Cunfang Zhang

Local adaptation of Gymnocypris przewalskii (Cyprinidae) on the Tibetan Plateau

Divergent selection among environments affects species distributions and can lead to speciation. In this article, we investigated the transcriptomes of two ecotypes of scaleless carp (Gymnocypris przewalskii przewalskii and G. p. ganzihonensis) from the Tibetan Plateau. We used a transcriptome sequencing approach to screen approximately 250,000 expressed sequence tags (ESTs) from the gill and kidney tissues of twelve individuals from the Ganzi River and Lake Qinghai to understand how this freshwater fish has adapted to an ecological niche shift from saline to freshwater. We identified 9,429 loci in the gill transcriptome and 12,034 loci in the kidney transcriptome with significant differences in their expression, of which 242 protein-coding genes exhibited strong positive selection (Ka/Ks > 1). Many of the genes are involved in ion channel functions (e.g., Ca2+-binding proteins), immune responses (e.g., nephrosin) or cellular water absorption functions (e.g., aquaporins). These results have potentially broad importance in understanding shifts from saline to freshwater habitats. Furthermore, this study provides the first transcriptome of G. przewalskii, which will facilitate future ecological genomics studies and aid in the identification of genes underlying adaptation and incipient ecological speciation.

Transcriptome profiling analysis of naked carp (Gymnocypris przewalskii) provides insights into the immune-related genes in highland fish.

The naked carp, Gymnocypris przewalskii, is one of the dominant aquaculture fish species in Qinghai Province, China. Its wild stocks have severely suffered from overfishing, and the farming species are vulnerable to various pathogens infections. Here we report the first immune-related tissues transcriptome of a wild naked carp using a deep sequencing approach. A total of 158,087 unigenes are generated, 2687 gill-specific gene and 3215 kidney-specific genes are identified, respectively. Gene ontology analysis shows that 51,671 unigenes are involved in three major functional categories: biological process, cellular component, and molecular function. Further analysis shows that numerous consensus sequences are homologous to known immune-related genes. Pathways mapping annotate 56,270 unigenes and identify a large number of immune-related pathways. In addition, we focus on the immune-related genes and gene family in Toll-like receptor signaling pathway involved in innate immunity, including toll-like receptors (TLRs), interferon regulatory factors (IRFs), interleukins (ILs) and tumor necrosis factors (TNFs). Eventually, we identify 5 TLRs, 4 IRFs, 3 ILs and 2 TNFs with a completed coding sequence though mining the transcriptome data. Phylogeny analysis shows these genes of naked carp are mostly close to zebrafish. Protein domain and selection pressure analyses together show that all these genes are highly conserved in gene sequence and protein domain structure with other species, and purifying selection underwent in these genes, implied functionally important features are conserved in the genes above. Intriguingly, we detect positive selection signals in naked carp TLR4, and significant divergence occurred among tested species TLR4, suggested that naked carp TLR4 function may be affected. Finally, we identify 23,867 simple sequence repeat (SSR) marks in this transcriptome. Taken together, this study not only contributes a large number of candidate genes in naked carp immunity, and also helps improve current understanding of immunogenetics basis and evolutionary history of immune related genes and gene family in highland fish species.

Ongoing Speciation in the Tibetan Plateau Gymnocypris Species Complex

Local adaptation towards divergent ecological conditions often results in genetic differentiation and adaptive phenotypic divergence. To illuminate the ecological distinctiveness of the schizothoracine fish, we studied a Gymnocypris species complex consisting of three morphs distributed across four bodies of water (the Yellow River, Lake Qinghai, the Ganzi River and Lake Keluke) in the Northeast Tibetan Plateau. We used a combination of mitochondrial (16S rRNA and Cyt b) and nuclear (RAG-2) genetic sequences to investigate the phylogeography of these morphs based on a sample of 277 specimens. Analysis of gill rakers allowed for mapping of phenotypic trajectories along the phylogeny. The phylogenetic and morphological analyses showed that the three sparsely rakered morphs were present at two extremes of the phylogenetic tree: the Yellow River morphs were located at the basal phylogenetic split, and the Lake Keluke and Ganzi River morphs at the peak, with the densely rakered Lake Qinghai morphs located between these two extremes. Age estimation further indicated that the sparsely rakered morphs constituted the oldest and youngest lineages, whereas the densely rakered morph was assigned to an intermediate-age lineage. These results are most compatible with the process of evolutionary convergence or reversal. Disruptive natural selection due to divergent habitats and dietary preferences is likely the driving force behind the formation of new morphs, and the similarities between their phenotypes may be attributable to the similarities between their forms of niche tracking associated with food acquisition. This study provides the first genetic evidence for the occurrence of convergence or reversal in the schizothoracine fish of the Tibetan Plateau at small temporal scales.

