Rosa sericea complex distributed in alpine regions of the temperate and subtropical zones in eastern Asia. We used three chloroplast (cp) DNA fragments  approximately 3,100 bp and nuclear microsatellite (nSSR) on eight sites to determine whether cold tolerant plants experienced expansion during the Pleistocene. The neutral test and mismatch distribution analysis (MDA) indicated that whole populations and major lineages of the Qinghai-Tibet Plateau (QTP) underwent expansion during the middle to late Pleistocene. Environmental niche modeling (ENM) indicates more suitable habitats during the Last Glacial Maximum (LGM) than at present. We concluded that the demographic history of R. sericea, which diverged in the middle Pleistocene, was mostly affected by climatic oscillations instead of by geographical barriers. The low genetic divergence, as well as the weak phylogenetic structure in the R. sericea complex both support treating this complex as a single taxon.While most temperate plants probably underwent glacial constriction to refugia and interglacial expansion, another type of interglacial refugia might have existed to maintain alpine plants during warm periods. To test this hypothesis, we applied phylogeographic methods to 763 individuals (62 populations) which belong to 7 taxonomically difficult species of the  Geographic changes, such as the uplifting of the Qinghai-Tibet Plateau and Rocky mountain systems, result in habitat fragmentation that forms barriers to gene flow, further leading to genetic diversification, accelerated variation and even speciation1234Two theories, named the pioneer model and the phalanx model, have been advanced to address the consequences of population expansion on genetic legacy791011Dryas integrifoliaPicea glaucaPinus kwangtungensis17Lagopus muta)In recent years, phylogeographic research has shown that glaciation may not only force population contractions in plants, but also promote expansion or persistence of cold adapted or cold tolerant species. For example, studies in Although recent phylogeographic studies have been conducted on various plant groups and many geographic regions21222324Rosa sericea complex comprises a group of shrubs in section Pimpinellifoliae DC. Ser. Sericeae (Crep.) Yu et Ku27R. sericea complex is a monophyletic group most closely related to R. hugonis Hemsl. and contains seven species: R. sericea, R. omeiensis, R. zhongdianensis, R. sikangensis, R. morrisonensis, R. taronensis, R. mairei or infraspecific taxaR. sericea complex has a broader eastward distribution. The complex may have had a continuous distribution in southeastern China in the past, and is a good candidate for a demographic study of cold adapted species.The R. sericea complex. In particular, we wanted to answer these questions: 1) Did range expansion exist in the demographic history of R. sericea? If it did, which factors  contributed to this expansion? 2) How were southwestern China – Taiwan disjunctions formed? Did continuous distributions once exist in eastern China? If so, then which part served as a bridge between the two areas? In addition, we also aimed to determine suitable species delimitations for the R. sericea complex.In this study, we used DNA sequences and a phylogeographic approach as well as niche modeling to determine the mechanism of range expansion and disjunction within the The three cpDNA-IGS surveyed across 691 individuals (belonging to 62 populations) were aligned. Of the 3057 bp, nucleotide substitutions occurred at 32 sites and 10 indels (from 1 to 24 bp) were present . After iπ) and haplotype (Hd) diversity across all populations was 0.00056 and 0.815. The QTP group exhibited the highest haplotype diversity (Hd = 0.779) while the largest group in Hengduan Mountains (region II in Hd = 0.522). Genetic diversity calculated for all populations (HT = 0.823) was higher than the average intra-population diversity (HS = 0.267). Relative high levels of genetic differentiation were observed . Permutation tests showed that NST was greater than GST in all groups and in the metapopulation  revealed a total of 254 alleles across the eight nSSR loci surveyed, along with high mean per-locus estimates of allele and gene diversity  was significant at each locus (P< 0.05) and averaged 0.298 across all loci , STRUCTURE yielded the highest likelihood when samples were clustered into three groups . The pre-defined taxa and the recognized clusters in detected . Three mdetected . Contactdetected .FCT increased with partitioning of species into 2 to 20 groups , and a large level of variation was due to differentiations between populations within geographic regions  and among geographical regions    . The AMO < 0.05) .Rosa sericea complex presented a radiation-shaped relationship, with a weak phylogeographic pattern (The tree topologies generated by Neighbor-joining (NJ) and Bayesian inference (BI) were congruent in terms of major clades, thus only the NJ tree is presented in Figure S2. Overall, 37 chlorotypes were clustered into three major lineages: 1) northern Hengduan Mountains and QTP, 2) southern Hengduan Mountains and Yungui Plateau, and 3) eastern China, named CPA to CPC, respectively . This wa pattern . We sepa−9 s/s/y, the divergence time for the Rosa sericea complex was estimated to be 0.482 Ma, with a 95% confidence interval (95% CI) of 0.281–0.722 Ma  and 0.352 Ma (95% CI 0.175–0.469 Ma), also espectively. Overall, all our results indicate a mid-Pleistocene divergence of populations within this complex.Assuming substitution rates of 2.0 × 100.722 Ma . We usedRosa sericea complex using a sudden demographic expansion model. The results showed that the whole populations and CPA lineage were unimodal, while lineages CPB and CPC were bimodal and multimodal, respectively. Accordingly, the two statistics –SSD and HRag – that assessed the fitness of the model only rejected the expansion model in lineage CPC. In neutrality tests, the whole group, lineage CPA and CPB showed negative values in Tajima’s D and Fu’s Fs test, which indicated population expansion. Only the whole lineage, however, showed significant negative value for Fu’s Fs , based on 10 replicates]. The model predicted suitable habitat for the species that was mostly coincident with its natural geographic distribution  except tf Taiwan . ObviousCercidiphyllumRosa sericea complex comprises no distinct geographic lineages as inferred from the chlorotype network and phylogenetic analysis Generally, if deep divergence occurred through paleogeological events, then a phylogeographic study should detect strong geographic clustering that coincides with known geographic boundaries. This scenario appears in some relict plants or relatively old species , and its major lineages diversified not earlier than the middle of Pleistocene  predicts rapid colonization of an area during glacial periods and fragmentation during interglacials accompanied by genetic drift and range expansions from interglacial refugia over short distances involving limited gene flow during glacial periodsAlthough the phalanx model may best explain the overall distributional patterns of the Rosa sericea complex is disjunct between southwest China and Taiwan   (R. sericea.We attempted to use our phylogenetic and population genetic data to delimit species within the  < 0.05) , compare 0.0001) . It is cR. sericea complex is comprised of a single species that underwent rapid geographic radiation during the LGM. The phylogeographic structure of modern populations is consistent with a phalanx model-type expansion during the LGM and subsequent pioneer model-type population histories. Our work supports the recognition of a single R. sericea species.In summary, our results show that the cold-tolerant Rosa sericea complex during 2010–2011 . Based on the results of amplify efficiency and amount of polymorphism, three IGS, ndhJ-trnFtrnL-trnFndhF-rpl32Rosa sericea were deposited in GenBank under accession numbers KF850715 – KF850751, KF850756 – KF850792, KF851050 – KF851086, and for three chloroplast sequences for four outgroup accessions, i.e. R. hugonis (2 accessions), R. banksiae and R. cymosa .Total genomic DNA was extracted from the dried leaf tissue by a modified cetyltrimethyl ammonium bromide (CTAB) methodRosaAll DNA samples were genotyped at eight nSSR loci using primers and amplification protocols developed for To verify the existence of significant heterogeneity in the phylogenetic signal among the three cpDNA fragments, we carried out a partition-homogeneity testπ) and haplotype (h) diversityNST) and unordered (GST) chlorotypes, both in the overall cpDNA data set and within population groups, using PERMUT1.0. The result of this program and the significant test by 1000 permutation were then employed to examine if NST > GST, which is a test for the presence of phylogeographic structure. The phylogenetic relationships among chlorotypes were investigated using the statistical parsimony procedure for phylogenetic network estimations implemented in TCS 1.21, with a 95% criterion for a parsimonious connection.Sequence variation was analyzed using MEGA 4, whereas nucleotide (FCT) as a result of differences between groups of populations. We set the number of initial condition to 100 with K = 2–20. To verify its consistency, we ran the analysis five times for each K value with 1000 independent annealing processes. Afterwards, analyses of the molecular variance (AMOVA)et al.Flora of ChinaTo identify population groups and genetic barriers, a spatial analysis of molecular variance (SAMOVA) was performed using SAMOVA 1.0. Based on a simulated annealing procedure, the SAMOVA algorithm iteratively seeks the composition of a user-defined number of groups (K) of geographically adjacent populations that maximizes the proportion of total genetic variance , allelic richness (RS), and gene diversity (HS). Differentiation between populations  was computed using FSTR. hugonis, R. cymosa and R. banksiae, as outgroups based on the framework of Rosa phylogenyThe phylogeny reconstruction based on haplotype was performed in both Neighbor-joining (NJ) and Bayesian inference (BI) using four accessions of three species, RosaRosa sericea is unknown, three different cpDNA substitution rates for angiosperms [the slowest (1.1 × 10−9 substitution/site/year), fastest (2.9 × 10−9 s/s/y) and mean (2.0 × 10−9 s/s/y)62Using the same matrix as above, tree topologies and node ages were determined in BEAST version 1.61 using a GTR+I substitution model, selected by jModelTest, and an uncorrelated lognormal relaxed clockSSD) between observed and expected mismatch distributions and Harpending’s raggedness index (HRag)For a single expanding subclade identified (see the Results section), demographic history was initially explored using mismatch distribution analysis (MDA), which represented the frequency distribution of pair-wise nucleotide differences among all chlorotypes in the population(s)Rosa sericea complex. First, the expansion time was estimated directly from the mismatch distribution with the statistic τ (tau) and translated into absolute time in years (t), using the equation T = τ/2u , where u is the neutral mutation rate for the entire sequence per generation and is calculated as u = μkg, where μ is the substitution rate in substitutions per site yr−1 (s/s/y), k is the average sequence length of the cpDNA region under study (k = 2974 in this study), and g is the generation time in yr . The substitution rate range of the three combined cpDNA-IGS regions was assumed as minimum and maximum mutation rates of 1.1 × 10−9 s/s/y and 2.9 × 10−9 s/s/y62R. sericea complex were inferred from Bayesian skyline plot (BSP) analyses using BEAST 1.6.1D testFS testIn such a context, two methods were used to estimate the time of expansion of the R. sericea complex during cold periods, species distribution models for the R. sericea complex were generated using MAXENT 3.3.3k. MAXENT is a program for maximum entropy modeling of the geographical distributions of species; it combines presence only data with ecological climatic layers to predict species occurrence in areas where data points are unavailable. We used MAXENT to predict species occurrence under both present day and LGM conditions. Although potentially useful for this study, we avoided making predictions for the last interglacial phase (about 135 000–115 000BP), because of a lack of detailed bioclimatic data for this period. Information on the geographic distribution of the R. sericea complex was based on present collection and herbarium specimens from the herbaria A, BM, CDBI, E, HNWP, IBSC, K, KATH, KUN, PE, TUCH and WUK. In total, 317 presence records of members of the R. sericea complex were obtained from throughout the species range, after removal of duplicate records within each pixel . Six bioclimatic variables  at 2.5 arc-min resolution were used to model the species’ niche: (i) annual mean temperature, (ii) mean temperature of the warmest quarter, (iii) mean temperature of the coldest quarter, (iv) annual precipitation, (v) precipitation of the wettest quarter and (vi) precipitation of the driest quarter. Model validation was performed using default settings with 100 replicates of cross-validation procedures with 25% of the data used for model testing. The area under the ROC curve (AUC) was then calculated for each run, which is an indicator of the accuracy of the model predictionTo reconstruct the past distribution of the http://www.worldclim.org). LGM paleo climate layers in 2.5 arc-min resolution were then prepared from these data following Sakaguchi et al.The established model was then projected onto the reconstructed climatic conditions during the LGM simulated by Community Climate System Model 3.0How to cite this article: Gao, Y.-D. et al. Pleistocene glaciations, demographic expansion and subsequent isolation promoted morphological heterogeneity: A phylogeographic study of the alpine Rosa sericea complex (Rosaceae). Sci. Rep.5, 11698; doi: 10.1038/srep11698 (2015).


