Anolis carolinensis) has been widely used as an animal model in physiology and neurobiology but has recently emerged as an important genomic model. The recent sequencing of its genome has shed new light on the evolution of vertebrate genomes and on the process that govern species diversification. Surprisingly, the patterns of genetic diversity within natural populations of this widespread and abundant North American lizard remain relatively unknown. In the present study, we use 10 novel nuclear DNA sequence loci (N = 62 to 152) and one mitochondrial locus (N = 226) to delimit green anole populations and infer their historical demography. We uncovered four evolutionarily distinct and geographically restricted lineages of green anoles using phylogenetics, Bayesian clustering, and genetic distance methods. Molecular dating indicates that these lineages last shared a common ancestor ∼2 million years ago. Summary statistics and analysis of the frequency distributions of DNA polymorphisms strongly suggest range-wide expansions in population size. Using Bayesian Skyline Plots, we inferred the timing of population size expansions, which differ across lineages, and found evidence for a relatively recent and rapid westward expansion of green anoles across the Gulf Coastal Plain during the mid-Pleistocene. One surprising result is that the distribution of genetic diversity is not consistent with a latitudinal shift caused by climatic oscillations as is observed for many co-distributed taxa. This suggests that the most recent Pleistocene glacial cycles had a limited impact on the geographic distribution of the green anole at the northern limits of its range.The green anole ( Anolis carolinensis, or the green anole lizard, is the first lepidosaurian reptile to have its entire genome sequenced Anolis genome has already provided insights into our understanding of how vertebrates have diversified since the split between reptiles and mammals more than 300 million years ago (Mya) Anolis genome Anolis genome Anolis “ecomorphs” on the Greater Antilles has brought much attention to the mechanisms governing adaptive radiations  on the basis of dewlap coloration and number of lamellae.While it belongs to a genus containing as many as 400 species across the tropical and semi-tropical New World, A. carolinensis remain unresolved. Eastern North America is geologically and topographically complex, and a number of common phylogeographic patterns have been reported across a wide range of co-distributed taxa The historical processes that account for the current distribution of Anolis genome database and one mitochondrial locus. We have characterized and delimited evolutionary distinct lineages within this species, estimated key demographic parameters associated with its history on the continent, and tested hypotheses postulated by other comparative phylogeographic studies of southeastern North America. The results of our analyses will have important implications for future studies of A. carolinensis, most notably shedding light on the effects that population structure may have on the maintenance of genetic variation in a genomic model organism.To test hypotheses regarding historical demography, the field of phylogeography has moved towards an emphasis on incorporating as many loci as practically possible. This is because stochastic differences between the coalescent histories of gene genealogies 2 exposure in a closed container followed by deep freeze. The IACUC at the American Museum of Natural History does not provide welfare assurance numbers or protocol numbers, however the approved protocol ensured that animals were euthanized humanely according to methods suggested by the AVMA as well as the American Society of Ichthyologists and Herpetologists via (1) intracoelemic injection of Tricaine Methanesulfonate (MS222) with a sodium bicarbonate buffer, and (2) once the animal was confirmed as inert, a second injection of unbuffered MS222.This study was carried out in accordance with the recommendations of the American Veterinary Medical Association (AVMA) for the euthanasia of ectotherms and every step was taken to avoid needless suffering. The following protocol was approved by the Queens College Institutional Animal Care and Use Committee (IACUC)  and was administered by the authors. After capture, animals were kept in the dark in fabric bags for a maximum of four hours and were sacrificed the same day. Euthanasia was carried out in the field via intra-abdominal injection of sodium pentobarbital at a dosage of 100 mg/kg of body weight. The euthanasia protocol approved by the University of Texas at Arlington IACUC  involved prolonged COWe collected 159 anoles in NC, SC, GA, FL, AL, TN, LA and AR during 2009 through 2011 and obtained their tail or liver tissues. We also obtained the tissues of nine Texan anoles from Andre Pires da Silva of the University of Texas at Arlington, and 31 blood samples from individuals collected at additional sites in SC, GA and FL given to us by Bryan Falk of the American Museum of Natural History. A map in in silico using the UCSC Genome Browser (http://genome.ucsc.edu/) Anolis genome database for introns of reasonable size (<2 Kb) for PCR amplification. An intron was chosen for further analysis if its presence was predicted by at least two of the tracks available for visualization on the browser . Although we selected introns for their length without respect to gene function, we recorded the putative function of each gene prediction  tion see . PrimersAnolis genome database for each nDNA locus and the ND2 region of mitochondrial sequence NC_010972 from GenBank. Putative heterozygous sites in nDNA sequence reads were assessed based on quality score. Less than 5% of all reads were unusable due to heterozygous indels, and were removed from further analyses. Each contig was edited manually and the consensus sequences were extracted and aligned using ClustalW After Sanger sequencing, we imported chromatograms into CLC Main Workbench version 5 and Geneious v5.5 Anolis genome sequence (NC_010972); 30 A. carolinensis sequences available in GenBank (accession numbers EU106323– EU106342); and three A. porcatus (AY654092 - AY654094), one A. isolepis (AY654022) and one A. altitudinalis (AY654023) to be used as outgroups. Phylogenies were reconstructed using the rapid bootstrap (RBS) Maximum Likelihood (ML) method in RAxML A. carolinensis with the phylogenetic information, we first conducted a preliminary BEAST analysis  and calculated the average Tamura-Nei and uncorrected pairwise genetic distances between recovered clades. Assuming a mutation rate of 1.3% per million years (Myr) for this region, as observed in other small lizards Anolis genus mrcat) of all A. carolinensis populations. We further calibrated our tree with knowledge from previous molecular phylogenetic analyses carolinensis anole subgroup  at 6Myr. The final BEAST analysis included all 226 sequences, two independent runs of MCMC length 100,000,000 with the evolutionary model and calibrations as stated above, a strict molecular clock, the known mutation rate, and a coalescent tree prior. Estimates of the posterior distributions of all parameters for each run were monitored with Tracer v1.4 The dataset used for phylogenetic inference included 226 ND2 sequences: 190 amplified by us; the homologous region available from the Structure 2.3.3Structure analyses were run with 100,000 steps for burn-in followed by 100,000 generations for K values ranging from 2 to 13. Each simulation was completed five times, and results files were compressed and submitted to HarvesterStructure has been shown to overestimate K (see the program documentation at http://pritch.bsd.uchicago.edu/structure.html); however, it allows the user to implement a model that includes admixture, which is a common feature of population genetic data sets. Structure provides estimates of the proportion of each individual’s genome derived from one of the K clusters. This differs from the STRUCTURAMA model we used, which estimates the posterior probability that each individual is a member of one of the K clusters using a no-admixture model. Therefore, similarities and differences between STRUCTURAMA and Structure results were interpreted such that STRUCTURAMA would recover genetic patterns on a larger geographic scale while Structure would be more sensitive to localized violations of Hardy-Weinberg equilibrium and therefore indicative of finer-scale population structure.We used several methods that allow us to delimit populations and infer their evolutionary history with the nDNA data. First, unrooted ML genealogies were constructed for each alignment in MEGA assuming the Tamura-Nei 1993 evolutionary model, and the topology of each genealogy was assessed in regard to congruence with each other and the mtDNA phylogeny. Multi-locus haplotypic data were entered into STRUCTURAMA 2.0 STF between 22 collecting localities of sample size four or greater from the concatenated dataset in Arlequin v 3.5 STF between STRUCTURAMA-inferred populations. In order to assess the degree to which the mtDNA and nDNA datasets can each explain the total genetic variation, we used Analysis of Molecular Variance (AMOVA) in Arlequin to partition groups of nDNA sequences in two ways: (1) assignment to their mtDNA clade from the phylogenetic analysis and (2) assignment to their STRUCTURAMA-inferred population. Specifically, we were most interested in the percentage of total genetic variation that is explained by differences between groups. Similar partitioning among these types of groups would suggest that both datasets recover the same phylogeographic patterns.Recently, the utility of Bayesian clustering methods as the sole source of evidence for determining population structure has come under scrutiny π), and average number of pairwise differences per sequence (k). Summary statistics were also calculated in Arlequin for: (1) the 22 collecting localities from which we obtained four or more individuals; (2) each inferred major mtDNA clade; and (3) each STRUCTURAMA-inferred population. We also measured the mean corrected Tamura-Nei distances within and between each mtDNA clade. The uncorrected pair-wise genetic distances per locus and the concatenated nDNA data set were measured within and between each STRUCTURAMA-inferred population. All corrected and uncorrected genetic distances were calculated in MEGA.We calculated standard diversity statistics for each locus in DnaSP including: number of polymorphic sites (s), number of haplotypes, haplotype diversity (Hd), nucleotide diversity , as not enough time will have passed for genetic drift to differentiate geographically separated subpopulations STF distance matrix and a geographic distance matrix generated in DIVA-GIS from the GPS points of each collecting locality. Significance was determined by 10,000 randomized permutations, and was used to accept or reject a null hypothesis of no correlation between geographic and genetic distances.In cases of evidence for population expansion, we tested for directionality using a series of linear regressions with nucleotide diversity as calculated with the concatenated nDNA data from collecting localities for which we collected four or more individuals. First, k, π, number of haplotypes, and Hd) than the nDNA. The