Understanding the genetic basis of adaptation to high altitude life is of paramount importance for preserving and managing genetic diversity in highland animals. This objective has been addressed mainly in terrestrial fauna but rarely in aquatic animals. Tibetan Schizothoracinae fish is the ideal model system in evolutionary biology, carrying key insights into evolutionary genetics of speciation and adaptation at high altitude. Gymnocypris przewalskii is the newly formed Schizothoracinae fish species in the Tibetan Plateau, inhabits chronic cold, extreme saline and alkaline aquatic environment in Lake Qinghai, thus evolving the unique genomic signatures to adapt extremely severe environments. To characterize its genomic features, we assembled de novo transcriptome of G. przewalskii from Lake Qinghai. Intriguingly, by comparative genomic analyses of G. przewalskii and 8 other fish species, we identified potential expansions in gene families related to energy metabolism, transport and developmental functions, possibly underlying the adaptation to these environmental stresses. Through comprehensive molecular evolution analyses, we found that sets of genes controlling mitochondrion, ion homoeostasis, acid-base balance and innate immunity show significant signals of positive selection. Compared to previous studies on highland fishes, we failed to identify any positively selected genes related to hypoxia response. Our findings provide comprehensive insights into the genetic basis of teleost fish that underlie their adaptation to extreme high altitude aquatic life on the Tibetan Plateau.

Environmental acclimation is important episode in wildlife occupation of the high-altitude Tibetan Plateau (TP). Transcriptome-wide studies on thermal acclimation mechanism in fish species are rarely revealed in Tibetan Plateau fish at high altitude. Thus, we used mRNA and miRNA transcriptome sequencing to investigate regulation of thermal acclimation in larval Tibetan naked carp, Gymnocypris przewalskii. We first remodeled the regulation network of mRNA and miRNA in thermal acclimation, and then identified differential expression of miRNAs and target mRNAs enriched in metabolic and digestive pathways. Interestingly, we identified two candidate genes contributed to normal skeletal development. The altered expression of these gene groups could potentially be associated with the developmental issues of deformity and induced larval death. Our results have three important implications: first, these findings provide strong evidences to support our hypothesis that G. przewalskii possess ability to build heat-tolerance against the controversial issue. Second, this study shows that transcriptional and post-transcriptional regulations are extensively involved in thermal acclimation. Third, the integrated mRNA and microRNA transcriptome analyses provide a large number of valuable genetic resources for future studies on environmental stress response in G. przewalskii and as a case study in Tibetan Schizothoracine fish.

Triplophysa stoliczkae is the most widespread species in the genus Triplophysa and may have originated from morphological convergence. To understand the evolutionary history of T. stoliczkae, we employed a multilocus approach to investigate the phylogenetics and the morphological evolution of T. stoliczkae on the Qinghai-Tibetan Plateau. All phylogenetic analyses (two mitochondrial and five nuclear loci), a genealogical sorting index and species tree inferences suggested that T. stoliczkae consists of distinct lineages that were not closest relatives. The time estimation indicated that the divergence events between “T. stoliczkae” and other Triplophysa species occurred from approximately 0.10 to 4.51 Ma. The ancestral state analyses supported the independent evolution of T. stoliczkae morphology in distinct lineages. The morphometric analysis and convergence estimates demonstrated significant phenotypic convergence among “T. stoliczkae” lineages. Triplophysa stoliczkae includes 4 different lineages with similar morphologies. The increasingly harsh environments that have occurred since the Pliocene have driven the occurrences of scrape-feeding fish in the genus Triplophysa. Morphological adaptations associated with scrape-feeding behavior resulted in convergences and the artificial lumping of four different species in the nominal taxon T. stoliczkae. A taxonomic revision for T. stoliczkae is needed.

Schizothoracine is the predominant wild fish subfamily of the Tibetan plateau (TP). Their scales, pharyngeal teeth and barbels have gradually regressed with increasing altitude. Schizothoracine have been divided into three groups: primitive, specialized and highly specialized. Ectodysplasin-A (Eda) has been considered as a major gene that contributes to the development of skin appendages. The present study cloned the Eda genes of 51 Schizothoracine fish species which represent the three groups and five Barbinae species. Phylogenetic analyses indicated that Eda may have acted as the genetic trigger for scale loss in the Schizothoracine. Furthermore, 14 single nucleotide polymorphisms (SNPs) and two deletions (18 bp and 6 bp in size), were also detected in the Eda coding sequence of the highly specialized group compared to the primitive group. The same SNPs and two indels result in four non-synonymous and two G-X-Y and 1 XY motif indels, which possibly contribute to significant structure changes in the Eda gene. The domain including (G-X-Y)_n motif in the Eda gene is relatively conserved amongst teleosts. Based on the above results, we hypothesize that the evolution of Eda gene might be associated with the scale loss in Schizothoracine fishes in response to the phased uplift of the TP.

Tibetan naked carp Gymnocypris przewalskii is an ideal model system to study highland adaptation of fish, because it evolved specific genetic and phenotypic characteristics to adapt to chronic cold and alkaline environments in Lake Qinghai. MicroRNAs (miRNAs) are small noncoding RNAs that regulating gene expression post-transcriptionally in a wide range of biological processes. In this study, we focus on the role of miRNAs in adaptation of G. przewalskii to extreme conditions in Lake Qinghai. We generate the first miRNAome of G. przewalskii in Schizothoracinae fish. Using several genomic resources, we inferred 341 conserved miRNAs belonged to 152 miRNA families and 43 novel miRNAs in G. przewalskii, and also identified 15 teleost-specific miRNAs. Using a large scale of conserved miRNAs, we constructed a high-confidence phylogenetic tree between teleost and mammals than mitochondria and nuclear genes. In addition, we found that several miRNA family (e.g. miR-10 and let-7) members highly expressed in G. przewalskii, which may function in multiple biological processes. Finally, we predicted a total of 34,258 miRNA targets genes. Conserved miRNAs target genes participating in signal transduction, cell differentiation and biosynthetic process, and showed signature of functional constraint. While novel miRNAs in a species displayed species-specific targets and involved in ion binding, transport and oxidoreductase activity, may affect the expression patterns of targets with signature of gene family expansion or positive selection under extreme environment. Taken together, this study demonstrated that miRNAs may involve into roles of adaptation of G. przewalskii to highland aquatic environment, and also provide insights into miRNA regulatory network in Schizothoracinae fish as a case study.

This study aimed to understand cold stress adaptations mechanism in fish. Thus, the transcriptional response to cold conditions in Gymnocypris eckloni was evaluated using RNA-seq and microRNA (miRNA)-seq analyses. Low-temperature (LT) group G. eckloni was cultivated outdoors in waters cooled to 2–4°C for 3 weeks, while individuals in the control temperature (CT) group were exposed to 14–16°C. Significantly different responses were observed in both mRNA and miRNA expression profiles, with more mRNAs (1,833 and 1,869 mRNAs were up- and downregulated, respectively) and fewer miRNAs (15 and 6 were up- and downregulated, respectively) observed in the LT group individuals relative to the CT group individuals. A miRNA-mRNA network involved in the regulation of G. eckloni responses to cold stress was constructed; this network included ubiquitin-mediated proteolysis, protein processing, and oxidative phosphorylation. These results provided new insights into mechanisms of cold tolerance by fish, including decreased metabolic activity in addition to proteolysis.