Creteuchiloglanis macropterus, is widely distributed in the main stem and tributaries of the Nujiang River basin. Here, we analyzed IIB restriction-site-associated DNA (2b-RAD) sequences and mitochondrial DNA sequences, to assess the genomic signature of adaptation by detecting and estimating the degree of genetic differentiation among ten Creteuchiloglanis macropterus populations from the Nujiang River. The analyses revealed significant population differentiation among the up-tributaries, main stem, mid-tributary and low-tributary. Annotation of contigs containing outlier SNPs revealed that the candidate genes showed significant enrichment in several important biological process terms between up-tributaries and low-tributary, and exhibited prominent enrichment in the term macromolecular metabolic process between all tributaries and the main stem. Population dynamics analyses indicated that the Late Pleistocene glaciations strongly influenced the demographic history of C. macropterus. Our results provide strong evidence for the utility of RAD-seq in population genetics studies, and our generated SNP resource should provide a valuable tool for population genomics studies of C. macropterus in the future.Advances in genome scanning using high-throughput sequencing technologies has led to a revolution in studies of non-model organisms. The glyptosternoid fish  However, when species have relatively narrow distributions, the detection of population differences is more difficult through classical methods, owing to insufficient data. In such case, classical methods often can-not meet the needs of researchers, who expect to detect intrinsic differences in population genetic studies. Recently, the rapid development of next-generation sequencing (NGS) has facilitated the identification of novel population genetic markers at an unprecedented scale, even in non-model organisms4. Thus, genome-wide patterns of diversity and differentiation can be explored without regard to whether the markers used are anonymous. Moreover, when thousands of loci across the genome are screened through high-throughput approaches, differentiation is generally detected even when only a small number of populations are considered8. These advances in genome scanning have allowed population genetics studies of non-model organisms9 and have improved understanding of evolution in ways that would not have been possible through classical methods10. Restriction-site-associated DNA sequencing (RAD-seq) has emerged as a powerful tool for genetic mapping and analyses of quantitative trait loci4, adaptation12 and phylogeography13. This method reduces the complexity of the genome, is less expensive and yields many more genetic markers than previous methods14.Population genetic studies are based on relatively wide distribution range. Such studies are helpful in detecting more significant differences to study their phylogeny and biogeography though they tend to focus on a low number of genetic markers17. However, these studies have typically been limited to a few loci (rarely more than five loci) investigated using mitochondrial or nuclear markers. Most previous genome-wide studies have exclusively focused on endothermic terrestrial vertebrates22. Before the present study, the only similar studies conducted by Ma23 and Yang24, who performed transcriptome analyses detecting genomic signatures of adaptation to high altitude in glyptosternoid fish and schizothoracine fish. However, the identified adaptive signatures were based on the comparison with different species that inhabits a low altitude environment, rather than on within-species comparisons among different geographical populations.To date, several population genetic studies of fish taxa have been conducted in the Nujiang RiverCreteuchiloglanis macropterus, a glyptosternoid fish  that inhabits in cold waters, is mainly distributed upstream of the Nujiang River, which originates in the Tibetan Plateau and flows into the Southwest Mountainous Region of China26. This region functions as a transition zone where the altitude decreases to 1,000–2,000 m in Yunnan Province, China. Documenting the genetic mechanisms across environmental gradients in this area, might provide new insights into the evolutionary process of adaptation to the extreme conditions of the Tibetan Plateau. Glyptosternoid fish inhabit shallow, rocky rivers with moderate currents, and do not migrate long-distances. They tend to remain attatching to rocks in the rapidly flowing water unless they encounter sudden danger28. These special behaviors may lead to population differentiation. Therefore, we sought to conduct a population genomic analysis determine whether genomic signatures of adaptation exist among geographical populations of C. macropterus. Powerful evidence regarding the mechanism of adaptation to the Tibetan Plateau might be provided through the analysis of C. macropterus population genomics along gradient altitudes in the Nujiang River.30. However, no study has investigated the population genetics and demographic history of C. macropterus. In our study, the population genetics of C. macropterus was assessed by using the IIB restriction-site-associated DNA (2b-RAD)31 tag technique. The objectives of this study were to (i) analyze the population genetic structure of C. macropterus across the Nujiang River; (ii) test whether adaptive signatures exist in the narrowly distributed populations, and, if so, identify the genomic regions of adaption by investigating genome-wide genetic variation and differentiation in ten populations; and (iii) explore the demographic history of C. macropterus and test whether the population dynamics has been influenced by climatic oscillations during the Quaternary.During the middle and late Pleistocene, glacial and interglacial periods caused repeated changes in the distributions of endemic fish on the Tibetan PlateauC. macropterus fish were sampled in the upper stream of the Nujiang River in Yunnan Province  yielded two main groups Fig. : a tribuSD = 1921,313, range 247–13456,369). Of these reads, an average of 79%  carried the complete recognition site of BcgI enzyme. Raw sequencing data are available at BioProject SRP079503.A total of 697.2 million reads were derived from three pooled libraries that included 196 individuals. The mean number of reads per individuals was 3,557,185 , with the mean coverage per stacks ranging from 21 to 161X per individual. After merging of all the stacks from each individual, the final catalog contained 222,116 loci, of which 148,909 contained at least one SNP . Four SNP datasets  revealed Δ= 3 Fig.  to have = 3 Fig. . Most in= 3 Fig.  using thST = 0.82 (P < 0.001), indicating a very high level of genetic structure. A signal for isolation by distance was detected  among the ten populations. The results of the AMOVA and isolation by distance test revealed a pronounced genetic structure among the clusters, and the clusters followed a geographical pattern. In addition, the differentiation among the populations exhibited a closely correlation with geographical distance.The hierarchical AMOVA among the four clusters  resulted in ΦFST) among the 12,125 SNPs of dataset3, which were detected in the six tributary populations  and almost all were positive in LT   in the nr database, and 22 had well-defined protein annotations  Fig. . And to P < 0.05, Fig. P < 0.05, Fig. Compared with genes with neutral SNPs, adaptive candidate genes showed a broad range of gene ontology (GO) annotations Fig. . The enrAll of the genes involved in the above processes might have been important in promoting differentiation among the groups.33 which included 1,351 putative hypoxia-related genes. Finally, we identified four genes  that were consistent with the list, and five genes  that might be homologous or analogues in contrast with the genes in the list  expression occurs in chronic hypoxic rat models of pulmonary arterial hypertension (PAH)35. DNAJB5 (dnaJ homolog subfamily B member 5-like) belongs to the DNAJ family, which is a heat shock protein 40 (Hsp40) family protein, their expression is significantly up-regulated under stresses of hypoxia37. These positive selection genes indicate that populations in UT are more adaptive with hypoxia than populations in LT.To identify genes that might be in correlation with the adaption to high altitude, we compared our candidate genes to an a priori list proposed38 to explore the demographic history of C. macropterus populations from the main stem and tributaries, respectively. It seems that the population demography has a correlation with the uplifting of the Tibetan Plateau40 , which started at approximately 0.075 and continued until 0.01 Ma45, seemed have no evident effect on C. macropterus populations.We estimated changes in effective population size by using a Pairwise Sequentially Markovian Coalescen (PSMC) methodu40 Fig. , Fig. S6u40 Fig.  during Keau Fig. . All popBayesian skyline plot (BSP) analyses also indicated that all of the populations experienced a decline during the glacial period Fig. . PopulatC. macropterus by using the RAD-seq approach. The key finding of this study was the high degree of genetic differentiation due to divergent selection among C. macropterus populations in different regions of the Nujiang River. This finding suggest that adaptive differentiations were apparent in local populations. Annotation of the outliers confirmed the existence of potential adaptive genomic regions. Furthermore, demographic analyses of the ten populations confirmed that the C. macropterus populations were influenced by Quaternary climatic oscillations. We discuss these findings and compare them with those of earlier studies on the genetic mechanisms of adaptation in the environment of the Tibetan Plateau below.In the present study, we identified a large number of SNPs for C. macropterus populations was fairly high  on a genome-wide scale , a result consistent with the mtDNA results   in dataset 2. In addition, despite extremely low FST values (−0.001) . These findings demonstrate the power and potential of RAD tags10 to detect subtle differentiations in population genetics16.Local adaptation analyses of value 0.03 , we found significant difference in two biological processes between the outliers and neutral SNP loci Fig. . These pC. macropterus. In the condition of lacking genome, lots number of loci would be filtered even they locate in non-coding region of positive selected genes. And in consideration of the low mapping ratio, we combined empirical method, Bayescan and Bayenv to detect outlier loci as much as possible. Therefore, the number of outliers were enormous which might be redundant. Our results suggest that the genetic structure of these populations is also driven by variation in environment conditions rather than by geographic alone, however, we only detected outlier loci that appeared to be driven by selection associated with spatial heterogeneity in altitude. The associations with environmental