number of individuals sequenced varies among nuclear loci, ranging from 62 to 74 sequences for the four introns and from 72 to 152 for the six anonymous loci , but never involved more than two events per locus, while the number of recombinant haplotypes per data set was limited to 2 or 3 individuals.An overview of the genetic data is featured in ous loci . IntroniA. carolinensis is strongly supported (1.0 bs and pp). As in the preliminary BI analysis, the final BI analysis recovered four major mtDNA clades with 100% pp. These clades are strongly correlated with geographic region . These estimates are close to estimates from past molecular phylogenetic studies carolinensis group and subgroup. The mrcat for all of our samples was estimated to be 2.1 Myr (95% HPD 1.3–2.9), pointing to a Late Pliocene-early Pleistocene origin for A. carolinensis.Given the mutation rate for the ND2 gene and the calibrations used, we estimated the age of the root of the BI tree to be 9.7 Myr (95% HPD 6.2–13.3), while the split between Structure simulations consistently encompassed the four STRUCTURAMA-inferred clusters. Using the delta-K method, Structure estimated a larger number of populations  than STRUCTURAMA, the main difference being that Structure detected a greater degree of clustering between GA, AL, TN, LA, TX and AR in the Gulf-Atlantic STRUCTURAMA group.We found some discordance between the mtDNA and nDNA phylogenies, due to lower variation in the nDNA and resulting multifurcations. Even so, the unrooted genealogies were useful in visualizing the concordance that did exist across most trees, showing bifurcations between FL populations and all others . STRUCTUSTF values between STRUCTURAMA-inferred populations are all significant . The relationship between π and longitude was positive and significant . When we removed the localities consisting of individuals from the Suwannee and Everglades lineages the relationship between π and longitude was greater . Therefore, a stronger signal exists for an east-to-west direction of expansion. With the Mantel Test, we could not reject a null hypothesis of no correlation between geographic and genetic distances in the wide-ranging Gulf-Atlantic population . Thus, evidence for a model of IBD is lacking in this wide-ranging population, suggesting that the east-west expansion was rapid and relatively recent.A strong pattern of genetic diversity loss in northern localities was not recovered, as the regression of Anolis genus are known to disperse a few hundred meters from their place of hatching The mtDNA phylogeny and STRUCTURAMA analysis both indicate that there are at least four distinct green anole populations. We identify these populations as (1) the Everglades in southern FL, (2) the Suwannee on the Gulf coast of FL, (3) the Gulf-Atlantic and (4) the NC clade (Carolinas for nDNA). The differences in population assignment using mtDNA and nDNA from localities in NC, SC and GA may arise in one of two ways: (1) male-biased dispersal leading to an introgression of NC nuclear haplotypes in the south and vice-versa, or (2) the retention of ancestral polymorphisms in more slowly evolving nuclear genes. Males in the STF supports three of the mtDNA and STRUCTURAMA-inferred groups, with subpopulations from the putative Suwannee population occurring in disparate regions of the tree. It is important to keep in mind that this is an unrooted tree derived from the genetic distances of populations and therefore not necessarily useful for inferring evolutionary relationships. However, that these subpopulations are not separated by very long branch lengths from the midpoint of the tree may be used as further evidence for the ancestral status of the Suwannee populations (see next section). As for the Structure analysis, the delta-K method points to a larger K (10) than inferred by STRUCTURAMA (4). This may be because demographic histories involving population size expansions will produce an excess of low frequency polymorphisms; thus the program will use evidence from slightly divergent haplotypes to add additional clusters with minimal cost, in the process overestimating K. Still, the Structure results do encompass all four STRUCTURAMA groups. That STRUCTURAMA agrees so closely with the mtDNA phylogeny, and that genetic variation partitions so closely in the AMOVAs, allows us to say with confidence that four populations most accurately reflect the distribution of individuals (in our sample) across this geographic scale.The topology of the NJ tree based on A. carolinensis colonized North America via overwater dispersal to FL from Cuba near the time of Plio-Pleistocene boundary mrcat of our mtDNA dataset is estimated at ∼2 Mya. Three lines of evidence suggest that populations on the mid- and northern Gulf coast of FL represent the most persistent remnants of this colonization event: (1) the Suwannee clade is the most basal A. carolinensis clade in the mtDNA phylogeny; (2) genetic diversity overall is highest in this population, suggesting long-term stability; and (3) Suwannee subpopulations tend to be closer to the midpoint of the STF NJ tree. There is a possibility that the current distribution of genetic variation represents refugial populations that were once more widespread. The genetic signatures of these populations could have been wiped out by rising sea levels and an insular history of FL during interglacial periods from the Miocene into the Pleistocene. Thus, we also cannot rule out the extinction of ancestral populations in southern FL followed by the more recent colonization of derived populations.It has previously been proposed that A. carolinensis entered the continental mainland, there were a number of geographic and topographical constraints that may have affected its dispersal patterns and thus the current distribution of individuals. Numerous studies of terrestrial fauna have described a common phylogeographic pattern showing genetic discontinuity between populations living along rivers that drain into the Atlantic Ocean and those which drain into the Gulf of Mexico A. carolinensis, as there appears to be extensive gene flow along the SC and GA portions where these regions overlap.Once Another important discontinuity existing on either side of the Appalachian Mountains has been observed in co-distributed taxa such as salamanders Whether or not Pleistocene glacial cycles have had region-wide effects on co-distributed taxa has long been a subject of debate in comparative phylogeographic studies focusing on eastern North America. Southern refugia during glacial maxima followed by subsequent northward dispersal and population size expansion has been cited as an explanation for observed genetic diversity across numerous studies Scincella lateralis) Anolis genus In addition to the inferred effects (or lack thereof) of late Pleistocene glaciation, the geography of genetic discontinuities in green anole populations differs from what is found in some co-distributed taxa in important ways. For instance, many of the riverine barriers that have strongly affected the cryptic fragmentation in the co-distributed common ground skink  mtDNA haplotypes essentially form a polytomy in the phylogenetic analysis; (2) Tajima’s D, Fu’s Fs and the mismatch distribution point to expansion; (3) a dramatic increase in population size inferred by the BSP, and (4) the lack of support for a model of isolation by distance.A statistically significant relationship between nucleotide diversity and longitude suggest that our data are most consistent with a hypothesis of westward expansion of A. c. seminolus, based on our analyses of the genetic data. While we have shown ample evidence for an independently evolving lineage at the southernmost reaches of the FL peninsula, we have detected an equally or even more divergent lineage endemic to the northern Gulf coast of FL. These lineages have been separate since the early to mid-Pleistocene, with minimal migration. When taken into account with previous morphological analyses that have concluded significant geographical differences, our genetic data point to significant polytypy in this species, although perhaps not as drastic as observed in ground skinks We can attend to the assertion of Vance (1991) that certain southern FL populations constitute the subspecies A. carolinensis genome and devised a multi-locus study of the demographic history of this species in North America. Combining phylogenetics, clustering methods, and population genetics, we have demonstrated the existence of at least four distinct evolutionary lineages of A. carolinensis, the most recent common ancestor of which may predate the Pleistocene. These lineages have been characterized by population expansions since the mid-Pleistocene, and do not seem to have been affected by more recent glacial periods at mid-latitudes. The most striking phylogeographic breaks separate subpopulations from the mainland Atlantic Seaboard, the Gulf Coastal Plain, and the Gulf coast and southern portions of FL. We propose that these genetic discontinuities may result from a combination of barriers provided by the Appalachian Mountains and dispersal patterns along waterways that drain towards either the Gulf of Mexico or the Atlantic Ocean. We have also inferred a rapid mid- to late Pleistocene westward expansion of A. carolinensis populations across the Gulf Coastal Plain, and into habitats that have expanded the traditional niche of their more tropical evolutionary progenitors. Surely, the resources provided by the sequencing of the Anolis genome will provide ample opportunity to investigate the adaptive differences that exist at the molecular level for a species that ranges across such a wide variety of habitats.We have taken advantage of the resource provided by the Figure S1Unrooted ML trees for intronic sequences. We performed phylogenetic inference using Maximum Likelihood (ML) in MEGA 5.0 with 1000 bootstrap replicates . Trees are unrooted due to lack of an outgroup. Circles are roughly proportional to the number of individuals present at a node, and pie charts reflect proportion of individuals at each node belonging to one of four major mitochondrial clades: Gulf-Atlantic (green), North Carolina (red), Suwannee (blue), Everlgades (magenta).(TIF)Click here for additional data file.Table S1Collecting localities and GPS coordinates.(XLS)Click here for additional data file.Table S2Predicted protein products for the intronic sequences used in this study.(XLS)Click here for additional data file.Table S3Primers for PCR products used in this study.(XLS)Click here for additional data file.Table S4Summary statistics for all nuclear loci for the Gulf-Atlantic population. #seqs – refers to phased haplotypes.(XLS)Click here for additional data file.Table S5Summary statistics for all nuclear loci for the Carolinas population.(XLS)Click here for additional data file.Table S6Summary statistics for all nuclear loci for the Suwannee population.(XLS)Click here for additional data file.Table S7Summary statistics for all nuclear loci for the Everglades population.(XLS)Click here for additional data file.Table S8Summary statistics of collecting localities for which we collecting four or more individuals using the concatenated nDNA. #seqs – refers to phased haplotypes.(XLS)Click here for additional data file.


