The Visual Opsin Gene Repertoires of Teleost Fishes: Evolution, Ecology, and Function.

Visual opsin genes expressed in the rod and cone photoreceptor cells of the retina are core components of the visual sensory system of vertebrates. Here, we provide an overview of the dynamic evolution of visual opsin genes in the most species-rich group of vertebrates, teleost fishes. The examination of the rich genomic resources now available for this group reveals that fish genomes contain more copies of visual opsin genes than are present in the genomes of amphibians, reptiles, birds, and mammals. The expansion of opsin genes in fishes is due primarily to a combination of ancestral and lineage-specific gene duplications. Following their duplication, the visual opsin genes of fishes repeatedly diversified at the same key spectral-tuning sites, generating arrays of visual pigments sensitive to the ultraviolet to red spectrum of light. Species-specific opsin gene repertoires correlate strongly with underwater light habitats, ecology, and color-based sexual selection.

Exon-based Phylogenomics and the Relationships of African Cichlid Fishes: Tackling the Challenges of Reconstructing Phylogenies with Repeated Rapid Radiations.

African cichlids (subfamily: Pseudocrenilabrinae) are among the most diverse vertebrates, and their propensity for repeated rapid radiation has made them a celebrated model system in evolutionary research. Nonetheless, despite numerous studies, phylogenetic uncertainty persists, and riverine lineages remain comparatively underrepresented in higher-level phylogenetic studies. Heterogeneous gene histories resulting from incomplete lineage sorting (ILS) and hybridization are likely sources of uncertainty, especially during episodes of rapid speciation. We investigate the relationships of Pseudocrenilabrinae and its close relatives while accounting for multiple sources of genetic discordance using species tree and hybrid network analyses with hundreds of single-copy exons. We improve sequence recovery for distant relatives, thereby extending the taxonomic reach of our probes, with a hybrid reference guided/de novo assembly approach. Our analyses provide robust hypotheses for most higher-level relationships and reveal widespread gene heterogeneity, including in riverine taxa. ILS and past hybridization are identified as the sources of genetic discordance in different lineages. Sampling of various Blenniiformes (formerly Ovalentaria) adds strong phylogenomic support for convict blennies (Pholidichthyidae) as sister to Cichlidae and points to other potentially useful protein-coding markers across the order. A reliable phylogeny with representatives from diverse environments will support ongoing taxonomic and comparative evolutionary research in the cichlid model system. [African cichlids; Blenniiformes; Gene tree heterogeneity; Hybrid assembly; Phylogenetic network; Pseudocrenilabrinae; Species tree.].

Expanded olfactory system in ray-finned fishes capable of terrestrial exploration.

BACKGROUND: Smell abilities differ greatly among vertebrate species due to distinct sensory needs, with exceptional variability reported in the number of olfactory genes and the size of the odour-processing regions of the brain. However, key environmental factors shaping genomic and phenotypic changes linked to the olfactory system remain difficult to identify at macroevolutionary scales. Here, we investigate the association between diverse ecological traits and the number of olfactory chemoreceptors in approximately two hundred ray-finned fishes. RESULTS: We found independent expansions producing large gene repertoires in several lineages of nocturnal amphibious fishes, generally able to perform active terrestrial exploration. We reinforced this finding with on-purpose genomic and transcriptomic analysis of Channallabes apus, a catfish species from a clade with chemosensory-based aerial orientation. Furthermore, we also detected an augmented information-processing capacity in the olfactory bulb of nocturnal amphibious fishes by estimating the number of cells contained in this brain region in twenty-four actinopterygian species. CONCLUSIONS: Overall, we report a convergent genomic and phenotypic magnification of the olfactory system in nocturnal amphibious fishes. This finding suggests the possibility of an analogous evolutionary event in fish-like tetrapod ancestors during the first steps of the water-to-land transition, favouring terrestrial adaptation through enhanced aerial orientation.

Visual Gene Expression Reveals a cone-to-rod Developmental Progression in Deep-Sea Fishes.

