The Phylogeny, Biogeograph And Adaptive Evolution Of Chinese Sisoridae Fishes

The Tibetan Plateau(the ‘‘Roof of the World’’) is the highest plateau on earth, with an average elevation of more than 4,000 m. The plateau, which covers more than 2,500,000 km2 of plateaus and mountains in central Asia and is surrounded by towering mountain ranges, has been designated as a global hotspot of biodiversity. The environment of the Tibetan Plateau is characterized by hypoxia and low temperatures. Despite its inhospitable environment, various adaptive responses that may be responsible for highland adaptation have been identified in several species. Among these adaptive processes, genes exhibiting signs of positive selection and expansion were significantly enriched in hypoxia and energy metabolism pathways.Glyptosternoids refer to catfishes in the family Sisoridae subfamily Glyptosterninae tribe Glyptosternina. Currently, there are around 10 genera and 71 species of glyptosternoids, which 9 genera and 31 species distributed in China. Chinese Glyptosternoids are found in the rivers around the Tibetan Plateau and eastern Himalayas, e.g., the Yaluzangbujiang(Brahmaputra River), Irrawaddy, Nujiang(Upper Salween), Lancangjiang(Upper Mekong River), Jinshajiang(Upper Yangtze), Yuanjiang(Red River), Nanpanjiang(Upper Pearl River) and the Brahmaputra basin. The Glyptosternoids(Siluriformes) represent one of the three broad fish lineages(including the schizothoracines and Triplophysa) commonly found on the Tibetan Plateau. Several previous studies have investigated the phylogeny, biogeography and evolution of glyptosternoids. However, these studies are limited by poor taxon and genomic samplings, as they only analyzed 8-10 glyptosternoid species and used only one or a few mitochondrial or nuclear genes as markers. As a result, some key nodes of glyptosternoid phylogeny were poorly supported and the phylogenetic relationships among Pseudecheneis, Euchiloglanis, Exostoma, Glaridoglanis, and Glyptosternon remain particularly unclear. Our study analysis the phylogeny, biogeography and adaptive evolution with the methods of PCR-based next-generation sequencing, cross-species gene capture and comprehensive transcriptome due to the advatage of big data. the mainly results are following:1. The original mitochondrial genome sequences of 10 species of Glyptosternoids were determined with PCR-based next-generation sequencing in this study, and the published mitochondrial genome sequences of 15 teleost species from Gen Bank were used to conduct phylogenetic analyses. The results of the Bayesian and ML nucleotide analyses for the 12 protein-coding gene sequence datasets showed monophyly of the Chinese Sisoridae and glyptosternoids with very high support values(PP = 1.00 and BP = 95). The Glyptosternoids and non-Glyptosternoids form a sister group with high support values. Exostoma labiatum was placed with other Glyptosternoids to form a sister group to Glyptosternon maculatum in the phylogenetic trees. However, G. maculatum was placed with other Glyptosternoid fishes to form a sister group to E. labiatum in the phylogenetic trees. Specialized Glyptosternoids diverged into three main lineages: The first lineage included Euchiloglanis kishinouyei and Pareuchiloglanis from the Upper Yangtze(P. anteanalis and P. sinensis); the second included the Creteuchiloglanis from Nujiang, Pareuchiloglanis gracilicaudata and Pseudexostoma; and the third lineage included the Oreoglanis, Pareuchiloglanis longicauda and P. macrotrem. The latter two lineages form sister groups separate from the Upper Yangtze lineage. Pareuchiloglanis was not resolved as monophyletic. Chinese Sisoridae were found to originate in the Late Miocene(c. 7.7 Ma), the Glyptosternoids later in the Late Miocene(c. 5.5 Mya), and the specialized Glyptosternoids, Pareuchiloglanis, Oreoglanis, Creteuchiloglanis and Pseudexostoma, between the Pleistocene and Holocene. These results also show that explosive speciation of the specialized Glyptosternoids occurred between the late Pliocene and the Quaternary. basal lineages for the Glyptosternoid in the Tsangpo drainages and then spread into drainages of the Tibetan Plateau. Averaged across all 12 protein-coding genes, the ratio of nonsynonymous to synonymous substitutions is significantly higher in most of the Glyptosternoids lineages than observed in other non-Glyptosternoid lineages, suggesting accelerated function evolution in the Glyptosternoids lineages. In addition, the basal species of Glyptosternoids had lower ratios of nonsynonymous to synonymous substitutions than the specialized species. Using the branch-site model, positively selected signals were detected. According to our estimates, ωpss was 10.73; thus, the ωpss value was significantly higher than 1(the p-value was 0.0002). Positively selected sites occurred in the COX1 gene. In the other branches, which represent specialized Glyptosternoids, positively selected sites occurred in most of the mitochondrial protein-coding genes. Thus, the mitochondrial genes of Glyptosternoid fishes may have experienced adaptively accelerated evolutionary rates to better adapt to the extreme environments of the Tibetan Plateau because accelerated evolution is usually driven by positive selection.2. By screening the genome alignments of zebrafish(Danio rerio), fugu(Takifugu rubripes) and green spotted puffer(Tetraodon nigroviridis) through the Evolmarker, we identified 3502 target single-copy protein-coding gene markers, which ranged from 200 bp to 5811 bp in length. Markers from zebrafish were used to design 16,852 enrichment RNA probes targeting sisorid fishes and zebrafish was used as a positive control. A total of 14,405,557 sequencing reads were generated via Illumina sequencing. These reads were then filtered and defective reads were removed for each species. High-quality sequencing reads and appropriate assembly methods are essential for obtaining a reliable de novo assembly, which serves as the foundation of all subsequent analyses. For