Through population genetic analysis, we found that Ningxia Hui received much more genetic contributions from East Asian populations than those from western Eurasian populations. Finally, we statistically identified the admixture signal of eastern and western Eurasians, although the latter is weak, in Ningxia Hui via the three-population test. All this evidence suggested that the formation of Ningxia Hui was mainly attributed to the cultural transformation of local Chinese residents with minor gene flow from western Eurasian populations.According to historical records, ethnic Hui in China obtained substantial genetic components from western Eurasian populations during their Islamization. However, some scholars believed that the ancestry of Hui people were native Chinese populations. In this context, the formation of Hui is due to simple cultural diffusion rather than demic diffusion. In this study, we examined the forensic and population genetic application of the 30 InDel loci in Hui population from Ningxia Hui Autonomous Region, Northwest China. Genotype analysis of 129 unrelated individuals revealed that all loci were in the Hardy–Weinberg equilibrium in Ningxia Hui. Forensic indices calculated from genotypes demonstrated that this panel, Qiagen DIPplex The term ‘Hui’ was derived from Mandarin word ‘Huihui’ which referred to Central Asians, Persians and Arabs residing in China in the age of the Yuan Dynasty (1271–1368). On the basis of Chinese historical records, Hui community originated from intermarriage between Han females and non-Chinese males [China is a unified multi-ethnic country with 56 officially recognized ethnic groups. Having a sizeable population of approximately 10.6 million, Hui is the largest one among 10 Muslim ethnic groups in China according to the 2010 national census , ranging from 1 to 10 000 bp in human genomes , is a tyanalyses .In this study, we assessed the forensic efficiency of this kit in a Hui population from the Ningxia Hui Autonomous Region. Combining the InDel data with that from other studies, we revealed the autosomal genetic relationships between Ningxia Hui and other populations. Although contributions have been made to the identification of admixture evidence in Hui, to the best of our knowledge, no unambiguous conclusions have been made in the literature. Using some population genetics analysis statistics, we identified an evident admixture signal of eastern and western Eurasians in this Hui population.2.2.1.® method  and the genotyping of 30 InDels was carried out using the DIPplex Investigator reagent , which followed established methods [We collected bloodstain samples of 129 unrelated healthy Hui individuals in the Ningxia Hui Autonomous Region, China according to their identity cards. No kinship existed among the samples within at least three generations, and no migration events happened in their family history as declared. All of the donors had been adequately informed and signed the informed consent before sample collection. This study was approved by the ethics committee of Xi'an Jiaotong University Health Science Center and conducted in accordance with the human and ethical research principles of Xi'an Jiaotong University Health Science Center, China. The DNA was extracted using the Chelex methods ,21. To p methods –24, Yi [ methods , Xibe [2 methods , South K methods , She [23 methods ,27–29 an2.2.FSTs that measure genetic distances between populations were computed using Arlequin v. 3.5.2 [K), the admixture analysis was performed using ADMIXTURE 1.23 [To assess the forensic efficiency of this panel, forensic parameters and allele frequencies were calculated using the modified powerstat (v. 1.2) spreadsheet . Pairwise v. 3.5.2  and visuv. 3.5.2 . A projev. 3.5.2 . By settURE 1.23  with defURE 1.23 . All figURE 1.23 .3.3.1.p-value being 0.102 (HLD77). The polymorphism information content (PIC) values ranged from 0.21 to 0.37. The power of exclusion (PE) ranged from 0.042 (HLD39) to 0.260 (HLD77) with a combined PE of 0.9940. The combined PE value was relatively low, suggesting that this panel can be used in combination with other markers, such as autosomal STRs, in paternity cases. The highest and lowest discrimination power (DP) was found at HLD6 (0.627) and HLD118 (0.396) respectively. Thus, the combined DP had a high enough value of 0.9999999999871, which provides a satisfactory level of discrimination for two randomly selected individuals in this group.Basic forensic indices and allele frequencies of the 30 InDel loci are summarized in 3.2.FSTs between these populations were calculated to show their genetic distances . For this Hui population, the minimal FST (0.00078) was observed for Han from Shanghai and the maximal value (0.21394) was observed for a group of Amerindian Mexicans. In general, the populations from East Asia, especially Han populations, had much closer relationships (FST < 0.023) with Hui than European populations (0.055 < FST < 0.075). As expected, Mexican populations had the most distant relationships (FST > 0.13) with Hui.To understand the genetic background of the Ningxia Hui population, we compiled obtained data with those from other 21 populations , Europeans  and Mexicans, the PCA plots roughly fell into three parts . The CenK  was, the pattern of Hui people resembled that of eastern Asians and was distinct from those of Central Asians and western Eurasians.We further conducted an admixture analysis on all individuals using ADMIXTURE  figure . Because3.4.Z-scores < −2, which resembles f3 and f3 . This result remained valid when Central Spanish was replaced with Dane (figure 4b). Therefore, we could reach a conclusion that western Eurasians had limited genetic contribution to Ningxia Hui, compared with their contributions to Uigur and Kazak.In the three-population, the so-called f3 statistic based on allele frequencies across populations can provide a clear evidence of admixture, and a negative value indicates a history of admixture . SpecifiIn the outgroup case, the three-population test can also be employed to measure genetic affinities between populations . For exa4.In this study, we tested the 30 InDels panel in the Hui population from Ningxia Hui Autonomous Region and found it highly efficient in the identification of unrelated individuals. However, this panel should be used in combination with other markers to achieve enough power in paternity cases. Since this panel was originally designed for individual identification and all loci were highly polymorphic in the majority of populations worldwide ,26,30,41FSTs, we found that Ningxia Hui people were genetically closest to East Asian, especially Han groups. Despite limited samples and loci, our results were consistent with that calculated from the 1000 Genome Data [FSTs between East Asian populations and European ranged from 0.08 to 0.11 in this study, which was in agreement with FST between Beijing Han and people of European ancestry (around 0.10) using genome-wide data. Besides, FSTs between populations in different continents are around 0.15 in this study and the genome-wide study [FSTs, results of the PCA and admixture analysis showed significant genetic homogeneity between Hui and East Asian populations. Although the genetic imprint of East Asian ancestry in Hui was not in question, these two non-quantitative methods could not prove whether or not Hui received any genetic contribution from populations outside East Asia with limited InDel loci.Nevertheless, this panel still could shed some light on the genetic relationships between populations . Based oome Data . For insde study . The PCAde study ,42,44. Set al. [Therefore, we attempted to apply the three-population test that was systematically introduced by Patterson et al.  to this et al. . AdditioEvidence from autosomal STR loci indicated that the spread of Islamic faith in India was predominantly cultural transformation associated with minor gene flow from West Asia . Like wh


Cinclosoma clarum and Chestnut Quail‐thrush C. castanotum, which together span the arid and semi‐arid zones of southern Australia, and primarily from specimens held in museum collections. We affirm the recent taxonomic separation of the two species and then focus on diversity within the more widespread of the two species, C. clarum. To guide further study of the system and what it offers to understanding the genomics of the differentiation and speciation processes, we develop and present a hypothesis to explain mitonuclear discordance that emerged in ourdata. Following a period of historical allopatry, secondary contact has resulted in an eastern mitochondrial genome replacing the western mitochondrial genome in western populations. This is predicted under a population‐level invasion in the opposite direction, that of the western population invading the range of the eastern one. Mitochondrial captures can be driven by neutral, demographic processes, or adaptive mechanisms, and we favor the hypothesized capture being driven by neutral means. We cannot fully reject the adaptive process but suggest how these alternatives may be further tested. We acknowledge an alternative hypothesis, which finds some support in phenotypic data published elsewhere, namely that outcomes of secondary contact have been more complex than our current genomic data suggest. Discriminating and reconciling these two alternative hypotheses, which may not be mutually exclusive, could be tested with closer sampling at levels of population, individual, and nucleotide than has so far been possible. This would be further aided by knowledge of the genetic basis to phenotypic variation described elsewhere.We surveyed mitochondrial, autosomal, and Z chromosome diversity within and between the Copperback Quail‐thrush  Cinclosoma clarum, a widespread bird of southern Australia's arid and semi‐arid zones, a mitochondrial capture is taking place driven by neutral demographic processes. This in turn reflects a likely range expansion of the species' western populations into the range of its eastern populations. We can see the need for further analyses to test this hypothesis as well as an alternative and so guide the system's contribution to the genomic study of differentiation and speciation.We present a case that within the Copperback Quail‐thrush  Our aim is to propose a hypothesis that this is an active, ongoing mitochondrial capture driven by neutral demographic processes. We acknowledge some issues hindering unambiguous interpretation of our data, but we hope that presenting the hypothesis here will guide further study of this system.Literature is growing rapidly on the genomics of speciation and introgression. A facet of this that we focus on here concerns mitochondrial DNA introgression and the mitonuclear discordance it can generate  and several zones of intergradation among them are currently recognized , the South Australian Museum (SAM), and the Australian National Wildlife Collection (ANWC) for mitogenomic study and single nucleotide polymorphism (SNP) analysis from toe pads; single specimens of another species of Cinclosoma, the Spotted Quail‐thrush C. punctatum and from a species in the sister genus, the Blue‐backed jewel Babbler Ptilorrhoa caerulescens, were included as outgroups  historical museum specimens. Reconstructions covered an estimated 98% of the complete mitochondrial genome, based on comparison with the reference T. guttata mitochondrial genome. Within this reconstructed region, completion varied between 82% and 100%. Appendix Genomic data have been lodged at 3.2MT and WestMT. Figure C. castanotum and C. clarum  and 1.40% (whole mitogenomes) and FST between them is high at 0.693 and 0.102, respectively.ND2 sequences were either extracted from the full mitogenome data or sequenced afresh from the 10 specimens