Development of Genetic Markers for Sex and Individual Identification of the Japanese Giant Flying Squirrel (Petaurista leucogenys) by an Efficient Method Using High-Throughput DNA Sequencing.

DNA markers that detect differences in the number of microsatellite repeats can be highly effective for genotyping individuals that lack differences in external morphology. However, isolation of sequences with different microsatellite repeat numbers between individuals has been a time-consuming process in the development of DNA markers. Individual identification of Japanese giant flying squirrels (Petaurista leucogenys) has been challenging because this species is arboreal and nocturnal and exhibits little to no morphological variation between individuals. In this study, we developed DNA markers for sex and individual identification of this species by an efficient method using high-throughput DNA sequence data. Paired-end 5 Gb (2 × 250 bp) and 15 Gb (2 × 150 bp) genome sequences were determined from a female and a male Japanese giant flying squirrel, respectively. We searched SRY and XIST genes located on Y and X chromosomes, respectively, from high-throughput sequence data and designed primers to amplify these genes. Using these primer sets, we succeeded to identify the sex of individuals. In addition, we selected 12 loci containing microsatellites with different numbers of repeats between two individuals from the same data set, and designed primers to amplify these sequences. Twenty individuals from nine different locations were discriminated using these primer sets. Furthermore, both sex and microsatellite markers were amplified from DNA extracted non-invasively from single fecal pellet samples. Based on our results for flying squirrels, we expect our efficient method for developing non-invasive high-resolution individual- and sex-specific genotyping to be applicable to a diversity of mammalian species.

Parallel evolution of opsin visual pigments in hawkmoths by tuning of spectral sensitivities during transition from a nocturnal to a diurnal ecology.

Light environments differ dramatically between day and night. The transition between diurnal and nocturnal visual ecology has happened repeatedly throughout evolution in many species. However, the molecular mechanism underlying the evolution of vision in recent diurnal-nocturnal transition is poorly understood. Here, we focus on hawkmoths (Lepidoptera: Sphingidae) to address this question by investigating five nocturnal and five diurnal species. We performed RNA-sequencing analysis and identified opsin genes corresponding to the ultraviolet (UV), short-wavelength (SW) and long-wavelength (LW)-absorbing visual pigments. We found no significant differences in the expression patterns of opsin genes between the nocturnal and diurnal species. We then constructed the phylogenetic trees of hawkmoth species and opsins. The diurnal lineages had emerged at least three times from the nocturnal ancestors. The evolutionary rates of amino acid substitutions in the three opsins differed between the nocturnal and diurnal species. We found an excess number of parallel amino acid substitutions in the opsins in three independent diurnal lineages. The numbers were significantly more than those inferred from neutral evolution, suggesting that positive selection acted on these parallel substitutions. Moreover, we predicted the visual pigment absorption spectra based on electrophysiologically determined spectral sensitivity in two nocturnal and two diurnal species belonging to different clades. In the diurnal species, the LW pigments shift 10 nm towards shorter wavelengths, and the SW pigments shift 10 nm in the opposite direction. Taken together, our results suggest that parallel evolution of opsins may have enhanced the colour discrimination properties of diurnal hawkmoths in ambient light.

Analysis of the Mitochondrial Genomes of Japanese Wolf Specimens in the Siebold Collection, Leiden.

The taxonomic status of extinct Japanese or Honshu wolves (Canis lupus hodophilax) has been disputed since the name hodophilax was first proposed by Temminck in 1839 on the basis of specimens stored in Leiden, the Netherlands. Points of controversy include whether the type specimen of hodophilax (Jentink c: RMNH.MAM.39181) and the other two specimens from Leiden (Jentink a: RMNH.MAM.39182 and Jentink b: RMNH.MAM.39183) represent different varieties or subspecies of Japanese wolves or not. Two Japanese names, ookami and jamainu, used to describe wild Canis species, further complicate the issue. In this study, the taxonomic status of Japanese wolves was clarified using mitochondrial DNA of the three specimens stored at the Naturalis Biodiversity Center in Leiden, in addition to three Japanese wolf specimens stored at the Museum für Naturkunde in Berlin and five new samples from Japan. The mitochondrial genomes of the type specimen of hodophilax (Jentink c) and another sample from Leiden (Jentink b) as well as Berlin specimens were included in the cluster of Japanese wolves distinct from other grey wolves. However, the other sample from Leiden (Jentink a) was identified as a domestic dog. A mitochondrial genome analysis suggested that Japanese wolves could be categorized into two distinct clusters. Studies of nuclear genomes are needed to further clarify the taxonomic status, divergence time, and population genetic structure of Japanese wolves.

