Untangling The Relative Contributions Of Geography And Climate In Driving Current Patterns Of Genetic Variation:A Case Study Of Hyla Annectans(Anura:Hylidae)

Geological events and climate fluctuations are two important factors in driving current patterns of genetic variation.Barriers,such as ridge,river,plain etc.contribute to population genetic divergence by causing low level gene flow between populations,which are the production of topographic change caused by geological events.Climate fluctuations also have effect on the population genetic structure and effective population size.In the Pleistocene period,especially,during the last glacial period when ice sheets were at their greatest extension,species migrate to refugia in southern or low-altitude areas due to glaciers and cold climate.Effective population size reduction and genetic differentiation caused by population habitat loss,low level of gene flow,genetic drift,natural selection etc.During interglacial periods where the warming climate causes ice bodies to recede,populations expanse from different refugia may be contacted in the same geographical area,with increasing in effective population size and second contact.The mode of contraction and expansion is reflected on demographic history and population genetic structure changing.The current genetic variation within a species is shaped by these two processes working singly or in combination Usually,the relative contribution of two factors can be explored by genetic imprints left by them.If geological events are the main driving factor,one would expect to show high levels of genetic differentiation among genetic groups which concurrently with geological events.Alternatively,if Pleistocene climatic fluctuations were the main driver of population differentiation,one would expect to see the time of genetic lineages differ among genetic concurrently with climatic events,as well as clear signs of population shrinkages or expansions during Pleistocene climatic fluctuations.Recently,researcher pay more attention on North America and Europe,reaching the conclusion that the regional consistency conclusions of geological events and climate fluctuations effect on the genetic variation pattern of species.However,few studies have focus on the mechanisms that shaped the current patterns of genetic variation in Southwest China,and the relative importance of these mechanism remains controversial In this region,the geomorphology and geological features have been remolded by the uplift of the Himalayas and the Qinghai-Xizang Plateau.Undoubtedly,the associated geographic events would have generated potential dispersal barriers,such as high mountains,deeply valleys and rivers.These barriers could have played a predominant role in shaping the evolutionary history and population structure of the species native to this region.In contrast to situation in Europe and North America,most areas in Southwest China were not covered by ice sheets during the Pleistocene,and characterized by a relatively mild Pleistocene climate.So,it evoked less stress for the species in this region.More studies are needed to focus on the mechanisms that shaped the current patterns of genetic variation in Southwest China,and the relative importance of these mechanism.The Hyla annectans restrict to a narrow range of elevations,i.e.,580-2500 m and breed in paddy fields located to mountain range.They are considered to be good indicators of research on shaping the current pattern of genetic variation due to their high sensitivity to environmental changes,low mobility,and high evolution rate.Most of the phylogeographic studies in south China have been based on a few genetic loci,usually mitochondrial DNA and few nuclear DNA fragments.Next-generation sequencing(NGS)methods provide opportunities to better understand the effects of past climate changes on species phylogeography being highly effective in tracing postglacial recolonization and reconstructing detailed demographic histories of species in Southwest China.We obtained 349 samples from 35 localities throughout its distribution range in China.This study based on Specific Length Amplified Fragment Sequencing with species distribution modeling to explore the current genetic variation pattern of Hyla annectans and determine the contributions of geological events and Pleistocene climatic changes to the contemporary genetic patterns of this species in South China.The results are as follows:(1)We sampled 349 Hyla annectans individuals from 35 localities of cover the distribution area in Southwest China and identified the genomic DNA sequence by SLAF.Sample was first digested with Haelll and Hpy166? restriction enzymes,and a size-selection window of 414-444bp were expected.The enzyme digestion efficiency was 89.38%.It showed good sequencing results due to the average Q30 of sequencing was 82.78%and the average GC content was 42.06%.In total,we got 478.65M reads,and the average sequencing depth was 5.53-fold.Subsequently,1075 515 tags(or SLAFs)were identified in total,of which 230 236 were polymorphic.A total of 2 303 646 biallelic SNPs were obtained from SLAFs.The SNPs with minor allele frequency lower than 0.05,and SNPs which were missing more than 20%in whole populations and 40%of the data at least in one of the 35 locations were filtered out.Eventually,8420 SNPs were retained,and used in population genetic analysis.(2)We evaluated genetic diversity by two indicators,which were expected heterozygosity(He)and observed heterozygosity(Ho).The observed heterozygosity and expected heterozygosity varied from 0.019(N2)to 0.103(C1),0.026(N1)to 0.169(C1),respectively.The highest heterozygosity occurred in the populations of Wuling Mountains and East Yunnan-Guizhou Plateau,and the lowest heterozygosity occurred in the populations of western Sichuan and Hengduan Mountains,indicating that the populations in different region have different evolutionary processes.