collected in 2017 (GenBank accession numbers MT296788–MT296797). The 72 in‐group specimens (62 museum specimens + 10 from 2017) from which data were recovered fell into two clades Appendix , which w3.3T. guttata genome was successful, yielding a mean read depth of 17,323,937 to 109,118,947 bp, and low read duplication levels of between 0.28 and 4.10  and so largely typical of western populations . All are therefore assigned as expected to their respective species.The DArTseq approach on cryofrozen material generated 70,627 SNPs. After filtering to remove loci with missing data, 16,602 SNPs were retained for further analyses. PCoA1 Figure  clearly C. clarum cluster tightly in Figure C. clarum samples in the lower right quadrant. These 10 specimens from 2017 are from the same region as the STRUCTURE analysis's Westnuc. They are from zones currently considered to be the range of either subspecies C. cl. fordianum or phenotypic intergrades between C. cl. clarum and C. cl. fordianum , given our sampling, has not been observed. One individual having the western mitochondrial genome (ANWC B51857) was inferred to be in the admixed nuclear genomic background. It is from a zone which all other phenotypic  has invaded the range of what is essentially the eastern population generated by that allopatry. These are represented taxonomically now most simply by what Black et al.  into the western . Conversely, its relaxation may lead to high variability in other habitats . Supporting this speculation, we note that quail‐thrush are primarily terrestrial birds. Different dorsal plumages of the various species in the genus, not just in populations of C. clarum, are vital for their crypsis against different substrates from rainforests to deserts. Further work might test whether relative consistency of dorsal plumage patterning in C. clarum reflects differing strengths of selection against different substrates. Similar alternatives may be considered in explaining other discordances between phenotypic and genomic patterns.An alternative hypothesis, though not necessarily mutually exclusive, is that et al.'s  hybrid set al.'s , which tet al.'s . If so, et al.'s  is unsurconcordance involve specimens spanning a century or more, for example, the western populations and the range of C. clarum morgani. And these specimens represent a very short temporal window in the species’ evolutionary history, notwithstanding their critical role in completing spatial sampling.Three possible limitations warrant discussion. First, it may be that the temporal spread of our samples renders it artifactual to analyze our data together as spatio‐temporally homogeneous. We counter with two points. Substantial zones of A related second concern might be that of error rates in data from older historical specimens. Most of the historical specimens we used came from the 1960s or later, only 11 coming from the 1920s or earlier, and none from the 1930s to 1950s Appendix . The parC. cl. clarum sensu Black et al. ; Formal analysis (lead); Methodology (lead); Visualization (lead); Writing‐original draft (supporting). Andrew Black: Conceptualization (supporting); Visualization (supporting); Writing‐review & editing (supporting). Gaynor Dolman: Formal analysis (supporting). Philippa Horton: Conceptualization (supporting); Writing‐review & editing (supporting). Lynn Pedler: Investigation (supporting). Catriona D. Campbell: Data curation (supporting); Formal analysis (supporting); Software (supporting). Alex Drew: Investigation (supporting). Leo Joseph: Conceptualization (lead); Visualization (supporting); Writing‐original draft (lead); Writing‐review & editing (lead).Appendix S1Click here for additional data file.


Banksia marginata, Proteaceae). We demonstrate significant genetic structuring among, and high level of relatedness within, fragmented remnant populations, highlighting imminent risks of inbreeding. Population simulations, controlling for effective population size (Ne), suggest that many remnant populations will suffer rapid declines in genetic diversity due to drift in the absence of intervention. Simulations were used to demonstrate how inbreeding and drift processes might be suppressed by assisted migration and population mixing approaches that enhance the size and connectivity of remnant populations. These analyses were complemented by niche suitability models that predicted substantial reductions of suitable habitat by 2080; ~30% of the current distribution of the species climate niche overlaps with the projected distribution of the species climate niche in the geographic region by the 2080s. Our study highlights the importance of conserving remnant populations and establishing new populations in areas likely to support B. marginata in the future, and adopting seed sourcing strategies that can help populations overcome the risks of inbreeding and maladaptation. We also argue that ecological replacement of B. marginata using climatically suited plant species might be needed in the future to maintain ecosystem processes where B. marginata cannot persist. We recommend the need for progressive revegetation policies and practices to prevent further deterioration of species such as B. marginata and the ecosystems they support.Habitat fragmentation imperils the persistence of many functionally important species, with climate change a new threat to local persistence due to climate niche mismatching. Predicting the evolutionary trajectory of species essential to ecosystem function under future climates is challenging but necessary for prioritizing conservation investments. We use a combination of population genetics and niche suitability models to assess the trajectory of a functionally important, but highly fragmented, plant species from south‐eastern Australia ( The species is now largely limited to small remnants in highly modified landscapes and is showing signs of climate stress. Population genetic analyses on remnant tree‐form populations spanning the Victorian Volcanic Plains region of western Victoria provide insights into patterns of contemporary gene flow and genetic structure. We use population simulations to assess risks of declining genetic diversity in small remnants, and discuss management interventions orientated towards enhancing connectivity and genetic diversity to reduce risks of inbreeding and maladaptation under climate change. We complement these analyses with climate niche modelling to investigate likely shifts in suitable habitat within and beyond the current distribution of Banksia marginata, providing a framework for conserving existing populations, establishing new populations, considering ecological replacement and identifying climate‐matched seed sources for restoration. We argue that studies of this nature are key to identifying the risk posed by habitat fragmentation and climate change to plant species, and management actions that foster resilience and improved biodiversity outcomes under climate change.Predicting the evolutionary trajectory of species essential to ecosystem function under future climates is challenging but necessary for prioritizing conservation investments. In this context, we use a combination of population genetic and climate modelling tools to assess the likely evolutionary trajectory of a functionally important plant species from south‐eastern Australia. The Silver Banksia (22.1Banksia marginata (Proteaceae) is endemic to forest, savanna and heathlands of south‐eastern Australia. The species varies widely in habit, ranging from a small shrub (<1 m) to a large tree (~12 m), although there is some uncertainty surrounding the taxonomic status of the different growth forms , with seeds being retained within the follicles for ~12 months prior to dispersal. Seed dispersal occurs predominantly by gravity, with seeds falling near the maternal plant; however, some occasional long‐distance dispersal is thought possible by cockatoos, which have been observed to carry cones . In contrast, inflorescences appear adapted to pollination by mobile vertebrates and invertebrates; this is thought to promote gene flow in other Banksia species .Hateley, . These sHateley, . Most re2.2B. marginata tree‐form stands from the Victorian Volcanic Plains were selected for genetic analysis  occurring in extensive and largely intact native habitats. To test for spatial autocorrelation, we genotyped between 80 and 100 individual plants, sampled from 8 to 10 sites (10 trees per site) separated by up to 2.2 km, at three locations from western Victoria: Claude Austin Reserve, Victoria Valley  and Durdidwarrah (Barwon Catchment) (Table We tested for inherent limitations to gene flow and propagule movement in t) Table . Field c2.4® 96 Plant II protocol (Macherey‐Nagel Inc.), and DNA quantitation was performed as per the QuantiFluor® dsDNA System (Promega Inc). Banksia marginata DNA samples were genotyped at 10 microsatellite loci using a composite of genetic markers developed by He, Krauss, & Lamont, Genomic DNA was extracted from 30‐mg tissue samples for individual specimens using the NucleoSpin2.5FIS) and global population differentiation (FST) with 95% confidence limits ; and (d) tests for linkage disequilibrium between loci using a log‐likelihood ratio test. The software Micro‐Checker ver. 2.2  including (a) allelic richness per population averaged over loci; (b) Weir and Cockerham's inbreeding coefficient ( includinHO) and expected (HE) heterozygosity were determined using the Excel Microsatellite Toolkit ) and regressed to the natural log of geographic distance between sites  analyses were performed to explore relationships between genetic differentiation and geographic distance between sites. Pairwise Rousset, , with stRousset, . Signifiadegenet package in R  was implemented in the Jombart,  to obtaiThe software package BOTTLENECK was used to test for evidence of recent reductions in the effective population size based on a comparison of allele numbers and observed heterozygosity at polymorphic loci  and putative relationships among individuals  using a maximum‐likelihood approach.Finally, relationships between individuals at each site were estimated with the program ML‐Relate  and the kinship coefficient of Loiselle, Sork, Nason, and Graham  of 20, 50, 100 and 500 (which are reflective of the distribution of current population size estimates for B. marginata). Simulations were undertaken in R Studio (version 1.2.1335) and loading libraries psych and Rlab (from www.r‐project.org). An R script was written to randomly sample two alleles with replacement at x loci for N number of individuals from a microsatellite data set (stored as a.csv file). The first step of the simulation created a panmictic population  from a data set of observed genotypes from a population of B. marginata. We chose two populations to simulate as examples of a current population with high diversity  and a population with low diversity . A constant population size  replicated 100 times was then created randomly (sampling with replacement) from the panmictic population of genotypes. These were then resampled independently for 200 nonoverlapping generations (t). At each generation, mean expected heterozygosity (He) and allelic richness over loci (Ar) were calculated as measures of genetic diversity.Using microsatellite genotypic data  generated for remnant N = 250 and 400, reflective of a medium and relatively large population that would be the approximate target size for creating a new “mixed” population or, alternatively, connecting several remnant populations through ongoing assisted migration events). Genotypes from 20 individuals were randomly sampled from each of four populations , all from a similar geographical area, creating a “mixed” population. Simulations were then run as above for N = 250 and 400, with Ar estimated