parameters and footprints of selection may be derived from some unmeasured factor 48, which might omit some outlier loci. It might be improved by measuring more environmental factors  of sample localities. C. macropterus is mainly distributed upstream  and scarce in low-elevation location , and we sampled the outlier loci with high value of overall π in the empirical method, these π value of loci might only exist in UT but not LT which would lead to omit some loci, it would be improved if we could increase the number of individuals in LT. In addition, when it exists the recent admixture between populations, some shared loci that should have high FST would be filtered because of their low values of FST.The finding that only small percentages of the outliers (10.45% and 8.47%) were mapped to the assembled transcriptome, may be due to the few BcgI enzyme recognition sites that occurred in the protein-coding regions of the DNA in 42. This uplift and the associated climate changes are considered to be the most important factors influencing the current spatial distributions, genetic diversities and population structures of local species51. In our study, we found that the effective population sizes of all C. macropterus populations might underwent decreases caused by uplift of The Tibetan Plateau. The patterns of coincidence population declines in these population might be a response of a common background following the intense uplift of the Plateau that may have been unfavorable to them52. It is strange to detected that all populations were maintained at a fairly low level while LMD has a relatively higher population size, which might be involved with the more feasible environment constructed by uplift of the Tibetan Plateau in LMD. Moreover, in contrast to the increase of MS effective population size after the period of the LGM, all tributaries were retaining a low level of effective population size after Kunhuang movement. In consideration of individuals of ML were sample in high mountain, the reason might lie in that C. macropterus migrated to low-altitude with warmer environment. Most temperate species appear to have dispersed to lower latitudes in response to Pleistocene climatic oscillations54.The demography of species around the Tibetan Plateau has drawn mach attention because of the region’s unique paleogeographical history and paleoclimatic oscillations. The Tibetan Plateau uplifted approximately 3000 m and experienced at least four major glaciations during the PleistoceneC. macropterus in the Nujinag river were mainly effected by the LGM rather than the LG. The reason might be the habitat of C. macropterus might not have been covered by ice during the LG. C. macropterus in our study dwell at 815–2048 m, while the snowline was 1800–3200 m56. However, the BSP analysis of the mtDNA dataset showed that all of populations along the Nujiang basin were suppressed during the time that spanned the LGM and LG. The reason for these results might be that the use of a limited number of loci decreased the resolution of the results57.In summary, all populations showed low effective population size or a decreasing tendency in the LGM, which have not detected in the LG, it indicated that Creteuchiloglanis macropterus) populations. Genomic heterogeneity at the level of genetic differentiation appears to be associated with adaptation to the high-elevation environment, we first detected the hypoxia adaption of fish in population genomic scale. This study also demonstrates that RAD-seq is a powerful tool for obtaining more genome information, it can provide much more genome information than is obtainable through classical methods. The SNP resources generated in this study might be valuable for future population genetics and genomics studies of C. macropterus. In addition, this study provides the first evidence that the population genetics and dynamics of C. macropterus were profoundly influenced by Quaternary climatic oscillations.The present study combined analyses of mtDNA and RAD-seq datasets, and provides the first exhaustive survey of genome-wide genetic variability and differentiation in Chinese glyptosternoid , Denglongba (DLB), Denggeng (DG), and Liuku (LK) populations were sampled in the main stem of the Nujiang River. The Qiunatong (QNT), Geza (GZ), Changwa (CW), Xiyuegu (XYG), Lumadeng (LMD), and Mangliu (ML) populations were sampled in the tributaries. Caudal fin or muscle tissue from each fish was preserved in 95% ethanol for DNA extraction. Then, the samples were maintained in 10% methanol for later identification and were deposited in the Freshwater Fish Museum of the Institute of Hydrobiology at the Chinese Academy of Sciences. Total genomic DNA was extracted using a standard procedure .Individual nce Fig. , Table 158 for COI, L14724 and H1591559 for Cytb, GEDL200 and GEDH86060 for CR, and ND5-F(GCATCCTGATACATACACTCCGA) and ND5-R (TGTTTGGAGGCTGTATTGGCT) for ND5. Each PCR tube contained approximately 100 ng of template DNA, 1 µL of each primer (10 pmol/µL), 3 µL of 10 × reaction buffer, 1.5 µL of dNTPs (2.5 mmol/L each), and 2.0 U of Taq DNA polymerase in a total volume of 30 µL. The PCR conditions were as follows: initial denaturation at 94 °C for 3 min, followed by 30 cycles of at 94 °C for 1 min and annealing at 58–64 °C for 1 min, followed by a final extension at 72 °C for 5 min. Finally, we obtained COI (accession number KY232646–KY232771), Cytb (accession number KY232772–KY232889), ND5 (accession number KY232890–KY233000) and CR (accession number KY233001–KY23319) sequences with a joint alignment length of 2,983 bp  in 102 C. macropterus individuals.Four regions of mitochondrial DNA sequence (cytochromec oxidase submit I (COI), cytochrome b (Cytb), control region (CR) and NADH dehydrogenase submit 5 (ND5)) were used as a mitochondrial concatenated data set to construct a phylogeny for verifying the genetic structure that was detected by RAD-seq. Polymerase chain reaction (PCR) amplifications were carried out using the following primers: FishF1 and FishR161. The type IIB restriction enzyme chosen for the experiment was BcgI (New England BioLabs), which excises a 36-bp fragment around the recognition site, cleaves genomic DNA upstream and downstream of its target site, and generates tags of uniform length that are ideally suited for sequencing on existing NGS platforms. Digestion reactions were performed in a total volume of 6 µL, using 4 µL of intact, high-quality genomic DNA sample, each containing a total of 0.25 to 1 µg of DNA in 4 µL, 1 unit of BcgI, 0.6 µL of 10 × NEBuffer 3 and 0.4 µL of 150 µM SAM. The reaction was inactivated at 65 °C for 20 min, and the reaction tubes were then incubated at 37 °C for 2 h. Then, 1 µL of the digested DNA was loaded to verify the effectiveness of the digest. Subsequently, 20 µL of ligation master mix containing 0.5 µL 10 mM ATP, 800 U of T4 DNA ligase (New England BioLabs), 2 µL of 10 × T4 ligase buffer, 1 µL of each adaptor (5 µM) and 14.5 µL nuclease-free water (NFW) was added to the remaining 5 µL of digested DNA. Each reaction tube was incubated at 16 °C for at least 2 h and then stored at 4 °C overnight.Three 2b-RAD sequencing libraries were prepared at the Institute of Hydrobiology at the Chinese Academy of Sciences , as described in WangThe ligation products (16 µL) were amplified in 80 µL PCRs, each containing 1.6 µL of each primer , 10 µL of 10 × dNTP, 1.2 mM Mpx primer, 0.8 µL (1.6 U) of Phusion high-fidelity DNA polymerase (New England BioLabs), 16 µL of 5 × HF buffer and 20.8 µL of NFW. The reaction protocol consisted of 17 cycles at 98 °C for 20 s, 65 °C for 2 min and 72 °C for 30 s, followed by a final extension of 72 °C for 10 min. Each individual reaction had a unique barcode to distinguish itself. The final genomic libraries were selected for 170 bp by gel extraction (Qiagen). A total of three multiplexed libraries, including 196 samples, were sequenced with 37, 86 and 72 samples in the three libraries. The resulting fragments were sequenced on an Illumina HiSeq 2500.de novo analysis, custom Perl scripts were used to obtain the reads with complete BcgI recognition sequences (https://github.com/z0on/2bRAD_denovo). First, we discarded individuals with obviously fewer reads than the others. Then, the reads were trimmed to a length of 32 bp, and quality filtering (Phred score more than 20 for at least 93% of the bases in the read) was performed using the FASTX TOOLKIT. The remaining reads were then analyzed in the software pipeline STACKS v.1.2362.Before the de novo assembly in ustacks (STACKS pipeline)62. We set a minimum stack size of five reads (m = 5) and excluded stacks with coverage lower than this threshold. Invariant stacks were then compiled into sample-specific loci if they differed by fewer than two nucleotides (M = 2). Then, sample-specific loci were assembled into homologous loci if they differed by fewer than three nucleotides (n = 3) between samples, by using cstacks63. Finally, the populations program64 was used to obtain the loci that satisfied the following conditions: (i) the loci were present in at least 80% of the individuals from each population, and (ii) at least five RAD tags were present per allele at each locus. To limit false single-nucleotide polymorphism (SNP) identification, we removed SNPs with a global minor allele frequency <0.0565. Furthermore, potential homologs were excluded by removal of markers that exhibited heterozygosity values >0.566. To avoid linkage bias for the SNP calling, only the first SNP per locus was included in the final analysis71. To maximize the detection of adaptive loci, a series of SNP datasets . In general, genomic regions exhibiting high FST values may represent signatures of local adaptation or islands of differentiation resistant to gene flow73. Loci with high FST and high diversity values were selected by two-model-contrasting analysis. Furthermore, potential adaptive loci were detected by using additional dataset, and these loci were eventually combined and contrasted with the neutral loci detected in Bayescan which was used to remove the potential adaptive loci.All of the reads were pooled and used for Pseudexostoma brachysoma mitochondrial genome were used as out-group sequences because of the close phylogenetic relationship of this species with C. macropterus47. The homogeneity test in PAUP* 4.0b1074 was conducted on the four concatenated gene haplotype sequences, and no significant heterogeneity was detected . Models of nucleotide substitution were tested using jModelTest 2.076, and a corrected Akaike information criterion (AICc) was used to determine the best-fit model. Bayesian trees were constructed with MRBAYES v.3.1.277 to generate Bayesian posterior probabilities for phylogenetic inferences. The closest general model was used when specific models selected by jModeltest were not implemented in MrBayes. Two independent Markov chain Monte Carlo (MCMC) runs, each with six chains for 5 million generations, sampled every 1000 steps, were