Actinidia chinensis complex, a widespread geographically woody liana with variable ploidy in subtropical China comprising two varieties, with three chloroplast fragments DNA . Macroevolutionary, microevolutionary and niche modeling tools were also combined to disentangle the origin and the demographic history of the species or cytotypes.Plant phylogeographic studies of species in subtropical China have mainly focused on rare and endangered species, whereas few studies have been conducted on taxa with relatively wide distribution, especially polyploid species. We investigated the cytotype and haplotype distribution pattern of the chinensis and deliciosa. Our results revealed that these two varieties inhabit significantly different climatic niche spaces. Ecological niche models (ENMs) indicate that all varieties’ ranges contracted during the Last Inter Glacial (LIG), and expanded eastward or northward during the Last Glacial Maximum (LGM).The ploidy levels of 3338 individuals from 128 populations sampled throughout the species distribution range were estimated with flow cytometry. The widespread cytotypes were diploids followed by tetraploids and hexaploids, whereas triploids and octoploids occurred in a few populations. Thirty-one chloroplast haplotypes were detected. The genetic diversity and genetic structure were found to be high between varieties (or ploidy races) A. chinensis complex. The polyploidization process also appears to have played an important role in the historical demography of the complex through improving their adaptability to environmental changes.Pliocene and Plio-Pleistocene climatic fluctuations and vicariance appear to have played key roles in shaping current population structure and historical demography in the The online version contains supplementary material available at 10.1186/s12870-021-03099-y. In the last several decades, many phylogeographic studies have focused on the Qinghai-Tibetan Plateau and adjacent areas  in China –4. As anActinidia chinensis complex is a geographically widespread woody liana with variable ploidy in subtropical China. It is widely planted around the world as an important horticultural fruit tree crop. In the newly revised infrageneric system of Actinidia, the complex comprises three varieties, A. chinensis var. chinensis (hereafter: var. chinensis), A. chinensis var. deliciosa (hereafter: var. deliciosa), and A. chinensis var. setosa (hereafter: var. setosa) [Actinidia based on chloroplast DNA sequences indicate that var. setosa should not be included in A. chinensis complex but rather treated as a separate species (A. setosa) [. chinensis and var. deliciosa can be distinguished by morphological characteristics such as the presence of overwintering buds or the hair types covering on flowering branchlets and fruits [Actinidia chinensis var. chinensis grows mainly in eastern and central China, whereas var. deliciosa is more inland in central and western China [chinensis being found at lower elevations. A cytogeographic analysis has revealed that ploidy types of the A. chinensis complex vary from diploid to hexaploid and that vars. chinensis and deliciosa comprise different cytotypes  [A. chinensis complex have been based on few sample sets and do not cover the entire geographic range of this species [A. chinensis complex and correlations based on ploidy level are still unclear.The  setosa) . However setosa) . Previoud fruits . Howeverd fruits –25. Actirn China . They co species , 25, 27.The development of ecological niche models (ENMs) is a more popular tool to predict the changes in species distribution in response to past or future climatic events and which has been used widely in recent phylogeographic studies . In combA. chinensis complex, and cpDNA markers were used to investigate the genetic diversity and population structure of the A. chinensis complex. We used mismatch distribution analysis (MDA) together with ENMs to investigate the evolutionary history and demographic structure. The main objectives of our study were to (a) reveal the pattern of cytotype distribution; (b) evaluate the level of genetic diversity and the degree of genetic differentiation among populations for each variety; and (c) test the origin of polyploidy and explore the demographic history of the A. chinensis complex. Our aim was to explore patterns and mechanisms of population diversity and historical demography of a typical polyploidy plant in the subtropical forest that generally correspond to the Metasequoia Flora (Sino-Japanese Kingdom), one of the two major floristic regions in the East Asian Floras [In this study, flow cytometric measurement (FCM)  was usedn Floras .. chinensis, whereas all the hexaploids belonged to var. deliciosa. The tetraploids were shared between both var. chinensis and var. deliciosa. Sixteen triploids having similar morphological characteristics with var. chinensis were observed in the diploid, tetraploid, and mixed populations , whereas rare octoploids were found in two hexaploid var. deliciosa populations and one mixed population , respectively.Ploidy was estimated for 3,338 individuals with fresh leaves harvested from hydroponic shoots as 248 individuals failed to obtain fresh leaves. Five ploidy types  across 608 sampled individuals  was higher than that of var. deliciosa . The coefficient of genetic differentiation NST was significantly larger than GST for var. chinensis, and vice versa for var. deliciosa . The inter-variant population fixation indices (FST) were 0.654 and 0.352 (P < 0.01) for var. chinensis and var. deliciosa, respectively. The isolation-by-distance (IBD) tests based on the pairwise FST/(1- FST) genetic distances against the geographic distances detected no significant correlation for the cpDNA  among the 50 populations of the A. chinensis complex.No spatial population structure was detected by the SAMOVA analysis of cpDNA haplotypes from 50 populations; the . chinensis and var. deliciosa, but other haplotypes only belong to the diploid population of var. chinensis. The haplotypes in lineage 3, which belong to var. chinensis (2x) were found in eastern China, such as the Wuyi mountains, Dabie mountains, and Luoxiao mountains. Finally, almost all polyploid haplotypes were found in lineage 2. The dominant haplotype H10 and H11 occurred in 154 and 111 individuals, respectively. Although these two haplotypes were shared by var. chinensis and var. deliciosa, they still mainly belong to var. deliciosa. Nearly half of the haplotypes (16/31) were restricted to a single population. For example, in lineage 3, H3, H6, and H8 were found exclusively in Dabie mountain and Huang mountain, whereas H28, H30, and H31 appeared only in the Zhejiang-Fujian Hilly Region.Phylogenetic analysis of the concatenated cpDNA haplotype data sets recovered three haplotype lineages  dates back to an estimated 11 million years ago (Ma) during the late Miocene. For the 31 cpDNA haplotypes of the A. chinensis complex, the mean crown-group age of the A. chinensis complex was ca. 4.43 Ma , thereby indicating the recent diversification of the cpDNA haplotypes of the A. chinensis complex during the Pliocene. Two polyploid haplotype lineages diverged at ca. 2.22 Ma .The time-calibrated evolutionary tree Fig. a showed Fs test and Tajima’s D test of group E4x and M4 × 6x were also negative, although only the tests for group E4x were significant  and tetraploid/hexaploid var. deliciosa in central-western (CE and WE) China was also recovered. Recent colonization events from the eastern region (EA) to the central region (CE) were inferred, as based on the genetic admixture  in the adjacent mountains (e.g. Qinling and Dabie Mountains) or between the distant mountains (e.g. Qinling and Wuyi Mountains), with multiple expansions at different times  analysis of ancestral distribution areas Fig.  revealedmes Fig. . For themes Fig. b identif. deliciosa was often higher than that of var. chinensis. The geographic areas of the two varieties overlapped in the mountainous regions of midwestern China. The predicted geographic range for var. deliciosa during the LIG  was more southward than that of the present  between 0.7 and 0.9 indicate good discrimination. The AUC values of ENM for each variety and climate scenario were high (0.978/0.982), indicating that all models performed well in predicting the suitable habitat for each variety. The current distributional predictions were good representations of the actual distribution of both varieties. The main geographic distributional areas for both varieties were predicted mainly in the mountains of the subtropical region in China. The elevations predicted for varent Fig. a, e. Thetly Fig. b, f. Theons Fig. , suggestD = 0.433 and I = 0.708, see Fig. chinensis vs. var. deliciosa, suggesting high niche differentiation between these two varieties. In addition, more detailed discrepancies can be found in the niches of three different cytotypes  across their entire geographic distribution. Var. deliciosa predominantly comprises tetraploids and hexaploids, whereas var. chinensis consists of diploids and tetraploids, which is largely consistent with the results of Li et al. [A. chinensis complex, hexaploids were centered in the western portion of their ranges and diploids and tetraploids in the eastern portions. For var. deliciosa, the most frequent and widespread cytotype is hexaploid, followed by tetraploid. For var. chinensis, diploid is geographically most widespread followed by tetraploid. This is the first study to detect octoploids in var. deliciosa. In addition, natural triploid var. chinensis is described for the first time. The low frequency of triploids within diploid-tetraploid populations suggest the probability of interaction between diploids and tetraploids in the contact zone and highlights the potential for contemporary gene flow from diploid to polyploid populations.To our knowledge, this study represents the most comprehensive investigation of the ploidy variation of i et al.  where onIn total, eight different mixed-ploidy populations were observed, including two majority cytotypes  and six minority cytotype  Fig. . The coechinensis is autoploid and hexaploid var. deliciosa is alloploid [A. chinensis complex may experience multiple origins regardless of whether they are autopolyploids or allohexaploids. In this study, 31 haplotypes were detected in A. chinensis complex, of which 19 were found in diploids only, six were found in polyploids (tetraploid or hexaploid) only, and six were shared by different ploidy. Moreover, none of these