Population Genetics And Phylogeography Of Dugesia Japonica In Four Major Mountains Of Henan Province And Taihang Mountains

Dugesia japonica is the model animal for studies of planarian regeneration and development in the world.This freshwater planarian species has widely distribution and a large population size in China.Because of its weak dispersal ability,it also serves as a good material for the study of population genetics and phylogeography.The Taihang Mountains,Funiu Mountains,Tongbai Mountains and Dabie Mountains in Henan Province are rich in animal and plant resources.They are the natural treasure houses of biodiversity and the key areas of biodiversity conservation in China.In this study based on the mitochondrial genes COI,Cytb and nuclear gene ITS-1,the genetic diversity,genetic structure,phylogeographic pattern and population dynamics of D.japonica from four major mountains in Henan Province and Taihang Mountains have been systematically explored using the method of single gene and/or multiple genes combination analysis.The following results were obtained:Genomic DNA of 116 D.japonica individuals from 20 populations across four major mountains in Henan Province(Dabie Mountains,Tongbai Mountains,Funiu Mountains and Taihang Mountains)were extracted.Then through PCR amplification,sequencing,sequence alignment and trimming,the final lengths of three fragments of Cytb,COI and ITS-1 are 310 bp,807 bp and 633 bp,respectively,and the total length of the concatenated sequence of three genes is 1750 bp.In 116 combined sequences,a total of 87 haplotypes were defined,including 5 shared haplotypes and 82 private ones.The overall haplotype diversity(Hd)and nucleotide diversity(?)were 0.979 and 0.00279,respectively,exhibiting a high Hd and low ? pattern typical for migratory species.Haplotype phylogeny reconstruction showed that the Bayesian inference(BI)tree and the maximum parsimony(MP)tree were similar in topology,and all haplotypes clustered into six haplogroups.Also,the haplotype network graph based on median-joining method revealed the genetic structure consistent with the phylogenetic analysis,and there was no strict correspondence between haplotypes and geographical locations,indicating no obvious phylogeography pattern.AMOVA results showed that the genetic variation of the 20 populations of D.japonica was very significant,and the variation mainly came from within the population;Pairwise FST analysis showed that there was obvious genetic differentiation among D.japonica populations.The positive neutrality test results and the multimodal arrangement of mismatch distribution collectively indicated that D.japonica from four major mountains in Henan Province might have been undergoing population shrinking.Twenty D.japonica populations collected from Taihang Mountains were analyzed based on single gene(COI)and combined genes(ITS-1-Cytb).By genomic DNA isolation,PCR amplification,sequencing,sequence alignment,trimming and concatenation,fragments of 791 bp COI and 986 bp of ITS-1-Cytb combined sequence were obtained.Thirty-two haplotypes were defined for the 116 COI sequences,including 8 shared haplotypes and 24 private haplotypes.The overall haplotype diversity(Hd)and nucleotide diversity(?)were 0.920 and 0.00083,respectively.As for the 120 ITS-1-Cytb combined sequences,a total of 46 haplotypes were yielded,including 10 shared haplotypes and 36 private ones.The overall haplotype diversity(Hd)and nucleotide diversity(?)were 0.920 and 0.00083,respectively.Both genetic diversity analysis presented typically high Hd and low ? pattern.The BI tree and ML tree based on COI and ITS-1-Cytb sequence showed similar topology,and all haplotypes were clustered into three haplogroups.Moreover,the median-joining network of all haplotypes revealed the genetic structure congruent with the phylogenetic analysis.However,the haplotypes of the various populations in Taihang Mountains are scattered in different geographical populations,showing no obvious pedigree geographic pattern.AMOVA results based on COI and ITS-1-Cytb showed that the genetic variation of D.japonica in Taihang Mountains was very significant,and the variation mainly derived from within the population;Pairwise FST analysis showed that there was significant genetic differentiation among D.japonica populations.The results of mismatch distribution and neutral tests indicated that D.japonica in Taihang Mountains might be in a state of population decline.In summary,the population genetic diversity of D.japonica in four major mountains of Henan Province and Taihang Mountains presented a pattern of high Hd and low ?,suggesting that D.japonica might have experienced rapid population growth,but the expansion history was short and the accumulation of nucleotide variation was insufficient.Although the results of AMOVA and Pairwise FST analysis indicated significant genetic differentiation among most populations,phylogenetic analysis did not find a clear pedigree geographic pattern.Isolation effect exerted by the special topography of the studied areas and the weak dispersal ability of benthonic freshwater planarians are probably the main reasons for the great genetic differentiation of D.japonica in four major mountains of Henan Province and Taihang Mountains.The positive values of neutral tests as well as the multimodal pattern of mismatch distribution together suggested that D.japonica in four major mountains of Henan Province and Taihang Mountains migtht have been experiencing population declines.Therefore,effective measures such as reducing human interference and reducing environmental pollution should be taken to strengthen the conservation of D.japonica in these areas.We hope these findings will arouse conservation and management strategy regarding freshwater planarians and contribute to the biodiversity in the long run.