Commiphora gileadensis (L.) C. Chr is a perennial plant existing mainly in the southern and western mountains of the Arabian Peninsula. In the Makkah province, the remaining populations are threatened by many factors such as overcutting, overgrazing, and urban developments. These dangers are expected to be aggravated by the progression of aridification factors arising from climate change. To overcome the decline in remaining populations of this valuable species, a timely evaluation of the population’s genetic variables and genetic structure is vital for the conservation of existing C. gileadensis populations. In this study, we used 61 SSR primers to achieve this objective. Only 50 loci showed polymorphisms, which led to further analysis of the population genetics for 600 genotypes that were collected from 50 populations of C. gileadensis found in 10 different sites in the Makkah region: Gebel Al Muliesaa, Wadi Albathna, Wadi Houra, Wadi Albaidaa, Wadi Elebiedia, Gebel Kniethl, Wadi Sayaa, Wadi Elbarasa, Wadi Alfawara, and Wadi Alkharar. The results showed an obvious decrease in genetic diversity variables in all studied populations. The range of PPL was between 8 and 40; additionally, the low HT value of 0.804 and the high value of inbreeding, Fis = 0.238, reflected a severe lack of heterozygotes. High levels of FST and GST and low gene flow indicate considerable segregation among the C. gileadensis populations, which creates a barrier to gene migration. Our data suggest the need for conservation planning for C. gileadensis in order to avoid the species’ forthcoming extinction. Efforts should be largely oriented around managing water consumption, prohibiting overcutting and overgrazing, and establishing appropriate seed banks. Commiphora are listed as endangered due to the overcollection of their populations for utilisation in medicinal and economical purposes (Burseraceae [Myrrh family]) [Commiphora gileadensis (L.) C. Chr is considered one of the most economically and medicinally valuable trees grown in the Northern Hemisphere. Its distribution is mainly centred in the Red Sea region in the southern and western mountains of Saudi Arabia and other mountainous habitats in neighbouring countries within the Southern Arabian Peninsula and East Africa [Many members of the genus family]) . Commipht Africa ,3. The wt Africa ,5 as welt Africa . Commiphora gileadensis is found in few remaining populations, and it is mainly associated with the bottom of rocky mountains in the south and western regions of Saudi Arabia where its distribution is centred in Makkah province . The species was originally estimated to have between 1800 and 3000 populations that declined to the current estimate of only 60 populations . The apparent reduction in the population numbers and population size of C. gileadensis could be attributed to progressive climate change conditions in the region [C. gileadensis populations and other associated plant taxa in arid habitats such as the Makkah region.e region  which, ee region , are antCommiphora gileadensis plants could be affected by a considerable decline in genetic diversity as a consequence of genetic drift, which is a key reason for the apparent low fitness and thus severe inability of many populations to adapt to ambient environmental confrontations [C. gileadensis is crucial to conserve and restore this valuable plant species [ntations ,11,12. T species  and may  species .C. gileadensis, self-incompatibility represents an extra stress that endangers its existence. This species has a floral structure like other members of Burseraceae and is recognizable by small, actinomorphic, and slightly odoriferous flowers; these features are considered to promote obligate outcrossing [One of the main limiting factors for plant species with low population sizes is their potential for outcrossing and performing successful seed setting, especially under the stress of arid conditions. For crossing . This recrossing ,17.C. gileadensis—are prone to the risk of extinction because of the ongoing decline in population size and potential loss of genetic diversity. Genetic analysis is pivotal for detecting genetic variation [C. gileadensis due to their high potential to detect repeat regions with variable sequences in the target genome. These loci are well characterized as co-dominant molecular markers [As a result of the ambient climate change conditions connected with human over-utilisation in the Makkah province, the existing plant taxa—including remaining populations of ariation  using SS markers ,21,22,23 markers ,25,26.C. gileadensis populations under the xeric conditions of the Makkah province; by applying microsatellite loci, we can extract the required data for proposing mandatory conservation plans that are crucial to prevent the imminent threat of extinction for C. gileadensis and other associated plant species grown in this region.Our research aims to elucidate the genetic diversity and genetic structure patterns of C. gileadensis, as the average inbreeding coefficient (Fis) was 0.238, verifying an obvious deficit of heterozygotes  varied between 1.48  and 1.08 (Welbi1 population), resulting in the mean number of effective alleles per locus (Ne) and the Shannon index (I). The highest number of private alleles was 0.087 and was calculated in the Gmul 3 population, while no private alleles were detected in Walb 1, Walb 4, Walbd5, Welbi2, Welbi3, Welbi5, Welbr2, Welbr3, Welbr5, Walk 2, and Walk 3 populations. Expected heterozygosity (He) ranged from 1.281, 0.217, and 0.138, respectively, in the Gmul 3 and Walb 5 populations to 1.037, 0.039, and 0.025, respectively, in the Welbi1 and Walbd2 populations (T) for all loci and populations was equal to 0.804.The mean number of alleles per locus , while the minimum value  was measured among individuals in the same population. The gene flow of Nm = 0.024 is a low value for the gene migration among population per generation. G-statistic measurements showed a higher value of FST = 0.913 and Gst value = 0.908 compared to the FST resulting from AMOVA, which confirmed high genetic differentiation among different populations.The AMOVA showed substantial genetic differentiation among the studied C. gileadensis showed a modest to extreme decline in measurements among all the enduring populations analysed and was in agreement with other studies on closely related plant species of Burseraceae. This indicates an extensive decline in gene diversity and meets the corresponding severe environmental circumstances [ST values, which measured for C. gileadensis (FST = 0.896) with other rare plant species in similar plant habitats in South Sinai, and they revealed considerable values of genetic differentiation, e.g., Primula boveana (FST = 0.737) [Cotoneaster orbicularis (FST = 0.634) [In this study, all the genetic diversity variables of mstances ,32,33,34= 0.737)  and CotoC. gileadensis populations are subjected to high genetic drift and inbreeding, which exacerbate the problem of decline in polymorphic alleles as an imminent result of encountering environmental conditions [The main reason behind the lack of genetic diversity in the existing populations may be their small population size. A leading factor underlying this decline could be that nditions ,37,38.The distribution outline of genetic diversity locations among the studied populations revealed a considerable range of variability, with relatively modest polymorphisms measured in the populations of Gebel Al Muliesaa, Wadi Alkharar, and Wadi Houra, which could be attributed to the relative abundance of water reserves in these regions. Wadi Albathna and Wadi Albaidaa are located at the foot of the Bany Ayoub Mountain region, where water reserves are more abundant due to frequent floods caused by rain in this location. Wadi Elbarasa has low-altitude valleys characterized by water aggregations that allow for the growth of a few plant populations. The slight gorges in Gebel Kniethl (750–1000 m a.s.l) facilitate the growth of plant populations , revealiC. gileadensis and the abundance of water reserves. The PCoA subdivided populations with relatively high polymorphisms, showing sites with water abundance on the right and left upper parts of the PCoA axis: Gebel Al Muliesaa, Wadi Alkharar, and Wadi Houra were in the upper-right group and populations from Wadi Albathna, Wadi Elebiedia, and Wadi Albaidaa were in the upper-left group. The STRUCTURE analysis results for the existence of only two subpopulations for the studied 500 genotypes of C. gileadensis and the notable values of genetic differentiation among populations, calculated using AMOVA and confirmed with G-statistics, indicated considerable isolation among the population sites. The main reason for this high isolation could be the increasing activities of human inhabitants, which include overcutting for cosmetic and medicinal purposes. This can also include excessive overgrazing by camels and sheep herds owned by local tribes in the area or other tribes inhabiting the southern region of Saudi Arabia with drier climatic conditions [The PCoA determined the association between the genetic diversity in nditions  that becnditions ,41.C. gileadensis and further isolation are anticipated with increasing temperatures and water deficiency conditions, as indicated by the fluctuations of rain frequency in these sites [C. gileadensis flowers—as well as have a negative impact on pollination potential—and is thus expected to increase selfing [is).Larger decreases in the population size of se sites ,9. Moreo selfing ,42, as rC. gileadensis is characterized by drupe-type fruit with one seed that is considered relatively heavy for wind dispersal (sizes range from 3.5 to 4.8 cm), which supports our computed low level of gene flow among C. gileadensis populations. For this reason, the extensive anthropogenic and climatic causes of isolation have promoted the elevation of the genetic differentiation value among the remaining populations of C. gileadensis. This phenomenon was clearly outlined in the PCoA. The values of the calculated gene flow decreased considerably from the values required for preventing an increase in genetic drift [C. gileadensis.ic drift . The conC. gileadensis in the mountains and plains of the Makkah province included fifty populations from ten different sites . Fifty loci revealing polymorphisms were recognized using sixty-one formerly published primers for other species belonging to the Burseraceae family ,29,30,31The products of the PCR reactions were sequenced using a 3130xl Genetic Analyzer  with LIZ500 as a size standard. The sequences of amplified fragments were determined using GeneMapper 4.0 , and the lengths of the amplified fragments ranged from 112 to 300 bp in accordance with .The variables of genetic diversity, genetic structure, inbreeding, and G-statistics were calculated using GenAlEx 6.5 , measuriC. gileadensis was conducted using the Bayesian clustering method in STRUCTURE version 2.3.4 [The genetic structure for 50 populations of on 2.3.4 , with thon 2.3.4 . The opton 2.3.4  as impleon 2.3.4 .Nm) was calculated