Vertebrates use cone cells in the retina for color vision and rod cells to see in dim light. Many deep-sea fishes have adapted to their environment to have only rod cells in the retina, while both rod and cone genes are still preserved in their genomes. As deep-sea fish larvae start their lives in the shallow, and only later submerge to the depth, they have to cope with diverse environmental conditions during ontogeny. Using a comparative transcriptomic approach in 20 deep-sea fish species from eight teleost orders, we report on a developmental cone-to-rod switch. While adults mostly rely on rod opsin (RH1) for vision in dim light, larvae almost exclusively express middle-wavelength-sensitive ("green") cone opsins (RH2) in their retinas. The phototransduction cascade genes follow a similar ontogenetic pattern of cone-followed by rod-specific gene expression in most species, except for the pearleye and sabretooth (Aulopiformes), in which the cone cascade remains dominant throughout development, casting doubts on the photoreceptor cell identity. By inspecting the whole genomes of five deep-sea species (four of them sequenced within this study: Idiacanthus fasciola, Chauliodus sloani; Stomiiformes; Coccorella atlantica, and Scopelarchus michaelsarsi; Aulopiformes), we found that they possess one or two copies of the rod RH1 opsin gene, and up to seven copies of the cone RH2 opsin genes in their genomes, while other cone opsin classes have been mostly lost. Our findings hence provide molecular evidence for a limited opsin gene repertoire in deep-sea fishes and a conserved vertebrate pattern whereby cone photoreceptors develop first and rod photoreceptors are added only at later developmental stages.

Developmental changes of opsin gene expression in ray-finned fishes (Actinopterygii).

Fish often change their habitat and trophic preferences during development. Dramatic functional differences between embryos, larvae, juveniles and adults also concern sensory systems, including vision. Here, we focus on the photoreceptors (rod and cone cells) in the retina and their gene expression profiles during development. Using comparative transcriptomics on 63 species, belonging to 23 actinopterygian orders, we report general developmental patterns of opsin expression, mostly suggesting an increased importance of the rod opsin (RH1) gene and the long-wavelength-sensitive cone opsin, and a decreasing importance of the shorter wavelength-sensitive cone opsin throughout development. Furthermore, we investigate in detail ontogenetic changes in 14 selected species (from Polypteriformes, Acipenseriformes, Cypriniformes, Aulopiformes and Cichliformes), and we report examples of expanded cone opsin repertoires, cone opsin switches (mostly within RH2) and increasing rod : cone ratio as evidenced by the opsin and phototransduction cascade genes. Our findings provide molecular support for developmental stage-specific visual palettes of ray-finned fishes and shifts between, which most likely arose in response to ecological, behavioural and physiological factors.

Molecular evolution and depth-related adaptations of rhodopsin in the adaptive radiation of cichlid fishes in Lake Tanganyika.

The visual sensory system is essential for animals to perceive their environment and is thus under strong selection. In aquatic environments, light intensity and spectrum differ primarily along a depth gradient. Rhodopsin (RH1) is the only opsin responsible for dim-light vision in vertebrates and has been shown to evolve in response to the respective light conditions, including along a water depth gradient in fishes. In this study, we examined the diversity and sequence evolution of RH1 in virtually the entire adaptive radiation of cichlid fishes in Lake Tanganyika, focusing on adaptations to the environmental light with respect to depth. We show that Tanganyikan cichlid genomes contain a single copy of RH1. The 76 variable amino acid sites detected in RH1 across the radiation were not uniformly distributed along the protein sequence, and 31 of these variable sites show signals of positive selection. Moreover, the amino acid substitutions at 15 positively selected sites appeared to be depth-related, including three key tuning sites that directly mediate shifts in the peak spectral sensitivity, one site involved in protein stability and 11 sites that may be functionally important on the basis of their physicochemical properties. Among the strongest candidate sites for deep-water adaptations are two known key tuning sites (positions 292 and 299) and three newly identified variable sites (37, 104 and 290). Our study, which is the first comprehensive analysis of RH1 evolution in a massive adaptive radiation of cichlid fishes, provides novel insights into the evolution of RH1 in a freshwater environment.

Protected area characteristics that help waterbirds respond to climate warming.

Protected area networks help species respond to climate warming. However, the contribution of a site's environmental and conservation-relevant characteristics to these responses is not well understood. We investigated how composition of nonbreeding waterbird communities (97 species) in the European Union Natura 2000 (N2K) network (3018 sites) changed in response to increases in temperature over 25 years in 26 European countries. We measured community reshuffling based on abundance time series collected under the International Waterbird Census relative to N2K sites' conservation targets, funding, designation period, and management plan status. Waterbird community composition in sites explicitly designated to protect them and with management plans changed more quickly in response to climate warming than in other N2K sites. Temporal community changes were not affected by the designation period despite greater exposure to temperature increase inside late-designated N2K sites. Sites funded under the LIFE program had lower climate-driven community changes than sites that did not received LIFE funding. Our findings imply that efficient conservation policy that helps waterbird communities respond to climate warming is associated with sites specifically managed for waterbirds.

