The translocation of wild animal species became a common practice worldwide to re-establish local populations threatened with extinction. Archaeological data confirm that chamois once lived in the Biokovo Mountain but, prior to their reintroduction in the 1960s, there was no written evidence of their recent existence in the area. The population was reintroduced in the period 1964–1969, when 48 individuals of Balkan chamois from the neighbouring mountains in Bosnia and Herzegovina were released. The main objective of this study was to determine the accuracy of the existing historical data on the origin of the Balkan chamois population from the Biokovo Mountain and to assess the genetic diversity and population structure of the source and translocated populations 56 years after reintroduction. Sixteen microsatellite loci were used to analyse the genetic structure of three source chamois populations from Prenj, Čvrsnica and Čabulja Mountains and from Biokovo Mountain. Both STRUCTURE and GENELAND analyses showed a clear separation of the reintroduced population on Biokovo from Prenj’s chamois and considerable genetic similarity between the Biokovo population and the Čvrsnica-Čabulja population. This suggests that the current genetic composition of the Biokovo population does not derive exclusively from Prenj, as suggested by the available literature and personal interviews, but also from Čvrsnica and Čabulja. GENELAND analysis recognised the Balkan chamois from Prenj as a separate cluster, distinct from the populations of Čvrsnica and Čabulja. Our results thus highlight the need to implement genetic monitoring of both reintroduced and source populations of endangered Balkan chamois to inform sustainable management and conservation strategies in order to maximise the chances of population persistence. Rupicaprarupicapra L.) is one of the examples of successfully translocated species  and Slovenia (Rupicaprarupicaprarupicapra)  . Since dpicapra) .Rupicaprarupicaprabalcanica) is one of the seven recognised subspecies of the Northern chamois. It is found both in the mountainous regions of Croatia and in the mountain ranges of the eight other countries of the Balkan Peninsula, from north to south: Bosnia and Herzegovina, Serbia, Montenegro, Kosovo, North Macedonia, Albania, Bulgaria, and Greece. The lack of continuity of these habitats and overhunting in the post-Neolithic period have severely fragmented the subspecies’ present distribution  and from remains of naturally dead animals (samples from Bosnia and Herzegovina).We collected 20 samples from Biokovo and 29 samples from three areas which serve as source populations for reintroduction and possible recent recolonisation . Details of sampling locations are given in Fig. N = 49) using the commercial peqGOLD Tissue DNA Mini Kit (PEQLAB Biotechnologie GmbH) following the manufacturer´s protocol in a final volume of 150 µL. DNA concentrations were measured with Qubit dsDNA BR Assay Kit  on a 3.0 Qubit Fluorimeter . Sixteen microsatellites were amplified using PCR multiplex sets previously investigated in studies with chamois (We extracted DNA from tissue samples (EM) algorithm implemented in FREENA (HWE). Values of null allele frequency greater than r ≥ 0.20 were reported  correction method, as described in STF values with the original STF values and to test the significance of null alleles in the analyses. The Wilcoxon Two Sample test was performed in R ver. 4.0.5 package stats  between all pairs of loci in all populations. A sequential Bonferroni procedure  and expected  and its confidence intervals. The number of private alleles was estimated using the GENALEX ver. 6.502 (ST) and calculate their respective p-values using 1000 permutations.GENETIX ver. 4.05.2  was used to calculate the mean number of alleles, observed  within the entire sample, and to estimate the proportions of each ancestral genotype in Balkan chamois individuals. We ran the analysis allowing for admixture and correlated allele frequency with ten independent runs for each K between 1 and 7 with a burn-in 500,000 steps followed by 105 Markov chain Monte Carlo (MCMC) iterations. The results of the repeated runs for each value of K were combined with the Greedy algorithm in CLUMPP v. 1.1.2 ] for each value of K. The same software was used to generate graphs for the mean log posterior probability of the data (mean ± SD).The Bayesian clustering program STRUCTURE ver. 2.3.4.  was usedv. 1.1.2 , and thev. 1.1.2 . To estieN) using the linkage disequilibrium-based method  and 10 (for locus BM1258) with an average value of 5.937 alleles per locus  but the deviation was significant at the 0.05 level only for locus SY434 after applying sequential Bonferroni adjustment  varied from 3.375 in the Čabulja population to 4.625 in the Biokovo population. All populations had private alleles (prN) and the highest number of private alleles (11) was observed in the Prenj population from Bosnia and Herzegovina  and expected (0.637) heterozygosity, and allelic richness 2.517). A similar pattern was recorded in the study of 7. A simieN, although very similar to those estimated for Prenj. The higher estimates for the Biokovo population should be taken with caution, considering our results suggest the presence of multiple funding sources. Additionally, p = 0.02.Effective population size was estimated for three sampled sites, excluding the Čabulja population due to small sample size Table . ČvrsnicSTF value was found between Čvrsnica and Čabulja (STF = 0.024), while the highest and significant STF value (0.084) was observed between two neighbouring populations from Bosnia and Herzegovina  without using the correction method and 0.071 (CI = 0.038–0.112) with the ENA correction method for null alleles. The Wilcoxon Two Sample test showed no significant differences between the corrected and original STF values (p = 0.734), indicating that the presence of putative null alleles did not affect the analysis , which for Prenj differ from Čvrsnica and Biokovo, while in the population from Čabulja the highest proportion of same ancestral genotype as in Čvrsnica was lower . One individual from Čvrsnica stood out from the others with a q = 0.23 proportion of ancestral genotype that was present mostly in Prenj population. As previously mentioned, natural migration between individuals from Prenj and Čvrsnica is currently unlikely, due to the presence of barriers, however this might not have been the case before the construction of the road infrastructure. STRUCTURE indicated that the reintroduced Balkan chamois population in the Biokovo Mt. is genetically more similar to the population from Čabulja, suggesting that the reintroduced individuals in the Biokovo Mt. may originate from this area, as well as from Čvrsnica, whereas animals translocated from Prenj did not leave detectable genetic signatures. According to The algorithm developed by MCMC chain  and which were declared protected areas . Jurić (Non-invasive monitoring of genetic parameters of both reintroduced and source populations of endangered Balkan chamois, together with demographic monitoring, is crucial for sustainable management practices and improving conservation strategies to maximise the chances of population persistence. Our genetic diversity results show that the Balkan chamois population from Biokovo can serve as a potential source for future translocations, especially to the source habitats, Čvrsnica and Čabulja, that are currently threatened by loss of genetic diversity due to unsustainable hunting and poaching, leading to inbreeding and genetic drift.