via the private allele method [Ho), the expected heterozygosity (He) under Hardy–Weinberg equilibrium, and Wright’s fixation index (F = 1 − Ho/He) were tested for each locus in each population to test deviations from the Hardy–Weinberg equilibrium and thereby determine inbreeding in existing populations of C. gileadensis. Principal coordinate analysis (PCoA) was performed using GenAlEx 6.5 [AMOVA was applied with 999 permutations to assess genetic differentiation among populations ,50. Genee method . The anaAlEx 6.5  based onC. gileadensis in habitats of the Makkah region; it was carried out in order to contribute to the conservation management and protection for C. gileadensis as an economically and medicinally valuable plant species. This species is confronted by the danger of forthcoming extinction due to an extreme loss of gene polymorphism associated with excessive interpopulation genetic differentiation and severe inbreeding.Our current research represents the first assessment of distribution patterns of population genetic variables and genetic structures among and within the remaining populations of C. gileadensis should be based on long-term and progressive actions and should be oriented mainly to prevent the continuous degradation of its populations and its habitats. Many efforts could be considered in this area. Firstly, the results of the present study suggest that we should establish enclosures of a wire-fence type to protect populations that are subjected to severe low genetic polymorphisms [The conservation plan of orphisms , as indiST) and higher selfing, potentially increasing the population fitness [C. gileadensis conservation efforts in its habitats. The recommended measures for the conservation of C. gileadensis could be applicable to the other rare species in the genus Commiphora which grow in similar arid habitats.Thirdly, the apparent decline in genetic diversity and the considerable genetic differentiation in these populations could be reclaimed via performing a test of interpopulation crosses among the populations with the highest genetic differentiation values (F fitness . Many fa fitness . Moreove fitness . The col


Centaurea L. is one of the most widespread, differentiated, and critical genera of Asteraceae in the Euro-Mediterranean area, with more than 100 currently recognized species inhabiting the region. The controversial C. tenorei group, narrowly endemic to the Peninsula of Sorrento , includes three weakly differentiated microspecies: C. tenorei Guss. ex Lacaita, C. montaltensis (Fiori) Peruzzi and C. lacaitae Peruzzi. However, their taxonomic distinctiveness and relationships with close or sympatric species are still unclear. In particular, the existence in several localities of individuals with intermediate morphology suggests inadequate taxonomic assessment within the group or hybridization and introgression with other species. In this study we aimed at defining population structure in this complex. With this objective, we sampled the three currently accepted species from their loci classici  and from other localities throughout the range, including populations of difficult identification occurring where the ranges of different taxa overlap. We employed a panel of SNPs obtained via genotyping-by-sequencing for investigations on genetic structure, admixture and ploidy inference, the latter also compared with chromosome counts. Our results showed that Centaurea tenorei s.l. is consistently tetraploid, contradicting the current taxonomy that was also based on ploidy level. Population structure analyses indicated the presence of four to seven clusters, most of which with clear evidence of admixture. Furthermore, contrarily to what previously supposed, we demonstrated a remarkable contribution of C. deusta, more that of C. cineraria in the genetic make-up of C. tenorei. However, we found a population of C. cineraria outside its ecological range, probably driven by climate change, which could be responsible in the future of further hybridization phenomena. Centaurea L. have been intensely investigated in the last years, from both a morphological and molecular point of view , C. montaltensis (Fiori) Peruzzi , and C. tenorei s.str. , all narrow endemic to the Sorrento Peninsula . As indicated in C. deusta Ten. subsp. deusta and C. cineraria L. subsp. cineraria  testing the occurrence of admixture within the C. tenorei species group or between these latter and C. cineraria or C. deusta, (c) detect a possible correlation between ploidy and populations \ taxa, (d) understanding whether genetic variation among our three taxa of interest is indeed sufficiently discontinuous to grant their taxonomic recognition.In this study, using a NGS approach, we aim at (a) describing genetic structure in populations of C. lacaitae [lacCA], C. montaltensis [monAV], and C. tenorei [tenMO] from the localities in which the taxa were originally described and are currently found. Some populations, difficult to ascribe to a single taxon, were included as well: some atypical individuals of C. cineraria from Valico di Chiunzi with glabrescent leaves [cixVA]; a single presumed hybrid between the former taxon and C. deusta [hybVA]; several populations showing, at various degrees, intermediate features between C. tenorei s.l. and C. cineraria  Del Guacchio, Cennamo & P.Caputo  [leuBA], occurring in the same region but not sympatric with our group of interest. We also added two individuals of C. ambigua Guss. subsp. ambigua [ambPC] because, even if C. ambigua s.l. is completely allopatric, it is often included in the taxonomic aggregate of C. tenorei (C. pandataria (Fiori & Bég.) Bég. [panVE] . We gathered a larger number of samples for monAV because this population extends broadly along an altitudinal cline (600–1000 m a.s.l.), and we aimed at checking whether any genetic difference might occur within it. The taxa were identified mainly by using We field-collected leaf material from 96 samples of the above-mentioned taxa Figure 1de novo SNP detection. Libraries were prepared using MsII restriction enzyme  and sequenced at LGC Biosearch Technologies  on a 2 × 150 bp Illumina NextSeq® 550 platform . Library groups were demultiplexed into samples using the Illumina bcl2fastq v2.17.1.14 software (available at https://support.illumina.com/downloads/bcl2fastq-conversion-software-v2-20.html) according to their inline barcodes and verification of restriction site . Sequencing adapters were clipped from all reads, reads with final length < 20 bp were discarded and reads with 5’ ends not matching the restriction site were removed as well. Reads were further filtered by quality, trimming at 3’ end to get a Phred quality score ≥ 20 over a window of ten bases and discarding reads with a final length < 20 bp. Forward and reverse reads were assembled in contigs using BBMerge v34.48 (We performed genotyping-by-sequencing (GBS) for e v34.48 . Unique e v34.48  allowinge v34.48 . SNP dise v34.48  at the fL is chosen as the true ploidy level. After the results of this analysis, we detected some triploids and, therefore, recalled SNPs in FreeBayes setting ploidy also to 3.To estimate the ploidy of single individuals, we analyzed the GBS data using the software nQuire . This soWe compared such estimated ploidy values with chromosome counts for the following populations: aeoST, cinML, intSM, intVA, leuBA, lacCA, monAV, panVE, and tenMO. For this purpose, we employed cypselae deposited in the seed bank of the Botanic Garden of Naples or collected in field. Chromosomal observations were made on root tips obtained from the germination of cypselae in 1% agar Petri dishes. After the appearance of root tips, plates were stored for 12-24 hours at 4°C. Then, seeds with attached root tip were immersed in 0.4% colchicine for 4 hours and subsequently fixed in Carnoy fixative solution for 1 hour. After hydrolysis in 1N HCl at 60° C for 7 min, the tips were stained with Schiff’s fuchsin-sulfite reagent . Root tips were then soaked in 45% acetic acid, macerated and squashed. Metaphasic plates were observed, whenever possible, from root tips of 3-5 different individuals from seeds of at least 3 different mother-plants, using a Nikon Eclipse Ci-L microscope .http://evomics.org/wp-content/uploads/2016/01/BayeScan_BayeScEnv_exercises.pdf.The final dataset containing mixed-ploidy individuals was further subject to filtering by missingness and linkage to reduce their impact of subsequent analyses. Specifically, we first calculated the missingness per locus, then ordered the loci by increasing missingness and retained, when multiple SNPs occurred in the same contig, only the one with less missing information across most of individuals. After this first selection, we filtered the dataset by missingness per individual, removing all the samples with missing data < 50%, and, per locus, removing all loci with ≥ 30% of missing data. To assess if the selected loci were neutral, in absence of a pipeline/software capable of handling data with mixed ploidy, we manually created an input file for BayeScan v2.1  making aAs first exploratory analysis of structure in our data, we carried out a principal component analysis (PCA) in adegenet v2.1.5 . After chttps://www.milan-malinsky.org/fineradstructure), also ordering loci according to linkage disequilibrium (script ‘sampleLD.R’) as suggested by the author for unmapped loci.To assess population structure and detect recent shared ancestry among population samples, we used fineRADstructure , a modelK), STRUCTURE was run with K varying from 2 to 17 for 100,000 generations, the first 50,000 of which were discarded as burn-in. Each value of K was evaluated using ten independent Markov Chain Monte Carlo (MCMC) replicates. To choose the best value of K, we looked at the plot showing the log probability of the data [ln Pr(X|K)] against a range of K values as provided by STRUCTURE HARVESTER , we carried out an analysis in STRUCTURE v2.3.4 . This soARVESTER  and seleARVESTER . We chosARVESTER  because ARVESTER . The resARVESTER  using LaARVESTER  and visuARVESTER .ST)   in the RST) . PopulatC. aeolica (2n = 36), C. ambigua subsp. ambigua , C. deusta subsp. deusta (2n = 18), C. deusta subsp. leucolepis (2n = 36), C. lacaitae , C. montaltensis , C. pandataria (2n = 18), C. tenorei . Intermediate populations  resulted tetraploid, or rarely triploids .We obtained from SNP data analysis (through nQuire software) the following estimation for each taxon  and one intermediate individual (from intVI), as well as all the triploid samples were discarded because afflicted by high individual missingness (mean = 0.68). BayeScan analysis identified only 37 loci out of 7473 under positive selection  . Of these loci, 130,411 were polymorphic and contained 466,078 SNPs. After filtering procedures described in Materials and Methods, we obtained two datasets: one, including 7,473 loci and 75 individuals and utilized for PCA, fineRADstructure, and FMean observed heterozygosity was 0.120, significantly lower (p < 0.001) than expected heterozygosity (= 0.285) 2. Most Centaurea pandataria was distant from all