at each generation. Confidence intervals (95%) for all simulations were calculated using the standard deviation from the 100 replicate populations at each N for each t.We also undertook a simulation where we combined individuals from several populations to see the effect this had on genetic diversity through time for two constant population sizes  was developed using presence data from the Atlas of Living Australia  and randomly generated pseudo‐absence point data. Presence data were filtered to choose only verified observations and datum with a spatial mapping accuracy of <500 m. This accuracy was chosen because we used climate data at 1‐km resolution. Presence and absence data points were only selected if they were >5 km apart to reduce spatial autocorrelation. The number of pseudo‐absence sites chosen was selected to ensure a balanced design of presence and pseudo‐absence points  bioclim variables were selected .A total of 497 individual i Tables . Marker p < .01; significant after corrections for multiple comparisons). For HDB, this estimate was influenced by a single locus only, while significant deviations were recorded at multiple loci for PSE. The estimate for PSE was supported by a significant inbreeding coefficient , and a high estimate of relatedness among individuals. This site was unique in being dominated by many relatively small individuals among very few larger plants, and it is likely that many of the plants sampled are siblings from a small number of recruitment events.All sites were found to conform to HWE suggesting random mating Table , except Relatedness analyses revealed that a significant number of individuals at each collection site are direct relatives, with an average of 21% of samples being half‐ or full siblings. The lowest number of related individuals was recorded at SHF (9%) and the highest at BRS and PSE (33%)  = 0.128–0.152) indicating genetic structure due to limited gene flow between sampling sites. Pairwise population comparisons of FST indicated significant genetic differentiation between all site pairs (231 pairwise comparisons), except for four comparisons which were not significant and were associated with sites in close proximity in the south‐west of the study area (Table p < .01).A global estimate of ea Table . AMOVA aB. marginata populations . Wilcoxon's signed‐rank test for heterozygote excess was significant after correction for multiple comparisons under the IAM and TPM models for most remnants, while significant excess was observed under all three models for remnants KAL and TRW only. In most cases, these results were further supported by evidence of mode shifts, suggesting that many B. marginata remnants are not in mutation‐drift equilibrium and have undergone significant reductions in effective population size in the recent past.BOTTLENECK analyses found evidence of recent reductions in effective population size (bottleneck events) in 12 of 22 FST and the natural log of geographic distance . Regression also showed no significant relationship .The Mantel test indicates no relationship between genetic distance and geographic distance among sites across the VVP sample distribution. Similarly, there was no relationship detected with regression analyses Figure . Based ox‐ and y‐axes, individuals from the BRH and FLB sites cluster separately from the main cluster, including all remaining remnant populations  was calculated for all pairs of individuals, involving 6,260 pairwise comparisons across eight distance classes, ranging from 0 to 2,100 m. Significant and positive spatial autocorrelation was at 0 m  from 3.3B. marginata highlight how quickly genetic diversity is likely to be lost from populations if they are small (Ne ≤ 100)  loses half of its allelic diversity within 65 generations  and half of its heterozygosity between 29 (Ne = 20) and 142 (Ne = 100) generations. Simulations with an Ne of ≥500 suggest the loss of genetic diversity over 200 generations is slowed. For the low diversity population BRS, simulations show that genetic diversity approaches fixation across loci within 200 generations when Ne = 20 or 50, and at Ne = 100, He = 0.14 and Ar = 1.41.The genetic simulations for the high and low diversity populations of ) Figure . At Ne Ne sizes (e.g. 240 or 400). We simulated combinations of individuals from four populations located in a similar region, and this resulted in a population with the highest allelic diversity (Ar = 7.9). However, over 200 generations, approximately half of the allelic diversity will still be lost at these effective population sizes . In many cases, Ne is likely to be significantly lower than the census population size meaning that declines in genetic diversity will occur more rapidly  can provide adequate gene flow to connect populations, slow the loss of genetic diversity, decrease genetic load and allow differences in allele frequencies between populations to persist  . The climate envelope of some Banksia species from south‐western Australia could therefore provide functional equivalence in areas of B. marginata's current range that become unsuitable in the future.Areas of predicted future climate suitability from the SDM could be used to select sites for assisted range expansion. This might be considered if in situ conservation efforts to maintain  Connell  quantifi5B. marginata will require significant interventions. This paper provides the data required to guide these interventions, highlighting the importance of enhancing the size, connectivity and genetic basis of remnant populations to help overcome risks of inbreeding and maladaptation. Populations must be maintained or created in areas with climates that remain suited to the species, and assisted migration to climatically suitable areas outside its current range may be needed in the future to help safeguard the species. As B. marginata is already showing signs of climate stress, seed mixing approaches that broaden the genetic basis of restoration plantings and include genotypes that will allow for adaptation to future climatic conditions will become increasingly important. Without such interventions, B. marginata is unlikely to persist in VVP region, and potentially other regions spanning its contemporary distribution, given the legacy of past demographic declines due to habitat fragmentation.Taken together, our results suggest that securing the future of None declared.This project was conceived by A.D.M, W.W, A.S and J.W.M, with all authors contributing to the study design. Field populations were identified and sampled by S.H, S.S. and W.W. A.D.M and O.H were responsible for genotyping, with genetic analyses led by A.D.M and A.R.W with contributions from L.B, S.E.H and C.D.H.S. Geospatial analyses were led by C.N with assistance from A.D.M, J.W.M and S.S. Writing of the manuscript was led by A.D.M with assistance from all authors.Supplementary MaterialClick here for additional data file.


Cimex hemipterus (F.), has now emerged as an important public health pest in the tropics. Despite its alarming infestation rate, the information on its population genetics remains scarce. Here, we described the infestation structure and population dynamics of C. hemipterus in the tropics, especially Malaysia and Singapore, based on eight novel microsatellites and two mtDNA markers, including cytochrome c oxidase I (COI) and 16S rRNA genes. Across populations, microsatellite data revealed high genetic diversity with significant genetic differentiation and restricted gene flow. Analysis within populations revealed evidence of a recent bottleneck. Nonetheless, elevated genetic diversity in nearly all populations suggests that the propagule in C. hemipterus populations were much diverse, distantly related (mean r = 0.373), and not significantly inbred (mean FIS = 0.24) than that observed in Cimex lectularius from previous studies. We observed seven mtDNA haplotypes across the 18 populations studied (Hd = 0.593) and several populations displayed more than one matrilineal descent. The two markers were generally congruent in suggesting a common, genetically diverse  source population with possibilities of multiple introductions for the bed bug populations in the present study.The tropical bed bug,  Bartonella quintana and Trypanosoma cruzi, that cause trench fever and Chagas disease, respectively [The bed bug is a nocturnal blood-sucking ectoparasite that has now emerged as an important public health pest globally . All mobectively ,6,7. HowBed bugs were largely eradicated in most parts of the world during the post-World War II era due to wide scale use of pesticides, especially DDT and malathion . NeverthCimex lectularius (L.) and Cimex hemipterus (F.). The common bed bug, C. lectularius, is now predominantly found in the temperate region, whereas C. hemipterus is more common in tropical and subtropical countries. In particular, the growing number of C. hemipterus infestations in Southeast Asia has gained public attention in the recent decade [Two species of bed bugs are now closely associated with humans, which are t decade ,12,13,14t decade , Australt decade , Taiwan t decade , Floridat decade , and Japt decade  indicatiC. lectularius [C. hemipterus [C. hemipterus, is necessary. Microsatellite markers are especially sensitive for assessing the population genetics of a target species at the molecular level due to their high level of polymorphism, reproducibility, and codominance inheritance. The advent of next-generation sequencing enables the development of microsatellite markers from the genome of non-model organisms such as bed bugs with relative ease [C. hemipterus for downstream population genetics analysis. We then investigated the infestation dynamics of C. hemipterus by understanding the genetic diversity and population structure of C. hemipterus in Malaysia and Singapore based on the developed microsatellite markers through fragment analysis. In addition, genetic variations of mtDNA were also assessed based on the partial sequences of 16S rRNA and cytochrome c oxidase I (COI) genes that are widely used for population genetic assessment [To date, studies on the population genetics and breeding strategy of the bed bugs were heavily focused on the common bed bug, tularius ,23,24,25mipterus . Severalmipterus ,28,29,30ive ease . To thissessment  to offerCimex hemipterus samples were collected from 18 infested units from Malaysia and Singapore, which includes various types of premises including hostels, hotels, and private residences  after being pulverized in liquid nitrogen. DNA quality was assessed by spectrophotometric absorbance in NanoDrop 2000 Spectrophotometer V1.0  and Qubit dsDNA BR Assay, followed by electrophoresis on 0.8% agarose gel. In total, ≈2 μg of gDNA extracted from a pool of 15 bed bugs nymphs was used for 1/16th of a 70-mm × 75-mm Titanium PicoTiter plate of sequencing on a Roche 454 GS FLX sequencer with titanium chemistry , performed by Macrogen Inc., South Korea. Sample preparation and analytical processing of sequence reads were performed according to manufacturer’s protocol for the Titanium series.The raw contigs data were processed using Msatcommander v0.8.2 . The motForty primer pairs were selected for preliminary tests of polymorphism across ten randomly selected bed bugs from different localities in Malaysia and Singapore. The primer pairs which produced unambiguous PCR products on 1% agarose gel were then evaluated on 6% ethidium bromide-stained polyacrylamide gel electrophoresis (PAGE). Primer pairs that produced scorable, unambiguous PCR products of the expected size, and that demonstrated allelic variation  on PAGE were selected for further testing. The resulting polymorphic loci were genotyped across a total of 351 bed bugs from 18 populations see  by usingHO and HE), allelic diversity, gene