polyploidy haplotypes were clustered together in the phylogenetic analysis  at the species level was 0.884. Among these haplotypes, the chloroplast genetic diversity (HT) of var. chinensis was 0.920, which was significantly higher than that the relict plants in this region, such as Cercidiphyllum japonicum (0.757) [Davidia involucrata (0.882) [Eupleea pleiosperma (0.893) [HT) of var. deliciosa was 0.552, which is significantly lower than that of other species distributed in the same region. Although the evolutionary rate of mutation of the chloroplast genome is generally slow and the level of intraspecific genetic diversity low [chinensis originated about 4.43 million years ago in China, whereas var. deliciosa originated about 2.22 million years . In addition, previous studies have also found that widespread species tend to have a higher level of genetic diversity than local species [. chinensis has a clear genetic structure , while var. deliciosa does not have such genetic structure . Although the above results indicate a significant genetic divergence between the two varieties. The finding of the lineage 1 haplotypes in the sympatric region suggested potential hybridization. In particular, the haplotype H16 was shared by the two varieties, indicating that genetic mixture occurred between these two varieties. Hybridization and introgression have been considered to be common in the evolution of Actinidia [In this study, 31 haplotypes were obtained by sequencing the cpDNA of 50 populations of  (0.757) , Davidia (0.882) , and Eupsity low , there a species . The samctinidia .A. chinensis complex was 4.43 Ma , polyploidization, and interspecific/intraspecific hybridization may also have contributed to its rapid spread and expansion [The BBM result of cpDNA and the distribution of an ancient haplotype H12) revealed that the most ancient area of var revealedon Figs. . The moixpansion .A. chinensis complex has experienced Last Interglacial contraction and Last Glacial Maximum expansion, which is mutually supported by the results of population dynamic analysis. This dynamic pattern is the same as that of Pinus kwangtungensis [Emmenopterys henryi [A. chinensis complex is a montane deciduous broad-leaved plant distributed throughout the subtropical zone of China with an elevation of 200–2500 m [. chinensis and var. deliciosa has been confirmed. Our field survey data directly show that the elevational distribution range of var. deliciosa (including tetraploid of var. chinensis) is higher than that of var. chinensis (mainly diploid). Therefore, based on the MDA analysis of demographics, two groups  showed a historical population expansion , the presence of over-wintering buds  and the color of the flesh of the mature fruit . Field collection followed the ethics and legality of the local government and was permitted by the government. The formal identification of the plant material was undertaken by the National Actinidia Germplasm Repository (NAGR) of China, and voucher specimens were deposited at NAGR. Each population consisted of either var. chinensis or var. deliciosa, and there were some populations containing both varieties as based on morphology in the sympatric regions. For each population, 6 to 72 individuals were sampled , whose chromosome number had been determined previously from chromosome counts and the estimated genome size of 750 Mb [The ploidy level of individuals were determined by estimating its relative DNA content with FCM. The detailed FCM method in this study was performed as in Li et al. . Brieflyf 750 Mb , was usef 750 Mb .A. chinensis complex. A modified CTAB method [For chloroplast DNA analysis, 50 out of 128 populations were selected to cover the entire geographic distribution of the B method  was used. chinensis and 12 var. deliciosa were randomly selected to undertake the preliminary screening of primer pairs for eight intron regions: ndhF-rpl132, rps16-trnQ, trnE-trnT, psbA-trnH, rpl16, trnL-trnF, trnL-trnT, and trnD-trnT , 0.5 μL each primer (0.2 μM), 1 μL template DNA (ca. 50–100 ng) and 8 μL ddH2O. The thermo-cycling conditions for PCR are given as follows: 94 °C, 4 min; 35 × ; and 72 °C, 10 min. PCR products were sequenced in an ABI 377XL DNA sequencer (Applied Biosystems). All haplotype sequences identified in this study were submitted to GenBank (MT812986-MT813020). For subsequent analysis, A. eriantha and A. polygama were used as the outgroup and these chloroplast sequences were downloaded from GenBank (KY100979.1 and KX345297.1).To identify chloroplast DNA (cpDNA) regions with sufficient variation, 12 individuals of varrnE-trnT , were seHd), and nucleotide diversities (π) for each variety were calculated with DnaSP 5.10 [HT) and within-population diversity (HS) were calculated with PermutCpSSR 2.0 [Three cpDNA fragments were aligned separately and trimmed in MEGA 6  and combaSP 5.10 . HaplotypSSR 2.0 .NST) and unordered (GST) haplotype with PermutCpSSR 2.0. A test of significance comprising 1000 permutations was used to determine if NST > GST. A significantly higher NST than GST usually indicates the presence of phylogeographical structure. The geographically and genetically distinguishable groups and the potential barriers between groups were analyzed with SAMOVA 2.0 [K groups until the percentage of explained variance among groups reached a limit (K = 2–20). In addition, a molecular variance (AMOVA) analysis was performed using Arlequin v3.5 [FST/(1 − FST) were regressed against the pairwise natural logarithm of the geographic distance by using a Mantel test with 999 random permutations in GENALEX 6.5 to assess IBD patterns among the populations [FST distances were calculated with Arlequin, and the pairwise natural logarithms of geographic distance were calculated with GENALEX 6.5.The phylogeographic structure was inferred by comparing population differentiation for phylogenetically ordered  with Markov chain Monte Carlo (MCMC) methods. A strict clock model in BEAST2 v2.5.0 was performed to estimate divergence time among lineages [Actinidia [A. polygama and the other two species , and another is the estimated split between A. eriantha and A. chinensis . The Monte Carlo Markov chain runs were performed every 1 × 109 generations, with sampling every 1 × 105 generations, following a burn-in of the initial 10% cycles. The convergence and effective sample size (ESS) > 200 for all parameters were assessed with Tracer version 1.7.1. After discarding the first 25% trees as burn-in, the rest of the trees were summarized in a maximum clade credibility tree with treeAnnotator version 2.6.0. Finally, the maximum clade credibility tree was visualized in FigTree version 1.4.3 (available at http://tree.bio.ed.ac.uk/software/figtree/).To determine phylogenetic relationships among haplotypes, median-joining networks were constructed with NETWORK v5.0 . Actinidlineages . A GRT mlineages . As a trctinidia  were use–8; refer to Gaut [A. chinensis under natural conditions [D [FS statistics of Fu [The mismatch distribution analysis was conducted with Arlequin 3.5  to test  to Gaut ), k is tnditions . Finallytions [D  and FS scs of Fu  using Arcs of Fu , which ics of Fu .A. chinensis complex using the Bayesian binary MCMC method (BBM) implemented in RASP v.4.0 [A. chinensis complex were defined four biogeographical regions: EA, East; SO, South; CE, Central; and WE, West . For all CVH records, the taxonomic identity was verified by specimen images and records of specimens taken from cultivated plants removed to ensure that geolocations were consistent with the known distribution. After removing duplicates, 150 sites for var. chinensis and 90 sites for var. deliciosa were identified. The current and past environmental datasets of 19 bioclimate variables with spatial resolutions of 2.5 arc minute were downloaded from the WorldClim database [To study the niche differentiation and distributional changes for each variety, the Maxent Version 3.4.0 program , 97 was database . To avoidatabase , strongldatabase , were exdatabase .D similarity index [I [D and Warren’s I ranged from 0 (no niche overlap) to 1 . One hundred pseudoreplicates of shuffling were conducted to generate null models for these statistics, and tested for significance; histograms were drawn using R 3.6.0 (http://www.rproject.org/). In addition, the same tests and analyses were also carried out for the three main different level ploidy  populations.To measure niche divergence, the environmental niches of the two varieties were compared using niche overlap and identity tests, in which the difference between the actual niches was contrasted with null models generated from randomly reshuffled occurrence points . Niche ity index  and Warrindex [I  implemenindex [I , 102. Bochinensis and var. deliciosa occur, principal components analyses (PCA) were conducted with the elevation variable and 19 other bioclimatic variables from WorldClim [To better understand and visualize the similarities and differences between the environments in which var. orldClim , 104. ThAdditional file 1: Figure S1. Schematic diagram of flow cytometric histogram of DAPI-stained nuclei of six type of ploidy levels  of the A. chinensis complex analyzed simultaneously with the internal standard Actinidia chinensis cv. ‘Hongyang’. Figure S2. Results of spatial analysis of molecular variance analysis  on the A. chinensis complex populations in subtropical China. K refers to the number of predefined groups used in the analyses. Figure S3. Observed and expected mismatch distributions for different groups of the A. chinensis complex. (a) group E2x; (b) group E4x; (c) group M2x; (d) group M4x6x. The solid line indicates observed distribution; The dashed line represents theoretically expected distribution under a population expansion model. Figure S4. The results of niche-identity tests by comparing the niches of three main ploidy level populations. (a) 2x vs. 4x (b) 2x vs. 6x (c) 4x vs. 6x.Additional file 2: Table S1. Characteristics of sampled populations and ploidy levels of the A. chinensis complex. Table S2. List of ploidy level and haplotype for 608 individuals in cpDNA analysis. Table S3. Description of haplotypes in the A. chinensis complex from three combined chloroplast DNA fragments. Table S4. Genetic characteristics of cpDNA for 31 var. chinensis populations and 25 var. deliciosa populations. Table S5. Results of AMOVA testing of the A. chinensis complex. Table S6. List of primers for the eight cpDNA loci. Table S7 The six environmental variables used for ecological niche modeling.