Las redes de áreas protegidas ayudan a las especies a responder al calentamiento climático. Sin embargo, se sabe muy poco sobre la contribución de las características ambientales y relevantes para la conservación de un sitio a estas respuestas. Investigamos cómo la composición de las comunidades no reproductivas de aves acuáticas (97 especies) en la red (3,018 sitios) Natura 2000 de la Unión Europea (N2K) cambió en respuesta a los incrementos de la temperatura durante más de 25 años en 26 países europeos. Medimos la reorganización comunitaria con base en series temporales de abundancia recolectadas durante el Censo Internacional de Aves Acuáticas en relación con los objetivos de conservación de los sitios N2K, el periodo de asignación de fondos y el estado del plan de manejo. La composición comunitaria de las aves acuáticas en los sitios con planes de manejo y designados explícitamente para su protección cambió más rápidamente en respuesta al calentamiento climático que en otros sitios N2K. Los cambios comunitarios temporales no se vieron afectados por el periodo de asignación a pesar de una mayor exposición al incremento de la temperatura dentro de los sitios N2K de asignación tardía. Los sitios financiados por el programa LIFE tuvieron menos cambios comunitarios causados por el clima que los sitios que no recibieron este financiamiento. Nuestros hallazgos sugieren que la política de conservación eficiente que ayuda a las comunidades de aves acuáticas a responder al calentamiento climático está asociada con sitios específicamente gestionados para las aves acuáticas.

Towards the phylogenetic placement of the enigmatic African genus Prolabeops Schultz, 1941.

The small cyprinid genus Prolabeops Schultz, 1941 is restricted to the Nyong and Sanaga River systems in Cameroon. In the past, the genus had been suggested to be either a member of the Labeoninae, Torinae or the Smiliogastrinae mainly on the basis of morphological similarities, and it is nowadays considered as incertae sedis within the Cypriniformes. This study provides the first attempt to reveal the phylogenetic position of Prolabeops using molecular data. For this purpose, the authors sequenced a large fraction of the mitochondrial genome (c. 13,600 bp), including all mitochondrial protein coding genes, of two Prolabeops melanhypopterus specimens and an additional four Enteromius specimens. The large-scale phylogenetic analysis was based on an alignment including all mitochondrial protein coding genes of 902 specimens representing c. 899 cypriniform species. Prolabeops was clearly recovered within the African Smiliogastrinae, forming a weakly supported clade together with Enteromius jae, Enteromius hulstaerti and Barboides gracilis. The study data underline the urgent need of a thorough taxonomic revision of the small African barbs collectively placed in the genus Enteromius.

One northward, one southward: Contrasting biogeographical history in two benthic freshwater fish genera across Southeast Asia (Teleostei: Cobitoidea: Nemacheilus, Pangio).

Southeast Asia is one of the world's biodiversity hotspots, and the high level of diversity and endemism was reached by colonisation events as well as internal diversification. We investigate the phylogenetic relationships and biogeographic history of the loach genus Nemacheilus, which is widely distributed and common across freshwaters of Southeast Asia. In addition we present the ancestral range reconstruction of the related loach genus Pangio that commonly occurs in the same region as Nemacheilus. Our results reveal that the species currently classified as Nemacheilus in fact are a polyphyletic assemblage; most species are now retaining in a monophyletic Nemacheilus sensu stricto and five species belong to different lineages. We further indicate the existence of hidden diversity within Nemacheilus in the form of several undescribed species. Three major clades (Selangoricus, Masyae and Ornatus) are found within the genus Nemacheilus sensu stricto. These clades generally correspond to the species groups formerly defined on the basis of their pigmentation pattern. The biogeographic analyses show that Nemacheilus most likely originated in mainland Southeast Asia and subsequently expanded in a southward direction to Borneo, Sumatra and Java and the southern Malay Peninsula. In contrast, the genus Pangio originated in Sundaland, from where it extended several times northwards into Indochina and to northern India. Our results demonstrate that small freshwater fishes with restricted dispersal ability are very helpful for the reconstruction of biogeographic history. The contrasting biogeographic history of these two groups of small, benthic and related fish show how complex and case-specific the processes that lead to the biodiversity richness of Southeast Asia are.