Evenks and Evens, Tungusic-speaking reindeer herders and hunter-gatherers, are spread over a wide area of northern Asia, whereas their linguistic relatives the Udegey, sedentary fishermen and hunter-gatherers, are settled to the south of the lower Amur River. The prehistory and relationships of these Tungusic peoples are as yet poorly investigated, especially with respect to their interactions with neighbouring populations. In this study, we analyse over 500 complete mtDNA genome sequences from nine different Evenk and even subgroups as well as their geographic neighbours from Siberia and their linguistic relatives the Udegey from the Amur-Ussuri region in order to investigate the prehistory of the Tungusic populations. These data are supplemented with analyses of Y-chromosomal haplogroups and STR haplotypes in the Evenks, Evens, and neighbouring Siberian populations. We demonstrate that whereas the North Tungusic Evenks and Evens show evidence of shared ancestry both in the maternal and in the paternal line, this signal has been attenuated by genetic drift and differential gene flow with neighbouring populations, with isolation by distance further shaping the maternal genepool of the Evens. The Udegey, in contrast, appear quite divergent from their linguistic relatives in the maternal line, with a mtDNA haplogroup composition characteristic of populations of the Amur-Ussuri region. Nevertheless, they show affinities with the Evenks, indicating that they might be the result of admixture between local Amur-Ussuri populations and Tungusic populations from the north.  Evenks and Evens are spread over a wide area of northern Asia from the Yenissey river in the west to the Chukotka and Kamchatka peninsulas in the east, and from the Taimyr Peninsula in the north to northern China in the south. They are linguistically and culturally closely related with a traditional life-style of highly nomadic hunting and gathering and reindeer herding; their languages belong to the North Tungusic branch of the Tungusic language family . Other Tth to 18th centuries CE. At this time Turkic-speaking cattle and horse pastoralists, the Yakuts, expanded over the large territory they occupy nowadays [Different hypotheses exist concerning the origins of the North Tungusic Evenks and Evens and their relations with the Amur Tungusic peoples. Vasilevič  proposednowadays ,8, in thnowadays .The spread of the ancestors of the North Tungusic groups over the large territory they occupy nowadays may have been accompanied by different degrees of intermarriage with local inhabitants. Thus, it is assumed that Yukaghir groups were assimilated by Evenks and especially Evens , a proceThe populations of northern Asia are characterized in the maternal line by high frequencies of mtDNA haplogroups C and D ,14–18; iThe historical expansion and the resulting fragmented settlement pattern of the Evenks and Evens raises the question to what extent different communities have intermarried with their geographic neighbours rather than their linguistic and cultural relatives. A previous investigation into the relationship of Evenks and Evens found evidence for shared ancestry in both the mtDNA and Y-chromosomal data, with subsequent isolation of men belonging to different subgroups as well as indications of intermarriage with neighbouring populations in the maternal line . In thisThe populations of the Amur-Ussuri region (Udegey and Nivkh) are characterized by low sequence diversity (0.88) and intermediate and low nucleotide diversity values , 8.4% of the variance is due to differences between (sub)populations. Only 5.7% of the variance can be explained by differences between groups when comparing the North Tungusic populations (Evens and Evenks) with the South Tungusic Udegey, with 6.8% of the variance being found between populations in each group. With respect to the North Tungusic populations, 7% of the variation is due to differences between subgroups. That these are due to differences among the individual Evenk and Even subgroups, and not to differences between the Evenk and Even populations, is demonstrated by the non-significant and negative proportion of variance due to between-group differences when comparing Evenks with Evens, while the proportion of between-population variance is highly significant and quite high when comparing only the Evenk subgroups (8.3%) and only the Even subgroups (6%) . ST values, the Nivkh and Udegey are the biggest outliers  based on the frequencies of basic haplogroups at the level of resolution shown in ST and geographic distances reveals that this correlation is not significant when considering all populations  with the Yakuts, four of these uniquely . Two of In contrast, the neighbouring Sebjan Evens do not show evidence of substantial amounts of Yakut gene flow in the maternal line: they are located at a distance from the Yakuts in the MDS plot and cluster with the Taimyr Evenks and the Tompo Evens in the CA analysis. Furthermore, they share only three of their eight haplotypes (37.5%) with Yakuts, none uniquely, and two of these shared haplotypes belong to haplogroups C4a1c and D4l2 characteristic of North Tungusic populations . These sThe Tompo Evens are close to Yukaghirs in the MDS analysis based on mtDNA sequences, as was seen previously  where t. In the The mtDNA haplogroup composition of the eastern Even subgroups, Berezovka and Kamchatka, with substantial frequencies of the characteristic eastern Siberian and Kamchatkan mtDNA haplogroups G1, Y1a and Z ,20, indith or 14th centuries CE [The mtDNA diversity values and Bayesian Skyline analyses  show suburies CE ,43. The Our analysis of complete mtDNA genome sequences and Y-chromosomal SNP and STR variation has revealed a differentiated picture of the relationships among Tungusic populations. Although the Amur Tungusic Udegey are linguistic relatives of the North Tungusic Evenks and Evens, in the maternal line most traces of their possible genetic relationship have been erased by the effects of drift and admixture both in the Siberian populations as well as in the Udegey. As to the Evenks and Evens, while they retain evidence of their shared ancestry, admixture with neighbouring populations, especially in the maternal line, has led to their genetic differentiation. In-depth investigations of the autosomal variation in these populations will enable deeper insights into their population history and relationships among each other and with other populations of Siberia.The collection of the samples was approved by the Ethics Committee of the University of Leipzig, the Ethics Committee of the Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow, and the Institutional Review Board of the University of Kansas. Written and/or oral consent to use their samples was obtained from all donors after explanation of the aims of the study. The Institutional Review Board of the University of Kansas approved the substitution of oral informed consent in lieu of written consent in the case of the Udegey, Koryaks, and Kamchatkan Evens, since donors were suspicious of signing written statements. The oral consent was witnessed by the Russian team members of the sampling expedition.We generated a total of 525 full genome mtDNA sequences from 130 Evenks belonging to four subgroups , 122 Evens belonging to five subgroups , 31 Udegey, 169 Yakuts belonging to three subgroups , 20 Yukaghirs, 15 Koryaks, and 38 Nivkh  using thST, standard mtDNA diversity indices, and Analysis of Molecular Variance (AMOVA) in Arlequin v3.5 [The sequences were generated with an Illumina Genome Analyzer IIx sequencer to an average coverage of 274x and full sequences were deposited in GenBank under accession numbers KF148067-KF148359 and KF148361-KF148592. Three different alignments were generated for analysis. For calculation of pairwise Φuin v3.5  all indeuin v3.5  in referuin v3.5 , using r-8 and 9.883E-8, respectively), using a strict clock model.For each population and subgroup the ideal substitution model was calculated using jModelTest v 2.1 . With thwww.fluxus-engineering.com), with transversions given a threefold higher weight than transitions. Correspondence analysis (CA) and multi-dimensional scaling (MDS) plots were generated with STATISTICA v10 [ST genetic distances between (sub)populations were performed in R using the “ade4” package [Median Joining (MJ) networks  were genTICA v10 . Mantel TICA v10  – and ΦS package .Published mtDNA genome sequences were downloaded from GenBank for inclusion in the network analyses; these included additional Yukaghir, Evenk, Even, Udegey, Koryak, and Yakut sequences as well as Mongolic-speaking Buryats and Khamnigans, Turkic-speaking Tofalar, Tubalar, Altai-Kizhi, Shor, Teleut, Tuvan, and Kazakh from South Siberia, Kets, who speak an isolate language, and Finno-Ugric-speaking Mansi from western Siberia, Samoyedic-speaking Nganasan from the Taimyr Peninsula, Tungusic-speaking Negidal and Ulchi from the Amur-Ussuri region, Chukchi from Chukotka who speak a Chukotko-Kamchatkan language, and Eskimos ,18,32,34To investigate the paternal relationships of the Evenks and Evens, Y-chromosomal SNPs were genotyped in the Sebjan, Berezovka, and Kamchatkan Evens as well as in the Taimyr Evenks following the protocol of de Filippo et al. ; subhaplThe age of the Y-STR diversity accumulated in Evenks and Evens belonging to haplogroup C3c1 was estimated with the rho statistic  implemenFigure S1MJ-network of mtDNA haplogroup D4.The North Tungusic haplotypes are coloured by population (Evenks and Evens) rather than subgroup. The size of the nodes is proportional to the number of individuals carrying that node, and the number of mutations is indicated along the branches. The haplotypes marked by arrows are discussed in the text.(EPS)Click here for additional data file.Figure S2MJ-network of mtDNA haplogroup D5a2.The North Tungusic haplotypes are coloured by subgroup. The size of the nodes is proportional to the number of individuals carrying that node, and the number of mutations is indicated along the branches. (EPS)Click here for additional data file.Figure S3MJ-network of mtDNA haplogroup A.The North Tungusic haplotypes are coloured by population (Evenks) rather than subgroup. The size of the nodes is proportional to the number of individuals carrying that node, and the number of mutations is indicated along the branches. Subhaplogroups discussed in the text are labelled.(EPS)Click here for additional data file.Figure S4Dimensions 1 vs. 3 of a three-dimensional MDS analysis.Based on pairwise ΦST values between populations; stress = 0.06. (Sub)population abbreviations as in (EPS)Click here for additional data file.Figure S5Dimensions 2 vs. 3 of a three-dimensional MDS analysis.Based on pairwise ΦST values between populations; stress = 0.06. (Sub)population abbreviations as in (EPS)Click here for additional data file.Figure S6Bayesian Skyline Analysis plots.Based on complete mtDNA genome sequences and a strict clock model. A: All Evenks, B: Nivkh, C: Koryaks, D: Yukaghir.(EPS)Click here for additional data file.Table S1List of mtDNA (sub)haplogroups found in the dataset and their frequencies in individual (sub)populations.(XLS)Click here for additional data file.Table S2Analysis of shared haplotypes.(XLS)Click here for additional data file.Table S3Y-chromosomal genotypes generated for this study.(XLS)Click here for additional data file.