diversity, number of alleles per locus, number of polymorphic loci and linkage disequilibrium among all pairs of loci. Evidence for large allelic dropout, scoring error because of stutter, and null alleles was determined using Microchecker v2.2.3 [FST) and levels of inbreeding (FIS) in each population were evaluated using FSTAT v2.9.3.2 [r) [We used the software Arlequin v3.01  to deterr v2.2.3 . The popv2.9.3.2 . Average.3.2 [r)  was esti.3.2 [r) . The det.3.2 [r) . Partiti.3.2 [r) . The aim.3.2 [r)  to ident.3.2 [r) . The all.3.2 [r) . * 4.10b [C. lectularius were used as the outgroup: C. lectularius 1 (KJ937992.1 for COI and KJ937977.1 for 16S rRNA) and C. lectularius 2 (KJ937991.1 for COI and KJ937976.1 for 16S rRNA). The pairwise genetic distance among the haplotypes was also estimated in MEGA v.6.0 using the Kimura-2-parameter model. A median-joining haplotype network was generated using the software Network v10.0 [A total of 90 bed bugs were subjected to mtDNA analysis targeting the COI and 16S rRNA regions. PCR was conducted using a newly designed internal forward primer CH-COI-F (5′-GGCAGGGATGCTGGGAAC-3′) and the reverse primer LepR (5′-TAWACTTCWGGRTGTCCRAARAATCA-3′)  for COI * 4.10b  was perf* 4.10b . A neigh* 4.10b  with 10,* 4.10b  implemen* 4.10b  based onrk v10.0  under dep > 0.0028). The mean polymorphic information content (PIC) across loci was 0.54, indicating highly polymorphic markers for downstream population genetic analysis  reads containing di-, tri-, and tetra-nucleotide repeat motifs, with an average length of 242 bp. The search using Msatcommander yielded 620 sequences suitable for primer design consisting of 507 di- (81.77%), 77 tri- (12.42%) and 36 tetra-nucleotide (5.81%) which contain tandem repeats of at least eight, six, and five respectively. Of these primer pairs suitable for primer design, 40 primer pairs were developed successfully  by using the stringent criteria set in Primer3. Despite having 40 unique microsatellite loci initially, only 23 loci produced unambiguous PCR product  and were subsequently screened for polymorphism using PAGE. A polymorphic locus is interpreted as a locus that has more than one allele (length variation of microsatellite repeated at a particular locus) found across the individuals tested within or across populations. A locus that exhibited a high number of alleles is more preferred since they have higher discriminating power in examining genetic differences within and between populations. Under this condition, eight loci were found to be polymorphic and consistent in their allelic profile and were therefore subsequently used for downstream population genetic analyses . A list analysis .p > 0.05) and there was no consistent deviation from HWE for any locus, we regarded the microsatellite markers to be unlinked and therefore suitable for genetic analysis of the study populations. A total of 351 individuals from 18 populations were genotyped at the eight microsatellite loci. Across all populations, a relatively high genetic diversity was observed with the number of alleles per locus ranging from 4 to 12 alleles . The obst-test; p < 0.05, Analysis within each populations showed that the observed and expected heterozygosities averaged across all loci ranged from 0.23 to 0.71 and 0.38 to 0.62, respectively, and did not deviate significantly from each other except one population, SJ, that displayed significant heterozygosity deficit (two-tailed The Garza–Williamson (G–W) index is a ratio of the number of alleles observed in a sample divided by the number of alleles expected under the observed size range . Generally, the values of G–W index which is greater than 0.80 are the representative of a stable population that has not suffered a known reduction in size. In the present study, the statistical test indicated a recent bottleneck or founder event in all the studied populations with respect to low G–W index values of less than 0.20 . A non-br) measures the degree of consanguinity between individuals. We found that the mean coefficient of relatedness in the study populations is 0.373 with a range from 0.124 in BLD to 0.844 in BPT , variations between samples within populations at 18.67% (p < 0.001), and the highest variations was within samples at 52.83% (p < 0.001) (p < 0.05) but low genetic differentiation (2.46%) between the two residential groups whereas genetic differentiation among populations within groups was significantly higher with 26.6% total variance (p < 0.05)  after Bonferroni corrections, except BLA-SJ (FST = 0.16)  suggesting populations that are geographically proximate to each other are no more similar to each other than populations that are distantly located  indicated a significant level of genetic differentiation at all population levels with variations between populations reported at 28.50% (< 0.001) . Additio< 0.001) . We foun < 0.05) . All pai.16) see . This su located .Bayesian analysis of population structure (BAPS) is a program for Bayesian inference of the genetic structure in a population whereby the algorithm will attempt to identify the a posteriori most probable partition and calculate the posterior probability for each of them. BAPS analysis returned 17 partitions (clusters) among the 18 study populations, suggesting that each population is genetically isolated, and each made up a unique cluster, except for KJO and PJO which grouped into a single cluster . Both ofp > 0.05) suggesting the two genes can be concatenated for subsequent downstream analysis. The concatenated dataset resulted in seven haplotypes consisting of seven variable sites, of which four were parsimony informative and with haplotype diversity, Hd = 0.5925 (see ). No obvious phylogenetic structuring was observed between the haplotypes and the bootstrap support was <70% to significantly support a clade structure [π, was 0.00079, and the average number of nucleotide differences, k, was 0.718 in the concatenated dataset  33.9%, Adenine (A) 29.0%, Guanine (G) 16.9%, and Cytosine (C) 20.2%. For the 16S rRNA gene, only two variable sites were detected, and both were parsimony informative within a 334 bp sequenced region. The nucleotide composition was similarly A-T rich consisting Thymine 43.7%, Adenine 28.7%, Guanine 17.4%, and Cytosine 10.2%. To improve the resolution of the findings, sequences from the two genes were concatenated resulting in a 909 bp aligned sequence. An ILD test resulted in homogeneity between the two gene loci . This finding is similar with Masran and Majid [C. hemipterus displayed >4 alleles in at least one microsatellite locus. This finding could be explained when the foundress of the population was mated to two or more unrelated males and/or their descendants [C. hemipterus, Saenz et al. [C. lectularius, populations from the eastern United States whereby 20 of the 21 populations studied had ≤4 alleles across all microsatellite loci, suggesting small propagule size with highly inbred individuals. Similar findings of reduced genetic diversity within aggregations were also found by Booth et al. [C. lectularius within apartment buildings in Raleigh, NC and Jersey City, NJ, followed by Fountain et al. [C. lectularius populations in London. While our study showed a significant reduction of population size as observed by the bottleneck parameters , the allelic diversity suggests that the founding propagule in C. hemipterus populations were much diverse and less genetically related than that observed in C. lectularius [C. lectularius populations are likely able to tolerate high levels of inbreeding or have mechanisms to purge deleterious mutations [Harmonia axyridis  that can purge deleterious alleles that causes inbreeding depression [C. hemipterus, as all the populations were not significantly inbred. More studies are therefore needed to understand the impact of inbreeding in both the bed bugs species to affirm these findings. Assessment of allelic diversity within the population level revealed that nearly all the nd Majid , where acendants . Anotherz et al.  observedn et al. , who invtularius ,25. Thistularius  as a restularius . It was utations . Such mepression . This coC. lectularius populations from an apartment building in Raleigh, NC would have a single origin resulting from a sequential founder effect whereas those of Jersey City, NJ apartment buildings would have infested from at least two established lineages as the location of the latter is expected to have high propagule pressure due to the proximity to New York City with a reportedly high level of bed bugs infestations. AMOVA analysis from our study revealed that the highest genetic variations were attributed to individuals in each aggregation more than that observed between populations. Such a scenario suggests that the C. hemipterus populations from the present study most likely share a common, genetically diverse source population, and with subsequent isolation and restricted gene flow resulting in higher genetic variations within aggregations. This observation was corroborated by BAPS analysis, which displayed high genetic clustering among the study populations. Two populations (BLA and BLD) also displayed significant admixture which suggests the possibility of further substructure within the aggregations. Masran and Majid’s [C. hemipterus populations in Malaysia reported two genetic clusters based on STRUCTURE analysis. Nonetheless, a significant proportion of the populations studied (nine populations) displayed a mixed membership of both clusters. Evanno et al. [K in STRUCTURE analysis cannot determine the true K  if K = 1. The membership to each cluster within the K = 2 runs in Masran and Majid [C. hemipterus samples collected from Malaysia and Singapore very likely share a common, genetically diverse source population.Alternatively, local variations due to geographical distribution of the sampling sites could have influenced the observed genetic differences between the two species. For instance, Booth et al.  suggeste Majid’s  study ono et al.  indicatend Majid  was incor = 0.373, which was consistent with the findings from Masran and Majid [r = 0.174, as opposed to C. lectularius with an overall r = 0.78 [r = 0.437, 0.432, and 0.719 in Raleigh, NC and Jersey City, NJ (JC-A and JC-B), respectively [C. lectularius populations in London was also reportedly high with a value of 0.566 [C. hemipterus infestation except BLD, which has r = 0.844, suggesting potential recent introduction. Interestingly, Narain et al. [C. lectularius [C. lectularius populations from Midwest, USA despite showing high relatedness of >0.5 across of the populations studied, which indicates local variation could have influenced the differences observed.Age structure was also thought to be a factor that influences the genetic diversity of a population whereby a more established infestation would favour multi-generational breeding, resulting in low relatedness among the individuals in an aggregation due to random mating, thereby elevating genetic diversity. We found that the overall relatedness was much lower in our study, nd Majid  with r =r = 0.78 , and r =ectively . The meaof 0.566 . This con et al.  also reptularius ,22,25 whC. hemipterus, we investigated the partial DNA sequences of two mtDNA genes, COI and 16S rRNA in understanding. We observed seven haplotypes among the 90 samples examined in the concatenated dataset. Hap01 was the most frequent haplotype that was found in two-thirds of the populations studied and is most likely to be the ancestral haplotype. The starburst pattern of the median-joining haplotype network suggests that all other haplotypes were likely to have derived from haplotype Hap01 with only 1–2 mutational step differences indicating recent expansion  [h = 0.7604, π = 0.00273 for both COI and 16S rRNA  [C. lectularius whereby