(3)We used 8 420 SNPs dataset for analysis of AMOVA,fixation index FST,phylogeny tree,Faststructure,PCA,DAPC and gene flow by Migrate-N software.AMOVA and FS-T results suggested that Hyla annectans had significant genetic differentiation,pairwise FS-T values were statistically significant ranging from 0.103 to 0.953(P<0.001).The results of genetic differentiation consist with phenotypic differentiation due to the seven genetic lineages(E,C1,C2,N1,N2,W1 and W2,see the details in the following section)shows significant differences both in phenotypic and genetic.For genetic divergence,pairwise FST values between seven genetic lineages were statistically significant ranging from 0.260 to 0.909(P<0.001)and suggested a low level of nuclear gene flow.For phenotypic divergence,the phenotypic traits of the number of black spots were significantly different between the lineages,although no significant differences were found in weight and length.70.98%(P<0.001)of the genetic variation was attributed to differentiation among populations.Combined the results of phylogeny tree,Faststructure,PCA and DAPC,that suggested there were seven genetic lineages within Hyla annectans,which distributed in different geological mountain systems and evolutionary branches.Lineage E from the Wuling Mountain Range,Lineages C1 and C2 co-occurred on the central Yunnan-Guizhou Plateau,Lineages Nland N2 occurred on the southwestern Sichuan Plateau.Lineage W1 located in the southern Hengduan Mountains and lineage W2 located in Ailao and Wuliang Mountains,respectively.In addition,we observed population substructure within lineages E,N2 and W1 in PCA analysis.(4)We used IBD(Isolation by Distance)model to reveal the correlation between geographic and genetic distance among populations.Our result suggested that the geological distance was an important factor in contribution to genetic differentiation among populations,since the IBD test revealed a significant correlation between geographic and genetic distance among populations(r2=0.453,P<0.001).(5)To reveal the relationship between the geological events and the divergence of lineages,we calculated the divergence time based on the age of the most recent common ancestor(tMRCA)of the H.annectans,which has been estimated to 4Mya,using 2130 SNPs with missing data less than 95%by the SNAPP software.Our dating estimates indicate that the diversification among different genetic groups of this species in Southwest China range from 3.89 to 1.73 Mya.The first divergence in H.annectans complex occurred ca.3.89 Mya with Group E in ancestral position.Group C diverged from Group N and W about 3.12 Mya,and group N from Group W occurred ca.2.67 Mya.The division between Group C1 and C2 occurred around 2.77 Mya,that between Group N1 and N2 ca.2.12 Mya and that between Group W1 and W2 ca 1.73 Mya.Which are linked to the three-phase rapid and drastic uplifting of the Qinghai-Tibetan Plateau.For example,major tectonic events in the Hengduan Mountains occurred 3.4 million years ago.And there was a strong activity during 2.5-1.2 million years ago in the southeastern of the Qinghai-Tibet Plateau including the Longmen,Daxiangling,Jinping,Yulong,Biluo Mountains.In addition,mountains in western Yunnan,such as the Daxue Mountain,Gaoligong Mountain,and Nu Mountain,reached their highest altitudes during the Pliocene.(6)We estimated the demographic history and distribution of Hyla annectans populations.Firstly,we performed a coalescent theory-based multilocus analysis Extended Bayesian Skyline Plot(EBSP)in BEAST for each of the seven phylogeographic clusters by choosing three groups of randomly selected 20 loci with 6-8 SNPs and checked for convergence in the inferred demography.The results suggested that the effective population sizes of all groups remained constant through evolutionary time,including LGM.Secondly,we calculated a Pearson correlations among all 19 bioclimatic variables in distribution area and deleted variables with correlation value>0.8.Therefore,seven variables were retained to create the SDM(Species Distribution Model)based on 134 occurrences to explore the distribution of this species by MAXENT model.Model indicated that the species current distribution area matched the sampling area and its distribution range has not been changed from the LIG and LGM to present.In conclusion,our genome-wide survey the pattern of genetic variation in widely distributed Hyla annectans show that geological events play an important role in shaping the genetic variation pattern of this species.The barriers caused by the processing of geological events leaded to differentiation between lineages due to the time of lineages divergence correspond to geological events timing.In contrast,climate events have had little impact on shaping the genetic variation pattern of this species.Since,the effective population sizes of all lineages kept stable and the species has not changed its distribution range over time.In the Pleistocene,the relatively mild Pleistocene climate combined heterogeneous topology of this region might have provided good opportunities for habitat choice,and thereby evoked less stress for the species in this region.Therefore,The complex geological events be responsible for driving the high level of genetic divergence and the long-term demographics of populations during the Pleistocene were consistent with earlier studies from Southwest China Thus,it appears that geological events,rather than Pleistocene climatic oscillations,might have been more significant drivers of the pattern of genetic variation might be a common pattern for organisms residing in Southwest China.These findings not only highlight the effects of geologic events and past climatic fluctuations as drivers of contemporary patterns of genetic variation,but also contribute to a better understanding of the mechanisms that contribute to species diversity in this biodiversity hotspot.