performed. We confirmed convergence to a stationary distribution by determining whether the deviation of split frequencies was below 0.01 between the two independent runs, and we discarded the first 1,250 trees as burn-in.COI, Cytb, CR and ND5 partial sequences from the 78 to estimate the population-genetic clusters by using dataset 1 with the hash term that all of the the SNP loci should be present in all of the ten populations. For this analysis, we assumed an admixture model and correlated allele frequencies with no prior information. We used a burn-in of 10,000, followed by another 10,000 MCMC steps. Then, we estimated the most likely number of clusters by estimating the maximum likelihood for K = 1–10, and ran each cluster option five times. The optimal K value was selected on the basis of the L(K) values; the individual assignment patterns using STRUCTURE HARVESTER Ver. 0.6.9379, which assesses the likelihood value at each K and selects the optimal value by using the ad hoc statistic ΔK; and the rate of change in the log probability of data between successive K values80. To visualize the multi-locus patterns of population differentiation, a PCO plot was generated using the R package labdsv81, on the basis of average FST values.We used STRUCTURE v.2.3.382, and NJ. Slatkin83 pairwise FST values were also estimated in Arlequin. The patterns of population differentiation were compared using a Mantel’ test84. Tests for isolation by distance were conducted using a Mantel’ test with linearized FST values [FST/(1 − FST)] and geographic distances separating sampling locations. Significance was tested after 1,000 permutations. Network 5.085 was used to reconstruct the phylogenetic relationships among the haplotypes with a median-joining network method.Hierarchical AMOVA was conducted among clusters of sampling localities identified by STRUCTURE and PCO using Arlequin v.3.586.File conversion of the STACKS output files to the desired formats was carried out with PGDSPIDER Ver.2.0.8.087. We calculated population fixation statistics (FST) and the nucleotide diversity (π) between UT and LT for each SNP loci in dataset 4. Similarly to the empirical outlier detection approach89, the putative selection targets were extracted on the basis of their representation in the top 5% of the odds ratio for both FST and π. In addition, Bayescan 2.190 was used to estimate the posterior probability that a given locus was affected by selection using dataset 3. Bayescan was run under the default settings, and the false discovery rate (FDR) was set at 0.05. In addition, we used the same method with dataset 1 and dataset 5 to identify genomic regions that showing marked genetic differentiation between the tributaries and MS.To identify genomic regions that showing marked genetic differentiation among the tributaries, we constructed dataset 4, in which the tributaries were divided into three groups . The allele frequencies of the variable sites were used to identify regions that were potentially affected by long-term selection, by using two complementary approaches91, to filter the SNP dataset 2 identified by STACKS, wherein all of the populations were taken into consideration; and any loci present in at least one population included in this dataset. Thus, we were able to maximize the detection of the SNP loci that were correlated with altitude. The altitude data were standardized by subtracting the mean and dividing by the standard deviation of the parameters across all of the sites in dataset 4.To investigate whether allele frequencies at any individual SNP locus were correlated with altitude and to prioritize the detect on of such SNP loci, a Bayesian approach was implemented in BAYENVC. macropterus reference transcriptome (unpublished) was used to annotate the protein-coding outlier loci. For each locus, a strict majority consensus sequence was generated and aligned to the de novo transcriptome using Bowtie 1.1.292 allowing up to one mismatch. Putative gene identities were determined by using homology searches with BLASTX against the nr database from the NCBI website; in the case of multiple hits, the best match was chosen. Then, functional annotation of these genes performed using the BLAST2GO suite (http://www.blast2go.com/b2ghome)93. Only those ontologies with an E-value < 1E-6, an annotation cut-off >55 and a GO weight >5 were considered for annotation.We used the empirical approach (dataset 5) and Bayescan (dataset 1) respectively to identify the differentiated genomic regions between tributaries and main stem populations. And to identify the putative functions of the outlier loci, a 38. Using the consensus loci created by the cstacks program, we concatenated the tags of each locus as the reference genome. Briefly, the RAD tags of each sample were aligned to the reference genome using BWA94 (version 0.7.3a-r367) with the default settings. The consensus sequences were called using SAMtools96 (version: 1.3.1). The ‘fq2psmcfa’ tool was used to create the input file for the PSMC modeling, with the option –q20. The resulting files were used as the input for the PSMC estimates using ‘psmc’ with the options −N25 −t15 −r5 −p’4 + 25*2 + 4 + 6’. The reconstructed histories of each population were plotted using ‘psmc_plot.pl’ with generation time (-g 4) and mutation rate (-u 2.5e-08). The mutation rate was selected according to the rates described in previous studies in medaka97 and channel catfish98. Historical population dynamics were also estimated from the mtDNA dataset by using coalescent-based Bayesian skyline plots (BSPs)99. BSPs were generated using BEAST 2.3.2100 to describe the demographic history by assessing the time variation of effective population size, and the results of the BSPs were visualized in Tracer 1.5.Demographic history was reconstructed on the basis of a Hidden Markov Model (HMM) approach using PSMCsupplementary informationDataset 1Dataset 2


Genetic diversity and variation among 11 populations of Chinese fir from Fujian province and Taiwan were assessed using inter-simple sequence repeat (ISSR) markers to reveal the evolutionary relationship in their distribution range in this report. Analysis of genetic parameters of the different populations showed that populations in Fujian province exhibited a greater level of genetic diversity than did the populations in Taiwan. Compared to Taiwan populations, significant limited gene flow were observed among Fujian populations. An UPGMA cluster analysis showed that the most individuals of Taiwan populations formed a single cluster, whereas 6 discrete clusters were formed by each population from Fujian. All populations were divided into 3 main groups and that all 5 populations from Taiwan were gathered into a subgroup combined with 2 populations, Dehua and Liancheng, formed one of the 3 main groups, which indicated relative stronger relatedness. It is supported by a genetic structure analysis. All those results are suggesting different levels of genetic diversity and variation of Chinese fir between Fujian and Taiwan, and indicating different patterns of evolutionary process and local environmental adaption. Taiwan, which is a typical continental island, is approximately 200 km east of Fujian province, China. Plants in Taiwan have a strong relationship with mainland China , whereasCunninghamia lanceolata (Lamb.) Hook.), a rapidly growing coniferous timber wood species, originated in the late Mesozoic Jurassic, was once widely distributed in Eurasia [C. Konishii (endemic species/subspecies of Taiwan) were carried out by several Taiwanese scholars. A phylogeographic investigation used noncoding spacer sequences of the cpDNA to reveal low differentiation between populations of Taiwan and China [C. konishii and the migration patterns proposed to be attributed to multiple factors such as events of long-distance dispersal indicated the derivation of C. konishii from Chinese fir [Chinese fir  analysis is a common technique for detecting the genetic variation and divergence. As a dominant marker ISSR has some disadvantages. A recently study reported that ISSR marker is less efficient than AFLP  for the lower degree of polymorphism . HoweverIn this study, ISSR amplification was utilized to evaluate the genetic divergence of wild Chinese fir populations from Fujian province and the Taiwan area to assess the different levels of genetic variation, reveal the relationships among all populations, especially between Fujian and Taiwan groups, and infer the possibility of a different evolutionary process of Chinese fir created by the separated continent-island environment.There were no specific permissions requirement in this study for the non-protected species involvement and the minimal invasive sampling methods.To represent the distribution in Fujian province and the Taiwan area, 11 populations (6 populations in Fujian province and 5 in Taiwan), including 225 wild individuals of Chinese fir, were sampled and detected in this research . More deWhereas confusion was brought by the large-scale seed source allocation in mainland China since 1978, a series of actions were used to ensure the regional characteristics could be represented. First, individuals from plantations were abandoned, and those from primary forests, natural reserves, and primitive villages were chosen. Second, age was estimated by diameter at breast height (DBH) and height to ensure that every wild individual tree was older than 35 years. In addition, the distance between any two individuals in a population was more than 1 km. For conservation purposes, cuttings shorter than 15 cm from each individual were brought to Fujian Agriculture and Forestry University to undergo cottage propagation and provide fresh young leaves for further experiments.Total DNA was extracted from young fresh leaves using a CTAB (Cetyl trimethyl ammonium bromide) method . The extSixty widespread ISSR primers from UBC  primer set #9 were used for this experiment. Those primers were synthesized by Shanghai Sangon Biological Inc., . A preliminary experiment using 2 donor samples as template was carried out to test the practicability of those primers. Based on the amplification bands that were clearly visible, polymorphic and stable, 18 primers  were sel2+), 1.5 mM Mg2+, 0.15 mM dNTPs, 0.4 μM primers, 1.5 U of TaqDNA polymerase and 75ng of template DNA. The reaction amplification program was: an initial denaturation of 5 min at 94°C, followed by 40 cycles of 45s at 94°C, 50s at 52°C, 90s at 72°C, and final extension at 72°C for 8 min. The PCR amplification products were separated by agarose gel electrophoresis, and all of the clear bands were counted for the subsequent analysis.The ISSR amplifications were carried out in 20 μl reactions which containing 2 μl 10×PCR buffer , effective number of alleles (Ne), Nei’s gene diversity (h), Shannon’s information index (I) and genetic distance (D) [ARLEQUIN version 3.5 software was utilized to estimate of gene flow (Nm). A dendrogram was drawn using UPGMA