two or more mitochondrial variants were found within a single individual [C. hemipterus, at least within the sampling limit of the current study. However, the PCR-Sanger sequencing approach can fail to detect mitochondrial heteroplasmy as standard sequencing methods may fail to detect the rare mtDNA molecules [C. hemipterus.In contrast, the mtDNA haplotype diversity and nucleotide diversity from the present study see  was muchalities)  and h = alities) . Szalansalities)  also foualities)  later prdividual . We founolecules . TherefoC. hemipterus populations from the current study could be accounted for by the limitations of the geographical scale and sample size used for the mtDNA analyses in the present study. A more extensive sampling strategy encompassing broader geographical scales is therefore necessary in future studies to further understand the mtDNA inheritance in the tropical bed bug species. Alternatively, the spread of maternally inherited microorganisms, such as Wolbachia bacteria, can induce indirect selective sweeps on host mitochondria, resulting in the reduction of effective population size that might lead to smaller mitochondrial diversity [Wolbachia is known to be a mutualistic endosymbiont for vitamin B2 provisioning in both C. lectularius and C. hemipterus [The reduced mtDNA diversity in iversity . Wolbachmipterus ,74; howeC. hemipterus based on two independently inherited molecular markers. The two markers were generally congruent in suggesting a common, genetically diverse  source population for the bed bug samples in the present study. We found a higher overall diversity in nuclear microsatellite markers than mtDNA regions owing to their high level of mutation rate and/or possibly due to selection pressure at the mtDNA regions. Interestingly, we found a contrasting scenario in the population genetic structure and breeding dynamics of the sympatric species, C. lectularius. Generally, C. lectularius displayed reduced genetic diversity in nuclear DNA  while d1 region . The dis


Banksia sessilis using three cpDNA markers and nine nuclear microsatellite markers. Sixteen cpDNA haplotypes were identified, comprising two major chloroplast DNA lineages that are estimated to have diverged in the Pliocene, approximately 3.3 million years ago. This timing coincides with major geomorphological processes in the landscape, including the separation of the Darling Plateau from the adjacent Swan Coastal Plain, as well as eustatic changes on the Swan Coastal Plain that are likely to have resulted in the physical isolation of historical plant lineages. Chloroplast lineages were broadly aligned with populations associated with older lateritic soils of the Darling Plateau and Geraldton sandplains or the younger sandy soils associated with the Swan Coastal Plain and Southern Coastline. This structural pattern of lateritic versus non‐lateritic division was not observed in the nuclear microsatellite data that identified three genetic clades that roughly corresponded to populations in the North, South, and Central portions of the distributions.The impact of Quaternary glaciation on the development of phylogeographic structure in plant species is well documented. In unglaciated landscapes, phylogeographic patterns tend to reflect processes relating to persistence and stochasticity, yet other factors, associated with the palaeogeographical history of the landscape, including geomorphological events, can also have a significant influence. The unglaciated landscape of south‐western Western Australia is an ideal location to observe these ancient drivers of lineage diversification, with tectonic activity associated with the Darling Fault in the late Pliocene attributed to patterns of deep phylogeographic divergence in a widespread tree from this region. Interestingly, other species within this region have not shown this pattern and this palaeogeographical boundary therefore presents an opportunity to examine age and historical distribution of plant species endemic to this region. In this study, we assess patterns of genetic diversity and structure across 28 populations of the widespread shrub  Banksia sessilis coincides with the uplift and separation of the Darling Plateau from the Swan Coastal Plain during the Pliocene.Phylogeographic structure in  The same pattern was observed in the mitochondrial DNA of the long‐lived tree Pinus pinaster  is an ideal location to search for signatures of palaeogeographical processes associated with geomorphological events in plant taxa. Species in this landscape including those that are widespread are often characterized by remarkably high levels of genetic diversity and divergence in the slowly evolving cpDNA genome  AR Mast & KR Thiele (formerly Dryandra sessilis) is a common and widespread woody shrub endemic to the SWAFR. The species occurs across multiple biogeographic regions including the sandy SCP and southern coastal sands, the lateritic duplex soils of the Darling Plateau, and the lateritic and non‐lateritic sands of the Geraldton Sandplains. Habitat types vary from Kwongan heath in the Geraldton Sandplains to Jarrah forest on the Darling Plateau and Banksia woodland on the SCP. The species straddles the biogeographical boundary created by the Darling Fault and so is an ideal candidate to assess hypotheses of phylogeographic structure resulting from palaeogeographical processes. Intrinsic factors, such as local distribution, mating system and fire response, may also influence genetic diversity. We have no a priori reasons to expect differences in these factors across the distribution, and so predict that the widespread distribution of the species in locally abundant populations, and visitation by mobile pollinators, will lead to little differences in genetic diversity and connectivity across the distribution. Using a combination of cpDNA and nuclear microsatellite markers, we investigated patterns of genetic diversity and structure at historical and contemporary time scales and test hypotheses that: (a) Banksia sessilis will show patterns of phylogeographical structure concordant with palaeographical processes associated with the uplift of the Darling Plateau; and (b) the current widespread distribution will reflect high levels of genetic connectivity across the landscape as revealed in contemporary genetic markers.22.1Banksia sessilis is a woody shrub that produces prolific bird‐pollinated flowers and seed that are a food source for parrots and cockatoos  are coastal and grow in alkaline sands over limestone, while two (var. sessilis and var. flabellifolia) occur further inland on acid, sandy or duplex soils over laterites. Morphological differences between varieties are limited to minor differences in the shape and color of leaves and presence of hairs on the stem. While plants from many populations sampled could be identified as belonging to one of the four recognized varieties, plants at several sites could not be readily ascribed variety status according to current morphological descriptions. We therefore conducted all analyses at the species level, Banksia sessilis.Four taxonomic varieties are currently recognized within the species George, . Two of 2.22 area, with care being taken to avoid sampling plants that were near‐neighbors and therefore potentially related. Collected leaves were immediately placed on silica gel and later freeze‐dried. DNA was extracted from approximately 50 mg of freeze‐dried material using a modified 2% CTAB method  universal primers by Macrogen (South Korea). Sequences were aligned using BIOEDIT v. 7.2  and B. hewardiana ((Meisn.) A.R. Mast & K. R. Thiele), as well as a specimen of Macadamia integrifolia were included as outgroups.Sequence variation in chloroplast DNA (cpDNA) regions known to detect intraspecific variation in Australian plants  used as an outgroup. A normal prior distribution was set around the divergence time with a standard deviation of 3.1 million years. Trees were visualized with FigTree v1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/). The relationships among haplotypes were also investigated with a maximum parsimony analysis (Median‐Joining method) implemented in NETWORK v.4.6.1.3 .Chloroplast haplotypes, haplotype diversity, and nucleotide divergence among clades were calculated using DnaSP v5.10  and pairwise population differentiation values (Weir and Cockerham's 1984 FST) were determined in GENEPOP with significance determined with 9,999 exact tests. Pairwise population FST values were displayed in R using the “pheatmap” package  and was used to analyze relatedness within populations. Simulations (n = 100) of dyads were performed to determine the distribution of R values expected for the data in Parent‐offspring, full sibling, half sibling, and unrelated relationships. The partitioning of genetic diversity among populations was tested with an analysis of molecular variance (AMOVA) implemented in ARLEQUIN v.3.5. One‐way ANOVAs were carried out to determine the effect of genetic cluster identification on mean genetic diversity estimates.For the nuclear microsatellite data, tests of genotypic disequilibrium among pairs of loci were conducted in GENEPOP v.4.0.11  was used to determine the most likely number of genetic clusters based on the Evanno method   in GENALEX.Genetic structure among individuals and populations was visualized using Bayesian clustering analysis implemented in STRUCTURE v.2.3.4 . This resulted in 16 haplotypes from a total of 138 sequences . Most populations (n = 21) possessed just one haplotype. Seven populations possessed more than one haplotype and six populations possessed population‐specific haplotypes  for Macadamia places the divergence of the two clades at 3.3 Ma (2.0–5.0 Ma) during the Pliocene  and those found on sand and limestone associated with the geologically younger coastal region of the SCP and Southern Coastline (hereafter non‐lateritic clade). There was one exception to this trend; haplotypes 11 and 12 from the Jarrahdale (JAR) population Figure . MoleculWithin clades, most haplotypes were restricted to single, or geographically proximate populations, although four were more widespread. Two of these were from the lateritic clade (Hap 6 and Hap 7) and were widespread across the Darling Plateau whereas Haplotype 9 and 15 from the non‐lateritic clade were widespread across the southern coastal sands and SCP respectively. Haplotype diversity was higher in the lateritic clade, but nucleotide diversity was higher in the non‐lateritic clade. Table . AMOVA iFs and R2) that may also predict population size changes were nonsignificant in both clades, as well as when considering all populations as a group  showed a high frequency of null alleles (36.8%) and was associated with populations where many or all individuals failed to amplify and was therefore removed from the dataset. The remaining nine loci showed no evidence of genotypic disequilibrium following Bonferroni correction. The average number of alleles per population ranged from 2.33 to 6 Table . The numK = 3) that did not reflect the lateritic/non‐lateritic division identified in the cpDNA data. Instead, the three groups broadly corresponded to populations occurring on the lateritic Darling Plateau (hereafter Plateau), those on sands, limestone, and laterite associated with the northern coastline (from PRE northward to WAG) , and those associated with sands and limestone of the southern coastline (from SUG southward to GAP)   = 13.67, p = <.005), mean observed heterozygosity (HO)  = 8.22, p = <.005), and mean expected heterozygosity (HE)  = 15.32, p = <.005). Estimates of FIS were often high, with significant estimates observed in 17 of the 28 populations  = 3.08, p = <.005). All populations except BUR showed significantly higher levels of relatedness than expected based on 500 randomized iterations  = 10.88, p = <.005) where the mean estimate indicated the presence of full sibling and parent–offspring relationships among individuals  Figure . The finls Table .FST values as low as 0.03 observed between GHR and NY, two North Coastal populations approx. 