Positive Darwinian selection within interferon regulatory factor genes of Gymnocypris przewalskii (Cyprinidae) on the Tibetan Plateau

Tibetan Plateau (TP) had experienced phased uplift, resulting in inhospitable environment of low temperature, hypoxia and high ultraviolet radiation for Tibetan wildlife. Many organisms can well adapt to TP, it is of ecological and evolutionary interest to untangle how organisms adapt to extreme environment on TP through evolution. Previous studies mainly focused on hypoxia and metabolism related genes, but we know little about the evolutionary history of immune genes in Tibetan wildlife. In this study, we first identified 10 interferon regulatory factor (IRF) genes from Tibetan naked carp Gymnocypris przewalskii. Within this gene family, IRF3, IRF5, IRF7 and IRF8 contained positive selection sites. Evidences indicated that positive selection may lead to IRF genes functional alternations, presumably driving genes towards adaptation to the environmental changes. Taken together, our results suggested 4 candidate genes as interesting targets for further experimental confirmation of their functional variations and contributions to high altitude adaptation in Tibet fish.

Characterization of two paralogous myostatin genes and evidence for positive selection in Tibet fish: Gymnocypris przewalskii

Myostatin (mstn) is an important member of TGF-β superfamily, a muscle growth inhibitor. Though mstn has been identified in many organisms, little is known about this gene in highland fish, Gymnocypris przewalskii endemic to the Qinghai-Tibetan Plateau. In this study, we first cloned two paralogous mstn genes (mstn1 and mstn2) from G. przewalskii through homologue cloning. The 3D structures of both Mstn proteins varied in the numbers of β-sheets and conformations of α-helices. The branch-site model showed that mstn1 has undergone positive selection, and two positively selected sites (107M and 181T) were located on the random coils of the 3D protein structure. Expression patterns indicated that the mstn1 expressed widely, while the mstn2 only expressed in the muscle and brain. During the early stage of embryo development, the expression levels of both mstn paralogous genes showed different increasing trends. These results suggest that it is diverging in two mstn paralogues of G. przewalskii via specific differences in gene structure, protein structure, selection pressure and gene expression patterns. Taken together, this study provides novel contribution on the research topics of growth related gene function and mechanism of highland fish in extreme aquatic environment on the Qinghai-Tibetan Plateau.

Comprehensive transcriptomic analysis of Tibetan Schizothoracinae fish Gymnocypris przewalskii reveals how it adapts to a high altitude aquatic life

BackgroundUnderstanding 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.ResultsTo 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.ConclusionsOur 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.

The microRNA repertoire of Tibetan naked carp Gymnocypris przewalskii: A case study in Schizothoracinae fish on the Tibetan Plateau

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.

Integrated mRNA and microRNA transcriptome analyses reveal regulation of thermal acclimation in Gymnocypris przewalskii: A case study in Tibetan Schizothoracine fish

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.

Damage to epitaxial GaN layer on Al 2 O 3 by 290-MeV 238 U 32+ ions irradiation

Micro-structural characteristics and electrical properties of an n-type GaN epilayer on Al2O3 irradiated by 290-MeV 238U32+ ions to various fluences were investigated using atomic force microscopy (AFM), scanning electron microscopy (SEM), high-resolution X-ray diffraction (HRXRD), and Raman scattering spectroscopy. AFM images show that the nano-hillocks generated, and the diameter and density of the nano-hillocks, increase obviously with increasing ion fluence, accompanied by an increase in surface roughness. SEM images display that the Al, O, and C elements appear on the GaN surface, along with a spiral-like, layered volcanic-cone structure formed at the highest-fluence irradiation. HRXRD reveals that the dislocation density increases, as the lattices gradually expand, and that Ga2O3 was produced with increasing ion fluence. Raman scattering spectra show that no N and Ga vacancies were produced, the free-carrier concentration decreases, while its mobility first increases and then exhibits a significant reduction with increasing ion fluence.

Adaptive Evolution of the Eda Gene and Scales Loss in Schizothoracine Fishes in Response to Uplift of the Tibetan Plateau

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.