the other populations, and therefore can be considered as the best outgroup for our system. Halfway between this group and the others, the OTUs relative to C. aeolica and C. ambigua subsp. ambigua are localized. The three microspecies of the C. tenorei group are very close and can be distinguished only in one projection (PC1/PC3); in all cases, they are very close to the intermediate populations , which effectively are halfway between them and the C. cineraria group  and showed that all the operational taxonomic units (OTUs) from the same population clustered together Figure 2C. tenorei s.l. For instance, C. montaltensis had high co-ancestry with the neighboring C. deusta (subsp. deusta and subsp. leucolepis), as well as with C. tenorei and C. lacaitae and, to a lower extent, with the intermediate populations. These latter ones had comparable levels of ancestry with C. tenorei s.l. and C. cineraria; C. cineraria showed high co-ancestry with C. pandataria and some individuals from intVI and intSM. Within C. cineraria, the sampled populations resulted highly structured: we observed four distinct sub-clusters, corresponding to cinCA, cinML (these two very close each to the other), cinMI, and cixVA , a second group encompassing C. cineraria  and specimens resembling it (cixVA), a third group with C. deusta , and a last one including populations attributable to C. tenorei s.l.  and its intermediates . The individual labelled as hybVA was confirmed as a hybrid between C. cineraria and C. deusta. At K = 5, the coastal population intVI was further separated from the others; similarly, at K = 6, the other coastal population, lacCA was segregated from the C. tenorei aggregate. At K = 7, the population cixVA was separated from the other C. cineraria samples. As a consequence, the specimen hybVA, which is sympatric with population cixVA, is obviously a hybrid between the latter population and C. deusta. For all Ks, C. montaltensis and C. tenorei appeared very similar to each other and remarkably more admixed with C. deusta than all the other populations of the complex.STRUCTURE analysis indicated K = 7 as the optimal number of clusters  a low genetic differentiation is present, altogether comparable to that between the two populations of C. deusta subsp. deusta (0.089). A slightly higher differentiation is observed between lacCA and monAV/tenMO . The plants with intermediate features  had FST values < 0.25 with C. tenorei s.l., with the only exception of lacCA vs. intVI (0.277); genetic structure was higher when comparing it with that of C. cineraria (~0.35) and C. deusta (~0.48). Within C. cineraria, the lowest differentiation was found between the populations from cinCA and cinML , a result also evident in the fineRADstructure analysis.FCentaurea tenorei s.l. also on account of their different ploidy . It is to be noted that the latter population corresponds to the tetraploid tenMO, whereas we could not find the former after several field trips. On Mt. Molare, we found diploid only the individuals belonging to C. deusta subsp. deusta, which is sympatric in that locality with C. tenorei and is widespread also elsewhere. Therefore, contrary to what previously supposed  and C. cineraria. Unexpectedly, our results revealed a greater involvement of C. deusta: according to STRUCTURE and fineRADstructure  and C. deusta subsp. deusta (2×)? This is difficult to explain by frequent hybridizations between these two latter taxa and diploid individuals of C. tenorei s.l.,  perform better in colonizing new habitats , a plasticity shared with C. deusta.These considerations entail an intriguing issue: as ei s.l.,  because habitats . This caC. tenorei s.l. remains obscure and might be clarified only by an extensive study involving the allied taxa of the group; this in turn is a rather insidious issue, because the obscure phylogeny of the section does not allow to identify them easily . It has to be noted, however, that the most widespread subspecies, i.e., C. ambigua subsp. ambigua, as far as it is known, is tetraploid  but different across them  did not observe it above 350 m a.s.l., even if he gathered on the pass as well. The plant might have been overlooked by him or confused with C. tenorei s.l. but this seems unlikely. Indeed, we cannot exclude that C. cineraria has reached the pass only recently, possibly also driven by climate change. The altitudinal shift upward has been documented for several mountain plants  and those indicated as ‘intermediate’  can be grouped in the PCA graphs , for example, did not accept var. maritima as a separate taxon, but was aware that f. cinerarioides would deserve recognition.The populations ascribed to ry close Figure 2V 0.092, ; also foes e.g., . The genuishable . Moreovee coasts , and we C. deusta group together, also including the subsp. leucolepis. This latter remarkable subspecies differs from the typical C. deusta on account of a combination of morphological features independently and sporadically occurring also in the subsp. deusta , of the maritime ecology and tetraploid chromosome number (C. corensis) suggested an allopolyploid origin of this taxon. Nevertheless, our results better support a direct differentiation from C. deusta by local autotetraploidy as an adaptation to coastal environments . Taxonomically, the results of the co-ancestry analysis with fineRADstructure  is entirely included in that of the other species . Indeed, a single hybrid between them is documented here for the first time . We may presume that C. deusta and C. cineraria, belonging to two different subsections (C. tenorei (mainly tetraploid) and C. deusta subsp. deusta, i.e., C. ×cavarae Guadagno ex Del Guacchio, Cennamo & Caputo, only known for a gathering in the same place . A form morphologically similar to C. ×cavarae still occurs in its locus classicus. Further and finer analyses are required to assess the precise identity of these forms.Hybrids between the homoploid taxa sections , are pooC. tenorei aggregate, i.e., C. tenorei, C. lacaitae, and C. montaltensis, including the intermediate forms, are chiefly tetraploid and that the current recognition of three microspecies is not supported by genetic data. These taxa appear of hybridogenous origin, with an important contribution of C. deusta (or its ancestor) and, to a lesser extent, C. cineraria. Some populations of this aggregate, however, have unique genetic features and may be regarded as evolutionary significant units (ESUs). We also find that the locality “Valico di Chiunzi” is particularly interesting from the evolutionary and ecological point of views because it harbors populations of C. cineraria out of their ecological range that hybridize with C. deusta and C. cineraria, creating an intricate pattern of relationships across species. Furthermore, this study confirms that SNPs markers are a powerful tool to analyze the genetic structure of difficult plant systems where mixed-ploidy, hybridization and introgression occur and to determine ploidy at individual level in the absence of chromosome counts.This study demonstrates that the taxa belonging to the The raw Illumina sequences used in the study are available in the Sequence Read Archive (SRA) repository under NCBI BioProject PRJNA928762. Voucher specimens are deposited at the Herbarium of the Botanical Garden of Naples (NAP) and available upon request. Supporting information to the paper is available as EDG and PCE contributed to conception and design of the study. Mainly EDG and PCE field-collected plant material, but all authors collected at least in one locality. DL carried out molecular work and data analysis. LP was responsible for chromosome counts. DDL, EDG, and PCA wrote the first draft of the manuscript. All authors contributed to the article and approved the submitted version. PCA supervised the whole work.


Paris polyphylla var. yunnanensis is a well-known medicinal plant that is mainly distributed in Southwest China; however, its genetic diversity and biodiversity processes are poorly understood. In this study, the sequences of cpDNA trnL-trnF fragments of 15 wild populations and 17 cultivated populations of P. polyphylla var. yunnanensis were amplified, sequenced, and aligned to study the population genetics of this species. Genetic diversity was analyzed based on nucleotide diversity, haplotype diversity, Watterson diversity, population-level diversity, and species-level genetic diversity. Genetic structure and genetic differentiation were explored using haplotype distribution maps and genetic distance matrices. A total of 15 haplotypes were identified in the 32 populations of P. polyphylla var. yunnanensis. Five unique haplotypes were identified from the fourteen haplotypes of the cultivated populations, while only one unique haplotype was identified from the ten haplotypes of the wild populations. The haplotype richness and genetic diversity of the cultivated populations were higher than those of the wild populations (TH = 0.900 vs. 0.861). In addition, there were no statistically significant correlations between geographic distance and genetic distance in the cultivated populations , whereas there was a significant correlation between geographical distance and genetic structure in the wild populations , indicating that there was a geographical and genetic connection between the wild populations. There was only 2.5% genetic variation between the wild populations and cultivated populations, indicating no obvious genetic differentiation between the wild and cultivated populations. Overall, the genetic background of the cultivated populations was complex, and it was hypothesized that the unique haplotypes and higher diversity of the cultivated populations were caused by the mixed provenance of the cultivated populations. P. polyphylla var. yunnanensis is a perennial herb whose stem is generally 20–30 cm in length and purplish red in color, and the plant has a membranous leaf sheath at the base that embraces the stem. The pedicel protrudes from the top of the stem and bears a terminal flower. The flowering period is from June to July, and the fruiting period is from September to October. This plant is mainly found in Southwest China ; prefers acidic or slightly acidic soil with rich humus layers; and is suitable for growing in evergreen broad-leaved forests, bamboo forests, or shrublands with temperatures in the range of 16–28 °C and elevations ranging from 1400 m to 3100 m [P. polyphylla var. yunnanensis have shown that the main functional components of this plant are Paris saponin and diosgenin, which have demonstrated antitumor, antibacterial, antiviral, and hemostatic functions [P. polyphylla var. yunnanensis has high medicinal and economic value and is the natural raw material that comprises 81 different Chinese patent medicines, such as “Yunnan Baiyao” and “Gongxuening Capsule” [P. polyphylla var. yunnanensis is listed in the Pharmacopoeia of the People’s Republic of China as one of the source plants of Paris polyphylla. However, wild P. polyphylla var. yunnanensis suffers from slow growth, and it has been overexploited, making it difficult