170 km apart, to a high FST of 0.61 between GAP and HES, populations from the South Coastal and Plateau clades, respectively, located approx. 210 km apart  in comparison with North Coastal populations  and Plateau populations . Across all populations, the mean pairwise differentiation was high (FST = 0.27 ± 0.00: Nei's DST = 0.326 ± 0.02)  of variation was maintained among populations. The mean number of effective migrants per generation (Nm) was 0.48 ± 0.001 across all populations, 0.443 ± 0.07 within North Coastal populations, 0.411 ± 0.09 within South Coastal populations, and 0.96 ± 0.05 within the Plateau cluster (Table Populations varied in their degree of genetic differentiation with rt Table . Within ) Tables . South C1 Figure . Consister Table .r2 = 0.037, p = .047). Within genetic groups, there was a pattern of increasing genetic differentiation with distance in the South Coastal cluster, but the relationship was not significant, likely a consequence of small sample size (r2 = 0.81 p = .09). There was no evidence of isolation by distance within the North Coastal and Plateau clusters, respectively .There was a weak signal of isolation by distance across all 28 populations . One lineage was restricted to lateritic soils of the Darling Plateau and Geraldton Sandplains and the other to non‐lateritic sands and limestones of the SCP and Southern Coastline. Interestingly, this structural pattern was not observed in the nuclear microsatellite data that showed three genetic clusters concordant with populations located on the Darling Plateau (lateritic), along the Northern Coastline (lateritic and non‐lateritic), and along the Southern Coastline (non‐lateritic). If the divergence of populations observed in the cpDNA were due to ecotype variation, where local adaptation  had resulted in incompatible lineages  identified was high in comparison to other south‐western Australian Banksia species (B. arborea: n = 5 and B. biterax: n = 7)  population occurs on laterite on the western edge of the Darling Scarp but is also close to non‐lateritic populations to the west. The Jarrahdale population possessed cpDNA haplotypes that were more closely related to non‐lateritic haplotypes on the SCP than to those on the lateritic Darling Plateau, suggesting the ancestor to this marginal population derived from the SCP. Subsequent gene flow with surrounding Darling Plateau populations would provide an explanation for the grouping of this population with other Plateau populations in the nuclear data.sessilis and var. flabellifolia) largely on lateritic soils and two (var. cordata and var. cygnorum) on coastal and sands over limestone. This suggests that occurrence on substrate is a more reliable identifier of historical relationship than morphology.Although we were not able to reliably assign populations to varietal status, we note that the separation between the lateritic clade and the non‐lateritic clade roughly corresponds to the substrate descriptions for the four varieties, two ; formal analysis (lead); investigation (lead); writing–original draft (lead). Sarah‐Louise Tapper: Data curation ; formal analysis ; investigation . David J. Coates: Conceptualization (lead); writing–review and editing (supporting). Shelley L. McArthur: Data curation ; formal analysis (supporting). Margaret Byrne: Conceptualization (lead); funding acquisition (lead); project administration ; resources ; writing–review and editing (supporting).Supplementary MaterialClick here for additional data file.


Agarophyton chilense were surveyed along the Chilean coast before, in the days after and 2 years after the 8.8 magnitude earthquake in 2010. The genetic diversity of sexual populations appeared sensitive to this massive perturbation, notably through the loss of rare alleles immediately after the earthquake. By 2012, the levels of diversity returned to those observed before the catastrophe, probably due to migration. In contrast, enhanced rates of clonality in cultivated populations conferred a surprising ability to buffer the instantaneous loss of diversity. After the earthquake, farmers increased the already high rate of clonality to maintain the few surviving beds, but most of them collapsed rapidly. Contrasting fates between sexual and clonal populations suggest that betting on strict clonality to sustain production is risky, probably because this extreme strategy hampered adaptation to the brutal environmental perturbation induced by the catastrophe.Catastrophic events can have profound effects on the demography of a population and consequently on genetic diversity. The dynamics of postcatastrophic recovery and the role of sexual versus asexual reproduction in buffering the effects of massive perturbations remain poorly understood, in part because the opportunity to document genetic diversity before and after such events is rare. Six natural  and seven cultivated  populations of the red alga  Indeed, most studies have focused on the consequences of a catastrophic event on genetic structure by analyzing the impacted populations a posteriori  represents a good candidate to tackle these predictions about the evolutionary shifts caused by catastrophic events in partially clonal species. This species, as other species of Gracilariaceae  dramatically affected the coast of Central Chile  with a similar morphology. In the field, only fertilized female gametophytes are easily detected by eye. Distinction among nonmature individuals, reproductive but nonfertilized female gametophytes, male gametophytes, and tetrasporophytes is not possible. The sampling was therefore blind, and it was not possible to control, a priori, the number of diploid individuals collected in each site. The sex and stage (ploidy level) of the individuals was determined a posteriori by observations of the reproductive organs under a binocular microscope  and farms (N = 6) began in 2002 and was undertaken during the Austral summer months . Before the earthquake, sites were sampled at one or two time steps between 2002 and 2009  probably because of the combined effects of the founding event (that likely took place at the end of the Quaternary) and the recent human impacts of harvesting and cultivation practices /(N − 1), G being the number of clonal lineages, and N the number of sampled individuals and (b) the slope β of the Pareto distribution, an index of evenness of clonal size   and unbiased gene diversity (E) Nei,  using Geockerham  and its 2.4c was assessed using ClonEstiMate, an efficient and robust Bayesian method to infer rates of clonality from populations genotyped at two time steps  explicitly taking into account partial clonality  as this time lapse probably involved more than two generations and could lead to underestimation of c  and from the region of Concepción for sampling dates prior to the earthquake .Impacted: This group encompassed the populations from the region of Concepción sampled in 2010 . Natural populations correspond to Dichato and Coliumo and farmed populations to Tubul, Lirquen, and Lenga , it is theoretically impossible to obtain p‐values of .05 or lower for some of the tests performed. For example, (a) when Na = Nb = 2, the minimal p‐value, obtained if the group A is ranked as  and the group B is ranked as , is of 0.33; (b) when Na = Nb = 3, the minimal p‐value, obtained if the group A is ranked as  and the group B is ranked as , is of 0.10. Theoretical minimal p‐values are provided with each boxplot. Despite the low statistical power expected for our dataset (few populations surveyed per group), this approach still provides a qualitative view of averaged genetic evolution observed between groups.We first performed Mann–Whitney Wilcoxon's rank tests to detect any differences among groups in the distribution of each descriptor of diversity. We compared nonimpacted versus impacted groups and impacted versus recovery groups at the population scale, using indices of genetic diversity averaged over all loci per population. Given the low size of impacted and recovery groups for both natural populations and farms , the probability of these transitions can be computed using CloNestiMate . In contrast, impacted populations should exhibit transitions with lower probabilities: The lower the transition probabilities, the higher the departures from the eWFM.Using the three groups described above, we aimed to test whether (a) transition probabilities estimated between 2009 and 2010 were lower in impacted populations than in nonimpacted populations and (b) transition probabilities of recovering populations  were higher than just after the earthquake .c : 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.98, 0.99, 0.999, and 1 . Five Fis values were significantly different from 0: two of them were positive and three were negative  and Lenga (P‐LEN) Figure HE), between sampling years, were generally small for these two sexual populations  and then an increase between the impacted and recovery groups . This variation in diversity was corroborated by the loss of rare alleles in 2010. Among the seven alleles occurring at a frequency of 0.12 or lower in P‐DIC and P‐COL before the earthquake, only one was resampled in 2010. The recovery of the polymorphism is explained by the emergence of seven new rare alleles in 2012 samples .Changes in genetic diversity  varied between 0.2 and 1.As expected, the farmed populations were highly clonal Table , often d β Table and 3. ThA. chilense farming beds still survived at the mouth of the estuary where both freshwater and saltwater influence exist . In addition, most of the planting efforts that followed were directed to the creation of new beds directly on the sandy beach that faces the Pacific Ocean . Of these new beds, only a few dying individuals were visible in 2013; all were dead in 2014.Aquaculture activities have now stopped in two farms (F‐TUB and F‐LIR) of the three studied in Concepción. In Tubul, a concession of 212 ha that was traditionally used for aquaculture since the 90s, a salt marsh crisscrossed by small channels of water, dried up almost completely after the earthquake due to coastal uplift  and departure from HWE (Fis) were uncovered. Clonal diversity, and consequently excesses in heterozygosity, was quite stable in the nonimpacted farms, while large variations occurred in the region of Concepción after the earthquake (Table p = .2) and was logically accompanied by an increase in the dominance of a few clones . Just after the catastrophic event, they reached values close to 0 in the farms of Concepción  and decreased again to significantly negative values in 2012  .Between 2010 and 2012, farms from both regions appeared highly clonal, with ke Table . The clo4A. chilense bore the mark of the major earthquake of 2010. This event impacted both their demography and genetic diversity. Reproductive modes modulated earthquake consequences on genetic diversity both less than a month and still 2 years after the catastrophe. In this area, the farming of seaweed is an important social activity for local communities. It is based only or mostly on clonal fragmentation of thalli  has been previously reported after earthquakes . These two small populations, of <1 mSurprisingly, genetic diversity in these two natural populations  recovered to pre‐earthquake levels after only 2 years. Extreme mortality events have been shown to lead to a decrease in genetic diversity due to drift  to parents . At the sampling date of March 2010, the surviving individuals thus only represented a subset of the original