Benefits of protected areas for nonbreeding waterbirds adjusting their distributions under climate warming.

Climate warming is driving changes in species distributions and community composition. Many species have a so-called climatic debt, that is, shifts in range lag behind shifts in temperature isoclines. Inside protected areas (PAs), community changes in response to climate warming can be facilitated by greater colonization rates by warm-dwelling species, but also mitigated by lowering extirpation rates of cold-dwelling species. An evaluation of the relative importance of colonization-extirpation processes is important to inform conservation strategies that aim for both climate debt reduction and species conservation. We assessed the colonization-extirpation dynamics involved in community changes in response to climate inside and outside PAs. To do so, we used 25 years of occurrence data of nonbreeding waterbirds in the western Palearctic (97 species, 7071 sites, 39 countries, 1993-2017). We used a community temperature index (CTI) framework based on species thermal affinities to investigate species turnover induced by temperature increase. We determined whether thermal community adjustment was associated with colonization by warm-dwelling species or extirpation of cold-dwelling species by modeling change in standard deviation of the CTI (CTISD ). Using linear mixed-effects models, we investigated whether communities in PAs had lower climatic debt and different patterns of community change than communities outside PAs. For CTI and CTISD combined, communities inside PAs had more species, higher colonization, lower extirpation, and lower climatic debt (16%) than communities outside PAs. Thus, our results suggest that PAs facilitate 2 independent processes that shape community dynamics and maintain biodiversity. The community adjustment was, however, not sufficiently fast to keep pace with the large temperature increases in the central and northeastern western Palearctic. Our results underline the potential of combining CTI and CTISD metrics to improve understanding of the colonization-extirpation patterns driven by climate warming.

Beneficios de las Áreas Protegidas para las Aves Acuáticas No Reproductoras que Están Ajustando su Distribución Debido al Calentamiento Climático Resumen El calentamiento climático está generando cambios en la distribución y en la composición comunitaria de las especies. Muchas de ellas tienen una deuda climática, es decir, los cambios en la distribución se atrasan con respecto a los cambios en las isoclinas térmicas. Dentro de las áreas protegidas (APs), los cambios comunitarios como respuesta al calentamiento climático pueden facilitarse mediante tasas mayores de colonización por especies de climas cálidos, pero también pueden mitigarse al reducir las tasas de extirpación de las especies de climas fríos. Se requiere una evaluación de la importancia relativa de los procesos de colonización-extirpación para orientar las estrategias de conservación que buscan la reducción de la deuda climática y la conservación de las especies. Analizamos las dinámicas de colonización-extirpación que participan en los cambios comunitarios como respuesta al clima dentro y fuera de las APs. Para realizar lo anterior, usamos datos tomados durante 25 años de la presencia de aves acuáticas no reproductoras en el Paleártico occidental (97 especies, 7,071 sitios, 39 países, 1993-2017). Usamos un marco de trabajo del índice de temperatura comunitaria (ITC) basado en las afinidades térmicas de las especies para así investigar la rotación de especies inducida por el incremento en la temperatura. Determinamos si el ajuste térmico en la comunidad estuvo asociado con la colonización por especies de climas cálidos o con la extirpación de especies de climas fríos al modelar el cambio mediante una desviación estándar del ITC (ITCDS ). Con los modelos lineales de efectos mixtos investigamos si las comunidades dentro de las APs tenían una deuda climática más baja y patrones diferentes de cambio comunitario que las comunidades localizadas fuera de las APs. Con la combinación del ITC y deL ITCDS , las comunidades dentro de las APs tuvieron más especies, una mayor colonización, una menor extirpación y una deuda climática más baja (16%) que las comunidades fuera de las APs. Por lo tanto, nuestros resultados sugieren que las APs facilitan dos procesos independientes que moldean las dinámicas comunitarias y mantienen la biodiversidad. Sin embargo, el ajuste comunitario no fue lo suficientemente rápido para mantener el paso de los grandes incrementos en la temperatura de las regiones central y noreste del Paleártico occidental. Nuestros resultados resaltan el potencial que tiene la combinación de las medidas del ITC y del ICTDS para mejorar el entendimiento de los patrones de colonización-extirpación causados por el calentamiento climático.

The round goby genome provides insights into mechanisms that may facilitate biological invasions.

