Oncorhynchus mykiss) are a partially migratory species wherein some individuals undergo long‐distance anadromous migrations, and others stay as residents in their native freshwater streams. The decision to migrate is known to be highly heritable, and yet, the underlying genes and alleles associated with migration are not fully characterized. Here we used a pooled approach of whole‐genome sequence data from migratory and resident trout of two native populations—Sashin Creek, Alaska and Little Sheep Creek, Oregon—to obtain a genome‐wide perspective of the genetic architecture of resident and migratory life history. We calculated estimates of genetic differentiation, genetic diversity, and selection between the two phenotypes to locate regions of interest and then compared these associations between populations. We identified numerous genes and alleles associated with life history development in the Sashin Creek population with a notable area on chromosome 8 that may play a critical role in the development of the migratory phenotype. However, very few alleles appeared to be associated with life history development in the Little Sheep Creek system, suggesting population‐specific genetic effects are likely important in the development of anadromy. Our results indicate that a migratory life history is not controlled by a singular gene or region but supports the idea that there are many independent ways for a migratory phenotype to emerge in a population. Therefore, conserving and promoting genetic diversity in migratory individuals is paramount to conserving these populations. Ultimately, our data add to a growing body of literature that suggests that population‐specific genetic effects, likely mediated through environmental variation, contribute to life history development in rainbow trout.Rainbow trout ( We find population‐specific genetic effects associated with life history development in two populations of rainbow trout. The figure shows a region of the genome—Chromosome 8—that seems to be especially important in migratory development in a population of trout from Alaska. This process, known as migration, is characterized by an onset of physiological and behavioral changes and is regulated by a combination of genetic factors and environmental cues , which is present in two distinct ecotypes; the rainbow (resident) and steelhead (migratory) trout. Heritability studies on O. mykiss have found strong evidence of an additive genetic component to migratory tendency  are an economically important group of fishes that are valued widely for both sportfishing and human consumption and play an important role in native subsistence fishing. One of the most abundant and well studied is the rainbow trout  parentage above two barrier waterfalls into Sashin Lake in 1926 . These data were analyzed to locate (1) regions of the genome associated with anadromy, (2) compare the locations of these regions between the two populations studied, and (3) link these polymorphisms within underlying protein‐coding genes. The results contribute to an increasing body of information dedicated to understanding the shared and population‐specific genetic control of migratory behavior in O. mykiss.Herein, we use whole‐genome pooled‐sequencing (pooled‐seq) approaches to evaluate both the shared and population‐specific genetic components of migratory and resident life histories among two geographically separated populations of 22.1Resident rainbow and anadromous steelhead trout were sampled from two populations: Sashin Creek, Alaska and Little Sheep Creek (LSC), Oregon. In the Sashin system, all residents were sampled from Sashin Lake and all sampled migrants were adults returning to spawn in Sashin Creek. In the LSC system, migrants and residents coexist, so accurate phenotype determination was critical in classifying life history type. The migratory phenotype was determined using physical traits including silvery coloration and body shape, whereas residents were determined by the expression of gametes . Pooled‐seq was used to provide a cost and time‐efficient way of sequencing many individuals with a high depth of coverage. The utility of pooled‐seq methods in nonmodel organisms has been discussed in detail in Micheletti and Narum . Pooled‐2.3PoolParty pipeline (https://github.com/StevenMicheletti/poolparty), as detailed in Micheletti and Narum  or that measured <50 base pairs in length. Quality‐filtered sequences were then aligned to the rainbow trout genome  using bwa mem with default settings , which summarizes total coverage information for each pool  values were calculated to evaluate genetic differentiation between resident and anadromous populations in both Sashin and LSC. Individual FST was calculated for each position within the genome using the FST‐slide script in Popoolation2 and the following parameters: ‐min‐count 8 ‐min‐coverage 25 ‐max‐coverage 250 ‐pool‐size 40. This value was calculated for each SNP individually to find SNPs with complete fixation (FST = 1) between migrant and resident pools. Manhattan plots were made using the program CMplot in R by plotting individual FST values at each position on the genome. Individual FST values were filtered to exclude any positions that produced FST values below 0.05 to reduce file size. Outlier loci were identified using the top 0.02% of FST values between migrant and resident pools in each population. To evaluate nucleotide diversity in each of the populations, a Tajima's D statistic was calculated over a 10,000 base pair window, with a step size of 10,000 for anadromous and resident Sashin and LSC pools using the variance program in PoPoolations . There were no fixed differences identified in the LSC collection (mean FST = 0.029 ± 0.039). The top 1% FST quantile began at 0.355 and 0.186 and we used a cut‐off value of 0.02% to identify outlier FST values. This more conservative percentile produced 33,215 outlier loci (minimum FST of 0.5) in the Sashin population compared with 8667 loci (minimum FST 0.256) in LSC. Sashin Creek contained a greater density of polymorphic sites throughout the genome, likely due to both a higher depth of sequencing coverage and reduced gene flow between the two ecotypes. Regions of high FST density in both populations were identified at Chr1:81.6 Mb, Chr2:9–10 Mb, Chr14:22–25 Mb, Chr18:8–9 Mb, and positions Chr22:44–46 Mb .Aligning the pooled sequencing to the rainbow trout genome produced an average depth per position of 30 reads for Little Sheep Creek and 115 reads for Sashin pools. A total number of quality‐filtered reads as well as average coverage depth are reported for each population—SM, SR, LSM, and LSR in Table D statistic was then calculated over adjacent 10,000 base pair windows across the genome of each of the four pools , and 8853 in the SM pool (of which eight were positive). Of these, 151 were shared in the same window in both pools and were all negative. There were additionally 21 locations where the Tajima's D value was below negative 2 in all four pools, leaving 73 regions of low Tajima's D unique to the resident groups, and 130 regions of low Tajima's D unique to the migrants.Tajima's s Figure  to deter Tajima, . This thD values in adjacent shared windows , which may indicate a larger target area for selection. Any genes in these regions were noted for their corresponding protein function and related pathways. We additionally used these data to identify trends in both resident and migrant phenotypes where large regions of significant Tajima's D values were found close together, indicating a shared selection event acting on the phenotype. For the resident groups, these large regions of selection were located near Chr2:29–30 Mb, Chr3:38–39 Mb, Chr5:46–47 Mb, Chr9:26–27 Mb, Chr12:53–55 Mb, Chr14:43–44 Mb, Chr15:36–37 Mb, and Chr17:28–30 Mb. In the migrant pools, we identified regions near Chr1:50–52 Mb, Chr2:28–30 Mb, Chr5:47–48 Mb, Chr9:26–27 Mb, Chr15:35–37 Mb, Chr17:28–30 Mb, and Chr27: 0.3–1 Mb.We also identified areas where there were significant Tajima's FST density from both Sashin and Little Sheep Creek individuals, suggesting a possible area of importance for retaining genetic diversity within both populations.The Watterson's theta estimate was calculated for each of the four populations, with a maximum theta value of 0.046 in the Sashin steelhead pool. Trends in diversity were compared between each of the populations to identify areas throughout the genome where increased polymorphic presence may contribute to a specific phenotype Figure . We did 4O. mykiss is different in Sashin Creek and Little Sheep Creek. Although genetic control is important in life history development of migratory behavior in trout from Sashin Creek  on chromosome 4. There could be a connection between SNPs within lepr and migration as leptin modulates appetite and energy use  suggesting this region of the genome may be under selection in O. mykiss more broadly. Although no SNPs were found in exon regions, these SNPs could be connected to differences in gene expression in lepr. Although previous RNA‐seq experiments using samples from Sashin Creek did not find lepr to be differentially expressed . However, this polymorphism was both synonymous and reversed in each pool, with the resident genotype in Sashin corresponding to the migrant genotype in LSC. Of the remaining 16 SNPs, six were found to have the same genotype distribution in both pools ) was found to segregate based on phenotypes in both LSC and Sashin. This gene encodes for phospholipase C involved in hydrolyzing phospholipids into fatty acids and other lipophilic molecules . In populations, where ecotypes overlap in space and time, such as LSC, phenotypic plasticity may play a larger role in life history development than in populations where ecotypes are separated  demonstrates purifying selection in both migrants and residents from Sashin Creek  and genes involved in photoperiod have been shown to be differentially expressed between life histories in Sashin Creek  and histone deacetylase 7 (hdac7) are connected to two genes (fgfrl1 and hdac11) previously shown by Hale et al.  that were not mirrored in residents  as a metabolic agent could be related to these adaptations and subsequent conversion of metabolic products. Several genes connected to ion binding and exchange were also found in regions of the genome with negative Tajima's D values . This gene is especially interesting as ion exchange is crucial during smoltification  to compensate for passive ion loss ; methodology (lead); visualization (lead); writing – original draft (lead); writing – review and editing (lead). Krista M. Nichols: Conceptualization (supporting); methodology (supporting); project administration (supporting); writing – review and editing (supporting). Frank P. Thrower: Methodology (supporting); writing – review and editing (supporting). Ewann A. Berntson: Methodology (supporting); writing – review and editing (supporting). Matthew C. Hale: Conceptualization (lead); funding acquisition (lead); methodology (supporting); project administration (lead); supervision (supporting); writing – original draft (supporting); writing – review and editing (supporting).Funding for this research was provided by two TCU grants awarded to Matthew Hale.The authors declare that they do not have any conflicts of interest.Figure S1.Click here for additional data file.