population collected in 2009. Without at least one reproductive event, the demographic effect of the earthquake only mimicked a low sampling effort, explaining why the probabilities of transitions were slightly lower than in the nonimpacted group.The two surviving natural populations departed more from the eWFM than nonimpacted ones during the short period of recovery, suggesting the violation of at least one of the underlying hypotheses of the model  before the earthquake disappeared in 2010 . Again, this suggests the violation of at least one hypothesis underlying the eWFM in farms during this period, the more obvious being the hypothesis of constant size. Near‐complete mortality of A. chilense was registered only days after the earthquake in F‐TUB . In F‐LEN, most of the algal production comes from the lower intertidal/high subtidal zone and is only extensively planted and harvested by local families. Farmers' response to the postcatastrophe socio‐ecological crisis splits into distinct scenarios: While activities in F‐LEN stayed somewhat unchanged, in F‐LIR the A. chilense farming was abandoned, while in F‐TUB farmers attempted to restock using survivors. In F‐LIR, only scattered thalli growing along the sandy banks that were previously farmed remained in 2012. An important reshuffling of clones was observed in F‐LIR , reaching 100% between 2010 and 2012 in F‐TUB and F‐LEN, exemplifies the “planting from cuttings” strategy by farmers that reinforces the dominance of local surviving clones  have proven highly informative and revealed subtle differences between farms probably linked with variation in amount and nature of the damage and postearthquake farmer's response. In the localities of Tubul and Lirquen (F‐TUB and F‐LIR), farmers lost practically everything after the destructive earthquake and tsunami's waves . The traditional farming practices allow the integration of a few spontaneous genotypes coming from sexual reproduction in farmed and natural populations each year. Coupled with the weeding of plants germinated from seed presenting low growth rate, this strategy maintains high levels of genotypic diversity and high heterozygosity in these crops  conferred a noticeable ability to buffer genetic impoverishment after a catastrophic event, it lowered the odds of survival at larger environmental and time scales. In contrast, populations reproducing sexually were more sensitive to the immediate genetic impacts of catastrophic events. Yet, genetic recoveries of these populations were fast, probably promoted by long‐distance migration and rapid integration, by sexual recombination, of new alleles into the surviving local genetic pool  were very similar to purely sexuals , many species of commercial or agricultural interest (tree‐species), or human parasites. The implications of the presented results and the recent theoretical developments in population genetics of partially clonal species go beyond the specific case of algal farming. Understanding the influence of this mixed reproductive mode on evolution is absolutely necessary for a world, where the frequency of dramatic fluctuations in population size is increasing. Notably, caricatural representations of clonality as a binary trait  should no longer persist. On the contrary, future studies devoted to partially clonal species should consider the rate of clonality as a continuous variable and more fully appreciate its variation among populations and species, as well as its evolutionary influences. It is a prerequisite for relevant management plans of harvested species, conservation actions of threatened species, or efficient measures in public health. Finally, while even a small amount of sex is evolutionarily advantageous, clonality also constitutes an opportunity for successful genotypes to spread and persist, notably the None declared.Table S1Click here for additional data file.


Montastraea cavernosa at two sites at Pulley Ridge, a mesophotic coral reef ecosystem in the Gulf of Mexico, and downstream sites along the Florida Reef Tract. We found differentiation between reefs at Pulley Ridge (~68 m) and corals at downstream upper mesophotic depths in the Dry Tortugas (28–36 m) and shallow reefs in the northern Florida Keys . The spatial endpoints of our study were distinct, with the Dry Tortugas as a genetic intermediate. Most striking were differences in population structure among northern and southern sites at Pulley Ridge that were separated by just 12km. Unique patterns of clonality and outlier loci allele frequency support these sites as different populations and suggest that the long‐distance horizontal connectivity typical of shallow‐water corals may not be typical for mesophotic systems in Florida and the Gulf of Mexico. We hypothesize that this may be due to the spawning of buoyant gametes, which commits propagules to the surface, resulting in greater dispersal and lower connectivity than typically found between nearby shallow sites. Differences in population structure over small spatial scales suggest that demographic constraints and/or environmental disturbances may be more variable in space and time on mesophotic reefs compared with their shallow‐water counterparts.Mesophotic reefs (30‐150 m) have been proposed as potential refugia that facilitate the recovery of degraded shallow reefs following acute disturbances such as coral bleaching and disease. However, because of the technical difficulty of collecting samples, the connectivity of adjacent mesophotic reefs is relatively unknown compared with shallower counterparts. We used genotyping by sequencing to assess fine‐scale genetic structure of  Montastraea cavernosa, even between nearby sites. Differences in population structure over small spatial scales suggest that demographic constraints and/or environmental disturbances may be more variable in space and time on mesophotic reefs compared with shallow‐water counterparts.Mesophotic reefs (30‐150 m) have been proposed as potential refugia that facilitate the recovery of degraded shallow reefs following acute disturbances at shallower depths, such as coral bleaching and disease. We find extensive population structure among mesophotic  Overfishing, pollution, and changing climate are major drivers of coral decline  and Pulley Ridge  and transported to the University of Miami for processing. Samples were extracted using a silica column and vacuum manifold following Ivanova, Dewaard, and Hebert . Alignment and SNP calling were performed using bwa .We removed low‐quality bases (Phred < 20) at the leading and trailing end of reads, and reads where a 4‐bp sliding window average read quality fell below 20 as an initial filtration step using Symbiodiniaceae reference following Manzello et al. , Cladocopium  and loci not in HWE (p < .01) were identified with VCFtools 0.1.13  for separate analysis, converting p‐values to q‐values and filtering at a 5% FDR. We then assessed clonality from hierarchical clustering of identity‐by‐state matrices generated by SNPrelate . Strong overlap of all Dry Tortugas sites , with the optimal K defined by the minimum cross‐validation error term  and loci called in an average of 97% of samples (range: 92%–100%). Average depth per call was 31.2 reads.3.2g/N) was highest in the Dry Tortugas, where we found no clones, and lowest at Pulley Ridge South, where 4 of 7 samples were from the same clonal group . This assignment showed a third ancestral group in the Dry Tortugas in addition to samples with shared assignments with the other two regions. When examining K = 4 populations, assignments were virtually the same as K = 3; however, Key Biscayne is assigned to a distinct additional ancestral group. The separation of Dry Tortugas assignments did not relate to the three “sites” that were pooled based on initial DAPC analysis , with 30% of variation explained by geographic distance (R2 = 0.30).Admixture results indicated s Figure , where Ps Figure ; howeverAllelic richness (Na) was similar across all sites Table , but Key3.3p < .001, R2 = 0.21). Of 182 outlier loci, 100 (54.9%) had blastx matches  and 62 has assigned gene ontologies (34.0%), most commonly involving ATP binding, G protein‐coupled receptor activity, ion binding, and various plasma membrane properties.The minor allele frequency  of the 182 outlier loci was highest within the Pulley Ridge region, where most loci had MiF > 0.25 Figure . Many ou3.4Symbiodiniaceae  from each other and from shallower downstream FRT populations. Moreover, these mesophotic populations appear to have variable horizontal connectivity with nearby mesophotic sites in the Dry Tortugas where there is infrequent but distinct signature of migration. Unique patterns of outlier loci and interindividual relatedness support differentiation among these populations.Connectivity is an important component of the health and resilience of contemporary reefs, and declining coral communities have prompted a focus on potential links between shallow and mesophotic populations. This study shows genetic structure distinguishing nearby mesophotic populations of M. cavernosa at the westernmost extent of the FRT (Dry Tortugas), at least at the depths sampled (28‐36 m). However, limited sampling and lack of paired shallow‐deep sampling locations  are distinctive, but that Dry Tortugas sites may have intermediate connectivity with each of these two endpoints, generally agreeing with the conclusions of Serrano et al.  who usedThe genetic disconnect between Pulley Ridge and the Dry Tortugas also indicates the Pulley Ridge mesophotic reef ecosystem is moderately isolated. Hierarchical clustering, DAPC, and admixture analysis suggest they are distinct but indicate some shared ancestry and migration between these mesophotic populations. However, admixture analysis for any number of populations does not discriminate between the two Pulley Ridge sites, while DAPC and fixation indices show strong separation and hierarchical clustering, suggesting relatedness is much higher within than between Pulley Ridge sites. In this case, admixture analysis could be a more comprehensive assessment of structure ; however, the interaction of demography and small sample sizes precludes us from rarefying our results and makes it difficult to determine whether this and other metrics are biased, even after clonal replicates were removed. Bayesian methods such as STRUCTURE and ADMIXTURE are generally restricted to larger sample sizes when using microsatellites  that this process is actually occurring in M. cavernosa. Results from simulated migration patterns also indicate common horizontal asymmetry, with more frequent “migration” in the downstream (east and north) direction of the Florida Current except in the Lower Keys, where westward transport is also common due to the complexity of oceanographic patterns in this area  may be driving these differences, distance‐based metrics explain only ~30% of variation. The minor allele frequency of these loci increases with depth, similar to the findings of Bongaerts et al. , explainSymbiodiniaceae from Caribbean scleractinian corals in every sample. Interestingly, Serrano et al. ; Formal analysis (lead); Investigation (lead); Visualization (lead); Writing‐original draft (lead); Writing‐review & editing (lead). Rocío Pérez Portela: Data curation (supporting); Methodology (supporting); Writing‐review & editing (supporting). Xaymara M. Serrano: Formal analysis (supporting); Visualization (supporting); Writing‐review & editing (supporting). Marjorie Oleksiak: Conceptualization ; Funding acquisition ; Writing‐original draft (supporting); Writing‐review & editing (supporting). Andrew C. Baker: Conceptualization ; Funding acquisition ; Writing‐original draft (supporting); Writing‐review & editing (supporting).Fig S1Click here for additional data file.