Toxabramis houdemeri, based on large-scale geographic sampling and mitochondrial and nuclear markers. Genetic endemism with clear geographic boundaries was formed in T. houdemeri populations due to river landscape changes, biogeographic barriers, and the species’ dispersal potential. Late Pleistocene demographic expansion had occurred in T. houdemeri populations. This study could help improve the monitoring and protection of this species.This study presents the first comprehensive view of the genetic structure of a widespread cyprinid, Toxabramis houdemeri, was chosen as an exemplar species with which to explore the effects of river configuration changes on spatial genetic structure using mitochondrial and nuclear markers. The results indicated that the T. houdemeri populations fell into four mitochondrial haplotype groups, each genetically endemic to a single river or two adjacent river systems. The mitochondrial haplotype network recovered a clear genetic boundary between Hainan Island populations and mainland populations. Notable genetic differentiation was observed within populations from distinct river systems in both mitochondrial and nuclear loci. River system separation, mountain barriers, and mobility were the key factors shaping the genetic structure of T. houdemeri populations. Late Pleistocene divergence and historical immigration were identified within the four mitochondrial haplotype groups, indicating that river rearrangements triggered by the Late Pleistocene glacial cycles were important drivers of the complex genetic structure and demographic history of T. houdemeri. Historical demographics suggested that T.houdemeri populations expanded during the Late Pleistocene. The present study has important consequences for the management and conservation of T. houdemeri.River dynamics have been hypothesized to substantially influence the genetic structure of freshwater fish taxa. Southern China harbors abundant independent river systems, which have undergone historical rearrangements. This river system is thus an excellent model with which to test the abovementioned hypothesis. In this study, a cyprinid widespread in many independent rivers in southern China,  For effectively managed conservation, it is critical to understand the spatial configurations of genetic diversity and to excavate genetic endemism (unique genetic resources in a particular geographic region) within species, particularly in the case of widespread species. In particular, since widely distributed populations of freshwater fish may frequently be isolated by mountains or salinity , gene flIn addition to the large Pearl River, many independent coastal rivers are distributed in southern China, such as the Moyang River and the Jian River on the mainland and the Nandu River, the Changhua River, and the Wanquan River on Hainan Island . These rHemibagrus guttatus populations in the Pearl River, Hanjiang River, and Jiulongjiang River [Megalobrama terminalis) in the Pearl River, the Moyang River, and the Wanquan River, and the authors argued that river rearrangements mediated by glacial cycles shaped this remarkable genetic pattern [Several genetic studies of freshwater fish in southern China have detected high levels of population differentiation ,9,10,13.ng River . Similarng River  found ma pattern . HoweverToxabramis houdemeri , a small cyprinid  found in numerous rivers in southern China, is a good exemplar species for explorations of the influences of river landscape alternations on the genetic patterns of freshwater fish taxa. T. houdemeri is a mesopelagic fish that is considered a poor disperser due to its small body size [T. houdemeri. Resolving the spatial genetic structure of T. houdemeri and revealing the processes influencing this structure will provide a solid theoretical basis for the development of effective conservation strategies.ody size . Given tody size ,17,18, iT. houdemeri populations from several independent coastal rivers in southern China and investigated population genetic structure using both mitochondrial and nuclear loci. We aimed (i) to investigate the spatial genetic structure of T. houdemeri populations and to identify genetic endemism in different rivers; (ii) to characterize population demographics and determine potential influencing factors; and (iii) to provide a relevant theoretical basis for the development of future conservation strategies.In this study, we sampled T. houdemeri from 26 localities in the rivers of southern China, including the Pearl River and eight independent coastal rivers  and the control region (CR) using universal primers L14724 and H15915 [RAG2) using the primers RAG2-f2a and RAG2-R6a [Total genomic DNA was extracted from fin or muscle tissue samples using the Axygen DNA Extraction Kit , following the manufacturer’s instructions. Two mitochondrial fragments were amplified: the partial mitochondrial cytochrome d H15915  and DL1 d H15915 . We alsoRAG2-R6a . All selRAG2-R6a ,20,21. TCytb and CR were concatenated into one mitochondrial locus (MCR) for subsequent analyses. For RAG2, alleles were unphased using the PHASE algorithm as implemented in DnaSP v5.0 [The nucleotide sequences were initially aligned using MUSCLE  and manuaSP v5.0  with defT. houdemeri populations. Instead, we used PopART v1.7.2 [RAG2 was used to build the allele network.Due to low intraspecific sequence divergence and low resolution in the phylogenetic topology, we did not build phylogenetic trees for the T v1.7.2  to inferT v1.7.2 . MCR seqT v1.7.2  for RAG2T. houdemeri populations, we calculated the number of haplotypes (h), haplotype diversity (Hd), and nucleotide diversity (π) using DnaSP v5.0. Genetic differentiation  was roughly measured as the straight-line distance using Google Earth v.4.3. Hierarchical and non-hierarchical population structures based on both types of loci were examined using analyses of molecular variance (AMOVAs) in ARLEQUIN v3.5. First, we estimated the overall differentiation of the complete data set without partitions. Second, we calculated the partitioning of genetic variation between the nine independent rivers and four defined genetic groups (see To characterize the genetic diversity of the ndex ϕST ) was assndex ϕST  by calcuoups see . All calCytb alone was 0.756. The estimated mean substitution rates for MCR (0.756% and 1.513%) were obtained by multiplying the Cytb rate by the K2P ratio for Cytb alone (0.756). The estimated substitution rates for MCR were used to measure the approximate divergence times between group pairs.We used pairwise net average sequence distances between species to estimate the approximate divergence times between the species in the four observed genetic groups. We calculated the net average sequence distance between genetic group pairs in MEGA X to estimate approximate divergence times. The average net Kimura-2-Parameter (K2P ) distancT. houdemeri populations. First, to test for departures from a constant population size, the summary statistics Tajima’s D [SF [Cytb and CR were separated in this analysis. Before conducting EBSPs, we chose the optimal model of nucleotide substitution for each locus  and the CR (772 bp) were successfully sequenced from 527 samples. In total, we obtained 527 MCR sequences (1877 bp), with 147 variable sites and 106 haplotypes. We yielded RAG2 sequences (1201 bp) from 509 specimens, with 74 variable sites and 168 alleles. All novel sequences obtained in this study have been deposited in GenBank  and RAG2 .The range of pairwise ϕnificant . The rannificant . Additioulations . Mantel d and π values of each population, overall samples, and the defined genetic groups were showed in d) and nucleotide diversity (π) values were 0.948 and 0.0044, respectively. The highest Hd and π values within the four defined groups were detected in G1. With respect to RGA2, the overall Hd and π values were 0.915 and 0.0036, respectively. The highest Hd and π values within the four genetic groups were examined in G3.The HD and Fu’s SF for the overall populations were significantly negative  or by the raggedness index of the distribution  (Cytb detected a population expansion from ~0.06 million years ago (Ma) to 0.120 Ma (Both Tajima’s negative . Additionegative . The modor RAG2) . Lastly,0.120 Ma .Using substitution rates of 0.756% and 1.513% per Myr for MCR, estimates of historical migration between the four groups showed symmetrical magnitude and direction between G1 and G2, between G1 and G3, and between G2 and G3. Additionally, asymmetrical bias in historical gene flow was detected between G1 and G4, between G2 and G4, and between G3 and G4. The median estimates of the number of effective migrants per generation originating from G1 and traveling into G4 was 0.175 (0.756%)/0.349 (1.513%), while the median number of migrants per generation traveling in the opposite direction was estimated at 0.044 (0.756%)/0.089 (1.513%). Estimates of migration between G2 and G4, as well as between G3 and G4, showed similar patterns a.Based on average sequence distances, the divergence time range for the four defined groups was 0.119–0.192 Ma based on a substitution rate of 1.513% and was 0.238–0.384 Ma based on a substitution rate of 0.756% b.T. houdemeri populations, suggesting long-term geographical separation. Divergence time estimates suggested that the four defined groups split at ~0.119–0.384 Ma, which indicated isolation during the Late Pleistocene. In addition, the historical gene flow between the four genetic groups indicated that, at some point in history, these independent rivers were connected, allowing overlapping dispersal and gene flow. River rearrangements during the Late Pleistocene glacial cycles [T. houdemeri populations. At the end of the glacial period, the rising sea levels [T. houdemeri populations. Similar population structure patterns have previously been documented in many other freshwater fish species in southern China, including Culter recurviceps [Megalobrama terminalis [Rhinogobius duospilus [Schistura fasciolata [Four mitochondrial haplotype groups with rigid geography were observed in the l cycles  appear ta levels ,43 isolarminalis , Rhinogouospilus , and SchCT = 0.253 for MCR and ϕCT = 0.322 for RAG2) between the nine independent river populations indicated that riverine isolation might have influenced the genetic architecture of T. houdemeri populations. Nowadays, these nine rivers are independent and unconnected, divided by both terrestrial and/or marine areas. Consequently, the T. houdemeri populations in these rivers are each restricted to single rivers, resulting in obvious genetic differentiation.Significant differentiation  in the Pearl River, which found no population structure among M. terminalis populations [T. houdemeri populations in the Pearl River was also greater than that between C. recurviceps populations