Introduction: The Tujia is the eighth most populous population in China, but its genetic structure has not been fully studied.Methods: In this study, we utilized 57 autosomal Insertion/deletion (InDel) loci to evaluate the genetic polymorphisms and efficiency of forensic applications in the Chinese Hubei Tujia group, and analyzed the genetic structure variances among the studied group and other 26 different reference populations from five continents in 1000 Genomes Project (1KG).Results: The results showed that 57 InDels have no significant deviations from Hardy–Weinberg equilibrium and linkage equilibrium. The combined power of discrimination (CPD) and the combined probability of exclusion (CPE) values for 57 InDels were 0.99999999999999999999999699822 and 0.999975177214539 in the Hubei Tujia group, respectively. In addition, the results of genetic structure analyses indicated that the Hubei Tujia group has close genetic relationships with the Chinese Han population and other East Asian populations.Discussion: These 57 autosomal InDels can be used as reliable tools for forensic individual identification and paternity testing, and are more suitable for East Asian populations. Furthermore, three InDels  are promising for inferring ancestral information. Insertion/deletion (InDel) polymorphisms refer to genetic markers with length polymorphisms formed by the insertion or deletion of DNA fragments of different sizes in the genome . The mosInDels have the advantages of both STR and single-nucleotide polymorphism (SNP) , which a® DIPplex kit  is the most widely used commercial InDel kit. However, it had a relatively low efficacy of forensic application in Chinese populations because the kit was not designed for East Asian or Chinese populations  and quantified using the NanoDrop 2000 instrument  following the recommendations of the manufacturers, and then diluted to about 1ng/ul. A total of 26 reference populations from the 1000 Genomes Project phase 3 database were selected as reference populations for population genetic analyses in this study  of 60 loci is shown in 2 bloodstain samples without extraction, and sterilized deionized water. The PCR parameters were as follows: initial denaturation at 95°C for 5 min; then, 28 cycles of denaturation at 94°C for 30s, renaturation at 60°C for 1 min, extension at 62°C for 1 min; and final extension at 72°C for 10 min; and stored at 4°C in the end. After centrifugation at 3000 rpm for 5 min, 1.0 μL amplification products or 1.0 μL InDel 60 Allelic Ladder was mixed with 12.0 μL deionized formamide and 0.5 μL AGCU Marker SIZ-500. The loading mixture was denatured at 95°C for 3 min and then chilled on ice for 3 min immediately. Finally, capillary electrophoresis detection was performed using an ABI 3500xL Genetic Analyzer , and genotyping was performed using GeneMapper® ID-X Software version 1.5 . DNA 9948 and deionized water were used as positive and negative controls for each detection.Multiplex PCR for 60 InDel loci was performed on the GeneAmp PCR System 9700 Thermal Cycler  according to the instructions on the AGCU InDel 60 kit. The multiplex PCR system was 25 μL in total which contained 10.0 μL reaction mix, 5.0 μL InDel 60 primers , 1.0 μL U-Taq enzyme, 1.0 ng DNA extracted from whole blood samples or 1mmR software ggalluvial package. The Hardy–Weinberg equilibrium (HWE) tests of the 57 InDel loci and linkage disequilibrium (LD) analyses of pairwise InDels in the Hubei Tujia group were assessed using STRAF v1.0.5 software  and fixation index (FST) among 27 populations. And phylogenetic tree was constructed based on the pairwise DA distances using the ggtree package  was used to draw the visual bar charts.The information of 60 InDels was visualized using the software ; in addi program  and Arle program  were use package . Principp-values for HWE tests at the 57 InDels in the studied Tujia group are shown in p > 0.05/57 = 0.00087719). LD tests among these 57 InDels were also evaluated in the studied Tujia group; all pairwise r2 values were higher than 0.0000313282, indicating that there were no significant LD in these pairs of 57 InDels.The As shown in The heatmap based on the insertion allele frequencies of 57 InDels for the studied Tujia group and 26 reference populations is shown in Cluster analysis showed that these 27 populations were mainly divided into two clusters, and seven AFR populations constituted one of the main clusters. As shown in Based on these 57 InDels, we compared the relevant forensic parameters  of the studied Tujia group and five continental populations. As shown in DA, and FST analyses. As shown in We evaluated the population genetic relationships of the studied Tujia group and 26 reference populations by PCA, structure, K = 2, the ancestral components of AFR populations were mainly light blue, while those of EAS populations were mainly dark blue. At K = 3, the ancestral components of AFR populations were mainly light blue, those of EAS populations were mainly green, and those of EUR populations were mainly dark blue. With the increase in the K values, the AMR and SAS populations also gradually showed different ancestral compositions compared with AFR, EUR and EAS populations. Regardless of the values of K, the studied Tujia group always showed similar ancestral compositions with the EAS populations.STRUCTURE analyses of the studied Tujia group and 26 reference populations at the individual level are shown in DA genetic distances, and FST heatmap could visualize the genetic relationships of the studied Tujia group and 26 reference populations. As shown in The phylogenetic tree on the basis of pairwise The Tujia group is one of the 56 populations in the People’s Republic of China, mainly distributed in the Wuling Mountain area at the junction of Hunan, Hubei, Chongqing and Guizhou provinces . AccordiIn this study, an InDel kit was used to analyze the genetic diversities of 57 autosomal InDels in 262 unrelated Tujia individuals living in Hubei province, China. The pairwise LD and HWE analyses of 57 InDels indicated that these loci could be used for CPD and CPE calculations in Tujia group and population genetic structure analyses. In order to evaluate the forensic application efficiencies of 57 InDels in the studied Tujia group, we calculated the forensic parameters of 57 InDels, and the results showed that all 57 InDels were polymorphic , and the® DIPplex kit for individual identification and paternity testing in East Asians. The current study also confirmed this conclusion by detecting 262 Tujia individuals using the AGCU InDel 60 kit. The CPD and CPE values of the Tujia group calculated by these 57 InDel loci were higher than those values calculated using the Investigator® DIPplex kit (0.99999999761 and 0.9860) in the previous literature (Consistent with the previous studies, the AGCU InDel 60 kit outperformed the Investigatorterature . Furtherterature , the genK, the studied Tujia group always showed similar ancestral components to EAS populations. When K = 3, the studied Tujia group and EAS, AFR, and EUR populations could be clearly distinguished based on their different ancestral components. However, the inability to distinguish AMR and SAS populations might be related to the lack of sufficient ancestral information in this panel. As shown in the heat map of the insertion allele frequencies (According to the STRUCTURE result , regardlquencies , the insNei’s DA and FST values and took advantage of PCA and STRUCTURE genetic analysis methods to evaluate the population genetic relationships between the studied Tujia group and 26 reference populations in the five continents. PCA can extract principal components from complex multivariate data and intuitively show the genetic relationships between various populations through the near and far relative positions of different populations. As shown in DA and FST values also confirmed the aforementioned viewpoint; in the EAS branch, the studied Tujia group and CHS population clustered first, then incorporated into the CHB population, and finally, the KHV, CDX, and JPT populations gathered in the branch. In addition, the FST values between the studied Tujia group and EAS populations were smaller than those of other continental populations. These results indicated that there were close genetic relationships between the studied Tujia group, the Chinese Han populations, and EAS populations. In particular, the Tujia group was closely related to the CHS population at the genetic level.Using the comparisons of insertion allele frequencies for the studied Tujia group and 26 reference populations in 1KG , we obseAs an ethnic group with a long history, the Tujia has its own national language Bifzivkar, without characters, which belongs to the Tibeto-Burman language family of the Sino-Tibetan languages and is close to the Yi language. The genetic origin of the Tujia is still inconclusive in academia . With thIn this study, 60 InDels from the AGCU InDel 60 kit were detected on 262 Hubei Tujia individuals using the capillary electrophoresis platform, and the genotyping results of 57 autosomal InDels were analyzed for revealing forensic efficiency. The high CPD and CPE values obtained in this study revealed that this AGCU InDel 60 kit can effectively compensate the deficiency of other commercial kits used in EAS populations and can be used as an effective tool for individual identification and parentage testing in the Hubei Tujia group. Moreover, the results of population genetic analyses indicated that the studied Tujia group has a closer genetic relationship with the CHS population.


Juglans sigillata Dode) is a deciduous nut tree crop endemic to southwestern China with a long history of cultivation. Due to the rapid expansion of the walnut industry, many natural populations are now being replaced by cultivars grafted onto wild rootstocks. However, little is known about the potential genetic consequences of such action on natural populations.Anthropogenic activities are causing unprecedented loss of genetic diversity in many species. However, the effects on genetic diversity from large-scale grafting onto wild plants of crop species are largely undetermined. Iron walnut (J. sigillata from Yunnan Province which is the center of walnut diversity and cultivation in China, and examined their genetic diversity and population structure using 31 microsatellite loci. Scions had lower genetic diversity than rootstocks, and this pattern was repeated in seven of the nine examined populations. Among those seven populations, AMOVA and clustering analyses showed a clear genetic separation between all rootstocks and all scions. However, the two remaining populations, both from northern Yunnan, showed genetic similarity between scions and rootstocks, possibly indicating that wild populations here are derived from feralized local cultivars. Moreover, our data indicated probable crop-to-wild gene flow between scions and rootstocks, across all populations.We sampled the scion and the rootstock from each of 149 grafted individuals within nine wild populations of J. sigillata within Yunnan. To mitigate these effects, we caution against the overuse of grafting in wild populations of iron walnut and other crop species and recommend the preservation of natural genotypes through in situ  and ex situ conservation.Our results indicate that large-scale grafting has been causing genetic diversity erosion and genetic structure breakdown in the wild material of The online version contains supplementary material available at 10.1186/s12870-023-04428-z. As a fundamental dimension of biodiversity, genetic diversity represents evolutionary potential, determining long-term maintenance of species , 2. In pFor cultivated plants, clonal propagation, including the regeneration of whole plants via cuttings or grafts, allows for phenotype uniformity, with the desired agronomic traits faithfully passed on to subsequent generations . This haGrafting is the fusion of two plant parts so that vascular function can establish between them , and theJ. regia L.) is mainly cultivated in the northwestern parts, whereas both wild and cultivated forms of iron walnut (J. sigillata Dode) are widely distributed in subtropical region of Yunnan. Iron walnuts are highly valued for their nutritious nuts and high-quality timber, as well as its religious and cultural significance )HO) and expected heterozygosity (HE) were 0.58 and 0.56, respectively. Mean effective number of alleles (NE), Shannon’s information index (I), and unbiased expected heterozygosity (uHE) among loci were 2.53, 1.06, and 0.56, respectively.A total of 298 samples Table  were sucNT)  than the scions . The number of private alleles (NP) was higher in the rootstocks (28) than among the scions (13). Similarly, other genetic diversity parameters, namely mean number of alleles (NA) (5.52/6.26), effective number of alleles (NE) (2.25/2.65), HE (0.51/0.58) and allelic richness (AR) (5.55/6.45) were lower in scions than in rootstocks. However, scions had a slightly higher observed heterozygosity (HO = 0.63) than rootstocks (0.53). At the population level (except for populations LH and YS), all genetic diversity indices other than HO were higher in rootstock than in scions. All loci were polymorphic within each dataset , but the proportion of polymorphic loci (PPL) ranged from 51.6% to 100% for populations. Seven scion populations had negative FIS values, while this was the case for only two of the rootstock populations. Finally, both the Wilcoxon and standardized difference tests independently indicated that two scion populations (YB-S and GB-S) had experienced significant bottlenecks (P < 0.05), whereas an additional four scion  and two rootstock (YS-R and CN-R) populations showed a bottleneck signal in one test but not the other , only rootstocks (R), and all samples (T). For each of the  datasets and populations, genetic diversity parameters were calculated across loci Table . A totalotstocks 8 than amFST = 0.350), then rootstocks (FST = 0.136), then all