Diospyros lotus is the one of the most frost-tolerant species in the Diospyros genera, used as a rootstock for colder regions. Natural populations of D. lotus have a fragmented character of distribution in the Northwestern Caucasus, one of the coldest regions of Diospyros cultivation. To predict the behavior of D. lotus populations in an extreme environment, it is necessary to investigate the intraspecific genetic diversity and phenotypic variability of populations in the colder regions. In this study, we analyzed five geographically distant populations of D. lotus according to 33 morphological leaf traits, and the most informative traits were established, namely, leaf length, leaf width, leaf index (leaf to length ratio) and the length of the fourth veins. Additionally, we evaluated the intraspecific genetic diversity of D. lotus using ISSR and SCoT markers and proposed a new parameter for the evaluation of genetic polymorphism among populations, in order to eliminate the effect of sample number. This new parameter is the relative genetic polymorphism, which is the ratio of polymorphism to the number of samples. Based on morphological and genetic data, the northernmost population from Shkhafit was phenotypically and genetically distant from the other populations. The correspondence between several morphological traits  and several marker bands  were observed for the Shkhafit population. Unique SCoT and ISSR fragments can be used as markers for breeding purposes. The results provide a better understanding of adaptive mechanisms in D. lotus in extreme environments and will be important for the further expansion of the cultivation area for persimmon in colder regions. Diospyros, and this species was first introduced here between 1880 and 1890. Natural populations of D. lotus have a fragmented character of distribution in this region. D. lotus is the most cold-tolerant species in the genus Diospyros and is important for the development of new cold-tolerant varieties and is often used as a cold-tolerant rootstock for D. kaki, allow them to survive extreme temperatures down to −20 °C, with good yields [The Northwestern Caucasus is one of the northernmost growing regions for d yields ,4,5,6.The adaptation of plant species to colder environment is believed to be due to phenotypic plasticity or adaptive evolution ,8. PhenoIn small populations or fragments, selection for local adaptation is less efficient because of the effects of genetic drift on individual loci, and further, on the associations of alleles at different loci ,25. HoweDiospyros, such as RAPD [Diospyros lotus.Several DNA marker systems were developed and used for population studies in  as RAPD , SSRs [2 as RAPD ,29,30,31 as RAPD , SRAP [2 as RAPD , IRAP [3 as RAPD , SCoT [3 as RAPD ,35,36, I as RAPD  or orgDN as RAPD . Among t as RAPD ,40. The  as RAPD ,42. ThusD. lotus according to their morphological traits and multilocus markers, in order to predict ontogenetic strategy for D. lotus in the Northwestern Caucasus. The obtained results provide a better understanding of the acclimation strategy of D. lotus and will be important for the further expansion of the cultivation area for persimmon in the colder regions.The aim of this study was to analyze the variability among the geographically distant populations of I.Bio-ecological indicators—traits with a high CV and high II.Biological indicators –traits with a low CV and high III.Genotype-specific indicators –traits with a low CV and low IV.Ecological indicators—traits with a high CV and low A low level of general variability was observed for several traits: leaf length, leaf width, petiole length, Leaf Index (LI), Leaf Blade Index (LBI), length of the fourth veins, length of the fifth right vein, the angles of second, third, fourth and fifth veins. The greatest general variability was observed in the following traits: the length of the first and second veins, the distance between the bases of the first, second, third, fourth and fifth veins. The low coefficient of determination was observed for the following traits: the angle of the first, second, third, fourth and fifth veins, the length of the petiole and the LBI. Based on these data, all morphological traits were specified to one of the four groups of indicators with different characters of morphological variability :I.Bio-ecD. lotus was constructed. Two populations (Gagra and Shkhafit) showed higher IVC and D. lotus under increasing environmental stresses  and 0.47 (by SCoT data)  .A total of 39 bands were detected with four ISSRs, ranging from Na = 7 (ISSR13) to 13 (ISSR880). The average number of polymorphic bands was 36.80%, ranging from 14.29% (for ISSR13) to 50.00% (for ISSR815) . The aveA total of 52 bands were detected with six SCoTs, ranging from 4 (for SCoT07) to 13 (for SCoT20). The average number of polymorphic bands was 31.68%, ranging from 10.00% (for SCoT05) to 50.00% (for SCoT32). The mean PIC was 0.41, ranging from 0.38 (for SCoT05) to 0.46 (for SCoT07).Generally, the ISSR markers detected a higher percentage of monomorphic bands, as compared to the SCoT markers. According to the SCoT data, Shkhafit and Gagra showed lower genetic polymorphism, as compared to the Gulripsh, Sochi and Sukhum populations . InteresD. lotus.The hierarchical clustering of 52 accessions based on SCoT and ISSR data, indicated four distant sub-branches . The bigD. lotus)  and observations (populations of . lotus) . The firAccording to PCA biplot, the accessions of Shkhafit were positioned on the positive sides of PC1 and PC2, with high loading. On the other hand, the accessions of Gagra were distributed on the positive side of PC1 and the negative sides of PC2. The accessions of Gulripsh, Sukhum and Sochi were grouped mostly on the positive loading of PC2 and the negative loading of PC1. In general, our previous results on relative genetic polymorphism among populations  correspoMost vectors of the morphological characteristics were distributed with high loading located on the positive side of the PC1. The proximity of the accessions to the vectors was in agreement with the strong influence of those characteristics (vectors) on the closest accessions. For example, the Shkhafit accessions were placed closely to the vectors of several morphological traits  and several marker bands , which corresponded to the highest leaf length and width and to bigger vein angles in this population, as compared with the other populations. On the other hand, morphological traits, such as the first to fourth veins base distances, the length of leaf veins, left vein angles and LI, as well as some marker bands (SCoT5_2250 and ISSR13_800), highly correlated to each other and were positioned with high positive loading in PC1. Interestingly, the length of the fifth right vein was loaded on the orthogonal position, together with the Sukhum, Gulripsh and Sochi data points. In addition, several ISSR and SCoT bands showed close relationships to these accessions; however, their PC scores were low. Finally, many DNA bands were positioned as outliers on the negative side of both PCs, indicating neither an association with a certain population, nor an association with a certain trait.D. lotus, different leaf traits were characterized by different characters of variability, indicating the different mechanisms underlying this variability, which is consistent with some other studies on several crops [D. lotus are leaf length, leaf width, Leaf index, and the length of the fourth leaf veins. Other studies on oak also showed that inter-population differences in leaf size were associated with the temperature at the sites of origin, suggesting local adaptation in response to diverging climates [Morphological variation is commonly influenced by genetic variation, environmental variation, or the interactions between them ,44,45,46al crops ,13,17. Aclimates . Howeverclimates . ProbablD. lotus. However, among populations, the different trends of ontogenetic strategies indicate the better state of plants in the Shkhafit and Gagra populations, as compared to those from Sochi, Sukhum and Gulripsh. The index of vitality of coenopopulations represents the state of these populations in the current, and the ratio of vitality indexes over time indicates the ecological plasticity of the coenopopulations [Generally, a depressive type of ontogenetic strategy was established for species of ulations . InteresD. lotus. On the other hand, ISSRs show better results than SCoTs when analyzing a diverse germplasm. This speculation is in accordance with recently published results on Aegilops tauschii [In our study, SCoT markers displayed greater polymorphism and discriminative power as compared with ISSRs. This is consistent with Gorji et al. 2011), who also demonstrated that the SCoT technique was more efficient than the ISSR technique  for pers011, who tauschii .D. lotus; many ISSR and SCoT bands were monomorphic. On the other hand, other researchers detected more than 90% genetic polymorphism in D. lotus [D. lotus varieties worldwide. The low level of genetic diversity could be a constraint to the further expansion of the cultivation area of D. lotus in North Caucasus. Thus, introduction of more D. lotus germplasm from other sources is necessary in order to increase heterozygosity in local populations.The level of genetic diversity among different regions could reflect the ability of species to adapt to the environment to some extent . GeneralD. lotus ,34,36. TThe main difficulty in comparing the level of polymorphism between studies and populations, is the different number of samples included in the different studies. A higher number of accessions usually shows a higher polymorphism in the sample set ,53. In tDiospyros introduction to the Western Caucasus [D. lotus could be due to both existing trait variation and new mutations, as supported by several other studies [Interestingly, according to genetic analysis, most accessions of the southernmost populations (Sukhum and Gulripsh) were placed on the greatest distance from the northernmost Shkhafit population. Along with this, it was indicated that the northernmost population from Shkhafit had the lowest level of genetic polymorphism. In contrast, the highest level of relative genetic polymorphism was detected in the southernmost Gulripsh population. Gulripsh, Sukhum and Gagra are the first locations of Caucasus ,5, and t studies ,44.Diospyros.Using PCA, we expected that the established biological indicators  would be strongly associated within the Shkhafit population. Interestingly, among these biological indicators, leaf length and leaf width were closely associated within Shkhafit population. In addition, the first to the fifth right vein angles were positively loaded with the Shkhafit data points. Vein angles had low CV and low iability . On the For morphological analysis, 52 trees of five populations with 48–50 leaves per tree were evaluated. All trees were fully developed, about 10–18 m high and 50–80 years old. The distances between the trees within each population were 50–600 m. The distances between populations were about 30–50 km. In total, 33 morphological leaf traits were evaluated . The minScanning of the leaves was performed during the day using the scanner HP PhotoSmart (600 × 600 dpi in JPEG). ImageJ  softwareLeaf index The structure of the morphological variability of the leaf traits was assessed by the general variability and coefficient of determination of leaf traits ,59,60,61The general variability is the coefficient of variability of the trait (CV) (%) counted as:he trait ,59,60,612, averaged over the entire matrix of morphological traits, as follows:The average coefficient of determination of the certain trait was calculated as a square of the correlation coefficient r numbers ,59,60,61i is the mean value of i-th trait for certain coenopopulation, The Index of Vitality of the Coenopopulation (IVC) was counted as:f traits ,59,60,61Defensive-type strategy: the increase of stress leads to the strengthening of morphological integrity