clustering to describe the relatedness of all 11 populations, and a 1000 times bootstrap test was carried out to verify the result of that clustering. The STRUCTURE version 2.3.4 software was applied to obtain the hierarchical organization of the genetic structure of the 11 populations and the admixture model was selected as the ancestry model because of the high heterozygous rate of Chinese fir. The number of genetic groups (K) was determined referring to Evanno’s method [MANTEL function from VEGAN package of R-STUDIO version 0.98.1091 software was utilized to carry out a mantel test to check the correlation of the matrixes of genetic and geographic distances.The ance (D) . The ARLs method ,22. The Genetic diversity analysis using 60 widespread ISSR primers was carried out. Eighteen ISSR primers generating clear, identifiable and polymorphic bands were selected and totally 266 polymorphic loci were generated from those primers. All the present/absent information of the polymorphic loci were listed in the h parameter ranged from 0.207 to 0.243 (group level = 0.325) in Fujian province’s populations and from 0.157 to 0.278 (group level = 0.263) in the Taiwanese area’s populations; meanwhile, the I value ranged from 0.321 to 0.374 (group level = 0.494) in populations from Fujian province and from 0.233 to 0.411 (group level = 0.410) in Taiwan, which indicated the probability of higher genetic diversity among Chinese fir in Fujian province. At the population level, the Nanping population  and Miaoli population  had the highest degrees of genetic diversity. The low Na  and Ne  values identified in this experiment reflected a number of recessive mutation loci that could not be detected by ISSR markers as the dominant markers they are.The main genetic parameters were reported in Nm value was calculated and is reported in Nms within populations from Taiwan (range from 1.843 to 14.151) are significantly higher than within Fujian (range from 0.343 to 0.716) and between populations from Fujian and Taiwan (range from 0.308 to 0.575), which indicated a stronger connection within populations from the Taiwan group. The strongest gene flow was found between Hualian and Nantou populations. However, the signal of gene flow may possibly be amplified by the limited sample sizes from the both populations. The Miaoli population exhibited a relatively stronger gene flow to other populations: its Nms range from 0.492 to 7.664, which was slightly higher than the other populations, thus partly confirming the high rate of diversity as analyzed by the genetic parameters.To ascertain the gene flow among the populations, the Cluster analysis and the UPGMA algorithm were used to generate a dendrogram, and the result is reported in STRUCTURE software. The number of genetic groups (K) showed a visible peak at 7  in coniferous species [S. verticillata (h varied from 0.282 to 0.450) [N. longibracteata (h varied from 0.291 to 0.851) [h = 0.394) [h = 0.325) and Taiwan area (h = 0.263). The different levels of diversity were probably due to the locations of the sample populations. A long distance, which is obviously greater between provinces than within a province, could affect the gene flow by seed dispersal. Similar levels of genetic diversity were reported by other researches [Coniferous species are known for the conserved genome structure and slow evolutionary strategy to fit the nature selection ,24. A re species , provideo 0.450)  and N. lo 0.851) , display= 0.394) , lower lC. Konishii [A. kawakamii [M. thunbergii [The geographical isolation of continental islands usually provides systems to study the evolutionary process ,31. NeveKonishii , A. kawaawakamii  and M. tunbergii , small gF. hodginsii, which was also showed a high level of genetic diversity and low level of gene flow among populations in Fujian province [Interestingly, an opposite pattern characterized by high genetic diversity , populatprovince , impliedprovince  in Fujiaprovince ,38,39.A. kawakamii [K. daviana var. formosana [To explain the different levels of genetic diversity between Fujian and Taiwan, a continent-island pattern for coniferous populations in evolution  could beawakamii  and K. dormosana , two pinormosana ,46. It iormosana .Geological evidence has revealed that several small patches across the southern part of China and Taiwan provided some possible migration routes , such asWild individuals were utilized as material in this study, which probably led to more reliable results. However, the limited population numbers collected in mainland China and individuals in Taiwan area probably created a negative effect. In further study, more wild populations of Chinese fir from mainland China, especially from typical geographic conditions, and larger size populations, especially in Taiwan, should be collected, and more types of molecular markers should be utilized to guarantee more accurate and reliable results.Overall, 266 polymorphic loci were developed from Chinese fir by using ISSR analysis. The results suggested different pattern of genetic diversity and variation of Chinese fir between Fujian province and Taiwan area. A possible extinct event and recolonization was revealed within Taiwan populations, whereas that might not occur within Fujian populations. The evaluation of genetic variation and structure is potentially useful for future research, which will probably focus on the evolutionary process of Chinese fir as affect by geographic events.S1 Text(TXT)Click here for additional data file.S1 FigK = 7 showed the highest DeltaK value for all values of K ranging from 1 to 9.(TIF)Click here for additional data file.


Loropetalum subcordatum is an endangered species endemic to China that is characterized by narrow distribution, small population size, and delayed fertilization. However, the genetic diversity of the entire extant natural and ex situ populations has not been assessed to date. In this study, we evaluated the genetic diversity and structure of six natural populations and a single ex situ population (the only known ex situ population of L. subcordatum) using sequence-related amplified polymorphism data.L. subcordatum. A Mantel test demonstrated that there was a positive correlation between genetic and geographic distances, indicating that significant genetic divergence was likely the result of geographic isolation among natural populations. Furthermore, based on genetic structure patterns, populations of L. subcordatum were divided into three clusters. Group 1 was composed of specimens from Libo, Guizhou Province (GZ) and Huanjiang, Guangxi Zhuang Autonomous Region (GX). Group 2 was composed of Mt. Wuguishan, Guangdong Province (GD). Group 3 was composed of three populations in Hong Kong Special Administrative Region. Additionally, clonal reproduction probably existed in GD population. According to the genetic information analysis and field survey, the ex situ population did not match its source population (GD) in terms of genetics, and its habitat was different from the original natural habitat. We observed that a few individual GD seeds were needed to improve ZS ex situ in the future.In total, 553 reliable DNA bands, of which 359 (63.28%) were polymorphic, were amplified by polymerase chain reaction with combinations of 15 primers. Low average gene diversity within populations and high genetic differentiation were detected in L. subcordatum had extremely low average gene diversity within populations and high genetic differentiation among populations. At present, the unique ex situ population has not been successful due to non-representative samples being taken, a smaller population size, and man-made changes in habitat. Potential strategies are suggested to improve the conservation of this species.Compared to previous SRAP-based studies of endangered plants, The online version of this article (10.1186/s12863-018-0599-6) contains supplementary material, which is available to authorized users. Genetic diversity plays a key role in the survival and succession process of plant species, and is usually considered to be an estimate of extinction risk –3. SpeciThe major techniques to protect rare and endangered plants include in situ conservation, reintroduction, and ex situ conservation . Ex situLoropetalum subcordatum (Benth.) Oliv. (Hamamelidaceae) is a shrub or small tree that grows in evergreen broadleaved forests in southern and southwestern China. This species is self-pollinating and endemic to China. It has a low fruit set (4.79 ± 1.45%) in nature and a long-period of arrested development between flower senescence and ovary enlargement, which indicates that delayed fertilization occurs in this species [L. subcordatum was distributed in Libo, Guizhou Province (GZ); Longzhou, Guangxi Zhuang Autonomous Region (LZ); Mt. Wuguishan, Guangdong Province (GD); and Bowen Road (BW) and Lantau Island (LT), Hong Kong Special Administrative Region  in China [Ht = 0.22606, Hw = 0.1771) and high genetic divergence among populations , while no significant correlation was detected between genetic distance and geographic distance  [ species . Accordit 4.79 ± .45% in nt 4.79 ± .45% in nin China  and assein China . Recentlin China . And com= 0.081) .Fig. 1DiL. subcordatum, including those found in nature and the one conserved ex situ population, because it is a highly reliable genetic marker [L. subcordatum to establish a better understanding of genetic information about this species, to estimate the validity of ex situ populations and to propose potential measures for further conservation.In this study, sequence related amplified polymorphism (SRAP) was conducted on populations of c marker –33. The In this study, six natural populations  and one ex situ population (ZS) were analysed Table ; Fig. 1.Total genomic DNA was isolated from dried leaf tissues following the modified hexadecyl trimethyl ammonium bromide (CTAB) method . The con2, 0.25 mM of dNTPs, 2.5 U of Exprime Taq, 0.3 μM of each primer, and ddH2O. The PCR cycling program was set to 94 °C for 5 min, followed by 5 cycles of 35 °C for 1 min and 72 °C for 1 min. Then, the process continued for 30 cycles of 94 °C for 1 min, 35 °C for 1 min, and 72 °C for 1 min, followed by a final extension cycle of 72 °C for 10 min.For PCR amplification, the procedure described by Li and Quiros  was usedAll of the readable amplified fragments with the same gel mobility, which ranged from 100 bp to 2000 bp, with an occurrence frequency of greater than 5%, manually scored as present (1) or absent (0) for entry into a binary data matrix.PPB), Nei’s genetic diversity (H) [I) [Ht), the average gene diversity within populations (Hw), gene flow (Nm), and Nei’s genetic distance.The binary data matrix was analyzed using POPGEN v.1.31  to estimsity (H) , Shannon (H) [I) , total gK) from 1 to 10 and 20 independent runs for each K, with a burn-in period of 1 × 105 and 1 × 105 Markov Chain Monte Carlo replicates after burn-in. Moreover, the admixture model and allele frequencies that correlated were