populations together (FST = 0.259) (Table FST = 0.115). Generally, we found a low to moderate pairwise genetic differentiation (FST) between all pairs of sampled populations with LH-R showing genetic distinctiveness from the rest of the populations of both rootstocks and scions , the among-population and within-population genetic variation for the whole dataset was 26% and 74% respectively Table . When thΔK had its maximum value at K = 2, making this the optimal K value  which grouped with the rootstocks. Three other scion populations, YG-S, FQ-S, and YD-S showed admixture between the two clusters. Most rootstock populations other than LH-R and GY-R showed some degree of admixture within populations, often with certain individuals belonging to the scion cluster. When K = 3, a third genetic cluster is formed containing both scions and rootstocks from populations LH and YS, plus scions of GB-S. At K = 4, another new admixed genetic cluster emerges containing mainly parts of individuals in YB-R, CN-R, YG-S and GB-S, plus some individuals of FQ-S.The STRUCTURE analysis Fig. a revealeK = 3  tree divided the 18 populations into three major groups Fig. b. Of theK = 3  and genetic structure (at K = 3)  Fig. . The res 3) Fig. .Fig. 4SpJ. sigillata provide a reservoir of genetic diversity, which is potentially under threat from the use of wild trees as rootstocks to graft cultivars for walnut industry. Therefore, the iron walnut is an ideal model to examine how grafting affects genetic diversity and population structure in fruit crops, and possibly other wild plants.Natural forests of NT) including 15 private alleles (NP) have been lost in the scions relative to the rootstocks, and all genetic diversity indices except HO are lower in scions than rootstocks  and extrinsic  factors , 4. Globks Table . The higHalwani”  and P. aP. avium . At the Citrus rootstocks in Tunisia [Olea europaea rootstocks in the Mediterranean [Prunus amygdalus in Lebanon [J. sigillata may already contain rootstocks originating from naturalized cultivars. Consistent with this, the common walnut (J. regia) exists as naturalized rather than native populations in most of Europe [J. sigillata. The genetic diversity among J. sigillata scions  Fig.  and betwJ. sigillata in the south; the two northern populations LH and YS grew near farmlands, and appeared to be semi-wild. This observation tentatively supports the hypothesis of northern rootstocks deriving from feralised cultivars (see above first point), providing a handy local grafting resource for farmers, and explaining the genetic similarity of rootstocks to scions in these populations.When interpreting unexpected genetic similarity between rootstocks and scions, as observed in the northern sites LH and YS Figs. , the posJ. sigillata is [Throughout Yunnan, we detected a relatively low genetic differentiation among rootstock populations, but a relatively high level of genetic differentiation among scion populations Figs. ; Table 3llata is , and so J. sigillata, wherein scions exhibit lower genetic diversity than rootstocks  and elsewhere. For future studies, we recommend inclusion of range-wide sampling with genomic, phenotypic and environmental data, to examine differences between rootstocks and scions and examine the impact of grafting upon adaptability to stresses such as pathogens and climate change.Detailed knowledge regarding the genetic diversity and differentiation of scions and rootstocks of a species provides important information for the conservation of genetic resources to prevent future loss of genetic diversity caused by global environmental change. Our results indicated that rootstocks had higher levels of genetic diversity than scions, suggesting that grafting onto wild trees causes genetic erosion. Meanwhile, contrasting genetic composition between scions and rootstocks, hinted that the population structure was distorted due to grafting. Therefore, conservation action is urgently needed, and we recommend both ex situ and in situ conservation of natural J. sigillata populations that include grafted individuals, distributed across the five well known walnut production areas of Yunnan Province  for data viewing and allele scoring. Null and stuttered alleles were examined by MICRO-CHECKER v2.2.3 , whereasAt the population level, scions and rootstocks at each site were treated as separate populations (e.g. CN-S and CN-R refer to the scion and rootstock populations from site CN (Goujie Xiang (耈街乡), Changning county (昌宁县)), respectively) (Tables 1 and 2). To allow for clear comparison and analysis, three datasets were compiled: dataset T comprised all populations, whereas datasets S and R comprised all scion, and all rootstock populations respectively.NA), effective number of alleles (NE), Shannon’s information index (I), observed (HO) and expected heterozygosity (HE), and unbiased expected (uHE) heterozygosity using GENALEX v6.51b2 [NT), private alleles (NP), and percentage of polymorphic loci (PPL) were also calculated in GENALEX. Rarefied allelic richness (AR) was computed using Hierfstat R package [FIS) was calculated using FSTAT v2.9 [The level of polymorphism of microsatellite loci for the current study was tested by calculating the number of alleles .FST) among populations involved a correction for null alleles using FreeNA [pheatmap R package [K) varying from 1 to 15 under the admixture model with correlated allele frequencies and without a priori grouping assumptions. To confirm the reliability of the results, we performed 10 independent runs per K value with a 10 000 burn-in period and 100 000 MCMC replications. The best K value was then determined in the web-based STRUCTURE HARVESTER v0.6.94 [https://itol.embl.de).The estimation of genetic differentiation was conducted for each of the three datasets. To minimize the impact of null alleles on this , estimatg FreeNA , with a  package . Analysi package  was used v0.6.94 . Further v0.6.94 . Finally v0.6.94  and visuAR and HE) and genetic structure (STRUCTURE result for K = 3) were subsequently mapped using ArcGIS v10.7 .To spatially link genetic diversity and genetic structure of scion and rootstock populations with geography, the genetic diversity indices : (a) Bimodal curve for scions, (b) Unimodal curve for rootstock genotypes. Fig. S2. Optimal K value of STRUCTURE selection graphs. (a) Delta (Δ)K for different numbers of subpopulations (K), maximum number of sub-populations were inferred at K=2 for STUCTURE analysis; (b) The average of log-likelihood value of K.


Phyllodactylus sentosus) is a critically endangered Peruvian species that shelters mostly in pre-Incan archeological areas called huacas, where the original environmental conditions are maintained. We used genotyping by sequencing to understand the population genomic history of P. sentosus. We found low genetic diversity (He 0.0406–0.134 and nucleotide diversity 0.0812–0.145) and deviations of the observed heterozygosity relative to the expected heterozygosity in some populations (Fis − 0.0202 to 0.0187). In all analyses, a clear population structuring was observed that cannot be explained by isolation by distance alone. Also, low levels of historical gene flow were observed between most populations, which decreased as shown in contemporary migration rate analysis. Demographic inference suggests these populations experienced bottleneck events during the last 5 ka. These results indicate that habitat modification since pre-Incan civilizations severely affected these populations, which currently face even more drastic urbanization threats. Finally, our predictions show that this species could become extinct in a decade without further intervention, which calls for urgent conservation actions being undertaken.Small species with high home fidelity, high ecological specialization or low vagility are particularly prone to suffer from habitat modification and fragmentation. The Lima leaf-toed gecko ( One of the primary consequences of these anthropogenic actions is the reduced connectivity of patches of natural habitat, which leads to decreased gene flow among isolated populations that can result in depleting their genetic diversity and increasing genetic drift5.Habitat loss, degradation and fragmentation stand out among the main anthropogenic threats to biodiversity6 and high sensitivity to human landscape modification8, has been often overlooked in studies on the effect of anthropogenic habitat alterations9. Although this is a growing field, there is currently a bias toward studies done on temperate countries and on mammals and birds9. Considering the recent assessment that 21% of reptile species are endangered10 and the current expansion of cities and human settlements, the scant studies of the effects of human landscape change on reptile population genetics, especially in the Neotropics, represent a knowledge gap that needs to be addressed in order to develop effective conservation strategies9.Reptiles are a highly diverse taxonomic group that, despite its ecological importance5. As a case in point, the Lima leaf-toed gecko (Phyllodactylus sentosus), endemic of the city of Lima (Peru) and classified as critically endangered in the IUCN Red List of Threatened Species11, is constrained to small patches mainly represented by pre-Incan archeological areas called huacas and hilltops15 , which allows the discovery of thousands of genome-wide single nucleotide polymorphisms (SNPs) without the need for a reference genome24. These techniques have also increased the opportunities to explore the demographic history of populations and to perform population viability analysis to inform conservation actions.In this study, we used GBS data to estimate genetic diversity and assess inbreeding to evaluate the current genetic status of Lima leaf-toed gecko populations. Also, we evaluated the genetic structure, historical and contemporary gene flow and demographic history to gain insights into the population genetics of the species. Finally, we performed a Population Viability Analysis (PVA) to simulate future scenarios of this species in absence of conservation actions.We generated two datasets of GBS data for a total of 50 individuals from 12 localities of the Lima leaf-toed gecko. In the first dataset (i), 147 856 SNPs were obtained using 0.85 as the clustering threshold and 33 as the minimum number of samples that must have information at a locus for it to be retained. From the second dataset (ii), which had no missing data, 10,869 SNPs were obtained.P. sentosus populations, while values for other four Phyllodactylus species analyzed ranged from 0.2933 to 0.3961  ranged between –0.0202 and 0.0187 (Table The results of Nucleotide Diversity (π) ranged between 0.0812 and 0.145. The population of Cerro Mulería has the highest value of Nucleotide Diversity (π) despite the fact that only 2 individuals were collected in this population. The deviation of the observed heterozygosity relative to the expected heterozygosity  to 1 (between San Marcos and Parque de Las Leyendas) Fig. A. The poOn the other hand, contemporary gene flow rates are low across all localities, with the proportion of migrants in most populations being lower than 0.07 (< 7%). Contemporary migration rates between localities range from 0.0149 to 0.166, the latter being the proportion of migrants from Parque de Las Leyendas present in San Marcos. Conversely, the proportion of migrants from San Marcos in Parque de Las Leyendas is 0.0604, showing a lower migration rate than the ones observed between SMP and Parque de Las Leyendas (0.071), and Muleria and San Marcos (0.0712).The demographic history estimation results for Parque de Las Leyendas, San Marcos, Mateo Salado and Huaca Pucllana revealed the same pattern in all populations, with an accentuated bottleneck and a posterior increase in population size Fig. . The effPVA using Vortex showed that all populations would experience a decrease in their population size resulting in extinction . Our analysis of He values for four other Phyllodactylus species revealed values around twice as high as those obtained for P. sentosus when considering all sampled individuals. This reduced genetic diversity could be explained by a recent bottleneck (see below) and by the fragmentation of small populations in restricted areas with limited resources28.The locations that host the largest populations of this species are San Marcos, Parque de Las Leyendas, Pucllana and Mateo Salado32. The threat of inbreeding depression and reduced genetic diversity in isolated wild populations has been well-documented, such as in wild house sparrows (Passer domesticus) (where inbreeding was associated with shorter telomeres)33, in Bengal tigers (Panthera tigris tigris) (where higher frequency of deleterious mutations were found in isolated populations)34, and even in the last woolly mammoth (Mammuthus primigenius) population35. Three out of four P. sentosus populations herein evaluated showed negative values of Fis which objectively indicate excess of heterozygotes than expected at random mating36, nevertheless this deviation can actually be due to genetic drift as these populations are small and retain low levels of genetic diversity37.Inbreeding in wildlife populations increases homozygosis, reduces genetic diversity, and can represent an important genetic threat if it derives into inbreeding depression, which decreases the overall fitness of individuals endangering the species survivalP. sentosus populations. High levels of population structuring have been previously observed in different reptile populations, including lizards and geckos of the family Phyllodactylidae39. Also, similar effects of urban fragmentation to those observed for P. sentosus have been reported in endemic geckos with restricted distribution such as the Mauritius lowland forest day gecko (Phelsuma guimbeaui) from the island of Mauritius, where its population was fragmented by urban expansion into small and isolated forest patches, resulting into populations with high genetic differentiation