to obtain sufficient numbers of seedlings. Many farms collect wild P. polyphylla var. yunnanensis and cultivate its samples to meet the growing market demand. Despite its popularity, few studies have analyzed the genetic diversity of wild and cultivated P. polyphylla var. yunnanensis, which is important to its future survival. As a consequence, it is urgent to understand the genetic diversity and genetic structure of P. polyphylla var. yunnanensis. Capsule” . P. polyP. polyphylla var. yunnanensis also underwent this migration, ultimately surviving and differentiating well in Southwest China. As a result, the genetic diversity and structure of P. polyphylla var. yunnanensis was deeply affected by the unique geographical conditions of Southwest China. Some studies have sought to identify the molecular markers that elucidate the genetic diversity of P. polyphylla var. yunnanensis. A total of 153 individuals from six populations (three wild populations and three cultivated populations) in the Sichuan and Yunnan provinces (China) were investigated based on ISSR molecular markers. The genetic diversity of the cultivated populations was higher than that of the wild populations (0.153 vs. 0.151), and there was no obvious genetic differentiation between the cultivated populations and the wild populations [P. polyphylla var. Yunnanensis [P. polyphylla var. Yunnanensis in the Yunnan province had a low genetic diversity [P. polyphylla var. yunnanensis [The harsh and unstable climate of the Quaternary Ice Age caused large-scale migration of organisms that led to disastrous survival rates. After the Ice Age, the glacial refugium provided a refuge for organisms to survive and ultimately rediffuse to other geographical regions because of its unique geographical conditions. ulations . The genulations . SSR molnanensis . Using aiversity . AFLPs wnanensis , which rP. polyphylla var. yunnanensis had been reported, earlier studies that focused on the Yunnan province had a narrow research area and only studied a small number of populations, which did not effectively reflect the genetic diversity and genetic structure of P. polyphylla var. yunnanensis at the entire species level. In addition, information regarding the phylogenetic structure of P. polyphylla var. yunnanensis based on cpDNA and nuclear genes has not yet been reported. Chloroplast genome (cpDNA) is maternally inherited in most angiosperms, while it is paternally inherited in most gymnosperms, meaning it undergoes almost no recombination. Compared to nuclear genomic markers inherited from both parents, cpDNA markers have the ability to reveal genetic differences in small population sizes. Therefore, cpDNA markers serve as an effective tool for studying phylogeography and elucidating the migration and diffusion routes of species in shelters and post-glacial periods ,12. AlthP. polyphylla var. yunnanensis using cpDNA fragments (trnL–trnF).The purpose of the present study was to analyze the genetic diversity and structure of 15 wild populations and 17 cultivated populations of trnL–trnF experiment using cpDNA fragments, the as-used materials were the same as AFLP markers [In the  markers . There wP. polyphylla var. yunnanensis leaves from each sample was extracted using cetyltrimethylammonium bromide (CTAB) according to a previously published (and improved) method [The DNA from approximately 0.5 g of dried ) method . trnL-trnF . PCR amplification of the trnL-trnF fragments was performed in a 20 µL reaction system using a K960 PCR apparatus with the following thermocycling program: 95 °C for 5 min, 95 °C for 1 min, 58 °C for 1 min, 72 °C for 1 min, and 33 cycles at 72 °C for 10 min; afterward, the PCR products were stored at 4 °C. The PCR products were then electrophoresed over a 1% agarose gel, and the clear bands with no impurities were selected and sent to Beijing Boyoushun Biotechnology Co., Ltd.  for sequencing.PCR amplification and sequencing were performed on 364 individuals using screened primers of cpDNA SeqMan 17.1 was used to check the peak map of all individual sequenced sequences. The positive and negative primers were spliced together and saved in the “.seq” format. Then, the chloroplast haplotypes were determined based on the variations in the sequences; the haplotypes in the population were named “H1”, “H2”, “H3”.... Then, all haplotype sequences saved in the “.seq” format were imported into SeqMan again and saved as a FASTA file for comparison and sequencing . Finally, the FASTA files were imported into the software for comparison, and the matched sequences were saved as FASTA and NEXUS files for subsequent sequence alignment analysis using MEGA-X .π), haplotype diversity (dH), Watterson diversity (θ), population-level genetic diversity (SH), and species-level genetic diversity (TH) [π, two sequences were randomly selected from the samples, and the average nucleotide variation was calculated based on the average unit site. dH referred to the frequency of two different haplotype sequences randomly selected from the samples. SH and TH referred to the haplotype diversity at the population level and species level, respectively. The parameters π and dH were statistically calculated using analysis of molecular variance (AMOVA) tests, while SH and TH were calculated using Permut v.1.2.1 with 1000 permutations [Genetic diversity parameters based on the DNA sequences included nucleotide diversity (ity (HT) ,16. To dutations ,18. The CTF); when the CTF value reached a maximum or stabilized at a specific value without a single population, the corresponding K value was considered the best group.The haplotype distribution maps of the wild and cultivated populations were edited using the ArcGIS software (v. 10.5) to visualize the distribution of the haplotypes in the wild and cultivated populations. The genetic distances between the wild and cultivated populations were calculated using MEGA-X based on Kimura’s 2-parameter model, which sorted out the geographic distance and genetic distance matrix, and the data were analyzed using GenAlEx (v. 6.503) to perform Mantel tests ,20. The STG and STN of the wild and cultivated populations were calculated using Permut v. 1.2.1 (1000 permutations). STG is the genetic differentiation coefficient of the frequency of haplotypes (range of 0–1). The smaller the coefficient, the lower the degree of genetic differentiation between populations, indicating that genetic variation mainly exists within each population. STN not only considers the frequency of the haplotypes but also the genetic differentiation coefficient related to the genetic distance between haplotypes. In many cases, these two values are not equal. One of the reasons for the difference may be the genetic and geographical structure of the populations. Therefore, the magnitude of the difference could be used to judge the genetic and geographical relationship between populations. When the value of STN is significantly greater than the value of STG, there is a clear differentiation in the geographical genetic structure between populations. However, when the value of STN is equal to or less than the value of STG, there is no geographical relationship and no differentiation in genetic structure between populations.The trnL–trnF intergenic spacer was used to classify 15 wild populations and 17 cultivated populations of P. polyphylla var. yunnanensis. Based on the sequence alignment, 15 haplotypes were detected in these 32 populations of P. polyphylla var. yunnanensis. Five unique haplotypes were identified from the fourteen haplotypes of the cultivated populations, while only one unique haplotype was identified from the ten haplotypes of the wild populations (P. polyphylla var. yunnanensis displayed high genetic diversity at the species level (TH = 0.861) (SH = 0.135), resulting in a high genetic differentiation among populations (STN = 0.919 and STG = 0.843). The cultivated populations showed the same trend as the wild populations of P. polyphylla var. yunnanensis . All populations of P. polyphylla var. yunnanensis displayed high genetic diversity at the species level (TH = 0.866), while the genetic diversity at the population level was low (SH = 0.181), resulting in a high genetic differentiation among populations (STG = 0.791 and STN = 0.860). Therefore, the total genetic diversity, haplotype diversity, and nucleotide diversity results indicated that the genetic diversity of the wild populations was slightly lower than the diversity of the cultivated populations, suggesting that artificial domestication in different planting regions increased the diversity of P. polyphylla var. yunnanensis.In this study, the cpDNA ulations . The wil= 0.861) , while ttrnL–trnF fragments were used to generate geographical distribution maps of the haplotypes of wild populations [p < 0.05)  indicated that haplotypes with a similar genetic distance appeared in the same population or populations with a similar geographic distance. Furthermore, out of 1000 replacements, the STN value for 990 of the replacements was 0.913. In this case, the value of STN was also higher than that of STG (p < 0.01), indicating a significant geographical genetic structure among the wild populations  . Similarulations . p > 0.05) , indicating no obvious geographical genetic structure between the populations of this group. Furthermore, after 990 replacements, the value of STN was 0.832, which was also higher than that of STG (p > 0.01)  . At the acements , which w > 0.01) , indicatCTF value did not stabilize. A maximum CTF value indicated that there was a single population group, which meant that there was no optimal group. Following the AMOVA analysis, genetic differentiation in the wild populations and cultivated populations (76.66% and 78.86%) was observed. However, there were no genetic differences between the wild populations and cultivated populations   shared between the wild populations and the cultivated populations, one unique haplotype (H13) in the wild populations, and five unique haplotypes  in the cultivated populations  . In termulations .TH = 0.861), which was higher than that of Cedrella odorata (TH = 0.700) [Juniperus przewalskii (TH = 0.700) [Alnus glutinosa (TH = 0.773), and Cyclobalanopsis glauca (TH = 0.681) [TH = 0.67) as measured using different molecular markers. However, Cycas taitungensis (TH = 0.998) [Cunninghamia lanceolate (TH = 0.952) [Scutellaria baicalensis (TH = 0.888) all had high cpDNA diversity [P. polyphylla var. yunnanensis as determined by sequencing the cpDNA trnL–trnF intergenic spacing was consistent with the diversity measured using AFLP markers [P. polyphylla var. yunnanensis was still higher than that of many other endangered plants, indicating that being endangered was not due to its own biological characteristics but rather by overexploitation or other factors.A higher genetic diversity of plants means they can more strongly adapt to environmental changes, which is important for their survival. Genetic diversity is affected by a number of natural and human factors, such as geographical distribution, habitat destruction, and