chosen. △K was calculated based on the rate of change in LnP (D) between adjacent K [Fst) as a measure of populations divergence was estimated using GenAlEx V.65 [G) and the ratio of genotypes to the number of sampled accessions (G/N). If the individuals in a population had the same band sequence that consisted of the presence (1) or absence (0) of SRAP fragments, they would have the same genotype.An unweighted pair-group method of the arithmetic (UPGMA) dendrogram tree was constructed based on Nei’s genetic distance using NTSYS 2.1 , and boojacent K . The STRlEx V.65 . For natlEx V.65 . In addilEx V.65 , calculaHt) was 0.2217, and the average gene diversity within populations (Hw) was 0.0469. Among the six natural populations, the percentage of polymorphic bands (PPB) ranged from 3.98% (in LT) to 21.7%  was 0.1604, and Shannon’s information index (I) was 0.2581. At the population levels, the average values were as follows: H = 0.0469 (range of 0.0145–0.0904) and I = 0.0684 (range of 0.0217–0.1308) (Table G/N = 0.913). The PPB, H and I of ZS were significantly less than those of GD  suggested that there was high genetic divergence among natural populations  and an additional file shows this information in more detail [see Additional file K was identified as 3  GZ s 3 Fig.  indicate12 Table . A ManteTetrastigma hemsleyanum  [Handeliodendron bodinieri  [Cibotium barometz  [Ht = 0.2217) and the average gene diversity within populations (Hw = 0.0469) of L. subcordatum were very low. According to the field survey, L. subcordatum was vulnerable to fragmentation due to human disturbance and its low densities in forest habitat, which corresponds to previous knowledge about this tropical endemic tree [L. subcordatum, self-pollination and low fruit set, were possible reasons for the constrained genetic diversity. The former feature usually reduces heterozygosity and decreases effective population sizes [L. subcordatum. However, the genetic diversity measured by SRAP in this study was lower than that measured by AFLP in Gong et al. [Ht = 0.22606, Hw = 0.1771, PPB = 11.36%~ 40%). A total of 47 samples and an average of 11 individuals of each population were used in Gong et al. [Compared to previous SRAP-based studies of endangered plants, such as  0.2402)  and Cibo 0.1354) , the coemic tree . Populaton sizes , while tg et al. , as wellL. subcordatum possesses extremely low dispersal ability due to low fruit set and gravity-dispersed seed. Therefore, the differences between the continental and island populations may be a result of differences in population size, the island’s geographic isolation, and poor dispersal ability.In addition, this study showed that the genetic diversity of the three continental populations  was higher than that of the island populations  was higher than the findings of an earlier study (Fst = 0.72) by Gong et al. [Gst = 0.523) [L. subcordatum are gravity-dispersed, so they cannot spread over long distances, which decreases effective population sizes. Moreover, in addition to their breeding strategy, genetic drift, and gene flow, geographic distribution is also a major factor affecting genetic variance among populations [In this study, the genetic differentiation among populations of g et al.  and the = 0.523) . General= 0.523) , 57, whi= 0.523) , 58. Unlulations .L. subcordatum could be divided into three clusters  and showed that significant geographic isolation existed among populations. Therefore, we hypothesized that the geographic isolation of L. subcordatum populations resulted in extremely low gene flow (Nm = 0.1342) among natural populations, which made the species more susceptible to genetic drift and genetic differentiation [L. subcordatum were likely its reproductive strategy (autogamous), the geographic isolation of its populations, and the narrow distribution of its range.rs Figs.  and 5, wrs Figs.  and thatntiation . OverallL. subcordatum in China and is used as a source for protecting and improving the GD population. It has been suggested that 95% of a species’ alleles with an occurrence frequency of more than 5% should be included in ex situ populations for successful genetic conservation [PPB = 7.59%; H = 0.0279; I = 0.0415) was much lower than that of GD , which suggests that the ex situ population does not adequately contain the genetic diversity of its source population. Additionally, the result of the UPGMA tree supported this analysis, and we found that ZS clearly derived from only part of the individuals of GD  was not successful and needs to be improved in the future due to its lower diversity, limited number of individuals, and unsuitable habitat. Efforts should be conducted to improve natural and ex situ populations in the future. All populations of L. subcordatum should be conserved in situ and ex situ, especially GD, BW, VT, and LT, by establishing protection zones to prevent interference introduced by human activity. Meanwhile, seed collection is a useful and effective method to build and maintain most ex situ populations [L. subcordatum should be collected as much as possible to store or use for future improvement of ex situ and in situ populations. Then, vegetative propagation can be used to effectively and rapidly expand ex situ population sizes. Additionally, a new habitat similar to the GD population’s living environment is required, and all of the existing individuals of the ZS ex situ should be transplanted into this new habitat. Finally, future studies should also focus on the mechanisms of the inherently low fruit set and delayed fertilization of L. subcordatum.As with other endangered species, another challenge to the survival of ulations , 68, andL. subcordatum, endemic to China, has low average gene diversity within populations and relatively high genetic differentiation. Based on an investigation of wild populations, we found that human activities impede the demographic expansion of L. subcordatum and fragment natural populations. The conservation of L. subcordatum is extremely urgent. The results of this study regarding the genetic information and UPGMA analysis between the ZS ex situ and its source population (GD) showed that this unique ex situ did not genetically represent GD and that the environment of the ex situ was different from its original habitat according to the field survey. Therefore, ZS ex situ conservation is not successful and needs to be improved in the future. Considering the previous discussion, more effective actions need to be taken to prevent further decline or extinction of L. subcordatum.The endangered species Additional file 1:Sequence of 15 SRAP selected primer combinations. (DOCX 14 kb)Additional file 2:Fst analysis. (DOCX 14 kb)Results of pairwise population


Musa basjoo var. formosana, a wild banana species, and test for adaptive evolution of amplified fragment length polymorphic (AFLP) loci underlying local adaptation in association with environmental variables. Postglacial range expansion was suggested by phylogeographical analyses based on sequence variation of the second intron of copper zinc superoxide dismutase 2 gene. Two glacial refugia were inferred by the average FST parameter (mean FST of a population against the remaining populations). Using variation partitioning by redundancy analysis, we found a significant amount of explained AFLP variation attributed to environmental and spatially-structured environmental effects. By combining genome scan methods and multiple univariate logistic regression, four AFLP loci were found to be strongly associated with environmental variables, including temperature, precipitation, soil moisture, wet days, and surface coverage activity representing vegetation greenness. These environmental variables may have played various roles as ecological drivers for adaptive evolution of M. basjoo var. formosana during range expansion after the last glacial maximum.Genetic variation evolves during postglacial range expansion of a species and is important for adapting to varied environmental conditions. It is crucial for the future survival of a species. We investigate the nuclear DNA sequence variation to provide evidence of postglacial range expansion of  During the last glacial maximum (LGM), most plant species in the Northern hemisphere would have retreated southward toward the tropics or warmer lowland areas, and survived in refugia2. Taiwan is a continental island situated off the coast of the Asian mainland and lies to the south of the Ryukyu Arc and north of the Philippines Archipelago. Although remnants of glaciations at the top of some peaks along the central mountain range (CMR) were found3, the lowlands of Taiwan were not covered with ice but were drier and colder, and the climate changes would have confined species to refugia in low elevations4. In Taiwan, many conifers escaped to the middle elevations during the LGM4. These species were originally distributed at middle and low elevations and would have migrated to the lowlands. A reverse course of events occurred since the LGM5, with species that were previously confined to refugia in the south expanded polewards and lowland forests colonizing at higher elevations6. Environmental gradients can potentially act as selective drivers during postglacial range expansion and result in locally adapted variants2. The current distributions of species are the results of a combinatorial effect by historical events, ecological factors, and stochastic or neutral mechanisms.In the Quaternary, temperature oscillations are an important historical factor influencing the current distributions of a plant species7. Studies have shown that natural population divergence is driven by variable environmental conditions and leads to the evolution of locally adapted lineages11. However, gene flow between closely related genetic lineages can be reduced by a combinatorial effect of geography and environment; and geographical isolation may play a larger role than environmental variation in shaping population structure13. Therefore, investigating the relative roles of geography and environment that influence genetic variation is critical to understand how environmental factors may act as selective drivers and lead to adaptive genetic variation underlying local adaptation in natural populations of a species12. Moreover, identifying environmental factors that play roles in driving adaptive divergence is particularly of interest in biological conservation and ecological restoration14.Population adaptive divergence is a central issue in evolutionary biology that focuses on understanding the correlations of population genetic variation with environmental heterogeneityCyclobalanopsis glauca15, Cunninghamia konishii16, Trochodendron aralioides17, Castanopsis carlesii18, and Cinnamomum kanehirae19. Although Taiwan spans a small range of latitude geographically, varied geographical topologies support vegetation from tropical to cool climates20. The dramatic topological differences combined with the influence of tropical