and plant development.Depressive-type strategy: the increase of stress leads to the weakening of morphological integrity and plant development.Defensive–depressive-type strategy: the increase of stress leads first to the strengthening and then to the weakening of morphological integrity.Depressive–defensive-type strategy: the increase of stress leads first to the weakening and then to the strengthening of morphological integrity.The ontogenetic strategy of the population represents the character of the morphological integrity of plants, assessed by the coefficient of determination of traits  and DEPC-treated water. Amplification was carried out in the MiniAmp thermal cycler  with the following program: primary denaturation for 5 min at 95 °C, then annealing during 40 cycles of 20 s at 53 °C, with elongations at 72 °C for 1 min 45 s and the final elongation at 72 °C for 7 min. The separation of ISSR fragments was performed on a 2% agarose gel for 2.5 h at 90 V in a 1 × TAE buffer. The data were recorded as a 1/0 matrix for the presence and absence of amplified fragments.Five ISSR ,64 and s−1) and DEPC-treated water, in a total PCR volume of 20 µL. Amplification was carried out in the MiniAmp thermal cycler  with the following program: primary denaturation 5 min at 95 °C, 35 cycles with denaturation at 95 °C—1 min, annealing at 52 °C—1 min, elongation at 72 °C for 2 min and final elongation at 72 °C for 5 min. The separation of SCoT-fragments was performed in a 2% agarose gel for 2.5 h at 90 V in a 1 × TAE buffer. The data were recorded as 1/0 matrix for the presence and absence of amplified fragments.The SCoT PCR reaction mixture consisted of a 10 μL 2× HS-TaqPCR reaction buffer, including Hot Start Taq-Polymerase , 0.4 μL of primer (10 µM), 2 μL of DNA —probability that the marker genotype of a given offspring will allow deduction, in the absence of crossing over, and which of the two marker alleles of the affected parents it received.D (Discriminating power)—the probability that two randomly chosen individuals have different patterns, and thus are distinguishable from one another.P to N) for comparing the polymorphism levels of different populations.Polymorphism level was also calculated. To eliminate the effect of sample number, we proposed the parameter of relative polymorphism  PCA type.Additionally, the XLStat software was used for hierarchical clustering, and dissimilarity was calculated using the DICE coefficient, with agglomeration by the Ward’s method. PCA analysis was used to find associations between traits. The joint matrix for these analyses contained phenotypic matrix data  and the genetic marker data (53 polymorphic ISSR and SCoT bands) in 52 accessions of D. lotus in the Northwestern Caucasus. However, a higher vitality index was observed in the northernmost population in Shkhafit, indicating a greater adaptive potential of this population, as compared to the other four populations.Among the different morphological traits, several traits were established as more informative with a low level of general variability: leaf length, leaf width, LI and length of the fourth veins. Generally, the Depressive-type ontogenetic strategy and a low level of intraspecific genetic diversity was observed in D. lotus are due to both existing variation or new mutations. Unique SCoT and ISSR fragments were detected for the Shkhafit population and can be used as markers for breeding purposes. The results provide a better understanding of the adaptive mechanisms of D. lotus in extreme environments, and will be important for the further expansion of the cultivation area for persimmon into colder regions.The strong associations of several morphological traits  and several marker bands  were detected for the Shkhafit population. Based on these results, it can be concluded that most adaptations in


Southern Thailand is home to various populations; the Moklen, Moken and Urak Lawoi’ sea nomads and Maniq negrito are the minority, while the southern Thai groups (Buddhist and Muslim) are the majority. Although previous studies have generated forensic STR dataset for major groups, such data of the southern Thai minority have not been included; here we generated a regional forensic database of southern Thailand. We newly genotyped common 15 autosomal STRs in 184 unrelated southern Thais, including all minorities and majorities. When combined with previously published data of major southern Thais, this provides a total of 334 southern Thai samples. The forensic parameter results show appropriate values for personal identification and paternity testing; the probability of excluding paternity is 0.99999622, and the combined discrimination power is 0.999999999999999. Probably driven by genetic drift and/or isolation with small census size, we found genetic distinction of the Maniq and sea nomads from the major groups, which were closer to the Malay and central Thais than the other Thai groups. The allelic frequency results can strength the regional forensic database in southern Thailand and also provide useful information for anthropological perspective. Most people are southern Thai Buddhists (66%) and southern Thai Muslims (33%), while the minorities, e.g. sea nomad and Maniq groups account for about 0.33%2. The populations of the three groups of sea nomads are 4000, 2000 and 3000 for Moklen, Moken and Urak Lawoi’, respectively, while only 250 were recorded for the Maniq2. The languages spoken in southern Thailand belonging to three linguistic families: Tai-Kadai (TK), Austroasiatic (AA) and Austronesian (AN). The AA-speaking Maniq who are scattered through the jungle are regarded as the indigenous people of Southeast Asia or often referred to “negritos” because of their phenotypic difference and traditional mode of subsistence practice that is hunter-gatherers3. The AN-speaking sea nomads were used to subsist through maritime foraging in most of the year, although nowadays they prefer to settle in the coastal area of Thailand and Myanmar4. Both Maniq and sea nomads were minority groups and thought to be native in southern Thailand since prehistorical time, together with the other groups, e.g. AA-speaking Mon and Khmer before the occupation of the AN-speaking Malays and TK-speaking Thais, thought the Mon and Khmer people are nowadays disappear in southern Thailand5.Southern Thailand lies on the Malay Peninsula, bordering the Gulf of Thailand to the East, the Andaman Sea to the West, and Malaysia to the South. A census size of ~ 9.16 million in southern Thailand is about 13.35% of the total census size of the country (68.61 million in 2020)7. In Thailand, studies on forensic microsatellites and also other markers have focused on northern, northeastern and central Thailand leaving the southern region understudied12. The only one study on autosomal STRs in southern Thailand indicated that the Thai-Malay Muslim and Thai Buddhist who lived in the five deep Southern Thai provinces had non-significant genetic difference13.The autosomal short tandem repeats (STRs) show a number of advantages for both population genetic and forensic studies, i.e. distribution across the human genome which led to escape from natural selection, highly polymorphism and informativeness to distinguish recently diverged populations4. The mitochondrial (mt) DNA investigation of the Moken showed ancient basal mtDNA haplogroup M21d and M46 with very low genetic diversity4. The basal mtDNA haplogroup M21a, R21 and M17a and Y chromosomal haplogroup K were also observed in the Maniq as well as close genetic affinity between the Maniq and other indigenous people of Southeast Asia in Malaysia, reflecting an ancient ancestry of the Maniq and common genetic ancestry of indigenous people of Southeast Asia in the Malay Peninsula3.In addition, there were some other genetic studies on southern Thai populations using uni-parentally inherited markers14. In addition, because the forensic database combined diverse southern Thai populations has not yet been established, we created a regional DNA database of 15 autosomal STRs of southern Thailand.To expand the genetic studies in southern Thailand, we reported genotypes of 15 autosomal STRs of seven southern Thai populations: one AA-speaking Maniq, four AN-speaking Moklen, Moken, Urak Lawoi’ and southern Thai Muslim and two TK-speaking southern Thai Buddhist and southern Thai Takbai. We explored genetic structure and relationships of southern Thai populations with other Thai and Malaysian populations3. When the genetic diversity calculated form the same marker set was compared between two hunter-gatherer groups in Thailand, the Maniq from the South had diversity value greater than the Mlabri from the North (0.547 ± 0.288)15 although the sample size of Maniq (n = 15) is lower than the Mlabri (n = 19). Also the genetic diversity results of these 15 STRs of ~ 70 Thai populations15 revealed that the Mlabri had the lowest genetic diversity, indicating a strong genetic drift of the Mlabri. Regarding the sea nomads and excluding the Moken due to their small sample size, the Moklen and Urak Lawoi’ showed lower genetic diversity than other Thai and Malaysian populations (Table Raw genotypic data of 15 STRs of 334 southern Thai samples are provided in Table D19S433 and D18S51) that depart from the Hardy–Weinberg equilibrium (HWE) even after applying Bonferoni adjustment (p < 0.0033). Although the forensic parameters show that both loci are highly discriminating (power of discrimination (PD) = 0.9246 for D19S433 and 0.9513 for D18S51) and power of exclusion (PE) = 0.5757 or D19S433 and 0.6873 for D18S51)), the lack of HWE must be taken into account in forensic investigation. A total of 157 alleles were detected, ranging from 6 alleles at TPOX to 21 alleles at FGA. The maximum allele frequencies is observed in TPOX (0.5472). The lowest expected heterozygosity (HE) was observed in the TPOX (0.6201), while the highest HE was in the FGA (0.8690) (Table TPOX) to 0.8529 (D2S1338) and matching probability (MP) values are from 0.0374 (FGA) to 0.2037 (TPOX) (Table TPOX) to 0.9673 (D2S1338) (Table D3S1358) to 0.7588 (FGA)  among 17 Thai and Malaysian populations showed that the Maniq (MN) and Urak Lawoi’ (UL) were genetically different from each other and from other populations , except with the pairs of newly generated southern Thai Muslim (MST) and Moken. However, due to the effect of very small sample size, the Moken did not differ from almost populations. In general, the Maniq and sea nomads from southern Thailand exhibited genetic differentiation from the other groups. Then, the matrix of Rst were constructed to multi-dimensional scaling (MDS) plots. The three-dimensional MDS result based on dimension 1 and 2 showed genetic distinction of Maniq (MN) and three sea nomads, i.e. Moklen (MLK), Moken (MOK) and Urak Lawoi’ (UL) from the other groups from Thailand and Malaysia. The MDS analysis based on dimensions 3 showed genetic differences of Urak Lawoi’ from other populations , as represented by orange, while the second clusters (purple) stood out in the sea nomads: Moklen (MLK) and Urak Lawoi’ (UL), supporting their genetic uniqueness , the Urak Lawoi’ and Moklen showed genetic dissimilarity with the latter displaying genetic admixture with other populations. According to ethnolinguistic background, the Moklen are more closely related to the Moken and both of them are more distant from Urak Lawoi’4. Although languages of sea nomads were grouped within Austronesian family and Malayo-Polynesian sub-family, different in dialects were spoken; the Urak Lawoi’ or Orang Laut speak Malayic that distantly related to both Moken and Moklen who shared many cultural connections. In addition, the Urak Lawoi’ was culturally isolated but the Moklen had frequently interacted with and influenced by other southern Thais18. Therefore, the unique genetic signature of Urak Lawoi’ and mixed ancestries of Moklen could be described by ethnolinguistic and cultural evidence. Third, we found more genetic similarity between major southern Thais and