BACKGROUND: The invasive benthic round goby (Neogobius melanostomus) is the most successful temperate invasive fish and has spread in aquatic ecosystems on both sides of the Atlantic. Invasive species constitute powerful in situ experimental systems to study fast adaptation and directional selection on short ecological timescales and present promising case studies to understand factors involved the impressive ability of some species to colonize novel environments. We seize the unique opportunity presented by the round goby invasion to study genomic substrates potentially involved in colonization success. RESULTS: We report a highly contiguous long-read-based genome and analyze gene families that we hypothesize to relate to the ability of these fish to deal with novel environments. The analyses provide novel insights from the large evolutionary scale to the small species-specific scale. We describe expansions in specific cytochrome P450 enzymes, a remarkably diverse innate immune system, an ancient duplication in red light vision accompanied by red skin fluorescence, evolutionary patterns of epigenetic regulators, and the presence of osmoregulatory genes that may have contributed to the round goby's capacity to invade cold and salty waters. A recurring theme across all analyzed gene families is gene expansions. CONCLUSIONS: The expanded innate immune system of round goby may potentially contribute to its ability to colonize novel areas. Since other gene families also feature copy number expansions in the round goby, and since other Gobiidae also feature fascinating environmental adaptations and are excellent colonizers, further long-read genome approaches across the goby family may reveal whether gene copy number expansions are more generally related to the ability to conquer new habitats in Gobiidae or in fish.

East African cichlid lineages (Teleostei: Cichlidae) might be older than their ancient host lakes: new divergence estimates for the east African cichlid radiation.

BACKGROUND: Cichlids are a prime model system in evolutionary research and several of the most prominent examples of adaptive radiations are found in the East African Lakes Tanganyika, Malawi and Victoria, all part of the East African cichlid radiation (EAR). In the past, great effort has been invested in reconstructing the evolutionary and biogeographic history of cichlids (Teleostei: Cichlidae). In this study, we present new divergence age estimates for the major cichlid lineages with the main focus on the EAR based on a dataset encompassing representative taxa of almost all recognized cichlid tribes and ten mitochondrial protein genes. We have thoroughly re-evaluated both fossil and geological calibration points, and we included the recently described fossil †Tugenchromis pickfordi in the cichlid divergence age estimates. RESULTS: Our results estimate the origin of the EAR to Late Eocene/Early Oligocene (28.71 Ma; 95% HPD: 24.43-33.15 Ma). More importantly divergence ages of the most recent common ancestor (MRCA) of several Tanganyika cichlid tribes were estimated to be substantially older than the oldest estimated maximum age of the Lake Tanganyika: Trematocarini (16.13 Ma, 95% HPD: 11.89-20.46 Ma), Bathybatini (20.62 Ma, 95% HPD: 16.88-25.34 Ma), Lamprologini (15.27 Ma; 95% HPD: 12.23-18.49 Ma). The divergence age of the crown haplochromine H-lineage is estimated to 22.8 Ma (95% HPD: 14.40-26.32 Ma) and of the Lake Malawi radiation to 4.07 Ma (95% HDP: 2.93-5.26 Ma). In addition, we recovered a novel lineage within the Lamprologini tribe encompassing only Lamprologus of the lower and central Congo drainage with its divergence estimated to the Late Miocene or early Pliocene. Furthermore we recovered two novel mitochondrial haplotype lineages within the Haplochromini tribe: 'Orthochromis' indermauri and 'Haplochormis' vanheusdeni. CONCLUSIONS: Divergence time estimates of the MRCA of several Tanganyika cichlid tribes predate the age of the extant Lake Tanganyika basin, and hence are in line with the recently formulated "Melting-Pot Tanganyika" hypothesis. The radiation of the 'Lower Congo Lamprologus clade' might be linked with the Pliocene origin of the modern lower Congo rapids as has been shown for other Lower Congo cichlid assemblages. Finally, the age of origin of the Lake Malawi cichlid flock agrees well with the oldest age estimate for lacustrine conditions in Lake Malawi.

Evolution of the visual sensory system in cichlid fishes from crater lake Barombi Mbo in Cameroon.