Visual adaptation of opsin genes to the aquatic environment in sea snakes.

BACKGROUND: Evolutionary transitions from terrestrial to aquatic life history cause drastic changes in sensory systems. Indeed, the drastic changes in vision have been reported in many aquatic amniotes, convergently. Recently, the opsin genes of the full-aquatic sea snakes have been reported. However, those of the amphibious sea snakes have not been examined in detail. RESULTS: Here, we investigated opsin genes and visual pigments of sea snakes. We determined the sequences of SWS1, LWS, and RH1 genes from one terrestrial, three amphibious and four fully-aquatic elapids. Amino acid replacements at four and one spectra-tuning positions were found in LWS and RH1, respectively. We measured or predicted absorption of LWS and RH1 pigments with A1-derived retinal. During their evolution, blue shifts of LWS pigments have occurred stepwise in amphibious sea snakes and convergently in both amphibious and fully-aquatic species. CONCLUSIONS: Blue shifted LWS pigments may have adapted to deep water or open water environments dominated by blue light. The evolution of opsins differs between marine mammals (cetaceans and pinnipeds) and sea snakes in two fundamental ways: (1) pseudogenization of opsins in marine mammals; and (2) large blue shifts of LWS pigments in sea snakes. It may be possible to explain these two differences at the level of photoreceptor cell composition given that cone and rod cells both exist in mammals whereas only cone cells exist in fully-aquatic sea snakes. We hypothesize that the differences in photoreceptor cell compositions may have differentially affected the evolution of opsins in divergent amniote lineages.

Presence-absence polymorphisms of single-copy genes in the stony coral Acropora digitifera.

BACKGROUND: Despite the importance of characterizing genetic variation among coral individuals for understanding phenotypic variation, the correlation between coral genomic diversity and phenotypic expression is still poorly understood. RESULTS: In this study, we detected a high frequency of genes showing presence-absence polymorphisms (PAPs) for single-copy genes in Acropora digitifera. Among 10,455 single-copy genes, 516 (5%) exhibited PAPs, including 32 transposable element (TE)-related genes. Five hundred sixteen genes exhibited a homozygous absence in one (102) or more than one (414) individuals (n = 33), indicating that most of the absent alleles were not rare variants. Among genes showing PAPs (PAP genes), roughly half were expressed in adults and/or larvae, and the PAP status was associated with differential expression among individuals. Although 85% of PAP genes were uncharacterized or had ambiguous annotations, 70% of these genes were specifically distributed in cnidarian lineages in eumetazoa, suggesting that these genes have functional roles related to traits related to cnidarians or the family Acroporidae or the genus Acropora. Indeed, four of these genes encoded toxins that are usually components of venom in cnidarian-specific cnidocytes. At least 17% of A. digitifera PAP genes were also PAPs in A. tenuis, the basal lineage in the genus Acropora, indicating that PAPs were shared among species in Acropora. CONCLUSIONS: Expression differences caused by a high frequency of PAP genes may be a novel genomic feature in the genus Acropora; these findings will contribute to improve our understanding of correlation between genetic and phenotypic variation in corals.

In vitro resynthesis of lichenization reveals the genetic background of symbiosis-specific fungal-algal interaction in Usnea hakonensis.

BACKGROUND: Symbiosis is central to ecosystems and has been an important driving force of the diversity of life. Close and long-term interactions are known to develop cooperative molecular mechanisms between the symbiotic partners and have often given them new functions as symbiotic entities. In lichen symbiosis, mutualistic relationships between lichen-forming fungi and algae and/or cyanobacteria produce unique features that make lichens adaptive to a wide range of environments. Although the morphological, physiological, and ecological uniqueness of lichens has been described for more than a century, the genetic mechanisms underlying this symbiosis are still poorly known. RESULTS: This study investigated the fungal-algal interaction specific to the lichen symbiosis using Usnea hakonensis as a model system. The whole genome of U. hakonensis, the fungal partner, was sequenced by using a culture isolated from a natural lichen thallus. Isolated cultures of the fungal and the algal partners were co-cultured in vitro for 3 months, and thalli were successfully resynthesized as visible protrusions. Transcriptomes of resynthesized and natural thalli (symbiotic states) were compared to that of isolated cultures (non-symbiotic state). Sets of fungal and algal genes up-regulated in both symbiotic states were identified as symbiosis-related genes. CONCLUSION: From predicted functions of these genes, we identified genetic association with two key features fundamental to the symbiotic lifestyle in lichens. The first is establishment of a fungal symbiotic interface: (a) modification of cell walls at fungal-algal contact sites; and (b) production of a hydrophobic layer that ensheaths fungal and algal cells;. The second is symbiosis-specific nutrient flow: (a) the algal supply of photosynthetic product to the fungus; and (b) the fungal supply of phosphorous and nitrogen compounds to the alga. Since both features are widespread among lichens, our result may indicate important facets of the genetic basis of the lichen symbiosis.