overexploitation . Because= 0.700) , Juniper= 0.700) , Alnus g= 0.681) . Petit e= 0.681)  determin= 0.998) , Cunning= 0.952) , and Scuiversity . In this markers . HoweverP. polyphylla var. yunnanensis, encompassing 10 haplotypes in the wild populations and 14 haplotypes in the cultivated populations (TH = 0.900 vs. 0.861). According to the UPGMA tree of all individuals in Yunnan constructed based on AFLP markers [P. polyphylla var. yunnanensis populations was relatively complex. Therefore, both the haplotype richness and genetic diversity of the cultivated populations were higher than those of the wild populations. However, the correlation between geographical distance and genetic distance was not strong in the cultivation populations, negating the existence of a geographical genetic structure and suggesting that the unique haplotype of the cultivation populations might have been obtained due to the mixed provenance of the cultivation populations. In this study, we identified 15 haplotypes in ulations . Among tulations . MoreoveP. polyphylla var. yunnanensis was considered an endangered species in the industrial age. Currently, P. polyphylla var. yunnanensis is classified as a national second-class endangered medicinal plant, and the development and sustainable applications of its wild resources are of far-reaching significance in order to formulate effective protection strategies. We found that P. polyphylla var. yunnanensis was widely distributed throughout Southwest China, but there were few wild individuals collected in some areas. For example, only six and four individuals were collected in Longli county (LL) and Qishe town (XY), respectively, in Xingyi city. Seven and nine individuals were collected in Yimen County and Changning County (CN), respectively, in Yunnan Province. The genetic diversity of 15 wild populations from three provinces was analyzed and compared by sequencing cpDNA fragments, the results of which showed that the wild populations in the Guizhou and Sichuan provinces had high genetic diversity; however, more studies need to be carried out in various populations.Wild P. polyphylla var. yunnanensis showed that the H1 and H6 haplotypes are ancient haplotypes [P. polyphylla var. yunnanensis in this study indicated that Guizhou and Western Yunnan provinces might have been two independent glacial refugia, suggesting that priority should be given to these areas to protect wild P. polyphylla var. yunnanensis in these regions and strictly prohibit the excavation of land harboring wild P. polyphylla var. yunnanensis to ensure the protection of its habitat.Previously published evolution network diagrams of wild plotypes ,21. The P. polyphylla var. yunnanensis is an important naturally growing TCM. In recent years, it has been rapidly cultivated in large quantities to meet the world’s demand for traditional Chinese medicine [P. polyphylla var. yunnanensis germplasm resource nursery for systematic relocation protection while taking local protection measures. Although we observed that these populations had high genetic diversity, hybrid provenances might produce an uneven quality of P. polyphylla var. yunnanensis. Therefore, to obtain high-quality raw materials for the development of the heavy building industry, the screening and purification of high-quality seed sources should be carried out in conjunction with relocation protection.medicine . It takemedicine . Traditimedicine ,32. FromP. polyphylla were analyzed by sequencing the cpDNA trnL-trnF fragments and comparing the results with those of AFLP markers. Based on the analysis of the trnL-trnF fragments, the wild populations of P. polyphylla had a moderate level of genetic diversity (TH = 0.861), which was consistent with the results of similar analyses using AFLP markers. A total of 15 haplotypes were identified in the 32 populations of Paris polyphylla; five unique haplotypes were identified in the cultivated populations, while only one unique haplotype was identified in the wild populations. Moreover, the genetic diversity of the cultivated populations was higher than that of the wild populations (TH = 0.900 vs. 0.861), which was also consistent with the results of the AFLP marker analysis. Combining the results shown in the UPGMA tree of the cultivated population grown in Yunnan province established based on AFLP markers with the results of the Mantel test of the cultivation group based on trnL-trnF fragment sequences, it was speculated that the unique haplotype of the cultivation group might have been obtained due to the mixed provenance of the cultivation populations.In summary, the genetic diversity and structure of 15 wild populations and 17 cultivated populations of


Blastocystis is a common intestinal protist with a wide range of hosts. Thus far, 38 subtypes have been identified. In recent years, wild animals have been confronted with habitat fragmentation as well as an increasing risk of zoonotic disease transmission due to human disturbance. Only limited data are available on Blastocystis infection and subtype distribution in wild rhesus macaques in China. The aim of the present study was to investigate the prevalence and genetic diversity of Blastocystis in nine wild rhesus macaque populations in China.A total of 276 faecal samples were collected from five high-altitude populations  and four low-altitude populations  of rhesus macaques. PCR-based analysis, using a new primer pair for the amplification of a 1690-bp sequence of the small subunit ribosomal RNA (SSU rRNA) gene, was used for prevalence and genetic diversity analysis.Blastocystis infection was common in rhesus macaques, with an infection positivity rate of 80.1% (n = 221/276 samples). There was no significant difference (P = 0.121) in positivity rate between the LAG (84.3%) and HAG (76.8%). Overall, 33 haplotypes were obtained and classified into four subtypes (STs), of which three were potentially zoonotic subtypes  and one that was first identified in this study and named ST39 (12.0%). The STs were distributed differently among the rhesus macaque populations, except for ST3, which was found in all populations. Phylogenetic analyses revealed two major divergent clades of ST3 for the HAG and LAG. Genetic diversity analysis showed a high genetic diversity of ST3  in the rhesus macaques, but a high genetic differentiation (FST > 0.25) and a low gene flow (Nm = 0.09) between the HAG and LAG.Analysis of faecal samples revealed that Blastocystis infection in multiple wild rhesus macaque populations in China, indicates a potential risk of zoonotic transmission of Blastocystis in the study areas. Blastocystis ST3 showed high genetic diversity in wild rhesus macaques and significant genetic differentiation between the HAG and LAG. Our results provide fundamental information on the genetic diversity and prevalence of Blastocystis in wild rhesus macaque populations.Our study, which is the first investigation on The online version contains supplementary material available at 10.1186/s13071-023-05691-7. Blastocystis is a genus of single-celled intestinal parasitic protists commonly found in humans  region), with large populations at altitudes in between  [Blastocystis in nine wild rhesus macaque populations in China . Five of these populations live in ecotourism areas and have close contacts with humans because of being fed by tourists and staff at the tourism site. We recorded information on the altitude of each rhesus macaque population in detail and analysed the differences in prevalence of Blastocystis between high-altitude and low-altitude populations. Genetic diversity was explored based on PCR amplification and sequencing of the SSU rRNA gene.In China, wild rhesus macaque (ovinces) . The aimBlastocystis in rhesus macaque, we collected 30–40 faecal samples from different individuals from each geographical population. The high-altitude group  included five populations: Baiyu , Heishui , Seda , Yajiang  and Daofu . The low-altitude group  contained four populations: Lingshui , Fengjie , Jiangjin  and Nanning  , following the instructions of the manufacturer. The obtained DNA specimens were stored at − 20 °C until PCR analysis.Blastocystis was identified by using nested PCR amplification. The primer pair RD3 (5’-GGG ATC CTG ATC CTT CCG CAG GTT CAC CTA C-3’) and RD5 (5’-GGA AGC TTA TCT GGT TGA TCC TGC CAG TA-3’) was used for the first PCR [irst PCR . Accordiirst PCR , novel shttps://www.dnastar.com/; DNASTAR, Madison, WI, USA). All sequences were compared with reference sequences available in GenBank, using the Basic Local Alignment Search Tool (BLAST). According to the recommendations for annotating new subtypes, Blastocystis subtypes were determined by exact match or an identity > 96% against reference subtypes, and all sequences obtained from this study were analysed by BLAST separately at each end [All positive PCR products were sequenced at Tsingke Biotech  by bidirectional sequencing. The raw sequences were edited manually using Lasergene Seqman software version 7 . Then, 2 µl of DNA was used with the pClone007 vector . Subsequently, between 5 and 10 colonies from each transformation were selected and sent to Tsingke Biotechnology for subculturing, plasmid purification and sequencing.All 33 representative sequences generated in this study were submitted to GenBank under accession numbers OP563821–OP563853.Blastocystis reference sequences of known valid subtypes  were downloaded from the reference database at http://entamoeba.lshtm.ac.uk/blastorefseqs.htm (updated 19 Nov 2021) and GenBank. Nucleotide sequences were aligned with the MUSCLE algorithm, phylogenetic analyses were performed using neighbour-joining (NJ) and maximum-likelihood (ML) methods based on 1690-bp sequences and genetic distances were calculated with the Kimura 2-parameter model using MEGA X, which was also used for evolutionary analysis to establish divergence between sequences (pairwise distance) (http://www.megasoftware.net/). Bootstrapping with 1000 replicates was used to determine support for the generated clades.The full-length Blastocystis and the genetic differentiation between the LAG and HAG, DnaSPv4 [Blastocystis ST3, which was found in all populations. We included these indices: number of segregating sites (S), haplotype diversity (Hd), nucleotide diversity (π), average number of pairwise nucleotide differences (K), gene flow (Nm), genetic differentiation index (FST), Fu's Fs and Tajima’s D test.To investigate the genetic diversity of  DnaSPv4  was usedBlastocystis among the HAG and LAG and among geographical populations were analysed by Chi-square test (χ2) in SPSS version 22.0 , and differences at P < 0.05 were considered to be statistically significant.Differences in the prevalence of Blastocystis was detected in 221 samples (80.1%) , DF (42.3%) and LS (64.5%) (P < 0.001). The highest prevalence of Blastocystis was found in population NN (97.2%), followed by JJ (96.6%) and YJ (96.4%). The prevalence rate of Blastocystis did not significantly differ between the HAG (76.8%) and LAG (84.3%) (P = 0.121)  between the HAG and LAG  confirmed 33 haplotypes from 221  