Pontoscolexcorethrurus  is an invasive tropical earthworm, globally distributed. It reproduces through parthenogenesis, which theoretically results in low genetic diversity. The analysis of the population structure of P.corethrurus using molecular markers may significantly contribute to understanding the ecology and reproductive system of this earthworm species. This work assessed the genetic diversity and population structure of P.corethrurus with 34 polymorphic inter simple sequence repeat markers, covering four populations in tropical and temperate pastures from Veracruz State. Nuclear markers distinguished two genetic clusters, probably corresponding to two distinct genetic lineages. The number of clones detected in the AC population was lower than expected for a parthenogenetic species. Also, the apparent lack of differences in population structures related to the geographic region among the populations studied may indicate that human-mediated transference is prevalent in these areas. Still, most individuals apparently belong to lineage A, and only a few individuals seem to belong to the lineage B. Thus, the admixture signatures found among the four populations of P.corethrurus may have facilitated a successful invasion by directly increasing fitness. In summary, addressing the genetic variation of P.corethrurus with ISSR markers was a suitable approach, as it evidenced the genetic diversity and relationships in the populations evaluated. Dendrobaenaoctaedra Savigny, 1826; Lumbricusterrestris Linnaeus, 1758), megascolecids , benhaminis , and rhinodrilids  the earthworms registered . In Mexico, specifically in Veracruz State, its distribution ranges from 0 to around 1600 m a.s.l., living at an average temperature of 17 °C , Actopan (AC), Ingenio La Concepción (LC) and Naolinco (NA) (Table P.corethrurus were collected (N = 10 per site). Earthworms were kept in plastic boxes with moistened soil and transported to the laboratory at INBIOTECA for taxonomical and anatomical identification  Table , each offication . SpecimeTail-wall tissue was used for extraction of genomic DNA. Total DNA was extracted using the DNeasy Blood & Tissue kit  following the manufacturer’s instructions. DNA was checked for quality by gel electrophoresis and quantified using a spectrophotometer .P.corethrurus (N = 10 per site) were used for ISSR screening. ISSR screening was based on five primers  and Eiseniafetida   according to the following conditions: an initial step at 95 °C for 3 minutes, followed by 35 denaturation cycles at 95 °C for 30 seconds, annealing at primer-specific temperature for 30 seconds, and elongation at 72 °C for 1 minute. A final extension was performed at 72 °C for 10 minutes. The PCR products were visualized in 2% agarose gels (with ethidium bromide at 1ul/ml). Although the initial screening used a total of 18 primers, only those primers that were polymorphic and reproducible were selected for subsequent analysis  . Each PCMLG) was constructed for each individual by pooling data of single ISSR fingerprints using the procedure available in the POPPR package in R for genetic analysis of populations , as reported previously . A multiulations . Isolatedatasets . The POPdatasets . To asseage in R  was usedMLGs was estimated through an analysis of molecular variance (AMOVA) using the GenAlEx v.6.5 software (st fixation index) for all populations, as well as regarding within-population genetic variance  implemented in GenAlEx v. 6.51  dendrograms were also created using the POPPR package   . STRUCTUDAPC) , which uDAPC) . RelativDAPC) . The appa-priori knowledge of parental allele frequencies, and also has the advantage of specifically assuming a mixture of parental and several hybrid classes  to assign them into categories. Individual posterior probabilities belonging to each hybrid category were estimated using the MCMC method in a Bayesian framework using Jeffreys-type priors and a burn-in period of 100000 iterations followed by 50000 sweeps from the posterior distribution sampling (IA) (IA (r̄d)  was further investigated using NEWHYBRIDS v1.1 , which asampling . Linkageing (IA)  and a stIA (r̄d) . The nulP.corethrurus, information on genetic variability is relevant to determine the selective forces that act on the reproductive system of this species. In that sense, the survey carried out in this study was aimed to reveal the population genetic structure of P.corethrurus in natural landscapes covering tropical and temperate pastures. For this, a set of ISSR markers were used to assess the genetic diversity and population structure of P.corethrurus genotypes from four locations in Veracruz State, Mexico.Beyond the ecological relevance of ISSR primers produced bands on agarose gel that were suitable for assessing the genetic diversity and genetic relationship between and across populations of P.corethrurus. The PCR products ranged between ~200 and ~2000 bp from genomic DNAs of P.corethrurus. The total number of bands and polymorphism rates are shown in Table MLGs among the 35 P.corethrurus individuals yielded reliable products. Overall, one MLG corresponding to AC was observed twice, whereas the rest of MLGs (31) were detected only once. As regards MLG diversity (H), this parameter varied across populations, with no correlation to any specific geographic location  were higher for LV, LC, and NA, in agreement with the fact that all genotypes found were unique to these populations (Table exp) values varied from the highest (LC = 0.39) to the lowest (NA = 0.29). Most populations exhibit low genetic diversity , there were significant differences between the LV, AC, and NA populations were found , most of the individuals scattered in a non-uniform clustering  and lineage B . Besides, the DAPC analysis confirmed the above genetic relationship between LC and LV, even when they belong to distant geographic locations  and NA (lineage B) are detached  and uncorrected (N = 35) reject the hypothesis of no linkage among markers  = -557.89 ± 0.31) distributed across the four geographic locations  was found within populations, and only 25% among populations. This finding is similar to those reported by Dendrobaenaoctaedra and P.corethrurus, respectively. In both cases, most of the genetic variability was found within populations, and only a minor proportion occurred between populations . It is likely that the lineages identified potentially correspond to those previously reported by Taheri and co-workers, namely lineages L1 and L3 , for which a single haplotype was found in all sampling stations  showed the best distinct cluster of individuals , soil rich in organic matter, or even interactions with other soil organisms , in which clonal lineages seem to remain close to their original areas, indicative of some level of local adaptation or strong interspecific relationships  may be acting as the main barrier to MGL dispersion . The lineages identified in this work likely correspond to lineages L1 and L3 identified previously by P.corethrurus populations occur in some sites but not in others, our work suggests that genetic variation is playing a key role in the invasion process. The association between the genetic variability of P.corethrurus and its success in invading new sites is counter-intuitive due to its parthenogenic reproduction, i.e., clonal multiplication. Additional genotyping of P.corethrurus individuals inhabiting diverse environments or different States like Tabasco, Puebla, and Tamaulipas, will be necessary, not only to confirm our results, but to track the dynamics of dispersal and diversification of lineages, as well as to identify new dominant genotypes or newly introduced lineages. All this information should be gathered before using P.corethrurus in biotechnology research, remediation, or fishing, or before estimating its effects on local crops, plants, or organisms.In this work, we assessed populations of