populations from central Thailand than other regions. The present result was in agreement with a recent genome-wide study19 that could be explained by historical evidence; there were movements from the central region to the south during the Ayutthaya Period (during 1350–1767 A.D.)20 and genetic admixture between the southern Thai and Malays after the settlement period might be possible13.Overall, there were three main observations according to genetic relationship results. First, the Maniq and sea nomads exhibited extremely genetic differences from other Thai and Malaysian populations. The distinct genetic structure coupled with low genetic diversity Table  is proba5. South Asian connections of southern Thai populations could be possibly driven by previous admixture, in agreement with previous study on genome-wide data19.A neighbor-joining (NJ) tree based on allele frequencies of 15 STR loci among 29 Asian populations reveals four clusters of populations. Cluster 1 consists of populations from Island Southeast Asia and Malaysia while the South Asian populations occupy cluster 2. Cluster 3 comprises of Mainland Southeast Asian populations and cluster 4 belongs to the Thai sea nomads, Maniq from Thailand and Indonesians from Bali, with the extreme divergence of Maniq  and Naresuan University Institution Review Board (COA No. 0464/2017). When combined with previously published southern Thai Buddhist and southern Thai Muslim dataes Table .Genomic DNA was extracted from buccal swabs using the Gentra Puregene Buccal Cell Kit  according to the manufacturer’s instructions. Each DNA sample was amplified for 15 STR loci in a multiplex PCR using a commercial AmpFlSTR Identifiler kit  according to the manufacturer’s protocols. The amplicons were genotyped by multi-capillary electrophoresis on an ABI 3130 DNA sequencer (Applied Biosystem), and allele calling was performed by the software GeneMapper v.3.2.1 (Applied Biosystem).21 was used to calculate allele frequency, Hardy–Weinberg equilibrium (HWE) P values, observed heterozygosity (HO), expected heterozygosity (HE), total alleles, and gene diversity (GD). Significant levels for the HWE were adjusted according to the sequential Bonferroni correction (α = 0.05/15)22. We used the Excel PowerStats spreadsheet23 to compute several forensic parameters, including power of discrimination (PD), matching probability (MP), polymorphic information content (PIC), power of exclusion (PE), and typical paternity index (TPI) as well as the combined PD (CPD), combined MP (CMP), and combined PE (CPE). To reveal population relationships and population structures, we also combined genotyping data of additional eight populations from northern Thailand (Yuan and Yong), northeastern Thailand (Khmer and Lao Isan) and central Thailand 37, and Malaysia 14 (Table Rst) was generated by Arlequin, and the matrix was then plotted in two dimensions by means of multidimensional scaling (MDS) using Statistica v.10 demo . The heatmap visualization of Rst and MDS values were obtained using R package (R Development Core Team).Arlequin v.3.5.2.226. We ran ten replications for each number of clusters (K) from 1 to 11 and used a burn-in length of 100,000 iterations, followed by 200,000 iteration running length. We used STRUCTURE Harvester27 to compute a second-order rate of change logarithmic probability between subsequent K values (△K) in order to identify the optimal K value in the data16. We used CLUMPAK28 and DISTRUCT29 to generate the final results of STRUCTURE. To evaluate genetic relatedness with other Asian populations, we used POPTREE v.230 to generate a neighbor-joining (NJ) tree based on Fst computation by allele frequency of 15 STR loci of 29 populations from South and Southeast Asia39.To delineate cryptic population structure using the Bayesian clustering method, we performed STRUCTURE version 2.3.4 under the following prior parameters: admixture, correlated allele frequencies, and assistance of sampling locations (LOCPRIOR model)The rights of participants and their identity have been protected during the whole process of this research. All experiments were performed in accordance with relevant guidelines and regulations based on the experimental protocol on human subjects which was approved by the Khon Kaen University Ethic Committee (Protocol No. HE622223) and Naresuan University Institution Review Board (COA No. 0464/2017).Supplementary Table S1.


The Laperrine’s olive is endemic to the Saharan Mountains. Adapted to arid environments, it may constitute a valuable genetic resource to improve water-stress tolerance in the cultivated olive. However, limited natural regeneration coupled with human pressures make it locally endangered in Central Sahara. Understanding past population dynamics is thus crucial to define management strategies. Nucleotide sequence diversity was first investigated on five nuclear genes and compared to the Mediterranean and African olives. These data confirm that the Laperrine’s olive has a strong affinity with the Mediterranean olive, but it shows lower nucleotide diversity than other continental taxa. To investigate gene flows mediated by seeds and pollen, polymorphisms from nuclear and plastid microsatellites from 383 individuals from four Saharan massifs were analyzed. A higher genetic diversity in Ahaggar  suggests that this population has maintained over the long term a larger number of individuals than other massifs. High-to-moderate genetic differentiation between massifs confirms the role of desert barriers in limiting gene flow. Yet contrasting patterns of isolation by distance were observed within massifs, and also between plastid and nuclear markers, stressing the role of local factors  in seed and pollen dispersal. Implications of these results in the management of the Laperrine’s olive genetic resources are discussed. Cupressus dupreziana A. Camus), the Nivelle’s myrtle (Myrtus nivellei Batt. & Trab.) and the Laperrine’s olive (Olea europaea L. subsp. laperrinei. (Batt. & Trab.) Cif.) ). DNA was extracted from the embryo as previously reported [.l2 [Here, we focused on the Central Saharan populations . Foul1 , 237 genl1 ,35, and l1 ,22,23). reported . This inted [.l2 ,34 and wCUL4 (cullin gene), FAD6 , MST2 (monosaccharide transporter-2 gene), OCO (constans-like gene), and OEW (lupeol synthase gene). For each locus, sequences of different alleles were obtained following the strategies previously described [Nuclear gene sequences are useful to detect recent admixture and also to compare patterns of nucleotide diversity among related species ,37. Partescribed . Nine inescribed ; Table SWe then genotyped all trees with microsatellites loci  for population genetics analyses. Twelve nuclear SSR loci were used to characterize 382 individuals . Nine oflaperrinei were combined to the 60 allelic sequences previously isolated from 17 Mediterranean olives and 13 African olives in the native range [Muscle implemented in Mega v7.0 [π [Mega as the average number of substitutions per site between two haplotype sequences. This parameter was estimated separately on subspp. europaea, cuspidata, and laperrinei, and then on the whole sampling. The standard error of these values was estimated by bootstrapping on haplotypes (100 replicates).For each locus, the 21 allelic sequences (or haplotypes) of subsp. ve range . All seqega v7.0 . The nuc v7.0 [π  was measNetwork v4.112 [OEW, but these two regions were excluded from our analyses because of an expected high homoplasy on these motifs .Network [For each individual, a plastid DNA haplotype (or chlorotype) was defined by the combination of alleles from the polymorphic loci. For the allele coding, we followed the procedure previously described . The geoNetwork .Fstat v2.9.3 [Fstat to depict genetic diversity patterns among populations: (i) First, the total heterozygosity HT [HT = 1 − ∑ i²p, where ip is the probability to meet the allele i at a given locus for the whole population. For a single nuclear SSR locus, this estimator represents the average proportion of individuals that carry two different alleles. HT is more rightly called genetic diversity in the case of haploid plastid markers. (ii) Second, the allelic richness RS [eN [eN = 1 / ∑ i²p. A different number of alleles can lead to the same heterozygosity according to the allele frequencies. eN represents the number of alleles of equal frequency required to lead to the same heterozygosity in a population. This estimator enables the comparison between populations or subpopulations based on their most frequent haplotypes.First, we tested for linkage disequilibrium between all pairs of nuclear SSR loci using t v2.9.3 . Then, tosity HT  was calchness RS  was meashness RS . (iii) As RS [Ne  was estiTo test the significance of the difference in both total heterozygosity and allelic richness between populations or subpopulations, a paired Wilcoxon test was carried out with a Bonferroni correction. We particularly tested whether the allelic richness was higher in Ahaggar than in other massifs  as previously reported .K) in the whole dataset and separately for the Hoggar and the Aïr populations (see below) was determined using two model-based clustering methods. We first used the snapclust clustering approach of the adegenet RStudio package [Structure v2.3.4 [K-means method  statistics [K value. The Structure analysis was run under the admixture model for 1,000,000 generations after a burn-in period of 200,000, assuming a correlation among allele frequencies. Analyses were run for K between one and ten clusters with ten iterations for each K value. The most likely number of clusters was determined using the ad hoc measure ΔK [K clusters (H’) was calculated with Clumpp v1.1.2 [K value, each accession was assigned to each cluster with a posterior membership coefficient (p). This analysis was done on the whole sampling  and the 226 genotypes without missing data.Populations and subpopulations used in this study are arbitrary delimited based on geographical criteria. To check the relevance of this separation, it is possible, using nuclear markers, to define different genetic clusters within the sampling without a priori on the origin of the trees. The number of genetically homogeneous clusters .To assess the potential effect of geographic distance on gene flow , we analeDi v1.5 . The p-vFST [Fstat between each pair of populations (massifs) or subpopulations (patches of trees in a given massif) on the nuclear and plastid data. These values reflect the intensity of isolation  between populations or subpopulations. The IBD was tested at two different geographic scales, i.e., across the Central Saharan massifs (from Aïr to Hoggar) and at the massif scale (considering 15 subpopulations from Ahaggar on an area covering ca. 3000 km2). Rousset [FST / (1 − FST) with the geographic distance (or the log of distance) provides an easier interpretation of the differentiation between subpopulations. In order to test the significance of the IBD pattern for the studied species, a Mantel test was computed with Genetix [In addition, differentiation measures based on FST  were als Rousset  develope Genetix . In this2), at the massif scale  and at the local scale . Gene flow in plants can be estimated by indirect methods [R):Finally, we investigated the relative contribution of pollen and seeds in the global gene flow at three different geographic scales, i.e., across the Central Saharan massifs  a very low mutation rate compared to migration rate, (ii) constant and equal sizes of populations, and (iii) a constant and equal migration rate between populations. The last two points represent what is called a drift/migration equilibrium. This equilibrium turns out to be rapidly reached in nature [This estimation is based on a model of island migration, where n nature . Howevern nature . Geneticn nature .MST2 and OEW, three on FAD6 and OCO, and six on CUL4. Several haplotypes were already identified in the Mediterranean olive and were named with codes used by [laperrinei  appeared to be