The Origin And Evolution Of Cave-dwelling Group Of Triplophysa Fishes(Teleostei,Cypriniformes,Nemacheilidae)

The genus Triplophysa is one of the largest groups in the family Nemacheilidae(Teleostei,Cypriniformes,Cobitoidea),and it constitute the mainpart of fish fauna in the Qinghai-Tibet Plateau(QTP)with schizothoracinae and glyptosternoid fishes.According to the distinctive morphological characteristics and distribution pattern,many researchers suggested the originand evolution of Triplophysa fishes were related to the uplift ofthe QTP.Therefore,this group of fishes plays an important role in zoogeographic study of the QTP.In China,this genus is widely spread in the QTP and its adjacent areas,but some Triplophysa species are restrictedly distributed in the karst region of Southwest China.These species are mostly cave dwellers which adapted to cave environments and usually show various degrees of morphological degradation characteristics,such as reduction of eyes or loss of skin pigmentation,so this group of species is very different from those distributed in the QTP or its adjacent areas in some ways.Up to now,a total of 27 valid cave-dwelling species are described in Triplophysa,they occur in the Yangtze River,Pearl River and Red River drainages,but the majority of cave species live in the Xijiang River,the upper reaches of Pearl River Basin.Till now,researchers have studied the phylogenetic relationships or the origin and evolutionary process of the genus Triplophysa,but few of them were related to the cavedwelling group,which is an important component of Chinese cave fishes that has important scientific value in the study of natural selection and adaptive evolution.The current studies on this group were still in the stage of description of new species,and a lot of fundamental research has not been carried out,such as the studies on their phylogeny,and their origin and evolution process.Therefore,we took the cave-dwelling group of Triplophysa fishes as the object of the presentstudy,and the main aims of the study were composed by three parts.Firstly,we use mitochondrial(COI and Cytb)and nuclear(IRBP,RAG1 and RH1)gene sequences as molecular markers to reconstruct the phylogenetic relationships among the cave-dwelling groupand to show their systematic position in the Triplophysa.Secondly,we use the Bayesian relaxed molecular clock methods and related fossil records to estimate the divergence time of the major Triplophysa lineages and to analyze its evolutionary patterns.Moreover,we choose the biogeographicmodel and geographical distribution data of this genus to reconstruct the ancestral geographicareas and to investigate the origin and evolution of these cave species of the genus.Finally,we compared the morphological characteristics of the cave and non-cave species of Triplophysa based on the existing studies and available specimens,which intended to provide the reference for further research.The main results were as follows:1.The sequences for 30 Triplophysa species and 65 individualswere obtained through PCR amplification and sequencing.We choose 59 COI sequences with 1116 bp,59 Cytb sequences with 1140 bp,59 IRBP sequences with 834 bp,57 RAG1 sequences within 1473 bp and 53 RH1 sequences with 769 bp for subsequent analysis.We reconstructed the phylogenetic trees by using Bayesian inference(BI)and maximum likelihood(ML)methods based either on a single gene or concatenated dataset.The phylogenetic trees based on the mitochondrial genes showed that the genus Triplophysa is not a monophyletic group,three species(T.longibarbatula,T.dongganensis and T.lihuensis)clustered with Yunnanilus,and other Triplophysa cave species and non-cave species formed monophyletic group,respectively.Moreover,Claea dabryi clustered with Triplophysa species and to be the sister group of cave species.Based on the analysis of nuclear genes,phylogenetic relationships were similar to those obtained from mitochondrial genes,but were slightly different.The results showed that Claea dabryi clustered with cave species in Triplophysa and to be the sister group of a cave species from Jinsha River.T.robusta was not grouping with other non-cave species but locating at the basal position in the genus Triplophysa.The results based on the combination of the mitochondrial and nuclear gene sequences were similar to the results of mitochondrial genes.According to the phylogenetic relationships of the cave-dwelling group of Triplophysa,we found that some species from the Yangtze River are more closely related to some species from the Pearl River,suggesting that the upper reaches of the Yangtze River and the upper reaches of the Pearl River have been connected in history.Overall,our analyses indicated that there is closer genetic relationship between species having closer distribution and collecting from the same river drainage.2.Molecular clock analyses showed that the divergence time of Triplophysa and outgroup is about 19.45millionyearsago(Ma),and this genus began to diversify at 17.91 Ma.The time of origin and differentiation was consistent with the rapid uplift of the QTP in the early Miocene(25-17 Ma).The results strongly supported the hypothesis that the origin and evolution of Triplophysa is closely related to the uplift of the QTP.The origin and differentiation of the cave species were also affected by the uplift of the QTP and the monsoon climate.The more immediate reason was that these factors change the formation and evolution of karst landform in southwest China.The results from the lineage numbers plotted over time indicated that Triplophysa lineages increased in an exponential mode with the equation expressed as y=66.835×e-0.19544 x.,showing the characteristics of nonuniform evolution.In the non-cave group,the general trend of growth rate was that it decrease with the increase of lineage number,and the fastest rate of growth occurred in the early stages of differentiation(15 to 12 Ma).The peak of the growth rate was found in the later stage of differentiation for cave-dwelling group.The results of the net growth rate analysis were the same as the growth rate,but the variation trend is more obvious.Based on the divergence time and ancestral geographic distribution,we speculated that uplift of the QTP cause drastic changes in environmental conditions,leading to the divergence among the ancestors of Triplophysa in the early Miocene,The intensive uplift of theTibetan Plateau has promoted the rapid differentiation of the cave species since the 3.6 Ma.The center of origin and divergence for ancestral of cave-dwelling group was ancient Jinsha River,and then gradually spread to the ancient Hongshui River or ancient Wujiang River.The distribution pattern of the cave species of Triplophysa resulted from dispersal and vicariance events,and vicariance was the main mechanism of their biogeographic process.3.In this study,we summarized the main adaptive morphological characteristics of the cave-dwelling group in Triplophysa and made a key to the existing valid cave species.The results suggested that we cannot over emphasize some morphological traits,such as body spot,skin color and barbel length,that strongly affected by environment during biological classification for Triplophysa species.