Genetic Structure, Suitability And Demographic History Of Scythropusyasumatsui Populations In China

The bud-eating weevil,Scythropus yasumatsui Kono et Morimoto（Coleoptera:Curculionidae）is a major pest of Chinese jujube（Ziziphus jujuba）trees in northern China.In1977,it was first reported in Xinzheng and Puyang of Henan Province.Influenced by global climate change,increased jujube cultivation area,human activities and other factors,its population density continues to increase,and its distribution area gradually moves northward.At present,Scythropus yasumatsui has occurred in part areas of Xinjiang and Ningxia,and the degree of its damage has become increasingly serious.In this study,we collected samples of Scythropus yasumatsui from 12 geographical populations in 5 provinces,and used molecular markers of the microsatellites,mitochondrial genes and nuclear genes to analyze its population genetic diversity and genetic structure.Suitable areas and key environmental factors for its occurrence in China were identified.The demographic history of different populations of Scythropus yasumatsui was analyzed,and the potential dispersal source and dispersal routes of its populations were predicted as well.The results were as follows:1.Screening of microsatellite loci from Scythropus yasumatsui based on transcriptome databaseThe next generation sequencing technology was used to sequence the transcriptome of Scythropus yasumatsui adult,and some microsatellite loci were screened for population genetic studies.In total,2613 microsatellite loci were identified from 171322 Unigenes of the transcriptome database,dinucleotide and trinucleotide repeats were the major repeat types,which accounted for 48.22%and 46.12%of total number of microsatellite loci,respectively.AT/TA was the most abundant binucleotide motif,and AAT/ATT was the most abundant trinucleotide motif.Of all microsatellite loci,the number of repeats was mainly between 5 and12.From all the identified SSR loci,50 pairs of microsatellite primers were designed by Primer Premier 5,and 16 polymorphic microsatellite loci were finally developed,which could be used for the analysis of population genetic diversity and genetic structure.2.The complete mitochondrial genome sequencing and phylogenetic analysis of Scythropus yasumatsuiThe next generation sequencing technology was used to sequence the complete mitochondrial genome of Scythropus yasumatsui.The length of the sequence was 16472 bp,and mitochondrial DNA was a closed circle with double strands,including 13 PCGs genes,22tRNA genes and 2 rRNA genes,as well as 2 long non-coding regions.The Bayesian phylogenetic tree was established using the nucleotide sequences of 13 protein-coding genes,and the phylogenetic relationship between Scythropus yasumatsui and 12 species within 7subfamilies was identified.The results showed that among 12 species,Scythropus yasumatsui had the closest relationship with Sympiezomias velatus that belonged to subfamily Entiminae.3.Genetic diversity and genetic structure of Scythropus yasumatsui populations based on microsatellitesGenetic diversity and genetic structure of 371 individuals from 12 geographical populations in 5 provinces（Shanxi,Shaanxi,Ningxia,Hebei,and Henan）of China were analyzed using 8 microsatellite loci.The results suggest that S.yasumatsui populations had a high level of genetic diversity.The effective number of alleles（N_e）ranged from 2.113 to 8.016,the allelic richness（A_r）ranged from 2.648 to 5.665,the polymorphic information content（PIC）ranged from 0.561 to 0.908,and the expected heterozygosity（H_e）was between 0.476 and 0.865.The average index of genetic differentiation(F_st)among populations was 0.151,and the average gene flow（Nm）was 1.594.Based on phylogenetic tree（UPGMA and NJ method）,PCA and STRUCTURE analysis,any of them supported the division of 12 geographic populations into3 groups,which included Group 1（SNSM,SNJX,SNQJ,SNYC,SXLX,SXLL and SXYH）,Group 2（HBCX and HNXZ）and Group 3（SNYL,HNLB and NXTX）.There was an obvious genetic differentiation between the groups due to the geographical isolation of Taihang Mountains and the Loess Plateau.The average N_m of the 7 populations within Group 1 was6.258,indicating that they had a high degree of genetic communication.Mantel test showed that there was a significant positive correlation between genetic differentiation and geographic distance among different