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More than ten species of the genus Sinocyclocheilus (Cyprinidae: Barbinae) have been found to exhibit striking variation related to cave-dwelling traits. Until now, the molecular phylogeny and evolutionary history of this genus have remained unclear. To understand the molecular phylogeny and biogeographic evolution of Sinocyclocheilus, we analyzed the complete mitogenomes of 12 Sinocyclocheilus species based on Bayesian Inference (BI) and Maximum Likelihood (ML) analyses. The results of phylogenetic analyses strongly suggest that the genus Sinocyclocheilus is a monophyletic group, and S. jii is the most basal species. Molecular clock estimates showed that the diversification of this genus occurred in the late Miocene (about 6.49 Ma), which was associated with the intensive uplift of the Tibetan Plateau. Selective pressure analyses showed the dN/dS ratios of all Sinocyclocheilus species were

The complete mitochondrial DNA genome of Sinocyclocheilus xiaotunensis was first reported by next-generation sequencing method. The entire length of mitochondrial genome is 16,588 bp and the nucleo...

The edible silver carp (Hypophthalmichthys molitrix) and bighead carp (H. nobilis), which are two of the "Four Domesticated Fish" of China, are cultivated intensively worldwide. Here, we constructed 837- and 845-Mb draft genome assemblies for the silver carp and the bighead carp, respectively, including 24,571 and 24,229 annotated protein-coding genes. Genetic maps, anchoring 71.7% and 83.8% of all scaffolds, were obtained for the silver and bighead carp, respectively. Phylogenetic analysis showed that the bighead carp formed a clade with the silver carp, with an estimated divergence time of 3.6 million years ago; the time of divergence between the silver carp and zebrafish was 50.7 million years ago. An East Asian cyprinid genome-specific chromosome fusion took place ~9.2 million years after this clade diverged from the clade containing the common carp and Sinocyclocheilus. KEGG and GO analyses indicated that the expanded gene families in the silver and bighead carp were associated with diseases, the immune system and environmental adaptations. Genomic regions differentiating the silver and bighead carp populations were detected based on the whole-genome sequences of 42 individuals. Genes associated with the divergent regions were associated with reproductive system development and the development of primary female sexual characteristics. Thus, our results provided a novel systematic genomic analysis of the East Asian cyprinids, as well as the evolution and speciation of the silver carp and bighead carp.

Cave‐dwelling animals evolve distinct troglomorphic traits, such as loss of eyes, skin pigmentation, and augmentation of senses following long‐term adaptation to perpetual darkness. However, the molecular genetic mechanisms underlying these phenotypic variations remain unclear. In this study, we conducted comparative histology and comparative transcriptomics study of the skin of eight Sinocyclocheilus species (Cypriniformes: Cyprinidae) that included surface‐ and cave‐dwelling species. We analyzed four surface and four cavefish species by using next‐generation sequencing, and a total of 802,798,907 clean reads were generated and assembled into 505,495,009 transcripts, which contributed to 1,037,334 unigenes. Bioinformatic comparisons revealed 10,629 and 6,442 significantly differentially expressed unigenes between four different surface‐cave fish groups. Further, tens of differentially expressed genes (DEGs) potentially related to skin pigmentation were identified. Most of these DEGs (including GNAQ, PKA, NRAS, and p38) are downregulated in cavefish species. They are involved in key signaling pathways of pigment synthesis, such as the melanogenesis, Wnt, and MAPK pathways. This trend of downregulation was confirmed through qPCR experiments. This study will deepen our understanding of the formation of troglomorphic traits in cavefishes., We conducted a comparative histology and comparative transcriptomics study on the skin of eight Sinocyclocheilus species, including four surface and four cavefish species. The result suggests that these differentially expressed genes (DEGs) may be responsible for pigmentation loss in the Sinocyclocheilus cavefish species.

The adaptive evolution of visual systems has been observed in many cavefish. However, little is known about the molecular mechanisms underlying these adaptations, which include regressive changes such as eye degeneration. Here, we analyzed phylogenetic and expression patterns of 6 eye-related genes (crx, foxg1b, opn1sw2, otx2, rho and sox2) in 12 Sinocyclocheilus species from China, including 8 stygobionts and 4 stygophiles, and examined photoreceptor cell morphology of these species. Those eye-degenerated species of Sinocyclocheilus were polyphyletic and showed different degrees of photoreceptor defects in responses to cave environments. The eye loss and degeneration are the result of convergent evolution. Although S. anophthalmus grouped with the eye-normal species, it displayed not only a high degree of eye degeneration but also significant expression differences in eye-related genes compared with the eye-normal species. The gene foxg1b, which was determined to be under positive selection, might play an important role in the process of eye degeneration in S. anophthalmus based on differential expression. Eye-related gene expression and selection may have contributed to the polyphyly of the cave species. We examined gene expression and duplication in 6 eye-related genes and revealed that these genes displayed considerable diversity in relative expression in Sinocyclocheilus fishes. Otx2 and sox2 were significantly up-regulated in individual cave species, while the other 4 genes (crx, foxg1b, opn1sw2 and rho) were significantly down-regulated. These findings provide a valuable resource for elucidating molecular mechanisms associated with visual system evolution in cavefish.

Cavefishes represent one of the most bizarre and intriguing life forms inhabiting groundwater environments. One‐third of the known cavefishes worldwide is endemic to China, and almost half of those belongs to a single genus, Sinocyclocheilus (Cypriniformes: Cyprinidae). Analyzing the morphometrics of three Sinocyclocheilus species, we aimed to assess whether variability among conspecific populations exists. We predict that populations inhabiting different subterranean habitats (shallow vs. deep) show divergences in specific morphological traits to better cope with the local ecological conditions. Our results showed that the populations showing bigger eyes and reduced humpback were those occurring close to the cave entrance (habitats with light and high food availability), while specimens with smaller eyes and increased humpback were collected from deeper groundwater areas (habitats laying in darkness with food scarcity). This explorative study paves the way for further researches aiming to collect novel data on Chinese cavefishes and highlights the usefulness of these species in evolutionary studies., Chinese cavefish are among the less studied vertebrates species. Conspecific populations show variability for some morphological traits, which are related to the ecological features of the inhabited environment.