X‐chromosome short tandem repeats (X‐STRs) with unique sex‐linkage inheritance models play a complementary role in forensic science. Guizhou is a multiethnic province located in southwest China and some genetic evidence focusing on X‐STRs for various minorities was reported. However, population data of Guizhou Tujia are scarce.A total of 507 Guizhou Tujia individuals were profiled using the AGCU X‐19 STR kit. Allele frequencies and forensic parameters were calculated. Additionally, population genetic relationships between Guizhou Tujia and other 19 populations were explored.A total of 257 alleles with the allele frequencies ranged from 0.0013 to 0.6098 were found. The combined power of discrimination in males and females and mean exclusion chances in all case scenarios were all greater than 0.99999. Population comparisons showed Guizhou Tujia had a homogeneity with all Han populations from different administrative regions, and other ethnic populations residing in Guizhou, while had obviously genetic heterogeneity with the Altaic family populations except Xibe.Nineteen X‐STRs can afford a reliable and informative database of Guizhou Tujia population for human identification and paternity testing, especially in complex biological relations. The genetic relationships of Chinese are significantly influenced by the geographic position and ethnolinguistic origin. 19 X‐STRs can afford a reliable and informative database of Guizhou Tujia population for human identification and paternity testing, especially in complex biological relations. The genetic relationships of Chinese populations are significantly influenced by the geographic position and ethnolinguistic origin. Tujia has its own language which belongs to the Tibeto‐Burman language branch of the Sino‐Tibetan language family but no script. Based on the Chinese historical sources, Tujia people are the main descendants of the Ba people, an ancient tribe in southwest China formed and named between the Xia and the Shang Dynasty. In the early period, the Ba people took Enshi as the center and lived in western Hubei. With the power increasing, the Ba people gradually expanded to the whole Wuling mountain areas, southward to the Guizhou and Hunan province, eastward to Sichuan. After the Ba was defeated by the Qin, its posterity survived and renamed “Tu” in the 1206 A.D. With the stability of the chieftain system, the Tujia people residing in Hunan, Hubei, Sichuan, and Guizhou province integrated and gradually consolidated. Since 1956, Tujia has become an officially recognized independent ethnic group (https://en.wikipedia.org/wiki/Tujia‐people). Guizhou, located in southwest China, is demographically one of China's most diverse provinces with a unique geographical environment and historical mass migration. Approximately 4.1% of the Tujia people in China have settled in Guizhou, and becoming the fifth chiefly ethnic group in the province (https://en.wikipedia.org/wiki/Guizhou).Tujia, one of the most ancient minorities in China, mainly live in the Wuling Mountains boarding Guizhou, Hunan, and Hubei Provinces, and Chongqing Municipality. According to the sixth national population census of the Chinese government, Tujia was the seventh largest minority in 55 Chinese minorities with a population of about 8 million people  Tujia populations using AGCU X‐19 STRs kit. With these highly polymorphic STRs’ allele frequencies data, we further evaluated the X‐chromosome forensic characteristics of AGCU X‐19 STRs kit in Guizhou Tujia populations and reconstructed the population genetic structure with previously reported populations  unrelated healthy Tujia individuals from Guizhou province (southwest China) were collected in accordance with the informed consent principle. Participants were all indigenous without immigration and interracial marriage at least three generations. This study was approved by the Biomedical Research Ethics Committee of Zunyi Medical University (No. 2014‐1‐044).2.22O (sterile deionized H2O). PCR condition consisted of predegeneration of 2 min at 95℃; 10 cycles of 30 s at 94℃, 1 min at 60℃, 1 min at 65℃, then 20 cycles of 30 s at 94℃, 1 min at 59℃, 1 min at 72℃; final extension of 30 min at 60 ℃, and hold at 4℃. The PCR products were detected and separated on an ABI 3500XL Genetic Analyzer . GeneMapper ID v1.4 software was employed to identify and assign the alleles determined by fragment sizes. Positive control (9947A DNA sample) and negative control (sdH2O) ran in each PCR reaction system.DNA extraction of Tujia individuals was performed from the EDTA anticoagulant tube using salting‐out method . SPSS v26.0 Hansen,  was usedp values of 19 X‐STRs between 20 populations, as well as pairwise Nei's genetic distances based on 19 shared X‐STRs, were calculated by Arlequin v3.5 software . Neighbor joining tree (N‐J tree) and multidimensional scaling plots (MDS) were visualized by Mega v7.0 , which explained that the selected sample could represent Guizhou Tujia population.In the present research, we used the AGCU X‐19 kit to genotype the 19 X‐STRs in 507 volunteers  residing in Guizhou province successfully. The Hardy‐Weinberg equilibrium (HWE) for the 19 X‐STR loci was tested using a Markov Chain with the dememorization steps of 100,000 and forecasted chain length of 1,000,000 testing based on the observed heterozygosity (Ho) and expected heterozygosity (He) in 249 unrelated healthy Tujia females Table  Nei & R. The valp > 0.05) in all 19 loci were detected between males and females , power of discrimination in males (PDm) and females (PDf), paternity exclusion chance (MEC) in the Trios  and Duos (MEC_Desmarais_Duos) of 19 X‐STRs based on pool frequencies are performed in Table 3.2p > 0.05/171 = 0.0003) in the females. In the male individuals, there were significant differences in 25 pairs, but only five pairs  still deviated from LD after Bonferroni correction (p > 0.05/171 = 0.0003).The linkage disequilibrium (LD) for 19 X‐STRs intercomparisons was calculated by permutation test using the EM algorithm  of females Table  and was 19 X‐STRs can be clustered into seven linkage groups (LG): LG1  after Bonferroni correction, which were identified between Guizhou Tujia and Xinjiang Kyrgyz at 12 loci, followed by Xinjiang Uyghur at 10 loci, Xinjiang Mogolian at eight loci, Tibet Tibetan and Xinjiang Kazakh at seven loci, Guangxi Zhuang, Sichuan Tibetan, and Sichuan