Taxonomy,Differentiation And Dispersal Of Earthworms In The Hengduan Mountains

The Hengduan Mountains(HMs)is a global biodiversity hotspot and key region for exploring some biology questions,which is located at the junction the western of Yunnan,Sichuan and southeastern Tibet provinces.Most part of this region are high mountains and deep gorges from north to south.Because of its unique geographical location,habitats,climates,complex topography and distinct geographical events,the level of biodiversity in this area is considerably high.However,the study of soil-dwelling animals such as earthworms in the HMs were largely ignored.Earthworms are an ancient group and play important functions in eco system.Because of their weak dispersal capability and susceptibility to the changes of environmental condition,they are considered as an ideal model for phylogeographical study.Therefore,understanding how earthworm in the HMs region form the species diversity,investigating its differentiation and dispersal pattern,and discussing the effect of paleogeographic events in this area on the geographic distribution pattern and evolution course of earthworm have important scientific significance of the biological resources evaluation and protection in this region,and also provide certain theoretical data and scientific reference for the future study regarding origin evolution and genetic diversity of earthworm in China.In this study,earthworm species were systematic collected from 157sampling sites in the HMs.Then we aim to:determine the species composition and geographic distribution;reveal the genetic variability and phylogenetic relationships of these species;estimate the divergence time and reconstruct ancestral areas;elucidate the genetic diversity of Amynthas.regularis sp.nov.populations and test gene flow among different lineages.The main results were:(1)A total of 64 new species of earthworm were first discoversed and recorded in China,and they were identified based on their difference of morphological characters and molecular information,which is important for improving the catalogue of earthworms in the HMs.(2)About 81.2%of the species belong to Oriental species,and a few species belong to Palaearctic species in this area.At the level of horizontal distribution pattern,the high latitude region of the HMs(northeast of the HMs)had the highest species richness,followed by the low latitude region of the HMs(south of the HMs).At the level of vertical distribution pattern,the species richness gradually decreased with the rise of altitude above 1000 m.The areas of mid-high altitudes has the high species richness.(3)The mean interspecific genetic distances of concatenated genes(3993bp:COI-COII-12S-16S-ND1)between Megascolecidae,Lumbricidae and Moniligastridae were 26.37%,37.24%and 36.55%,respectively.The mean interspecific genetic distances of five genes at species level ranged from 10%to 30%,the mean interspecific genetic distances of five genes at population level ranged from 0.0%to 5.0%,(4)The topologies of the BI and ML trees were basically identical.For the large data set were clustered different clades into eight species groups and one single species(M405)according to the“sequencing convention”and the species groups of Chinese Megascolecidae earthworm,with high posteriori probability and non-parametric bootstrap(PP>0.9,BP>70).The evolution relationships among different species of the Family Moniligastridae,Lumbricidae and Megascolecidae revealed by molecular phylogeny analysis.(5)Molecular phylogeny and divergence times analysis based on concatenated genes suggest that the divergence time of Moniligastridae and Lumbricidae was 215.84 MYA(95%HPD:200.03-226.31 MYA),the divergence time of Lumbricidae and Megascolecidae was 211.03 MYA(95%HPD:190.74-220.15 MYA).About 44.42 MYA(95%HDP:38.14–52.62MYA),the dispersal of Megascolecidae from the southern of the HMs which is bordering Xishuangbanna and Burma to this region coincides with Indian-Eurasian collision between Eocene and Oligocene.The reconstruction of ancestral-area reveals the clades in Megascolecidae(Group 2-8)may have experienced different evolutionary histories following their divergence time during Cenozoic,which suggests that paleogeographical events(Indian-Eurasian collision,the uplift of Qinghai-Tibetan Plateau,the movement of Himalayan tectonic,the uplift of the HMs,et al.)might promote the dispersal and diversification of earthworm in the HMs.(6)Here,we applied 769,957 single-nucleotide polymorphisms(SNPs)gathered by RAD-seq to unearth the genetic variability of populations of the earthworm A.regularis sp.nov.collected from the HMs.The neighbor-joining(NJ)tree,PCA and population structrue methods revealed that 30 individuals strongly clustered into three main lineages(G2,G3,and G5 lineages)which coincided with the geographic distribution proximity of the sampling locations.There was a low level of genetic differentiation among these lineages with the average genetic differentiation(F_ST)value of 0.105,0.135 and 0.080,respectively.The genetic divergence(??=0.118±0.037)of G3 lineage was significantly higher than the other two lineages.There was only one migration edge in the population from the G3 lineage to the G2 lineage.Therefore,we presume that a reduction in gene flow caused by geographic isolation influences the population genetic differentiation of A.regularis sp.nov.Additionally,the presence of river as geographical barrier is likely to limit gene flow.