The symbiosis between a host and its microbiome is essential for host fitness, and this association is a consequence of the host’s physiology and habitat. Sinocyclocheilus, the largest cavefish diversification of the world, an emerging multi-species model system for evolutionary novelty, provides an excellent opportunity for examining correlates of host evolutionary history, habitat, and gut-microbial community diversity. From the diversification-scale patterns of habitat occupation, major phylogenetic clades (A–D), geographic distribution, and knowledge from captive-maintained Sinocyclocheilus populations, we hypothesize habitat to be the major determinant of microbiome diversity, with phylogeny playing a lesser role. For this, we subject environmental water samples and fecal samples (representative of gut-microbiome) from 24 Sinocyclocheilus species, both from the wild and after being in captivity for six months, to bacterial 16S rRNA gene profiling using Illumina sequencing. We see significant differences in the gut microbiota structure of Sinocyclocheilus, reflective of the three habitat types; gut microbiomes too, were influenced by host-related factors. There is no significant association between the gut microbiomes and host phylogeny. However, there is some microbiome related structure at clade level, with the most geographically distant clades (A and D) being the most distinct, and two geographically overlapping clades (B and C) being similar. Microbes inhabiting water were not a cause for significant differences in fish-gut microbiota, but water quality parameters was. Transferring from wild to captivity, the fish microbiomes changed significantly and became homogenized, signifying adaptability and highlighting the importance of environmental factors (habitat) in microbiome community assembly. The core microbiome of this group closely resembled that of other teleost fishes. Our results suggest that divergent selection giving rise to evolutionary novelties also includes the microbiome of these fishes, which provides a functional advantage for life in the resource-depleted cave environment.SIGNIFICANCE STATEMENTThe largest diversification of cavefishes of the world, Sinocyclocheilus, not only show that habitat, and phylogenetic clade is important in determining their gut microbiome, but also that they reach a common microbiome in captivity irrespective of their phylogenetic position, region of origin and habitat, indicating that they are adaptable in the context of microbe related changes in their environment.

Troglomorphism— any morphological adaptation enabling life to the constant darkness of caves, such as loss of pigment, reduced eyesight or blindness, over-developed tactile and olfactory organs—has long intrigued biologists. However, inferring the proximate and ultimate mechanisms driving the evolution of troglomorphism in freshwater fish requires a sound understanding of the evolutionary relationships between surface and troglomorphic lineages. We use Restriction Site Associated DNA Sequencing (RADseq) to better understand the evolution of theSinocyclocheilusfishes of China. With a remarkable array of derived troglomorphic traits, they comprise the largest cavefish diversification in the world, emerging as a multi-species model system to study evolutionary novelty. We sequenced a total of 120 individuals throughout theSinocyclocheilusdistribution. The data comprised a total of 646,497⍰bp per individual, including 4378 loci and 67,983 SNPs shared across a minimum of 114 individuals at a given locus. Phylogenetic analyses using either the concatenated RAD loci (RAxML) or the SNPs under a coalescent model (SVDquartets, SNAPP) showed a high degree of congruence with similar topologies and high node support (> 95 for most nodes in the phylogeny). The major clades recovered conform to a pattern previously established using Sanger-based mt-DNA sequences, with a few notable exceptions. We now recognize six major clades in this group, elevating the blind cavefishS. tianlinensisand the micro-eyedS. microphthalmusas two new distinct clades due to their deep divergence from other clades. PCA plots of the SNP data also supports the recognition of six major clusters of species congruent with the identified clades based on the spatial arrangement and overlap of the species in the PC space. A Bayes factor delimitation (BFD) analysis showed support for 21 species, recognizing 19 previously described species and two putative new cryptic ones. Two species whose identities were previously disputed,S. furcodorsalisandS. tianeensis, are supported here as distinct species. In addition, our multi-species calibrated tree in SNAPP suggests that the genusSinocyclocheilusoriginated around 10.5 Mya, with most speciation events occurring in the last 2 Mya, likely favored by the uplift of the Qinghai-Tibetan Plateau and cave occupation induced by climate-driven aridification during this period. These results provide a firm basis for future comparative studies on the evolution ofSinocyclocheilusand its adaptations to cave life.

An adult Sinocyclocheilus maitianheensis, a surface-dwelling golden-line barbel fish, was collected from Maitian river (Kunming City, Yunnan Province, China) for whole-genome sequencing, assembly, and annotation. We obtained a genome assembly of 1.7 Gb with a scaffold N50 of 1.4 Mb and a contig N50 of 24.7 kb. A total of 39,977 protein-coding genes were annotated. Based on a comparative phylogenetic analysis of five Sinocyclocheilus species and other five representative vertebrates with published genome sequences, we found that S. maitianheensis is close to Sinocyclocheilus anophthalmus (a cave-restricted species with similar locality). Moreover, the assembled genomes of S. maitianheensis and other four Sinocyclocheilus counterparts were used for a fourfold degenerative third-codon transversion (4dTv) analysis. The recent whole-genome duplication (WGD) event was therefore estimated to occur about 18.1 million years ago. Our results also revealed a decreased tendency of copy number in many important genes related to immunity and apoptosis in cave-restricted Sinocyclocheilus species. In summary, we report the first genome assembly of S. maitianheensis, which provides a valuable genetic resource for comparative studies on cavefish biology, species protection, and practical aquaculture of this potentially economical fish.

Cave adaptation has given rise to a diversity of unusual morphologies. One prominent character in Chinese cavefishes of the genus Sinocyclocheilus is the presence of a skull horn and a dorsal hump. These characters are present only in species that are troglobitic. Here we examine the osteological growth that underlies the horn and the hump of the species S. furcodorsalis using micro computed tomography. We tested the hypothesis that all structures grow isometrically, and found that the skull and the bony shelf that supports the horn grow isometrically in all dimensions. The neural spine of the first vertebrae, on the other hand, grew allometrically, getting taller and thinner with time. When comparing each structure with skull growth, the horn grew isometrically in the anterior-posterior and the medial-lateral dimensions. But the horn grew faster in height than the skull. The neural spine grew isometrically with the skull in the rostral-caudal dimension, but allometrically in the dorsal-ventral and medial-lateral dimensions. Even though the function of the horn and the hump are not known, our results suggest that heterochronic changes has led to allometric growth in these structures.

A blind fish of Sinocyclocheilus (Cypriniformes: Cyprinidae)was caught in open water in the Three Gorges (Sanxia)reservoir, at a depth of 20 m in the mainstream of YangtzeRiver in Zigui County, Hubei Province, China. This fish can beeasily distinguished from all other congeners by externalmorphological characteristics, and is estimated to havediverged from its sister group about 0.55 million years ago(Ma). The geologically well separated locality of this specieshas expanded the distribution of Sinocyclocheilus cavefishfrom around N25° (latitude) to above N30° . Herein, wedescribe this new species as Sinocyclocheilus sanxiaensis sp.nov ., and discuss the possible reasons why the speciesappears, surprisingly, in the Three Gorges reservoir.