cross-species captures, we were able to obtain full sequences for 1514 of the 3502 target sequences in the worst case and 2416 of the 3502 target sequences in the best case. The average identity between baits and successfully captured target sequences ranged from 79.5% to 100%. To obtain credible phylogenetic relationships for sisoridae fishes, we assembled and analyzed two different datasets and compared the results. For the first dataset, we extracted target genes present in all of 28 species and concatenated them(“all species” dataset). The second dataset(“partial species” dataset) included all of the target genes present in more than two species, and we concatenated these genes and treat gaps as missing data. After trimming using Gblocks, the “all species” dataset contained 232 captured orthologs(6.6% of the initial markers), whereas the “partial species” dataset contained 2494(71.2% of the initial markers). The “partial species” dataset was used in the subsequent analyses. Results of the Bayesian inference and maximum-likelihood(ML) analyses all converged to the same topology, differing only in the support values for specific nodes. The obtained phylogenies indicated non-monophyly of the Glyptosternina catfishes and the controversial group(G. maculatum and G. andersonii), located between Pseudecheneis, Bagarius, and Glyptothorax, were recovered with high bootstrap support(PP = 0.97 and BP = 99). The genus Pseudecheneis was placed at the base of the Chinese Sisoridae. The genera Bagarius and Glyptothorax constituted a monophyletic group and then formed a sister group with all Glyptosternina catfishes less G. maculatum and G. andersonii. All of the non-controversial groups(Glyptothorax, Oreoglanis, Pseudexostoma, and Creteuchiloglanis) were recovered with unambiguous bootstrap support(BP = 100%) and Bayesian posterior probabilities(PP = 1.0). The specialized Glyptosternina diverged into two main lineages: the first lineage included Creteuchiloglanis, Pseudexostoma, and Pareuchiloglanis(P. gracilicaudata, P. kamengensis, and P. myzostoma), while the second lineage included Oreoglanis, Pareuchiloglanis longicauda, and P. macrotrem. These two lineages formed sister groups that were separate from the Upper Yangtze lineage(E. kishinouyei, P. sinensis, and P. anteanalis). Pareuchiloglanis was not resolved as monophyletic. To test Stability of phylogenetic relationships within the Sisoridae, a likelihood framework to evaluate topology and the maximum pseudolikelihood estimation of the species tree method, analysis of long-branch attraction(LBA) artifacts and compositional biases and saturation plot were used. It also shows the Glyptosternina catfishes were not a monophyly group.3. We sampled three glyptosternoid fishes(G. maculatum, P. sinensis, and P. macrotrema) from the Yaruzampbo River in Tibet(3800 m-4000 m), Daduhe River in Sichuan(1,000 m-2,000 m), and the Yuan River in Yunnan(<1,000 m), as well as three additional catfish species as control. A total of 20,659,183- 37,166,756 sequence reads were generated from the six catfishes liver tissue by Illumina sequencing. First, we filtered these reads and removed the defective reads for each species. De novo assembly of the cleaned reads was performed using Trinity with various combinations of K-mer lengths and coverage cut-off values. In total, 6 raw assemblies were obtained for the six catfishes and further merged by integrating sequence overlaps and eliminating redundancies for each species. Blastx(a blastx search against zebrafish proteins with significant hits to zebrafish genes), Blast2 GO with the Gene Ontology(GO) annotation database and clusters of orthologous groups of proteins(COGs) and the non-redundant(NR) databases were used to annotate these Unigenes and to produce good results for putative proteins. Hamstr and the reciprocal blast best-hit method with an E-value cutoff of 1e-10 were used to identifiction for putative orthologs among six catfishes. A total of 708 putative orthologs were identified by comparing all six transcripts sets used hamstr and 170 were retained after alignment and trimming for quality control. After alignment and trimming for quality control, a total of 1,656 orthologs ranging from 150 to 7155 bp were retained through reciprocal blast best-hit method. these orthologs were used in the subsequent evolutionary analysis. The free-ratio model(M1 model) in PAML was used to examining the Ka/Ks ratios for 1,656 orthologous genes in the glyptosternoid lineage, we found that 480 genes had higher Ka/Ks values in all three glyptosternoid fish lineages. The Ka/Ks of P. sinensis was highest within the glyptosternoid lineage. A number of GO categories were involved in the 480 genes in the glyptosternoid lineage that underwent rapid evolution compared to the yellowhead catfish. For example, genes associated with energy metabolism, hypoxia response, and DNA repair showed significantly accelerated evolution in glyptosternoid fish compared to the yellowhead catfish. To detect genes that might have evolved due to lineage-specific adaptation, two types of gene sets were compiled: 1) fast-evolving genes(FEGs) that exhibited a significantly higher Ka/Ks ratio in specific lineages compared with other lineages and 2) positively selected genes(PSGs) that were influenced by positive selection only on a few codons along a particular lineage. In total, we identified 121-178 FEGs in living glyptosternoid fish and 63 FEGs in yell owhead catfish and 58-244 PSGs in living glyptosternoid fish and 48 PSGs in yellowhead catfish. This finding suggests that the living glyptosternoid fish lineages have higher numbers of FEGs and PEGs compared to yellowhead catfish. To identify genes that might directly contribute to the adaptation to high altitude, we used the 244 PSGs in G. maculatum as the candidate genes. Finally, we identified 13 candidate PSGs in glyptosternoids that may be involved in the hypoxia response: Slc2a8, Igfbp7, C2, Cp, Ndc1, Hspa5, Ttr, Gapdh, Prmt5, Srebf1, Perp, Map3k14 and Fam162 a.