even in the absence of natural barriers40.The genetic structure analyses indicated high levels of population structuring, which can be due to the impact of anthropogenic habitat modifications on the huaca could disperse to another is minimal, since the populations are isolated due to the presence of buildings, parks and roads acting as barriers for a species characterized by low vagility and dispersal capability11.The historical gene flow results are largely consistent with this scenario, showing low relative migration rates between populations, particularly those at the extremes of the species distribution. This situation got established thousands of generations ago (see below), accelerating the population differentiation by genetic drift. Contemporary gene flow rates are lower than historical ones, showing that currently, the probability that an individual from a Qhapaq Ñan (the Andean road system that expanded across six Andean countries), these ancient routes have been destroyed during Lima’s urban expansion, with the connection between Parque de Las Leyendas and San Marcos being one of the last remnants until 195041.On the other hand, high levels of historical gene flow (0.99–1) were found between San Marcos and Parque de Las Leyendas populations, which is in agreement with population structure analyses where both populations clustered together. Interestingly, both localities were part of the pre-Hispanic Maranga archeological complex and were connected by unpaved areas until the 1950s, when the process of asphalting an avenue was completed and there was an increase in infrastructure in the area including the establishment of a zoo and two of the largest universities in Lima. Contemporary gene flow between these two localities is significantly lower (0.06–0.166), although higher than between most other localities, showing that connectivity between Parque de Las Leyendas and San Marcos stopped more recently. While during pre-hispanic settlement these huacas were connected by the In addition, when considering the four largest populations, the observed historical and contemporary migration values to and from Mateo Salado were lower, which is consistent with the high coefficients of genetic group membership (q) previously recorded for this locality Fig. b,c, despHomonota uruguayensis) in the Brazilian and Uruguayan pampas42, in rodent populations43 and lizards44. Another explanation to the population structuring observed here could be an isolation by distance model; however, the Mantel test results indicate that geographic distance does not explain genetic differentiation among populations. Thus, the scenario of population structuring observed among the studied populations of P. sentosus cannot be a product of geographic distance alone. Therefore, the results obtained indicate that anthropogenic habitat modifications may represent an important barrier to the dispersal of this gecko.The genetic population structuring, and low gene flow observed among populations could be explained by geographical reasons, since the species distribution is interrupted by the Rímac and Chillón rivers Fig. . HoweverP. sentosus has been modified constantly by the human occupancy during more than 5000 years. The city of Lima has been inhabited by several human cultures over time. The pre-Inca cultures developed aquifer systems that allowed them to sustain large areas of cultivated lands45. This water and soil management modified the landscape of Lima and, therefore, the habitats of local fauna and flora47. In the Late Archaic period (~ 4720 to ~ 3520 years ago), the domestication of plants and animals was already recorded in the Rímac valley, as well as the first irrigation canals built in the area47. Subsequently, in the Formative Period (~ 3520 to ~ 1920 years ago), large-scale hydraulic works and an expansion of intensive agriculture were built47  event, which can produce flooding, variations in temperature and precipitation. However, climate simulations during the Holocene in South America show that in the central coast of Peru there was no major variation in precipitation and vegetation in the last 8000 years, unlike what happened in northern and southern PeruP. sentosus conservation does not seems to be favorable. Unlike the previous PVA57, in which no initial genetic diversity value was included, we included a starting initial genetic diversity value of 0.15 based on our genetic diversity results. PVA results showed a mean extinction time of less than 10 years, in contrast with the previous one, in which the mean extinction time was longer than 33 years . The addition of an initial genetic diversity value causes the effects of this low diversity to be evident from the beginning and as the simulations progress58 these effects include inbreeding depression and the increased probability of lethal alleles occurrences59. This implies that, in a period of 10 years, if no changes are made in favor of its conservation and considering that there are no populations with more than 1000 individuals, all populations of P. sentosus would become extinct.The whole scenario for P. sentosus57 and that they are not definitive results but are a prediction that may vary as more is known about this species and actions are taken to preserve it. The drastic decline in the following years is plausible because the species could enter an extinction vortex in which both a deterioration of population and genetic characteristics occur gradually61. In addition, the rate of population decline increases the closer the population gets to extinction62. These predictions represent a red flag for a species that need urgent conservation measures.We must mention that these results are based on simulations performed by the Vortex program based on the information that is available about the natural history of P. sentosus is only found in a few huacas and hills that maintain sandy substrate with rocks11. Most of these sites are legally protected for cultural reasons63, but still face several threats in practice such as illegal land appropriation and informal garbage dumps. Furthermore, protection measures may not necessarily align with the goal of conserving wildlife inhabiting the huacas and can either not address threats relevant to the Lima leaf-toed gecko  or directly endanger them with the removal of the gecko’s retreat sites during archeological restoration.Nowadays, P. sentosus that incorporates the recovery of gene flow between populations through the translocation of individuals between localities. Management actions should be coordinated with national biodiversity authorities as with local archeological institutions, considering that most populations of P. sentosus live in archeological areas that are protected by the national government authorities. Also, our attention should be drawn to the populations in areas that do not have protection and that are at risk of disappearing. Another important future step should be producing a high-quality genome assembly, as this would provide more information about the genomic health of these populations , and about genomic signatures of local adaptation64. Finally, a management plan should be integrated with the ongoing efforts in captive breeding that have already produced protocols for artificial incubation for this species65 and future ex-situ conservation programs that include zoo-kept gecko populations to build insurance colonies for this endangered taxon66. This would benefit from implementing more monitoring, ecological and reproductive studies of P. sentosus. These studies are crucial for incorporating more precise information into population genetic analyses and population viability simulations, to gain a deeper understanding of the natural history of this species.Based on the results obtained, we recommend a management plan for the conservation of P. sentosus localities in Lima, Peru . We used sterilized surgical steel scissors to cut the tail tip up to 15 mm from the tip. In a few cases autotomized tail parts were collected. Geckos were all safely released after processing. Tissue samples were stored in 96% ethanol at − 18 °C prior to DNA extraction. We then extracted genomic DNA using an innuPREP DNA Mini Kit  and quantified the extracted DNA with NanoDrop Lite .We collected 50 tissue samples from 12 https://genomics.umn.edu/services/gbs). Here we briefly describe the protocol, which is based on the one proposed by Elshire et al.24. A total of 100 ng of DNA was digested with 10 units of the restriction enzymes PstI-HF and MspI ; and incubated at 37 °C for 2 h. DNA samples were ligated with 200 units of T4 ligase  and phase adaptors with -TGCA and CG overhangs at 22 °C for 1 h and heat killed. Ligated libraries were purified with SPRI beads and then amplified for 18 cycles with 2 × Taq Master Mix  to add the barcodes. GBS libraries were then purified, quantified using a NanoDrop™ Lite Spectrophotometer, and pooled. Fragments with the 300–744 bp size region were selected and diluted to 2 nM for sequencing on the Illumina NovaSeq 550  using 1 × 150 single-end reads.GBS libraries were generated via labor contract with University of Minnesota Genomics Center  with no missing data  and a second one (ii) with loci shared by at least 33 individuals .First, cutadapt70: (1) nucleotide diversity (π) defined as the mean number of differences per nucleotide site between two sequences from a pair of individuals randomly selected from a population72; and (2) expected heterozygosity (He). To facilitate a meaningful comparison with our results, we also calculated expected heterozygosity for four other Phyllodactylus species using data from a study conducted by Ramírez-Reyes et al. in 202073. The inbreeding coefficient (FIS) indicating the deviation of the observed heterozygosity relative to the expected heterozygosity36, was also obtained using the Stacks tool set. Fis was calculated only for the four largest populations  within the field of artificial intelligence and has been proposed as a better method for visualizing SNPs than PCA76. Also, a discriminant analysis of principal components (DAPC) was conducted using the function dapc of the R package adegenet74, and the number of principal components that were retained for the DAPC was chosen with the function xvalDapc using the cross-validation method as recommended by Jombart and Collins77. Finally, a STRUCTURE78 analysis was performed using the ipyrad packages and the admixture model with a correlated allele frequency. Also, we used two different mincov options, filtering all missing data or maintaining only SNPs with 0.9 of sample coverage. The number of genetic groups (K) was determined by the largest ΔK79.To analyze the genetic structure, a principal component analysis (PCA) was performed using the glPCA function of the R package adegenet80 format from the vcf file using unlinked_snps_to_genepop from ipyrad tools (github.com/brunoasm/ipyrad_tools). We then examined the pairwise historical migration patterns of local populations using the divMigrate method25 implemented in the R package diveRsity81. This method estimates relative directional migration based on asymmetric distributions of allele frequencies25. Results are presented in relative migration rates with values between 0 and 125. We made a modification in the code to allow the incorporation of populations that had a single individual as a sample ) and the pairwise geographic distances (log transformed) was performed using the Mantel function of the R package vegan86 with 10,000 permutations.We used the Mantel test to assess if there was a correlation between geographical and genetic distances between populations, following the isolation by distance model (IBD)27, called BA3-SNPs v. 3.0.426, which accepts large SNP datasets and estimates migration rates as proportion of migrants using a Bayesian framework and Markov chain Monte Carlo (MCMC). We used BA3-SNP-autotune v 3.0.426 to determine the optimal mixing parameters for our dataset for migration rates (deltaM = 0.9991), allele frequencies (deltaA = 0.7750), and inbreeding coefficients (deltaF = 0.0250). We ran BA3-SNPs for 100 million iterations, with a burn-in of 10 million. Chain convergence was assessed in Tracer v 1.7.187.Contemporary gene flow was estimated using a modification of BayesAss 3.04https://github.com/isaacovercast/easySFS). We then used Stairway plot v. 2.188 to estimate the historical effective population size changes of the largest gecko populations in Lima18: Huaca San Marcos, Parque de Las Leyendas, Mateo Salado and Huaca Pucllana. We did not analyze the gecko population as a whole, since high levels of population structure are considered factors that artificially modify the results56.To infer the demographic history of the Lima leaf-toed gecko we first generated a folded SFS file with easySFS . The mutation rate was assumed to be 2.4 × 1058 which performs simulations, based on given conditions, for different populations. Five populations were simulated, each one with a different initial census population size  and a carrying capacity (K) that corresponds to 125% of the initial census population size. The maximum age was 4 years old. These conditions were chosen based on previous ecological population studies18 and previous PVAs57. Simulation parameters are summarized in Supplementary Table P. sentosus57, and through in-depth consultations with experts actively engaged in the study of this species.To carry out the Population Viability Analysis (PVA) we used Vortex (v. 10.2.13.0)Field protocols for the capture, handling, release of Lima leaf-toed geckos, and tail tips sampling were approved by the competent authority: the National Forestry and Wildlife Service (SERFOR) of the Ministry of Agriculture and Irrigation of Peru (permit: SERFOR N° AUT-IFS-2018-40). All methods were performed in accordance with the relevant SERFOR guidelines and regulations, as well as in compliance with the IUCN Policy Statement on Research Involving Species at Risk of Extinction.Supplementary Information.