Evolution sometimes proceeds by loss, especially when structures and genes become dispensable after an environmental shift relaxes functional constraints. Subterranean vertebrates are outstanding models to analyze this process, and gene decay can serve as a readout. We sought to understand some general principles on the extent and tempo of the decay of genes involved in vision, circadian clock, and pigmentation in cavefishes. The analysis of the genomes of two Cuban species belonging to the genus Lucifuga provided evidence for the largest loss of eye-specific genes and nonvisual opsin genes reported so far in cavefishes. Comparisons with a recently evolved cave population of Astyanax mexicanus and three species belonging to the Chinese tetraploid genus Sinocyclocheilus revealed the combined effects of the level of eye regression, time, and genome ploidy on eye-specific gene pseudogenization. The limited extent of gene decay in all these cavefishes and the very small number of loss-of-function mutations per pseudogene suggest that their eye degeneration may not be very ancient, ranging from early to late Pleistocene. This is in sharp contrast with the identification of several vision genes carrying many loss-of-function mutations in ancient fossorial mammals, further suggesting that blind fishes cannot thrive more than a few million years in cave ecosystems.

Cavefish evolved a series of distinct survival mechanisms for adaptation to cave habitat. Such mechanisms include loss of eyesight and pigmentation, sensitive sensory organs, unique dietary preferences, and predation behavior. Thus, it is of great interest to understand the mechanisms underlying these adaptability traits of troglobites. The teleost genus Sinocyclocheilus (Cypriniformes: Cyprinidae) is endemic to China and has more than 70 species reported (including over 30 cavefish species). High species diversity and diverse phenotypes make the Sinocyclocheilus as an outstanding model for studying speciation and adaptive evolution. In this study, we conducted a comparative transcriptomics study on the brain tissues of two Sinocyclocheilus species (surface-dwelling species – Sinocyclocheilus malacopterus and semi-cave-dwelling species – Sinocyclocheilus rhinocerous living in the same water body. A total of 425,188,768 clean reads were generated, which contributed to 102,839 Unigenes. Bioinformatic analysis revealed a total of 3,289 differentially expressed genes (DEGs) between two species Comparing to S. malacopterus, 2,598 and 691 DEGs were found to be respectively, down-regulated and up-regulated in S. rhinocerous. Furthermore, it is also found tens of DEGs related to cave adaptability such as insulin secretion regulation (MafA, MafB, MafK, BRSK, and CDK16) and troglomorphic traits formation (CEP290, nmnat1, coasy, and pqbp1) in the cave-dwelling S. rhinocerous. Interestingly, most of the DEGs were found to be down-regulated in cavefish species and this trend of DEGs expression was confirmed through qPCR experiments. This study would provide an appropriate genetic basis for future studies on the formation of troglomorphic traits and adaptability characters of troglobites, and improve our understanding of mechanisms of cave adaptation.

Background Natural model systems are indispensable for exploring adaptations in response to environmental pressures. Sinocyclocheilus of China, the most diverse cavefish clade in the world (75 species), provide unique opportunities to understand recurrent evolution of stereotypic traits (such as eye loss and sensory expansion) in the context of a deep and diverse phylogenetic group. However, they remain poorly understood in terms of their morphological evolution. Therefore, we explore key patterns of morphological evolution, habitat utilization and geographic distribution in these fishes. Results We constructed phylogenies and categorized 49 species based on eye-related condition (Blind, Micro-eyed, and Normal-eyed), habitat types (Troglobitic—cave-restricted; Troglophilic—cave-associated; Surface—outside caves) and existence of horns. Geometric-morphometric analyses show Normal-eyed morphs with fusiform shapes segregating from Blind/Micro-eyed deeper bodied morphs along the first principal-component axis; second axis accounts for shape complexity related to horns. The body shapes showed a significant association with eye-related condition and horn, but not habitat types. Ancestral reconstructions suggest at least three independent origins of Blind morphs, each with different levels of modification in relation to their ancestral Normal-eyed morphs; Sinocyclocheilus are also pre-adapted for cave dwelling. Our geophylogeny shows an east-to-west diversification spanning Pliocene and Pleistocene, with early-diversifying Troglobitic species dominating subterranean habitats of karstic plains whereas predominantly Surface forms inhabit hills to the west. Evolutionary rates analyses suggest that lineages leading to Blind morphs were characterized by significant rate shifts, such as a slowdown in body size evolution and a 5–20 fold increase in rate of eye regression, possibly explained by limited resource availability. Body size and eye size have undergone reversals, but not horns, a trait entailing considerable time to form. Conclusions Sinocyclocheilus occupied cave habitats in response to drying associated with aridification of China during late Miocene and the Pliocene. The prominent cave-adaptations (eye-regression, horn-evolution) occur in clades associated with the extensive subterranean cave system in Guangxi and Guizhou provinces. Integration of morphology, phylogeny, rate analyses, molecular-dating and distribution show not only several remarkable patterns of evolution, but also interesting exceptions to these patterns signifying the diversification of Sinocyclocheilus as an invaluable model system to explore evolutionary novelty.

Evolution sometimes proceeds by loss, especially when structures and genes become dispensable after an environmental shift relaxing functional constraints. Gene decay can serve as a read-out of this evolutionary process. Animals living in the dark are outstanding models, in particular cavefishes as hundreds of species evolved independently during very different periods of time in absence of light. Here, we sought to understand some general principals on the extent and tempo of decay of several gene sets in cavefishes. The analysis of the genomes of two Cuban species belonging to the genusLucifugaprovides evidence for the most massive loss of eye genes reported so far in cavefishes. Comparisons with a recently-evolved cave population ofAstyanax mexicanusand three species belonging to the tetraploid Chinese genusSinocyclocheilusrevealed the combined effects of the level of eye regression, time and genome ploidy on the number of eye pseudogenes. In sharp contrast, most circadian clock and pigmentation genes appeared under strong selection. In cavefishes for which complete genomes are available, the limited extent of eye gene decay and the very small number of loss of function (LoF) mutations per pseudogene suggest that eye degeneration is never very ancient, ranging from early to late Pleistocene. This is in sharp contrast with the identification of several eye pseudogenes carrying many LoF mutations in ancient fossorial mammals. Our analyses support the hypothesis that blind fishes cannot thrive more than a few millions of years in cave ecosystems.