populations.4.Genetic diversity and genetic structure of Scythropus yasumatsui populations based on mitochondrial and nuclear genesGenetic diversity of S.yasumatsui individuals from 9 geographical populations in China were analyzed using mitochondrial（COI,COII and Cytb）and nuclear genes（ITS1 and ITS2）.The average mitochondrial haplotype diversity of different populations was 0.527,and the average nucleotide diversity was 0.0018.The average haplotype diversity of ITS was 0.318,and the average nucleotide diversity was 0.0005.The high diversity of haplotypes（H_d>0.5）and the low nucleotide diversity（π<0.005）indicated that S.yasumatsui was an ancient species and may have experienced bottlenecks during the Quaternary glacial period.Based on haplotype data,the genetic structure of different populations was analyzed by phylogenetic tree,adjacent network and BAPS.The results showed that the systematic geographical structure was formed among populations.The mitochondrial haplotype supported the division of 9geographical populations of S.yasumatsui into 5 clades,while the ITS haplotype results were the same as microsatellites,supporting the division of different populations into 3 clades.The genetic differentiation and gene flow analysis of 9 geographical populations showed that there was no genetic differentiation between various populations in the Yellow River valley of Shaanxi and Shanxi,and Yellow River didn′t become a geographical barrier to gene flow between populations,which was the same as the results of microsatellites.5.Analysis of suitability and environmental factors for the distribution of Scythropus yasumatsuiUsing the MaxEnt niche model,combined with the distribution of Scythropus yasumatsui and Worldclim global environment data for four periods（LIG,LGM,current and future）,potential suitable areas of Scythropus yasumatsui in different historical periods in China were predicted.It was also predicted that North China Plain,Weihe Valley of Shaanxi Province and Fenhe Valley of Shanxi Province were suitable areas for S.yasumatsui,and these areas may be glacial refugia for S.yasumatsui in the Quaternary Ice Age.The analysis of the suitability using the climatic factors in 2070 indicated that the suitable areas of S.yasumatsui will continue to move northward in the future,and Inner Mongolia,Liaoning,Xinjiang,Gansu and Ningxia will become suitable areas for S.yasumatsui.According to the Jackknife analysis,the most important environmental factor affecting the distribution of S.yasumatsui was temperature.The temperature variance between different seasons and the dry climate were most suitable for its occurrence.6.Demographic history and potential dispersal routes of Scythropus yasumatsui populationsThe initial divergence time of Scythropus yasumatsui populations was estimated to be about 2.54 Ma using BEAST.The mismatch distribution analysis and neutral test results showed that the mismatch distribution of five geographical populations was a unimodal pattern.Tajima’s D and Fu’s Fs neutral tests reached significant levels in these populations（P<0.05）,which implied that Scythropus yasumatsui had experienced population expansion.The expansion time started from the last interglacial period and ended at the end of the last glacial period（0.012-0.15 Ma）.The Bayesian Skyline Plot（BSP）had also drawn similar conclusions.The historical migration rates between populations were analyzed using Migrate-n.It was concluded that there were two possible dispersal sources:SXLV and HNLB,while three populations（HBCX,SNYU and NXTX）were the dispersal ends of populations.Using the lowest cost path method（LCP）in the SDMtoolbox in ArcGIS,combined with the molecular marker data and geographical distribution data of Scythropus yasumatsui,the potential dispersal routes of its populations in China were predicted.The results showed that the valley region of Yellow River was the most likely population dispersal corridor for Scythropus yasumatsui.This study provided a theoretical basis for clarifying the genetic structure of Scythropus yasumatsui populations,and understanding their distribution and dispersal trends in China.It also provided an important reference for revealing disaster situations of this pest and formulating comprehensive prevention strategies,and helped to understand the adaptability to environments,hosts and human activities and evolutionary mechanisms of this pest.