Characterization of the Pristionchus pacificus "epigenetic toolkit" reveals the evolutionary loss of the histone methyltransferase complex PRC2.

Comparative approaches have revealed both divergent and convergent paths to achieving shared developmental outcomes. Thus, only through assembling multiple case studies can we understand biological principles. Yet, despite appreciating the conservation-or lack thereof-of developmental networks, the conservation of epigenetic mechanisms regulating these networks is poorly understood. The nematode Pristionchus pacificus has emerged as a model system of plasticity and epigenetic regulation as it exhibits a bacterivorous or omnivorous morph depending on its environment. Here, we determined the "epigenetic toolkit" available to P. pacificus as a resource for future functional work on plasticity, and as a comparison with Caenorhabditis elegans to investigate the conservation of epigenetic mechanisms. Broadly, we observed a similar cast of genes with putative epigenetic function between C. elegans and P. pacificus. However, we also found striking differences. Most notably, the histone methyltransferase complex PRC2 appears to be missing in P. pacificus. We described the deletion/pseudogenization of the PRC2 genes mes-2 and mes-6 and concluded that both were lost in the last common ancestor of P. pacificus and a related species P. arcanus. Interestingly, we observed the enzymatic product of PRC2 (H3K27me3) by mass spectrometry and immunofluorescence, suggesting that a currently unknown methyltransferase has been co-opted for heterochromatin silencing. Altogether, we have provided an inventory of epigenetic genes in P. pacificus to compare with C. elegans. This inventory will enable reverse-genetic experiments related to plasticity and has revealed the first loss of PRC2 in a multicellular organism.

Improved Reference Genome Uncovers Novel Sex-Linked Regions in the Guppy (Poecilia reticulata).

Theory predicts that the sexes can achieve greater fitness if loci with sexually antagonistic polymorphisms become linked to the sex determining loci, and this can favor the spread of reduced recombination around sex determining regions. Given that sex-linked regions are frequently repetitive and highly heterozygous, few complete Y chromosome assemblies are available to test these ideas. The guppy system (Poecilia reticulata) has long been invoked as an example of sex chromosome formation resulting from sexual conflict. Early genetics studies revealed that male color patterning genes are mostly but not entirely Y-linked, and that X-linkage may be most common in low-predation populations. More recent population genomic studies of guppies have reached varying conclusions about the size and placement of the Y-linked region. However, this previous work used a reference genome assembled from short-read sequences from a female guppy. Here, we present a new guppy reference genome assembly from a male, using long-read PacBio single-molecule real-time sequencing and chromosome contact information. Our new assembly sequences across repeat- and GC-rich regions and thus closes gaps and corrects mis-assemblies found in the short-read female-derived guppy genome. Using this improved reference genome, we then employed broad population sampling to detect sex differences across the genome. We identified two small regions that showed consistent male-specific signals. Moreover, our results help reconcile the contradictory conclusions put forth by past population genomic studies of the guppy sex chromosome. Our results are consistent with a small Y-specific region and rare recombination in male guppies.

Molecular Evolutionary Analysis of Nematode Zona Pellucida (ZP) Modules Reveals Disulfide-Bond Reshuffling and Standalone ZP-C Domains.

Zona pellucida (ZP) modules mediate extracellular protein-protein interactions and contribute to important biological processes including syngamy and cellular morphogenesis. Although some biomedically relevant ZP modules are well studied, little is known about the protein family's broad-scale diversity and evolution. The increasing availability of sequenced genomes from "nonmodel" systems provides a valuable opportunity to address this issue and to use comparative approaches to gain new insights into ZP module biology. Here, through phylogenetic and structural exploration of ZP module diversity across the nematode phylum, I report evidence that speaks to two important aspects of ZP module biology. First, I show that ZP-C domains-which in some modules act as regulators of ZP-N domain-mediated polymerization activity, and which have never before been found in isolation-can indeed be found as standalone domains. These standalone ZP-C domain proteins originated in independent (paralogous) lineages prior to the diversification of extant nematodes, after which they evolved under strong stabilizing selection, suggesting the presence of ZP-N domain-independent functionality. Second, I provide a much-needed phylogenetic perspective on disulfide bond variability, uncovering evidence for both convergent evolution and disulfide-bond reshuffling. This result has implications for our evolutionary understanding and classification of ZP module structural diversity and highlights the usefulness of phylogenetics and diverse sampling for protein structural biology. All told, these findings set the stage for broad-scale (cross-phyla) evolutionary analysis of ZP modules and position Caenorhabditis elegans and other nematodes as important experimental systems for exploring the evolution of ZP modules and their constituent domains.