The genus Sinocyclocheilus is a representative group of cave creatures. However, genetic studies on Sinocyclocheilus are rare. The primary objective of this study was to explore the structure and feature of the complete mitochondrial genome of S. multipunctatus, and reconstruct the mitogenomic phylogeny of Sinocyclocheilus. The mitochondrial DNA of S. multipunctatus was amplified by overlapping PCR fragments. The mitogenome was assembled by the SeqMan and annotated using MitoAnnotator. The phylogenetic tree was established using the Bayesian inference and Maximum likelihood methods. The mitogenome of S. multipunctatus is a typical circular molecule of 16,586 bp with base composition A (31.25%), T (25.90%), G (16.35%), and C (26.50%), and consists of 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs) genes, two ribosomal RNAs, and a 931 bp control region. Phylogenetic analysis reveals two clades in the Sinocyclocheilus with robust support. S. multipunctatus is close to a newly discovered cavefish, S. ronganensis. We obtained and described the complete mitogenome of S. multipunctatus, and investigated its phylogenetic status, which may provide a valuable resource for future phylogenetic analyses and population genetic studies in Sinocyclocheilus.

Sinocyclocheilus longibarbarus and Sinocyclocheilus punctatus were collected from a karst cave Libo County, southwest of China. The two Sinocyclocheilus species can be distinguished obviously by external morphological characteristics. In this study, the complete mitochondrial genome sequences of two species were assembled, and both sequences reflected gene organization typical for mitochondrial DNA of the genus Sinocyclocheilus, comprising of 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs), and a large non-coding control region. Phylogenetic analysis showed that S. punctatus was first clustered together with S. mutipunctatus, and S. longibarbarus was closely related to S. yishanensis. The complete mitogenome of two species may provide useful information for the further taxonomic and phylogenetic studies.

Sinocyclocheilus lingyunensis is endemic to Youjiang River in Guangxi, Southwestern China. In this study, the complete mitochondrial genome of S. lingyunensis was sequenced. It was determined to be 16,572 bases. The overall base composition was 31.9% A, 25.3% T, 26.9% C, 15.9% G with 42.8% GC content. The nucleotide sequence data of 12 heavy-strand protein-coding genes of S. lingyunensis and other 16 Sinocyclocheilus species were used for phylogenetic analyses. Trees constructed using Bayesian inference showed strong support for a topology indicating Sinocyclocheilus species as a monophyletic group.

We describe the complete mitochondrial genome of the endangered fish Sinocyclocheilus aluensis. It is a circular molecule of 16,583 bp in size with a D-loop region and contains 22 transfer RNA (tRN...

Length‐weight relationships (LWRs) were estimated for four cavefish species belonging to the genus Sinocyclocheilus (Cypriniformes: Cyprinidae) occurring in the Nanpanjiang River and Hongshuihe River, which constitute the upstream of the species, S. angularis, S. furcodorsalis, S. rhinocerous, and S. tileihornes, were collected from 2011 to 2017. The b values of LWRs of these four species ranged from 2.830 to 3.719 and the correlation coefficient values (R²) were high for all species. This is the first record of LWRs for cavefish species with the horn in FishBase database.

Chinese cavefishes are a bizarre and interesting vertebrate taxa, but one with relatively little research. China holds the highest global cavefish diversity, accounting for about one-third of known species. Sinocyclocheilus is the largest genus of cavefishes in the world and is endemic to the south of China. The distribution of Sinocyclocheilus species is very narrow, and sometimes they inhabit just a single cave; this feature increases the vulnerability to extinction. With this study we provide the first comprehensive dataset related to the morphometrics of eight Sinocyclocheilus species. In addition to enhancing our knowledge on these poorly known species we aim to provide a dataset useful for future comparative analyses aiming to better understand the adaptive ability of cavefishes., Measurement(s)morphometryTechnology Type(s)digital camera • computational modeling techniqueFactor Type(s)species • Museum collectionSample Characteristic - OrganismSinocyclocheilusSample Characteristic - EnvironmentgroundwaterSample Characteristic - LocationChina Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.9963206

Cavefish, with sensitive lateral lines, can swim freely and locate preys in invisible and complex cave environments, though their eyes are greatly degenerated. Investigations on the morphology and distribution characteristics of their lateral line systems would benefit our understanding of the high-sensitivity mechanism of the fish. In this study, the arrangement and morphology of the lateral lines are described for two species of Sinocyclocheilus: S. macrophthalmus and S. microphthalmus, which live in the karst caves in Guangxi, China. The behavior experiments indicate that the lateral line system of the S. macrophthalmus is more sensitive at a low vibration frequency range from 20 Hz to 70 Hz. The cephalic and trunk lateral line systems both contribute to the efficient object-locating capability. For both of the two species of cavefish, the diameter of the lateral canal nearby the neuromasts is narrower than that nearby the canal pores. This variation can increase the normal pressure to the surface of the cupula, and increase the sensitivity of the canal lateral line system.

Cave animals possess remarkable phenotypes associated with existence in their dark environments. The Chinese cavefish Sinocyclocheilus tileihornes shows substantial eye degeneration, a trait shared by most cave species. The extent to which independent evolution of troglomorphic traits uses convergent molecular genetic mechanisms is as yet unknown. We performed transcriptome-wide gene expression profiling in S. tileihornes eyes and compared results with those from the closely related surface species S. angustiporus and an independently derived congeneric cavefish, S. anophthalmus. In total, 52.85 million 100 bp long paired-end clean reads were generated for S. tileihornes, and we identified differentially expressed genes between the three possible pairs of species. Functional analysis of genes differentially expressed between S. tileihornes and S. angustiporus revealed that phototransduction (KEGG id: dre04744) was the most significantly enriched pathway, indicating the obvious differences in response to captured photons between the cavefish S. tileihornes and the surface species S. angustiporus. Analysis of key genes regulating eye development showed complete absence of otx5b (orthodenticle homolog 5) expression in S. tileihornes eyes, probably related to degradation of rods, but normal expression of crx (cone-rod homeobox). The enriched pathways and Otx5 are involved in phototransduction, photoreceptor formation, and regulation of photoreceptor-related gene expression. Unlike the S. tileihornes reported here, S. anophthalmus has reduced crx and otx5 expression. These results show that different species of cavefish within the same genus that independently evolved troglodyte characteristics can have different genetic mechanisms of eye degeneration. Electronic supplementary material The online version of this article (10.1007/s00239-019-09901-8) contains supplementary material, which is available to authorized users.