Mikania micrantha H.B.K. (Asteraceae), one of the world's most invasive weeds, has adapted rapidly in response to novel environments since its introduction to southern China. In its genome, we expect to find outlier loci under selection for local adaptation, critical to dissecting the molecular mechanisms of invasiveness. An explorative amplified fragment length polymorphism (AFLP) genome scan was used to detect candidate loci under selection in 28 M. micrantha populations across its entire introduced range in southern China. We also estimated population genetic parameters, bottleneck signatures, and linkage disequilibrium. In binary characters, such as presence or absence of AFLP bands, if all four character combinations are present, it is referred to as a character incompatibility. Since character incompatibility is deemed to be rare in populations with extensive asexual reproduction, a character incompatibility analysis was also performed in order to infer the predominant mating system in the introduced M. micrantha populations. Out of 483 AFLP loci examined using stringent significance criteria, 14 highly credible outlier loci were identified by Dfdist and Bayescan. Moreover, remarkable genetic variation, multiple introductions, substantial bottlenecks and character compatibility were found to occur in M. micrantha. Thus local adaptation at the genome level indeed exists in M. micrantha, and may represent a major evolutionary mechanism of successful invasion. Interactions between genetic diversity, multiple introductions, and reproductive modes contribute to increase the capacity of adaptive evolution.Why some species become successful invaders is an important issue in invasive biology. However, limited genomic resources make it very difficult for identifying candidate genes involved in invasiveness.  Why some species become successful invaders is an important issue in invasive biology. When species are introduced into a new region, they face two fates. Some species quickly go extinct, whereas others persist and finally become highly competitive invaders, posing a serious threat to native diversity and ecosystems FST estimates FST distribution for the simulation Genomic scans are useful to identify potential adaptive loci under selection at the genomic level Mikania micrantha H. B. K. (Asteraceae) is one of the top ten worst weeds in the world M. micrantha was deliberately introduced into Asia as early as 1900s M. micrantha is subjected to new selective pressures, including such abiotic and biotic stresses as new climate, soils, pathogens, herbivores, pollinators, and competitors M. micrantha survived and reproduced in new environments using strikingly different strategies from those employed in its original niche M. micrantha became a dominant plant across most regions, influencing ecosystems, biological diversity, and natural communities M. micrantha grows on dry soils as well as shady sites M. micrantha in new environments M. micranthaM. micranthaM. micrantha populations, which is helpful to predict their adaptability in response to management practices M. micrantha (M. micrantha and (2) to understand contributions of interactions between genetic diversity, reproductive modes, bottlenecks, and multiple introductions to the adaptive evolution of M. micrantha.To test our prediction that outlier loci can be found in southern China populations of icrantha ; Table 1M. micrantha was optimized by determining which selective primer sets produced the clearest DNA fragments based on the eight primer pair combinations. The eight primer pairs were polymorphic in each population. A total of 483 clear polymorphic fragments were detected. The proportion of polymorphic loci within a population ranged from 10.57% to 73.47%, with an average value of 41.84%  and character compatibility among the 483 AFLP loci. Each region was observed to possess significant LD and matrix compatibility obtained by testing the index of association (io (IER) . Likewisio (IER) .Alleles were defined as rare when they occurred at a frequency of less than 0.05 in the examined populations M. micrantha appeared subject to a significant bottleneck (Using the stepwise mutation model (SMM) and the infinite allele model (IAM), we identified bottleneck signatures in each population with a heterozygosity excess/deficiency ratio that significantly deviated from the expected ratio (1∶1) at mutation-drift equilibrium , P<0.05.ttleneck .ΦST measured by AMOVA analysis was 0.3335; 66.65% of the variation was partitioned within populations, 28.61% was attributed to differences among populations within regions, and only 4.74% of the variation was due to regional differences  , whereas only five values derived from SZ64 vs. SZ75, SZ64 vs. SZ77, SZ64 vs. DG91, SZ75 vs. SZ77, and SZ75 vs. DG91 were not (P>0.05), highlighting remarkable differences among populations.The genetic differentiation P<0.001) . When inf-free model chosen based on the smallest mean DIC was more suitable than other models for the AFLP data set of the 28 populations. Therefore, Bθ = 0.2927  was determined to be an unbiased Bayesian estimate of all population genetic differentiation.The Bθ were observed in the full AFLP data set . A similar pattern was derived at the regional level. Hence no evidence of “isolation by distance” No significant associations between geographical distances and pairwise estimates of ΔK criterion of Evanno et al.ΔK was K = 24, indicating the need to divide the samples into 24 clusters. Bar plots showed varying extents of admixture among populations  were identified as outlier loci under directional selection at the 99.5% probability level (10 PO>2.0 (posterior probabilities higher than 0.99), corresponding to 7.87% of the 483 investigated loci. This approach identified 38 outlier loci potentially under selection or linked to a locus under selection  representing different habitats  located on Hainan Island  were deposited at the herbarium of Sun Yat-sen University (SYS), Guangzhou, China.For habitats ; Table 1n Island ; Table 1Total genomic DNA was extracted from ground tissue, following the modified CTAB protocol et al.2O, then 2 µl of each sample was used as a template to conduct the pre-amplification in a final volume of 20 µl that contained 1×PCR buffer, 100 nM each of EcoRI+A and MseI+C primers, 0.24 mM dNTPs, and 1.5 U Taq DNA polymerase. The preamplification reaction was carried out for 20 cycles of 30 s at 94°C, 1 min at 56°C and 1 min at 72°C. After that, preamplified product was diluted 1∶50 with ddH2O, and then used as template for the selective PCR amplifications to generate AFLPs.AFLP analyses were performed according to Vos Taq DNA polymerase and 2 µl diluted pre-amplified DNA sample. The reaction was conducted for 13 cycles of 30 s at 94°C, 30 s at 65°C and 1 min at 72°C. The annealing temperature was reduced by 0.7°C per cycle. Then 23 cycles consisting of 30 s at 94°C, 30 s at 56°C, and 1 min at 72°C were performed.Selective amplification was performed in a final volume of 10 µl containing 1× PCR buffer, 125 nM EcoRI primer, 125 nM 6-FAM-EcoRI primer, 250 nM MseI primer, 0.2 mM dNTPs, 0.75 U An initial screening of 48 combinations of selective primers, in which six were EcoRI  and eight were MseI , were performed. Eight combinations were selected that generated clear and evenly distributed bands: EcoRI+ACT/MseI+CAG, EcoRI+AAC/MseI+CAG, EcoRI+ACT/MseI+CTA, EcoRI+AAC/MseI+CTC, EcoRI+AAC/MseI+CTA, EcoRI+ACT/MseI+CTC, EcoRI+AGC/MseI+CAC, and EcoRI+ACC/MseI+CAC. Therefore, the eight primer combinations were chosen for selective amplifications of all samples. PCR reactions were conducted on a PTC-100 Peltier Thermal Cycler . The selective PCR products were separated by electrophoresis on 6.5% polyacrylamide gels on an ABI 377 automated sequencer . An ROX-500-labeled internal size standard (Applied Biosystems) was added to each sample to size fragments.http://hordeum.oscs.montana.edu/genographer) were utilized to collect and score raw fluorescent AFLP data. The presence (1) or absence (0) of data from unambiguous AFLP bands was used to establish the matrix of genetic identity of the sampled individuals. Genetic diversity statistics, including percentage of polymorphic loci and Nei's gene diversity Software GeneScan 3.7 (Applied Biosystems) and genographer  Bθ estimates.Analysis of Molecular Variance (AMOVA) based on a Euclidean squared distance matrix was hierarchically calculated to estimate the allocation of genetic variation among and within populations by ARLEQUIN 3.0  and the fixation index (FST) of F-statistics, respectively. The posterior distributions of f and Bθ were estimated through Markov Chain Monte Carlo (MCMC) methods by HICKORY v1.0, with a burn-in of 50 000 iterations and a sampling run of 250 000 iterations from which every fiftieth sample was retained for posterior calculations f-free” model, where f was not estimated but was chosen at random from the prior distribution, was decided due to its smaller DIC than for other models. Alleles were deemed rare if their frequencies were ≤0.05 in the sampled populations Holsinger K (K = 1 to 30) were performed with an admixture model at 100 000 MCMC iterations and a 10 000 burn-in period. We used ΔK, the second-order rate of change in ln P (X|K) for successive values of K to determine the number of clusters K value at the highest log likelihood. Moreover, based on Nei's genetic distance To gain further perspectives on genetic structure, we also employed the Bayesian clustering method to infer the pattern of population structure by implementing the STRUCTURE 2.2.3 program We further tested whether populations have suffered a bottleneck using the program BOTTLENECK IAhttp://www.bio.ic.ac.uk/evolve/software/multilocus/).Linkage disequilibrium, which is an association of alleles at different loci on chromosomes in a population, is mainly affect by population size A character incompatibility analysis was carried out to probe the predominant mating system in the populations at the molecular level M. micrantha and M. cordata, two complementary methods were applied to our AFLP data set. We first used Program Dfdist, which is the most popular software for detecting candidate loci http://www.rubic.rdg.ac.uk/~mab/stuff) was recently modified from Beaumont and Balding FST values for each locus against a null distribution of FST values expected from a neutral drift model FST close to the empirical distribution was acquired by 50 000 coalescent simulations. Simulations were computed with a mean FST similar to the trimmed mean FST, which was calculated by excluding 30% of the most extreme FST values observed in the empirical dataset M. micrantha and M. micrantha using 28 populations and 2 populations, respectively. The threshold for outliers was set to the more conservative 0.005, estimated from simulated FST values to control for false positives http://www-leca.ujf-grenoble.fr/logiciels.htm), which better handles dominant marker data by directly estimating the posterior probability of a given locus to be under selection FST coefficients, but also considers all loci in the analyses 10 PO>2.0 was considered a threshold value for determining loci under selection according to Jeffreys' interpretation 10 PO>0.5 ; log10 PO>1.0 (strong); log10 PO>1.5 (very strong); and log10 PO>2.0 (decisive support for accepting a model) 10 PO>2.0, which provides decisive support for acceptation of the model. Outliers identified by both Dfdist and Bayescan are likely to be truly adaptive regions of the genome, because the two approaches differ in algorithms and assumptions To detect outlier loci under selection for local adaptation of


