| As a small cold-fish,Rhynchocypris lagowskii Dybowski,1869 is widely distributed in East Asia with weakly ability to spread.It is an excellent bioindicator taxon to study fish phylogeography in precisely revealing how the geological changes and climate oscillation affect population distribution in the Qinling Mountains.Recently,large range sampling is less in these mountains,it is difficult to elucidate the distribution pattern and speciation mechanism for this species.As fish ectoparasite,there is merely morphological and taxonomic study in parasite Gyrodactylus konovalovi Ergens,1976,while other studies are few in biological characteristics and phylogeography in China.Due to shorter generation and higher evolutionary rate,there are more genetic diversities and variations in gyrodactylids species.Therefore,parasite G.konovalovi being a good marker in studying coevolution can be used as proxy for comprehending phylogeography and evolutionary history of host R.lagowskii.This study was applied phylogeographic method and mitochondrial and nuclear marker genes to reveal the distribution patterns and speciation mechanism of 48 populations of host and 21 populations of its parasite G.konovalovi in the Qinling Mountains,including genetic diversity,genetic structure,population history,divergence time and coevolution.The following was the main results:First,we sequenced mitochondrial Cytb dataset(539 bp)and D-loop control region dataset(416 bp),nuclear RAG2 dataset(641 bp)without heterozygous locus and the concatenated dataset of Cytb and D-loop and RAG2(1596 bp)for 464 individuals from 48 localities populations across most of its distribution in the Qinling Mountains.The ratio of nonsynonymous(n)and synonymous(s)substitution sites(dN/dS)of coding gene Cytb and RAG2 was 0.028 and 0.418 and showed purifying selection.In total,the numbers of haplotypes from datasets of Cytb,D-loop,RAG2 and concatenated of Cytb and D-loop and RAG2 were 69,44,24 and 137,the genetic diversity of R.lagowskii was higher.Drainage capture was significant in the watershed between south and north slopes of the Qinling Mountains,accompanying frequent gene flow and lower differentiation in genetic diversity.The genetic distances within and between populations were consistently largest in D-loop dataset with distinct differentiation,while,the smallest was in RAG2 dataset with little differentiation.The lower genetic distance in south and north slopes of the Qinling Mountains implied that population differentiation was not remarkably affected by natural physical barrier,existing extensive gene flow among populations in same river,the mean distances between haplotypes were more than 0.01.Phylogenetic analyses revealed that all haplotypes were clustered monophyletic group and well-supported different lineages,the results of network analysis of haplotypes were basically consistent with phylogeny,indicating a significantly geographic distribution patterns,different lineages were appeared highly frequent haplotype in evolutionary network center.Analysis of molecular variance(AMOVA)identified significantly genetic structures among different populations,linear correlation analysis showed that there was no significantly correlated between genetic differentiation fixed index(Fst)and geographic distance and verified that isolation by distance did not affect the population distribution pattern of R.lagowskii.The molecular clock calibration inferred that the most common ancestor of R.lagowskii complex was estimated to have emerged during the Miocene to early Pleistocene.The results of the mismatch distribution and neutrality test(Tajima's D and Fu's Fs)of four datasets showed that the lineage in the Nanluo River underwent population expansion ranging from the early to middle Pleistocene,while,other lineages and total population were in according with the hypothesis of neutral evolution.The analyses with Bayesian skyline plot(BSP)supported that the population expansion was spanning from 0.05 to 0.25 Mya and 0.15 to 1.15 Mya and also showed that the lineage in the Nanluo River existed as glacial relics and underwent population expansion at 0.15 Mya,0.075 Mya and 0.05 Mya during the middle to late Pleistocene.We therefore considered that the phylogeographic distribution pattern of R.lagowskii may be related with geological events such as orogenesis and drainage capture and climate oscillation during the Pleistocene in the Qinling Mountains,while,the population subdivision was not affected by isolation by geographic distance.Second,we sequenced mitochondrial COI dataset(536 bp),nuclear ITS dataset(518 bp)without heterozygous locus and the concatenated dataset of COI and ITS(1054 bp)for 109 individuals from 21 localities populations and ND5 dataset(528 bp)for 79 individuals from 11 localities populations across most of its distribution in the Qinling Mountains.The ratio of dN/dS of coding gene COI and ND5 was 0.015 and 0.068 and showed negative selection.In total,the numbers of haplotypes from datasets of COI,ITS,concatenated COI and ITS,ND5 were 46,6,48 and 25,the genetic diversity of G.konovalovi was higher.Drainage capture was significant in the watershed between the south and north slopes of the Qinling Mountains,accompanying frequent gene flow and lower differentiation in genetic diversity.The genetic distances within and between populations were largest in ND5 dataset with distinct differentiation,while,the smallest was in ITS dataset with little differentiation.The lower genetic distance in south and north slopes of the Qinling Mountains implied that population differentiation was not remarkably affected by natural physical barrier,existing extensive gene flow among populations in same river,the mean distances between haplotypes of four datasets were more than 0.01 except ITS.Phylogenetic analyses revealed that all haplotypes were clustered monophyletic group and well-supported different lineages,the results of network analysis of haplotypes were consistent with phylogeny,indicating significantly geographic distribution pattern,different lineages were appeared highly frequent haplotype in evolutionary network center with star-like pattern in the network of ITS and network patterns in others.AMOVA identified significantly genetic structures among different populations,linear correlation analysis showed that there was significantly positive correlated between Fst and geographic distance and verified that isolation by distance affected the population distribution pattern of G.konovalovi.The molecular clock calibration inferred that the most common ancestor of G.konovalovi complex was estimated to have emerged during the early to middle Pleistocene.The results of the mismatch distribution and neutrality test(Tajima's D and Fu's Fs)of four datasets showed that all lineages and total population were in according with the hypothesis of neutral evolution without population expansion.The analyses with BSP supported that the lineages of three datasets underwent expansion at 0.005 Mya and 0.01 Mya,while the lineages and total population of ITS dataset underwent population contraction at 0.15 Mya.We therefore considered that populations were stable without expansion during the Pleistocene,the phylogeographic distribution pattern of G.konovalovi may be related with geological events such as orogenesis and drainage capture and isolation by geographic distance in the Qinling Mountains,while,the population subdivision was hardly affected by climate oscillation during the Pleistocene.Last,the result of comparative phylogeography between the R.lagowskii and its parasite G.konovalovi showed that the Hd,? and Fst were not consistently higher in the parasite than in the host,rejecting the hypothesis of magnifying glass for this host-parasite system.Drainage capture in the watershed between the south and north slopes of the watershred of the Qinling Mountains increased frequently gene flow between host populations and reduced population differentiation in these mountains,finally decreasing the differences of genetic diversity.The lineages of whole phylogenetic tree of G.konovalovi were compared with the corresponding lineages in the host and supported significant distribution pattern of the parasite and without exactly identical.The phylogeographic distribution pattern of R.lagowskii showed that the lineage in the Nanluo River existed as relics underwent population expansion,spreading to higher elevation in the Qinling Mountains at interglacial stage during the late Pleistocene,finally promoting the diffusion and differentiation of G.konovalovi. |
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