Light is completely absent in cave habitats, causing a shortage or lack of autochthonous photosynthesis. Thus, understanding the mechanisms underlying the ability of organisms to adapt to the unique cave habitat is of great interest. We used high-throughput sequencing of the 16S ribosomal RNA gene of intestinal microorganisms from 11 Sinocyclocheilus (Cypriniformes: Cyprinidae) species, to explore the characteristics of intestinal microorganisms and the adaptive mechanisms of Sinocyclocheilus cavefish and surface fish. We found that the α-diversity and richness of the intestinal microbiome were much higher in cavefish than in surface fish. Principal coordinate analysis showed that cavefish and surface fish formed three clusters because of different dominant gut microorganisms which are generated by different habitats. Based on PICRUSt-predicted functions, harmful substance degradation pathways were much more common in cavefish intestinal microorganisms than in those from surface fish. The intestinal microbiota of surface fish group 1 had a higher capacity for carbohydrate metabolism, whereas protein and amino acid metabolism and digestive pathways were more abundant in microorganisms from the cavefish group and surface fish group 2. Combined analysis of the intestinal microbial composition and functional predictions further revealed the structures and functions of intestinal microbial communities in Sinocyclocheilus cave and surface species. Moreover, based on their habits and intestinal microbial composition and intestinal microbial functional predictions, we inferred that the three fish groups were all omnivorous; however, surface fish group 1 preferred feeding on plants, while surface fish group 2 and cavefish preferred meat. This study improves our understanding of mechanisms of adaptation in cave habitats and may contribute to the protection of these habitats from water pollution.

The genus Sinocyclocheilus (golden-line barbel) includes 25 species of cave-dwelling blind fish (cavefish) and more than 30 surface-dwelling species with normal vision. Cave environments are dark and generally nutrient-poor with few predators. Cavefish of several genera evolved convergent morphological adaptations in visual, pigmentation, brain, olfactory, and digestive systems. We compared brain morphology and gene expression patterns in a cavefish Sinocyclocheilus anophthalmus with those of a closely related surface-dwelling species S. angustiporus. Results showed that cavefish have a longer olfactory tract and a much smaller optic tectum than surface fish. Transcriptomics by RNA-seq revealed that many genes upregulated in cavefish are related to lysosomes and the degradation and metabolism of proteins, amino acids, and lipids. Genes downregulated in cavefish tended to involve “activation of gene expression in cholesterol biosynthesis” and cholesterol degradation in the brain. Genes encoding Srebfs (sterol regulatory element-binding transcription factors) and Srebf targets, including enzymes in cholesterol synthesis, were downregulated in cavefish brains compared with surface fish brains. The gene encoding Cyp46a1, which eliminates cholesterol from the brain, was also downregulated in cavefish brains, while the total level of cholesterol in the brain remained unchanged. Cavefish brains misexpressed several genes encoding proteins in the hypothalamus–pituitary axis, including Trh, Sst, Crh, Pomc, and Mc4r. These results suggest that the rate of lipid biosynthesis and breakdown may both be depressed in golden-line cavefish brains but that the lysosome recycling rate may be increased in cavefish; properties that might be related to differences in nutrient availability in caves.

Sinocyclocheilus microphthalmus is endemic to Youjiang River and Hongshuihe River systems in Guangxi, Southwestern China. In this study, the complete mitochondrial genome of S. microphthalmus was sequenced. It was determined to be 16,589 bases. The overall base composition was 31.87% A, 24.96% T, 27.19% C, 15.99% G with 43.17% GC content. The nucleotide sequence data of 12 heavy-strand protein-coding genes of S. microphthalmus and other 13 Sinocyclocheilus species were used for phylogenetic analyses. Trees constructed using Bayesian inference showed a topology demonstrating that Sinocyclocheilus species clustered as one monophyletic clade with strong supports.

In this study, the complete mitochondrial genome of Sinocyclocheilus huizeensis has been determined by polymerase chain reaction method for the first time. The overall base composition of Sinocyclo...

Background: Chinese clearhead icefish, Protosalanx hyalocranius, is a representative icefish species with economic importance and special appearance. Due to its great economic value in China, the fish was introduced into Lake Dianchi and several other lakes from the Lake Taihu half a century ago. Similar to the Sinocyclocheilus cavefish, the clearhead icefish has certain cavefish-like traits, such as transparent body and nearly scaleless skin. Here, we provide the whole genome sequence of this surface-dwelling fish and generated a draft genome assembly, aiming at exploring molecular mechanisms for the biological interests. Findings: A total of 252.1 Gb of raw reads were sequenced. Subsequently, a novel draft genome assembly was generated, with the scaffold N50 reaching 1.163 Mb. The genome completeness was estimated to be 98.39 % by using the CEGMA evaluation. Finally, we annotated 19 884 protein-coding genes and observed that repeat sequences account for 24.43 % of the genome assembly. Conclusion: We report the first draft genome of the Chinese clearhead icefish. The genome assembly will provide a solid foundation for further molecular breeding and germplasm resource protection in Chinese clearhead icefish, as well as other icefishes. It is also a valuable genetic resource for revealing the molecular mechanisms for the cavefish-like characters.

In order to obtain reliable results of gene expression, appropriate internal control gene(s) were required for normalization before real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Here, we selected eight candidate housekeeping genes as the potential reference genes in normalizing qRT-PCR data in ten tissues of the juvenile and adult stages of a naturally tetraploid cyprinid fish Sinocyclocheilus grahami. Before qRT-PCR, appropriate primers were designed and compared for distinguishing different copies in some duplicate genes. Candidate reference genes were evaluated for their stability using online software RefFinder, which is integrated by four normal software of the comparative delta-CT, BestKeeper, NormFinder and GeNorm. According to the rankings produced by these analyses, Eef2, ACTB and G6PD were the most stable reference genes as internal controls for qRT-PCR in juvenile, while B2M, Eef2 and RPL17 were shown to be most appropriate in adult fish. All the analyses show that GAPDH was the least stable expression gene which would be unsuitable as reference gene at both stages. This is the first report about reference gene selection in Sinocyclocheilus and we think that is beneficial for the future gene expression studies in golden-line barbel.

Sinocyclocheilus oxycephalus is a freshwater cyprinid fish of high nutritional value, endemic to Shilin County, Southwestern China. In this study, we first sequenced the complete mitochondrial genome (mitogenome) of S. oxycephalus. The whole length of mitogenome is 16,585 bp, which contains 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, and a control region. The gene arrangement and structure is identical to other previously reported Sinocyclocheilus fishes. The overall base composition is 31.26% A, 16.42% G, 25.41% T and 26.90% C, with AT content of 56.67%. Phylogenetic analysis using mitogenomes of 13 cyprinid fishes showed that S. oxycephalus are closely related to S. anophthalmus, S. grahami, and S. wumengshanensis, and 11 Sinocyclocheilus species are grouped as a monophyletic clade with strong supports.