Yi at six loci, Guizhou Sui at five loci, Guangxi Mulao at four loci, Xinjiang Xibe at three loci, Ningxia Hui at two loci, and Shaanxi Han, Guizhou Han, Guizhou Miao at only one loci. No significant differences were observed between Guizhou Tujia and Xinjiang Han, Southern Han, Sichuan Han, and Guizhou Gelao. Significant differences were observed at DXS10159 locus of Guizhou Tujia and 19 reference populations, while no differences were found at DXS10074, DXS10134, and DXS10164 loci.Locus by locus Fst and corresponding Pairwise Nei's genetic distances among 20 populations were estimated and listed in Table Then, phylogenetic relationships between Guizhou Tujia and other reference populations were explored using N‐J tree and MDS plot. As shown on the N‐J tree Figure , three mThe MDS Figure  showed tFinally, PCA was also constructed with the exception of Xinjiang Mogolian populations, which can be prominently separated from others in the N‐J tree and MDS. As shown in Figure 4Herein, genotypes of 507 unrelated Tujia individuals  from Guizhou were successfully obtained using AGCU X‐19 STRs PCR amplification kit. To explore the capacity of 19 X‐STRs in individual identification and forensic complex paternity testing, a series of forensic parameters were calculated, such as PDm, PDf, and four paternity exclusion chance . MEC_Krüger  in Guizhou and other provinces are needed.5In this study, we first analyzed the genetic polymorphisms of the Guizhou Tujia population based on AGCU X‐19 PCR kit. All loci in this population can be used to establish a reliable and informative database of X chromosome markers for human identification and paternity testing, especially in complex biological relations. Additionally, population comparisons indicate that Guizhou Tujia has genetic homogeneity with populations who reside in geographically adjacent regions and share the same language. Besides, Guizhou Tujia as a Tibeto‐Burman‐speaking population has an intimate relationship with geographically close Guizhou Gelao and the Han populations. Additional studies with other provinces and genetic markers of Tujia populations are needed for further understanding of the genetic structure of the Tujia populations.No potential conflict of interest was reported by the authors.PYC and LMY conceived the idea for the study. LL, HYG, LLY, HZ, and FL performed or supervised laboratory work. LL, LSZ, and LLY analyzed the data. PYC, LL, JY, and MLY wrote and edited the manuscript.Fig S1Click here for additional data file.Table S1‐S10Click here for additional data file.


Tor putitora (GM) is an endangered and sensitive cyprinid species. It is considered an indicator of a healthy freshwater ecosystem and is found in cold-water habitats. Therefore, it is crucial to understand how longitudinal connectivity and anthropogenic factors affect the diversity and population genetic structure of GM. The population genetic structure, gene flow and demography of the GM in four Himalayan rivers were investigated by mitochondrial cytochrome b gene (cyt b) as well as microsatellite genotyping. The results showed overall high mtDNA diversity (hd: 0.795) couple with low nucleotide diversity (π: 0.0012) in all GM populations. We also found significant levels of observed heterozygosity (ranging from 0.618 to 0.676), with three genetic clusters. The mtDNA and microsatellite analysis suggested that there are close genetic relationships between the Bhagirathi and Ganga populations; whereas, significant level of genetic differentiation was observed with that of Alaknanda and Yamuna populations. Haplotype distribution, unimodal distribution graph and results of the neutrality test indicated a sign of recent population growth in the GM population. Analysis of molecular variance (AMOVA) and spatial molecular variance (SAMOVA) revealed existence of genetic structures in GM populations. In addition, spatial genetic analysis detected a significant correlation between the pairwise genetic and geographical distances for the entire study area . Considering the significant level of heterozygosity, high rate of unidirectional migration and the intra-population structuring in Alaknanda and Yamuna, it is crucial to propose an effective conservation plan for the GM populations. In general, dams obstruct continuous water flow and create isolated microhabitats. Therefore, we recommend the establishment of microscale protected areas near GM breeding sites and construction of fish pass to maintain the genetic connectivity of fish species that enhance viable populations.In many aquatic species, alteration of habitats and human-induced barriers shape the population’s genetic structure in rivers with longitudinal connectivity. The golden mahseer,  The Himalayan riverine region has a rich diversity of species and unique bio-geographic features. The Ganga and Yamuna, the major Himalayan rivers, have been designated a lifeline of India. They flow from the Western Himalaya and converge at Triveni Sangam, Prayagraj, Uttar Pradesh and drain into the Bay of Bengal. Hydro-power development and construction of dams and barrages in the Himalaya have significantly affected the natural habitat, abundance, and population structure of aquatic animal species over the last 45–50 years –4. The bTor putitora, GM), is a flagship species, belongs to the family Cyprinidae [b gene and microsatellite markers to describe the population genetic structure, gene flow, and demography of the GM in the Bhagirathi, Alaknanda, Ganga, and Yamuna rivers. We have also attempted to address the impact of dams and barrages on the genetic structuring of the GM.The golden mahseer . After sequence alignment, we obtained 27 variable sites among the populations, accounting for 2.36% of the entire number of sites. Of these, 14 sites were singletons, and 13 were parsimony informative. The estimated transition/transversion bias (R) was 1.55. The typical nucleotide composition was T = 27.7%, C = 28.3%, A = 30%, and G = 14%; these values indicate A+T rich region among the mitochondrial cyt b gene.We obtained the complete mtDNA cyt The sequences were grouped into 38 haplotypes (hap) . The hapSequences of GM from seven other different Indian rivers, namely the Ravi , Beas , Satluj , Chenab , Kosi , Jia Bhoreli , and Walwan dam on the Indrayani  were taken from GenBank to determine the inclusive mtDNA structure and to carry out network analysis [The median-joining (MJ) network of the 83 haplotypes represented the distribution pattern of haplotypes among the GM populations . The hapP = 0.285) and the raggedness index of the distribution   .HO) was comparable (0.67) in the Ganga, Bhagirathi and Yamuna populations, whereas it was low in Alaknanda (0.618). However, the expected heterozygosity (HE) was highest in Ganga and Bhagirathi (0.692), followed by the Alaknanda (0.668) and Yamuna (0.626). The estimated inbreeding coefficient (FIS) value of the populations ranged from -0.070 in the Yamuna to 0.095 in Alaknanda, and the overall value was 0.028  showed very less heterozygosity; hence those were excluded from analysis; therefore, nine loci were used to calculate the genetic diversity of the