In deep-water animals, the visual sensory system is often challenged by the dim-light environment. Here, we focus on the molecular mechanisms involved in rapid deep-water adaptations. We examined visual system evolution in a small-scale yet phenotypically and ecologically diverse adaptive radiation, the species flock of cichlid fishes in deep crater lake Barombi Mbo in Cameroon, West Africa. We show that rapid adaptations of the visual system to the novel deep-water habitat primarily occurred at the level of gene expression changes rather than through nucleotide mutations, which is compatible with the young age of the radiation. Based on retinal bulk RNA sequencing of all eleven species, we found that the opsin gene expression pattern was substantially different for the deep-water species. The nine shallow-water species feature an opsin palette dominated by the red-sensitive (LWS) opsin, whereas the two unrelated deep-water species lack expression of LWS and the violet-sensitive (SWS2B) opsin, thereby shifting the cone sensitivity to the centre of the light spectrum. Deep-water species further predominantly express the green-sensitive RH2Aα over RH2Aß. We identified one amino acid substitution in the RH2Aα opsin specific to the deep-water species. We finally performed a comparative gene expression analysis in retinal tissue of deep- vs. shallow-water species. We thus identified 46 differentially expressed genes, many of which are associated with functions in vision, hypoxia management or circadian clock regulation, with some of them being associated with human eye diseases.

Adaptive Radiation from a Chromosomal Perspective: Evidence of Chromosome Set Stability in Cichlid Fishes (Cichlidae: Teleostei) from the Barombi Mbo Lake, Cameroon.

Cichlid fishes are the subject of scientific interest because of their rapid adaptive radiation, resulting in extensive ecological and taxonomic diversity. In this study, we examined 11 morphologically distinct cichlid species endemic to Barombi Mbo, the largest crater lake in western Cameroon, namely Konia eisentrauti, Konia dikume, Myaka myaka, Pungu maclareni, Sarotherodon steinbachi, Sarotherodon lohbergeri, Sarotherodon linnellii, Sarotherodon caroli, Stomatepia mariae, Stomatepia pindu, and Stomatepia mongo. These species supposedly evolved via sympatric ecological speciation from a common ancestor, which colonized the lake no earlier than one million years ago. Here we present the first comparative cytogenetic analysis of cichlid species from Barombi Mbo Lake using both conventional (Giemsa staining, C-banding, and CMA3/DAPI staining) and molecular (fluorescence in situ hybridization with telomeric, 5S, and 28S rDNA probes) methods. We observed stability on both macro and micro-chromosomal levels. The diploid chromosome number was 2n = 44, and the karyotype was invariably composed of three pairs of meta/submetacentric and 19 pairs of subtelo/acrocentric chromosomes in all analysed species, with the same numbers of rDNA clusters and distribution of heterochromatin. The results suggest the evolutionary stability of chromosomal set; therefore, the large-scale chromosomal rearrangements seem to be unlikely associated with the sympatric speciation in Barombi Mbo.

Bayesian Phylogenetic Estimation of Clade Ages Supports Trans-Atlantic Dispersal of Cichlid Fishes.

Divergence-time estimation based on molecular phylogenies and the fossil record has provided insights into fundamental questions of evolutionary biology. In Bayesian node dating, phylogenies are commonly time calibrated through the specification of calibration densities on nodes representing clades with known fossil occurrences. Unfortunately, the optimal shape of these calibration densities is usually unknown and they are therefore often chosen arbitrarily, which directly impacts the reliability of the resulting age estimates. As possible solutions to this problem, two nonexclusive alternative approaches have recently been developed, the "fossilized birth­death" (FBD) model and "total-evidence dating." While these approaches have been shown to perform well under certain conditions, they require including all (or a random subset) of the fossils of each clade in the analysis, rather than just relying on the oldest fossils of clades. In addition, both approaches assume that fossil records of different clades in the phylogeny are all the product of the same underlying fossil sampling rate, even though this rate has been shown to differ strongly between higher level taxa. We here develop a flexible new approach to Bayesian age estimation that combines advantages of node dating and the FBD model. In our new approach, calibration densities are defined on the basis of first fossil occurrences and sampling rate estimates that can be specified separately for all clades. We verify our approach with a large number of simulated data sets, and compare its performance to that of the FBD model. We find that our approach produces reliable age estimates that are robust to model violation, on par with the FBD model. By applying our approach to a large data set including sequence data from over 1000 species of teleost fishes as well as 147 carefully selected fossil constraints, we recover a timeline of teleost diversification that is incompatible with previously assumed vicariant divergences of freshwater fishes. Our results instead provide strong evidence for transoceanic dispersal of cichlids and other groups of teleost fishes.

Ancestral duplications and highly dynamic opsin gene evolution in percomorph fishes.