In our previous work, we reported comparative genomics studies on 3 Sinocyclocheilus fishes, including the surface-dwelling S. graham (Sg), the semi-cave-dewlling S. rhinocerous (Sr) and the cave-restricted S. anshuiensis (Sa). Here, we performed a genomic survey on the immunity differences among the 3 fishes by combination of the genome and transcriptome data. Our results demonstrate that immune-related genetic pathways in Sa and Sr present more similarities than Sg. The innate immune activity in Sa seems to be higher than Sg and Sr, while Sr owns more copy numbers of MHC-related genes (related with the acquired immune system) than Sg and Sa. These differential immune activities in the 3 Sinocyclocheilus species may be due to their differential habitats. Generally speaking, most immunity genes transcribe the lowest levels in Sa when compared with Sg and Sr, which may be associate with a less various microbial environment in the cave-restricted habitat. However, Sr has more members of MHC-related genes than those in Sg and Sa, suggesting a great contribution from the semi-cave-dwelling condition.

A new species of the genus Sinocyclocheilus is described based on specimens collected from Zuojiang River drainage, Guangxi, China. The new species, named Sinocyclocheilus jinxiensis, is distinguished from all congeners by its vestigial eyes, short barbels, long pectoral fin, eight to nine branched dorsal-fin rays, last unbranched dorsal-fin ray soft with serrations on posterior edge of its lower part, and 38–41 lateral line scales.

The complete mitochondrial genome (mitogenome) sequence of Sinocyclocheilus wumengshanensis from Yunnan Province in China was analysed using next-generation sequencing. The complete mitogenome was 16,585 bp in length and consisted of 13 protein-coding genes, two ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, and one D-loop region. Nucleotide composition of the whole mitogenome was 30.5% A, 25.2% T, 27.0% C, and 17.2% G. The gene arrangement and nucleotide composition of the mitogenome of S. wumengshanensis were similar to those of other Sinocyclocheilus species. Phylogenetic analyses using mitogenomes of 12 species showed that 10 Sinocyclocheilus species clustered as one monophyletic clade with strong supports and S. wumengshanensis was closely related to S. grahami.

In this study, we first determined the complete mitochondrial genome sequence of Sinocyclocheilus jii, which is an endemic species to Southwestern China. The complete mitochondrial genome is 16,577 bp in length, consisting of 37 genes coding for 13 proteins, two rRNAs, 22 tRNAs, and one control region. Its gene arrangement pattern was identical to that of most vertebrates. Phylogenetic analysis using mitochondrial genomes of 11 species showed that nine Sinocyclocheilus species clustered as one monophyletic clade and S. jii was the most basal species on the phylogenetic tree of the Sinocyclocheilus fishes.

Caves and karsts are among the most threatened ecosystems in the world. They are very fragile, balanced habitats with high levels of endemic species that are extremely sensitive to environmental changes. In recent decades, however, threats from rapid economic growth have increased the need for conservation efforts for cave-dwelling communities. In addition, difficulties in accessing and sampling these habitats mean that they remain as one of the least known ecosystems in the world with modern studies of cave fishes only starting in China during the 1980s. Here, the current status of cave fishes in China is reviewed. China is host to the highest number of cave fish species in the world, with 48 troglobite species out of a total of 101 cave fish species. All of these cave fish species (one order and three families) and half of the genera are endemic to China with Sinocyclocheilus being the most speciose cave fish genus. Species from this genus possess horns and humpbacks resulting from processes of parallel evolution, but the function of these features remains unknown. With the exception of Onychostoma macrolepis distributed in north China, all other species are found in the karst environment of the Yunnan-Guizhou Plateau. Sympatric distribution is common, and sometimes several different cave fish species can be found in the same cave or subterranean river. For this reason, Chinese cave fishes represent an important evolutionary framework.

The northwestern part of Guangxi is a typical karst landform. There are about ten species of the genus Sinocyclocheilus distributed in the region. To investigate the phylogenetic relationships and resolve the taxonomic status of these species, we determined nucleotide sequences of complete mitochondrial cytochrome b gene of 11 ingroup species and one outgroup species Barbodes laticeps. Phylogenetic trees were reconstructed using maximum parsimony and maximum likelihood analyses. Phylogenetic results showed that these species cluster as one clade with strong support. The 95% credibility interval (CI) of the divergence time between the species in the northwest part of Guangxi and other species in the genus Sinocyclocheilus is 8.28–12.40Mya. For the karst environment, these species show marked, recent genetic differentiation. From the overall consideration of morphology and molecular data Sinocyclocheilus tianeensis should be a valid species.

The fish genus Sinocyclocheilus contains many different species that inhabit diverse natural environments, such as surface water layer, cave, or intermediate. As a result of these different habitats there are some differences in their sensory systems. Microscopic and submicroscopic structures of olfactory systems in six representative species of Sinocyclocheilus were studied, including one surface-dwelling species (S. grahami), two intermediate species (S. jii and S. macrophthalmus) and three cave-dwelling species (S. brevibarbatus, S. anshuiensis, and S. tianlinensis). Due to adaptive evolution under extreme environmental conditions, cave-dwelling species have more developed olfactory systems. We observed that, compared with surface-dwelling species, the olfactory sac of the cave-dwelling Sinocyclocheilus species has the following characteristics: higher density of cilia, greater length of sensory cilia, many other special structures (micro-ridge, olfactory islet, rod cilia). These results reveal different levels of olfactory system development, consistent with the view that that cave-dwelling species have more developed olfactory systems than intermediate and surface-dwelling species.

The cyprinid fish genus Sinocyclocheilus, as the most cavefish rich genus, includes many species showing striking adaptation to caves and convergent reduction or even loss of eyes and pigmentation. RH1 is responsible for dim vision. In order to explore the evolution of RH1 gene in this genus, we sequenced the complete gene from 28 individuals of 16 representative species of Sinocyclocheilus, with cave and surface species included. Phylogenetic analyses supported the monophyly of Sinocyclocheilus and polyphyly of the cave species. Codon models implemented in PAML were used to infer the evolution of RH1. We found that Sinocyclocheilus had a significantly higher evolutionary rate for amino acids than other cyprinid fishes compared, which might be the result of relaxation of purifying selection and could be ascribed to cave habit of this genus. In contrast to previous hypotheses, both cave and surface lineages exhibited a similar rate of molecular evolution, so the RH1 of cave species may still be functional, although these species were highly adapted to cave environment. Two amino acid substitutions (D83G and E122V) that were not reported before were found, which may be useful for site-directed mutagenesis in the future.

The genus Sinocyclocheilus is distributed in Yun-Gui Plateau and its surrounding region only, within more than 10 cave species showing different degrees of degeneration of eyes and pigmentation with wonderful adaptations. To present, published morphological and molecular phylogenetic hypotheses of Sinocyclocheilus from prior works are very different and the relationships within the genus are still far from clear. We obtained the sequences of cytochrome b (cyt b) and NADH dehydrogenase subunit 4 (ND4) of 34 species within Sinocyclocheilus, which represent the most dense taxon sampling to date. We performed Bayesian mixed models analyses with this data set. Under this phylogenetic framework, we estimated the divergence times of recovered clades using different methods under relaxed molecular clock. Our phyloegentic results supported the monophyly of Sinocyclocheilus and showed that this genus could be subdivided into 6 major clades. In addition, an earlier finding demonstrating the polyphyletic of cave species and the most basal position of S. jii was corroborated. Relaxed divergence-time estimation suggested that Sinocyclocheilus originated at the late Miocene, about 11 million years ago (Ma), which is older than what have been assumed.