in the Pearl River [T. houdemeri, considering T. houdemeri is a small fish  and has been deemed a poor disperser.Unexpectedly, AMOVA and ϕulations . Furtherrl River . This diG. orientalis and Channa gachua, both which recovered deeply divergent lineages among the Hainan Island river populations [T. houdemeri and the other two species. T. houdemeri is a mesopelagic fish, while G. orientalis and C. gachua are benthic species [T. houdemeri may have relatively higher dispersal potential than either G. orientalis or C. gachua. Indeed, the five rivers are spatially close , and some river sections may occasionally be physically connected by seasonal flooding. Consistently, previous studies have suggested that seasonal flooding may promote migration in some fish taxa, such as Arapaima gigas [Marcopodus [The low levels of genetic differentiation identified between populations from the five independent rivers on Hainan Island suggested that the Hainan Island populations might constitute a single panmictic population, even though these rivers have been thought to be distinct river systems separated by known barriers, such as the Yinggeling and Wuzhishan Mountains ,47. Our ulations ,47. This species ,48. Therma gigas  and the rcopodus . MoreoveT. houdemeri. EBSP analyses suggested the expansion event had occurred ~0.060–0.120 Ma, during the Late Pleistocene. Geological reports have argued that southern China experienced an interglacial period during the Late Pleistocene (0.126–0.018 Ma) [T. houdemeri populations, as has previously been proposed in C. recurviceps [T. houdemeri populations. However, we found that T. houdemeri populations remained stable during the Last Glacial Maximum (~0.021 Ma), suggesting that the Last Glacial Maximum did not impact the demography of this species. The regions in southern China are temperate and tropical zones, which might be less influenced by the Last Glacial Maximum. Similar patterns were also reported in other fish species in southern China [Hemiculter leucisculus [Ochetobius elongatus [Neutrality tests, mismatch distribution analyses, and EBSP analyses supported the recent population expansion of .018 Ma) . During rn China ,51,52, sd and low levels of π observed in T. houdemeri populations using both MCR and RAG2 were indicative of a population bottleneck followed by rapid population expansion [T. houdemeri populations was also demonstrated by different analytical methods. The global genetic diversity level using concatenated Cytb and CR of T. houdemeri populations was slightly lower than that of M. terminalis populations [RAG2 was higher than that of M. terminalis populations [T. houdemeri members within the four genetic groups were derived from the ancestral populations through a recent population expansion.High levels of Hxpansion . A signaulations . Howeverulations . FurtherT. houdemeri populations were comprised of four mitochondrial haplotype groups that were distributed in separate river systems. The genetic endemism uncovered in these independent rivers deserves specific consideration. Special attention should also be paid to the Moyang River, the Jian River, and the Lian River because these small rivers harbor two unique mitochondrial haplotype groups. The low genetic diversity and fragmented distributions of T. houdemeri in these three rivers increase the susceptibility of these populations to adverse effects, such as inbreeding depression and diseases, leading to a high threat of extinction [Delineating the population structures of widespread taxa remains inherently difficult, as many populations of widespread species have dwindled or even disappeared in certain sections of their distribution ranges due to human interference and/or environmental degradation . It is etinction ,56. Impotinction ) due to T. houdemeri populations based on wide geographic sampling and both mitochondrial and nuclear markers. Genetic endemism with clear geographic boundaries was identified in the T. houdemeri populations. River landscape changes during the Late Pleistocene, independent river systems, biogeographic barriers, and dispersal potential have played key roles in shaping the genetic structure and demographic dynamics of T. houdemeri. The results of this study may help improve monitoring and protection efforts targeting T. houdemeri.Our study provides the first comprehensive view of the genetic structure


Quercus acutissima (n = 418) and Q. chenii (n = 183) and compiled previously published sequence data of Q. variabilis (n = 439). The phylogenetic relationships among haplotypes were examined using a median‐joining network. The geographic patterns of interspecifically shared haplotypes were assessed to test whether nearby populations have a higher degree of interspecific cpDNA sharing than distant ones. We identified a total of 27 haplotypes that were grouped into three non‐species‐specific lineages with overlapping distributions. Ancestral haplotypes were extensively shared and randomly distributed across populations of the three species. Some young haplotypes were locally shared in mountainous areas that may have been shared refugia. The local exchange of cpDNA resulted in an excess of similar haplotypes between nearby populations. Our study demonstrated that the haplotype sharing pattern among East Asian Cerris oaks reflected the imprints of both shared ancestral polymorphism and introgression. This pattern was also associated with the relatively stable climates and complex landscapes in East Asia, which not only allowed the long‐term persistence of ancestral lineages but also connected the survived populations across refugia.Shared ancestral polymorphism and introgression are two main causes of chloroplast DNA (cpDNA) haplotype sharing among closely related angiosperms. In this study, we explored the roles of these two processes in shaping the phylogeographic patterns of East Asian Cerris oaks by examining the geographic distributions of randomly and locally distributed shared haplotypes, which coincide with the expectations of shared ancestry and introgression, respectively. We sequenced 1340 bp of non‐coding cpDNA from  We demonstrated that the haplotype sharing pattern among East Asian Cerris oaks reflected the imprints of both shared ancestral polymorphism and introgression. This pattern was also associated with the relatively stable climates and complex landscapes in East Asia, which not only allowed the long‐term persistence of ancestral lineages but also connected the survived populations across refugia. Some Ilex oak species form the first diverging plastid lineage of the ‘Old World oak’ clade (subgenus Cerris), while the others are clustered with either Cerris oaks (sect. Cerris) or ring‐cupped oaks . A fourth lineage has been found in the westernmost populations of the Mediterranean Q. ilex , which share six lineages partitioned along a longitudinal gradient, reflecting a common history of long‐term isolation among separate refugia and massive introgression during the post‐glacial northward recolonization  to examine the geographic patterns of chloroplast diversity in Q. acutissima and Q. chenii. The results were compared to cpDNA data previously obtained for Q. variabilis  and 19 populations of Q. chenii , encompassing the majority of the distributions of both species in China  sampled throughout its entire distribution in China , haplotype diversity (Hd), and nucleotide diversity (π) were calculated for each population using DnaSP 5.10. Average within‐population gene diversity (hS) and total gene diversity (hT) were computed for each species using Permut 2.0  and the equivalent coefficient of differentiation considering similarities among haplotypes (NST). A higher NST than GST usually indicates the existence of a phylogeographic structure, that is, closely related haplotypes occur more often in the same populations than less related ones  in Permut 2.0. To further investigate interspecific differentiation, a hierarchical analysis of molecular variance (AMOVA) was performed with Arlequin 3.5  was assessed by 10,000 random permutations.The number of haplotypes  analyses to infer the past demographic dynamics of each species using the BDSKY package in BEAST 2.6.7  was tested for each distance class using a permutation test (n = 9999) that randomly shuffles all the individuals among sites. If the observed r‐value lies beyond the upper 95% bound of the null distribution of permuted r values, a positive spatial genetic structure is inferred. The significance of r was also assessed by bootstrap resampling (n = 9999) with replacement from the original dataset for a specific distance class. When the 95% confidence intervals (CIs) do not overlap zero, a significant spatial genetic structure is inferred. These analyses were conducted for each species pair and all three species using GenAlEx 6.5 , a measure of between‐population genetic similarity, for all pairs of populations belonging to different oak species. We then compared the distributions of J among three groups of population pairs: (1) population pairs separated by <300 km and sharing haplotypes (J1); (2) population pairs separated by <300 km, regardless of whether haplotypes were shared or not (J2); and (3) population pairs separated by ≥300 km (J3). We used 300 km as a threshold distance because we found that, for each species pair, the spatial autocorrelation coefficient declines smoothly when the size of the distance class exceeds 300 km, suggesting that the oak trees separated by <300 km were more genetically similar to each other. The measures of interspecific gene identities and their means  were computed according to Dumolin‐Lapègue et al.  were detected in the atpB‐rbcL region , followed by Q. variabilis (15) and Q. chenii (14). Eight haplotypes  were shared by all three species, three  were shared by two species, and the remaining 16  were private to a single species  and two reconstructed members of western Eurasian Cerris oaks (Q. cerris and Q. suber) shared the haplotype closest to the lineage C . The other nine outgroups were separated from H28 by one to three mutational steps in the trnH‐psbA (Q. bawanglingensis) or atpB‐rbcL region (Q. phillyraeoides and Q. ilex), or by three to 10 mutational steps in both regions (the other six outgroups).The median‐joining network grouped the 27 haplotypes of East Asian Cerris oaks into three non‐species‐specific lineages Figure . Among tn Figure . Haplotyn Figure . In contn Figure . The linn Figure . Among tThe three lineages of East Asian Cerris oaks presented an overlapping distribution Figures . The lin3.2h), haplotype diversity (Hd), and nucleotide diversity (π) in each population ranged from 1 to 6 (mean 1.65), zero to 0.842 (mean 0.163), and zero to 0.00263 (mean 0.00032), respectively . Among the three East Asian Cerris oaks, the total gene diversity (hT) was