Convergent evolution of small molecule pheromones in Pristionchus nematodes.

The small molecules that mediate chemical communication between nematodes-so-called 'nematode-derived-modular-metabolites' (NDMMs)-are of major interest because of their ability to regulate development, behavior, and life-history. Pristionchus pacificus nematodes produce an impressive diversity of structurally complex NDMMs, some of which act as primer pheromones that are capable of triggering irreversible developmental switches. Many of these NDMMs have only ever been found in P. pacificus but no attempts have been made to study their evolution by profiling closely related species. This study brings a comparative perspective to the biochemical study of NDMMs through the systematic MS/MS- and NMR-based analysis of exo-metabolomes from over 30 Pristionchus species. We identified 36 novel compounds and found evidence for the convergent evolution of complex NDMMs in separate branches of the Pristionchus phylogeny. Our results demonstrate that biochemical innovation is a recurrent process in Pristionchus nematodes, a pattern that is probably typical across the animal kingdom.

Adaptive Patterns of Mitogenome Evolution Are Associated with the Loss of Shell Scutes in Turtles.

The mitochondrial genome encodes several protein components of the oxidative phosphorylation (OXPHOS) pathway and is critical for aerobic respiration. These proteins have evolved adaptively in many taxa, but linking molecular-level patterns with higher-level attributes (e.g., morphology, physiology) remains a challenge. Turtles are a promising system for exploring mitochondrial genome evolution as different species face distinct respiratory challenges and employ multiple strategies for ensuring efficient respiration. One prominent adaptation to a highly aquatic lifestyle in turtles is the secondary loss of keratenized shell scutes (i.e., soft-shells), which is associated with enhanced swimming ability and, in some species, cutaneous respiration. We used codon models to examine patterns of selection on mitochondrial protein-coding genes along the three turtle lineages that independently evolved soft-shells. We found strong evidence for positive selection along the branches leading to the pig-nosed turtle (Carettochelys insculpta) and the softshells clade (Trionychidae), but only weak evidence for the leatherback (Dermochelys coriacea) branch. Positively selected sites were found to be particularly prevalent in OXPHOS Complex I proteins, especially subunit ND2, along both positively selected lineages, consistent with convergent adaptive evolution. Structural analysis showed that many of the identified sites are within key regions or near residues involved in proton transport, indicating that positive selection may have precipitated substantial changes in mitochondrial function. Overall, our study provides evidence that physiological challenges associated with adaptation to a highly aquatic lifestyle have shaped the evolution of the turtle mitochondrial genome in a lineage-specific manner.

Unexpected sex-specific post-reproductive lifespan in the free-living nematode Pristionchus exspectatus.

Patterns of senescence (or aging) can vary among life history traits and between the sexes, providing an opportunity to study variation in the aging process within a single species. We previously found that females of the nematode Pristionchus exspectatus outlive males by a substantial margin under laboratory conditions. Here, we show that sex-specific reproductive senescence unfolds in the opposite direction in this species, resulting in a prolonged period of female-specific post-reproductive survival: females lost the ability to reproduce at approximately 4.7 weeks despite a median lifespan of about 12.3 weeks under lab conditions, whereas males lost the ability to reproduce at approximately 6.6 weeks, roughly in line with their median lifespan of around 7.6 weeks. Interestingly, somatic senescence (declining crawling speed) only explained reproductive senescence in males, whereas females lost the ability to reproduce regardless of condition. However, we found that housing females with males significantly increased their mortality rate, indicating that female-specific post-reproductive survival is unlikely to occur in the wild. We discuss our results in light of evolutionary theories of post-reproductive survival and previous studies of nematode behavioral ecology, arguing that premature reproductive senescence may stem from sex-specific condition-dependent survival during the reproductive period. Given the proven lab tractability of Prisitonchus nematodes, our findings provide a foundation for integrative research that combines evolutionary ecology and molecular genetics in the study of sex-specific senescence and post-reproductive survival.