Durvillaea antarctica) has a broad distribution along much of the Chilean coast. This species represents an ideal model taxon for studies of coastal marine connectivity and of palaeoclimatic effects, as it grows only on exposed rocky coasts and is absent from beaches and ice-affected shores. We expected that, along the central Chilean coast, D. antarctica would show considerable phylogeographic structure as a consequence of the isolating effects of distance and habitat discontinuities. In contrast, we hypothesised that further south - throughout the region affected by the Patagonian Ice Sheet at the Last Glacial Maximum (LGM) - D. antarctica would show relatively little genetic structure, reflecting postglacial recolonisation.South America's western coastline, extending in a near-straight line across some 35 latitudinal degrees, presents an elegant setting for assessing both contemporary and historic influences on cladogenesis in the marine environment. Southern bull-kelp (rbcL) DNA analyses of D. antarctica from 24 Chilean localities  revealed two deeply divergent  clades from the centre and south of the country, with contrasting levels and patterns of genetic structure. Among populations from central Chile (32° - 44°S), substantial phylogeographic structure was evident across small spatial scales, and a significant isolation-by-distance effect was observed. Genetic disjunctions in this region appear to correspond to the presence of long beaches. In contrast to the genetic structure found among central Chilean populations, samples from the southern Chilean Patagonian region (49° - 56°S) were genetically homogeneous and identical to a haplotype recently found throughout the subantarctic region.Mitochondrial (COI) and chloroplast  Chile has been recolonised by  Oceanic ecosystems have historically been considered relatively open, with high connectivity among populations . Recent Genetic patterns of isolation-by-distance are common in the marine realm, with neighbouring populations generally more closely related to each other than to those further away, as the chance of successful dispersal decreases with increasing distance ,6. Gene Fucus vesiculosus: [Hormosira banksii: [Macrocystis pyrifera: [Durvillaea antarctica), found evidence of recent  recolonisation of much of the subantarctic, a feat almost certainly achieved by trans-oceanic rafting of adult specimens [Although most macroalgae have only short-lived gametes or dispersive zygotes, some - particularly highly buoyant species - are hypothesised to be capable of long-distance dispersal via rafting of fertile adults . A broapecimens . Recolonpecimens -24). Butpecimens  suggestepecimens ,25. In dDurvillaea antarctica currently dominates the rocky shores of Chile from Cape Horn [D. antarctica included few samples from central Chile , and none from Chilean Patagonia, and was therefore unable to assess phylogeographic contrasts along the Chilean coast. For the current study, we analysed samples from a total of 24 localities, encompassing the full range of D. antarctica in Chile, to test the predictions that (i) D. antarctica populations in central Chile would show strong phylogeographic structure, and that (ii) D. antarctica populations in southern Chile would show little genetic structure, reflecting postglacial recolonisation.Chile presents an intriguing system for assessing the relative phylogeographic impacts of isolation-by-distance, habitat discontinuities, and historic glaciations. Central Chile has an almost straight coastline characterised by numerous extensive stretches of beach that isolate rocky-shore habitats ; this coape Horn , at arouape Horn . Fraser ape Horn  recent bD. antarctica samples from 24 localities along the coast of Chile yielded 14 distinct haplotypes for mitochondrial COI, with 34 variable sites detected across the 629 bp fragment. For a chloroplast marker, rbcL, 78 sequences from nine localities  were restricted to populations in central Chile, whereas all sampled Chilean Patagonian populations south of 44°S were fixed for a single haplotype (C-I), which was highly divergent from the others  haplotypes detected in Chile were 1.4% divergent (uncorrected distance), with one (R-I) restricted to all sampled Patagonian populations, and the other (R-II) found only in central Chile . More broadly, the Patagonian rbcL haplotype R-I has a circum-subantarctic distribution, including the Falkland Islands and South Georgia .Network analyses of Chilean COI haplotypes recovered two parsimonious networks that could not be joined at ≥90% confidence limits, with one comprising only the Patagonian haplotype C-I. All other Chilean haplotypes (CII-CXIV) formed a single network   beaches , whereas total coastal distance was not . The outlier formed by Lebu/Tirua . With or without the outlier, these results indicate that habitat discontinuity influences genetic connectivity among populations of central Chilean bull-kelp.The Mantel test revealed a significant relationship between genetic and geographic distance (ns) Fig. . DetaileD. antarctica in Chile. First, substantial phylogeographic structure was observed in central Chile (hypothesis i). Genetic isolation among populations in this region appears driven by habitat discontinuities. Second, and in direct contrast to the pattern observed for central Chile, samples from Chilean Patagonia showed complete genetic homogeneity within and among localities (hypothesis ii). These Patagonian populations were deeply divergent to those from more northern  localities, and appear to have trans-oceanic ancestry.Two broad findings were revealed by our phylogeographic research of D. antarctica , suggesllospora ,31, dispD. antarctica is in direct contrast to the broad evidence of long-distance, trans-oceanic dispersal of this species in the subantarctic [D. antarctica zygotes would suppress settlement of zygotes from such migrants. Conversely, shores swept clean by ice [D. antarctica is clearly an effective long-distance disperser, there may be differences in reproductive viability of rafts across latitudinal gradients. Numerous reports suggest that D. antarctica has an extended period of fertility in the subantarctic versus mainland New Zealand or Chile , and thences in ), perhapences in  suggest r et al. . If D. ania Fig. .D. antarctica populations could additionally be influenced by a range of processes such as seasonal variation and patchy recruitment [not show a genetic disjunction (Lebu-Tirua) is in the immediate vicinity of a large coastal island (Isla Mocha) that may effectively connect the two populations despite the beach barrier. Interestingly, Constitución, flanked by long beaches on both sides, was genetically distinct from adjacent localities but not from some more distant localities . These populations, on the northernmost edge of the range of D. antarctica in Chile, may be sensitive to any changes in water temperature. Changes to the northern range limit of an Australian congeneric, D. potatorum, have been linked to warming waters in recent years [D. antarctica in Chile  may thus be somewhat ephemeral in the long term, and could have been relatively recently colonised by kelp drifting from southern populations  in the north-flowing Humboldt Current.Local genetic structure within and among central Chilean ruitment  or harveruitment -36. Whilruitment  and Castnt years . The norD. antarctica populations indicate a biogeographic break in northern Chilean Patagonia (44° - 49°S), with both mitochondrial and chloroplast DNA phylogenies revealing a deep genetic disjunction between samples from southern Patagonia and those from central Chile  and wind (primarily eastward) through the fiords. Taitao Peninsula, however, projects across the dense system of channels that could otherwise potentially allow mixing and north-south transport of drifting D. antarctica within the fiords.Our phylogeographic analyses of Chilean ile Fig. . Althougile Fig.  has prevD. antarctica samples from southern Patagonia and those from central Chile raises the question of the taxonomic status of these two divergent lineages. A recent study of D. antarctica revealed that this taxon comprises two ecologically, morphologically and genetically distinct forms in New Zealand [D. antarctica (4.5 - 6.1% for COI) exceeds the divergence observed between the co-occurring, genetically-distinct forms in New Zealand , it is D. antarctica from all localities south of latitude 44°S  ice during the most recent ice age may also have driven kelp population extinctions. If populations of D. antarctica in southern Chile were indeed destroyed by the Patagonian Ice Sheet during the last glacial period, numerous other coastal taxa may have been similarly affected. Intriguingly, a broad-scale phylogeographic study of the rocky-shore gastropod Concholepas concholepas [Although genetic evidence of postglacial recolonisation has previously been identified from a variety of Patagonian terrestrial and freshwater taxa, including fish  is capable of long-distance dispersal via rafting of fertile adults, as evidenced by the trans-oceanic ancestry of populations throughout postglacially-recolonised Chilean Patagonia . IntrigD. antarctica were collected from 24 localities along the coast of Chile, from Pichicuy (32°27'37"S) to Cape Horn (55°59'31"S)  pieces of tissue cut from the tip of healthy fronds of attached D. antarctica, and were preserved by desiccation in either 96% ethanol or silica gel beads. DNA extraction, amplification, purification and sequencing were performed as described in Fraser et al. [c oxidase I (COI) was amplified using primers GazF1 and GazR1 [rbcL was amplified using primers KL2 and KL8 [164 samples of r et al. . A 629 bnd GazR1  for all  and KL8  for sampDurvillaea willana , Durvillaea potatorum  and Fucus (GenBank accessions: F. vesiculosus AY494079 for COI and F. gardneri AF195515 for rbcL). To place the new Chilean sequence data in a wider context, published D. antarctica DNA sequences  as selected by the AIC of Modeltest 3.06 [D. antarctica COI and rbcL sequences used in this study: FJ550093-FJ550095; FJ550097; FJ550099; FJ550119; HM103936-HM104173).Phylogenies for each DNA dataset were constructed using maximum-likelihood (ML) and Bayesian analyses, and included outgroup sequences from r et al.  with a Tr et al.  model foest 3.06 , with heest 3.06  and are D) average pairwise differences [FST values could not be used in the Mantel test: several localities were genetically monomorphic for different haplotypes, and pairwise comparisons between such fixed populations gave an FST of 1.0; hence for these locality pairs no linearised FST values (FST/(1 - FST), [Analysis of isolation-by-distance was performed on data from the 19 localities in central Chile (north of 44°S) via the Mantel  test, imferences   versus  - FST), ) could bhttp://earth.google.com/ to assess the continuity of suitable rocky shore habitat for D. antarctica among localities. As Chilean shores south of about 43°S are almost completely rocky, with few sandy beaches and, moreover, many wide oceanic channels between islands and peninsulas, measurements of beaches were only made for continental Chile and the coast of the Great Island of Chiloé (north of 43°S). The following analysis was performed only on data from sampled localities in this region . The isolating effects of beach length and total coastal distance between localities in central Chile were assessed by logistic regression. Adjacent locality pairs were first classified binomially as either genetically disjunct (D ≥1.0) or not (D < 1.0). Under this definition, locality pairs not sharing any haplotypes were considered genetically disjunct, whereas those with shared haplotypes were not considered disjunct. Logistic regression was then carried out using R [Approximate measurements of the lengths of all beaches  between pairs of adjacent rocky coastal localities in central Chile were made using the 'path ruler' tool in Google Earth  using R , using tC.I.F., J.M.W., M.T. and H.G.S. conceived the ideas; C.I.F. obtained and analysed the data; C.I.F. led the writing; and J.M.W., M.T. and H.G.S assisted with the writing. All authors read and approved the final manuscript.