Network , the nov690 bp coMixed infection with different subtypes was found in 27 samples , mainly from LAG populations (23/27) , with 95.3–95.4% nucleotide identity, which is in compliance with the proposed guidelines that new subtypes should normally differ by ≥ 4% from previously known subtypes [The novel subtype ST39 had eight haplotypes, ST39A–H. The most closely related reference sequence for ST39 was ST1-AB107968  and full-length reference sequences , with  D and Fu’s Fs values were positive, and the differences in the neutrality indices were not statistically significant. The FST and Nm values of ST3 between the LAG and HAG were 0.732 and 0.09, respectively, revealing a high degree of genetic differentiation and a low gene flow.The results of the genetic analysis showed a high level of genetic diversity in all populations Table , with 78Blastocystis is distributed worldwide as a common intestinal protozoan parasite that can infect a range of hosts; wild and domestic animals are potential reservoirs for human infection [Blastocystis in wild NHPs in China has been poorly studied, only one study reporting the infection by Blastocystis ST5 in a golden snub-nosed monkey (Rhinopithecus roxellanae) in a natural reserve [Blastocystis in multiple wild rhesus macaque populations in China. Our results provide data on the prevalence and distribution of Blastocystis subtypes, and data on the genetic diversity of Blastocystis ST3.nfection . The pre reserve . The preBlastocystis is highly prevalent in wild rhesus macaques , with significant differences in prevalence among the nine wild rhesus macaque populations. A previous study reported that Blastocystis prevalence in captive macaques in China ranged from 5.6% to 96.6% [Blastocystis in wild populations of macaques. In Thailand, the prevalence of Blastocystis in 14 wild Macaca fascicularis populations was 41.9% [Blastocystis may be attributed to the difference in environmental and geographical factors [The findings of the present study indicate that to 96.6% , but onlas 41.9% , which i factors . Various factors . For wilBlastocystis between the LAG (84.3%) and HAG (76.8%). Normally, the warm and humid climate of low-altitude areas may be suitable for parasite survival, leading to more chances for the parasite to colonise the host. The high prevalence of Blastocystis in the HAG indicates that at high-altitude areas, the parasite may be not largely impacted by environmental stress. At present, there are no relevant studies on the impacts of environmental factors on Blastocystis infection. Research estimating the effects of natural environmental factors, human interventions and socioeconomic conditions on parasitic diseases may inform disease prevention and control strategies [Blastocystis or other zoonotic parasite infections, with the aim to provide suggestions for protecting wild rhesus macaques from parasite infections.In our study, there was no significant difference in the prevalence of rategies . In our P < 0.05) between the HAG and LAG, most likely because of the different distributions of subtypes: ST3 was found in all populations, whereas ST1 was only present in the LAG. A previous study suggested that water and soil were the main reservoirs of Blastocystis in a community in Thailand and that Blastocystis might be transmitted by contaminated food and water [Blastocystis infection in humans or livestock was not investigated, considering that ST1–ST3 are common subtypes in human infection [Blastocystis subtypes may cause differences in the prevalence of subtypes. Such interactions can be mediated by factors such as the abundance of host resources, host immune responses and specific parasite strategies for survival, growth and reproduction [Blastocystis subtypes.In the present study, we identified three known subtypes, ST1–ST3, and one novel subtype, ST39, from wild rhesus macaques. Previous studies reported infections with ST1–ST5, ST7–ST11, ST13, ST15 and ST19 in NHPs, with ST2 being the most common subtype in wild NHPs, followed by ST1 [nd water . Since ooduction . HoweverBlastocystis subtypes, indicating the remarkable genetic diversity of Blastocystis in rhesus macaque populations. In most populations, the number of haplotypes was limited. By comparison, NN had more haplotypes of ST1 and ST2, which may indicate multiple introduction events of Blastocystis by infected tourists. However, the sequences published in GenBank are not the same as those of the haplotypes obtained from rhesus macaques, and the existing data could not prove zoonotic transmission in rhesus macaques. Another speculation is that this could be the result of population founder effects. Regarding the finding of the same haplotypes in different populations, we speculate that the same haplotypes may have colonised rhesus macaques before the migration of the rhesus macaque population.In the present study, 33 haplotypes were obtained from the four Blastocystis ST3 has a high genetic diversity, with significant genetic differentiation between the LAG and HAG. This leads us to infer that isolation is caused by a large geographical distance. Results from previous studies suggest that the high molecular diversity of Blastocystis may be correlated with high host multiplicity and a coevolutionary history with the host [ D and Fu’s Fs values suggest a recent demographic expansion of ST3 in Asia [Blastocystis. Furthermore, genetic diversity and population diversification might play a key role in the generalist profiles and cross-species infections of some parasites [Blastocystis. Recently, MinION long-read sequencing was used in the generation of accurate full-length sequences of the SSU rRNA gene [Blastocystis.The results of the genetic analysis showed that the host , 37. The in Asia . Howeverarasites , 39. TheRNA gene . AnalysiRNA gene . The resBlastocystis biology and its relevance to the host.In this study, we identified and validated one novel subtype, which we named ST39. In addition, phylogenetic analysis based on the SSU rRNA gene indicated that ST39 and ST1 have a close genetic relationship. ST1 is a zoonotic subtype that is associated with asymptomatic infection . At presBlastocystis infection and genetic diversity in multiple wild rhesus macaque populations in China. The studied populations were located in high- and low-altitude areas. Infections with the zoonotic subtypes ST1–ST3 are common in rhesus macaques, and we assume that there may be a potential risk of zoonotic transmission of Blastocystis in the study areas. We observed different distributions of Blastocystis subtypes and genetic differentiation of ST3 between LAG and HAG. The novel subtype ST39 was identified and validated, and its host adaptation and pathogenicity should be issues of concern. The present results provide fundamental information on genetic data and the prevalence of Blastocystis in wild rhesus macaque populations.This is the first study on Additional file 1: Table. S1. Chi-square test for the prevalence between HAG and LAG.Additional file 2: Table. S2. Distribution, positive number and accession numbers of subtype haplotypes.Additional file 3: Figure S1. Phylogenetic relationships among Blastocystis SSU rRNA gene sequences (1690 bp) in the present study (represented with a black circle) and representative reference sequences. Proteromonas lacertae was used as outgroup taxon to root the tree. Analysis was conducted by a maximum likelihood method. Genetic distances were calculated using the Kimura two-parameter model.Additional file: 3 Fig. S1. Phylogenetic relationships among Blastocystis SSU rRNA gene sequences (1690 bp) in the present study (represented with a black circle) and representative reference sequences. Proteromonas lacertae was used as outgroup taxon to root the tree. Analysis was conducted by a maximum likelihood method. Genetic distances were calculated using the Kimura two-parameter model.


Background: The major histocompatibility complex (MHC) plays a key role in the adaptive immune response to pathogens due to its extraordinary polymorphism. However, the spatial patterns of MHC variation in the striped hamster remain unclear, particularly regarding the relative contribution of the balancing selection in shaping MHC spatial variation and diversity compared to neutral forces. Methods: In this study, we investigated the immunogenic variation of the striped hamster in four wild populations in Inner Mongolia which experience a heterogeneous parasitic burden. Our goal was to identify local adaptation by comparing the genetic structure at the MHC with that at seven microsatellite loci, taking into account neutral processes. Results: We observed significant variation in parasite pressure among sites, with parasite burden showing a correlation with temperature and precipitation. Molecular analysis revealed a similar co-structure between MHC and microsatellite loci. We observed lower genetic differentiation at MHC loci compared to microsatellite loci, and no correlation was found between the two. Conclusions: Overall, these results suggest a complex interplay between neutral evolutionary forces and balancing selection in shaping the spatial patterns of MHC variation. Local adaptation was not detected on a small scale but may be applicable on a larger scale. The term “local adaptation” refers to the phenomenon when a population evolves to be better adapted to its specific local environment compared to migrants from nearby populations . AdaptatIn host–parasite systems, the genetic variation among hosts and parasites creates dynamic environments that require adaptation. The degree of local adaptation is influenced by gene flow, as well as the variation in genes associated with fitness. Studies on host–parasite adaptation often prioritize examining the ability of pathogens to adapt to their hosts, as the shorter generation times of pathogens make them more likely to exhibit patterns of local adaptation. However, parasites can engender significant ecological and evolutionary interactions with both hosts and host populations . ParasitThe major histocompatibility complex (MHC) plays a crucial role in adaptive immunity, primarily due to its ability to recognize antigens and initiate the immune response . MHC claCricitulus barabensis) frequently causes significant agricultural damage and has the potential to act as a vector for numerous severe zoonotic diseases [The striped hamster  and the north–south direction (temperature gradient) . TemperaWe conducted a total of six sampling events over a two-year period, which took place three times per year in May (spring), July (summer), and September (autumn). We deployed a total of 600 traps, covering an area of approximately 0.05 square kilometers during each sampling event. To ensure sufficient sample sizes for each population, we left the traps in place for one to two days at each location. To minimize potential parasite influence, we placed each captured striped hamster in a clean cloth bag every morning after checking the traps. The bags were securely fastened to prevent parasites from escaping. The impact of dead hamsters