Patterns of genomic differentiation between two Lake Victoria cichlid species, Haplochromis pyrrhocephalus and H. sp. 'macula'.

BACKGROUND: The molecular basis of the incipient stage of speciation is still poorly understood. Cichlid fish species in Lake Victoria are a prime example of recent speciation events and a suitable system to study the adaptation and reproductive isolation of species. RESULTS: Here, we report the pattern of genomic differentiation between two Lake Victoria cichlid species collected in sympatry, Haplochromis pyrrhocephalus and H. sp. 'macula,' based on the pooled genome sequences of 20 individuals of each species. Despite their ecological differences, population genomics analyses demonstrate that the two species are very close to a single panmictic population due to extensive gene flow. However, we identified 21 highly differentiated short genomic regions with fixed nucleotide differences. At least 15 of these regions contained genes with predicted roles in adaptation and reproductive isolation, such as visual adaptation, circadian clock, developmental processes, adaptation to hypoxia, and sexual selection. The nonsynonymous fixed differences in one of these genes, LWS, were reported as substitutions causing shift in absorption spectra of LWS pigments. Fixed differences were found in the promoter regions of four other differentially expressed genes, indicating that these substitutions may alter gene expression levels. CONCLUSIONS: These diverged short genomic regions may have contributed to the differentiation of two ecologically different species. Moreover, the origins of adaptive variants within the differentiated regions predate the geological formation of Lake Victoria; thus Lake Victoria cichlid species diversified via selection on standing genetic variation.

Physiological responses of ionotropic histamine receptors, PxHCLA and PxHCLB, to neurotransmitter candidates in a butterfly, Papilio xuthus.

Histamine is the only known neurotransmitter released by arthropod photoreceptors. Synaptic transmission from photoreceptors to second-order neurons is mediated by the activation of histamine-gated chloride channels (HCLs). These histaminergic synapses have been assumed to be conserved among insect visual systems. However, our understanding of the channels in question has thus far been based on studies in flies. In the butterfly Papilio xuthus, we have identified two candidate histamine-gated chloride channels, PxHCLA and PxHCLB, and studied their physiological properties using a whole-cell patch-clamp technique. We studied the responses of channels expressed in cultured cells to histamine as well as to other neurotransmitter candidates, namely GABA, tyramine, serotonin, d-/l-glutamate and glycine. We found that histamine and GABA activated both PxHCLA and PxHCLB, while the other molecules did not. The sensitivity to histamine and GABA was consistently higher in PxHCLB than in PxHCLA. Interestingly, simultaneous application of histamine and GABA activated both PxHCLA and PxHCLB more strongly than either neurotransmitter individually; histamine and GABA may have synergistic effects on PxHCLs in the regions where they co-localize. Our results suggest that the physiological properties of the histamine receptors are basically conserved among insects, but that the response to GABA differs between butterflies and flies, implying variation in early visual processing among species.

Visual adaptation in Lake Victoria cichlid fishes: depth-related variation of color and scotopic opsins in species from sand/mud bottoms.