Mating System Transitions Drive Life Span Evolution in Pristionchus Nematodes.

Interactions between the sexes influence evolution at many scales, but not all animal species conform to the familiar male-female (dioecious) mating system; such taxa are powerful tools for studying the evolutionary importance of sexual selection and conflict on all manner of life-history traits, including longevity. We tested for an effect of mating system on adult life span in Pristionchus nematodes, where self-fertile hermaphrodites have replaced females multiple times independently throughout the genus (androdioecy). By measuring adult life span for 11 species (6 dioecious, 5 androdioecious), we found that life span is considerably shorter in hermaphrodites relative to closely related females. This effect is not a cost of reproduction; brood size did not reliably trade off with life span in self-fertilizing hermaphrodites or in mated females. Furthermore, we found that sexual dimorphism in life span varied among dioecious species, with females generally outliving males. Finally, we documented intraspecific variation for life span and cuticular disease (blistering) prevalence in Pristionchus pacificus, a model system for evolutionary-developmental biology. This work demonstrates that mating system transitions and life span evolution are linked in Pristionchus nematodes and provides a foundation for future comparative and mechanistic studies of aging in this genus.

Molecular evolutionary analysis of vertebrate transducins: a role for amino acid variation in photoreceptor deactivation.

Transducin is a heterotrimeric G protein that plays a critical role in phototransduction in the rod and cone photoreceptor cells of the vertebrate retina. Rods, highly sensitive cells that recover from photoactivation slowly, underlie dim-light vision, whereas cones are less sensitive, recover more quickly, and underlie bright-light vision. Transducin deactivation is a critical step in photoreceptor recovery and may underlie the functional distinction between rods and cones. Rods and cones possess distinct transducin α subunits, yet they share a common deactivation mechanism, the GTPase activating protein (GAP) complex. Here, we used codon models to examine patterns of sequence evolution in rod (GNAT1) and cone (GNAT2) α subunits. Our results indicate that purifying selection is the dominant force shaping GNAT1 and GNAT2 evolution, but that GNAT2 has additionally been subject to positive selection operating at multiple phylogenetic scales; phylogeny-wide analysis identified several sites in the GNAT2 helical domain as having substantially elevated dN/dS estimates, and branch-site analysis identified several nearby sites as targets of strong positive selection during early vertebrate history. Examination of aligned GNAT and GAP complex crystal structures revealed steric clashes between several positively selected sites and the deactivating GAP complex. This suggests that GNAT2 sequence variation could play an important role in adaptive evolution of the vertebrate visual system via effects on photoreceptor deactivation kinetics and provides an alternative perspective to previous work that focused instead on the effect of GAP complex concentration. Our findings thus further the understanding of the molecular biology, physiology, and evolution of vertebrate visual systems.

Complex patterns of divergence among green-sensitive (RH2a) African cichlid opsins revealed by Clade model analyses.