Lobaria pulmonaria (L.) Hoffm., low frequency of sexual reproduction is likely to limit the potential of populations to adapt to changing environmental conditions. Our study provides direct evidence of self-incompatibility  in L. pulmonaria. It can thus be hypothesized that sexual reproduction in small populations might be limited by an unbalanced distribution of mating-type genes. We therefore assessed neutral genetic diversity (using microsatellites) and mating-type ratio in 27 lichen populations . We found significant differences in the frequency of the two mating types in 13 populations, indicating a lower likelihood of sexual reproduction in these populations. This suggests that conservation translocation activities aiming at maximizing genetic heterogeneity in threatened and declining populations should take into account not only presence of fruiting bodies in transplanted individuals, but also the identity and balanced representation of mating-type genes.Fungal populations that reproduce sexually are likely to be genetically more diverse and have a higher adaptive potential than asexually reproducing populations. Mating systems of fungal species can be self-incompatible, requiring the presence of isolates of different mating-type genes for sexual reproduction to occur, or self-compatible, requiring only one. Understanding the distribution of mating-type genes in populations can help to assess the potential of self-incompatible species to reproduce sexually. In the locally threatened epiphytic lichen-forming fungus  Sexual reproduction is an important factor for introducing genetic variation in populations, thus providing the basis for long-term adaptation and population survival Lichens, symbioses between a fungus and one or more algal photobiont species, have often developed a complex reproductive strategy that combines clonal and sexual reproduction MAT) locus MAT1-1 and MAT1-2, of the single regulatory MAT locus MAT locus are referred to as “idiomorphs” rather than alleles MAT genes is the presence of conserved DNA binding motifs in the encoded proteins, which might serve as transcription regulators MAT1-1 encodes a protein with conserved alpha box motif, and MAT1-2 protein contains conserved DNA binding motif called HMG box In the fungi, almost the entire sexual cycle starting from the recognition of a potential mate to the development of sexual structures, is regulated by a specialized region of the genome known as the mating-type  Hoffm. This is one of the best-studied lichen species L. pulmonaria to be heterothallic. However heterothallism in L. pulmonaria has not yet been demonstrated by molecular analyses.In this study we investigate mating-type loci in the epiphytic lichen-forming fungus L. pulmonaria symbiosis and to infer the relative contribution of sexual reproduction to the genetic composition of lichen thalli in natural populations Microsatellites are the marker of choice for estimating genetic diversity and population structure of highly clonal organisms such as lichens. The high mutation rate of microsatellite loci and thus high variability gives them a far greater resolving power than previous, sequence- based method. They are valuable tools for detecting linkage disequilibrium associated with the absence of sexual reproduction Lobaria pulmonaria, 2. Assess the distribution and frequencies of the two mating types in 27 populations of L. pulmonaria with newly developed, species-specific mating-type multiplex markers. 3. Investigating the genetic structure and linkage disequilibrium pattern using fungal and algal-specific microsatellite markers for these populations.Our objectives here were to 1. Determine the mating system  in the lichen model organism L. pulmonaria specimens  were cultivated according to Denison (2003) Mycelia of two L. pulmonaria was extracted as described in Photobiont DNA from L. pulmonaria specimens from populations previously described in In addition, total genomic DNA, containing both fungal and algal DNA, was extracted using DNeasy Plant Mini Kit (Qiagen) according to the manufacturer’s recommendations. This involved isolation from single lobes of L. pulmonaria was selected to be shotgun sequenced (1/4th run) using a Roche 454 Genome Sequencer FLX with the Titanium Sequencing kit XLR 70 at Microsynth AG . The 454 sequencing was done with the culture 10161 . We obtained 73.2 Mbp of raw sequence data, from the 1/4 run of 454 sequencing, containing 181’541reads/sequences with an average length of 363 bp. The number of assembled sequences was 139937 with an average length of 369 bp. The raw sequences represented large numbers of individual sequence reads, which were then assembled into contigs. A total of 181541 reads were assembled into 41692 contigs with an average length of 685 bp.The DNA of the mycobiont culture of MAT1-1 and MAT1-2 genes of the fungal DNA were first amplified using several sets of degenerate primers (MAT1-2 were amplified by inverse PCR The conserved regions of  primers . Based oMAT1-1 with degenerate primers: (1) 2 min 94°C; (2) 40 cycles of 30 sec 94°C, 45 sec 50°C, 1 min 72°C; (3) 10 min 72°C final extension. The amplification of MAT1-2 with degenerated primers used cycle conditions that differ only in two points: 35 sec at 72°C elongation time in each cycle and a total of 35 cycles. Finally, digested DNA prepared for inverse PCR was amplified under the following conditions: (1) 2 min 94°C; (2) 35 cycles of 40 sec 94°C, 45 sec 50°C, 90 sec 72°C; (3) 10 min 72°C for the final extension. In the end, all products obtained from degenerate primer PCR or inverse PCR were excised from the agarose gel and purified using the MinElute PCR Purification Kit (Qiagen) following the manufacturer’s recommendations.All amplifications were performed with 20 ng of DNA extract in a total volume of 50 µl containing 18 µl of JumpStart REDTaq ReadyMix PCR Reaction Mix (Sigma-Aldrich), 500nM of each degenerate primer or 250 nM of the specific ones. The following cycling conditions were used for For sequencing the regions flanking the conserved regions of mating-type genes, three rounds of TAIL-PCR were performed with thermal cycling conditions as described in All PCR products were labeled with the Big Dye Terminator v3.1 Cycle Sequencing Kit  and cycle sequenced as follows: (1) 1 min 96°C; (2) 26 cycles of 20 sec 96°C, 5 sec 50°C and 2 min 60°C. Products were then purified using the Big Dye XTerminator Purification Kit (Life Technologies). Sequences were detected on 3130×l Genetic Analyzer (Life Technologies) and analyzed with the Sequencing Analysis Software 3.4 (Life Technologies).MAT1-1 specific primers amplified a product of ca. 380 bp, and MAT1-2 primers amplified an 180 bp fragment. Axenic fungal culture DNA was used as positive control, whereas algal DNA and no template reactions served as negative controls.Specific primers were designed using Primer 3 plus online tool Samples used in this study were collected in the framework of an earlier project see  [35], [4SAI) as implemented in LIAN 3.1 http://genepop.curtin.edu.au/; To check for recombination or clonality in the fungal symbiont within populations, we performed two tests: i) the overall index of association (Expected heterozygosity (H) for each population was estimated for the fungal symbiont using Arlequin software For each population we calculated the number of unique multilocus genotypes (MLG) and their ratio in the population . The calculations were performed for fungal MLGs i.e. FG (FG:N) and algal MLGs i.e. AG (AG:N). Samples having identical MLGs were treated as clones. The degree of clonality of the fungus or percent clonality (CF) was derived from FG:N as follows: one hundred minus the percent of unique genotypes. As clonally derived lichen offspring are expected to share fungal and algal components genetically identical to their mother thallus , we calculated the number of unique lichen MLG i.e. LG  for each population.MAT gene imbalance and clonality, by performing a linear regression analysis MAT gene ratio  versus percent clonality.We analyzed the ratio of MAT1-1, MAT1-2 isolates in all the populations . As clonMAT1-1 resulted in the amplification of approximately 500 bp fragment. Combining this sequence with the 454 sequence data we obtained a total of 900 bp sequence of MAT1-1. tBLASTx showed the presence of a conserved alpha box motif and high homology with MAT1-1 protein of various ascomycetes fungi: Xanthoria polycarpa (32%), Fusarium pseudograminearum, Fusarium lunulosporum, Fusarium culmorum (30%), Aspergillus parasiticus, A. flavus, A. oryzae (34%). The L. pulmonaria-specific MAT1-1 α-box primers target a segment of approximately 380 bp. No band of this size was amplified in axenic photobiont DNA. Sequences obtained by degenerate PCR were then searched in the 454 data from the cultured L. pulmonaria mycobiont. Combining the degenerate PCR sequence with the 454 data and TAIL-PCR we obtained a 2666 bp fragment of the MAT1-1 gene. No MAT1-2 sequences were retrieved from the 454 data.Degenerate primers (RsMat1-1F&R) MAT1-2, i.e. the HMG box, was amplified from genomic DNA as well as from axenic mycobiont culture of L. pulmonaria with degenerate primers (ChHMG1 and ChHMG2). A band of the expected size (280–300 bp) was excised from the gel, purified and sequenced. Combining degenerate PCR and inverse PCR we obtained a total of 450 bp of the MAT1-2 gene of L. pulmonaria. The sequenced region showed high similarity with lichen-forming ascomycetes and other fungi, e.g., Xanthoria polycarpa, Xanthoria parietina and Xanthoria elegans (83% similarity), Xanthoria flammea (79%), Arthroderma simii (75%), Cladonia galindezii (74%), Aspergillus parasiticus (70%). The translated protein BLAST showed the presence of a conserved HMG box. No band of this size was amplified in axenic photobiont DNA. The L. pulmonaria MAT1-2 specific primers were then designed from this 450 bp region for multiplex PCR and TAIL-PCR. The newly-designed L. pulmonaria-specific MAT1-2 primers amplified a fragment of 180 bp and were used in the multiplex PCR along with L. pulmonaria specific MAT1-1 primer. We obtained a total of 1400bp of MAT1-2 from L. pulmonaria by combining the sequencing results of degenerate, inverse and TAIL PCR.The conserved region of MAT1-1 and MAT1-2 mating-type genes in L. pulmonaria are deposited in GenBank Accession Nos. JX520967 (MAT1-1), JX520966 (MAT1-2).Sequences for the L. pulmonaria, i.e., single thalli without apothecia amplified either MAT1-1 or MAT1-2, hence confirming L. pulmonaria to be heterothallic. The multiplex primers were used to determine the frequency of MAT1-1 and MAT1-2 in different populations of L. pulmonaria . We tested the null hypothesis of an equal number of MAT1-1 and MAT1-2 isolates within 27 L. pulmonaria populations from a broad geographic range for a total of 933 lichen thalli. Both mating types were found in all populations tested. However, in several populations we found significant deviations from the