and subtropical climates have fostered high habitat diversity and may serve as a driving force for adaptive evolution12. During the postglacial recolonization process, a species would have evolved with adaptive variation invoked by differential selection along environmental gradients occurring in species’ distribution ranges.In Taiwan, genetic signatures of postglacial expansion into ranges of habitats from refugia were observed in many tree species such as Musa basjoo Siebold is a cold hardy banana species and is thought to be originated from southern China22 and is genetically differentiated from other Musa species23. M. basjoo Siebold & Zucc. ex linuma var. formosana (Warb. ex Schum.) Ying is a variety of M. basjoo endemic to Taiwan24. M. basjoo var. formosana is distributed in low elevations in Taiwan25 and has a diploid chromosome number of 2n = 2226. The generation time of bananas under cultivation can be up to 18 months25 and could be longer for natural populations of M. basjoo var. formosana. Fruit from M. basjoo var. formosana is edible but has numerous large and hard seeds. This species is an important germplasm for banana breeding due to its characteristics of cold tolerance and disease resistance. For conservation of this species, it is important to identify the genetic relationships of individuals among populations and to understand the potential for evolutionary adaptation because biodiversity is increasingly threatened by human-induced anthropogenic climate change27.SOD2) was sequenced for 46 individuals from eight populations 29 to assess the relative influences of geographical and ecological isolation contributes to AFLP variation. FST outliers were identified using genome scan methods. In light of understanding local adaptations associated with environmental gradients, multiple univariate logistic regression was used to correlate environmental variables with AFLP loci that have potentially evolved under selection. We hypothesized that frequencies of AFLP outliers may display correlations with environmental gradients underlying local adaptation because the distribution of M. basjoo var. formosana in different geographical regions represents habitat environmental heterogeneities across the CMR. The main goals of this study were to: (i) demonstrate postglacial expansion of M. basjoo var. formosana; and (ii) find evidence for potential adaptive evolution associated with environmental gradients.In this study, the second intron of copper zinc superoxide dismutase 2 gene  was found between nucleotide sites 15 and 510 when total samples were analyzed. In addition, 2 of 46 samples collected from populations Shouka and Wufeng were heterozygotes and one single-base pair indel was found in the aligned sequences. Sixteen haplotypes were identified in the 92 sequences analyzed  against the remaining populations  and nucleotide diversity θS and θπ. Hd ranged between 0 (population Sandimen) and 0.848 (population Shitou), θπ ranged between 0 (population Sandimen) and 0.00173 (population Beishi), and θS ranged between 0 (population Sandimen) and 0.00168 (population Wufeng). The levels of Hd, θπ, and θS were not correlated with the number of sequences (sample size) via Pearson’s correlation test .Population pairwise ons Fig. , which m FST =0.54 againsD30 and Fu’s Fs31 revealed non-significant negative values in many populations and even positive values were observed  and Fu’s Fs  were found. Moreover, non-significant raggedness index (rg)32 was found for all individual populations and the pooled samples   and significant R2 index  were found for pooled samples, indicating spatial range expansions34. The mean demographic expansion factor (τ)35, representing the time since the beginning of an expansion, was 1.6167 ). The time at which the expansion event took place was dated with a mean of 21317  years before present for M. basjoo var. formosana, which corresponded roughly to the time frame at the end of the LGM, from 25,000–18,000 years before the present37.Neutrality tests based on Tajima’s ed Table . In contHE)38 averaged 0.276 and ranged from 0.231 (population Shouka) to 0.336 (population Wufeng)  = 0 model, which estimated θB  with f = 0 best fitted the data by having the lowest deviance information criterion (DIC) and f = 0 model and the next best model (full model) (13771.7 vs. 13772.4) . These results suggested that the degree of inbreeding was low in M. basjoo var. formosana. The M. basjoo var. formosana populations were moderately structured according to HICKORY estimates based on the f = 0 and full model . The moderate level of genetic differentiation between populations estimated with HICKORY was consistent with the values estimated with AFLP-SURV40 . AMOVA ΦST value based on the three DAPC clusters was 0.2091 (P < 0.001).In AFLP data, the northern population Wulai had the largest average pairwise 41, the maximal ΔK value (change in the log probability) occur at K = 2 ) was obtained when K = 8. In the LEA analysis42, the minimal cross-entropy was lowest when K = 7  were higher when K = 2, 3, and 4  and lower when K ≥ 5 , normalized difference vegetation index (NDVI), soil pH, soil moisture index (TMI)45, and wet days , and the proportion of explained variation was 19.06% (fraction [a+b+c]) (Supplementary Table P < 0.001) explained by pure environmental variables (fraction [a]) and 8.25% (P < 0.001) by geographically structured environmental variables (fraction [b]). No AFLP variation attributed to pure geographical difference (fraction [c]) was found.Seven environmental variables, including annual mean temperature (BIO1), annual precipitation (BIO12), mean wind speed  and five (0.96%) AFLP loci that potentially evolved under selection  and locus P3_24 with annual mean temperature and NDVI . Significant negative relationships were found between locus P3_23 and NDVI  and between locus P6_12 and annual mean temperature . Samβada analysis reported several types of pseudo-R2 values, we adopted the Nagelkerke pseudo-R2 because its calculation is based on log likelihoods rather than on residual deviance and scaled approximately from 0 to 1 equivalent to the unadjusted R2 in linear regression49.We performed outlier detection for AFLP loci potentially evolved under selection with two neutrality test methods: DFDST and BAYESCANon Table . Samβadaon Table . We consd six 1.1% and fivSOD2 and AFLP variation to investigate whether the postglacial range expansion occurred in M. basjoo var. formosana and adaptive evolution in association with the environmental gradients of contemporary populations in this species. Sequence variation in the second intron of Cu/Zn SOD2 suggested range expansion since the LGM. A vast amount of unaccounted variation was observed, which is typical for RDA multivariate analysis50. In addition, a considerable amount of unexplained variation could also be attributed to random processes triggered by ecological drift and dispersal, non-spatially structured biological, and/or unmeasured environmental differences51. In addition, the natural landscapes of Taiwan have been dramatically altered by humans, particularly in the lowlands, which may have played a role in influencing genetic variation surveyed in M. basjoo var. formosana. Nevertheless, a significant amount of AFLP variation can be explained by the environmental variables. Local adaptation in M. basjoo var. formosana populations was suggested by the AFLP loci that were potentially evolved under selection in association with environmental variables, including annual mean temperature, annual precipitation, surface coverage activity (NDVI), soil moisture, and number of days with > 0.1 mm of rain per month (wet days).In this study, we surveyed sequence variation in the second intron of Cu/Zn 2. The discrepancy in detecting population expansion for individual populations could be because of either population reduction, population subdivision, a recent bottleneck, or migration52. Our data revealed that the mismatch distribution for the frequency of pairwise differences among haplotypes did not fit tightly with a population in the expansion model  32, non-significant SSD 56, and significant R2 33  parts of Taiwan for tree species6. Populations served as refugia in the north-central part of Taiwan were found for tree species, including T. aralioides17, Cu. koinishii57, Ca. carlesii18, Machilus thunbergii58, Ma. Kusanoi58, and Ci. kanehirae6 based on chloroplast DNA variation; and Ci. kanehirae19 and Quercus glauca59 based on nuclear DNA variation. Species with southern glacial refugia include Cy. glauca15, T. aralioides17, Ca. Carlesii18, Ma. thunbergii58, Ma. Kusanoi58, and Ci. kanehirae6 based on chloroplast DNA variation; and Ci. kanehirae19 and Q. glauca59 based on nuclear DNA variation. Our results in M. basjoo var. formosana reflect recolonization events after the latest glacial period that probably originated from the southern population Sandimen due to its highest mean pairwise FST (=0.154) against the remaining populations based on sequence variation of the second intron of Cu/Zn SOD2  compared to introns of chalcone synthase (Chs) and leafy (Lfy) genes in Ci. kanehirae 19 and intron of glyceraldehyde-3-phosphate dehydrogenase gene in Q. glacuca (Hd = 0.840 and θπ = 0.00500)59. However, these comparisons may not be appropriate based on only one gene and the gene sequences compared between species were different. Nevertheless, the level of sequence nucleotide diversity reflects the past demographic events in natural populations. Although populations Sandimen and Wulai may have been the glacial refugia for M. basjoo var. formosana, populations Beishi, Shitou, and Wufeng located in north and central Taiwan across the CMR had higher levels of nucleotide diversity  and between adjacent populations except for the population Wulai; and scarcely southward from the northern refugium (population Wulai). This is because the population Wulai had a high average FST (=0.274) in comparison with all other populations based on AFLP variation  in M. basjoo var. formosana compared with other broadleaf tree species occurred in Taiwan, such as Rhododendron oldhamii (average HE = 0.216)11 and species in the genus Salix (average HE = 0.166)12. M. basjoo var. formosana also had a relatively high level of AFLP variation compared to the average HE (=0.230) for 13 plant species summarized in Nybom60. Moreover, the level of AFLP variation of M. basjoo var. formosana was comparable to that of M. balbisiana, another wild banana species, occurred in China (average HE = 0.241)61. Patterns of genetic variation in contemporary populations of a species are influenced by the historical processes that shaped the distribution of a species2, by the landscape ecological properties62, and by life history traits63. High levels of HE in M. basjoo var. formosana may have been related to the low degree of inbreeding revealed by the HICKORY analysis 73, which was not retained as explanatory variable in this study (Supplementary