on parasite count was relatively consistent among individuals, and therefore did not significantly affect the final results.We meticulously extracted ectoparasites from the hamsters’ fur using a comb. We meticulously and comprehensively enumerated fleas and gamasid mites for each specimen. We microscopically identified parasites to the species level based on morphological characteristics. Our focus was on the average parasite burden per individual at the population level. To account for the low population sizes in certain groups, we chose a subset consisting of N2, N3, W2, and W4 for comparing parasite pressure across populations using the Kruskal–Wallis test in R 4.1.1. We collected multiple tissues, including lung, liver, spleen, and kidney, from each individual. Before DNA extraction, all tissue samples were rapidly frozen in liquid nitrogen and stored at −80 °C. Total genomic DNA was extracted from liver samples using the Qiagen DNeasy Blood and Tissue Kit, following the manufacturer’s instructions. We genotyped the total genomic DNA extracted from each individual using a set of 7 highly polymorphic microsatellite markers . The forNa), number of effective alleles (Ne), Shannon’s information index (I), observed heterozygosity (Ho), expected heterozygosity (He), and Fixation index (F). [We assessed the genetic diversity of each locus and population using GenAlEx . The foldex (F). . We assedex (F). . We conducted a series of analyses to identify the genetic structure among populations of striped hamsters. Initially, we employed a Bayesian-model-based algorithm implemented in STRUCTURE  to deterWe targeted the MHC class II DRB gene exon 2, as it harbors the majority of functionally important antigen binding sites that have been extensively studied . AmplifiSubsequently, raw sequence data was processed into individual genotypes using the Stepwise Threshold Clustering program implemented in the amplisas web software . The sofdN) to synonymous (dS) base pair substitutions (ω) using the Nei and Gojobory method [To assess the historical selection pressure on DRB, we calculated the relative ratio of non-synonymous , the number of segregating sites (S), and nucleotide diversity (π) using DnaSP [An) across hamsters.Vertebrates commonly exhibit variation in the number of MHC loci within individuals, posing challenges in assigning MHC alleles to specific loci. To assess MHC allelic diversity at the intra-population level, we quantified the number of alleles per population . We constructed a neighbor-net network using SplitsTree  to visuaWe conducted Bayesian Clustering analysis in STRUCTURE using correlated allele frequencies, without incorporating prior location information. For each K value, we performed ten independent Markov Chain Monte Carlo (MCMC) runs, each consisting of 1,000,000 generations with a burn-in of 500,000. The input file for structure analysis assigned a value of 1 if the corresponding alleles were present for each individual, 0 if they were not present, and −9 if the data was missing. To further examine the population structure exhibited at the MHC DRB locus, we performed Principal Component Analysis (PCA) to maximize the differences between populations and minimize the variations within populations. The inter-population differentiation at the MHC locus was quantified using pairwise Rho values. To consider nonlinear population distribution and migration patterns, the pairwise Rho values were transformed to Rho/(1-Rho). The analyses were conducted using SPAGeDi . To compare the substructuring levels exhibited by two different marker types, we conducted Mantel tests and partial Mantel tests using the Vegan package in R . To examPopulation genetic differentiation at MHC loci is expected to be more pronounced than that resulting from neutral genetic variation due to selective pressure. We chose to perform a co-inertia analysis (CoA)  to assesThe ectoparasite burden of these four study populations was shown in p < 0.05), gamasid mite richness , and flea load . Precipitation was positively correlated with gamasid mite burden . However, we did not observe any significant effect of precipitation on flea burden , as shown in Temperature increase was positively correlated with gamasid mite load  to 21.429 (W4). The Ne in all populations was lower than the Na, with an average value of 8.781. The lowest Ho was 0.477 at N3, while the highest was 0.600 at W4. Observed heterozygosity was higher than expected heterozygosity at all sites. The HWE test indicated that all populations were in equilibrium, except for W4. No significant departures from LD were observed within global regions after Bonferroni correction. This study utilized 7 pairs of fluorescent primers to amplify 181 samples from 4 populations, resulting in polymorphic loci for all markers. The overall polymorphic characteristics of microsatellite markers were summarized in Although the Bayesian clustering analysis with STRUCTURE identified the most likely number of clusters as 2, there was no significant population genetic structure observed among the populations A. In theR2 = 0.001, p = 0.733), indicating that our sequencing was adequate for reliable genotyping. Our validation process identified a total of 89 unique sequences. The MHC01 allele was the most prevalent among nearly all individuals. The average depth of individual sequencing coverage for MHC DRB exon 2 was 4381 reads (ranging from 3331 to 4826). There was no significant correlation between the number of alleles and sequencing depth (dN/dS) ratio for residues . Positive selection was detected in all four nested models based on likelihood ratio test (LRT) p values . The number of segregating sites (S) varied from 66 to 77 across populations, and the haplotype diversity (Hd) exceeded 0.97 for all populations  or MHC-DRB . Controlling for neutral diversity, the correlation between the MHC-DRB locus and geographic pairwise distances remained non-significant . Additionally, no association was found between MHC-DRB and microsatellite inter-population diversity when controlling for geographic distance. Despite the dissimilarity in cluster results between PCA-based microsatellite and MHC, the CoA plot  genes in relation to neutral microsatellite loci across four populations of striped hamsters in Inner Mongolia. We observed significant variation in parasite pressure among sites, with parasite burden showing correlation with temperature and precipitation. Molecular analysis revealed a similar co-structure between MHC and microsatellite loci. We observed lower genetic differentiation at MHC loci compared to microsatellite loci, and no correlation between the two. Our findings suggest that MHC genetic variation is primarily influenced by neutral processes, with a lesser contribution from balancing selection, and no evidence of local adaptation.Despite extensive research devoted to studying parasite burden, our understanding of parasite distribution remains limited. In this study, we observed significant variation in gamasid mite burden among different sites, indicating that environmental heterogeneity may influence gamasid mite distribution. However, there was no significant difference in flea burden, possibly due to a dilution effect caused by a high number of uninfected host individuals in our study. It should be noted that, since we used clips to catch striped hamsters, some fleas migrated to find new hosts after the hamsters died, despite our efforts to minimize sampling time. Nevertheless, this migration had a consistent impact on all individuals and did not significantly affect our results. We observed a significant correlation between the annual temperature levels at each sampling site and the parasite burdens found in striped hamsters. Specifically, we noted that flea load, gamasid mite load, and gamasid mite richness increased with higher temperatures. Climate factors can influence parasite development and transmission , abundanPrecipitation can have significant effects on parasite pressure. This is because the free-living stages of parasites are directly exposed to the environmental conditions in their respective microhabitats ,65. Our The MHC represents one of the most genetically variable regions in vertebrate genomes. Most species that have been investigated exhibit high levels of MHC diversity ,73, whicThe ability of populations to adapt across spatially heterogeneous and temporally variable landscapes is influenced by the competing forces of natural selection, gene flow, and genetic drift . AlthougIn contrast to the genetic structure observed in microsatellite loci, MHC-DRB exhibited lower genetic differentiation among the four populations, indicating weaker genetic structure. Pairwise Rho/(1-Rho) values for microsatellites displayed greater magnitude than those for MHC, and no correlation was found between them. This disparity may stem from limitations in uncovering patterns of genetic structure when transforming data into binary-encoded format. Nevertheless, this approach does offer a means to compare multi-locus MHC and microsatellite data . Indeed,Rhabdomys pumilio) can switch from group to solitary living with the onset of the breeding season to avoid competition for reproduction within groups [Gene flow among the four populations is expected due to the relatively small geographical scale of this study, in contrast to the extensive range of the striped hamster throughout northern Asia. It is noteworthy that, although we did not observe any local adaptation of MHC in Inner Mongolia, the MHC allele frequencies of striped hamsters in this region differ from those in Shandong Province. This suggests that patterns of local adaptation may vary depending on the spatial scale. While local adaptation has been frequently demonstrated, our understanding of its spatial scale remains limited. Generally, the prevalence of local adaptation tends to increase with geographical distance . As the n groups . AdditioMeriones unguiculatus plague focus in the Inner Mongolian Plateau, the Microtus brandti plague focus in the Xilin Gol Grassland, the Spermophilus dauricus plague focus in the Song-Liao Plain, and the Marmota sibirica plague focus in the Hulun Buir Plateau. Recent years have seen reported cases of plague in this region [Citellophilus tesquorum mongolicus, Amphipsylla primaris mitis, Frontopsylla luculenta, and others [Four plague foci are located in Inner Mongolia: the s region . Previoud others . Based oOur findings indicate that the genetic variation at MHC DRB exon 2 in striped hamsters is influenced by the interplay of neutral processes (gene flow) and, to a lesser extent, balancing selection. Additionally, we emphasize the importance of considering spatial scale in investigations of local adaptation. Collectively, this study contributes to our understanding of MHC gene evolution in wild populations. Furthermore, given the significant gene flow observed, efforts aimed at preventing the spread of plague should prioritize monitoring inter-animal plague transmission and controlling rodent density, particularly in areas affected by plague outbreaks.