BACKGROUND: For Lake Victoria cichlid species inhabiting rocky substrates with differing light regimes, it has been proposed that adaptation of the long-wavelength-sensitive (LWS) opsin gene triggered speciation by sensory drive through color signal divergence. The extensive and continuous sand/mud substrates are also species-rich, and a correlation between male nuptial coloration and the absorption of LWS pigments has been reported. However, the factors driving genetic and functional diversity of LWS pigments in sand/mud habitats are still unresolved. RESULTS: To address this issue, nucleotide sequences of eight opsin genes were compared in ten Lake Victoria cichlid species collected from sand/mud bottoms. Among eight opsins, the LWS and rod-opsin (RH1) alleles were diversified and one particular allele was dominant or fixed in each species. Natural selection has acted on and fixed LWS alleles in each species. The functions of LWS and RH1 alleles were measured by absorption of reconstituted A1- and A2-derived visual pigments. The absorption of pigments from RH1 alleles most common in deep water were largely shifted toward red, whereas those of LWS alleles were largely shifted toward blue in both A1 and A2 pigments. In both RH1 and LWS pigments, A2-derived pigments were closer to the dominant light in deep water, suggesting the possibility of the adaptation of A2-derived pigments to depth-dependent light regimes. CONCLUSIONS: The RH1 and LWS sequences may be diversified for adaptation of A2-derived pigments to different light environments in sand/mud substrates. Diversification of the LWS alleles may have originally taken place in riverine environments, with a new mutation occurring subsequently in Lake Victoria.

Physical contact and carbon transfer between a lichen-forming Trebouxia alga and a novel Alphaproteobacterium.

Recent progress in molecular techniques has begun to alter traditional recognition of lichens as symbiotic organisms comprised of a fungus and photosynthetic partners (green algae and/or cyanobacteria). Diverse organisms, especially various non-photosynthetic bacteria, are now indicated to be integral components of lichen symbiosis. Although lichen-associated bacteria are inferred to have functions that could support the symbiosis, little is known about their physical and nutritional interaction with fungi and algae. In the present study, we identified specific interaction between a lichen-forming alga and a novel bacterium. Trebouxia alga was isolated from a lichen, Usnea hakonensis, and kept as a strain for 8 years. Although no visible bacterial colonies were observed in this culture, high-throughput sequencing of DNA isolated from the culture revealed that the strain is composed of a Trebouxia alga and an Alphaproteobacterium species. In situ hybridization showed that bacterial cells were localized on the surface of the algal cells. Physiological assays revealed that the bacterium was able to use ribitol, glucose and mannitol, all of which are known to exist abundantly in lichens. It was resistant to three antibiotics. Bacteria closely related to this species were also identified in lichen specimens, indicating that U. hakonensis may commonly associate with this group of bacteria. These features of the novel bacterium suggest that it may be involved in carbon cycling of U. hakonensis as a member of lichen symbiosis and less likely to have become associated with the alga after isolation from a lichen.

Speciation through sensory drive in cichlid fish.

Theoretically, divergent selection on sensory systems can cause speciation through sensory drive. However, empirical evidence is rare and incomplete. Here we demonstrate sensory drive speciation within island populations of cichlid fish. We identify the ecological and molecular basis of divergent evolution in the cichlid visual system, demonstrate associated divergence in male colouration and female preferences, and show subsequent differentiation at neutral loci, indicating reproductive isolation. Evidence is replicated in several pairs of sympatric populations and species. Variation in the slope of the environmental gradients explains variation in the progress towards speciation: speciation occurs on all but the steepest gradients. This is the most complete demonstration so far of speciation through sensory drive without geographical isolation. Our results also provide a mechanistic explanation for the collapse of cichlid fish species diversity during the anthropogenic eutrophication of Lake Victoria.

B chromosomes have a functional effect on female sex determination in Lake Victoria cichlid fishes.

The endemic cichlid fishes in Lake Victoria are a model system for speciation through adaptive radiation. Although the evolution of the sex-determination system may also play a role in speciation, little is known about the sex-determination system of Lake Victoria cichlids. To understand the evolution of the sex-determination system in these fish, we performed cytogenetic analysis in 11 cichlid species from Lake Victoria. B chromosomes, which are present in addition to standard chromosomes, were found at a high prevalence rate (85%) in these cichlids. In one species, B chromosomes were female-specific. Cross-breeding using females with and without the B chromosomes demonstrated that the presence of the B chromosomes leads to a female-biased sex ratio in this species. Although B chromosomes were believed to be selfish genetic elements with little effect on phenotype and to lack protein-coding genes, the present study provides evidence that B chromosomes have a functional effect on female sex determination. FISH analysis using a BAC clone containing B chromosome DNA suggested that the B chromosomes are derived from sex chromosomes. Determination of the nucleotide sequences of this clone (104.5 kb) revealed the presence of several protein-coding genes in the B chromosome, suggesting that B chromosomes have the potential to contain functional genes. Because some sex chromosomes in amphibians and arthropods are thought to be derived from B chromosomes, the B chromosomes in Lake Victoria cichlids may represent an evolutionary transition toward the generation of sex chromosomes.