BACKGROUND: Gene duplications play an important role in the evolution of functional protein diversity. Some models of duplicate gene evolution predict complex forms of paralog divergence; orthologous proteins may diverge as well, further complicating patterns of divergence among and within gene families. Consequently, studying the link between protein sequence evolution and duplication requires the use of flexible substitution models that can accommodate multiple shifts in selection across a phylogeny. Here, we employed a variety of codon substitution models, primarily Clade models, to explore how selective constraint evolved following the duplication of a green-sensitive (RH2a) visual pigment protein (opsin) in African cichlids. Past studies have linked opsin divergence to ecological and sexual divergence within the African cichlid adaptive radiation. Furthermore, biochemical and regulatory differences between the RH2aα and RH2aß paralogs have been documented. It thus seems likely that selection varies in complex ways throughout this gene family. RESULTS: Clade model analysis of African cichlid RH2a opsins revealed a large increase in the nonsynonymous-to-synonymous substitution rate ratio (ω) following the duplication, as well as an even larger increase, one consistent with positive selection, for Lake Tanganyikan cichlid RH2aß opsins. Analysis using the popular Branch-site models, by contrast, revealed no such alteration of constraint. Several amino acid sites known to influence spectral and non-spectral aspects of opsin biochemistry were found to be evolving divergently, suggesting that orthologous RH2a opsins may vary in terms of spectral sensitivity and response kinetics. Divergence appears to be occurring despite intronic gene conversion among the tandemly-arranged duplicates. CONCLUSIONS: Our findings indicate that variation in selective constraint is associated with both gene duplication and divergence among orthologs in African cichlid RH2a opsins. At least some of this variation may reflect an adaptive response to differences in light environment. Interestingly, these patterns only became apparent through the use of Clade models, not through the use of the more widely employed Branch-site models; we suggest that this difference stems from the increased flexibility associated with Clade models. Our results thus bear both on studies of cichlid visual system evolution and on studies of gene family evolution in general.

Visual pigment molecular evolution in the Trinidadian pike cichlid (Crenicichla frenata): a less colorful world for neotropical cichlids?

The Trinidadian pike cichlid (Crenicichla frenata) is a major predator of the guppy (Poecilia reticulata), a model system for visual ecology research, and visual predation by the pike cichlid is known to select for male guppies with reduced short-wavelength reflectance. However, an early study of the pike cichlid's visual system suggested a lack of short-wavelength-sensitive cone photoreceptors, a surprising finding as many African cichlids have highly developed short-wavelength vision. In this study, we found evidence for only four expressed cone opsins (LWS, RH2a, SWS2a, and SWS2b), plus one pseudogene (RH2b). Taken together with our microspectrophotometry data, which revealed the presence of three types of cone photoreceptor, including one sensitive to short-wavelength light, this would indicate a broader spectral capacity than previously believed from earlier visual studies of this fish. Relative to the highly diverse African cichlids, however, this Neotropical cichlid appears to have a greatly reduced opsin complement, reflecting both gene loss along the Neotropical lineage (lacking functional RH2b and, possibly, SWS1 opsins) and gene duplication within the African clade (which possesses paralogous RH2aα and RH2aß opsins). Molecular evolutionary analyses show that positive selection has shaped the SWS2b and RH1 opsins along the Neotropical lineage, which may be indicative of adaptive evolution to alter nonspectral aspects of opsin biology. These results represent the first molecular evolutionary study of visual pigments in a Neotropical cichlid and thus provide a foundation for further study of a morphologically and ecologically diverse clade that has been understudied with respect to the link between visual ecology and diversification.

Seeing orange: prawns tap into a pre-existing sensory bias of the Trinidadian guppy.

Sensory bias, a predisposition towards certain signals, has been implicated in the origin of mate preferences in some species. A risk associated with these biases is that they can be co-opted by predators as sensory lures. Here we propose that the orange spots on the brown pincers of a diurnal, predatory species of prawn function as lures for Trinidadian guppies, which have a sensory bias for orange. We exposed female guppies to (i) a life-like model of this Trinidadian prawn with orange, green or no spots on the pincers or (ii) a live, novel (non-Trinidadian) crustacean (crayfish), also with spotted pincers. First, we provide evidence that guppies sympatric with the prawn recognized our model as a potential predator. Next, we found that guppies spent more time in the dangerous head region of the model prawn with orange-spotted pincers compared with unspotted pincers. Finally, we show that allopatric, but not sympatric, guppies spent more time in the vicinity of the head of a live crayfish when orange spots were added to its pincers than when brown spots were added. Our results suggest that the orange spots on prawn pincers can act as a sensory lure.

An improved likelihood ratio test for detecting site-specific functional divergence among clades of protein-coding genes.

