| Tetrix japonica Bolivar,1887,belonging to the Family Tetrigidae,Order Orthoptera,Class Insecta,Phylum Arthropoda,is a small pygmy grasshopper in eastern Asia.Generally,This species is surviving at regions where with a certain degree disturbed by human,such as streamsides,roadsides and farmlands.T.japonica is a widely distributed species in eastern Asia,however,less studies were assigned on this species previously,especially about the biological information,such as interpopulation morphological variation,population structure and population history are have not unveiled.In present study,32 geographical populations,including 428 females and 311 males individuals,were sampled across China.Multiple analyses,such as traditional morphometrics,geometric morphometrics,molecular technology and statistics,were used to analyse the morphological variation,relationship between morphology and bioclimate factors,genetic diversity,population history and phylogeography of this species.In traditional morphometrics analysis,8 linear eigenvalues of morphological traits,body length(BL),pronotum length(PL),pronotum width(PW),hind femurlength(HFL),forewing length(FW1),expanded forewing length(FW2),expanded forewing width(FW3)and hindwing length(HWL),were measured and 18 bioclimate factors were extracted from DIVA-GIS.Multiple analyses,such as descriptive statistics,correlation analysis,principal component analysis,clustering analysis,regression analysis and Mantel test between the Euclidean distances of morphological variation and linear geographic distances between populations,were performed and results were showed as follows:1)body size of T.japonica with significant sexual dimorphism and females larger than males.2)Eight linear eigenvalues are with significantly positive correlation to each other and significant difference between 32 geographic populations.The largest variation is FW2(10.14%)in females and HWL(11.71%)in males,respectively;however,the smallest one is HFL,as 6.96%and 7.05%of females and males,respectively.3)Three morphological principal components(MPCs)of 8 linear eigenvalues were extracted of both sexes of T.japonica,and representing body size(MPC1),forewing size(MPC2)and hindwing size(MPC3),respectively,and explained 82.151%and 85.148%of total variances of females and males,respectively.Four climate principal components(CPCs)were extracted from 18 bioclimate factors,and explained 96.126%of total variation.4)Four classes were clustered by agglomerative hierarchical clustering(AHC)according to the PCs of 8 linear eigenvalues and no clear geographic population pattern was detected.Among them,the population ZJDC was clustered as a single class of both sexes T.japonica.5)Regression analysis between PCs of morphological eigenvalues and the bioclimate factors revealed that MPC1 with significantly negative correlation to CPC1 and CPC2 of females;MPC1 with significantly negative correlation to CPC2 and MPC2 with significantly positive correlation to CPC4 of males.6)BL,PL and PW of both sexes T.japonica are significantely positive correlate to altitude and gradually larger with altitude raising.The body size of T.japonica following Bergmann's rule.7)Mantel test revealed that the Euclidean distance of morphological variation with significantly positive relationship to geographic distances of populations in females,but the coefficient was small;nevertheless,males without significant relationship between them(p=0.496).In geometric morphometrics analysis,with 46,16,55,21 and 45 landmarks and/or semilandmarks of dorsal pronotum(DP),lateral pronotum(LP),forewing(FW),hindwing(HW)and hind femur(HF)were extracted.Multiple software,such as XTSTAT 2015,SPSS,MorphJ and PAST,were executed for clustering analysis,morphological variation analysis and the correlation analysis between morphology and bioclimate factors,and results were showed as follows:1)Both traditional and geometric morphometrics unveiled homogeneous clustering pattern of T.japonica.2)In general,population mixture and without clear spatial pattern were detected by clustering analysis based on 5 morphological traits.3)Population ZJDC with clear morphological differentiation of pronotum and was clustered as single clade according to the morphology of pronotum.4)Regression analysis between the PCs of the reference morphology of pronotum and bioclimate factors revealed that the positively significant relationship between PC1_DP and CPC2,PC2_LP and CPC1,CPC2 in females.However,without significant relationship was detected in males.5)The Euclidean distance of morphological variations and geographic distances between populations with significant relationship according to the Mantel test on five different morphological traits,except forewing(p>0.05).In molecular phylogeographic studies,mitochondrial DNA cytochrome c oxidase subunit I(COI,676 bp),internal transcribed spacer between 18S and 28S rDNA(ITS,483 bp)and elongate factor 1?(EF-1?,889)of T.japonica were sequenced from 496,463 and 402 individuals,respectively.Multiple analyses,such as genetic diversity,neutrality test,phylogeny and population history were executed based on the three gene sequences,and results showed:1)T.japonica with high genetic diversity.The mean haplotype diversity(Hd),nucleotide diversity(?)and sequence diversity(k)of COI,ITS,EF-1? are 0.9858,0.03,20.303;0.9797,0.039,17.456 and 0.9892,0.01,9.649,respectively.2)According to COI sequences,neutrality test and mismatch analysis revealed that the observed curve obviously smooth and with single peak,and the Tajima's D and Fu's Fs as significantly negative value(-1.77 and-34.14),the SSD and Raggedness as significantly small value(0.001 and 0.001).COI supported that T.japonica have undergone recent population rapid expansion history.Also,the expansion history were supported by ITS and EF-1?.3)T.japonica began to population expansion at 0.18 Ma(95%CI:0.155-0.207 Ma),and reached stable in population size at 0.05 Ma(95%CI:0.033-0.072 Ma)based on COI.This phases is during MIS6 of Penultimate glaciation.4)AMOVA showed that 61%variation from intrapopulations based on COI and ITS sequences,but less than 50%according to EF-1?.5)The smallest and largest FST between populations are 0.0001(ZJZJ/GXLS)and 0.803(ZJDC/AHGNJ),0.0041(GSTS/JXJGS)and 0.891(HLJ/SCDJY),0.031(SXHP/SXTB)and 0.942(HBBD/HNAH),respectively.Mantel test showed that insignificant correlation between the Euclidean distances of genetic divergence,gene flow and population geographic distances.6)Haplotype phylogeny(NJ,ML,Bayesian)and network analyses showed that the clades of T.japonica are not according to the geographic distribution.Haplotypes from same population were mostly clustered in different clades.7)The most recent common ancestor(TMRCA)of the Chinese T.japonica is 3.65 Ma(95%CI:1.91-6.29 Ma).In conclusion,body size of T.japonica with significant sexual dimorphism and females larger than males,and body length following Bergmann's rule.Pronotum is the major structure with morphological variation between populations,especially in insular population(ZJDC).Morphology of T.japonica was significantly affected by the lowest temperature and the least precipitation.Traditional morphometrics,geometric morphometrics and molecular biology are support the consistent conclusion that Chinese T.japonica populations without clear geographic population structure.Meanwhile,three genes are support that multiple locality recently rapid expansion.T.japonica began to population rapid expansion from 0.18 Ma(95%CI:0.155-0.207 Ma)based on COI.TMRCA of Chinese populations is 3.65 Ma(95%CI:1.91-6.29 Ma).Combining the biological characteristics of T.japonica,we could speculate that passive dispersal may significant affect the current population structure of this species. |
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