found to be three to nine times greater than the average within‐population gene diversity (hS). The highest value of hT was observed in Q. chenii (0.902), followed by Q. variabilis (0.793) and Q. acutissima (0.747). The largest value of hS was detected in Q. acutissima (0.252), followed by Q. variabilis (0.120) and Q. chenii (0.100) . NST and GST were not significantly different when the three datasets were combined . The results of AMOVA indicated a relatively low level of cpDNA differentiation between each species pair  as well as among the three species   Table .3.3p‐values <.01). The highest value of the spatial autocorrelation coefficient (r) was observed for the distance class of 0–50 km (0.529 ≤ r ≤ 0.683). The r values decreased with the increasing size of the distance class and began to decline smoothly when the size of the distance class exceeds 300 km  and that for population pairs separated by ≥300 km (M3) , but such a difference was not significant for the ‘Q. chenii‐Q. variabilis’ pair (P23 = 0.920) (Table M1) was found to be always significantly larger than M2 and M3 for all species pairs (P12 and P13 ≤ 0.004), regardless of whether H1 were considered or not  interspecifically sharing eight cpDNA haplotypes except for H1 Figure . Among tQ. acutissima and Q. variabilis populations in the Funiu Mountains, central China (Henan and Hubei Provinces); its direct ancestor H14, derived from H2, was restricted to populations of the three species in the Tianmu, Jiuhua, and Lu Mountains, southeastern China . Lineage A H18, a less frequent satellite of H2, was mainly found in populations of the three species in the Wuling Mountains, central China (Hunan Province). Lineage B H15, a satellite of H1, was shared by Q. acutissima and Q. variabilis populations in the Liaodong Peninsula, northeastern China (Liaoning Province), as well as Q. acutissima and Q. chenii populations in the Tianmu and Huangshan Mountains, southeastern China (Anhui and Zhejiang Provinces). Lineage B H16, another satellite of H1, was only detected in one ‘Q. acutissima‐Q. chenii’ pair in the Nanling Mountains, southern China (Hunan and Guangdong Provinces).Thirteen of the 39 population pairs shared five haplotypes with a narrow distribution ; H6 was shared by five Q. acutissima, five Q. variabilis, and one Q. chenii population in the mountainous areas of central and southeastern China .In these analyses, we also considered high‐frequency haplotypes H2/H7 (lineage A) and H6 (lineage C) because the possibility of introgression‐induced sharing of them cannot be ruled out. Twenty‐six of the 39 population pairs shared these three haplotypes Figure . Among t4FCT = 0.03; Table FCT ≥0.15; Chen, Zhang, et al., We investigated the cpDNA variation in East Asian Cerris oaks based on the sequence data from 93 wild populations sampled throughout China. We found that the level of interspecific differentiation at cpDNA markers (4.1trnS(GCU)‐trnG(UCC) and trnS(GCU)‐trnT(GGU) regions. These non‐species‐specific lineages hint that the initial differentiation of plastid sequences of East Asian Cerris oaks is independent of the speciation process that formed the modern‐day species; the three modern‐day East Asian Cerris oaks share an ancestral plastid gene pool, which may have split into three lineages before the formation of modern species , H1 (lineage B), and H6 (lineage C), presented a relatively high frequency and a wide geographic distribution across populations of the three species Figure . AccordiQ. variabilis in Japan  are less frequent Table . PreviouCerris and species of closely related oaks of the sister sect. Ilex . Thus, it represents an ancient plastid lineage of East Asian Cerris oaks. Notably, the haplotypes of this lineage, including the ancestral haplotype H6, were not observed in southwestern China, suggesting that the ancestor of the lineage C, hence, all East Asian Cerris plastomes, originated outside this area. Secondary introgression/hybridization with sym‐ or parapatric sect. Ilex oaks (chloroplast capture) as found in the case of Mediterranean oaks  that could point to chloroplast capture, that is, chloroplast of one species being transferred to another through introgression Figure . The chlQ. acutissima  individual of the Q. acutissima population at the Tianmu Mountain, a forest rich in Tertiary relicts , where it's again shared by one out of 12 individuals of nearby Q. acutissima populations. That the same satellite haplotype evolved in a northeastern Q. variabilis and two ~1100 km afar southeastern Q. chenii populations may be due to fixation of a convergent mutation. However, in the overall conservation of plastid DNA in oaks, it may as well represent a common geographic origin. Once more widespread, the now‐extinct northeastern Q. chenii population was introgressed by Q. variabilis in northeastern China and its private plastome was captured. Q. acutissima captured this potential Q. chenii plastome when introgressing at a large scale into its sibling species.A clear case of recent and past introgression from the most widespread ts Wang, . The samThe local sharing of haplotypes may also be influenced by demographic history and the complex landscapes of East Asia. During the Pleistocene, local plants like oaks underwent repeated range contractions and expansions  and our dataset did not include any pair of populations that belong to these two species and are separated by less than 1 km. In contrast, mixed stands of Q. acutissima and Q. chenii or Q. variabilis are commonly observed in the field. The interspecific gene identities for sympatric populations of these two species pairs ; data curation (lead); formal analysis (lead); investigation ; writing – original draft (lead). Lu Wang: Formal analysis (supporting); investigation ; writing – review and editing . Xingwang Zhang: Formal analysis (supporting); investigation ; writing – review and editing . Hongzhang Kang: Resources ; writing – review and editing . Chunjiang Liu: Resources ; writing – review and editing . Lingfeng Mao: Supervision ; writing – review and editing . YanMing Fang: Conceptualization ; funding acquisition (lead); supervision (lead); writing – review and editing .None declared.Appendix S1 Supplementary FiguresClick here for additional data file.Appendix S2 Supplementary TablesClick here for additional data file.


Lycium ruthenicum, a plant species adapted to the climate in northwestern China. We identified 20 chloroplast haplotypes of which two were dominant and widely distributed in almost all populations. The species has high haplotype diversity and low nucleotide diversity based on the cpDNA data. The EST-SSR results showed a high percentage of total genetic variation within populations. Both the cpDNA and EST-SSR results indicated no significant differentiation among populations. By combining the evidence from ENM and demographic analysis, we confirmed that both the last interglacial (LIG) and late-glacial maximum (LGM) climatic fluctuations, aridification might have substantially narrowed the distribution range of this desert species, the southern parts of the Junggar Basin, the Tarim Basin, and the eastern Pamir Plateau were the potential glacial refugia for L. ruthenicum during the late middle Pleistocene to late Pleistocene Period. During the early Holocene, the warm, and humid climate promoted its demographic expansion in northwestern China. This work may provide new insights into the mechanism of formation of plant diversity in this arid region.Climactic oscillations during the Quaternary played a significant role in the formation of genetic diversity and historical demography of numerous plant species in northwestern China. In this study, we used 11 simple sequence repeats derived from expressed sequence tag (EST-SSR), two chloroplast DNA (cpDNA) fragments, and ecological niche modeling (ENM) to investigate the population structure and the phylogeographic history of  Picea crassifolia , continuous uplift of the QTP, the Tianshan, and the Himalayas coupled with weakening of the southwest monsoon and intensification of the plateau winter monsoon caused extreme drought and low temperatures  is diploid, monoecious, and cross-pollinated and its root propagates via clones  and phylogeographical analyses based on the simple sequence repeats (SSRs) derived from expressed sequence tag (EST-SSR) and chloroplast DNA (cpDNA) fragments (rps16–trnK and trnH–psbA). We investigated the genetic diversity patterns of L. ruthenicum in northwestern China, established its historical population demography in response to climatic oscillations, and provided critical genetic information required to conserve it. The present work will serve as an empirical model for the study of desert plant phylogeography in northwestern China.The aim of this study was to clarify the phylogeography of the taxa by determining how the distribution of L. ruthenicum in Xinjiang, Qinghai, Gansu, and Inner Mongolia, China, were collected and dried with silica gel  according to the manufacturer’s protocol. DNA product quality and concentration were evaluated by 1% agarose gel electrophoresis. The DNA was stored at −20°C until use. For the EST-SSR, 11 polymorphic EST-SSR markers were screened and their corresponding primer pairs were developed for 540 individuals . The prirps16–trnK, trnH–psbA, matK, and rp132 from representative samples of L. ruthenicum were used in preliminary screening to detect variations in the cpDNA fragments , observed heterozygosity (HO), expected heterozygosity (HE), and inbreeding coefficient (FIS) for each population were calculated by FSTAT v. 2.9.3.2  value. To this end, the online program STRUCTURE HARVESTER was used.The number of alleles, allelic richness ( 2.9.3.2 .2 The si 2.9.3.2 . The pop 2.9.3.2 . The K vrps16–trnK and trnH–psbA) sequences were assembled and manually checked in Geneious v. 11.0.2.Lycium dasystemum was sequenced and served as the outgroup in the chloroplast genealogy analysis . Haplotypes were determined using DnaSP v. 5.1  values (Wright’s fixation index) were calculated by analyses of molecular variance (AMOVA) in Arlequin v. 3.1  and Atropa belladonna (NC004561) species. Lycium dasystemum, L. cestroides, and A. belladonna served as the outgroups. An uncorrelated lognormal relaxed clock model with a Yule process was used for the speciation model. The GTR + I model was selected by jModelTest v. 2.1.5  and L. dasystemum and L. cestroides (4.85 Mya) as secondary calibration points  for all parameters were tested by Tracer v. 1.5  was applied to measure population expansion for all samples. All expansion tests were implemented in Arlequin v. 3.1 (SSD) between the observed and expected mismatch distributions as well as Harpending’s raggedness index  units, k is the average sequence length used for the analyses , and g is the generation time in years  