Molecular Adaption Of The Schizothoracine Fishes In The Phased Uplifting Of Qinghai-Tibetan Plateau Revealed By Transcriptome Comparisons

Many species of Schizothoracine,a sub-family of Cyprinidae,are highly endemic to the Qinghai-Tibetan Plateau(QTP).Current transcriptome studies are limited to single highland species with comparisons made to distantly related teleosts with available genomic information.Comparisons among closely related Schizothoracine species were absent.In this study,we compare the transcriptome of Cyprinidae fished from three level,and then we performed evolutionary analyses on these data.Firstly,we sequenced transcriptomes of eight tissues from a primitive Schizothoracine fish Schizothorax nukiangensis Tsao,which lives at about 1,000 m a.s.l,and a highly specialized one Gymnocypris dobula which lives at >4,000 m a.s.l.Their gene expression profiles are compared with those of a primitive cyprinid specie,Danio rerio.Raw reads were cleaned by removing adapter sequences using sequence pre-processing tool Trimmomatic.The transcriptomes of the tissues were pooled to generate an improved de novo assembly using the Trinity package,and open reading frames(ORFs)were predicted using the transdecoder.The assembled unigenes were first annotated by NR,KOG,GO and KEGG.Orthologs among the two Schizothoracine fishes and zebrafish were determined using OrthoMCL.After sequence alignment and trimming,11,007 one-copy orthologous genes were retained for further analyses.The lineage-specific evolutionary rates were estimated by measuring dN/dS ratios using the free-ratio model in PAML.We found that both high-and subhighSchizothoracines demonstrated elevated dN/dS ratios in the protein coding genes compared to lowland cyprinids.These results indicated that Schizothoracinaes have undergone rapid evolution to adapt to the extreme environments of the Tibetan Plateau.The improved branch-site model in PAML was used to detect positively selected genes(PSGs).PSGs in G.dobula and S.nukiangensis covered similar GO categories,including “response to hypoxia”,“Metabolic process”,“cardiovascular system development”.The disparate distribution of the PSGs suggested divergent adaptation strategies deployed by the highland and sub-highland Schizothoracines in evolution.Furthermore,we sequenced transcriptomes of five tissues from four Schizothoracine fishes,including two high-altitude and specialized ones,Gymnocypris dobula and Ptychobarbus kaznakovi(termed as highland species),and two sub-high altitude and primitive ones,Schizothorax gongshanensi and Schizothorax prenanti(termed as subhighland species).Unigenes from the Schizothoracines were compared with two lowland,non-Schizothoracine fishes,zebrafish(Danio rerio)and grass carp(Ctenopharyngodon idella).After multiple sequence alignment and trimming of low quality alignment,7,532 single-copy orthologous genes were retained for further analysis.They were concatenated into a single supergene and four-fold degenerate sites were extracted for phylogenetic tree reconstruction.Then,we used Modeltest to select the best substitution model(GTR + gamma + I).Maximum likelihood method was applied for phylogenetic tree reconstruction using PAUP.The two specialized species G.dobula and P.kaznakovi formed one lineage(highland lineage)and the two primitive species,S.gongshanensis and S.prenanti formed another lineage(subhighland lineage).All nodes were supported with bootstrap values of 100.The MCMCtree program implemented in PAML package was used to estimate the divergence times among the species using a maximum likelihood method.Phylogenetic tree reconstruction and divergence time estimation indicated that the common ancestor of Schizothoracine fishes lived around 32.7 million years ago(MYA),coincided with the timing of the first phase of QTP uplifting.The common ancestor of P.kaznakovi and G.dobula diverged about 8.1 MYA ago,when QTP was in the second phase of uplift.The Schizothoracine fishes exhibited significantly higher dN/d S values than the lowland cyprinid species.The GO categories such as “metabolic process”,“response to hypoxia” and “DNA repair” were found to have elevated dN/dS ratios in the highland species.We identified 208 and 147 PSGs from the highland and subhighland branches respectively.The two PSG sets overlapped by only 12 genes.The highland and subhighland lineages presented drastically divergent landscapes of positively selected genes(PSGs),enriched with very different gene ontology profiles,including those in “sensory organ morphogenesis”,“regulation of protein ubiquitination”,“blood circulation” and “blood vessel development”.These results indicated different selection pressures imposed to the highland and subhighland lineages of the Schizothoracine subfamily.We compared PSGs identified in our study with a hypoxia response gene list.In the highland and sub-highland species.There were 28 and 21 PSGs found to be hypoxia responsive,respectively,and the two sets overlapped by zero genes.Some PSGs are involved in various metabolic processes.We compared the expression pattern between the highland and subhighland lineages.We compared the expression pattern between the highland and subhighland lineages.We found liver,muscle and spleen,which involved in metabolic process,immune response and hemopoiesis,had a large number of different expression genes.There were 27 genes appeared in both PSGs list and DEGs list.These genes may play an important role in the highland adaptation.