related to African olive haplotypes. Six of the most frequent alleles (>5 occurrences) detected in the Laperrine’s olive were shared with the Mediterranean olive , which was also shared with Mediterranean oleasters (π = 0.179 ± 0.090) compared to subspecies europaea (π = 0.798 ± 0.212) and cuspidata .HO and HT values were not significantly different regardless of the considered locus or massif  and Bagzane . The other comparisons between massifs were not significant. On the other hand, there was no significant difference between massifs in the total heterozygosity (HT), with average values ranging from 0.62 (Bagzane) to 0.67 (Tassili n’Ajjer). Finally, the highest effective number of alleles was observed in Ahaggar (eN = 4.57) and the lowest was observed in Bagzane (eN = 3.50). In Ahaggar, HT and SR were higher in the Adrar Heggueghene subpopulations  than in northernmost subpopulations sampled at an elevation above 1700 m , although these values were not significantly different given the limited number of loci . A trend of SR reduction with increasing elevation was also revealed in all trees sampled in the Ahaggar massif  are connected by a single mutation step and three of them were located at the center of the network. In contrast, 10 of the 15 chlorotypes with a frequency equal or inferior to 2% were at tip position, and 7 of them may derive from the most frequent chlorotypes. The relationships between E1-l1.11, E1-l1.12 and E1-l1.13 were not resolved and may be due to a homoplasious mutational change  that were specific to this region. Individuals from Tamgak, which was geographically close to Bagzane-Egalah (70 km), harbored three frequent chlorotypes  that were all shared with the Ahaggar population. This latter contained the highest plastid DNA diversity with 15 chlorotypes, among which 11 were unique to this location. Lastly, four chlorotypes  were detected in the Tassili n’Ajjer population, but only one rare chlorotype  was unique to this massif. The allelic richness , while the lowest values were found in Tassili n’Ajjer . In Ahaggar, a particularly high plastid DNA diversity was observed in subpopulations of Adrar Heggueghene (SR = 13.53 (computed for 72 genotypes), HT = 0.77), which contrast with subpopulations sampled at higher elevation . Such a trend of chlorotype diversity reduction with increasing elevation was also found in all the trees sampled in the Ahaggar massif , while others were restricted to a site or a limited region . In BagzFST) was detected between Tassili n’Ajjer and Bagzane (11.9%), whereas the lowest value was observed between Ahaggar and Tassili n’Ajjer (2.5%). The differentiation values were higher on plastid markers, with FST ranging from 9.4% between Tamgak and Ahaggar to 55.4% between Tassili n’Ajjer and Bagzane  was observed  was still observed in Tamgak (2 < 0.01) but still significant IBD pattern was revealed in Ahaggar, probably thanks to a denser sampling in this massif.Patterns of IBD were first investigated on nuclear data. At the Central Sahara scale, a highly significant IBD pattern  than for nuclear loci , and was intermediate at the massif scale (R = 17.08 in Ahaggar). In the Mediterranean Basin, a similar trend was revealed, since R reached 7.06 on the whole area, while the value tended to increase both in the western and central regions  but remained similar in the eastern area  was found in five nuclear gene regions among nine trees sampled over the whole distribution area of the Laperrine’s olive  supports the hypothesis of gene introgression from subsp. cuspidata into the Laperrine’s olive gene pool, but the contribution of this taxon seems minor in comparison to the Mediterranean olive.Relatively low variation . Moreover, measures of differentiation on each genome could be differently biased due to variable mutation and homoplasy rates [At the scale of Adrar Heggueghene, a low and nonsignificant geographic structure was revealed based on nuclear SSRs , suggestsy rates ,70. In asy rates  dependinsy rates . But untsy rates .Knowledge of the genetic structure of olive populations and their dispersal ability is key to identify taxonomic units or gene pools and then to define appropriate conservation actions . PopulatThese genetic resources should also be conserved in gene banks. Individuals of each main gene pool have to be collected to get a representative sampling of the diversity of the Laperrine’s olive. Until now, the only collection of Laperrine’s olive was constituted with seedlings sampled from very few places in the Ahaggar massif . The genetic diversity of this collection was, however, shown to be significantly reduced when compared to the initial population, as the consequence of seeds collected on a limited number (12) of mother trees ,31. To l


Eucryptorrhynchus brandti (Harold) and the tree‐of‐heaven root weevil (TRW), E. scrobiculatus (Motschulsky) on the tree‐of‐heaven across their native range of China. We analyzed the population genetics of the two weevils based on ten highly polymorphic microsatellite markers. Population genetic diversity analysis showed strong population differentiation among populations of each species, with FST ranges from 0.0197 to 0.6650 and from −0.0724 to 0.6845, respectively. Populations from the same geographic areas can be divided into different genetic clusters, and the same genetic cluster contained populations from different geographic populations, pointing to dispersal of the weevils possibly being human‐mediated. Redundancy analysis showed that the independent effects of environment and geography could account for 93.94% and 29.70% of the explained genetic variance in TTW, and 41.90% and 55.73% of the explained genetic variance in TRW, respectively, indicating possible impacts of local climates on population genetic differentiation. Our study helps to uncover population genetic processes of these local pest species with relevance to control methods.Increasing damage of pests in agriculture and forestry can arise both as a consequence of changes in local species and through the introduction of alien species. In this study, we used population genetics approaches to examine population processes of two pests of the tree‐of‐heaven trunk weevil (TTW),  Eucryptorrhynchus brandti (TTW) and E. scrobiculatus (TRW) populations based on 10 microsatellite markers.Genetic structure of 13 and 11 populations of  These pPopulation genetics approaches can help to reveal the population history of species and then infer whether the species is local or introduced outside of their native range  and the tree‐of‐heaven root weevil (TRW), E. scrobiculatus (Motschulsky), are the only two wood‐boring species of Eucryptorrhynchus in China  Swingle and its varieties A. altissima Qiantouchun in China and cause serious damage. They spend their larvae stage in the trunk (TTW) or root (TRW) of the host tree. There is a strict reproductive isolation between these two species of weevil. TRW lay eggs in the soil near the roots of Ailanthus altissima, while TTW lay eggs in the trunk of Ailanthus altissima , ina Liu, . The twoIn this study, we examined the population genetic diversity and structure of the two weevils to infer their population history. Comparative study of the two weevils allows us to check if the two co‐occurring pests show similar population genetic processes. We hypothesized that the increasing damage by the two species in China is related to human‐mediated dispersal of seedings, based on the low flight ability of the two species and their life cycle. We also tested whether local climate can have an impact on the population genetic structure of the two species.22.1We collected 13 TTW and 11 TRW populations across their native range of China , and polymorphism information content (PIC) were analyzed by the macros in Microsatellite Tools , and inbreeding coefficients (FIS) were estimated in GENEPOP v4.0.11  and allelic richness of private alleles (PAR) of each site. We used GENCLONE v2.0  and the unbiased expected heterozygosity (HE). We compared the number of alleles (AS) and heterozygosity (HES) among samples with different sample sizes in GENCLONE.The number of alleles, observed heterozygosity  associated with the optimal K were processed using CLUMPP v1.12  was used to analyze population genetic structure under default settings ) with geographic distance (km) and climate data using the R package ade4 v 1.7‐15, with 10,000 permutations . The standard (19) WorldClim Bioclimatic variables for WorldClim v2 at a 10‐min spatial resolution were used.Geographic and climatic effects on population genetic variation were examined with two methods. First, the Mantel test was used to test the presence of isolation by distance (IBD) and isolation by environment (IBE) by correlating pairwise genetic differentiation . The first half of the positive feature vector as the explanatory variable of the population structure was kept. To avoid high linearity among the PCNM and climate variables, we excluded variables with a variance inflation factor (VIF) exceeding 15 calculated in vegan v2.5‐6. We used the R package vegan v2.5‐6 to analyze the full model (environment and geography) and partial model (environment or geography) of RDA. The independent environmental effect was calculated by constraining the effect of geography, while the independent geographic effect was calculated by constraining the effect of the environment in a partial RDA analysis. The collinearity effect was calculated by subtracting the independent effects of environment and geography from the total variance explained in the full RDA analysis.Second, multivariate redundancy analysis (RDA) was used to examine the variance of microsatellite genotypes which can be explained by climate and geography and their collinear part . RDA is the PCA analysis of the fitted value matrix of the multiple linear regression between the variable matrix and the explanatory variable matrix  for each population ranged from 0.15 to 0.36. The inbreeding coefficient (FIS) ranged from −0.24 to 0.31, with an average value of 0.06; the polymorphism information content of each population (PIC) ranged from 0.15 to 0.30; the allelic richnesses (AR) of six populations were not quite significantly different, varying from 1.50 in LNDL to 2.47 in SDRZ. The private allelic richness was also low for the 13 populations. The standardized total number of alleles (AS) ranged from 20.70 in NXLW to 42.03 in SDRZ. The standardized expected heterozygosity (HES) varied from 0.28 in BJSY to 0.69 in TJTJ (Table FST) ranged from 0.0197 to 0.6650  of loci scored for TTW and TRW are shown in Tables HO) for each population ranged from 0 to 0.17. The inbreeding coefficient (FIS) ranged from −0.14 to 0.78, with an average value of 0.43; the polymorphism information content of each population (PIC) ranged from 0.10 to 0.23; the allelic richness (AR) of 11 populations was not significantly different, varying from 1.30 in BJCY to 1.84 in SDRZ. Private allelic richness was also low for the 11 populations. The standardized total number of alleles (AS) ranged from 14.00 in SXYC to 35.51 in BJSY. The standardized expected heterozygosity (HES) varied from 0.13 in SXYC to 0.67 in BJSY  in TTW Figure  and from3.4p = .47, r = −0.01; TRW, p = .15, r = 0.14), or environmental conditions , TTW and TRW can be spread through seedling transfer. This suggests migration of TTW and TRW mainly passively through human activities due to seedling transfer. Similar passive and human‐assisted ways of spread occur in other pests, such as the Frankliniella occidentalis ; Formal analysis ; Resources (lead); Writing – original draft . Wei Song: Formal analysis ; Writing – original draft . Li‐Jun Cao: Data curation ; Formal analysis . Jin‐Cui Chen: Resources . Ary A. Hoffmann: Writing – review & editing . Junbao Wen: Funding acquisition ; Project administration ; Writing – review & editing . Shu‐Jun Wei: Conceptualization ; Writing – original draft ; Writing – review & editing .Supplementary MaterialClick here for additional data file.