set to 3.0 × 10−9 s/s/y was used for the estimation (7 generations under the GTR + I substitution model. Both the MCMC convergence and the ESS (>200) were tested using Tracer v. 1.5.The selective neutrality indices Tajima’s D  and Fu’sD , the late-glacial maximum , the Mid-Holocene , and current conditions (1970–2000). It was based on 7 years (2013–2019) of geographical distribution records for the L. ruthenicum populations included in the present study  was used to select eight climate factors with Pearson’s correlation coefficient < 0.8 for the subsequent modeling . The parL. ruthenicum individuals. The average number of alleles (NA), allelic richness (AR), observed heterozygosity (HO), expected heterozygosity (HE), and inbreeding coefficient (FIS) values are listed in NA, AR, HO, HE, and FIS were 28.975, 1.357, 0.384, 0.359, and − 0.053, respectively. The highest genetic diversity was found in the AL (Alashan) population  located in the northern parts of the Tianshan Mountains. AL was followed by GS  and SC  populations located in the eastern and southern proportions of the range of the species , respectively.The characteristics of 11 polymorphic microsatellite loci were identified in 540 K reached its maximum value at K = 3  and low species-level nucleotide diversity (Pi = 0.00152). The phylogeographic structure between haplotypes did not significantly contribute to population differentiation . Hence, the species had no significant phylogeographical structure. The results of AMOVA showed that a large proportion of the variation (65.64%) occurred within population; this result was consistent with the high genetic differentiation within population of this species  = 1.17–2.74 Mya] to the middle Pleistocene  and Fu’s FS . The unimodal mismatch distribution, the positive SSD values , and the HRag  displayed a steady growth between ca. 1.95 and 0.12 Mya while the species-level rapid expansion events occurred since the past ca. 0.12 Mya  and Late Glacial Maximum (LGM) than they are at present  but low species-level nucleotide diversity (Pi = 0.00152) for L. ruthenicum  were not conducive to the expansion of L. ruthenicum. For this reason, this species retreated to the refugia situated on the edges of the Junggar Basin, the Tarim Basin, the Tianshan Mountains, the Pamir Plateau, the Qaidam Basin, and near the Qilian Mountains during the LIG and LGM  and five unique haplotypes  negative values for the neutrality test statistics, unimodal mismatch distribution, and relatively low SSD and HRag  and LGM , the climate of northwestern China has fluctuated between humid and dry conditions, resulting in the enlargement of deserts while the desert margin  and arid piedmont grassland areas seem to have provided stable habitats for desert plants  period, snow and glacial ice melted off the Tianshan, the Altai, the Kunlun Mountains, and Qilian Mountains surrounding the Junggar, Tarim Basins, and Qaidam Basin irrigating the surrounding deserts and increased the humidity on the edge of these regions  repository, accession numbers, ON055439, ON055440, ON390854–ON390893.JL, PL, and X-MT conceived the ideas. GY and JL contributed to the sampling. GY performed the experiments and analyzed the data. GY, PL, and X-MT wrote the manuscript. C-XW, M-QX, and HC revised the manuscript and provided useful suggestions. All authors contributed to the article and approved the submitted version.This research was supported by the National Natural Science Foundation of China (grant nos. 31970225 and 31760102), the Zhejiang Provincial Natural Science Foundation (LY19C030007), the “Xinjiang Key Laboratory of Special Species Conservation and Regulatory Biology,” and the “13th Five-Year” Plan for Key Discipline Biology, Xinjiang Normal University.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Camellia is one of the four woody oil crops in the world and has high ecological, economic and medicinal values. However, there are great differences in the classification and merging of tea-oil Camellia Sect. Oleifera species, which brings difficulties to the innovative utilization and production of tea-oil Camellia resources. Here, ISSR, SRAP and chloroplast sequence markers were analyzed in 18 populations of tea-oil Camellia Sect. Oleifera species to explore their phylogenetic relationships and genetic diversity. The results showed that their genetic diversity were low, with mean H and π values of 0.16 and 0.00140, respectively. There was high among-population genetic differentiation, with ISSR and SRAP markers showing an Fst of 0.38 and a high Nm of 1.77 and cpDNA markers showing an Fst of 0.65 and a low Nm of 0.27. The C. gauchowensis, C. vietnamensis and Hainan Island populations formed a single group, showing the closest relationships, and supported being the same species for them with the unifying name C. drupifera and classifying the resources on Hainan Island as C. drupifera. The tea-oil Camellia resources of Hainan Island should be classified as a special ecological type or variety of C. drupifera. However, cpDNA marker-based STRUCTURE analysis showed that the genetic components of the C. osmantha population formed an independent, homozygous cluster; hence, C. osmantha should be a new species in Sect. Oleifera. The C. oleifera var. monosperma and C. oleifera populations clustered into two distinct clades, and the C. oleifera var. monosperma populations formed an independent cluster, accounting for more than 99.00% of its genetic composition; however, the C. oleifera populations contained multiple different cluster components, indicating that C. oleifera var. monosperma significantly differs from C. oleifera and should be considered the independent species C. meiocarpa. Haplotype analysis revealed no rapid expansion in the tested populations, and the haplotypes of C. oleifera, C. meiocarpa and C. osmantha evolved from those of C. drupifera. Our results support the phylogenetic classification of Camellia subgenera, which is highly significant for breeding and production in tea-oil Camellia.Tea-oil  Camellia of family Theaceae, the more than 30 species of tea-oil Camellia are woody, oil-bearing tree species with a high content of seed oil that is widely processed into skin and health care products and edible oil  in 2004. Tea oil is rich in squalene, vitamin E, Camellia glycosides and unsaturated fatty acids  and sequence-related amplified polymorphism (SRAP) markers are two types of dominant markers that have been widely used in the identification and evaluation of plant populations . BecauseC. vietnamensis and two populations of C. oleifera. var. monosperma, two populations of C. oleifera, two populations of C. gauchowensis, one population of C. osmantha and seven populations of tea-oil Camellia resources on Hainan Island, all of which are old seedling forest populations. The JXP and FY6 populations of C. sinensis from Jiangxi and Fujian, respectively, China, were used as an outgroup.We collected samples from 18 populations, including four populations of In the sampling of each population, the individual interval was greater than 15 meters. A total of 8 healthy young leaves were selected from each sample, washed with 75% alcohol, and immediately placed in silica gel for drying and sealing for subsequent molecular experiments. See Camellia spp. using 14 primers  of each primer was calculated. The MXComp program in 02 software was used for the Mantel statistical test (1967) to analyze ISSR and SRAP data to compare the correlation between the two genetic similarity coefficient matrices.h), haplotype diversity (Hd), nucleotide diversity (π) and average number of nucleotide differences (K) were recorded.Assuming valid Hardy–Weinberg equilibrium HWE; , we usedp values of the 18 populations were analyzed based on the coefficient of variation (CV), CV = (SD/mean) × 100%. To compare and evaluate the differences in genetic diversity among the populations, the Pearson correlation coefficients between ISSR markers and SRAP markers were analyzed using SPSS20 to verify the consistency of the results.To determine the degree of variation in genetic diversity among the test populations, the Na, Ne, H, I, and Camellia. we performed the following steps: (1). Assuming that marker A was in Hardy–Weinberg equilibrium (HWE), POPGENE1.32 software was used to calculate the Nei’s genetic distance (GD) between the tested populations. (2). GenAlEx 6.5 software was used to analyze Fst and Nm based on cpDNA. (3). NTSYS-PC (2.02j) and MEGA6 were used to cluster the test populations by principal component analysis (PCA) and phylogenetic tree  analysis. (4) Based on the Bayesian selection allele frequency association model, STRUCTURE 2.3.2 was used to analyze the genetic composition of different individuals in the 18 tea-oil Camellia populations. After running this analysis, STRUCTURE 2.3.2 software was used to obtain the average L(K) value corresponding to each K value to determine the optimal K value  tools to map the sites of each population. NETWORK 5.001To assess historical population dynamics, the mismatch distribution was estimated using DnaSP 5.0. For Tajima’s neutrality test, Tajima’s D values were tested for significant deviations from the null hypothesis of neutral evolution and constant population size.A total of 198 bands were obtained from 14 ISSR primers, and the number of bands generated with each primer ranged from 11 to 17 (average 14.14), with an average percentage of polymorphism of 97.96% (91.67–100%). For SRAP markers, 291 bands were obtained, with an average PPB of 95.08% (76.92–100%), and the number of bands generated with each primer ranged from 8 (ME12-EM3) to 18 .h of the five aligned cpDNA sequence fragments was 53, ranging from 3 to 19, with an average of 10.60 . A correlation analysis (Pearson test) of the genetic diversity indexes of each population calculated based on the two marker types was carried out, and it was found that there was a significant positive correlation between the genetic diversity index data of the two markers, with Pearson correlation coefficients of 0.81 (p < 0.01) for Na and I with P, and 0.79 (p < 0.01) for both Ne and H. This showed that there was high consistency in the analyses of the 18 tested populations based on ISSR and SRAP markers. In the analysis and the discussion presented later in this paper, the comprehensive ISSR and SRAP data are used for analysis.The results of the Mantel test showed that there was a positive correlation between the genetic similarity coefficient matrices of ISSR and SRAP  showed the lowest genetic variation, with H, I and p values lower than 0.14, 0.22 and 43.15%, respectively. The genetic variation levels among the tested populations were quite different, and the CV values of H, I and P were 16.32, 15.66 and 15.17%, respectively.The results of ISSR and SRAP analyses showed that , under HThe results of analyzing the five chloroplast sequences showed  that theThe results of genetic differentiation analysis among the 18 tested populations  showed tr = 0.72), with high consistency. In the ISSR and SRAP analyses (C. oleifera var. monosperma showed the most distant genetic relationship from the tea-oil Camellia resources on Hainan Island (MP and FH), with GDs of 0.198 and 0.199, respectively. In the cpDNA analysis, the GD between the tested populations varied from 0 to 0.009, with an average GD of 0.0028, while the GD between the populations on Hainan Island was only 0 or 0.001, and the genetic distance between the C. oleifera. var. monosperma populations and other populations was relatively large .The Mantel test results showed that there was a significant positive correlation between the nuclear ribosomal DNA (ISSR and SRAP) and cpDNA genetic distance matrices  method, and the 18 tested populations were grouped into four categories (C. oleifera. var. monosperma (GD = 0.060) and two populations of C. oleifera (GD = 0.081) were clustered into group a and group b, respectively. C. osmantha constituted group c, and its average GD from the other groups was 0.155, ranging from 0.129 to 0.182. Group d was composed of 13 populations of C. gauchowensis and C. vietnamensis and from Hainan Island. Seven populations on Hainan Island were first clustered into subgroup 4–1, and the genetic distance between the populations was relatively small, with an average GD of 0.065. Subcategory 4–2 was composed of a mixed cluster of the populations of C. gauchowensis and C. vietnamensis, with an average GD value of 0.107.Based on the ISSR and SRAP data, a phylogenetic tree among the 18 tested populations was constructed tegories . Among tC. oleifera. var. monosperma constituted Group A. C. oleifera populations HZP, YML and TPY were included in Group B . Lastly, Group C consisted of C. osmantha, C. gauchowensis, C. vietnamensis, and populations on Hainan Island (GD = 0.0009), among which seven populations on Hainan Island were first clustered into a small subgroup.According to UPGMA tree clustering based on cpDNA, the 18 tested populations were clustered into three groups . Two popC. oleifera (HZP and YML) and C. oleifera var. monosperma (HCX and CKX) were aggregated into Cluster a and Cluster b, respectively (C. osmantha (SNH); Cluster d included all of the Hainan Island populations ; and Cluster e consisted of the C. vietnamensis and C. gauchowensis populations. In the two-dimensional plot, Cluster d was found to be located in the middle of all the classification groups, which is the closest to Cluster e. The clustering results were basically consistent with the UPGMA trees constructed for all 18 tested populations.PCA of the ISSR and SRAP data showed that the tested populations could be divided into five clusters. The cumulative contribution of the first three principal components was 69.01% , indicating that the clustering results were reliable. Among these populations, ectively ; ClusterC. oleifera. Var. monosperma (HCX and CKX) were grouped into Cluster A (C. osmantha (SNH), C. vietnamensis and C. gauchowensis.The results of PCA based on cpDNA showed that the tested populations could be divided into four clusters. The cumulative contribution of the first three principal components was 82.50% , indicating that the clustering results were reliable. The two populations of luster A ; ClusterC. vietnamensis cluster based on the ISSR and SRAP markers but to the C. oleifera cluster (cluster C) according to the cpDNA data.According to PCA clustering, the TPY population tended to belong to the K = 2 was the best value and that all the tested materials could be divided into two clusters: Cluster 1 and Cluster 2 , showing significant complexity; the HCX and CKX individuals of C. oleifera. var. monosperma were highly homozygous, and the majority of the genetic material of C. oleifera HZP and YML individuals came from the same cluster as C. oleifera. var. monosperma but was mixed with other cluster components. When K = 4 and K = 5, the seven tested populations on Hainan Island showed a high degree of homozygosity, and 6 populations presented more than 97.5% homozygosity; JX represented the sole exception, showing 73.0% (K = 4) and 74.4% (K = 5) homozygosity. C. osmantha presented significant complexity when K = 2–4, as nearly 50% of the genetic components came from two different clusters, whereas when K = 5, it was 99.2% homozygous.For the ISSR and SRAP data, when luster 2 . A totalK = 2, while the genetic material of C. oleifera individuals (HZP and YML populations) were diverse and accounted for between 23.1 and 76.9% of the material in Cluster 1 and Cluster 2, respectively, the genetic components of the remaining 16 populations came from a single source. The individuals of HCX, CKX and TPY accounted for more than 99.7% of the genetic material from Cluster 1, and those of BQY, WSG, TJG, LJTL, HTDL, JX, ZX, LS, XYL, MNL, MP, FH and SNH accounted for more than 86.8% of the genetic material from Cluster 2. When K = 3, the genetic components of the HCX, CKX and TPY populations, the Hainan populations, and the C. osmantha population came from three different clusters, showing a high degree of homozygosity . The genetic components of the C. oleifera population came mainly from the populations of C. oleifera. var. monosperma and C. osmantha, while the genetic components of the BQY, WSG, TJG, LJTL, and HTDL populations came mainly from the Hainan and C. osmantha populations, all of which showed significant complexity. When K = 4 and K = 5, the population genetic material of C. oleifera, C. gauchowensis and C. vietnamensis was observed to be complex and contained genetic components from the Hainan Island, C. oleifera. var. monosperma and C. osmantha populations. Among the Hainan Island populations, genetic exchange was observed only between C. gauchowensis and C. vietnamensis. Similarly, the populations of C. oleifera. var. monosperma and C. osmantha maintained relatively high homozygosity of their genetic components. When K = 4, the genetic material of HCX, CKX and SNH accounted for 99.6, 99.6 and 96.2%, respectively, whereas when K = 5, the genetic material of HCX, CKX and SNH accounted for 97.5, 97.5 and 96.4%, respectively.Based on the cpDNA data , when K C. oleifera var. monosperma HCX and CKX populations, located in northern Guangxi  and Hainan (H10) populations could be the origin of the ancestral haplotype.According to the positions of the haplotypes in the network , H2 locaD value of −0.41555 (p > 0.10) and an Fu’s Fs value of −0.51948, and the Tajima’s D values were not significantly negative. These results indicated that all of the tested loci presented neutral evolution and that the tested populations had not experienced a rapid expansion event. This was completely consistent with the results of the mismatch distribution analysis.Based on cpDNA sequence data, DnaSP software was used to analyze the mismatch distribution of the tested populations to determine whether the tested population experienced a rapid expansion event . The resC. oleifera. var. monosperma is a variant of C. oleifera. In Flora of China , C. oleifera. var. monosperma exhibits small leaves, small flowers, small fruit, mostly single seeds, a thin peel and a high oil content of its fruit, which are significantly different from the characteristics of C. oleifera. C. oleifera. var. monosperma is the second most commonly cultivated tea-oil Camellia species in China after C. oleifera and is mainly distributed in Fujian, Jiangxi, and Guangxi. Most producers and researchers refer to this species as C. meiocarpa.According to the Zhang Hongda system in Flora of China . Thus, it should not be simply classified within C. oleifera , oleifera . In thisme group –5, with me group , 3. The C. gauchowensis and C. vietnamensis in morphological taxonomy is whether or not current-year branchlets are glabrous. Under the Zhang Hongda system in Flora of China (Chinese version), the current-year branchlets and leaves of C. gauchowensis are glabrous, while they are gray-brown pubescent for C. vietnamensis. Under the Min Tianlu system in Flora of China (English version), the current-year branchlets of both C. gauchowensis and C. vietnamensis (both of which are revised names of C. drupifera) are glabrous  was significantly larger than that among the C. drupifera populations  according to ISSR and SRAP data ; at K values of 3 to 5, C. osmantha formed an independent cluster . Further analysis based on cpDNA, ISSR and SRAP data showed that the populations from Hainan Island  clustered together first and were located at the very edge in both the phylogenetic tree and PCA results . Accordingly, we suggest that the tea-oil Camellia resources on Hainan Island belong to C. drupifera.The phylogenetic tree and PCA results of this study showed that all of the Hainan populations were clustered with the  results –5. A STRH of only 0.138, average π of 0.0047) and high gene flow . These populations exhibited significant genetic differentiation from and limited gene flow with the populations of C. drupifera, C. oleifera, C. meiocarpa, with cpDNA-based Nm values of 0.55, 0.298 and 0.057, respectively  was higher than that between the C. oleifera and C. meiocarpa  low. Further analysis showed that the genetic distance between the tested populations was positively correlated with geographic distance (r was 0.7590 for ISSR and SRAP markers and 0.5488 for cpDNA markers), revealing that geographic distance factors play a role in population genetic analysis and gene exchange.It is generally believed that an Fst > 0.25 indicates relatively high genetic differentiation among populations . In the K value of 2, more than 86.8% of the genetic components of all individuals of C. drupifera in the BQY, WSG, TJG, LJTL, HTDL, JX, ZX, LS, XYL, MNL, MP, and FH populations belonged to Cluster 2; similarly, more than 99% of the genetic components of individuals in the C. meiocarpa populations (HCX and CKX) came from Cluster 1, and 47.3 and 23.1% of the genetic components of the C. oleifera HZP and YML populations, respectively, came from Cluster 2. When K = 3 and K = 4, the populations of C. oleifera still showed a heterozygous composition of their genetic material, while the C. meiocarpa populations and the populations of Hainan Island presented a more homozygous composition , the Project of Sanya Yazhou Bay Science and Technology City (SCKJ-JYRC-202258), the Science and Technology Innovation Project in the Hainan Academy of Agricultural Sciences (HAAS2022KJCX01), and the Project for Technology Development of Hainan Provincial Scientific Research Institutions (SQ2021JSKF0004 and SQKY2022-004).