Single-gene and whole-genome duplications are important evolutionary mechanisms that contribute to biological diversification by launching new genetic raw material. For example, the evolution of animal vision is tightly linked to the expansion of the opsin gene family encoding light-absorbing visual pigments. In teleost fishes, the most species-rich vertebrate group, opsins are particularly diverse and key to the successful colonization of habitats ranging from the bioluminescence-biased but basically dark deep sea to clear mountain streams. In this study, we report a previously unnoticed duplication of the violet-blue short wavelength-sensitive 2 (SWS2) opsin, which coincides with the radiation of highly diverse percomorph fishes, permitting us to reinterpret the evolution of this gene family. The inspection of close to 100 fish genomes revealed that, triggered by frequent gene conversion between duplicates, the evolutionary history of SWS2 is rather complex and difficult to predict. Coincidentally, we also report potential cases of gene resurrection in vertebrate opsins, whereby pseudogenized genes were found to convert with their functional paralogs. We then identify multiple novel amino acid substitutions that are likely to have contributed to the adaptive differentiation between SWS2 copies. Finally, using the dusky dottyback Pseudochromis fuscus, we show that the newly discovered SWS2A duplicates can contribute to visual adaptation in two ways: by gaining sensitivities to different wavelengths of light and by being differentially expressed between ontogenetic stages. Thus, our study highlights the importance of comparative approaches in gaining a comprehensive view of the dynamics underlying gene family evolution and ultimately, animal diversification.

Comparative transcriptomics of anal fin pigmentation patterns in cichlid fishes.

BACKGROUND: Understanding the genetic basis of novel traits is a central topic in evolutionary biology. Two novel pigmentation phenotypes, egg-spots and blotches, emerged during the rapid diversification of East African cichlid fishes. Egg-spots are circular pigmentation markings on the anal fins of hundreds of derived haplochromine cichlids species, whereas blotches are patches of conspicuous anal fin pigmentation with ill-defined boundaries that occur in few species that belong to basal cichlid lineages. Both traits play an important role in the breeding behavior of this group of fishes. Knowledge about the origin, homology and underlying genetics of these pigmentation traits is sparse. RESULTS: Here, we present a comparative transcriptomic and differential gene expression analysis of egg-spots and blotches. We first conducted an RNA sequencing experiment where we compared egg-spot tissue with the remaining portion of egg-spot-free fin tissue using six individuals of Astatotilapia burtoni. We identified 1229 differentially expressed genes between the two tissue types. We then showed that rates of evolution of these genes are higher than average estimated on whole transcriptome data. Using quantitative real-time PCR, we found that 29 out of a subset of 46 differentially expressed genes showed an analogous expression pattern in another haplochromine species' egg-spots, Cynotilapia pulpican, strongly suggesting that these genes are involved in the egg-spot phenotype. Among these are the previously identified egg-spot gene fhl2a, two known patterning genes (hoxC12a and bmp3) as well as other pigmentation related genes such as asip. Finally, we analyzed the expression patterns of the same gene subset in two species that feature blotches instead of egg-spots, one haplochromine species (Pseudocrenilabrus philander) and one ectodine species (Callochromis macrops), revealing that the expression patterns in blotches and egg-spots are rather distinct. CONCLUSIONS: We identified several candidate genes that will serve as an important and useful resource for future research on the emergence and diversification of cichlid fishes' egg-spots. Only a limited degree of conservation of gene expression patterns was detected between the egg-spots of the derived haplochromines and blotches from ancestral haplochromines, as well as between the two types of blotches, suggesting an independent origin of these traits.

From crypsis to mimicry: changes in colour and the configuration of the visual system during ontogenetic habitat transitions in a coral reef fish.

Animals often change their habitat throughout ontogeny; yet, the triggers for habitat transitions and how these correlate with developmental changes - e.g. physiological, morphological and behavioural - remain largely unknown. Here, we investigated how ontogenetic changes in body coloration and of the visual system relate to habitat transitions in a coral reef fish. Adult dusky dottybacks, Pseudochromis fuscus, are aggressive mimics that change colour to imitate various fishes in their surroundings; however, little is known about the early life stages of this fish. Using a developmental time series in combination with the examination of wild-caught specimens, we revealed that dottybacks change colour twice during development: (i) nearly translucent cryptic pelagic larvae change to a grey camouflage coloration when settling on coral reefs; and (ii) juveniles change to mimic yellow- or brown-coloured fishes when reaching a size capable of consuming juvenile fish prey. Moreover, microspectrophotometric (MSP) and quantitative real-time PCR (qRT-PCR) experiments show developmental changes of the dottyback visual system, including the use of a novel adult-specific visual gene (RH2 opsin). This gene is likely to be co-expressed with other visual pigments to form broad spectral sensitivities that cover the medium-wavelength part of the visible spectrum. Surprisingly, the visual modifications precede changes in habitat and colour, possibly because dottybacks need to first acquire the appropriate visual performance before transitioning into novel life stages.