Japanese wolves are most closely related to dogs and share DNA with East Eurasian dogs.

Although the domestic dog's origin is still unclear, this lineage is believed to have been domesticated from an extinct population of gray wolves, which is expected to be more closely related to dogs than to other populations of gray wolves. Here, we sequence the whole genomes of nine Japanese wolves (7.5-100x: Edo to Meiji periods) and 11 modern Japanese dogs and analyze them together with those from other populations of dogs and wolves. A phylogenomic tree shows that, among the gray wolves, Japanese wolves are closest to the dog, suggesting that the ancestor of dogs is closely related to the ancestor of the Japanese wolf. Based on phylogenetic and geographic relationships, the dog lineage has most likely originated in East Asia, where it diverged from a common ancestor with the Japanese wolf. Since East Eurasian dogs possess Japanese wolf ancestry, we estimate an introgression event from the ancestor of the Japanese wolf to the ancestor of the East Eurasian dog that occurred before the dog's arrival in the Japanese archipelago.

Correlation between nuptial colors and visual sensitivities tuned by opsins leads to species richness in sympatric Lake Victoria cichlid fishes.

Reproductive isolation that prevents interspecific hybridization between closely related coexisting species maintains sympatric species diversity. One of the reproductive isolations is mate choice based on color signals (breeding color perceived by color vision). This is well known in several animal taxa, yet little is known about its genetic and molecular mechanism. Lake Victoria cichlid fishes are thought to be an example of sympatric species diversity. In the species inhabiting different light environments in rocky shore, speciation by sensory drive through color signals has been proposed by analyses of the long wavelength-sensitive (LWS) opsin gene and the male nuptial coloration. However, the genetic and molecular mechanism of how diversity of sympatric species occurring in the same habitat is maintained remains unknown. To address this issue, we determined nucleotide sequences of eight opsins of six sympatric species collected from a sandy-muddy shore--an ideal model system for studying sympatric species. Among eight opsins, the LWS and RH1 alleles were diversified and one particular allele is dominant or fixed in each species, and we propose that this is due to natural selection. The functions of their LWS alleles were also diversified as shown by absorption measurements of reconstituted visual pigments. To analyze the relationship between nuptial coloration and the absorption of LWS pigments, we systematically evaluated and defined nuptial coloration. We showed that the coloration was species specific with respect to hue and significantly differentiated by the index values of hue (dominant wavelength: λ(d)). The λ(d) value of the male nuptial coloration correlated with the absorption of LWS pigments from all the species, suggesting that reproductive isolation through mate choice using color signals may prevent sympatric interspecific hybridization, thereby maintaining the species diversity in sympatric species in Lake Victoria.

Reverse evolution in RH1 for adaptation of cichlids to water depth in Lake Tanganyika.

Reverse evolution is a widespread phenomenon in biology, but the genetic mechanism for the reversal of a genetic change for adaptation to the ancestral state is not known. Here, we report the first case of complete reverse evolution of two amino acids, serine and alanine, at a single position in RH1 opsin pigment for adaptation to water depth. We determined RH1 sequences of cichlid fishes from four tribes of Lake Tanganyika with different habitat depths. Most of the species were divided into two types: RH1 with 292A for species in shallow water or 292S for species in deep water. Both types were adapted to their ambient light environments as indicated by the absorption spectra of the RH1 pigments. Based on the RH1 locus tree and ecological data, we inferred the ancestral amino acids at position 292 and the distribution of the depth ranges (shallow or deep) of ancestral species of each tribe. According to these estimates, we identified two distinct parallel adaptive evolutions: The replacement A292S occurred at least four times for adaptation from shallow to deep water, and the opposite replacement S292A occurred three times for adaptation from deep to shallow water. The latter parallelism represents the complete reverse evolution from the derived to the ancestral state, following back adaptive mutation with reversal of the RH1 pigment function accompanied by reversal of the species habitat shift.

Functional divergence of the pigmentation gene melanocortin-1 receptor (MC1R) in six endemic Macaca species on Sulawesi Island.