GM populations of the four rivers. The comparison of allele size for each markers obtained in the present study to the original source has been provided in the as 0.028 . The negpulation .We used DAPC to analyze the genetic structure of the GM population. We found that there are three genetic clusters. Cluster 1 consisted of the populations of the Ganga and Bhagirathi, and Cluster 2 and Cluster 3 were the populations of the Alaknanda and Yamuna, respectively. The scatterplots and stacked bar graphs of the assignment probabilities of GM individuals indicated that the Yamuna population was significantly differentiated from the Bhagirathi, Alaknanda, and Ganga populations. Additionally, the Alaknanda’s GM exhibited a distinct but closer genetic relationship with the GM of the Ganga and Bhagirathi. The assignment probability of the GM indicated that individuals of the Bhagirathi and Ganga have a similar genetic structure and cluster with each other Figs  and 5.b was lower between the Ganga and the Bhagirathi (FST = 0.0077) than the Ganga and the Alaknanda (FST = 0.0584), whereas it was slightly higher between the Alaknanda and the Bhagirathi (FST = 0.0772). A higher genetic distance was observed between the Yamuna to Ganga (FST = 0.1008), Alaknanda (0.292), and Bhagirathi (0.109). A similar FST pattern was also observed with the microsatellite loci. The low pairwise genetic distance was obtained between Ganga and Bhagirathi (FST = 0.007), whereas it was higher between the Alaknanda and Yamuna (FST = 0.032) (The level of pairwise genetic differentiation was significantly different from zero in the GM population. The genetic differentiation at Cyt = 0.032) . The nei= 0.032) .b gene) rather than among the populations (0.480% for the microsatellite markers and 1.053% for the mtDNA Cyt b gene), suggesting that there is significant genetic structuring within the group. The fixation index among groups (FCT) except microsatellite, among populations within groups (FSC) and within populations (FST) were statistically significant (P<0.05) . FCT value was highest at K = 3,  with Fsc  in this case, GM from Jia Bhoreli, Assam and Walwan dam, MH became separated, and leaving other populations. With an increase of K value up to K = 8, Ganga and Bhagirathi GM were still attribute to one group, whereas Alaknanda, Yamuna, and the rest of the population created a separate group  . The SAMte group .rM = 0.126; P = 0.010)  . However) FST in  and the The Garza–Williamson index  test showed values lower than 0.68 , indicated that GM had suffered a historical reduction in population size. Besides, to examine the recent demographic scenario, we analysed the bottleneck test. We did not detect signs of the recent bottleneck in any of the studied GM populations with non-significant values in the Wilcoxon sign-rank test and two mutational models test: Two-phase model (TPM) and step-wise mutation (SMM) that correspond to a one-tailed test of heterozygosity excess. The mode-shift test suggests that there is no distortion of the allelic frequency, and a normal L-shaped distribution was observed in our data set . Four inWe have assessed the genetic diversity, population genetic structure, gene flow, and demography of GM based on mtDNA and microsatellite markers. GM is restricted to cold water habitats and is very sensitive to change in water temperature; therefore, small climatic fluctuation may lead to a change in its genetic structure. Construction of dams/barrages and high anthropogenic activities in Himalayan rivers leading to limited dispersal ability, unidirectional migration, and isolation of habitats might have shaped the genetic structure and driven differentiation among the GM populations. The population genetic studies on endangered species are dominant tools for understanding the demographic pattern, which has resulted in the development of conservation and management strategies for species , 26.b from 201 samples from four major populations of Himalayan rivers exhibited 38 haplotypes. Our data supplement those relating to seven other Indian rivers. In a previous study, 134 GM individuals were sequenced into 47 distinct haplotypes [b gene was reported from the GM samples from the Ravi and Chenab [Our analysis of the variations in the mtDNA Cyt plotypes . Strikind Chenab . The higFurthermore, the microsatellite data indicated the significant level of allelic diversity in all the four populations. The mean numbers of alleles were slightly higher in the Ganga in comparison with the Bhagirathi, Alaknanda, and Yamuna rivers. These results were ecologically supportive, where two rivers Bhagirathi and Alaknanda merge and form the mighty Ganga river. Thus, the admixing of these tributaries populations leads to an increase in the allelic diversity of GM in Ganga. In the Hathini Kund barrage of the Yamuna river, heterozygosity excess was detected. It might be due to the human-mediated non-random mating strategy or presence of small reproductive population size. Another possible explanation could be that we collected samples from a single location, i.e. the upper Hathini Kund barrage of Yamuna; therefore, for better insight, more samples from different locations need to be collected for further study.We found a high rate of unidirectional migration of GM from the Tehri dam across the Bhagirathi replenishes the population in the Ganga river. It was also confirmed by the scatterplot, DAPC structure, and BayeAss analysis. The DAPC results clearly revealed that most of the genetic characters of the Bhagirathi’s GM were found in the Ganga, whereas clear structuring was observed in the Alaknanda and Yamuna. The BayeAss results indicated a high self-association rate in the Yamuna (98.7%) followed by Bhagirathi (91.5%) and Alaknanda (73.7%), which showed limited migration rates. The proportion of nonmigrants from Ganga was 69%, indicates that the majority of individuals were unimigrated from Bhagirathi and other populations. The result is in concordance with the earlier study on European grayling in a Danish river system, where the main stem population continuously receives individuals and alleles from upstream situated tributaries . TherefoFST value obtained with mtDNA and microsatellite markers indicated that Alaknanda exhibits a significant level of genetic differentiation from Ganga and Bhagirathi populations. It is also supported by microsatellite-based phylogenetic analysis and AMOVA. A significant subdivision among populations was revealed due to variance within populations. The SAMOVA analyses also indicated the existence of genetic structures in GM that have formed in response to barriers to gene flow by fragmentation of habitat. The SAMOVA first unglued the Assam GM, subsequently with increasing numbers of groups, other populations of GM from the highly diverse region were also separated. The fact that the unique haplotypes were also detected in single locality indicates limited gene flow among populations. The limited gene flow in the GM could increase genetic differentiation due to the restricted nature of the habitat. The fine-scale population genetic structuring seems common in a species