The endemic Chinese cavefish genus Sinocyclocheilus possesses very rich species diversity. Specimens of this genus collected from Guilin, Guangxi, China, were originally described in an unpublished document in 1982. Later, they were recognized as Sinocyclocheilus jii. Comparison to the type specimens of S. jii leads to the conclusion that, though similar, they represent two different species. Herein we describe them as a new species, Sinocyclocheilus guilinensis sp. nov. It can be distinguished from all congeners by its possession of soft, unbranched dorsal ray without serrations, 19–20 (11–12) scale rows above (below) the lateral line, 34–36 circumpeduncular scales and 8–9 pre-dorsal vertebrae. Sinocyclocheilus guilinensis sp. nov. is most similar to S. jii. In addition to the scale count differences, a morphometrics-based principal component analysis (PCA) also supports designation of S. guilinensis and S. jii as two distinct species.

A new species of the cavefish genus Sinocyclocheilus is described based on six specimens collected from a subterranean river in Du’an County in the Guangxi Zhuang Autonomous Region of southern China. Sinocyclocheilus brevibarbatus sp. nov. can be distinguished from all congeners by the following combination of characteristics: a completely scaled body with well-developed eyes and a strongly-humped back; an almost straight lateral line possessing 49–51 perforated scales; seven soft rays on the dorsal fin; a relatively large eyeball (3.3–5.7% of SL) and short maxillary and rictal barbels (1.7–5.7% and 4.5–7.0% of SL, respectively).

Sinocyclocheilus donglanensis, a new cyprinid species from a subterranean river in Donglan County in the Guangxi Zhuang Autonomous Region of southern China, is described. It is distinguished from all congeners by the following combination of characteristics: a completely scaled body with well-developed eyes; a curved lateral line possessing 57–64 scales; pectoral fin not reaching pelvic fin origin and last unbranched ray of the dorsal fin clearly serrated along its posterior edge; 8–9 predorsal vertebrae; 8–9 gill rakers; joints of dentary-angulars not close to each other at the isthmus; and a slightly inferior mouth with the upper jaw (6.2–7.4% in standard length: SL) protruding slightly beyond the lower one (5.7–6.7% SL). Sinocyclocheilus donglanensis is sympatric with the peculiarly shaped, hunchbacked S. altishoulderus.

More than 10 species within the freshwater fish genus Sinoncyclocheilus adapt to caves and show different degrees of degeneration of eyes and pigmentation. Therefore, this genus can be useful for studying evolutionary developmental mechanisms, role of natural selection and adaptation in cave animals. To better understand these processes, it is indispensable to have background knowledge about phylogenetic relationships of surface and cave species within this genus. To investigate phylogenetic relationships among species within this genus, we determined nucleotide sequences of complete mitochondrial cytochrome b gene (1140 bp) and partial ND4 gene (1032 bp) of 31 recognized ingroup species and one outgroup species Barbodes laticeps. Phylogenetic trees were reconstructed using maximum parsimony, Bayesian, and maximum likelihood analyses. Our phylogenetic results showed that all species except for two surface species S. jii and S. macrolepis clustered as five major monophyletic clades (I, II, III, IV, and V) with strong supports. S. jii was the most basal species in all analyses, but the position of S. macrolepis was not resolved. The cave species were polyphyletic and occurred in these five major clades. Our results indicate that adaptation to cave environments has occurred multiple times during the evolutionary history of Sinocyclocheilus. The branching orders among the clades I, II, III, and IV were not resolved, and this might be due to early rapid radiation in Sinocyclocheilus. All species distributed in Yunnan except for S. rhinocerous and S. hyalinus formed a strongly supported monophyletic group (clade V), probably reflecting their common origins. This result suggested that the diversification of Sinocyclocheilus in Yunnan may correlate with the uplifting of Yunnan Plateau.

Neighbour-joining and parsimony analyses identified five lineages of cyprinids: (1) European leuciscins (including Notemigonus)+North American phoxinins (including Phoxinus phoxinus); (2) European gobionins+Pseudorasbora; (3) primarily Asian groups [cultrins+acheilognathins+ gobionins (excluding Abbotina)+xenocyprinins]; (4) Abbottina+Sinocyclocheilus+Acrossocheilus; (5) cyprinins [excluding Sinocyclocheilus and Acrossocheilus]+barbins+labeonins. Relationships among these lineages and the enigmatic taxa Rhodeus were not well-resolved. Tests of monophyly of subfamilies and previously proposed relationships were examined by constraining cytochrome b sequences data to fit previous hypotheses. The analysis of constrained trees indicated that sequence data were not consistent with most previously proposed relationships. Inconsistency was largely attributable to Asian taxa, such as Xenocypris and Xenocyprioides. Improved understanding of historical and taxonomic relationships in Cyprinidae will require further morphological and molecular studies on Asian cyprinids and taxa representative of the diversity found in Africa.

This work was funded by the Marine Alliance for Science and Technology for Scotland pooling initiative and Scottish Funding Council grant number HR09011. Teleost fish underwent whole-genome duplication around 450 Ma followed by diploidization and loss of 80-85% of the duplicated genes. To identify a deep signature of this teleost-specific whole-genome duplication (TSGD), we searched for duplicated genes that were systematically and uniquely retained in one or other of the superorders Ostariophysi and Acanthopterygii. TSGD paralogs comprised 17-21% of total gene content. Some 2.6% (510) of TSGD paralogs were present as pairs in the Ostariophysi genomes of Danio rerio (Cypriniformes) and Astyanax mexicanus (Characiformes) but not in species from four orders of Acanthopterygii (Gasterosteiformes, Gasterosteus aculeatus; Tetraodontiformes, Tetraodon nigroviridis; Perciformes, Oreochromis niloticus; and Beloniformes, Oryzias latipes) where a single copy was identified. Similarly, 1.3% (418) of total gene number represented cases where TSGD paralogs pairs were systematically retained in the Acanthopterygian but conserved as a single copy in Ostariophysi genomes. We confirmed the generality of these results by phylogenetic and synteny analysis of 40 randomly selected linage-specific paralogs (LSPs) from each superorder and completed with the transcriptomes of three additional Ostariophysi species (Ictalurus punctatus [Siluriformes], Sinocyclocheilus species [Cypriniformes], and Piaractus mesopotamicus [Characiformes]). No chromosome bias was detected in TSGD paralog retention. Gene ontology (GO) analysis revealed significant enrichment of GO terms relative to the human GO SLIM database for "growth," "Cell differentiation," and "Embryo development" in Ostariophysi and for "Transport," "Signal Transduction," and "Vesicle mediated transport" in Acanthopterygii. The observed patterns of paralog retention are consistent with different diploidization outcomes having contributed to the evolution/diversification of each superorder. Publisher PDF