Ailanthus altissima to gain new insights into the effects of these pressures on genetic variability in undisturbed and disturbed forests. We analyzed the genetic diversity and population structure of A. altissima using nuclear (EST-SSR) and chloroplast (cpSSR) microsatellite markers. The genetic diversity across the 34 studied populations based on EST-SSRs was found to be moderate to high (nHE = 0.547–0.772) with a mean nHE of 0.680. Bayesian clustering, principal coordinate analysis (PCoA), and discriminant analysis of principal component (DAPC) consistently divided the populations into three distinct groups based on EST-SSRs. Allelic combinations of 92 different chloroplast size variants from 10 cpSSR loci resulted in a total of 292 chloroplast haplotypes. The mean haplotype diversity was relatively high (cpHE = 0.941), and the mean haplotype richness was 2.690, averaged across the 34 populations of A. altissima. Values of FST in A. altissima from chloroplast and nuclear markers were 0.509 and 0.126, respectively. Modeling results showed evidence for population range contraction during the Last Glacial Maximum with subsequent population expansion in the Holocene and the future. Although genetic variation did not differ substantially across disturbed and undisturbed sites, there were small trends indicating higher genetic diversity and population bottlenecks in disturbed forests. As a result, disrupted ecosystems might display surprising genetic patterns that are difficult to predict and should not be overlooked.Understanding how anthropogenic disturbances affect the genetics of tree species is crucial; however, how tree populations in the wild can tolerate these activities remains unexplored. Given the ongoing and intensifying anthropogenic disturbances, we conducted a study using  Swingle, commonly known as the tree of heaven, belongs to the family Simaroubaceae, and is a deciduous tree species native to Eastern Asia -derived microsatellite markers (EST-SSR) and 10 cpSSR markers. The study aimed to determine if disturbance would reduce, maintain, or increase the genetic diversity of A. altissima across undisturbed and disturbed populations in China. Additionally, niche modeling was used to explore probable shifts in habitat suitability of A. altissima within and beyond the present distribution to provide a framework for establishing new populations. This study helps to unravel the population genetic diversity and structure of A. altissima in a human-impacted forest system and provides necessary information for its management and use.Simple sequence repeat (SSR) markers are highly polymorphic and widely distributed in eukaryotic genomes. Chloroplast microsatellites (cpSSRs) provide useful information about ancient historical relationships among maternal lines, while nuclear microsatellites are preferred in the evaluation of patterns of genetic structure due to their codominant and hypervariable nature. The present investigation aimed to estimate genetic variation and test for evidence of past bottlenecks on disturbed and undisturbed populations of 22.1A. altissima, mostly growing in or near natural reserves of China, and 302 wild trees from disturbed habitats, such as semi-natural forests close to roads, villages, or towns, that have experienced varying levels of disturbance over the past 60 years  of template DNA, 0.5 µL of each primer, 0.2 µL of Taq polymerase enzyme, 0.5 µL of dNTPs, 2.5 µL of Taq buffer, and 11.3 µL of ddH2O. PCR amplification was performed as follows: an initial denaturation phase at 95°C for 5 min, followed by 35 cycles of denaturation for 30 s at 95°C, annealing for 40 s at 52–54°C, and extension at 72°C for 40 s, with a final extension step at 72°C for 10 min. We determined the primers that produced a single brilliant band using 2% agarose gel electrophoresis. The forward sequence of the primers that produced clear bands were chosen, their 5’ ends were tagged with 6-FAM fluorescent dye, and they were then employed for genotyping. We examined the microsatellite marker profiles of all A. altissima individuals using GeneMapper 4.0 (Applied Biosystems). We sized PCR amplicons using GeneScan 500 ROX (Applied Biosystems) on an ABI 3730 XL capillary electrophoresis sequencer, and calculated allele lengths using GeneMapper v.4.0 .Our study sampled 432 trees from 34 populations (defined by the geographic distribution) across most of the native range in China, with each population consisting of 4–20 individuals spaced at least 50 m apart Table 1.2.2FST by region and the inbreeding coefficient (FIS). It is important to note that the estimate of genetic differentiation can be affected by null alleles (FST (FREENA) values] using the FREENA software (nHe/nHE and nHo/nHO), total number of alleles (Nt), allelic richness (Ne/NE), mean allelic number (Na/NA), Shannon’s Information Index (I), and number of private alleles (AP), across all loci and populations using GenAlEx v.6.5 , effective number of haplotypes (cpNE), haplotype diversity (cpHE), number of private haplotypes (P), haplotypic richness (Rh), and mean within-population genetic distance between haplotypes (D2sh). We calculated the number of alleles using FSTAT software , we used genetic and geographic distances to perform the Mantel test, executed in GeneAlEx 6.5 (K). The first method was performed in the STRUCTURE software v 2.3.4  was evaluated using 10 independent runs per K, by running K values from 1 to 34. Burn-in period was set to 50,000 and Markov Chain Monte Carlo (MCMC) length to 500,000 for each run. We utilized a web-based STRUCTURE HARVESTER 0.6.94  as well as the maximum delta K value  . To deteAlEx 6.5 , with 10AlEx 6.5 , and to AlEx 6.5 , which wAlEx 6.5 . Two app v 2.3.4  among thilibrium , the nonR 0.6.94  to find  K value . CLUMPP  K value  and DIST K value  were use K value . We applgram 1.3  with 10,2.4A. altissima using the BOTTLENECK v.1.2.02 program . The study area was defined by geographical coordinates of 20.9°–43.6°N and 91.5°–126°E. We obtained A. altissima locality data from the Chinese Virtual Herbarium  and field data collected by We used MaxEnt 3.4.0  to generhttp://www.worldclim.org/) (r < 0.85) to remove correlated variables. For the LGM, Mid-Hol, and Future periods, we used calibrated global climate model (GCM) data from the Community Climate System Model “CCSM4”. The future period was represented by the representative concentration pathway 8.5 (RCP8.5) to represent pessimistic scenarios  data with a resolution of 2.5 arc-min from WorldClim (im.org/) . We perfcenarios . All ENM33.1p < 0.001) except loci Ail 29  observed in all populations. A total of 255 alleles were yielded using the 10 EST-SSRs, varying from 7 to 36 in locus Ail 22 to locus Ail 06, respectively (average = 19.615). The mean expected heterozygosity (nHe) 0.807 and mean observed heterozygosity (nHo) 1.000 were highest at locus Ail 09 and Ail 26, respectively. The PIC values varied from 0.632 (locus Ail 11) to 0.908 (locus Ail 09) with a mean of 0.771  and observed heterozygosity (nHO) values were moderate to high ranging from 0.547 to 0.772, and from 0.725 to 0.930, with an average value of 0.680 and 0.848, respectively. A total of 62 private alleles were realized in 34 populations, all with varying frequencies. Population XXS recorded the highest number of private alleles. For each population, the values of NA, NE, and inbreeding coefficient (FIS) ranged from 2.700 to 9.100, 2.344 to 5.330, and −0.459 to −0.097, respectively. Moreover, we found no significant difference in FST values  of the recorded haplotypes were private haplotypes. The most abundant haplotype was H1, which was found in 16 individuals, followed by H2 with 9 individuals; H3 to H64 were identified in 2 to 8 individuals, while the remaining haplotypes (H65 to H292) had single individuals each with a complex network , followed by LA and CZ populations with 18 haplotypes each, while three populations  had the lowest number of detected haplotypes (A = 4). We detected a number of private haplotypes in 34 populations with a maximum of 20 in PX, followed by LA and CZ populations with 18 haplotypes each, and NJ and XXS with 15 and 14 haplotypes, respectively. Population MEK lacked the private haplotypes, while a minimum number of one haplotype was recorded in the XC population, and two for HD. The effective number of haplotypes (cpNE) was lowest in population SFS (3.273) and DLL (3.571), and highest in PX (cpNE = 19.174), followed by populations LA and CZ (cpNE = 16.667). Populations  had the highest haplotype richness (Rh = 3.000), whereas MEK, and SFS populations had the lowest (Rh = 1.859 and Rh = 1.879), respectively. The highest genetic diversity values were observed in populations ZJ, CY, DY, HP, LX, XX, and LH (cpHE = 1.000), and the lowest in population SFS (cpHE = 0.758). The average genetic distance between individuals ranged greatly from D2sh = 343.435 (population WYS) to D2sh = 1.800 (LX)   significantly deviated from the three-phase model (TPM) equilibrium Table 2.3.4A. altissima after conducting a Pearson correlation analysis  period  compared to the LGM, indicating the presence of fragmented refugia during this period as well. Future projections for the year 2070 suggested a reduction in habitat suitability for A. altissima in the northern and southern regions, with a slight northeastern shift and a decrease in the number of suitable pixels.Eight of the 19 variables were selected for constructing the ecological niche models (ENMs) for analysis Table S34A. altissima within its native range. Compared to neutral nuclear SSRs, EST-SSRs were designed to amplify the microsatellite loci that are tightly associated with the transcribed regions of gene(s) (p < 0.001) except only one loci (Ail 29), suggesting that these EST-SSRs are mostly not neutral markers. Thus, these markers represent suitable molecular tools for assessing the impact of human disturbance (selection) on the genetic diversity of A. altissima across undisturbed and disturbed forests in China. Our study, utilizing nuclear EST-SSR markers, revealed moderate to high levels of genetic variation in both disturbed and undisturbed populations. The overall level of genetic diversity (nHE = 0.680) found in this study is comparable to the A. altissima populations in Eastern United States . The overall genetic diversity of A. altissima is also similar to that of several tree species found in subtropical China, with their distribution ranges partly overlapping with that of A. altissima, such as Castanopsis fargesii   and very gene(s) . In our cpHE) of the 10 cpSSR loci was found to be 0.921. Our findings provide support for the idea that habitat disturbance in wind-pollinated trees promotes pollen movement, leading to increased genetic variation in the remaining populations  than overall haplotypic variation (HT = 0.993); this was supported by slightly higher differentiation values of GST = 0.493 compared to RST = 0.434, suggesting a weak phylogeographic structure. On the other hand, an increased number of microsatellites led to a higher haplotypic diversity than a previous study by A. altissima is comparable with other plant species studied with cpSSR markers, such as Quercus semiserrata    , three E64–0.92) , and HSndifolia . The lifnditions . It is ner 200 m . Interesividuals .nFST values of 0.2 or less, with high genetic diversity within the populations, especially dioecious species . As a dioecious species, A. altissima possess an outcrossing mating system and a long life cycle, which may have resulted in the low among-population and high within-population genetic variation. The current genetic differentiation results are similar to those reported by other studies that have used nuclear SSR markers, for instance, Q. semiserrata (nFST = 0.12) (Q. castanea (nFST = 0.13) (Prunus africana (nFST = 0.12)  (Quercus section Lobatae subsection Racemiflorae (nFST = 0.12)  , Q. cast = 0.13) , Prunus  = 0.12) , Q. infe = 0.12) , and Que = 0.12) .FST) at a range-wide scale . Therefore, the differences in genetic variation observed in this study may be shaped by various factors, such as human disturbances and climatic conditions.Bottleneck analyses based on EST-SSRs indicate that the genetic diversity of some ltissima Table 3.ng error . PreviouA. altissima has expanded, while it contracted during the Mid-Holocene  and the Last Glacial Maximum . Therefore, this study supports the hypothesis that plants were influenced to some extent by the glaciation period and were able to persist during inter-glacial cycles. Furthermore, the findings suggest the presence of fragmented refugia during the Last Glacial Maximum and Mid-Holocene. This finding aligns with previous research indicating that the Qinghai–Tibet plateau acted as a protective barrier, allowing plants to survive the ice age by seeking refuge in central China , followed by population expansion in the Holocene  and projected future . In conclusion, the information regarding the extent of differentiation and genetic variation between and within A. altissima populations can aid in the future monitoring of this species in the face of ongoing environmental changes and human impacts.To the best of our knowledge, no study has yet analyzed the population structure and genetic diversity of ed areas . Furtherhttps://www.ncbi.nlm.nih.gov/genbank/, MN531147 - MN531169.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: Y-YL and J-MC conceived and designed the experiment; JS performed the experiments, analyzed data, and visualized and wrote the first draft of the manuscript. Z-ZL and BN assisted in data analysis. RG, Y-YL, J-MC, and Z-ZL reviewed, edited, and commented on previous versions of the manuscript. All authors contributed to the article and approved the submitted version.