Population Genetic Structure And Phylogeography Of Machilus Thunbergii,a Tree Of Continental And Island Distributions In East Asia

Unraveling the dynamic changes in species distribution is a core issue in ecology and evolution.Historical changes in environmental factors mainly include paleoclimate fluctuations and geologic topography changes.These factors are considered to play an essential role in shaping the current species distribution.However,it is still unclear how they affect the population genetic structure and historical population dynamics.Climate fluctuations can drive plants to migrate along latitude and altitude gradients.In addition,they can also cause the rise and fall of sea levels,substantially affecting the distribution pattern of plant species on continental edges and islands and forming isolation-contact dynamics of plant populations.Such isolation-contact dynamics may facilitate population differentiation and even speciation but is also likely to mitigate genetic differentiation among populations.Thus,it crucially contributes to the formation and maintenance of regional species diversity and endemism.The Sino-Japanese floristic region(SJFR)harbors rich plant diversity with complex and diverse geological and climatic history.It is,therefore,a hotspot region for studying ecological and evolutionary issues.Machilus thunbergii(Lauraceae)is endemic to East Asia and widely distributed on both the mainland and the islands in SJFR.Chloroplast DNA markers are maternal inheritance and thus are appropriate for tracing the historical seed dispersal pattern,which can be used to infer refuges during glacial periods and post-glacial migration routes.In this study,we collected 709samples of M.thunbergii from 64 locations and used five pairs of chloroplast primers to determine the haplotype diversity and distribution.We constructed a phylogenetic tree and analyzed the population demography based on haplotype sequences(Chapter2).We obtained the genotypes of 1,378 M.thunbergii samples from these 64 locations using ten nuclear microsatellite loci and determined the population genetic structure and the best population demographic model.With the help of ecological niche modeling,we assessed the influences of the last glacial maximum(LGM)on its population differentiation(Chapter 3).Moreover,as the relatively high mutation rate of nuclear microsatellites can only reflect the impacts of recent geological and climatic events.Combining the above research conclusions using Sanger sequencing,we acquired the whole-genome resequencing data of 34 M.thunbergii samples from 18 locations to analyze the population genetic structure and the best population demographic model.Genomic analysis was conducted based on single nucleotide polymorphism(SNP)loci using the existing de novo assembled genome of Phoebe bournei as the reference genome to infer the influences of periodic Pleistocene glacial-interglacial cycles on genetic differentiation among populations(Chapter 4).The results according to three kinds of molecular markers are as follows.1)The refuges of M.thunbergii and its post-glacial migration routes based on chloroplast DNA:analysis based on chloroplast DNA markers showed 22 haplotypes within all samples.We found high haplotype diversity of M.thunbergii in the mainland China(MC)and Taiwan–Ryukyu archipelago(TR)(haplotype diversity:h _MC=0.561,h _TR=0.658;nucleotide polymorphism:?_MC=3.9×10^-4,?_TR=2.8×10^-4).However,there was only one haplotype in the Zhoushan archipelago and the Japanese archipelago.Across the distribution area in M.thunbergii,the haplotype diversity decreased with increasing latitude significantly(P<0.001).Combined with the distribution of haplotypes,we suggested that Nanling in South China,Wuyi Mountain in East China,and the Chinese Taiwan–Ryukyu archipelago were the refuges of M.thunbergii during the last glacial period.There was northward migration after glacial.The chloroplast haplotype phylogenetic tree support two lineages found within M.thunbergii.The divergence time(4.4 million years ago(Ma))between the above two lineages was later than the Taiwan strait's formation time.The results of mismatch analysis,neutrality test,and historical population dynamics based on the coalescent approach showed a significant post-glacial expansion in the effective population size(EPS)of M.thunbergii.2)Genetic structure of M.thunbergii based on nuclear microsatellites:analysis based on nuclear microsatellites showed the distribution area of M.thunbergii in East Asia comprised two regions:the mainland and the island regions divided by the coastline of mainland China.The mainland region was further partitioned into two subregions:South China and East China,with the Luoxiao Mountains as the boundary;the island region could be divided into three subregions,i.e.,Chinese Taiwan–Ryukyu archipelago,Zhoushan–Kyushu archipelago and Honshu Island,with the Tokara Gap and the Seto Inland Sea as boundaries.There was high genetic diversity in populations across the mainland and island.In East Asia,there were no significant correlations between genetic diversity and latitude and longitude(longitude:P=0.489,latitude:P=0.179);however,in mainland China and the Japanese archipelago,the genetic diversity gradually decreased with increasing latitude significantly(China,P=0.009,Japan,P=0.004).These patterns further supported the three southern refugia inferred based on cp DNA and implied that the East China Sea Land Bridge and the Japanese Archipelago near the Pacific coast were potential refugia,supporting that M.thunbergii experienced a post-glacial population expansion towards high latitude regions.Results of approximate Bayesian analysis supported a secondary contact between the Chinese and Japanese populations,which occurred in Zhoushan–Kyushu area about 15,000years ago,and M.thunbergii populations there may be the relics of the refuge in the East China Sea land bridge.The coalescence time between populations on mainland China and those on the Taiwan–Ryukyu archipelago was about 14,000 years ago,indicating that these two regions contacted during LGM.The consistency,as mentioned above,of contact times in different regions collectively reflected that the sea level decline during the Last Glacial Maximum provided a connecting corridor for mainland and island populations.Analysis based on the Bayesian approach showed the presence of historical gene flow among M.thunbergii populations in different subregions but the absence of recent gene flow(within three generations).Moreover,the results of ecological niche modeling showed that the East China Sea and the Taiwan Strait were the potential distribution areas of M.thunbergii during LGM.This further confirmed that the land bridge created by the sea level decline during LGM provided a channel for the contact of M.thunbergii populations,supporting the genetic admixture of the populations from the mainland and the island regions during LGM.3)Historical population dynamics and genomic differentiation of M.thunbergii using resequencing data:the population genomic analysis also supported the division of M.thunbergii distribution range into two regions including the mainland and the island regions.The EPS of M.thunbergii started to decrease from the early Pleistocene.The lowest EPS of M.thunbergii(c.10,000)on the mainland was found during LGM;in the island region,the EPS of M.thunbergii(c.30,000)reached its minimum at about0.5 million years ago and then increased to a peak value(c.50,000)during the last interglacial period with an after declining until LGM(less than 10,000)and a recent rapid increase.Results from the approximate Bayesian model showed that the three-time contact model could better explain the distribution of current genetic variation data than the single-divergence model,and the contact time(0.99,0.37,and 0.06 Ma)was consistent with the formation time of the Pleistocene land bridge.The coalescence time between the mainland and the island region of M.thunbergii was about 2.13 Ma,and the coalescence time between the Taiwan–Ryukyu archipelago and the Japanese archipelago subregion was about 0.30 Ma.The post-divergence gene flow could be found between different M.thunbergii populations.Genomic islands of divergence were found between the mainland and the island populations of M.thunbergii,with high genetic differentiation in three genomic regions where only a moderate level of differentiation was detected among island populations.Our results indicate that the population genetic structure and demography of M.thunbergii in East Asia have been strongly affected by historical geological and climatic changes since the Pleistocene,especially the periodic rises and falls of sea level caused by the Pleistocene glacial-interglacial cycles,which resulted in isolation-contact events between the mainland and island populations of M.thunbergii.However,such events failed to drive the speciation between M.thunbergii populations on the mainland and island.