promoter and the first part of the gene AtFOLT1 are totally unmethylated but they are in sha. The homologous region of AtFOLT2 is also methylated in sha, the gene being transcribed (black arrow) from the unmethylated upstream promoter (green box). Modified from Durand et al. (1).

Therefore, they examined SNPs that might be responsible for the suppression of AtFOLT1 in sha and identified 29 such SNPs between sha and col. However, another strain named Ishikawa was shown to express AtFOLT1 even if the nucleotide sequence of AtFOLT1 is exactly the same as that of sha. This indicates that epigenetic changes cause the silencing of AtFOLT1. Indeed, AtFOLT1 in sha was highly methylated.
They further investigated how the methylation is induced and found that AtFOLT2 in sha generates small RNAs which directly induce DNA methylation on promoter regions of AtFOLT1 and AtFOLT2. Yet, because AtFOLT2 in sha has an irregular promoter region which is not methylated, sha can express AtFOLT2 and therefore be fertile (Fig. 1). Interestingly, this small RNA is sufficient to induce de novo DNA methylation in the AtFOLT region, because after several generations the unmethylated AtFOLT1 region was methylated (Fig. 2).

Fig. 2. De novo DNA methylation of AtFOLT1 by small RNAs in the AtFOLT2 region. In F1, AtFOLT1 from col is expressed because the region is not methylated. However, small RNAs at the AtFOLT2 region derived from sha stochastically and progressively induce DNA methylation on the AtFOLT1 region. Consequently, AtFOLT1 is not expressed after seven generations.

In summary, this study represents the first case of a natural epiallele that has strong deleterious phenotypic consequences steadily maintained in the progenies of crosses between strains, which play roles in establishing reproductive isolation. I think this type of processes might be much more frequent than currently appreciated.

1. Durand S, Bouche N, Strand EP, Loudet O, and Camilleri C. (2012) Rapid establishment of genetic incompatibility through natural epigenetic variation. Curr Bio 22: 326-31.