that is having poor disperse ability [S. nukiangensis in continuous water habitat [The genetic differentiation between the Ganga and Bhagirathi was weak, whereas it was comparatively higher in the Alaknanda and Yamuna populations. The pairwise  ability ; thus, m habitat .FS test. Although, analysis of population expansion based on mismatch distribution shows non-significant p-values and unimodal distribution graph, indicates the possibility of allopatric divergence followed by stable population growth in GM. The past demographic fluctuations in population size over time (100 generations) were analysed using the G-W index and bottleneck analysis. The G-W indices of four populations were lower than the critical Mc value of 0.68, indicating that GM had experienced a historical reduction in population size. However, it was also suggested that the value of the G-W index was very sensitive for the detection of a population bottleneck because the number of alleles is frequently more reduced than the range by a reduction in population size [HEQ) within the past 2Ne to 4Ne generations [FST values, our findings indicate that Alaknanda’s GM has a significant level of genetic differentiation as compared to the Bhagirathi population. It might be due to tunneling of Alaknanda river water after construction of the Srinagar dam leading to a lowering of water level in the mainstream. Similar results were observed in brown trout, where an increase in genetic differentiation is most likely a result of fragmentation by hydro-power dam [To check the excess of rare mutations as evidence for population expansion, the test of neutrality Tajima’s D statistic was non-significant, whereas it was significant negative for Fu’s ion size , 30. Theion size ,30. It werations . The modower dam . It highower dam ,34.The GM is a flagship species that is confined to the cold water habitats of Himalayan rivers. Various anthropogenic activities in these sensitive habitats have led to a sharp reduction of the population that was present in the natural environment , 35. AnyThe Alaknanda originates from two glaciers, the Satopanth and Bhagirath Kharak glaciers (elevation 3800 m). The Bhagirathi originates from Gomukh, (elevation 3920 m), the snout of the Gangotri glacier. After flowing for approximately 225 km, these two rivers join at Devprayag and are subsequently known as the Ganga . The GanA total of 201 individuals’ fin and tissue samples were obtained from four populations: Ganga (n = 42), Bhagirathi (n = 78), Alaknanda (n = 26), and Yamuna (n = 55). These samples were collected from local fishermen from river banks being harvested for selling in the fish market, and no animals were captured explicitly for this study. Therefore, Institutional Animal Ethics Permission was not required for this research. All the experiments were carried out in accordance with relevant guidelines and regulations. The samples from the Yamuna river were collected from upstream of Hatnikund barrage and compared with the connected populations of the Bhagirathi–Ganga–Alaknanda river system for their genetic parameters and the level of gene flow. Daily (4:30 to 9:00 am and 5:30 to 8:00 pm) efforts were made to collect biological samples from local fishermen at 13 sites . The finb gene using the primers L14724 and H15915 [2, 0.2 mM of each dNTP, 2 pmol of each primer, 5 U of Taq DNA polymerase and 1 μl (~30 ng) of the template DNA. The PCR conditions were initial denaturation at 95°C for 10 minutes, followed by 32 cycles of denaturation at 95°C for 45 seconds, annealing at 52–56°C for 45 seconds, and extension at 72°C for 75 seconds. The final extension was at 72°C for 10 minutes. The effectiveness and consistency of the PCR reactions were monitored using positive controls. The amplified PCR amplicons were visualized in UV light on 2% agarose gel stained with ethidium bromide. Exonuclease I (EXO-I) and shrimp alkaline phosphatase (SAP) treatments were given to the amplified PCR products  for 15 minutes each at 37°C and 80°C, respectively, to eliminate any residual primer. The amplified PCR products were directly sequenced using the BigDye® Terminator Kit (v3.1) and analyzed on an ABI 3500XL Applied Biosystems Genetic Analyzer. All the products were sequenced in both directions. The sequences were aligned and edited using Sequencer 4.7 (Gene Code Corporation). All the raw sequences were aligned using CLUSTAL W, as implemented in the BioEdit v 7.2.5 software (http://www.mbio.ncsu.edu/BioEdit/bioedit.html).For PCR amplification, we targeted the complete Cyt d H15915 . PCR reaEleven microsatellite markers were selected for the analysis: MFW 11, MFW 17 and MFW 26 ; BARB 37® version 4.9 . The analysis of each sequence was performed separately using the CLUSTAL X multiple sequence alignment program [The sequences obtained from the forward and reverse directions were aligned and edited using SEQUENCHER program , and the program  was used program .P-values were obtained from 1000 simulations on the basis of a selective neutrality test.To determine whether the GM populations carried a signal of spatial range expansion or a stationary population history, Tajima’s D  and Fu’sHo), and expected heterozygosity (HE) were estimated using the GenAlEx v6.5 program [FIS) was calculated in Fstat 2.9.3.2 for each population, and its significance was tested, assuming no Hardy-Weinberg equilibrium within the samples and using 1,000 permutations [FST values (gene flow) among the populations were calculated using GenAlEx v6.5 [adegenet package in R was used to determine the genetic structure of the population [The number of alleles per locus (Na), the number of effective alleles (Ne), observed heterozygosity ( program . Inbreedutations . The polutations . The suputations . All theutations . The pailEx v6.5 . DAPC impulation . DAPC isHE) from the heterozygosity expected as the drift-mutation equilibrium (Heq). BOTTLENECK v. 1.2.02 [The recent population bottleneck signature for microsatellite markers was obtained by testing the deviations of the expected heterozygosity  among the populations were calculated using GenAlEx v6.5 [Da, and the non-rooted tree was generated using the neighbour-joining (NJ) method with 1,000 bootstrap replicates in POPTREE2 [The pairwise lEx v6.5 . PairwisPOPTREE2 .FCT), among populations within groups (FSC), and within populations (FST) for the mtDNA and microsatellite markers in ARLEQUIN v3.5 [FCT and minimal positive FSC values obtained after the 100 independent simulated annealing processes was retained as the best grouping of populations. Additionally, Isolation-by-distance (IBD) was evaluated for the golden mahseer populations using a Mantel test with 1000 permutation as implemented in Alleles In Space version 1.0 based on microsatellite markers [The analysis of molecular variance (AMOVA) was run to estimate the source of the variation found among groups  was used as outgroup. Asterisk represent the core haplotypes (Hap1 to Hap4).Posterior values are provided at their respective nodes. The (DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 TableN, number of samples.(DOCX)Click here for additional data file.


