| Objective:Pomacea spp.(Ampullariidae:Pomacea)is an aquatic mollusk,and P.canaliculata is recognized as a global invasive organism and causes serious damage.The Pomacea spp.invaded into Yunnan Province in the mid-to-late 1980s and then spread to 33 counties in 10 districts,destroying the original ecology of the invaded land.It is also the main intermediate host of Angiostrongylus cantonensis,threatening human public health safety.In recent years,there have been many cases of Angiostrongyliasis cantonensis in Yunnan Province.This paper takes the three major water systems in Yunnan Province(Lancang River,Nujiang,and Yuanjiang)as the research scope,and investigates the distribution and types of the snails based on the mitochondrial coxidase ? gene(COI)marker.At the same time,all samples were analyzed for haplotype and molecular evolution.It is proposed to reveal the molecular composition of the three major water systems,and to analyze the laws of invasion,colonization and diffusion.Methods:In this study,the regional sampling method was used to select 9 states/districts(districts/counties)flowing through the Lancang River,Nujiang River and Yuanjiang River systems in Yunnan Province,with a total of 5 regions and 27 sampling points.For the above-mentioned survey,the farmer's market in Point City,as well as the surrounding area,the land,the pond,the vegetable plot,the ditch,the fish pond,etc.,are there traces of the Pomacea spp.If the Pomacea spp.are found,the samples are taken back to the laboratory.The whole genomic DNA was extracted from the foot tissue,and the COI sequence of the sample was amplified by conventional PCR and sequenced.The phylogenetic tree of all sequencing samples was constructed by MEGA 6.0 using the maximum likelihood method.Then,the haplotype calculation was performed using Dna SP 5.10,and the haplotype network was constructed by using TCS 1.21 and Netwalk4.0.Finally,the haplotype was drawn by ArcGIS 10.2.Type distribution map.The haplotype COI sequence in the haplotype analysis was selected,and the COI sequence of the snail was published in the public database.After sequence alignment using MEGA 6.0,the phylogenetic tree was constructed based on the maximum likelihood method.Use DnaSP 5.10 to calculate Pi,k,Hd and other indicators reflecting population genetic diversity.Results:The Pomacea spp.were found at the 22 of the 27 sampling points in the Lancang River,Nujiang River and Yuanjiang River,accounting for 81.48%.At the same time,a total of 344 COI sequence information of the Pomacea spp.samples were obtained.Phylogenetic analysis indicated that there were three invasive species of Pomacea spp.in five regions of Yunnan's three water systems:P.canaliculata(84.59%),P.maculata(1.16%)and Pomacea sp.(14.24%).For the P.canaliculata,the region of the upstream water system in the Lancang River,middle reaches of the water system?downstream water system area?Nujiang River System,a total of 7 sampling points existed P.canaliculata and Pomacea sp.In the Yuanjiang River area,Yuanjiang County was accounting for 4.07%and Luxi County was accounting for 4.07%,there were only existed P.canaliculata.In this study,there were only four samples of P.maculata,which were from the middle reaches of the Lancangjiang River and the downstream water system areas(Aihua Town and Gengma Town of Linyi City,Nanping Town of Pu'er City,Yuanjiang County of Yuxi City),and all were single samples.The haplotype analysis of the P.canaliculata of the three rivers in Yunnan Province showed that there were 14 haplotypes(PcH1,PcH2,PcH 19,PcH39?PcH49),including:6 shared haplotypes and 8 unique haplotypes.PcH19,PcH47 and PcH1 were the most important shared haplotypes,accounting for 28.51%,26.12%and 22.34%of the sample size,respectively.There are 13 haplotypes in the three regions of the Lancang River system,and the main shared haplotypes are PcH19 and PcH47,accounting for 29.60%and 29.15%of the samples of the Pomacea spp.in the Lancang River region,respectively,in the middle reaches of the water system,there are a maximum of 10 haplotypes of the P.canaliculata in the Lincang city of Gengma County.There are 4 haplotypes in the Nujiang River and Yuanjiang River system.The main shared haplotypes are PcH1,PcH19.PcH1 is also the most important shared haplotype in the global P.canaliculata