Caulokaempferia coenobialis, which does not use anemochory or zoochory for seed dispersal; such studies could contribute to an improved understanding of the effect of rivers or streams on population genetic diversity and structure in riparian plants. Using polymorphic ISSR and cpDNA loci, we studied the effect at a microgeographic scale of different stream systems  in subtropical monsoon forest on the genetic structure and connectivity of C. coenobialis populations across Dinghu Mountain (DH) and Nankun Mountain (NK).Rivers and streams facilitate movement of individuals and their genes across the landscape and are generally recognized as dispersal corridors for riparian plants. Nevertheless, some authors have reported directly contrasting results, which may be attributed to a complex mixture of factors, such as the mating system and dispersal mechanisms of propagules (seed and pollen), that make it difficult to predict the genetic diversity and population structure of riparian species. Here, we investigated a riparian self-fertilizing herb C. coenobialis within each stream system. Furthermore, downstream local populations do not accumulate genetic diversity, whether in the linear streamside local populations across DH (H: 0.091 vs 0.136) or the dendritic streamside local populations across NK . Our results show that the connectivity of local C. coenobialis populations across DH and NK can be attributed to historical gene flows, resulting in a lack of spatial genetic structure, despite self-fertilization. Selfing C. coenobialis can maintain high genetic diversity  through genetic differentiation , which is intensified by local adaptation and neutral mutation and/or genetic drift in local populations at a microgeographic scale.The results indicate that the most recent haplotypes  are not shared among local populations of C. coenobialis, and conservation strategies for maintaining genetic diversity of selfing species should be focused on the protection of all habitat types, especially isolated fragments in ecosystem processes.We suggest that streams are not acting as corridors for dispersal of The online version contains supplementary material available at 10.1186/s12870-021-03101-7. Primula sieboldii, [Heliconia metallica, [Genetic variation in a plant species is determined by mating system, natural selection, evolutionary history, life-history characteristics, and mechanisms of gene flow , and these factors can lead to complex genetic structuring of populations within the species , 2. Thereboldii, ; Helicontallica, ), are raHibiscus moscheutos, [Primula sieboldii, [Mauritia flexuosa, [Rivers or streams facilitate movement of individuals and genes across the landscape and are generally recognized as corridors for riparian plants , 28. Thecheutos, ; Primulaeboldii, ; Mauritilexuosa, ). HoweveCaulokaempferia coenobialis (Hance) K. Larsen  data, focusing on the following questions: (1) does selfing C. coenobialis show low genetic diversity and significant spatial genetic structure on a microgeographic scale, as theory predicts? (2) do streams facilitate the dispersal of C. coenobialis seeds among local populations along the longitudinal course of a stream, thus leading to an accumulation of genetic diversity in downstream local populations, as theory predicts?sen Fig.  is a decH) and the Shannon indices (I) of C. coenobialis were 0.251 and 0.382, respectively. The genetic diversity index of NK was greater than that of DH . At the local population level, genetic diversity index ranged from 0.058 to 0.136  and 0.089 to 0.211. Local populations JLS (DH) and TYSZ (NK) showed the highest levels of genetic diversity , while local populations FST  and SLCX  exhibited the lowest levels. Among all local populations except JLS (DH) and TYSZ (NK), common loci  accounted for the highest proportion of amplified fragments (34.3–60.4%), while low-medium gene frequency loci (5% < gene frequency ≤ 50%) accounted for a higher proportion of amplified fragments (23.6–41.9%) than medium–high gene frequency loci (50% < gene frequency < 100%) (13.2–24.1%)  from the 13 local populations in metapopulations DH and NK, of which 213 (97.7%) were polymorphic and 6 (2.8%) were specific (Table h = 0.869) and average nucleotide diversity (π = 2.375 × 10–3). At the local population level, haplotype diversity and nucleotide diversity ranged from 0 to 0.800 (average 0.489) and from 0 to 3.170 × 10–3 (average 0.845 × 10–3), respectively. All local populations except SH and XXPB (NK) were polymorphic, while local populations JLS (DH) and SLCX (NK) contained four and three different haplotypes, respectively.The concatenated and aligned cpDNA sequences for metapopulations DH and NK were a total of 1262 and 1191 base pairs (bp) long, respectively. The polymorphisms identified 20 haplotypes in DH and NK, of which eight haplotypes (H1-H8) resided in DH and 12 haplotypes (h1-h12) resided in NK (Table GST) for the local populations of C. coenobialis in DH and NK was 0.5034 and 0.6796, respectively, which indicates that 49.66% and 32.04% of total genetic variability was distributed within local populations in DH and NK, respectively. The estimated gene flow (Nm) per generation among local populations in DH and NK was 0.4933 and 0.2357, respectively. In NK, the genetic differentiation among the transverse hydrological system local populations in NK-II were greater than among the dendritic streamside system local populations in NK-I (GST: 0.6589 vs 0.5982); accordingly, the mean estimated gene flow (Nm) among local populations in NK-II is lower than that in NK-I (0.2588 vs 0.3359). AMOVA results  among local populations was estimated as 0.556 and 0.770 for DH and NK, respectively . AMOVA  trees based on ISSR Nei’s genetic distance  and ISSR  data    or ISSR  data  Fig.  B-2, altZingiber species at both the macrogeographic and microgeographic levels [Z. corallinum was significantly lower than that in outcrossing Z. nudicarpum, the level of species/metapopulation genetic diversity of selfing Z. corallinum was comparable to that of outcrossing Z. nudicarpum. Our results are consistent with those of the above comparative study [C. coenobialis is comparable to that of outcrossing Z. nudicarpum  despite the within-population genetic diversity (Nei’s H = 0.098) being significantly lower than that in outcrossing Z. nudicarpum . This implies that, similarly to outcrossing species, selfing plant species can maintain a high level of species genetic diversity—albeit by using different strategies—on both the microgeographic and macrogeographic scales.The mating system is a major factor affecting the genetic variability of plant species. Numerous studies have shown that selfing plant species have less genetic diversity at both the population and species levels than outcrossing species , 68–70. c levels , 56 reveC. coenobialis metapopulations, any mutation that arises in a particular local population may fix in that local population and cannot spread to other local populations. The present results based on ISSR and cpDNA data confirmed that the genetic differentiation among local populations was relatively high in both DH  and NK  metapopulations. The proportion of ISSR common loci was highest within local populations of C. coenobialis, but common loci and low-to-medium gene frequency loci accounted for the lowest proportion and the highest proportion within metapopulations DH and NK, respectively . Evolutionary theory predicts that genetic drift will result in substantial local differentiation if Nm < 1 [C. coenobialis in our study. The estimates of ISSR gene flow (Nm) between local populations of C. coenobialis in DH and NK were only 0.4933 and 0.2357 on average (Nm < 1), respectively, which can be attributed to a lack of pollen migration within and among local populations [C. coenobialis are highly heterogeneous, thus causing an increase in genetic variation [C. coenobialis can maintain high genetic diversity through differentiation intensified by local adaptation and neutral mutation and/or by the stochastic force of genetic drift in local populations on a microgeographic scale. Thus, conservation strategies for maintaining and improving genetic diversity in selfing species should be focused on the protection of all habitat types, especially isolated fragments in ecosystem processes.Unlike outcrossing species, the high species genetic diversity level of selfing plant species may result from a low pollen migration rate, which leads to high levels of genetic differentiation among populations , 72. Witely Fig. . Togethef Nm < 1 , 74, sinf Nm < 1 . This isulations , 65, as ulations , 67, as ariation . Based or = 0.007, p = 0.428; cpDNA: r = -0.344, p = 0.423) among local populations of the DH metapopulation, implying that historical dispersal and/or contemporary gene flow by seed dispersal and pollen movement might occur among local populations [C. coenobialis and thus lack of pollen migration within and among local populations, together with limited seed dispersal, which was confirmed by the low gene flow (Nm = 0.4933) between local populations of C. coenobialis across DH, contemporary gene flow should not occur among local populations. Moreover, our results showed that some ancestral haplotypes  were shared in DH local populations, but not the most recent haplotypes . This confirmed that historical dispersal, but not contemporary gene flow, has occurred among local populations of C. coenobialis across DH.Self-fertilizing species, with their reduced genetically effective population size and gene flow processes, should be characterized by strong spatial genetic structure , 77–79. ulations , 80. Duer = 0.262, p = 0.152) and that some ancestral haplotypes  were shared among local populations. These results