| Purpose:1.Using the 16S rRNA and 28S rRNA gene combination sequences,the genetic structure among the five geographical populations of Potamiscus motuoense in the Medog county,Yarlung Zangbo suture zone was analyzed.At the same time,the role of rivers and mountains in the evolution of different populations was initially understood.2.Obtaining the mitochondrial genome data of P.motuoense,combining the mitochondrial genomes of brachyura in the Genbank database,and constructing phylogenetic trees based on 13 protein-coding genes of mitochondrial genomes,exploring the phylogenetic relationship of P.motuoense in brachyura.Method:1.According to the only species distribution clues of Potamiscus motuoense reported by Dai Aiyun（1999）,combined with two field trips to the Grand Canyon that conducted in April 2013 and August 2014 in this study,defining the detailed and true geographical distribution of P.motuoense in the Yarlung Zangbo suture zone;acquisition of 16S and 28S rRNA gene sequence data for 128 P.motuoense samples from 5 different geographic groups in the Medog segment of the Yarlung Zangbo suture zone.The two genes were sequence aligned and conserved regions were selected;Sequence Matrix software formed a combined sequence with a total sequence of 981 bp.Based on the joint sequences,the gene flow,genetic distances between populations and groups,neutral tests,and haplotype network analysis were calculated.2.The full-length mitochondrial genome data of P.motuoense was measured and uploaded,and its total length was 17971 bp.The location of 22 tRNAs,the presence of mismatches,and the codon usage of 13protein-coding genes were analyzed,and combining with the genomic data of brachyura mitochondrial clusters published in the Genbank database,the phylogenetic analyses was constructed based on 13 protein-coding genes,and the taxonomic status of P.motuoense in brachyura was discussed.Conclusion1.Preliminary studies have confirmed that Potamiscus motuoense is the only species that distributed in the minor tributaries of the Yarlung Zangbo River,such as Medog Town,Damu Township,Dexing Township,Beibeng Township,Beibeng Village,Badeng Village,etc.in the Medog County of the Grand Canyon.The altitude of its habitat is from 812m to 1548m.Only a single species,P.motuoense,in Potamiscus,was found and collected in the above section.The reasons of this need further studies.2.The male first gonopod and other important morphological features of mature freshwater crab individual collected from the above areas were compared with the holotype photo of Potamiscus motuoense,and the result is consistent,indicating that the collected freshwater crab specimens were P.motuoense.The genetic distance between the five geographical populations calculated with MEGA software was in the range of 0.001-0.006.The genetic distance within the population was 0.001,and was less than the genetic distance between species of 0.02,indicating that the five geographic populations were the same species.This study confirms that the collected freshwater crabs are P.motuoense from both morphological and molecular biological perspectives.3.Seventeen haplotypes were found in 128 sequences of five geographical populations of Potamiscus motuoense.Only haplotype 1（Hap1）and haplotype 2were shared haplotypes,and the total population haplotype diversity index was0.857±0.014.The glycoside diversity index was 0.00277±0.00016.The genetic diversity of the entire population of P.motuoense was low haplotype diversity and low nucleotide diversity.4.Potamiscus motuoense has a gene flow（Nm）value of 0.00550-0.51912among the 5 geographic populations,which is small.It shows that there is a lack of genetic exchange among different geographical populations,and factors such as mountains and rivers may hinder gene exchange between different geographical groups to a certain extent.However,there are still some channels among various groups that make it possible to carry out weak gene exchange,suggesting that mountain and river play a role as barrier in the evolution of P.motuoense,but it did not cause complete reproductive isolation.5.The AMOVA analysis of five geographical populations of Potamiscus motuoense showed that the genetic variation among populations accounted for79.91%,and the genetic variation within the population accounted for 20.09%.It revealed that the genetic variation of P.motuoense’s five geographical groups mainly resulted from inter-group variation,within population variation is only a small part.Neutral test results showed that the total population Tajima’s D test and Fu’s Fs were negative and there was no significant difference（Tajima’s D=-0.70222,P<0.5;Fu’sFs=-0.90547,P<0.5）.This shows that P.motuoense has not experienced population expansion;Combined with the mismatch distribution,the nucleotides showed a multi-peak curve,suggesting that the population of P.motuoense was in a stable state and the results were consistent.The Yarlung Zangbo Grand Canyon may play a role in ice evacuation sites during the historical evolution of P.motuoense,hindering the complicated and changeable environment,protecting P.motuoense from the effects of large environmental changes,making it stable and conducive to the state of population dynamic balance.6.The mitochondrial genome of Potamiscus motuoense is 17971 bp in length and encodes 37 genes as commonly found in metazoans.The study found that P.motuoense mitochondrial genomic data share tRNAHisis translocation with other brachyura in gene arrangement(ie,between ND5 and ND4 to tRNAGlulu and tRNAPhe).This phenomenon supports the ubiquity of brachyura from the mitochondrial genome level.In addition,there is also a rearrangement of two tRNA genes,that is,tRNAGlnln is transferred from tRNAIlele and tRNAMetet to rrnL and rrnS,and tRNAValal is transferred from rrnL and rrnS to CR and tRNAIle.Comparing the genetic arrangement of five freshwater crabs that have published mitochondrial genome data,the results showed that compared with the original pancrustacea,fewer gene rearrangements occurred in P.motuoense,Geothelphusa dehaani,Huananpotamon lichuanense,Somanniathelp-husa boyangensis and Sinopotamon xiushuiense occurred.Larger gene recombination suggests that P.motuoense,G.dehaani and H.lichuanense are more primitive freshwater crabs,and S.boyangensis and S.xiushuiense have undergone a higher degree of evolution.7.An ML-tree constructed from the combined sequence data of 13 protein-coding genes showed that 5 species of freshwater crabs gathered together,and 4species of freshwaters,Sinopotamon xiushuiense,Geothelphusa dehaani,Huananp-otamon lichuanense and Potamiscus motuoense,clustered together,and finally clustered with Somanniathelphusa boyangensis,which is consistent with the results of traditional morphological classification.This study believes that P.motuoense,H.lichuanense,and G.dehaani are closely related.8.According to the INCU Red List Criteria（version 3.1）,freshwater crabs that are at risk of extinction are evaluated and the Potamiscus motuoense assessment is classified as a“Vulnerable/VU”rating in the red list.In view of this,this study proposes that the natural habitat of Potamiscus motuoense should be protected in the Medog County at the downstream of Yarlung Zangbu Grand Canyon. |
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