The strange case of East African annual fishes: aridification correlates with diversification for a savannah aquatic group?

BACKGROUND: Annual Nothobranchius fishes are distributed in East and Southern Africa and inhabit ephemeral pools filled during the monsoon season. Nothobranchius show extreme life-history adaptations: embryos survive by entering diapause and they are the vertebrates with the fastest maturation and the shortest lifespan. The distribution of Nothobranchius overlaps with the East Africa Rift System. The geological and paleoclimatic history of this region is known in detail: in particular, aridification of East Africa and expansion of grassland habitats started 8 Mya and three humid periods between 3 and 1 Mya are superimposed on the longer-term aridification. These climatic oscillations are thought to have shaped evolution of savannah African mammals. We reconstructed the phylogeny of Nothobranchius and dated the different stages of diversification in relation to these paleoclimatic events. RESULTS: We sequenced one mitochondrial locus and five nuclear loci in 63 specimens and obtained a robust phylogeny. Nothobranchius can be divided in four geographically separated clades whose boundaries largely correspond to the East Africa Rift system. Statistical analysis of dispersal and vicariance identifies a Nilo-Sudan origin with southwards dispersion and confirmed that these four clades are the result of vicariance events In the absence of fossil Nothobranchius, molecular clock was calibrated using more distant outgroups (secondary calibration). This method estimates the age of the Nothobranchius genus to be 8.3 (6.0 - 10.7) My and the separation of the four clades 4.8 (2.7-7.0) Mya. Diversification within the clades was estimated to have started ~3 Mya and most species pairs were estimated to have an age of 0.5-1 My. CONCLUSIONS: The mechanism of Nothobranchius diversification was allopatric and driven by geographic isolation. We propose a scenario where diversification of Nothobranchius started in rough coincidence with aridification of East Africa, establishment of grassland habitats and the appearance of the typical African bovid fauna of the savannah. Although confidence intervals for the estimated ages of the four Nothobranchius clades are quite large, this scenario is compatible with the biology of extant Nothobranchius that are critically dependent on savannah habitats. Therefore, Nothobranchius diversification might have been shaped by the same paleoclimatic events that shaped African ungulate evolution.

The evolution of the green-light-sensitive visual opsin genes (RH2) in teleost fishes.

Vertebrates have four visual cone opsin classes that mediate sensitivity from ultraviolet to red wavelengths of light. The rhodopsin-like 2 (RH2) opsin is sensitive to the central mostly green part of the spectrum. While lost in some terrestrial vertebrates (mammals), the RH2 opsin gene has proliferated during the evolution of teleost fishes. Here, we investigated the genomes of 132 extant teleosts and found between zero and eight RH2 gene copies per species. The RH2 gene shows a dynamic evolutionary history with repeated gene duplications, gene losses, and gene conversions affecting entire orders, families, and species. At least four ancestral duplications provided the substrate for today's RH2 diversity, with duplications occurring in the common ancestors of Clupeocephala (twice), Neoteleostei, and likely Acanthopterygii as well. Despite these evolutionary dynamics, we identified conserved RH2 synteny in two main gene clusters; the slc6A13/synpr cluster is highly conserved within Percomorpha and also present across most teleosts, including Otomorpha, Euteleostei and in parts in tarpons (Elopomorpha), and the mutSH5 cluster, which is specific for Otomorpha. When comparing the number of visual opsin genes (SWS1, SWS2, RH2, LWS, and total cone opsins) with habitat depth, we found that deeper-dwelling species had less (or none) long-wavelength-sensitive opsins. Using retinal/eye transcriptomes in a phylogenetic representative dataset of 32 species, we show that if present in the genome, RH2 is expressed in most fishes except for some species within the tarpons, characins, and gobies (and Osteoglossomorpha and some other characin species have lost the gene). Those species instead express a green-shifted long-wavelength-sensitive LWS opsin. Our study applies modern genomic and transcriptomic tools within a comparative framework to elucidate the evolutionary history of the visual sensory system in teleost fishes.