Methods). NDVI and/or fPAR can be influential factors acting on the genetic variation of a species in response to interactions with other species in a local ecological community74; and has been shown to be correlated with population adaptive divergence12 and adaptive divergence between species13.Soil properties can explain a non-negligible proportion of the spatial distribution of tree speciesM. basjoo var. formosana from experienced postglacial range expansion is important. Our results suggest that there are two glacial refugia located in the northern and southern part of the M. basjoo var. formosana distribution range. Postglacial expansion confronting ecological discontinuity may have triggered the evolution of environmentally associated AFLP variation underlying local adaptation. Although a vast amount of AFLP variation was attributed to residual effects, a significant amount of explained variation attributed to environmental effects was found. In conclusion, environmental variables include temperature, precipitation, soil moisture, surface coverage activity, and wet days may have been the most important ecological drivers for adaptive evolution of postglacial expanded M. basjoo var. formosana populations, and have played roles in shaping the current distributions of this species.Understanding environmental variables acting as ecological drivers and playing roles in shaping the contemporary gene pool structure of M. basjoo var. formosana were collected , and amplified using RACE kit (Invitrogen) with degenerate primers . RACE products were then ligated into pGEM-T vector (Promega) and transformed to E. coli. Two full-length Cu/Zn SOD coding sequences were cloned and sequenced (mbCSD1: ABI34606 and mbCSD2: ACY24898).The extraction of genomic DNA and total RNA from leaves followed the methods of Dellaporta BamHI-, BglII-, EcoRI-, or SacI-digested genomic DNA was self-religated and used for the first inverse polymerase chain reaction (IPCR) with CSD-F2 and CSD-R2 primers (5′-GGTGATACCACCAACGGCTGC-3′ and 5′-GCAGCCGTTGGTGGTATCACC-3′). The first IPCR was performed at 94 °C for 6 min, 25 cycles of 20 sec at 94 °C, 15 sec at 60 °C, 2 min at 72 °C, followed by 5 min at 72 °C. The first IPCR amplified fragments were used as template in the second IPCR with CSD-F3 (5′-CACGTGATGAGGAACGACATGC-3′) and CSD-R3 (5′-GCAGCCGTTGGTGGTATCACC-3′) primers in a PCR reaction at 94 °C for 4 min, 25 cycles of 30 sec at 94 °C, 30 sec at 60 °C, 2 min at 72 °C, followed by 5 min at 72 °C. The second IPCR products were subcloned to pUC119 and sequenced .The SOD1 and Cu/Zn SOD2 except the second intron of Cu/Zn SOD2 in several samples from different populations. Specific CSD2-F1 (5′-CTCCACTGGTAAACCCTCG-3′) and CSD2-R1 (5′-GAGGTCCTGCATGACAACAAG-3′) primers were used to amplify Cu/Zn SOD2 second intron sequences of 46 individuals in PCR reactions with 6 min at 94 °C for 6 min, followed by 30 cycles of 30 sec at 94 °C, 30 sec at 57 °C, 1.5 min at 72 °C, and 5 min holding at 72 °C. The sequential PCR fragments were ligated into pGEM-T vector, transformed into E. coli, and double sequenced using M13 primers and haplotype sequences deposited (GenBank accessions: KX688826~KX688841).An initial screening found no variation in introns of Cu/Zn 77 and nucleotide diversity θπ and θS, haplotype diversity (Hd), R2 index, and pairwise mismatch distribution estimated using DnaSP v5.078. θπ and θS were calculated based on the average pairwise number of differences between sequences and the number of segregating sites per sequence, respectively. Population pairwise FST, raggedness (rg) index of observed mismatch distribution, and neutrality statistics (Tajima’s D and Fu’s Fs) were estimated using Arlequin v3.579. Moreover, goodness-of-fit of the observed mismatch distribution to that expected under the spatial expansion model was tested using the sum of square deviations (SSD) statistic with Arlequin. We generated a haplotype network with the R “pegas” package81.Sequences were aligned with Clustal XR2 value and significance of coalescent simulation against neutral model indicate population expansion. The raggedness index is a measure that quantifies the smoothness of the observed mismatch distribution and small rg values represent a population that has experienced sudden expansion32. A significant SSD value indicates departure from spatial expansion34.Neutrality statistics are nearly zero in constant-size populations, whereas a significant negative value and significant positive value reflect processes such as population subdivision or recent bottlenecks. Positive t = τ/2μk, where t is the time elapsed between initial and current population sizes, τ is the estimated number of generations since the expansion, μ is the mutation rate, and k is the sequence length. A mutation rate of 1.5% per 106 year per site was used19. Demographic expansion factor (τ) was estimated using Arlequin. We assumed a mean generation time of 2 years for M. basjoo var. formosana.To estimate the time since the beginning of an expansion, we used EcoRI and 5 U MseI  for AFLP genotyping28. Digested products were ligated with EcoRI (0.5 μM) and MseI (5 μM) adaptors in a 10-μl ligation reaction mixture using 30 U T4 DNA ligase (Yeastern Biotech) at 22 °C for 1 hr. Preselective amplification was performed in a total volume of 10-μl reaction buffer, including 1 μl of the digested sample (1:9 dilution), 100 nM of EcoRI (E00: 5′-GACTGCGTACCAATTC-3′) and MseI (M00: 5′-GATGAGTCCTGAGTAA-3′) primers . The thermal cycling parameters for preselective amplification were as follows: 2 min at 72 °C and 3 min at 94 °C, followed by 25 cycles of 30 sec at 94 °C, 30 sec at 56 °C, and 5 min holding at 72 °C. Twelve EcoRI (labeled with FAM) and MseI selective primer combinations  selective primers, 0.25 mM dNTPs, 0.75 U Taq with an initial holding at 94 °C for 3 min, followed by 12 cycles of 30 sec at 94 °C, 30 sec at 65 °C with a 0.7 °C touchdown per cycle and 1 min at 72 °C, followed by 24 cycles of 30 sec at 94 °C, 30 sec at 56 °C, 1 min at 72 °C, with a final 5 min holding at 72 °C. Selective amplification were visualized on an ABI PRISM 3100 sequencer . AFLP fragments were scored with Peak Scanner v2.0 (Applied Biosystems) for each individual in the range of 150–500 bp with relative fluorescent unit threshold set at 100, and genotyping error rate estimated (Supplementary Table Genomic DNA (200 ng) was digested with 5 U mean), normalized difference vegetation index (NDVI), soil pH, and soil moisture index (TMI) were retained as explanatory variables , annual precipitation (BIO12), number of days with > 0.1 mm of rain per month (wet days), mean wind speed (WSHE)38 and pairwise FST based on a Bayesian method with non-uniform prior distribution of allele frequencies82 was calculated using AFLP-SURV40. The numbers of private and fixed private bands were calculated using FAMD83. Hierarchical analysis of molecular variance (AMOVA) was performed with the “poppr” package of R84 and significance tested based on 9999 permutations with the “ade4” package of R85. The Bayesian program, HICKORY v1.139, was used to estimate an FST analogue (designated θB) from dominant markers accounting for inbreeding coefficient (f ) using default settings for sampling and chain length parameters . Four models, including a full model, f = 0 model, θB = 0 model, and f-free model were fitted with the AFLP data (Supplementary Methods).Nei’s genetic diversity (M. basjoo var. formosana was carried out with two assignment test methods: Bayesian clustering41 and sparse non-negative factorization (sNMF)42 methods. Bayesian clustering method adapted for dominant markers implemented in STRUCTURE41 was used to estimate an individual’s probability of belonging to a homogeneous cluster (K populations). An admixture model was adopted and tested with ten runs, for K ranging from 1 to 9, with 106 iterations and 105 burn-in steps. R package “pophelper”86 was used to summarize mean log likelihood (LnP(D)), change in the log probability (ΔK)87, and symmetric similarity coefficient (SSC)88 to evaluate the fit of different clustering scenarios. Genetic assignment of individuals was also inferred for K values ranging from 1 to 9 based on sNMF algorithm and least-squares optimization with the “LEA” package of R42. The settings for the LEA analysis were: regularization parameter = 100, iterations = 200, and repetitions = 10 with other arguments set to defaults, and the best K evaluated with the means of minimal cross-entropy.Initial detection of genetic structure of M. basjoo var. formosana were also visualized based on the discriminant analysis of principal components (DAPC) using the R “adegenet” package89. DAPC first performed a principal component analysis (PCA), and an optimal number of PCs was estimated and retained for further discriminant analysis.The relationships between individuals and populations of 90 and significance tested with 9,999 permutations. Proportions of AFLP genetic variation (adjusted R2) explained by pure environmental variables, geographically structured environmental variables, pure geographical variables, and residual components were estimated29. The longitude and latitude of sample localities were used as geographical variables.Redundancy analysis (RDA) was used to partition AFLP variation and explained by environmental and geographical variables using the R “vegan” packageFST outlier detection methods (DFDIST and BAYESCAN) were used to identify AFLP loci that potentially evolved under selection. DFDIST implemented in Mcheza software46 estimated allele frequencies based on the Bayesian approach82 and the highest and lowest 30% of the initial FST were removed for calculating the mean FST. Outliers were identified by observed FST and HE compared to simulated neutral distributions generated using 105 iterations of coalescent simulations. Loci with unusually high FST values at the 95% confidence level by applying a false discovery rate of 5% were considered potentially evolved under selection. BAYESCAN uses a Bayesian likelihood approach via a reversible-jump Markov Chain Monte Carlo algorithm in comparing the selection versus neutrality model to identify AFLP loci that are potentially evolved under selection47. Posterior odds (PO), the ratio of posterior probabilities of selection over neutrality, was estimated with the settings of a sample size of 50,000 and thinning interval of 20 among 106 iterations, following 100 pilot runs of 50,000 iterations. When an AFLP locus with log10(PO) > 1.591 was considered to have strong evidence for selection.Two 48 was further used to evaluate the associations between frequencies of AFLP loci and values of environmental variables using logistic regression model; and significant fit was identified based on the Wald and likelihood ratio tests with Bonferroni correction at P < 0.01. Given the relationships between values of environmental variables and frequencies of AFLP outlier loci, logistic regression plots were depicted.SamβadaSUPPLEMENTARY INFORMATION