We found that this area likely hosts a caribou metapopulation historically characterized by high levels of gene flow, which indicates that multiple sources of founders would be appropriate for initiating a breeding program. However, population structure and adaptive divergence analyses indicate that JNP caribou are closest to populations in the BC Columbia range, which also have suitable genetic diversity for conservation breeding. We suggest that collaboration among jurisdictions would be beneficial to implement the program to promote recovery of JNP caribou and possibly other caribou populations in the surrounding area, which is strategically at the periphery of the distribution of this endangered species.Conservation breeding programs are increasingly used as recovery actions for wild animals; bringing founders into captivity to rear captive populations for future reintroduction into the wild. The International Union for the Conservation of Nature recommends that founders should come from genetically close populations and should have sufficient genetic diversity to avoid mating among relatives. Genomic data are highly informative for evaluating founders due to their high resolution and ability to capture adaptive divergence, yet, their application in that context remains limited. Woodland caribou are federally listed as a Species at Risk in Canada, with several populations facing extirpation, such as those in the Rocky Mountains of Alberta and British Columbia (BC). To prevent local extirpation, Jasper National Park (JNP) is proposing a conservation breeding program. We examined single nucleotide polymorphisms for 144 caribou from 11 populations encompassing a 200,000The online version contains supplementary material available at 10.1007/s10592-023-01540-3. Bison bonasus) in Poland  in Oklahoma (Kleiman Oryx leucoryx) in Arabia  in China  such as microsatellites and/or mitochondrial DNA (mtDNA) (Russello and Jensen Rangifer tarandus), also known as reindeer, are a Holarctic species that is globally declining, and listed as a Species at Risk in Canada   and 96-well plates (QIAamp 96 DNA QIAcube HT kit). DNA samples were then quantified using either the BioTek Synergy LX Multimode Reader or the Thermo Fisher Qubit 3 Fluorometer following Thermo Fisher with Qubit and Quant-iT 1X dsDNA high sensitivity (HS) or broad range (BR) Kits. One hundred and forty-four samples that yielded ≥ 400 ng of DNA were selected for analyses. Samples were normalized to a quantity of 400 ng, dried on a Thermo Scientific Savant SpeedVac DNA 130 Integrated Vacuum Concentrator System, and shipped at room temperature to Genome Québec Ltd  where genotyping using the Illumina Caribou 60 K SNP array  and Southern Mountain DU   Fig. . In addi60) Fig. .FIS, with 95% Confidence Intervals (CI) of bootstrap values) for each herd as well as pairwise fixation index values (FST) between herds. We then used FST values to derive the number of migrants per generation  also to sample sizes. Due to low sample size, we therefore combined individuals from the Banff herd (n = 3) with those from JNP (n = 15). We also combined individuals from the Purcell South herd (n = 4) with those from South Selkirk herd (n = 3). The pooling of these samples was warranted due to known genetic similarities , we performed a Mantel test to detect the potential correlation between the geographic and genetic distances calculated between individuals pairwise. We calculated the geographic distances with the function “distGeo” within the R package GEOSPHERE Karney , whereas, which uses a Bayesian iterative algorithm to place samples into clusters (K) whose members share similar patterns of genetic variation  to a model in which selection explains the difference in allele frequencies better than a null model. A positive alpha indicates population group-specific directional selection, while a negative alpha suggests balancing or purifying selection. We ran analyses using a prior odd of 10, where SNPs were considered to be under selection when below the false discovery rate (FDR) threshold of 0.05.We identified putatively adaptatively divergent SNPs between population groups determined with population structure analyses (see results) with BAYESCAN v.2.1 (Foll and Gaggiotti FIS) ranged from 0.003 to 0.047 (mean 0.019 + SD 0.015). Pairwise fixation index (FST) between herds ranged from 0.01 to 0.09 (mean 0.06 + SD 0.02). FST values between JNP and the other herds varied from 0.06 to 0.09  of caribou herds in the study area ranged from 0.33 to 0.39 . JNP and Banff individuals grouped together in the neighbor-joining tree and were most similar to caribou from Columbia North, Purcell South, and South Selkirk. JNP and Banff individuals then grouped together with caribou from À La Pêche, and then caribou from Redrock-Prairie Creek  . We also detected 68 SNPs diverging between JNP and CR, with FST values ranging between 0.15 and 0.24 (average 0.19). Lastly, we found only 53 outlier SNPs between JNP and COL with FST values ranging between 0.21 and 0.29 . We identified 89 outlier SNPs divergent between JNP and ALP, with 2 study area we found that, despite wide-spread recent declines , which provide information on relatedness among individuals (Crow and Kimura The levels of heterozygosity we identified (including He and Ho) were aligned with other reported values for caribou and approaching levels considered as “high” in wild mammals  were all greater than one, indicating significant historical and/or recent gene flow among all herd pairs. One migrant per generation, as a minimum, is typically considered sufficient to offset genetic deterioration within subunits Wright . It is tPopulation structure results indicate that caribou individuals could be grouped in two major genetic clusters, which is consistent with both past and current studies conducted in western Canada Below is the link to the electronic supplementary material.