also indicate that historical dispersal has occurred in local populations across NK. However, a weak relationship between local populations was detected in the ISSR data  and the gene flow was very low (Nm = 0.2357), implying that contemporary gene flow between local populations is limited by restricted seed dispersal and absence of pollen movement [C. coenobialis [C. coenobialis across NK may have undergone shrinking and fragmentation relatively recently, and that this was accompanied by a greatly decreased gene flow and increased genetic differentiation among local populations. Numerous studies have shown that fragmentation decreases habitat size and increases habitat isolation of populations in many terrestrial ecosystems, and that this reduces gene flow and genetic variation, while increasing the inter-population genetic divergence of plant populations [C. coenobialis across NK was substantial. In fact, as a result of increasing tourism in the NK region, habitat connectivity among local populations of C. coenobialis has been destroyed by roads, farm lands and holiday villages, which have likely hindered propagule dispersal. The cluster and STRUCTURE results reveal a clear pattern of population structure in C. coenobialis, with two clusters corresponding to two areas in NK, the West-North area  and the East-South area . The two areas are separated by the G355 National Road along a valley. Mantel tests based on cpDNA and ISSR data applied to the two clusters separately suggest that there is no significant correlation between geographical and genetic distance in both the dendritic streamside system local populations  and the transverse hydrological system local populations . This is also consistent with historical dispersal , but not recent gene flow.Similarly, Mantel tests based on cpDNA data showed that there was no significant spatial genetic structure in local populations across NK  or in the dendritic streamside local populations across NK . This implies that downstream local populations do not accumulate genetic diversity, which we attribute to the absence of movement of seeds and propagules from upstream to downstream local populations. This was confirmed by a lack of accumulation of particular cpDNA haplotypes in downstream local populations , whether in the linear streamside local populations across DH  or in the dendritic streamside local populations across NK . In addition, the high degree of differentiation and low level of gene flow (Nm) among local populations of C. coenobialis also suggests that seed dispersal among streamside local populations has been restricted across both DH and NK. The limited seed dispersal in C. coenobialis is apparent from the significant positive autocorrelation of spatial genetic structure being detected only at 0.20—0.92 m. Our field observations showed that the small seeds of C. coenobialis, which adhere to the axial placentation in an unilocular capsule that opens via a large oval slit in the upper part  and NK  indicate that the connectivity of selfing C. coenobialis local populations in the two mountain areas studied could be attributed to historical gene flows, which occurred between the Holocene and Pleistocene. Further study is needed to investigate these hypotheses in more detail.Studies have shown that gene flow between populations is largely congruent with river basins and the direction of water flow within and among them, suggesting that rivers and streams are important for seed dispersal , 90. Indart Fig. , can be Caulokaempferia coenobialis (Hance) K. Larsen  were deposited in the Herbarium of School of Life Science, South China Normal University (SN). The field work permits were obtained from the Dinghu Mountain National Nature Reserve Administration and the Nankun Mountain Provincial Nature Reserve Administration. The sample collection work and molecular experiments complied with local legislation, national and international guidelines, and did not involve protected species. We also abide by the Convention on the Trade in Endangered Species of Wild Fauna and Flora.To investigated the patterns of genetic diversity and structure within and between populations in subtropical monsoon forest at a fine scale, two metapopulations with different streamside local population systems were studied in the Dinghu Mountain National Nature Reserve  and Nankun Mountain Provincial Nature Reserve , both in Guangdong, but separated by nearly 160 km  were conducted on a Bio-Rad T100™ Thermal Cycle  under the following conditions: initial denaturation at 94 °C for 5 min, followed by 39 cycles of 45 s at 94 °C, 45 s at a primer-specific annealing temperature, extension for 90 s at 72 °C, and a 10-min final extension step at 72 °C. The PCR products were sequenced with an ABI 3730XL automated sequencer .We screened eight non-coding plastid DNA regions that have revealed substantial levels of polymorphism based on chloroplast genome sequence of ectabile  and then2, 0.2 mM dNTPs, 1.0 μM primer, 1.5 units Taq polymerase, 50 ng template DNA and double-distilled water. ISSR PCR conditions were consistent with cpDNA PCR conditions. Amplification products were electrophoretically separated in 1.8% agarose gels, together with a 100 bp ladder as a size marker, and visualized on a UV transilluminator . All clear and reproducible amplified fragments were scored as presence (1) or absence (0) and converted into a binary data matrix. The annealing temperatures for cpDNA and ISSR primers are given in Table For ISSR analysis, ten polymorphic primers  were selected from 100 ISSR primers obtained from the University of British Columbia. Reactions were performed in a total volume of 20 μL containing 2.0 μL 10 × PCR buffer, 1.5 mM MgClh), nucleotide diversity (π) and gene flow (Nm) were calculated using DNASP v5.1 [FST) based on 1000 random permutations [For the cpDNA dataset, sequence data were aligned using MEGA X  and wereASP v5.1 . The perH) [I) [GST) [Fis = 1) because C. coenobialis plants are self-fertilization [For the ISSR dataset, Nei’s gene diversity (H) , ShannonH) [I) , percentI) [GST) , and genI) [GST) , assuminlization , 65. PaiR- and Φ-statistics, respectively. The significance of fixation indices was tested using 1,000 and 999 permutations, respectively [In order to quantify the variation in cpDNA sequences and ISSR gene frequency among local populations, we performed analyses of molecular variance (AMOVA) in ARLEQUIN v3.1  and GENAectively .Pyrgophyllum yunnanense and Kaempferia rotunda were used as the outgroup. ML analysis was carried out using MEGA X and the appropriate model (T92 + G + I) of DNA substitution was determined using the Akaike information criterion (AIC) as the selection criterion. The ModelFinder in PhyloSuite v.1.1.15 [Caulokaempferia, branch lengths of the clock-constrained ML tree were transformed into absolute time by assuming the substitution rates of these spacers to be 1.2–1.7 × 10–9 substitutions per site per year (s/s/y) in MEGA X. This is a rough estimate of the substitution rate in non-coding chloroplast regions of seed plants, which can be used to estimate the divergence time of taxa without fossil records in the same study area  and Bayesian inference (BI). Two individuals of v.1.1.15  was applv.1.1.15  with thexandrum, ; Rosa soulieana, ).q) for each genetic cluster were then averaged using Clumpp software [For the ISSR datasets, a Bayesian cluster was implemented using STRUCTURE version 2.2 . Five insoftware  to corresoftware  with 1,0software , and priTo test the relationship of geographical distance and genetic structure between local populations on a microgeographic scale, a Mantel test was performed in GENALEX ver.6.5 for ISSR data and ARLEQUIN v3.1 for cpDNA data, respectively. To detect spatial genetic structure (SGS) within a local population, the genetic relatedness of individuals relative to their spatial position within the TXL local population in DH was analyzed by spatial autocorrelation analyses in GenAlEx. The even sample classes were chosen because this was particularly useful for reducing noisy confidence limits when sample sizes were very uneven. Two-tailed probability values were calculated and bootstrap resampling was performed 999 times.Caulokaempferia coenobialis populations, which does not use anemochory or zoochory for seed dispersal, across Dinghu Mountain and Nankun Mountain. Such studies could contribute to an improved understanding of the effect of rivers or streams on population genetic diversity and structure in riparian plants. Our results show that streams are not acting as corridors for dispersal of C. coenobialis and the connectivity of local populations can be attributed to historical gene flows, resulting in a lack of spatial genetic structure, despite self-fertilization. Selfing C. coenobialis can maintain high genetic diversity through genetic differentiation, which is intensified by local adaptation and neutral mutation and/or genetic drift in local populations at a microgeographic scale.Using polymorphic ISSR and cpDNA loci, we studied the effect at a microgeographic scale of different stream systems  in subtropical monsoon forest on the genetic structure and connectivity of a riparian self-fertilizing herb Additional file 1:Fig. S1.Caulokaempferia coenobialis. a: plant, flower and habitat; b: seeds adhered to the axial placentation in an unilocular capsule opening by a large oval slit; c: seeds dispersed by rain splash. Fig. S2. Line graph of genetic cluster (K) vs. Delta K for metapopulations DH (A) and NK (B). Fig. S3. UPGMA dendrogram based on Nei's genetic identity for individuals of Caulokaempferia coenobialis in metapopulations DH (A) and NK (B). Table S1. Inter Simple Sequence Repeat (ISSR) data for Caulokaempferia coenobialis.