The horned Golden-line barbel, Sinocyclocheilus rhinocerous (Cypriniformes, Cyprinidae) is a species of ray-finned fish of the genus Sinocyclocheilus with specific features. For example, its forehead protrudes forward like a rhinoceros horn. Here, we first reported the complete mitochondrial genome of this semi-caved teleost. The whole mitochondrial genome was 16 588 bp, and composed of 13 protein-coding genes, 22 transfer RNAs, 2 ribosomal RNAs, and a 933 bp control region. A phylogenetic tree with the complete mitochondrial genome sequences of S. rhinocerous together with 10 other affinis species was constructed. The newly achieved mitochondrial genome sequence seem to be useful for addressing taxonomic issues and studying related evolution events, which would contribute to enrich the fish mitochondrial genome resource and promote the biological research.

In adaptating to perpetual darkness, cave species gradually lose eyes and body pigmentation and evolve alternatives for exploring their environments. Although troglodyte features evolved independently many times in cavefish, we do not yet know whether independent evolution of these characters involves common genetic mechanisms. Surface-dwelling and many cave-dwelling species make the freshwater teleost genus Sinocyclocheilus an excellent model for studying the evolution of adaptations to life in constant darkness. We compared the mature retinal histology of surface and cave species in Sinocyclocheilus and found that adult cavefish showed a reduction in the number and length of photoreceptor cells. To identify genes and genetic pathways that evolved in constant darkness, we used RNA-seq to compare eyes of surface and cave species. De novo transcriptome assemblies were developed for both species, and contigs were annotated with gene ontology. Results from cave-dwelling Sinocyclocheilus revealed reduced transcription of phototransduction and other genes important for retinal function. In contrast to the blind Mexican tetra cavefish Astyanax mexicanus, our results on morphologies and gene expression suggest that evolved retinal reduction in cave-dwelling Sinocyclocheilus occurs in a lens-independent fashion by the reduced proliferation and downregulation of transcriptional factors shown to have direct roles in retinal development and maintenance, including cone-rod homeobox (crx) and Wnt pathway members. These results show that the independent evolution of retinal degeneration in cavefish can occur by different developmental genetic mechanisms.

Cave dwelling animals that live in perpetual darkness have evolved a number of striking troglomorphic characters. The most common phenotypes are the reduction or loss of eyesight and pigmentation (Jeffery, 2009; Poulson and White, 1969; Protas et al., 2006). Among vertebrates, most research has focused on the regressive evolution of the teleost Astyanax mexicanus in North America. The eye degeneration of cave dwelling Astyanax is induced by lens apoptosis caused by the over-expression of sonic hedgehog (shh) (Yamamoto et al., 2004). Researchers also found a mutation in the oca2 gene that blocked development of melanin-containing pigment cells (Protas et al., 2006). However, the general importance of these phenomena and the genetic basis of eye degeneration remains unclear. Sinocyclocheilus (Cypriniformes, Cyprinidae), a freshwater teleost genus, is endemic to China and includes many cave-dwellers among its 55 known species. This genus is only found in the central eastern Yungui Plateau and its surrounding region, Guangxi, in southwestern China (Xiao et al., 2005; Chen et al., 2009; Wu et al., 2010). Sinocyclocheilus is remarkable in its high species diversity and troglomorphic character variation within the genus. Thus, Sinocyclocheilus is an ideal model system for investigating the evolutionary biology of, and natural selection in troglobionts. Most vertebrate retinas consist of two types of photoreceptor cells, rod and cone cells, which contain different visual pigments (Swaroop et al., 2010). The primary components of visual pigments are opsins, a group of light-sensitive membrane-bound G-protein-coupled receptors that mediate the conversion of energy in photons into an electrochemical signal (Fain et al., 2010). Opsins include rhodopsin (Rho) and cone opsins, which are responsible for dim vision in rod cells and color vision in cone cells, respectively (Shichida and Matsuyama, 2009). The cone opsins contain four subgroups, which form primarily red-sensitive (long-wavelength), green-sensitive (middle-wavelength), blue-sensitive (shorter wavelength, SWS2), and UV/violet-sensitive (shorter wavelength, SWS1) pigments. (Chinen et al., 2003; Terakita, 2005). Li and colleagues analyzed the molecular evolution of rhodopsin and two cone opsin genes in several Sinocyclocheilus cavefishes (Li and He, 2009; Li et al., 2009). They found that rhodopsin was subject to relaxed purifying selection, while LWS1 (long wavelength sensitive-1) had experienced strong positive selection that increased the substitution rate in the pocket-forming residues. No positive selection was observed in SWS2 (short wavelength sensitive-2). During eye degeneration in cavefish, morphological and functional changes may result from both divergence in coding DNA sequences and by differential levels of gene expression. While the expression of rhodopsin, SWS1, MWS (middle wavelength sensitive), and LWS are reduced in the eye of cave populations relative to surface populations of Astyanax mexicanus (Strickler and Jeffery, 2009; Tobler et al., 2010), it is unclear whether opsin gene expression is reduced in Sinocyclocheilus cavefishes. To address this, we detected morphological differences between a surface dwelling species S. angustiporus and a cavefish S. tileihornes using antibody markers to label rod and cone cells. In addition, we prepared eye cDNA samples from surface- and cave-dwelling species and identified differentially-expressed opsin genes in cave species relative to surface species.

A diverse group of animals, including members of most major phyla, have adapted to life in the perpetual darkness of caves. These animals are united by the convergence of two regressive phenotypes, loss of eyes and pigmentation. The mechanisms of regressive evolution are poorly understood. The teleost Astyanax mexicanus is of special significance in studies of regressive evolution in cave animals. This species includes an ancestral surface dwelling form and many con-specific cave-dwelling forms, some of which have evolved their recessive phenotypes independently. Recent advances in Astyanax development and genetics have provided new information about how eyes and pigment are lost during cavefish evolution; namely, they have revealed some of the molecular and cellular mechanisms involved in trait modification, the number and identity of the underlying genes and mutations, the molecular basis of parallel evolution, and the evolutionary forces driving adaptation to the cave environment.