Aedes albopictus, is an invasive species that spreads disease and has recently become prevalent in its habitat due to human interference. In this study, we analyzed the genetic diversity and population structure of Ae. albopictus on the basis of two mitochondrial genes  and sixteen polymorphic microsatellites sourced from localities in Republic of Korea (hereafter Korea), Japan, and Laos. Mitochondrial analysis showed that the genetic diversity of the Korean and Japanese populations was low, while the Laos population did not share haplotypes with the former two groups, indicating that the Korean Ae. albopictus were not derived from the Laos population. On the basis of microsatellite results, the Korean, Japanese, and Laos populations showed higher diversity, although there were mixed groups, which implies that the Korean cluster is distinct. Continuous international trade and overseas travel require constant monitoring of species that can transmit mosquito-borne diseases. In addition, genetic studies would provide a useful basis for understanding the genetic status of vectors and for managing vector-borne diseases.The Asian tiger mosquito, Aedes albopictus is native to Southeast Asia and has emerged as a major vector for vector-borne diseases that are spreading rapidly worldwide. Recent studies have shown that Ae. albopictus populations have different genetic groups dependent on their thermal adaptations; however, studies on Korean populations are limited. In this study, we analyzed the genetic diversity and structure of two mitochondrial genes (COI and ND5) and sixteen microsatellites in mosquitoes inhabiting Korea, Japan, and Laos. The results indicate that the Korean population has low genetic diversity, with an independent cluster distinct from the Laos population. Mixed clusters have also been observed in the Korean population. On the basis of these findings, two hypotheses are proposed. First, certain Korean populations are native. Second, some subpopulations that descended from the metapopulation (East Asian countries) were introduced to Japan before migrating to Korea. Furthermore, we previously demonstrated that Ae. albopictus appears to have been imported to Korea. In conclusion, the dengue-virus-carrying mosquitoes could migrate to Korea from Southeast Asian epidemic regions, where they can survive during the severe winter months. The key findings can be used to establish an integrated pest management strategy based on population genetics for the Korean Ae. albopictus population. Invasive insect pests have recently increased in prevalence owing to climate change and increased trade, resulting in a global phenomenon . DespiteAedes, Anopheles, and Culex are the 3 genera that mostly serve as carrier of mosquito-borne diseases [Aedes genus are major carriers of arboviruses, such as dengue fever, yellow fever, chikungunya, and zika virus, and such diseases have recently emerged in Southeast Asian countries [Aedes-mediated diseases are hazardous since various virus types occur, and there are cases where there are no vaccines or therapeutic agents available [Ae. aegypti, which originates from Africa, has spread rapidly in places with a climate similar to that of Africa, Europe, and Southeast Asia, including Laos [Ae. aegypti has been studied extensively as one of the most notorious exotic species in the Aedes genus [Mosquito-borne viruses, including dengue fever, are mostly arthropod-borne viruses transmitted to animals and humans by blood-sucking invertebrates . There adiseases ,10. Mosqountries ,11. Aedevailable ,11. Mosqvailable ,12. Sevevailable ,13. For ing Laos  and Japaing Laos , causinging Laos . Furtheres genus . Additioes genus ,19,20,21Ae. albopictus [Ae. albopictus was first conducted on individuals found in Jeju Island [Malaria and Japanese encephalitis frequently occur in Korea. Studies in Korea have focused primarily on mosquitoes that transmit pathogens and cause diseases, whereas there is a general lack of investigations on bopictus ,20,22,23Ae. albopictus in Korea is expanding, owing to recent climate change [Ae. albopictus [The abundance of e change . Indigene change ,26,27. He change . As a rebopictus .COI and ND5 as reliable markers to identify whether a species has been imported or to assess genetic variation in a population [COI and ND5 sequences, we previously demonstrated the genetic diversity of two mosquito species, Ae. albopictus and Ae. flavopictus. Ae. flavopictus is native to the Korean Peninsula, whereas Ae. albopictus seems to be an introduced species [Genetic markers can be used to identify the origins of locations and paths of invading species from one country to another ,29. Molepulation . On the  species .Ae. albopictus is one of the notorious exotic species that has recently expanded its distribution areas . Ae. albopictus has expanded its distribution areas through human activities in Southeast Asia, which is its place of origin, and is currently found in all parts of the world except Antarctica [Ae. albopictus [tarctica ,36,37. Ttarctica ,39. So fbopictus . Ae. albopictus in Korea using sixteen microsatellite markers developed in addition to the two mt markers (COI and ND5) from our recently published study [Ae. albopictus.In this study, we aimed to elucidate the genetic status of ed study . Such reAe. albopictus has been studied in the Korean population [Samples of 211 mosquitoes from 21 populations were captured in Korea, Japan, and Laos between 2017 and 2020 , Table 1pulation . Adult mCOI and ND5) were amplified using PCR using two primer pairs: albCOIF (5′-TTTCAACAAATCATAAAGATATTGG-3′) and albCOIR (5′-TAA ACTTCTGGATGACCAAAAAATCA-3′) for COI gene [ND5 gene [COI, PCR amplifications were performed with a 25 μL reaction volume containing 0.5 μL of isolated DNA, 2.5 μL of 10 × Taq buffer, 2.0 μL of MgCl2 (25 mM), 0.7 μL of dNTP solution (2.5 mM each), 0.5 μL of each primer, and 0.3 μL of Taq DNA polymerase . The PCR cycling conditions were as follows: an initial denaturation step at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 45 °C for 30 s, and elongation at 72 °C for 45 s, and a final extension at 72 °C for 7 min. For ND5, PCR mixture was the same as COI. The amplification conditions were as follows: initial denaturation at 98 °C for 5 min, followed by 10 cycles at 95 °C for 1 min, 45 °C for 1 min, 72 °C for 1 min 30 s, 30 cycles at 95 °C for 1 min, 46 °C for 1 min, 72 °C for 1 min 30 s, and a final extension at 72 °C for 3 min. PCR products were separated using 2% agarose gel electrophoresis  and sequenced by Cosmo Genetech  using an ABI 3730xl DNA Analyzer .The two regions of the mt gene , elongation step at 72 °C for 30 s, and a final extension step at 72 °C for 20 min. Genotyping was performed using an ABI 3730xl DNA Analyzer , and the data were analyzed using GeneMapper software v.5 .COI and ND5) were aligned using Clustal W [https://www.geneious.com accessed on 29 June 2021; Biomatters, New Zealand). Concatenated sequences were derived from two mt genes using gene flow and genetic diversity analyses. The number of haplotypes, number of segregating sites (S), haplotype diversity (Hd), nucleotide diversity (π), and average number of nucleotide differences (k) were analyzed for genetic diversity using DnaSP.6.12 [FST) were calculated to investigate genetic differentiation among populations using Arlequin v.3.5 [FST values. To determine the population structure, analyses of molecular variance (AMOVA) were conducted using Arlequin v.3.5 [The sequences of two mt genes , allelic range (AR), observed heterozygosity (Ho), expected heterozygosity (HE), and Hardy–Weinberg equilibrium (HWE) using Arlequin v.3.5 [P-values were adjusted using Bonferroni correction in R [https://genepop.curtin.edu.au accessed on 29 June 2021) was used to assess for linkage disequilibrium (LD) between pairs of loci using probability tests on 1000 dememorizations with 100 batches (1000 iterations per batch). Arlequin v.3.5 [FST and RST with 1000 permutations, and the AMOVA was employed with locus-by-locus and 1000 permutations to determine the genetic differentiation among populations. FSTAT 2.9. [IS) and allelic richness (r) in each population. GenAlEx was used to generate visual data to facilitate an easier comparison of the mean allelic patterns of the entire population [The genetic diversity of the 16 loci was characterized by estimating the mean number of alleles  was 1.426 overall. The Vientiane, Laos (VL), population had the most significant number of haplotypes, with 11 haplotypes from 28 individuals, and 9 individuals had a single haplotype. The Yangsan (YS) population had the highest haplotype diversity (0.758), followed by VL population (0.706)  .FST) of the tested specimens showed that Geoje, Korea (GJ), population showed the highest level, followed by VL and TJ  populations that distinguished themselves from the other populations  using Arlequin software. The AMOVA was used to compare genetic variations within and between the populations. In this study, the AMOVA was performed by grouping each country . The highest genetic variation (56.02%) was observed among groups. Within populations, however, a relatively low variation (27.82%) was observed. The least variation (16.15%) was found among populations within groups . The AMOD and Fu’s) were employed to analyze the signatures of historical demographic events. The goal of the Tajima’s test is to identify sequences that do not fit the neutral theory model when mutation and genetic drift are in equilibrium. Fu’s Fs is built upon the Ewens’ sampling distribution based on haplotype (gene) frequency distribution, which is different from the Tajima’s D. Tajima’s D and Fu’s Fs neutrality tests showed that the VL population had a statistically significant negative value , indicating that the VL population has an excess of alleles (D (p > 0.05) and Fu’s Fs (p > 0.05) in most geographic groups. All populations under study had unimodal mismatch distributions, which supported the standard neutral model of a panmictic population of constant size   and Japan (TJ) populations formed lone haplotypes in their haplotype networks . Japanes clades) . This re network .CO1 and 423 bp ND5), 131 out of the 211 entities, which corresponded to 19 populations, were selected and analyzed using 16 microsatellites , population had the highest Ho (0.545), while JD population had the lowest oH (0.117). In most populations, Ho was lower than HE, indicating an excess of homozygote genotypes at most loci. In addition, most populations deviated considerably from HWE after Bonferroni correction. The inbreeding coefficient (FIS) ranged from 0.385 to 0.828, whereby a high FIS value indicates a high level of inbreeding in the population, while a low FIS value indicates a low level of inbreeding. The allelic richness (r) ranged from 1.846  to 2.471 . Allelic richness (r) of microsatellites was high in indigenous Korean populations; however, there was no significant difference between the Laos and Korean populations. The linkage disequilibrium test assessed the independence of the 16 loci, which showed no significant results in all samples.Based on the partial mt sequence analysis  result supported the presence of two clusters for the nineteen populations, suggesting that the population can be divided into two clusters/subpopulations. However, the two clusters did not differ significantly from one another. At the highest value (K = 4), eight Korean populations  had distinct genetic groups (orange color), whereas the other regions of Korea  shared genetic clusters with TJ (Japan) and VL (Laos).STRUCTURE analysis can reveal historical population structures and a mix of populations in the past by reconstructing the ancestry of individuals in the population and determining the contributions of various ancestral populations . Two metK values . The EvaFST and RST to assess the genetic distance between different populations were 0.117~0.438 and −0.028~0.581, respectively, which were generally lower for the mt data. The pairwise FST and RST values were slightly different; however, the GJ population of Korea and the VL population had a greater distance compared with the other groups (FST was conducted to determine the cause of the genetic variations observed within the studied populations (Pairwise r groups . An AMOVulations . The varThe Wilcoxon test (two-tailed Wilcoxon signed rank test for heterozygosity excess) and Garza–Williamson index (M-ratio) test were conducted to assess the recent bottleneck phenomenon, which showed that most populations suffered from a genetic bottleneck in the past . The GarClimate change has created suitable climatic conditions, especially in terms of temperature changes, for many insects to adapt to new habitats . Insect Ae. albopictus. The COI and ND5 sequences were used to identify the three clades in COI sequences with NCBI obtained  analyses. However, this genetic structure was not notably different from that of other populations in Laos and Japan  or AR   . This fiAe. albopictus population in Korea. However, mixed clusters were also observed, which is consistent with the previous studies showing that Japan and China were considered the origin of Ae. albopictus [Ae. albopictus [Ae. albopictus [Ae. aegypti study also showed mixed results, suggesting that the Geoje population in Korea, in particular, may