Coat color is often highly variable within and between animal taxa. Among hundreds of pigmentation-related genes, melanocortin-1 receptor (MC1R) plays key roles in regulating the synthesis of the dark eumelanin and the red-yellow pheomelanin. The six species of macaques that inhabit Sulawesi Island diverged rapidly from their common ancestor, M. nemestrina. Unlike most macaques, Sulawesi macaques commonly have a dark coat color, with divergence in shade and color pattern. To clarify the genetic and evolutionary basis for coat color in Sulawesi macaques, we investigated the MC1R sequences and functional properties, including basal cAMP production and α-MSH-induced activity in vitro. We found fixed non-synonymous substitutions in MC1R in each species. Furthermore, we found that six species-specific variants corresponded with variation in agonist-induced and basal activity of MC1R. Inconsistent with the dark coat color, four substitutions independently caused decreases in the basal activity of MC1R in M. hecki, M. nigra, M. tonkeana, and M. ochreata. Selective analysis suggested MC1R of M. nigra and M. nigrescens underwent purifying selection. Overall, our results suggest that fixed differences in MC1R resulted in different functional characteristics and might contribute to divergence in color among the six Sulawesi macaque species.

Two divergent haplogroups of a sacsin-like gene in Acropora corals.

Reef-building corals are declining due to environmental changes. Sacsin is a member of the heat shock proteins and has been reported as a candidate protein associated with the stress response in Acropora corals. Recently, high nucleotide diversity and the persistence of two divergent haplogroups of sacsin-like genes in Acropora millepora have been reported. While it was not clear when the two haplogroups have split and whether the haplogroups have persisted in only A. millepora or the other lineages in the genus Acropora. In this study, we analyzed a genomic region containing a sacsin-like gene from Acropora and Montipora species. Higher nucleotide diversity in the sacsin-like gene compared with that of surrounding regions was also observed in A. digitifera. This nucleotide diversity is derived from two divergent haplogroups of a sacsin-like gene, which are present in at least three Acropora species. The origin of these two haplogroups can be traced back before the divergence of Acropora and Montipora (119 Ma). Although the link between exceptionally high genetic variation in sacsin-like genes and functional differences in sacsin-like proteins is not clear, the divergent haplogroups may respond differently to envionmental stressors and serve in the adaptive phsiological ecology of these keystone species.

De novo assembly and annotation of the mangrove cricket genome.

OBJECTIVES: The mangrove cricket, Apteronemobius asahinai, shows endogenous activity rhythms that synchronize with the tidal cycle (i.e., a free-running rhythm with a period of ~ 12.4 h [the circatidal rhythm]). Little is known about the molecular mechanisms underlying the circatidal rhythm. We present the draft genome of the mangrove cricket to facilitate future molecular studies of the molecular mechanisms behind this rhythm. DATA DESCRIPTION: The draft genome contains 151,060 scaffolds with a total length of 1.68 Gb (N50: 27 kb) and 92% BUSCO completeness. We obtained 28,831 predicted genes, of which 19,896 (69%) were successfully annotated using at least one of two databases (UniProtKB/SwissProt database and Pfam database).

Heterotypy in the N-terminal region of growth/differentiation factor 5 (GDF5) mature protein during teleost evolution.

Heterotypy is now recognized as a generative force in the formation of new proteins through modification of existing proteins. We report that heterotypy in the N-terminal region of the mature growth/differentiation factor 5 (GDF5) protein occurred during evolution of teleosts. N-terminal length variation of GDF5 was found among teleost interfamilies and interorders but not within teleost families or among tetrapods. We further show that increase of proline and glutamine to the N-terminal region of mature GDF5 occurred in Eurypterygii, the higher lineage of teleosts. Because the basic amino acids, believed to control diffusion, are conserved in this region across all species examined, we suggest that the N-terminal elongation of the mature GDF5 protein during evolution has altered the protein diffusion in Eurypterygii, leading to high concentrations of the protein in the joint of the pharyngeal skeleton, the location of cartilage formation during development.

Divergent selection on opsins drives incipient speciation in Lake Victoria cichlids.

Divergent natural selection acting on ecological traits, which also affect mate choice, is a key element of ecological speciation theory, but has not previously been demonstrated at the molecular gene level to our knowledge. Here we demonstrate parallel evolution in two cichlid genera under strong divergent selection in a gene that affects both. Strong divergent natural selection fixed opsin proteins with different predicted light absorbance properties at opposite ends of an environmental gradient. By expressing them and measuring absorbance, we show that the reciprocal fixation adapts populations to divergent light environments. The divergent evolution of the visual system coincides with divergence in male breeding coloration, consistent with incipient ecological by-product speciation.