Tetraena mongolica Maxim. (TM), belongs to Zygophyllaceae family, a rare and endangered plant with narrow distribution. However, for the last decade, due to excessive logging, urban expansion, industrial and tourism development, habitat fragmentation and loss of natural habitats have become major threats to the population of endangered plants.Studying population genetic structure and gene flow of plant populations and their influencing factors is of particular significance in the field of conservation biology, especially important for species such as rare and endangered plants. In this study, genetic diversity, population genetic structure and gene flow of TM populations were evaluated by reduced representation sequencing technology, and a total of more than 133.45 GB high-quality clean reads and 38,097 high-quality SNPs were generated. Analysis based on multiple methods, we found that the existing TM populations have moderate levels of genetic diversity, and very low genetic differentiation as well as high levels of gene flow between populations. Population structure and principal coordinates analysis showed that 8 TM populations can be divided into two groups. The Mantel test detected no significant correlation between geographical distances and genetic distance for the whole sampling. Moreover, the migration model indicated that the gene flow is more of a north to south migration pattern in history.This study demonstrates that the present genetic structure is mainly due to habitat fragmentation caused by urban sprawl, industrial development and coal mining. Our recommendation with respect to conservation management is that, all 8 populations should be preserved as a whole population, rather than just those in the core area of TM nature reserve. In particular, the populations near the edge of TM distribution in cities and industrial areas deserve our special protection. Population genetic structure is the result of interaction between ecological and genetic processes. The study of genetic structure and gene flow play an important part in understanding the genetic characteristics and the dynamics of population, especially, those rare and endangered species with a limited geographical range. This is mainly because their populations are more vulnerable to damage, and gene flow is considered one of the most important determinants of the genetic structure of plant populations . In addiEarly studies relating to genetic diversity, population genetic structure and gene flow studies have largely relied on traditional molecular markers . However, these markers have their own limitations in resolving the geographical differentiation of population, especially when using few genetic markers. In addition, developing traditional molecular markers not only takes time and effort, but also involves complicated steps, and most importantly they are costly. At present, whole genome sequencing is still pretty expensive, especially sequencing of hundreds samples. With the rapid development of high-throughput sequencing technology, so far, scientists have developed various reduced-representation genome sequencing by restriction enzyme digestion of genomic DNA –7, and tTetraena mongolica Maxim. (TM) which belongs to Zygophyllaceae family, is an endangered species. It is only distributed in the Alashan Ordos area of Inner Mongolia in china, and its habitats is defined as ecologically fragile areas due to the extremely low annual rainfall, TM which is dependent on its well-developed roots can thrive in these places and has gained considerate attention for being extremely resistant to drought and cold as well as windbreak and sand-fixation. However, owing to excessive logging (for firewood), overgrazing, urban expansion, industrial development, coal mining and gas exploration, together with habitat fragmentation, the number of TM have declined significantly in recent decades. The area of TM decreased by 225.2 km2 (from 629.5 km2 in 1990 to 404.3 km2 in 2010) declined by up to 35.8%, and the reduced area was mainly transformed into mining areas, sandy land, farmland and urban area [ban area . Currentban area , 16, anaban area , chemicaban area , 19, ecoban area , influenban area , salt-toban area , geneticban area –26 and sTo understand the geographic patterns of genetic variation, population structure and gene flow of this plant will be crucial for their effective conservation. Reduced representation sequencing technology (SuperGBS method) is utilized in this study to evaluate patterns of genetic diversity, structure and gene flow of TM population across its entire geographic distribution range. Based on a genome-wide SNPs analysis, a novel understanding of genetic structure and gene flow of TM under urbanization, apparent hydrographical and geological barriers  were revealed. Furthermore, this study also characterized biogeographic patterns of TM population and discovered several possible factors for its formation. Also, we assessed “isolation by distance” based on large-scale spatial genetic structure. According to the results, at the last, we put forward some constructive suggestions for better protection and follow-up management.One hundred twenty TM accessions were sequenced using Illumina Hiseq Xten, PE150 Platform, after the primary quality filtering step, a total of 981,376,270 reads, 133.45 Gb clean reads were generated, average of per accession were 1.11 Gb reads. According to the comparison results, the average sequencing depth of all samples was 104.17×, and the coverage ranged from 77.89 to 89.66%. With a minimal set of initial quality filters, we obtained 40,931 SNPs. After rigorously screening , 38,097 SNPs genotyping data were obtained, and several values of genetic diversity were calculated. With all 120 individuals, as a whole, it is observed that the Ne varied from 1.020 to 2 and average was 1.616, the levels of Ho and He ranged from 0.067 to 0.933 and 0.019 to 0.500 with an average of 0.446 and 0.348 respectively. Also, the PIC values varied from 0.019 to 0.375 with an average of 0.274  software was used to evaluate genetic structure, the K-value represent the number of clusters of all 120 TM individuals during the analysis, and the optimal K-value was selected according to CV. In this study, CV ranged from 0.436 to 0.575  and medium levels of gene flow (Nm of 0.04–0.71) between TM populations [Although the analysis of phylogenetic relationship and population structure showed that the TM populations were divided into two groups clearly, surprisingly, we detected very low levels of genetic differentiation and high levels of gene flow (Nm) between populations. And, genetic differentiation coefficient (Fst) ranged from 0.010 to 0.026 among 8 populations, and with an average of 0.020 within 120 TG individuals  = 0.094)  among different populations. Findings also indicated geological landforms such as Yellow River, Menggu Mountains, Zhuozi Mountains were not as barrier to gene flow, Thus, we speculated that it is this high level of gene flow that can sustain the current level of moderate diversity in this species. Additionally, a lot of admixtures were observed among in all populations in population structure. Some of the theories claim that admixture, reflecting allele sharing, can result from incomplete lineage sorting of historically contiguous populations . It is aThe direction of the gene flow was evaluated by migration model. According to our data, north to south migration model has the highest probability based on the marginal likelihood and the Bayes factor among the five speculative models Table , this suIn order to explore the spatial genetic structure of different source populations of species, Mantel test is often used to verify whether the species conform to the model of IBD, and to analyze the correlation between their genetic distance and spatial distance . The resCombining genetic variation, gene flow, population genetic structure and IBD analysis, we can conclude that the existing genetic variation and spatial genetic structure of TM population were caused by rapid development of urbanization and industrialization. Although the TM population maintained a certain level of genetic diversity and a high level of gene flow, current populations has shown subtle spatial genetic structure, Wuhai city is in the middle of the north and south TM population, to some extent, the dense urban population can affect the routes of pollinators, insect-mediated gene flow over several generations that generated the current genetic structure, It is a signal that TM populations which are disturbed seriously by human activities beginning to started gradually forming genetic divergence. Compared with previous studies, our results of genetic diversity and population structure present a new perspective, and can provide valid information for TM management and conservation. To avoid genetic degradation and to maintain the TM population, we offer the following recommendations for protective measures: (1) Mining of opencast coal mines in TM distribution areas should be strictly forbidden, the establishment of new industrial zones in the TM distribution areas should be prohibited. (2) The 8 populations should be protected as a whole unit, as a bridge and link between the north and south populations. Also, the HN population should be continuously monitored and protected. In addition, because the populations have edge effects, those populations near urban and industrial areas also needs to be well-protected. (3) Collection of Germplasm resources, establishment of seed Banks and transplanting experiments are extremely necessary to expand TM population. Furthermore, as an insect-pollinated plants, and those pollinators should also be given adequate attention.In this study, we investigated roundly geographic distribution of TM before sampling, 144 individuals of TM were collected from all eight extant populations  following the manufacturer’s introduction. After DNA quality and concentration were tested by Agarose Gel and NanoDrop, we selected 15 samples with the highest DNA quality from each population as our experimental samples.we adopted modified GBS methods  to conshttp://hannonlab.cshl.edu/fastx_toolkit/). Through the steps above, clean reads were obtained. Because the public databases does not contain full genome information for TM, and even there is also no the genome data of relative species of zygophyllaceae family to reference. Under these circumstances, de novo generation of a GBS reference was constructed following [To ensure the accuracy of analysis, ‘FastQC’ (v. 0.11.4)  was usedollowing . Clean Rollowing , and theollowing  to prediSeveral population genetics general statistics to describe genetic variation were estimated by R package genepop (Version1.0.5) as described . Mean nu2 threshold) to filter the obtained SNP, the SNP without close linkage was selected, and then we used the ADMIXTURE (version 1.3.0) software [For further elucidation of the genetic structure of the eight TM populations, we used the PLINK (version 1.9) software  , and thePhylogenetic trees are tree branch structures representing near or far relationships between individuals, for further elucidation of genetic distance between samples and clusters. As the first step, each individual SNP marker was end to end, and if the corresponding locus was missing, the - was used instead. Neighbor-joining method was used to build phylogenetic tree of all 120 individuals, Treebest software (v1.9.2)  was usedhttps://peterbeerli.com/migrate-html5/index.html) according to Beerli & Palczewski [Genepop program (Version1.0.5) in R soft was used to analyze Fst of each SNP locus in all individuals. We also analyzed the gene flow level among eight populations and the parameter Nm was used evaluated using formula Nm = (1-Fst)/4Fst. The directions of the gene flow between different populations were estimated using MIGRATE (lczewski  and Blanlczewski . The estIn order to understand whether there is a correlation between genetic distribution and spatial location of TG from different populations, Mantel test is performed using GenAlEx v6.5  with 10,