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Nuphar shimadai Hayata) is a critically endangered species in Taiwan. Here, we examined genetic structures of four extant populations, WP, GPa, GPb and GPn, using 39 simple sequence repeat (SSR) markers. Positive genetic correlation was observed within 50 m, beyond which no correlation was detected due to isolation by distance according to Mantel correlogram. This suggests a significant genetic structuring of the species. Besides, multilocus genotype (MLG) analysis revealed that GPa was a panmictic population and the species’ putative center of origin. Genetic exchange was observed between GPa and GPb populations, which likely resulted from their geographic proximity. Nevertheless, there was a strong asymmetric migration detected from GPa to WP, but a recent genetic barrier prevented dispersal further northward (WP). Geneland estimated the best number of clusters as K = 2, where WP distinctly separated from the rest of the populations. In STRUCTURE output of K = 3, a third cluster was abundant only in WP. We suggest to consider GPn and WP as separate conservation units, being far from GPa. There is indeed a need to investigate these populations; as predicted, Ne = 1.6 to 3.0 is considered low and that may put the species at risk of extinction.Yellow water lily ( Nuphar Sm. (Nymphaeaceae), which consisted of 11 species, originated from East Asia, and dispersed throughout the temperate region of the Northern Hemisphere, including North America, Cuba, Europe, northern Asia, and northern Africa [Nymphaeaceae is one of the basal groups in the ancestral lineage of the phylogeny of angiosperms . This ean Africa .Nuphar pumila (Timm.) DC, which is considered as a synonym to Nuphar shimadai Hayata [Nuphar shimadai with Nuphar pumila without description and type specimen comparison. Nuphar shimadai was morphologically different from N. pumila by the stigmatic disks, which, in the former, are usually dark red, and the latter are usually yellow [N. shimadai in Taiwan under a subtropical climate adaptation compared to N. pumila in temperate regions, and the geological separation and isolation, further strengthen the morphological divergence of N. shimadai in Taiwan from other closely related species.According to nomenclature, the yellow water lily is named as i Hayata . Howevery yellow . In addiN. pumila  has a slightly larger genome size than N. shimadai . Using a 66 protein-coding gene dataset for the phylogenetic analysis, four Nuphar species  are monophyletic and basal among all the species in the Nymphaeaceae family. N. pumila and N. shimadai are sister branches with a very high statistical support for both Maximum Likelihood and Bayesian Inference [pumila and N. shimadai are sister taxa based on plastid phylogenomics [A study that analyzed chloroplast genome showed that dai 160,65 kb. UsiN. shimadai has been categorized as a “critically endangered” species in Taiwan [N. shimadai in northern Taiwan, which resulted in the extant populations surviving in few ponds.n Taiwan   Sm. [N. japonica DC. [N. shimadai Hayata [matK and nuclear ribosomal internal transcribed spacer (ITS) regions [Several studies have proposed to evaluate the population structures of these closely related species, (L.) Sm. , N. japonica DC. , and N. i Hayata , by usin regions .Microsatellites  are common molecular markers useful in investigating population structures. SSRs are highly variable and are classified as neutral variations which are co-dominant and widely distributed throughout the whole genome ,13.N. shimadai, i.e., populations in the four ponds in Taoyuan City, Taiwan, and to understand their population structures brought about by environmental perturbations.To be able to protect and preserve this critically endangered species, understanding its genetic structures is urgently needed. Therefore, this study aims to use SSRs to evaluate the levels of genetic diversity of Taiwanese N. shimadai. The diversity indices indicate that observed heterozygosity (Ho) values were generally lower than expected heterozygosity (He) in all populations, which indicated the isolation of population and habitat fragmentations of the species  in Taoyuan City, Taiwan . We inve species . It was  species .Based on the multilocus genotype (MLG) analysis where expected MLG (eMLG) estimates the number of genotypes based on the largest sample size (N = 22 in our study) , only thIS), inbreeding coefficient among populations (FST), and overall inbreeding coefficient (FIT). We observed similar results with AMOVA tests: the fixation indices within (FIS) and among population (FST) are 0.3736 and 0.1707, respectively, while the overall coefficient is 0.4805. Taken together, the results of AMOVA and F indices suggest that there is population structuring likely due to inbreeding.We further investigated the sources of variations that contribute to population differentiation using the hierarchical AMOVA and fixation indices on polymorphisms of SSRs. These tests can measure the variations at the population level such as among or within populations, and further between individuals within population or within individuals. The AMOVA results indicate that about 17% and 31% of the variations are differentiated among populations and among individuals in the populations, respectively, while more than half (52%) of these variations are within individuals . The fixTo determine the mechanism that leads to this excess of homozygosity, we evaluate isolation-by-distance using Mantel test. Results detected significant positive correlation (r = 0.3715), which indicates that there was an isolation-by-distance, as shown in Among the 62 samples, three clusters were identified by principal coordinates analysis (PCoA) . The majGeneland  were determined using STRUCTURE. The results showed that two clusters (K = 2) may describe the population differentiation the best A,B, wherGeneland E,F, whicGeneland D.Genetic barriers between the four populations were further examined by Monmonier algorithm. We found that a significant barrier was detected between WP and the three other populations A. Under Nm) as a measure of genetic differentiation. No significant migration events were detected in other populations.According to the relative migration pattern, there was a statistically significant asymmetric migration from GPa to WP based on the estimated number of migrants   .Ne) of the four populations was estimated as high as infinite and 23.1 in GPa and GPn, respectively. We hypothesized that the high level of Ne contributed to the current genetic diversity for GPn populations. The northernmost population, WP, obtained the least Ne. In general, the Ne estimates based on the temporal method between 0 and 1.5 generations indicated also very low with Ne = 1.6 to 3.0  (0)-generation immigrants between GPa and GPn populations  was much lower compared to the extant N. shimadai populations in Taiwan from the current study.Particularly GPb, which is significant to all heterozygosity excess, might have diminished bottleneck footprints due to recent genetic exchange with GPa based on proximity . GPa may= 0.291) . None waulations . In a siulations . A similal Japan . The aveSurprisingly, there was a strong relative migration from GPa as a source to WP. It is likely that this strong directional gene flow between these two populations occurred in the distant past that influenced the composition of individuals in the populations recently. The footprints can either enhance or obscure the signs of recent migration in the genetic data . Based oNe = 2.3)  . If thisto drift . Simply,As STRUCTURE analysis indicated that either two (K = 2) or three (K = 3) estimated clusters can well differentiate clusters involving 4 extant populations within 1 km in still water or ponds, we thus suggest to consider populations far apart as separate management units. There is a reason for GPa and GPb to be considered as one, not only physically, but by proximity based on genetic exchange. As Mantel correlogram indicated that correlation was only detected within 50 m, GPn and WP can be treated as separate units because of the distance from GPa, as a putative center of diversity.Nuphar shimadai existed had gone extinct due to urbanization, as we mentioned in the introduction, except for the four populations found in the protected ponds in Taoyuan City, northern Taiwan. There were 62 samples collected at 20 m apart to avoid clones from the same individuals , 0.2 mM of both primers, and sterile ddH2O. PCR amplification was performed using a Labnet Multigene 96-well Gradient Thermal Cycler . The amplicons were checked with 1% agarose gel electrophoresis (please see ® software (Bio-Rad) to create a data matrix manually.A microsatellite-enriched library was conducted through magnetic bead procedure ,22. Out poppr in R, where departure from linkage disequilibrium indicates a panmictic population [Data analyses and visualizations were performed using R Stats Package  unless opulation .adegenet in R [The proportion of each hierarchical variation was evaluated using the Analysis of Molecular Variance (AMOVA) by Arlequin , while tnet in R . Additionet in R .5 generations after 2 × 106 MCMC replicates per run. The results were illustrated by STRUCTURE HARVESTER to determine the best K according to the Evanno method [Geneland is another Bayesian-model clustering analysis, but it runs georeferenced genotype data [5 MCMC iterations under spatial and nonspatial models in uncorrelated assumption of allele frequency. The output of the Poisson–Voronoi tessellation was determined based on 200 × 200 pixels with a burn-in of 200.Clustering analysis was also performed by using STRUCTURE  in 10 ruo method . Like SType data . The mosdiveRsity implemented in R based on relative migration network using Nm as a measure of genetic differentiation [p = 0.01 [Nm as an estimate of the effective number of migrants per generation based on Slatkin’s private allele method was estimated using GENEPOP as an R Package [adegenet in R to visualize the population clustering in northern Taiwan [The directional migration pattern was simulated by ntiation . Moreovep = 0.01 . On the  Package . The phyn Taiwan .Ne) was performed by the software BOTTLENECK. In this test, the heterozygosity excess was compared to expected equilibrium heterozygosity (He > Heq) under mutation-drift equilibrium. It was determined statistically by Wilcoxon’s signed rank test [Ne, as a contemporary effective population size, was estimated by NeEstimator v 2.1 based on linkage disequilibrium under random mating with a critical value (Pcrit ≥ 0.02) on allele frequency for rare alleles. Both the single-sample method and the two-sample or temporal method under a chi-squared parametric 95% confidence interval were performed for the Ne estimate [The bottleneck test examining populations that experienced a severe reduction in effective population size (ank test . The Ne,estimate .