have been invaded by multiple introductions [Ae. albopictus populations have shown that admixture is confirmed across Asian countries [Second, genetic structure was observed in the Korean population, which showed that the Korean populations were concentrated on one focal haplotype . Howeverbopictus ,82,87. Obopictus ,80. Korebopictus ,39,64,87bopictus ,90. A prductions . Other sountries ,91.Ae. albopictus mosquito is further supported by genetic diversity, variations in haplotypes and shared haplotypes with Japan  of the Korean peninsula) th Japan , networkAnopheles and Culex genera, such as malaria and Japanese encephalitis [Aedes genus [Ae. albopictus [Ae. albopictus inhabiting Korea can transmit the zika virus under laboratory conditions [Ae. albopictus has sufficient capacity to transmit and establish these diseases, a basic vector control strategy is needed [Every year, Korea suffers from persistent outbreaks of mosquito-borne diseases mediated by the phalitis ,22,92. Tes genus ,34,93,94es genus ,95,96. Ibopictus ,26. A prnditions . To dates needed ,26.Ae. albopictus were analyzed mainly in Korea, including samples from two neighboring countries. The results confirmed that the Korean Ae. albopictus population had low genetic diversity but had distinct clusters from overseas populations and mixed clusters. It was observed that there was the possibility of introduced populations due to globalization and increased international trade and travel. Increased temperature due to climate change, domestic patients, and increased population density present a risk of mosquito-borne diseases in Korea. In addition, the increasing temperature and humidity in Korea make it a suitable environment to which mosquitoes can adapt, making it more likely that mosquito-borne diseases will spread. The results of this preliminary investigation of the potential vector may be used to better understand the geographic range of Ae. albopictus species, population size, and monitoring, which may have an impact on human arbovirus transmission and may aid in the development of a national-level policy control strategy.Genetic diversity and population structure of


Spodoptera frugiperda or commonly known as fall armyworm (FAW) poses serious menace to the global agricultural production. Owing to the revamped invasion of FAW in 2018 in India, present study was undertaken for precise assessment of its genetic identity and pesticide resistance to aid in pest-management strategies.The invasive and calamitous polyphagous pest COI sequences were used which revealed a low nucleotide diversity. Analysis of molecular variance indicated significant genetic variation between four global geographical FAW populations with lowest differentiation between India and Africa suggesting a present-day and shared origin of FAW. The study demonstrated existence of two different strains (‘R’ strain and ‘C’ strain) based on COI gene marker. However, discrepancies between COI marker and host plant association of FAW was observed. Characterization of Tpi gene revealed abundance of TpiCa1a followed by TpiCa2b and TpiR1a strains respectively. The FAW population showed higher susceptibility towards chlorantraniliprole and spinetoram than cypermethrin. Insecticide resistance genes depicted marked upregulation although with lot of variance. Chlorantraniliprole resistance ratio (RR) exhibited significant correlation with 1950 , 9131  and 9360 (CYP) genes, while spinetoram and cypermethrin RR was found to correlate with 1950 (GST) and 9360 (CYP) genes.To evaluate the diversity in FAW population across Eastern India, mitochondrial S. frugiperda.This study manifests Indian subcontinent as the potential new hotspot for the growth and distribution of FAW population that can be effectively controlled using chlorantraniliprole and spinetoram. This study also adds novel significant information on FAW population across Eastern India for developing a comprehensive pest management approach for The online version contains supplementary material available at 10.1186/s12896-023-00786-6. Spodoptera frugiperda, J.E Smith, Lepidoptera: Noctuidae) is a polyphagous pest natively growing in tropical and sub-tropical America by feeding on 353 plants from 76 plant families  and were undertaken for comparative sequence analysis and inter-population variation. The details of these sequences are provided in Table SIn the present study, 42 FAW specimens collected from eight different provinces across Eastern India , i.e., TpiCa1a (n = 23), TpiCa2b (n = 16), and TpiRa1a (n = 3)  were C indicating the dominance of corn strain in this region  belonged to COI-RS and two (H4 and H5) belonged to COI-CS   were identified based on the For detailed understanding of the haplotype diversity of FAW, 460 bp of COIA barcode region for 42 specimens collected from Eastern India was analysed. 14 polymorphic sites (15 mutations) and a nucleotide diversity of 0.0075 were observed as shown in Table SStudying Indian FAW genetic variation in contrast to other global FAW populations is an intriguing aspect of the current study. This will help uncover the demography of FAW and the relationships among subpopulations. The global FAW population was split into four different groups wherein population-level studies were feasible. These groups included 1. America (containing population from North and South America); 2. Africa, 3. India (considering the population under study) and 4. Asia-II . Sequences obtained from these particular groups were used for polymorphism study  of haplotypes. On the other hand, Indian and Asia-II subgroups had only 5 haplotypes, and African sub group consisted of 4 haplotypes. The number of polymorphic sites was higher in American population (46 polymorphic sites and 8 singleton variable sites) followed by Indian (14 polymorphic sites and 8 singleton variable sites) and Asia-II population (12 polymorphic sites and 8 singleton variable sites). As shown in Table SMismatch distributions analysis conducted to understand further demographic processes revealed distributions were distinctly unimodal for the four groups under study Fig. . This isST value indicate genetic homogeneity and lack of genetic structuring between the population of India and Africa.The present study also focused on the population structure of four geographical groups using AMOVA. Primarily the four geographical groups were compared and the same analysis was individually performed between India and America; India and Africa; India and Asia-II populations Table S. The res., chlorantraniliprole, spinetoram and cypermethrin  level varies between 17.60–89.05 mg/L for chlorantraniliprole  was found to be the most toxic followed by spinetoram (23.87 mg/L) and cypermethrin (39.98 mg/L). The populations belonging to Assam, Bihar, Meghalaya and Odisha followed a similar toxicity trend for these insecticides. However, the FAW population collected from West Bengal showed higher toxicity towards spinetoram (LC50 = 53.45 mg/L) followed by chlorantraniliprole (89.05 mg/L) and cypermethrin (105.75 mg/L).The susceptibility of different populations of FAW was evaluated against three insecticides vizrin Fig. . The resrin Fig. 0 level vrin Fig. 0 level vrin Fig. 0 level vThe insecticide susceptibility comparison between the different populations indicated that the resistance factor  varies between 1.97–5.06, 1.95–5.50 and 1.93–3.74 for chlorantraniliprole, spinetoram and cypermethrin, respectively (Table SExpression of six EST genes (four GST genes and two P450 genes) was compared in 5 FAW populations against a susceptible reference population . As compared to the susceptible population, all the experimental FAW populations displayed upregulation of all the six genes, although at varying degrees. In case of 1950 (GST), Assam, Odisha and Bihar populations showed maximum upregulation 11–16-fold) followed by Meghalaya and West Bengal populations depicting ninefold and fourfold upregulation respectively , 0.64 (p = 0.05) and 0.80 (p = 0.01) respectively. Similarly, both the resistant ratio of spinetoram and cypermethrin exhibited a positive correlation with the transcript level of 1950 (GST) and 9360 (CYP). The correlation coefficient was 0.71 (p = 0.03) and 0.76 (p = 0.02) for spinetoram while, 0.65 (p = 0.05) and 0.71 (p = 0.03) for cypermethrin respectively  values of FAW field population and relative expression of detoxifying genes is presented in Fig. Tpi and COI genes. This study also observed a disagreement between Tpi and CO1 gene markers for the estimation of host association. Varying proportions of different genetic groups were seen using markers (Tpi and CO1 gene) individually. However, this observation aligns well with the reports from other regions of the world  [On one hand, amplification of a partial sequence (444 bp) of the Tpi and COI genes [Tpi gene was termed as “g” (genomic), whereas the COI gene was labelled by “mt” . In order to classify the C and R strains of FAW by marking the polymorphic nucleotides in Tpi and COI gene, the sequences of the collected FAW specimens were aligned with the earlier reported NCBI sequences of C-strain and R-strain FAW in CLUSTAL W. The alignments of the COI gene  fragment having single nucleotide polymorphism (SNP) often nucleotides  were strain-specific and used to indicate the difference between ‘R’ and ‘C’ sister strains of FAW in the Western Hemisphere [Tpi-E4) and intron region (Tpi-I4) of the Tpi gene. While, the gTpi183  helped to identify C- and R-strains [Tpi-I4 aided in the understanding of genetic variation of FAW population.Diagnostic markers to differentiate between strains were based on single nucleotide substitution of OI genes . The TpiCOIA followed the expectations of neutrality by DnaSP. Mismatch distribution graphs for each geographic groups were created through DnaSP Ver. 5.10.01 [ST) and analysis of molecular variance (AMOVA) were calculated by Arlequin v.3.5 [Tpi and COI genes were submitted to the GenBank (http://www.ncbi.nlm.nih.gov) with accession no. as detailed in Table SCOI gene sequences were studied via a phylogenetic tree construction using MEGA X software [The demographic history of each geographical group of FAW was analysed by different neutrality statistics. Tajima’s D  and Fu’s 5.10.01  to inferin v.3.5 . The seqsoftware , verifieIn order to evaluate the insecticide susceptibility and expression status of resistant genes, the field FAW populations from five most significant provinces  were chosen, wherein, FAW were identified on fields based on the larval morphological features . The acc2) were dipped in the respective insecticide dilutions for 30 ± 2 s and air-dried. The control leaves were dipped in water. After complete drying of the solution on the leaf surface, the treated leaves were then shifted to 2% agar layer. Five replicates of each treatment were used and six larvae were released per replication (thirty in each treatment) in the experiment being performed under laboratory conditions . Following every 24 h, the treated leaves were changed with freshly treated leaves. The mortality counts (moribund larvae were considered as dead) were noted at 96 h after feeding (HAF).Insecticides viz., chlorantraniliprole 18.5% SC (Coragen) obtained from Du Pant Co., Ltd., cypermethrin 10% EC (Super killer) from Dhanuka Chemical Ltd. and spinetoram 11.7 SC (Delegate) from Dow Agro Sciences served as the test agent in bioassay studies of the FAW larval population. A modified leaf dip bioassay method of Insecticide Resistance Action Committee was followed . For toxnd instar) population were used for total RNA extraction using Insect RNA Isolation Kit (Thermo Fisher Scientific) following the manufacturer's protocol, followed by amount estimation using Qubit Flex Fluorometer. 1 μg of total RNA was converted to cDNA according to manufacturer’s instruction .The field-collected FAW , 9360 (CYP), 1950 (GST), 3423 (GST), 801 (GST), 968 (GST) using SYBR Green Master Mix  in Agilent Technologies Stratagene (Model- Mx3000P). Primer details including name and sequences along with annealing temperatures have been enlisted in Table St method  has been50 of the test population/LC50 of the susceptible population [p < 0.01, **p < 0.001, and ***p < 0.0001. The functional relationship between the resistance level of the population and the mean normalized expression value of each gene in different populations was determined by Linear Model II regression analysis in SPSS .The logged mortality counts corrected using Abbott’s formula  were usepulation , and thepulation . The qRTAdditional file 1: Table S1. Collecting information of Spodoptera frugiperda from different prefectures in 2020. Table S2. COIA gene sequences across different geographical regionused in the present study. Table S3. Summary of genetic diversity of FAW populations analysed on the basis of partial mt-COIA gene from four different geographical location i.e., India, America, Africa and Asia-II.. Table S4. Comparison between genetic diversity of FAW sister strains in India.. Table S5. Result of AMOVA analysis among the different FAW geographical groups. Table S6. Susceptibility of Spodoptera frugiperda field populations to three insecticides. Table S7. Primers used in the current study. The primer name, PCR type, primer sequences and the annealing temperature are listed in the table below.