Maximum likelihood codon substitution models have proven useful for studying when and how protein function evolves, but they have recently been criticized on a number of fronts. The strengths and weaknesses of such methods must therefore be identified and improved upon. Here, using simulations, we show that the Clade model C versus M1a test for functional divergence among clades is prone to false positives under simple evolutionary conditions. We then propose a new null model (M2a_rel) that better accounts for among-site variation in selective constraint. We show that the revised test has an improved false-positive rate and good power. Applying this test to previously analyzed data sets of primate ribonucleases and mammalian rhodopsins reveals that some conclusions may have been misled by the original method. The improved test should prove useful for identifying patterns of divergence in selective constraint among paralogous gene families and among orthologs from ecologically divergent species.

Sequencing and characterization of the guppy (Poecilia reticulata) transcriptome.

BACKGROUND: Next-generation sequencing is providing researchers with a relatively fast and affordable option for developing genomic resources for organisms that are not among the traditional genetic models. Here we present a de novo assembly of the guppy (Poecilia reticulata) transcriptome using 454 sequence reads, and we evaluate potential uses of this transcriptome, including detection of sex-specific transcripts and deployment as a reference for gene expression analysis in guppies and a related species. Guppies have been model organisms in ecology, evolutionary biology, and animal behaviour for over 100 years. An annotated transcriptome and other genomic tools will facilitate understanding the genetic and molecular bases of adaptation and variation in a vertebrate species with a uniquely well known natural history. RESULTS: We generated approximately 336 Mbp of mRNA sequence data from male brain, male body, female brain, and female body. The resulting 1,162,670 reads assembled into 54,921 contigs, creating a reference transcriptome for the guppy with an average read depth of 28×. We annotated nearly 40% of this reference transcriptome by searching protein and gene ontology databases. Using this annotated transcriptome database, we identified candidate genes of interest to the guppy research community, putative single nucleotide polymorphisms (SNPs), and male-specific expressed genes. We also showed that our reference transcriptome can be used for RNA-sequencing-based analysis of differential gene expression. We identified transcripts that, in juveniles, are regulated differently in the presence and absence of an important predator, Rivulus hartii, including two genes implicated in stress response. For each sample in the RNA-seq study, >50% of high-quality reads mapped to unique sequences in the reference database with high confidence. In addition, we evaluated the use of the guppy reference transcriptome for gene expression analyses in a congeneric species, the sailfin molly (Poecilia latipinna). Over 40% of reads from the sailfin molly sample aligned to the guppy transcriptome. CONCLUSIONS: We show that next-generation sequencing provided a reliable and broad reference transcriptome. This resource allowed us to identify candidate gene variants, SNPs in coding regions, and sex-specific gene expression, and permitted quantitative analysis of differential gene expression.

Duplicate dmbx1 genes regulate progenitor cell cycle and differentiation during zebrafish midbrain and retinal development.

BACKGROUND: The Dmbx1 gene is important for the development of the midbrain and hindbrain, and mouse gene targeting experiments reveal that this gene is required for mediating postnatal and adult feeding behaviours. A single Dmbx1 gene exists in terrestrial vertebrate genomes, while teleost genomes have at least two paralogs. We compared the loss of function of the zebrafish dmbx1a and dmbx1b genes in order to gain insight into the molecular mechanism by which dmbx1 regulates neurogenesis, and to begin to understand why these duplicate genes have been retained in the zebrafish genome. RESULTS: Using gene knockdown experiments we examined the function of the dmbx1 gene paralogs in zebrafish, dmbx1a and dmbx1b in regulating neurogenesis in the developing retina and midbrain. Dose-dependent loss of dmbx1a and dmbx1b function causes a significant reduction in growth of the midbrain and retina that is evident between 48-72 hpf. We show that this phenotype is not due to patterning defects or persistent cell death, but rather a deficit in progenitor cell cycle exit and differentiation. Analyses of the morphant retina or anterior hindbrain indicate that paralogous function is partially diverged since loss of dmbx1a is more severe than loss of dmbx1b. Molecular evolutionary analyses of the Dmbx1 genes suggest that while this gene family is conservative in its evolution, there was a dramatic change in selective constraint after the duplication event that gave rise to the dmbx1a and dmbx1b gene families in teleost fish, suggestive of positive selection. Interestingly, in contrast to zebrafish dmbx1a, over expression of the mouse Dmbx1 gene does not functionally compensate for the zebrafish dmbx1a knockdown phenotype, while over expression of the dmbx1b gene only partially compensates for the dmbx1a knockdown phenotype. CONCLUSION: Our data suggest that both zebrafish dmbx1a and dmbx1b genes are retained in the fish genome due to their requirement during midbrain and retinal neurogenesis, although their function is partially diverged. At the cellular level, Dmbx1 regulates cell cycle exit and differentiation of progenitor cells. The unexpected observation of putative post-duplication positive selection of teleost Dmbx1 genes, especially dmbx1a, and the differences in functionality between the mouse and zebrafish genes suggests that the teleost Dmbx1 genes may have evolved a diverged function in the regulation of neurogenesis.