haplotype analysis.Seven unique haplotypes of three water systems in Yunnan represent 19 haplotypes in the Americas,Asia,and Oceania.Together with the haplotype PcH1,a star-like structure is formed;while the two major haplotypes of PcH2 and PcH19 are shared with the previously reported P.canaliculata population in mainland China.For the Pomacea sp.,there are 4 haplotypes(HaP1?4),including 2 major shared haplotypes(HaP1,55.10%;HaP4,40.82%)and 2 unique haplotypes.HaP1 exists only in the Lancang River system and is shared by four geographical populations in the upstream,middle and downstream reaches,including:Dali Prefecture of Eryuan County(5%),Aihua Town of Lincang City(50%),and Gengma Town(20%)and Ximeng County(25%)in Pu'er City;HaP4 is mainly shared by three geographical populations in the lower reaches of the Lancang River and the Nujiang River Basin,including:Jinghong Menghan Town(3.70%),Gadong Township(22.22%);Ruili City(74.07%).The phylogenetic tree analysis results constructed by the maximum likelihood method are consistent with the haplotype analysis results.It is also indicated that the three water system P.canaliculata are divided into two lines:lineage A and lineage B.Among them,lineage A includes:shared haplotype PcH2,PcH19,and the remaining 10 unique haplotypes of this study,and several haplotypes of the domestic P.canaliculata are reported in a branch with a self-expansion value of 80%.Lineage B includes:shared haplotype PcH1,and unique haplotype PcH49 are associated with published haplotypes,including South America(Argentina,Uruguay),Southeast Asia(Myanmar,Thailand,Philippines,Indonesia,Japan,etc.),North America(the United States),Oceania(Papua New Guinea),Taiwan and China,etc,and clustering into large branches with a self-spreading value of 64%.Based on the diversity analysis of the five regional COI sequences of the three water systems,the five regional populations are all polymorphic.Among them,the nucleotide diversity and the haplotype diversity of the P.canaliculata in the middle reaches of the Lancangjiang River was the highest(Pi=0.03761>0.005,Hd=0.703±0.029>0.5),and the Yuanjiang water system area was the second(Pi=0.02067>0.005,Hd=0.548±0.045>0.5),while the nucleotide diversity of the P.canaliculata in the upper and lower reaches of the Lancang River and the water system of the Nujiang River were not high,and the pi values were 0.01119?0.00403?and 0.01766,respectively,which were less than the middle reaches of the Lancang River and the Yuanjiang River region,and the haplotype diversity of these three regions is low(Hd<0.5).The lower haplotype diversity suggests that the population of the P.canaliculata at the the upstream,downstream reaches in the Lancang River,and the Nujiang River system may be affected by the founder effect.Conclusion(s)1?There are a large number of invasive Pomacea in the Lancangjiang,Nujiang and Yuanjiang water systems of Yunnan Province.The main invasive species are P.canaliculata,P.maculata,however only a small amount of Pomacea sp.are distributed in the Lancangjiang and Nujiang water systems.2?There are 14 haplotypes of the invasive P.canaliculata at the three water systems in Yunnan Province.The most important haplotypes are PcHl and PcH19.PcH1 and PcH19 were found in all three water systems,but PcH1 was not found in the downstream reaches of the Lancangjiang River.There is an invasive species of the Pomacea sp.in the Lancangjiang and Nujiang water systems of Yunnan Province.There are 4 haplotypes in this Pomacea sp.species,and the most important haplotypes are Hap 1 and Hap4.Hap 1 is mainly distributed in three regions of the upstream,middle and downstream water systems of the Lancangjiang River.Hap4 is mainly distributed in the lower reaches of the Lancangjiang River system and the Nujiang River system.There are two main P.canaliculata,molecular populations in the three water system of Yunnan Province:Lineage A and Lineage B.Lineage A is only found in China and the Philippines,and mainland China is the main population.This population includes the main haplotypes PcH19 and PcH47.Lineage B is homologous to Argentina(the origin of the P.canaliculata),which can be considered as the introduced population at that time.This population includes the major haplotype PcH1. |
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