Saussurea involucrata, a perennial alpine species mainly distributed in the Tianshan Mountains, is famous for its medicinal value but has become endangered due to over-exploitation. In the present study, we employed both nuclear and chloroplast DNA sequences to investigate the genetic distribution pattern and evolutionary history of S. involucrata. A total of 270 individuals covering nine S. involucrata populations were sampled for the amplification and sequencing of nrDNA Internal Transcribed Spacer (ITS) and chloroplast trnL-trnF, matK and ndhF-rpl32 sequences. Via calculation, we identified 7 nuclear and 12 plastid haplotypes. Among the nine populations, GL and BA were characterized by high haplotype diversity, whereas BG revealed the lowest haplotype diversity. Molecular dating estimations suggest that divergence among S. involucrata populations occurred around 0.75 Ma, coinciding with the uplift of Tianshan Mountains. Our results reveal that both isolation-by-distance (IBD) and isolation-by-resistance (IBR) have promoted genetic differentiation among populations of S. involucrata. The results from the ecological niche modeling analyses show a more suitable habitat for S. involucrata in the past than at present, indicating a historical distribution contraction of the species. This study provides new insight into understanding the genetic differentiation of S. involucrata, as well as the theoretical basis for conserving this species.Rare and vulnerable endemic plants represent different evolutionary units that occur at different times, and protecting these species is a key issue in biological protection. Understanding the impact of the history of endangered plant populations on their genetic diversity helps to reveal evolutionary history and is crucial for guiding conservation efforts.  Saussurea involucrata is only distributed in the Tianshan Mountains and Altai Mountains in China. The study of conservation genetics can provide a reference for its protection research.The Tianshan Mountains and the Altai Mountains, located in the arid periphery of the Junggar Basin in northwestern China, are part of the Central Asian and High Asian Mountain systems ,2. The QSaussurea involucrata is an endemic alpine plant, just distributed in the Tianshan Mountains and Altai Mountains with an altitude of about 2400–4100 m. It has been listed as a national secondary protected plant [http://www.Iplant.cn/bhzw/info/1102 (accessed on 7 September 2021)). As a traditional medicinal plant, S. involucrata has been used for dispelling wind to eliminate dampness, eliminating inflammation, promoting blood circulation and relieving pain [S. involucrata in anti-cancer has been gradually discovered and deeply explored by researchers [Saussurea involucrata has suffered from excessive excavation and habitat destruction for a long time and has not been effectively and reasonably protected, resulting in a sharp decline in its resources [S. involucrata distributed in the western Tianshan Mountains, we should protect them in situ and give priority to the protection of S. involucrata in the Bayinbuluke area [ed plant ,19  and isolation-by-resistance (IBR). Our analyses reveal the differentiation process of S. involucrata across and within geographic regions. This study provides new insights into the differentiation of S. involucrata and the theoretical basis for the establishment of effective protection strategies for this species. In order to further analyze the distribution pattern and population history of S. involucrata were sampled. A total of 270 samples were collected from each population in the Tianshan Mountains and Altai Mountains. The longitude, latitude and altitude were recorded during collection. Fresh leaves were collected, immediately dried on silica gel and stored at room temperature for DNA extraction. DNA was extracted from silica gel dried leaves via the CTAB method [Nine populations of B method .trnS-trnG, atpB-rbcL, trnQ-rps16, rpl32-trnL, psbK-psbI, psbA-trnH, rps16, rps12-rpl20 and ycf6-psbM) were screened for genetic variations [trnL-trnF, matK and ndhF-rpl32 were found to have effective levels of variation. For the amplification of nuclear regions, we selected the gene ITS1–4. ITS1–4 was successfully amplified and exhibited sufficient variation in preliminary experiments. PCR amplification and sequencing of both strands were performed using previously published methods for these regions. PCR amplification and sequencing of the ITS region were implemented with ITS1-4 primers. All PCR products were stained with SYBR on 1.5% agarose gel electrophoresis to check the amplification. PCR amplification and sequencing of cpDNA were performed using three primers: trnL-trnF, matK and ndhF-rpl32. In addition to the annealing temperature of 55 ℃ and the total volume of 50 μL, the PCR protocol was the same as the ITS amplification protocol.Initially, ten plastid DNA segments  and nucleotide diversity (π) . HaplotycpDNA  and isolation resistance (IBR). The normalized pairwise genetic distance FST/(1-FST) between populations was calculated using ‘hierfstat’. The geographical distance was calculated by using the R package ‘land boundary’  accordinS. involucrata [DnaSP v 5 was used to construct the mismatch distribution curve of olucrata  to test olucrata .S. involucrata populations. The chloroplast haplotype H1 was the most widely distributed haplotype, which was found in seven S. involucrata populations  population had the highest Hd value  of cpDNA was 1.26, ranging from 1.24 to 1.28. The haplotype diversity (Hd) in the ITS1-4 region was 0.813, ranging from 0.804 to 0.822, and π was 6.13, ranging from 5.98–6.28, which was much higher than that of the chloroplast genes.A total of 12 chloroplast haplotypes  and 7 nuulations . Each haulations . Each ofaplotype a and nucaplotype b. AccordHd value a, and thHd value b. Based Hd value a, and thHd value b. The toS. involucrata has not experienced rapid expansion. The neutral test results show that the population of S. involucrata had a history of a stable population size and had not experienced population expansion and a continuous growth pattern  and nrDNA (ITS1-4) of S. involucrata, our results show that the nine natural populations of S. involucrata had high haplotype diversity  [Saussurea medusa (He = 0.2757) [Based on the iversity a and nuciversity b at the ion size . In genelination . Saussur= 0.275)  and Saus 0.2757) . DiffereS. involucrata were collected from the Tianshan Mountains and Altai Mountains. The genetic diversity of S. involucrata in the western Tianshan Mountains was higher and had special haplotypes. Specifically, the S. involucrata population in the Bayinbuluke area in the western Tianshan Mountains had higher genetic diversity and had special haplotypes   . Therefotectorum . Based otectorum . Due to tectorum . In thiss higher  and had s higher . TherefocpDNA and nrDNA mismatch distribution curves of S. involucrata were multi-peak , and another lineage was mainly distributed in the western Tianshan Mountains   a. The reS. involucrata began in the Middle Pleistocene. The estimated differentiation time of its lineage is later than the maximum glacial period. The extremely low temperature during the glacial period may have caused obstacles to gene flow between geographically isolated S. involucrata populations, and gene flow was cut off or restricted. It promoted the generation of regional special haplotypes [S. involucrata rapidly diversified, and the divergence time was roughly the same as that of many plants, such as Cyananthus delavayi (0.49 Ma) and Pomatosace filicula (0.53 Ma), affected by the Qinghai–Tibet Plateau glacial period [S. involucrata. Many studies on the phylogenetic structure differentiation of alpine plant populations have proved that the uplift of the Qinghai–Tibet Plateau and Quaternary glacial oscillation have had a great impact on the distribution and evolution history of plant species [S. involucrata survived in the eastern Tianshan Mountains, Altai Mountains and western Tianshan Mountains when the Quaternary glacial climate repeatedly oscillated. At the same time, the uplift of Tianshan Mountains seriously hindered gene exchange between populations, resulting in the fragmentation of the distribution of S. involucrata in the current habitat.The arrival of glaciers during the Pleistocene 0.01–2.59 Ma) made the geological age officially enter the cold Quaternary. Large-scale iceberg movements occurred in the high, middle and low latitudes of the Northern Hemisphere. The alternation of glacial and interglacial cycles accelerated the intraspecific differentiation of plants ,50,51,52plotypes . We belil period . In the l period ,58,59, w Ma made  species ,61. We sS. involucrata also shrinks sharply. Due to excessive excavation and the destruction of the natural environment, the distribution area and population size of S. involucrata have decreased sharply, resulting in the loss of the germplasm resources of S. involucrata. In view of the fact that S. involucrata is a perennial one-time flowering and fruiting plant, it is difficult to restore its genetic diversity after it is disturbed by human activities. The protection of S. involucrata germplasm resources are important. Habitat reduction and human destruction will cause population fluctuations, and ultimately showing a low level of diversity, high population differentiation and a high level of inbreeding in genetics. Inbreeding in small-scale and fragmented populations may lead to the loss of genetic variation, which seriously affects the adaptability of threatened species to environmental changes [S. involucrata should be a priority in the conservation plan. This study increases our understanding of the possible main factors affecting the genetic composition of the S. involucrata population, thus benefiting its population protection. The cpDNA haplotypes of S. involucrata were closely related, but some haplotypes were missing, indicating that there may be a loss of genetic diversity. Due to geographical isolation, each S. involucrata population should be protected as an independent unit. Among the nine populations of S. involucrata in the Tianshan and Altai Mountains, the genetic diversity of S. involucrata in the Bayinbuluke area is high, and there are many special haplotypes that should be given priority protection. Based on in situ conservation, seed preservation can also be carried out, and a few individuals in the unique haplotype population and plants with special traits can be collected and transferred to the resource nursery for protection. The natural growth cycle of S. involucrata is 5–6 years. The devastating root digging and flowering of the snow lotus has seriously affected its natural reproduction. We need a comprehensive artificial propagation strategy for S. involucrata, including seed germination, large-scale seedling production, grafting and tissue culture  programs, to avoid collecting plants from natural populations. Finally, all the protection work is inseparable from the support of the residents‘ community, and it is also necessary to enhance residents’ awareness of natural protection.In order to develop targeted conservation management strategies, it is necessary to understand the genetic structure of threatened species . Maintai changes . TherefoS. involucrata is proposed by exploring the factors affecting the population history and genetic structure of S. involucrata. The strong uplift of the Tianshan Mountains in the Pleistocene and the alternation of the Quaternary glacial and interglacial periods played an important role in the genetic differentiation of S. involucrata populations. The results of the niche model analysis show that geographical isolation could promote the genetic differentiation of S. involucrata compared with IBR. The geographical distance between populations of S. involucrata is far, which can easily lead to a sharp decrease in gene flow between populations, thus increasing the genetic distance between populations. The S. involucrata distributed in the Tianshan Mountains and Altai Mountains is affected by the uplift of the Tianshan Mountains, resulting in the fragmentation of its distribution in the current habitat. Saussurea involucrata is divided into two lineages, the Tianshan Mountains and Altai Mountains, which seriously hinders gene exchange between populations. The study of the gene differentiation and genetic structure of S. involucrata provides evidence for future research on the response of alpine endemic plants to Quaternary geological and climatic events in the Tianshan and Altai regions. The population of S. involucrata distributed in the western Tianshan Mountains has high genetic diversity. In order to avoid the loss of endangered species of S. involucrata germplasm resources, priority should be given to the protection of S. involucrata in this area. In order to further protect S. involucrata effectively, S. involucrata in the Bayinbuluke area deserves further study.In this study, the effective protection of