Molecular evolution of the betagamma lens crystallin superfamily: evidence for a retained ancestral function in gamma N crystallins?

Within the vertebrate eye, betagamma crystallins are extremely stable lens proteins that are uniquely adapted to increase refractory power while maintaining transparency. Unlike alpha crystallins, which are well-characterized, multifunctional proteins that have important functions both in and out of the lens, betagamma lens crystallins are a diverse group of proteins with no clear ancestral or contemporary nonlens role. We carried out phylogenetic and molecular evolutionary analyses of the betagamma-crystallin superfamily in order to study the evolutionary history of the gamma N crystallins, a recently discovered, biochemically atypical family suggested to possess a divergent or ancestral function. By including nonlens, betagamma-motif-containing sequences in our analysis as outgroups, we confirmed the phylogenetic position of the gamma N family as sister to other gamma crystallins. Using maximum likelihood codon models to estimate lineage-specific nonsynonymous-to-synonymous rate ratios revealed strong positive selection in all of the early lineages within the betagamma family, with the striking exception of the lineage leading to the gamma N crystallins which was characterized by strong purifying selection. Branch-site analysis, used to identify candidate sites involved in functional divergence between gamma N crystallins and its sister clade containing all other gamma crystallins, identified several positively selected changes at sites of known functional importance in the betagamma crystallin protein structure. Further analyses of a fish-specific gamma N crystallin gene duplication revealed a more recent episode of positive selection in only one of the two descendant lineages (gamma N2). Finally, from the guppy, Poecilia reticulata, we isolated complete gamma N1 and gamma N2 coding sequence data from cDNA and partial coding sequence data from genomic DNA in order to confirm the presence of a novel gamma N2 intron, discovered through data mining of two pufferfish genomes. We conclude that the function of the gamma N family likely resembles the ancestral vertebrate betagamma crystallin more than other betagamma families. Furthermore, owing to the presence of an additional intron in some fish gamma N2 crystallins, and the inferred action of positive selection following the fish-specific gamma N duplication, we suggest that further study of fish gamma N crystallins will be critical in further elucidating possible ancestral functions of gamma N crystallins and any nonstructural role they may have.

Reconstruction of ancestral FGLamide-type insect allatostatins: a novel approach to the study of allatostatin function and evolution.

Allatostatins (ASTs) are a class of regulatory neuropeptides, with diverse functions, found in an array of invertebrate phyla. ASTs have complex gene structure, in which individual ASTs are cleaved from a precursor peptide. Little is known about the molecular evolution of AST structure and function, even in extensively studied groups such as cockroaches. This paper presents the application of a novel technique for the analysis of this system, that of ancestral reconstruction, whereby ancestral amino acid sequences are resurrected in the laboratory. We inferred the ancestral sequences of a well-characterized peptide, AST 7, for the insect ancestor, as well as several cockroach ancestors. Peptides were assayed for in vitro inhibition of JH production in Diploptera punctata and Periplaneta americana. Our results surprisingly, indicate a decrease in potency of the ancestral cockroach AST7 peptide in comparison with more ancient ones such as the ancestral insect peptide, as well as more recently evolved cockroach peptides. We propose that this unexpected decrease in peptide potency at the cockroach ancestor may be related to the concurrent increase in peptide copy number in the lineages leading to cockroaches. This model is consistent with current physiological data, and may be linked to the increased role of ASTs in the regulation of reproductive processes in the cockroaches.