Zaprionus indianus, have less genetic diversity than conspecific populations in their native range and that diversity is proportionally lower in regions of the genome experiencing low recombination rates. This result suggests that selection may have played a role in lowering diversity in the invasive populations. We next use interspecific comparisons to show that genetic diversity remains relatively high in invasive populations of Z. indianus when compared with other closely related species. By comparing genetic diversity in orthologous gene regions, we also show that the genome-wide landscape of genetic diversity differs between invasive and native populations of Z. indianus indicating that invasion not only affects amounts of genetic diversity but also how that diversity is distributed across the genome. Finally, we use parameter estimates from thermal performance curves for 13 species of Zaprionus to show that Z. indianus has the broadest thermal niche of measured species, and that performance does not differ between invasive and native populations. These results illustrate how aspects of genetic diversity in invasive species can be decoupled from measures of fitness, and that a broad thermal niche may have helped facilitate Z. indianus’s range expansion.During biological invasions, invasive populations can suffer losses of genetic diversity that are predicted to negatively impact their fitness/performance. Despite examples of invasive populations harboring lower diversity than conspecific populations in their native range, few studies have linked this lower diversity to a decrease in fitness. Using genome sequences, we show that invasive populations of the African fig fly,  Populations of invasive species can experience extreme demographic histories. For example, processes such as bottlenecks, inbreeding, hybridization, and multiple introductions can all operate in populations of invasive species . More geAlthough studies have shown that some invasive populations experience a loss of genetic diversity relative to populations in the species’ native range , these fIn cases where invasive populations show less genetic diversity than native populations, at least two general arguments have been made against the idea of a genetic paradox in the invasive populations. First, lower genetic diversity at neutral loci is not equivalent to a loss of adaptive genetic variation: invasive populations may show reduced genetic variation at neutral loci but retain variation at loci that are important for maintaining fitness and adapting to novel environments . Second,Theory predicts that changes in population size will affect both the overall amount and also the type of genetic variation found within a population. Genetic drift in small populations can, for example, lead to the fixation of weakly deleterious mutations segregating at low frequencies in source populations , and popWhen explicit links between genetic variation and adaptive phenotypic variation have not been made, a comparative approach can be used to gain insight into general effects that invasion has on genetic variation. First, genome-wide data can be used to gain a more nuanced understanding of patterns of genetic variation within populations found in invasive versus native parts of a species’ range. For example, genetic diversity varies across the genome  and genoSecond, although studies of genetic variation in invasive species tend to focus on comparisons between invasive and native populations, comparisons between other invasive species and closely related noninvasive species can reveal whether the reduction in genetic diversity observed in an invasive population results in levels of genetic diversity that are below broadly observed levels. This type of comparison can be informative if populations that show comparable genetic diversity to the invasive population of interest also show evidence of contemporary adaptation to different environments. However, as mentioned above, summary statistics of genome-wide trends in genetic diversity may not reflect amounts of additive-genetic variation, and we require additional information to understand the effects of invasion on performance and adaptation in invasive populations.From a phenotypic perspective, quantifying performance across different environments can be used to assess whether range expansions associated with biological invasions have affected the fitness of individuals in invasive populations. If inbreeding and small population size had led to a decrease in fitness, one prediction is that between-population crosses will display higher fitness than crosses carried out within an inbred population  in Z. indianus’s native range may have acted to buffer invasive populations against critical losses of genetic diversity.Here, we analyze whole-genome sequences collected from 93 individuals sampled across 7 species and 16 populations of African fig fly  showed that invasive populations were closely related to one another and genetically differentiated from African populations. Despite broad geographic sampling in Africa, these analyses did not indicate that invasive populations were more closely related to any one African population (median FST ranged from 0.14 to 0.19 between invasive and African populations), as would be expected if they were recently derived from that population, or an unsampled, but closely related one. Below, we focus on aspects of genetic diversity within each population rather than differentiation among them.We estimated genetic differentiation among populations of invasion  online. invasion b, princiinvasion c, and geZ. indianus relative to its native range , the number of segregating sites (S), and Tajima’s D in nonoverlapping 5,000-bp windows across the genome for 16 populations across 7 species of Zaprionus. Among populations of the invasive Z. indianus, populations sampled in the invasive part of the species’ range display significantly less genetic diversity than populations sampled in their native range: the median number of segregating sites (S) in 5-kb genomic windows was between 120 and 160 across North American and Hawaiian populations , consistent with a recent contraction in population size, and native populations showing negative median estimates of Tajima’s D (−0.48 to −0.72), consistent with range expansion  is comparable to S in invasive populations of Z. indianus (median S = 120–140). By contrast, populations of Z. nigranus, Z. taronus, and Z. inermis all show markedly less genetic diversity  than any of the Z. indianus populations we sampled. The broadly distributed species Z. africanus (the closest relative to Z. indianus included in this study) showed amounts of genetic diversity comparable with those in native populations of Z. indianus. It is also worth noting that the level of genetic diversity we observe in invasive and native populations of Z. indianus is similar to the distantly related invasive Drosophilid fly Drosophila suzukii  within a given genomic window varied depending on whether that window overlapped-with, was adjacent-to, or was distant-from an annotated gene . Genetic diversity tended to be lower in windows that overlap an annotated gene compared with windows that were either within or outside of 5-kb from an annotated gene  and window-location relative to annotated genes and the median amount of genetic diversity (S) observed across windows (GLM: P = 0.99). The amount of genetic diversity within a genomic region is therefore affected by the presence (or absence) of genes, but broad scale differences in diversity between “genic” and “nongenic” regions is not systematically altered—for example, due to selection preferentially maintaining genetic diversity in or around genes—during the course of invasion.Across all genomic windows, we found that the amount of genetic diversity (ted gene  online. S) and mean estimates of population-scaled recombination rates in 5-kb genomic windows . Consistent with previous studies ; however, the mean difference in S between invasive and native populations of Z. indianus was correlated with local recombination rate  within annotated BUSCO genes was correlated between populations and species of Zaprionus  altering the genome-wide landscape of diversity; however, explicit tests of these mechanisms are still needed.As a second test of whether the genome-wide distribution of diversity is altered during biological invasions, we compared genetic diversity between species in genomic windows that spanned annotated BUSCO genes. Consistent with a genome-wide landscape of genetic diversity, we found that genetic diversity (aprionus  online. parisons a. The stS) was significantly correlated across all genomic windows between populations of Z. indianus . By contrast, Tajima’s D was significantly correlated across genomic windows between all populations of Z. indianus  between Z. indianus and other species of Zaprionus . Native populations of Z. indianus also never showed consistently weaker correlations than invasive populations  and their native range in Africa .We estimated thermal performance curves using a hierarchical Bayesian