| The northeastern part of The Qinghai-Tibet Plateau is one of the regions where the number of the Triplophysa species in the plateau is the largest.the Triplophysa fishes is one of the three major fish groups distributed in the Qinghai-Tibet Plateau,which has very important ecological value.However,their taxonomy and systematics are still controversial,and a large number of new species have been reported in recent years.This phenomenon was caused by the low degree of morphological variation and the relatively poor phylogenetic information provided by morphological and anatomical features for species identification,and the existence of a large number of cryptic species.Based on the setting of high-density sampling sites,this study evaluated the hidden diversity of the Triplophysa fishes in Gansu Province by morphology,DNA barcoding and multiple species demarcation methods,and described the new species discovered.The effects of uplift and climate oscillation on the evolution of loach population in the Qilian Mountains were explored.The adaptation mechanism of different ecotypes of the Triplophysa fishes to salt-alkali stress was compared and analyzed.1.Morphological characteristics and DNA barcodes of 1630 specimens were identified and compared.A total of 22 morphological species were identified,including20 species that were considered valid native species and 2 new species that have not been described.A total of 24 indigenous species were estimated by integrated molecular methods,including Triplophysa robusta sp1 and Triplophysa minxianensis sp1.Fourteen species formed distinct DNA barcoded monophyletic groups,which allowed them to be reliably identified using molecular species demarcation methods.The remaining 10 closely related species could not be distinguished by DNA barcoding due to the short time of species differentiation,disputed taxonomic status or hybridization.These results emphasize the necessity of combining traditional taxonomies with molecular methods to correctly identify cryptic species.2.Eleven species of plateau loach were collected in the Qilian Mountains,among which the T.hsutschouensis and T.papillosolabiatus are widely distributed in all rivers on the Northern Slope of the Qilian Mountains.The genetic structure,phylogenetic relationship and phylogeographical characteristics of the Triplophysa species in the Qilian Mountains were studied by using mitochondrial DNA gene,3 nuclear genes and11 pairs of microsatellite markers.It was found that there was a high degree of lineage differentiation among species and the genetic diversity of endemic species was low.The different geographical groups of the plateau loach presented some degree of allogenetic adaptation and differentiation,which was closely related to the changes of river system.Except for the population expansion event of the T.hsutschouensis during the last glacial period of the Tibetan Plateau(0.025 Ma),the population size of other species remained stable in recent history without significant population expansion.The T.hsutschouensis and T.papillosolabiatus showed two species dispersal routes in opposite directions from the west and east sides of Qilian Mountains respectively.The tectonic events of the uplift of the Qinghai-Tibet Plateau and the repeated climatic fluctuations during the Quaternary glaciation had a great influence on the genetic structure of the plateau loach in the Qilian Mountains,which promoted the genetic differentiation of the plateau Loach and formed some endemic species.3.According to morphological and molecular biological analysis,Triplophysa jingheensis sp.nov.,a new species of the genus,was identified.The Triplophysa jingheensis is distinguished from other species by the following morphological characteristics: the number of medial gill rakes of the first gill arch was 7-8;the head length/eye spacing ranged from 2.6 to 3.3;the number of vertebrae was 4+38～39;the caudal peduncle length was 2.0-2.6 times of the caudal peduncle height;scales are rare,and residual scales are distributed from the caudal peduncle along the lateral line to the front of the dorsal fin,and are hidden under the skin,which is not easy to be observed;the lateral line is incomplete and there are few lateral line holes near the base of caudal fin;posterior margin of dorsal fin slightly concave;the base of the pelvic fin is slightly in front of the base of the dorsal fin;posterior margin of caudal fin deeply concave or forked;Back in the dorsal fin before and after each 4～5 black large transverse spots;The pelvic fin ends far beyond the anus.The K2 P genetic distance between T.jingheensis and other homologues ranged from 0.6-20.8%.The genetic distance between T.jingheensis and T.pappenheimi and T.siluroides was much lower than that of the other nine loach species(0.6% and 0.8%,respectively),but there were significant differences in external morphological characteristics,ecological preferences and distribution areas of these three species.The genetic distance of K2 P between the T.jingheensis and T.robusta was 11.7%,which was much higher than the threshold of 2%among different species.Phylogenetic analysis supported the monophyletic character of the T.jingheensis,and the genetic differences between the T.jingheensis and other fish reached the specie-level differentiation level.The morphological and molecular differences between the T.jingheensis and other members of the same genus indicate the taxonomic status of the species,so the T.jingheensis is considered to be an independent species.4.A total of 8002 differentially expressed genes were detected in the two different ecotypes of the T.papillosolabiatus,among which 3558 genes were up-regulated and4444 genes were down-regulated.These differentially expressed genes include osmotic pressure regulation genes(such as KCNG1,P2RX7,TTC26,ITM2BB),and genes involved in fat metabolism(MOGAT2,acetyl-Coa carboxylase-like,fatty acid desaturase 2-like),and genes related to immune stress(C-type lysozyme,LIM binding protein,metallothione gene,? defensin 2 gene,AND MHC gene).Eight genes were screened for RT-QPCR validation,which proved the reliability of transcriptome data.Through GO functional classification and KEGG metabolic pathway analysis,it was found that multiple life activities of the T.papillosolabiatus showed adaptability,and multiple biological pathways were involved in the stress response of salt-alkali stress,such as stress response,ion regulation,lipid metabolism,energy metabolism,reproductive metabolism and immune stress.These results suggest that the T.papillosolabiatus adapted to saline-alkali water ecotype may cope with saline-alkali stress from osmotic pressure regulation,metabolic response,immune response and other aspects.In conclusion,the species diversity level of the Triplophysa fish in Gansu Province has been underestimated,and the discovery of hidden species reveals the need to revise the classification of the Triplophysa.The species differentiation of the Triplophysa fish in the Qilian Mountains is closely related to the geological events of plateau uplift and the Quaternary ice age.the T.papillosolabiatus adapts to saline-alkali environment from osmotic pressure regulation,metabolic response and immune response. |
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