Pigments, patterns, and fish behavior.

Color patterns in fish are often multicomponent signals, composed of pigment-based and structural color patches that can be used to communicate within species, in both inter- and intrasexual interactions, and between species. In this review, we discuss some of the roles played by pigment-based elements of color pattern. We begin by discussing general forms of coloration, classifying them by appearance (e.g., cryptic vs. conspicuous) and apparent function (e.g., conspicuous coloration and mating displays, stripes and cooperation, and bars and aggression). We then briefly discuss the roles pigments play in the perception of these color patterns via their presence in the eye. In the last section, we look at the relative importance of carotenoid versus melanic coloration in situations where honest signals to potential rivals and potential mates might be required. In this survey, we have highlighted some recent research, especially studies that consider both the physiological and behavioral processes underlying the evolution and expression of pigment-based color patterns in fish. The nature of pigmented color patterns depends not just on the dynamics of pattern development and physiological regulation, but also on the behavioral roles played by these patterns, both now and in the past. As such, advances in particular fields of study on pigment patterns (physiology, developmental biology, behavioral ecology, evolutionary biology, etc.) will increasingly depend on insights from other fields.

Long-wavelength sensitive visual pigments of the guppy (Poecilia reticulata): six opsins expressed in a single individual.

BACKGROUND: The diversity of visual systems in fish has long been of interest for evolutionary biologists and neurophysiologists, and has recently begun to attract the attention of molecular evolutionary geneticists. Several recent studies on the copy number and genomic organization of visual pigment proteins, the opsins, have revealed an increased opsin diversity in fish relative to most vertebrates, brought about through recent instances of opsin duplication and divergence. However, for the subfamily of opsin genes that mediate vision at the long-wavelength end of the spectrum, the LWS opsins, it appears that most fishes possess only one or two loci, a value comparable to most other vertebrates. Here, we characterize the LWS opsins from cDNA of an individual guppy, Poecilia reticulata, a fish that is known exhibit variation in its long-wavelength sensitive visual system, mate preferences and colour patterns. RESULTS: We identified six LWS opsins expressed within a single individual. Phylogenetic analysis revealed that these opsins descend from duplication events both pre-dating and following the divergence of the guppy lineage from that of the bluefin killifish, Lucania goodei, the closest species for which comparable data exists. Numerous amino acid substitutions exist among these different LWS opsins, many at sites known to be important for visual pigment function, including spectral sensitivity and G-protein activation. Likelihood analyses using codon-based models of evolution reveal significant changes in selective constraint along two of the guppy LWS opsin lineages. CONCLUSION: The guppy displays an unusually high number of LWS opsins compared to other fish, and to vertebrates in general. Observing both substitutions at functionally important sites and the persistence of lineages across species boundaries suggests that these opsins might have functionally different roles, especially with regard to G-protein activation. The reasons why are currently unknown, but may relate to aspects of the guppy's behavioural ecology, in which both male colour patterns and the female mate preferences for these colour patterns experience strong, highly variable selection pressures.

Phylogeographic analysis of the Bufo gargarizans species complex: a revisit.

Using mtDNA sequencing and allozyme electrophoresis data, we tested the "vicariance followed by dispersal" hypothesis of the Bufo gargarizans species group and re-evaluated the species status in the general lineages species concept. A phylogenetic analysis suggested that dispersal, instead of vicariance, dominated the history of the species group. There was a general trend of west to east dispersal, while some lineages from the east subsequently returned to the west. The secondary admixture of those previously allopatric lineages produced substantial levels of sympatric genetic diversity, often as high as 7.0% pairwise difference within populations. The phylogenetic hypothesis does not support the current two species designation. Neither B. andrewsi nor B. gargarizans represents an independent evolutionary lineage, and monophyletic groups did not correspond to geographically discrete groups. Allozyme data also failed to reveal any fixed allelic difference among the populations. Therefore, we recommend regarding the complex as a single species, Bufo gargarizans, without subspecies division.