framework  online. Zaprionus species with estimates of genetic diversity and thermal performance, thermal niche breadth (B50) and the total area of the thermal performance curve (Ac) were positively associated with genetic diversity . By contrast, there was no relationship between levels of genetic diversity and thermal optima . Linear models fit to our complete data set should however be interpreted with caution because of phylogenetic nonindependence and uneven sampling across species . We therefore also fit models that only included a single population from each of the seven species sampled from São Tomé , but no relationship between genetic diversity and Ac or thermal optimum (both P > 0.1). These results are consistent with genetic diversity being higher in species that are able to exploit a broad range of thermal environments, potentially due to these species maintaining large population sizes.Among populations of São Tomé b. This rZ. indianus tended to have the highest estimated B50  is a more important factor differentiating Z. indianus from noninvasive species of Zaprionus than the absolute maximal fitness/performance that individuals in a population can achieve.In addition to comparisons among populations of measured . The popd Zambia . Three oZ. indianus, has resulted in a loss of diversity in invasive populations, but that levels of diversity in invasive populations remain as high or higher than those observed in other species in the genus Zaprionus. The level of genetic diversity we observe in Z. indianus  part of the species’ range show geographic and temporal changes in allele frequency that are consistent with adaptation to novel environments  genetic diversity in invasive populations could also help explain the high levels of genetic diversity in invasive populations of ulations . High leindianus  may alsoZ. indianus  in native populations prior to range expansion, reductions in genetic diversity would not remove those high-frequency adaptive alleles. This scenario is related to the idea that adaptation to anthropogenically disturbed environments in the native portion of a species’ range can facilitate subsequent range expansions . For example, we collected Z. indianus near cities or rural human settlements in rainforest, savannah, and desert environments, and Z. indianus is among the first and most abundant species to visit fruit traps across these environments. If the traits that underlie a broad thermal niche evolved in African populations prior to range expansion, then lower diversity in invasive populations may not have negatively affected the fitness of Z. indianus in the invasive part of their range.The broad thermal niche of pansions . AlthougZ. indianus in North America have not evolved to tolerate more temperate climates that African populations  than at African sites, populations of Z. indianus is that the lower level of genetic diversity in North American populations of Z. indianus is constraining potentially adaptive niche evolution in these populations. Comparing levels of diversity that segregate in Z. indianus to those in other Drosophilid populations suggest this is unlikely: invasive populations of Z. indianus still harbor as much or more genetic diversity than other species of Zaprionus and Drosophila. However, future work testing levels of additive-genetic variation for traits involved in thermal tolerance are needed to quantify the adaptive potential of the different populations and species.An alternate interpretation for the lack of differences in thermal performance curves between invasive and native populations of Drosophila). Measuring performance in the same populations and species analyzed here, across a wide range of diets, would be useful to determine the extent to which invasive populations of Z. indianus have adapted to successfully colonize the broad range of environments they now occupy.Traits associated with thermal performance are only one suite of traits potentially under selection during colonization and range expansion into novel environments. Other traits such as competitive ability  and the Z. indianus is lower than in native populations . Our sequencing, assembly, and annotation approaches resulted in genome assemblies with scaffold N50s between 336 kb and 2.45 Mb, complete single-copy BUSCO annotations of 90.7–97.4%, and 9,275–11,071 annotated transcripts , sorted, and filtered mapped reads using SAMTOOLS (v1.4), marked duplicates using the PICARDMarkDuplicates tool (v2.2.4), and realigned around indels using GATK’s RealignerTargetCreator and IndelRealigner tools . To facilitate comparisons across populations where we sampled different numbers of individuals, joint genotyping and filtering were carried out on randomly selected groups of four individuals (eight chromosomes) per population, except for the population of Z. africanus sampled from São Tomé, where we only sampled three individuals.To quantify genetic diversity within populations of ls v3.8; . We estiVCFtools with coverage-masked sites excluded using the “–exclude-positions” filter option. Because πSNP and S were highly correlated in all populations (r > 0.963), we focus primarily on S: the number of sites with segregating variation within a given 5-kb window.Population genetic metrics of genetic diversity were computed using glm function in R) to model the number of segregating sites (S) within a genomic window as a function of the position of that window relative to a gene annotation. For populations of Z. indianus, we were also interested in whether aspects of biological invasion had a different effect on levels of genetic diversity depending on the proximity of a genomic region to a gene. We therefore used a GLM to test the interaction between gene region type  and invasion status  on median levels of genetic diversity across genomic windows.We used GLMs with Poisson distributed error (ρrec = 2Nr) across the genome, we used the maximum likelihood method implemented in LDhelmet , which allowed us to use haplotype information to estimate recombination rates across the genome. All analyses below are conducted on mean recombination rates within 5,000-bp genomic windows that were generated from median posterior estimates provided by LDhelmet.To estimate fine-scale population recombination rates  using Spearman’s rank correlation tests. We found no evidence for a relationship between recombination rate and gene density—either across all windows  or windows containing at least one gene . We therefore conducted independent tests for relationships between genetic diversity and recombination rate and genetic diversity within and around gene annotations.We tested for a relationship between recombination rate and gene density across the 40 largest scaffolds of the rcorr function from the Hmisc R package. To test whether comparing BUSCO windows produced correlations greater than expected by chance, we performed randomization tests where we randomly selected 2,714 genomic windows (100 iterations) and computed all pairwise Spearman’s rank correlation coefficients for those windows across species.To facilitate interspecific comparisons, we analyzed genomic regions that spanned the first and last exon of an annotated BUSCO ortholog. In total, we quantified the correlation in diversity (or lack thereof) across 2,714 genomic windows spanning BUSCO orthologs (hereafter “BUSCO windows”) annotated across all seven species’ genome assemblies. Correlation coefficients were estimated using the dist.alignment function from the seqinr R library. We then tested whether the correlation in S across the genome of two species was related to their genetic distance . We also calculated Spearman’s rank correlations for S and Tajima’s D between populations of Z. indianus  using diversity estimates across all 5-kb genomic window.The genomic landscape of diversity may diverge between species with increasing phylogenetic distance due to aspects of genome evolution. To quantify whether this would affect interspecific comparisons of genetic diversity, we computed phylogenetic distances between each species using a concatenated alignment of 1,709 BUSCOs shared across all species genome assemblies and representing 4,616,644 sites. Genetic distances were computed from the identity matrix of the aligned BUSCOs using the Tmin and Tmax parameters, respectively). The height , the total area under the estimated performance curve (Ac), and the breadth of the curve between the lower 25% and the upper 25% of the curve (B50). We compared performance curves between species by generating posterior draws of parameter estimates. Two populations were considered to differ with respect to parameters describing their thermal performance curve when a given parameter estimate had a probability >0.95 of being different between the two populations. To quantify differences in performance across populations, we fit a model that jointly describes the thermal performance curve to data from all populations using a hierarchical Bayesian framework . This moMolecular Biology and Evolution online.msaa050_Supplementary_DataClick here for additional data file.