Hopea hainanensis (Dipterocarpaceae) is an endangered tree species restricted to Hainan Island, China, and a small part of Northern Vietnam. On Hainan Island, it is an important indicator species for tropical forests. The wood of Hopea hainanensis has a very high utilization value in nature since it is compact in structure, hard in texture, not easily deformed after drying, durable, and resistant to sunlight and water. As a result of its high quality, it has been felled and mined by humans without restraint, resulting in a reduction of its population size, severe habitat fragmentation, and a sharp decline in its population. Therefore, its conservation biology needs to be researched urgently. Researchers are currently focusing on the ecological factors and seed germination in the habitat of Hopea hainanensis to determine its endangered status. In the literature, there are no systematic analyses of the endangered mechanism of Hopea hainanensis in terms of genetic diversity. It focuses especially on the systematic genetic diversity of Hopea hainanensis in fragmented habitats. Using single nucleotide polymorphism (SNP) and genotyping-by-sequencing (GBS) technology, 42 samples from seven different cohabitation groups were genotyped. The results showed that the average heterozygosity of the seven populations of Hopea hainanensis was 19.77%, which indicated that the genetic diversity of Hopea hainanensis was low. Genetic diversity research is essential for rare and endangered plant protection research. We can find a scientific basis for protecting endangered plants on slope bases by analyzing genetic differences and relationships among populations. Around Hopea hainanensis research is primarily concerned with the impacts of various environmental conditions in the habitat on seed germination and seedling development, ex situ conservation, and better seed selection and cultivation techniques in artificial propagation and cultivation  in simplified genome sequencing technology has become increasingly popular as next-generation high-throughput sequencing technology has developed . These i22.1https://www.hainan.gov.cn). Hainan Island is hailed as the largest “natural museum” by biologists, and 102 rare animals in Hainan Island have been included in the list of national first- and second-class key protected animals  is located in southern China , and it  animals . There a animals .2.2H. hainanensis sample materials were divided into seven regional populations according to their geographical sources for population sampling. The bawangling population includes nine subpopulations, namely BWL P1-P9, including samples BWL1-BWL_13. Limushan population consists of 7 subpopulations, LMS P1 and LMS P3-P8, including samples LMS1-LMS_10, respectively. The Jianfengling population included seven subpopulations JFL X1-X2, JFL P1, JFL P3-P6, which included samples JFL1-JFL_10. Wuzhishan population included two subpopulations (WZS P1-P2), including samples WZS_1-WZS_2, respectively. Yinggeling population included two subpopulations YGL P1-P2, including YGL1-YGL_3, respectively. The Diaoluoshan population included one subpopulation, DLS P1, including samples DLS_1-DLS_3. Fanyang population included one subpopulation FY1, including sample FY_1. The Fanyang population belongs to the Wuzhishan region. Regional population division, longitude, latitude, and sample number are shown in In the early field investigation, field investigation and actual sampling possibilities are based on the natural distribution of the levee population. The 2.32.3.1H. hainanensis leaf samples were extracted with a Biotech  DNA extraction kit (Plant no. DP305) under the standard operating procedure. After DNA extraction, the quality and concentration of DNA samples should be tested. Qubit was used to determine the concentration of DNA samples, and AGE was used to detect the quality of DNA samples  > 0.05 and data integrity > 80% .2.3.4The phylogenetic tree is a diagram used to describe the genetic differentiation relationship between species. According to the evolutionary relationship between different sources and different types of organisms, all kinds of organisms are placed on the branching tree diagram. The evolution process and the relationship between these organisms are succinctly described. Based on SNP, 1000 replicates of the PDIST model were obtained as phylogenetic trees of all samples based on the neighbor-joining algorithm of MEGA software .2.3.5Principal Components analysis (PCA) was performed based on SNP to obtain the clustering of Principal components of all samples. Through PCA analysis, it can be known which samples have short genetic distances and close relatives. The samples with long genetic distances and distant relatives are helpful for population genetic evolution analysis.2.3.6Population genetic structure analysis can provide information on the origin and composition of individual lineage. Based on SNP, the population structure of all samples was analyzed by ADmixture software, and the number of clusters  was assumed to be 1-10, respectively. Different K values represent the distribution of ancestral genetic material of different populations under the assumption that there are K ancestral groups.2.3.7Based on SNP, the genetic distance between pairs of all samples was calculated. We can get the relative distance between samples by analyzing genetic distance data, which can assist the evolution analysis. In the phylogenetic heat map, the redder the color, the closer the relationship between the two individuals on the horizontal and vertical axes, the large area of red in the phylogenetic heat map between multiple individuals indicates that these individuals may constitute a closely related family group. Conversely, the bluer the heat map, the more distant the relatives.33.1H. hainanensis samples. After removing low-quality sequences, sequences containing more than 5% N (N represents undetermined base information), and adapter sequences, 27.85 Gb of high-quality sequencing data (Clean data) was obtained. The average size of each sample is 0.66 Gb. The average proportion of base error rate below 1% (Q20) was 96.66%, and the average proportion of base error rate below 0.1% (Q30) was 91.34%, indicating the high quality of sequencing. The average ratio (GC content) of guanine (G) and cytosine (C) among the four bases of DNA was 47.84%, indicating that the distribution was reasonable. The data overview of each sample is shown in After GBS sequencing, 28.09 Gb of Raw READ data were obtained from 42 3.2S with polymorphisms. After filtering the SNPs obtained, 430376 high-quality SNPs were finally obtained for subsequent analysis. It can be seen from the following After comparing the data to the consensus sequence, GATK and SAMTOOLS software were used for mutation detection . SNPs co3.33.3.1H. hainanensis resources. After 1000 repetitions based on the PDIST model, the neighbor-joining algorithm of MEGA software was used to perform evolutionary analysis on all samples, and the phylogenetic tree of 42 H. hainanensis sample resources was obtained  was 1-10. Different K values represent the distribution of ancestral genetic material of different populations under the assumption that there are K ancestral groups. Since K=1 cannot represent the distribution of ancestral genetic material of different populations, it is not shown in the figure. As shown in H. hainanensis populations in Hainan Island came from two different ancestors, and there was less gene exchange among them. In the table of genetic differentiation coefficients among populations  between FY, WZS, and DLS populations. The genetic differentiation between DLS and BWL, WZS populations, and FY and BWL populations was significant (0.15 < Fst < 0.25). There was a moderate genetic differentiation between BWL and JFL, YGL, FY and LMS, JFL and DLS, and the other three populations (0.05 < Fst < 0.15). In addition, the genetic differentiation among other populations was low, so differentiation could not be considered (Fst < 0.05).To further verify the evolutionary genetic relationship between the samples and infer that the ulations Table 4.3.3.3H. hainanensis were clustered to form three independent clusters, among which eight samples from Jianfengling , Limushan  and Yinggeling (YGL_1) populations with similar genetic backgrounds were clustered together to form cluster 1. Fanyang (FY_1), Wuzhishan(WZS_1-WZS_2), Bawangling (BWL1-BWL_13), Yingge Mountain , Jianfeng Mountain  and Limushan . The 31 samples from the six populations of MS_7, LMS_9, and LMS_10 were clustered together with similar genetic backgrounds to form cluster 2. The population of DLS_1-DLS_3 was far away from the other 2 clusters, showing a long genetic distance, so the population of DLS_1-DLS_3 formed a cluster alone. Principal component analysis (PCA) was carried out on H. hainanensis population samples from Hainan Island to determine the evolutionary relationship among the populations further. When the geographical distance between groups is relatively close, PCA can better reflect the relationship between groups. Samples with similar genetic backgrounds will gather in the figure to form a cluster. The farther the distance between the two samples in the figure, the greater the genetic background difference between the two samples. As shown in the following figure Figure 53.3.4In the relatedness heatmap Figure 644.1H. hainanensis field, the ecological range of the population is wide. The seeds are winged nuts, and the germination rate is high. Still, the seeds have higher requirements for germination conditions in the natural environment, which restricts the development of the population . In the 20th century, Hainan Island’s deforestation increased rapidly. About 80% to 95% of the primary forests have been destroyed because of logging for wood on a large scale. Furthermore, transitions to rubber trees and Eucalyptus plantations, and the growth of cities , the second principal component (PC2), and the third principal component (PC3) were 28.78%, 11.2%, and 6.29%, respectively. The contribution rates of the three principal components selected in this analysis were all low, and the total contribution rate was less than 50%. Therefore, there may be a deviation (difference) between the cluster results of PCA and the analysis results of other groups. In the principal component analysis, the genetic distance of the Diaoluoshan population was far from the other populations, and a single cluster was formed. Except for principal component analysis, the population structure of all samples, K value selection of population structure, and phylogenetic evolutionary tree analysis results showed that the cluster division was the same and supported the division of seven populations into two populations. Therefore, it is more reasonable to divide the 42 H. hainanensis samples from seven populations into two groups: Group 1  and Group 2 . In this study, high-throughput GBS sequencing was performed based on SNPs, and the analysis results may be limited due to the lack of reference genomes covering the whole genome of SNP.Based on SNP, simplified genome sequencing analysis was performed on 42 4.2st = 0.09211), the existing gene flow between populations may have originated from the common ancestor of genetic exchange, carried by man-made factors, animals or other factors such as geological factors into the other group.Genetic structure is influenced by many factors, such as breeding system, genetic drift, population size, seed dispersal, gene flow, evolutionary history, and natural selection . The ter5H. hainanensis populations, the H. hainanensis population resources of endangered plants should be effectively protected and developed. In order to protect H. hainanensis species, H. hainanensis seedlings may be protected ex situ due to their weak competitive ability and easy inhibition by mother trees. By conserving H. hainanensis seedlings ex-situ, we can reduce competition within the population and increase competition between poke stack populations. Genetic drift can also be reduced by increasing gene flow among small populations. Additionally, cross-introduction and breeding among the seven populations can improve genetic diversity.In order to improve genetic diversity among 6H. hainanensis populations because seedlings and saplings grow slowly and are frequently unable to establish themselves in heavily shaded conditions. To help restore endangered H. hainanensis populations on Hainan Island, select populations with high genetic diversity .Because the loss of genetic variation is a major threat to endangered species, preserving and restoring genetic variation is an important conservation action . We discThe datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.Conceptualization, LZ, MMN; methodology, YKC, HLZ, XTZ; software, HLZ, YKC, and MMN; validation, MMN; formal analysis, HLZ, LZ and MMN; investigation, YKC, and XTZ; resources, YKC; data curation, YKC; writing—original draft preparation, LZ, HLZ, HYZ, TTL, XTZ, YKC, and MMN; writing—review and editing, LZ, HLZ, HYZ, TTL, XTZ, YKC, and MMN; visualization, YKC; supervision, YKC; project administration, YKC; funding acquisition, YKC All authors have read and agreed to the published version of the manuscript. The data presented in the study are deposited in the Dryad repository.