expected 1∶1 idiomorph ratio. Four populations were mostly formed by MAT1-1 individuals and nine populations of MAT1-2 individuals, out of the 27 investigated populations . Three populations were excluded from these analyses because 37 individuals  amplified simultaneously with both MAT1-1 and MAT1-2 primers. These populations will be the subjects of further investigations .In majority of the samples (96.03%) only one mating-type gene per specimen was amplified in the multiplex PCR of ulations . DeviatiL. pulmonaria from 27 populations  was significantly different from 0 in most of the populations  to 88.88% (CHS) with an overall average of 0.4450 . The indulations . Investiulations . These fulations . Linear L. pulmonaria is a heterothallic species L. pulmonaria, i.e. the existence of a dimictic mating system with two idiomorphs, MAT1-1 and MAT1-2 located on a single locus. The majority of isolates (96.03%) of L. pulmonaria thalli amplified with either MAT1-1 or MAT1-2 specific primers.Previous studies suggested that MAT1-1) and HMG box motif (MAT1-2). The sequenced regions for both MAT1-1 and MAT1-2 showed presence of respective conserved motifs and also displayed high sequence similarity with mating-type genes of lichen-forming ascomycetes and other fungi.The two idiomorphs were amplified using degenerate primers designed against conserved alpha box motif  to 0.584 (pop: MO1) with an average of 0.4233. The percentage of clonality ranged from 10.14% in RXS to 88.88% in CHS. This indicates that all of the studied populations utilize sexual as well as asexual reproductive strategies for their propagation. However the prevalence varies in different populations, with some populations mostly asexual and others more recombinant. For example, the Swiss population CHS, German population DS1 and Greek population GRR have high index of association with 88.88%, 70.42% and 77.14% clonality respectively. Mating gene analysis in these populations showed skewed MAT genes ratio. Since L. pulmonaria is a heterothallic species and our analyses showed significantly unbalanced MAT gene distribution in these populations, the observed high clonality in these populations may be caused by the lack or paucity of compatible spermatia , SA6 (South Africa), EG (Spain) and CA (Ukraine) displayed low clonality but medium to high genetic diversity (H = 0.538–0.402), low LD and low index of association (Ia), suggesting past sexual history. One population MO1 (Norway), showed average LD and Ia, in-spite of low clonality and high genetic diversity. However current mating-type gene ratio in these populations was profoundly unbalanced. This may be the result of stochastic events such as genetic drift or population bottleneck resulting in deficit of a particular mating type in a population in recent past, thus still having high genetic diversity. The present scenario of biased representation of either mating type in such populations may hinder sexual reproduction in this heterothallic species. Special attention for conservation should be given to such populations. Thallus translocations aiming to restore the mating-type gene balance should be tested in future as a conservation measure.MAT gene ratio. It is sensitive to decline or even extinction because of the scarcity of compatible MAT type isolates , combined with small population size and low genetic diversity. The appropriate conservation strategy for such populations would be thallus translocations aiming not only to increase the population size but also to balance the MAT gene ratio.Population EGR showed the lowest genetic diversity (H) and low Ia, with 44.44% clonality. This population is small  and has biased MAT gene imbalance in natural populations. This is probably due to a number of factors that i) alter the genetic makeup of populations over time independently of changes in their MAT gene composition  L. pulmonaria sexual reproduction usually occurs at later life stages (15–25 years) MAT genes can be balanced or dis-balanced independent of the observed clonality of the population. Significance of MAT gene frequency is apparent only after many years of population establishment and long clonal history, which might have resulted in current disproportionate representation of mating-type isolates in the population. Availability of reliable markers for mating type identification in such species thus can be used to estimate current MAT isolate distribution in such populations. Furthermore thallus transplants of scarce mating-type isolate in a population will increase the probability of successful sexual reproduction and hence long distance spore dispersal. The main conclusion we can draw from these results is that, at least in case of natural populations of lichen-forming fungi such as Lobaria pulmonaria with long generation time and clonal history along with delayed sexual reproduction, clonality cannot be used to predict MAT imbalance.Our results show that clonality in populations cannot be used to predict MAT1-1 and MAT1-2 in the same thallus, i.e. local switch to homothallism. Concerning the first two points, we can exclude the amplification of a DNA mix from different thalli or vegetative diaspores as this would have resulted in double peaks at some or all L. pulmonaria-specific microsatellite loci. None of the samples from the three populations having both MAT1-1 and MAT1-2 showed the presence of double peaks at any of the eight fungal-specific microsatellite loci.A few samples  from three populations showed amplification with both mating-type markers. We exclude primer or sample contamination, as PCRs were double-checked in the presence of positive - (mycobiont culture DNA), negative - (photobiont DNA) and no-template controls. Since all apothecia were removed prior to DNA extraction, we can exclude the amplification of DNA from intra-thalline spores as an explanation. Therefore we envision three possible explanations to account for this observation: i) presence of conidia , or vegetative diaspores  of an individual with opposite mating type on the lobe that was used for extraction; ii) fusion of thalli with two opposite mating types during the formation of a new lichen thallus Click here for additional data file.Figure S2MAT gene ratio  and percent clonality in 27 natural populations of L. pulmonaria.Scatter plot displaying the relationship between (PDF)Click here for additional data file.Table S1Linkage disequilibrium map for the eight fungal microsatellite loci in 29 populations used in this study. Significantly linked loci pairs (p<0.05) are indicated by shaded boxes.(XLSX)Click here for additional data file.


Michelia coriacea, a critically endangered tree, has a restricted and fragmented distribution in Southeast Yunnan Province, China. The genetic diversity, genetic structure and gene flow in the three extant populations of this species were detected by 10 inter-simple sequence repeat (ISSR) markers and 11 simple sequence repeat (SSR) markers. Examination of genetic diversity revealed that the species maintained a relatively high level of genetic diversity at the species level (percentage of polymorphic bands) PPB = 96.36% from ISSRs; PPL (percentage of polymorphic loci) = 95.56% from SSRs, despite several fragmental populations. Low levels of genetic differentiation among the populations of M. coriacea were detected by Nei’s Gst = 0.187 for ISSR and Wright’s Fst = 0.090 for SSR markers, which is further confirmed by Bayesian model-based STRUCTURE and PCoA analysis that could not reveal a clear separation between populations, although YKP was differentiated to other two populations by ISSR markers. Meanwhile, AMOVA analysis also indicated that 22.84% and 13.90% of genetic variation existed among populations for ISSRs and SSRs, respectively. The high level of genetic diversity, low genetic differentiation, and the population, structure imply that the fragmented habitat and the isolated population of M. coriacea may be due to recent over-exploitation. Conservation and management of M. coriacea should concentrate on maintaining the high level of genetic variability through both in and ex-situ conservation actions. The ultimate goal of conservation biology is to maintain the evolutionary potential of species by maintaining natural levels of genetic diversity –4. To acMichelia coriacea (Magnoliaceae) is a critically endangered species, restricted to karst areas of Southeast Yunnan Province, China, bordering northern Vietnam [The recently described  Vietnam –15. This Vietnam ,17, but M. coriacea occurs as scattered individuals in evergreen woods on limestone slopes or roadsides at altitude of 1300–1600 m, and has a potential distribution range of approximate 4190 km2 [M. coriacea are over 100 years old, having been protected as symbols of “good fortune” by local villagers; however, they are surrounded and isolated by farmland and villages. In DT population, the maximum age of sampled trees was less than 30 years, and in YKP population the average tree age was 10–50 years [M. coriacea is urgently needed for planning its conservation action.Currently, 4190 km2 . These iM. coriacea, we employed ISSR and SSR markers to address the key issue: what are the levels of genetic diversity and differentiation within, and among, populations of M. coriacea? In addition, implications for conserving this critically endangered species are discussed.The use of molecular analysis as an integral component in the conservation of rare and endangered species is becoming more widely used ,11,20. TH) was estimated to be 0.226 within populations and 0.283 at the species level, and the average Shannon information index (Hpop) within populations and species levels was 0.345 and 0.436, respectively  were polymorphic . At the ectively .K was 903.1 for K = 2, 412.9 and 233.5 for K = 3 and K = 4, respectively, and <155 for all values of K higher than 4. Therefore K = 2 best represents the data. Structure analysis run with K = 2 showed a clear separation between YKP and the other two populations  was estimated at 1.086 (Nm > 1), indicating no differentiation among populations.The two-dimensional PCoA plot  shows thM. coriacea. The number of alleles per locus ranged from 2 at locus MC64 to 7 at locus MC49, with an average of 4.091 per locus (Ho) ranged from 0.172 at locus MC45 to 0.759 locus MC8 (average 0.412), and for expected heterozygosity (He) from 0.247 at locus MC45 to 0.782 at locus MC8 (average 0.505). PIC values ranged from 0.417 to 0.750, with an average value of 0.502 per locus  and that for He was 0.470 (ranging from 0.429 to 0.498). Unlike private bands detected from ISSRs, private SSR alleles were present in all three populations: DS, DT and YKP contained 2, 3, and 2 private alleles respectively (Fst) was 0.090 and the estimated level of gene flow (Nm) among populations was 2.543. AMOVA analysis showed that 13.90% of the variance existed among populations  to 91.90% (DT) with an average of 82.15%. The average value for ectively . The estulations .K was 61.2 for K = 3, 59.3 for K = 4, and <51 for all values of other K. The scores for K = 3 and K = 4 were very similar; therefore we performed runs with each K value. However, results either from K = 3 or K = 4 failed to differentiate between populations , whereas individuals from DT and YKP tended to have higher and lower scores respectively for coordinate 2 . However, all three populations overlapped for both coordinates, meaning that discrimination based on this data and PCoA analysis of this data was not possible .Within each population, certain individuals appeared highly distinct from certain others, according to the STRUCTURE analysis of SSR data. In population DS, individuals DS1 and DS2 were highly distinct from DS5, DS6, DS8, and DS14; in DT population, DT2, DT3, DT4, DT5, DT6, DT7 and DT10 were most distinct from DT9, DT12, DT14, DT17, DT19 and DT21; in YKP population YKP1, YKP2, YKP7, YKP8, YKP9, YKP10, YKP12, YKP15, YKP16, YKP17, YKP21 and YKP22 were most distinct from YKP3, YKP4, and YKP6 .M. coriacea is very narrow and fragmented, but it retains high levels of genetic diversity, mostly at the within-population level. The genetic diversity in the species overall  is higher than that of its relatives Manglietia deciduas  and Mag= 0.366; ). In gen= 0.366; ,20,25,26i.e., are naturally rare; [High genetic diversity can be maintained in rare plants that are adapted to an existence comprising small isolated populations . Howevely rare; ,20,27,28M. coriacea, as evidenced both by witness accounts of local people and the literature record. The species was first discovered in 1986 and then described as a new species in 1987 [i.e., DS, DT, YKP and a fourth, Guangnan [M. coriacea, indicating its extinction from Guangnan within the last 30 years. However, there is no evidence for range contraction during the same period for the three extant populations of M. coriacea in this study. In addition, our molecular data clearly indicated apparently high levels of gene flow for M. coriacea , which would account for low differentiation between populations (Fst = 0.090 for SSRs and Gst = 0.187 for ISSRs). It should be noted that indirect estimates of Nm values must be interpreted with caution [Habitat destruction appears to be recent for  in 1987 . InitialGuangnan . Despite caution , and thiMichelia formosana [M. coriacea also has animal-dispersed seeds, and insect pollination may also have contributed to gene flow over distance in this species. However, seed or pollen flow appears unlikely between the modern, isolated populations. Therefore, gene flow between plants at the three extant sites via seeds and/or pollen was probably extensive and relatively unhindered in the past, before the populations became isolated.Low genetic differentiation and high gene flow between populations can result from long-distance gene dispersal either by pollen or by seed . Long raormosana . M. coriM. coriacea populations. The fact that heterozygosity for SSR alleles was slightly higher in DS than the other two populations indicates that lower diversity here had not yet brought about inbreeding when the current generation was founded. However, the absence of seedlings from all three populations has prevented us from assessing whether range contraction will lead to an immediate inbreeding effect in the next generation.Populations DT  and YKP  contained relatively higher genetic diversity than did population DS . Because trees at DS are usually >100 years old, whereas those at DT and YKP are < 50 years old , range cFicus carica [M. coriacea; and (3) ISSR and SSR have different modes of inheritance . In addition, some marker loci could be under selection, or tightly linked to loci under selection, meaning that a small minority of markers for either system might not be truly neutral, although neutrality of both ISSR and SSR markers is often assumed. To test directly for these possibilities is beyond the scope of the current study, but using two types of marker, and interpreting results of each with a degree of caution, can give a reasonable assessment of the true genetic situation for the examined populations. In the current study, population YKP was clearly separable from the other populations based on ISSR, but not SSR, markers. Hence, YKP might be the most distinct population, but SSR data indicates that this conclusion must be treated with caution.It should be noted that there are some discrepancies between the results  from different markers, suggesting that the manner of polymorphism differs because of marker specificity. Contrasting patterns are commonly found when multiple markers are used to detect genetic structure, as for example in soybean , cashew s carica . Such coM. coriacea, AMOVA results shows that each of the three examined populations currently maintains a high level of within-population genetic diversity. Therefore, the endangered status of this species currently reflects a small number of extant individuals and very poor natural regeneration. Conservation measures should therefore focus on establishing large numbers of seedlings, both in and ex situ, involving many different individuals as parents, to preserve as much as possible of the existing genetic diversity in subsequent generations. Given the current lack of recruitment, establishing seedlings in situ will require management and co-operation from local communities.The maintenance of a maximal amount of genetic diversity is one of the major objectives for conserving endangered and threatened species , and theIn addition, the observed high diversity among relatively few adult trees also places a conservation value on each individual adult tree as a mini-reservoir of genetic diversity, in a way that would not apply for genetically depauperate, relict populations. Hence, conservation efforts to preserve each existing tree would also have a value in maintaining genetic diversity.Conservation strategies may differ between sites. In DS, the oldest trees are protected by villagers as good luck symbols , so encoWithin each population, however, SSR data also indicated clusters of individuals more similar to one another than they were to others. For example, individuals DS1 and DS2 were genetically highly distinct from others is DS, and likewise YKP3, YKP4 and YKP6 were genetically distinct from others in YKP. Therefore, seedlings with a higher degree of heterozygosity could be generated by crossing individuals that, according to our results, are genetically dissimilar.in-situ efforts to conserve remaining habitats need to be combined with ex-situ research on seed propagation, with a view to establishing a new generation of plants both in cultivation and the wild. Should in-situ conservation be more successful at one site than others, then seedlings from all three sites could be established at the protected site.In the short term, conservation priorities must be to preserve as many as possible of the surviving individuals. In the medium term, new generations must be ensured, and in the long term, genetic health must be maintained. Therefore, Michelia coriacea is diploid (2n = 38) [2n = 38) . The mat2n = 38) ,19,40, a2n = 38) ,14.The plant sampling and material collection were carried out in March and July 2007, and in August 2008. Prior to this, comprehensive field surveys had located only 15, 33 and 53 individuals at DS, DT and YKP, respectively . It shouTotal DNA was extracted from the dried leaves following the modified CTAB method described by Doyle . The pur2O, 0.4 μL formamide, 1.5 μL 10× PCR buffer (Mg2+ Plus), 0.25 mM of each dNTP, 0.9 U Taq polymerase, 0.6 μM primers, 0.6 μL template DNA (approximate 50 ng). Cycling conditions consisted of an initial denaturation step at 97 °C for 4 min, followed by 35 cycles of denaturation at 94 °C for 1 min, annealing temperature for 1 min (One hundred ISSR primers from the University of British Columbia (UBC) were initially screened on eight randomly selected individuals for PCR. Of these, ten primers which consistently amplified well, and produced polymorphic bands, were selected for analyzing . To ensuor 1 min , extensiet al. [2), 20–50 ng genomic DNA. The amplification profiles included initial denaturation at 94 °C for 3 min; followed by 35–40 cycles of 30 s at 94 °C, 30 s at 54–68 °C and 1 min at 72 °C , effective allele number per locus (Ae), Nei’s gene diversity (H) and Shannon index of diversity (Hpop) [Amplified ISSR fragments showing consistent amplification were scored manually as present (1) or absent (0) for each sample to form a binary matrix. The POPGENE program Version 1.31  was emplNm = (1 − Gst)/4Gst, where Nm is the number of migrants per generation [et al. [K), we ran STRUCTURE with K varying from 1 to 10, with five runs for each K value. Previous studies have found that, in many cases, the posterior probability for a given K increases slightly, even after the real K is reached [ad hoc statistic, ΔK, to determine the true value of K [Genetic differentiation among populations was analyzed using Nei’s gene diversity statistics . The genneration . To infeneration , followi [et al. . We adop reached . Therefolue of K . Our parlue of K .The AMOVA  was performed through Arlequin 3.0 program  to descrAo), effective allele number per locus (Ae), observed heterozygosity (Ho), expected heterozygosity (He), and the number of private loci, i.e., those found only in a single population. The same program was used to test for deviations from Hardy-Weinberg equilibrium (HWE) and pair-wise linkage disequilibrium (LD). To measure the marker polymorphism, the polymorphism information content (PIC) for each SSR Marker was calculated according to the formula PIC = 1 − ∑Pi2, where Pi is the frequency of the allele for each SSR marker locus [To estimate overall genetic diversity, the following measures were calculated for each SSR primer locus and each population using POPGENE program Version 1.31 : observeer locus .Fst was estimated by a weighted analysis of variance with the GENEPOP 4.0 program [Nm) among populations was estimated by an indirect genetic differentiation method based on Fst value, Nm = (1 − Fst)/4Fst. Partitioning of genetic variation within and among populations was further performed by analysis of molecular variance using the Arlequin 3.0 program [Wright’s  program . In addi program . For theMichelia coriacea. Furthermore, Bayesian model-based STRUCTURE and PCoA analysis could not reveal a clear separation between populations, although YKP was differentiated from the other two populations by ISSR markers. We therefore presume that the fragmented habitat and the isolated populations of M. coriacea may be due to very recent contraction of its range. Based on this data, conservation strategies for this critically endangered species were also proposed.In summary, our results from both ISSR and SSR markers show that a relatively high level of genetic diversity and low levels of genetic differentiation among populations exist in the critically endangered plant