Lasionycteris noctivagans populations suggest they could be genetically isolated, but this has rarely been assessed using genetic data. Here, we evaluate this possibility by sequencing the complete mitochondrial genome of four silver-haired bats from eastern and western populations. The three usable mitogenomes were closely associated with other Vespertilionid bats and the phylogenetic tree revealed the two western individuals grouping together to form their own clade. Our results support the idea of small but significant genetic differences between eastern and western populations of these bats, but this should be tested further.The geographic distributions of eastern and western  Qiagen DNeasy blood tissue extraction was performed and was sequenced using Illumina NovaSeq. DNA is currently stored at the Museum of Biological Diversity at The Ohio State University . Raw reads were trimmed using Trimmomatic , one possible explanation for the differentiation is sex-biased migration. Whitaker and Hamilton  state th


P. scolopendrifolia is considered endangered and highly threatened. Like many endangered species, it is vulnerable to biological and anthropogenic threats that can lead to the loss of genetic diversity and, ultimately, extinction. Therefore, the assessment of genetic diversity and population genetic structure is imperative for conservation. In this study, we newly developed 15 polymorphic microsatellite markers. Analyses of genetic diversity and population genetic structure that included 182 samples from 11 populations were conducted using microsatellite markers and four noncoding regions of chloroplast DNA. Our study revealed a relatively low level of genetic diversity , albeit harboring with private alleles based on microsatellite genotyping data, and high haplotype diversities based on chloroplast DNA sequences data. The results of STRUCTURE and PCoA based on microsatellite genotyping data showed population differentiations. An AMOVA based on chloroplast DNA sequence data further corroborated these conclusions, indicating about 70% of variations found among populations. Low genetic diversity and divergence among the population might have been caused by factors, such as asexual reproduction, demographic events (bottleneck and population expansion), geographic isolation, and low gene flow. The development and implication of conservation strategies and management of P. scolopendrifolia are proposed based on these results.Due to substantial population decline, the Korean orchid  Species with small population size or small geographic ranges subsequently tend to have reduced gene flow and increased genetic divergence, due to genetic drift and inbreeding . In partThe family Orchidaceae is one of the most species-rich families, and this high diversity along with species-specific pollination has attracted numerous biologists –9. PolliPelatantheria scolopendrifolia (Makino) Aver. [= Cleisostoma scolopendrifolium (Makino) Garay] is an epiphytic or lithophytic evergreen perennial orchid, belonging to subtribe Aeridinae (Orchidaceae), and occurs across Korea, China, and Japan. In Korea, P. scolopendrifolia is highly restricted to southwestern corner of the Korean Peninsula, including coastal  and inland  regions as well as islands of the southwest . With a narrow extent of occurrence  and substantial population decline due to anthropogenic activities, such as illegal collection and habitat disturbances, the species is assessed as EN B2bc on Korean Red list and protected by law as Endangered Wildlife [P. scolopendrifolia. For example, given the fact that genetic diversity is particularly important to the conservation of rare or endemic species [He = 0.002) based on allozymes [He = 0.4162) based on microsatellite markers [Megachile yasumatsui) as a pollinator and self-incompatibility of this species. Furthermore, Son et al. [P. scolopendrifolia using various markers and extensive sampling are necessary to provide more comprehensive genetic information and also to develop conservation guidances based on this genetic perspective.Wildlife . The rar species , allozymnce EOO, ,000 km2  species . Their s markers . In addi markers  reportedn et al.  also preP. scolopendrifolia and to determine the genetic diversity, population genetic structure, and the factors that contribute to genetic patterns using the newly developed markers and four non-coding chloroplast DNA regions. Based on the findings of this study, we proposed several baseline guidelines or suggestions for conservation and management strategies of highly threatened species of P. scolopendrifolia in Korea.Thus, the present study aimed to develop additional microsatellite markers of P. scolopendrifolia from 11 populations, including two in Japan, to analyze genetic diversity and structure , and a minimum collecting distance of 20 cm between sampled individuals was used to prevent resampling the same individual. All leaf samples were dried with silica gel, and voucher specimens for each population were deposited in the Chonnam National University Herbarium (CNU).We sampled tructure . In KoreP. scolopendrifolia. According to the MiSeq platform, an Illumina paired-end genomic library was constructed using the TruSeq DNA LT Sample Prep Kit  and sequenced 11,173,080 and 8,146,500 paired-end reads from MP-2 and NJ individuals, respectively. Each paired-end read was 301 base pairs (bp) in length after trimming. The adaptor sequences and low-quality reads (< 36 bp) were trimmed by Scythe v0.994 and Sickle v1.33, respectively. The trimmed sequencing data were registered at NCBI Sequence Read Archive (SRA BioProject accessions MP-2: PRJNA565547 and NJ: PRJNA625297) and pair-end reads were merged into one read using the BBMerge program in Geneious ver. 10.2.2 [Total DNA was extracted using the DNeasy Plant Mini Kit , following the instructions of the manufacturer. The leaves sampled from two representative populations, coastal population Mokpo  and inland population Naju  were specifically selected to develop the microsatellite markers of  reads <  bp were . 10.2.2  and Prim. 10.2.2  was then2) of PCR buffer, 0.2μL (each 10 mM) of dNTPs, and 0.05μL (5 U/μL) of Taq DNA polymerase . PCR conditions were an initial denaturation at 95°C for 3 min; followed by 35 cycles at 95°C for 1 min, annealing at 60°C for 1 min, extension at 72°C for 2 min, and a final extension at 72°C for 5 min. PCR amplicons were then separated with a GeneScan 500 LIZ Size Standard  at the Macrogen Company  utilizing ABI 3730XL DNA Sequencer . Allele size was scored using GeneMapper v5.0 (Applied Biosystems).PCR amplifications were performed in a total volume of 15μL containing 0.5μL of genomic template DNA, 0.2μL of 10 pmol forward primer with fluorescence dye, 0.2μL of 10 pmol reverse primer, 1.0μL  [accD- psaI, petL-psbE, psbA-trnH, and rps12-rpl20) with variations within and between populations were ultimately selected. PCR amplifications were performed in a total volume of 50μL containing 1.0μL of genomic template DNA, 1.0μL of 10 pmol forward primer, 1.0μL of 10 pmol reverse primer, 5.0μL (with 2.5mM MgCl2) of PCR buffer, 1.0μL (each 10mM) of dNTPs, and 0.25μL (5U/μL) of Taq DNA polymerase . The PCR conditions were initial denaturation at 95°C for 2 min, 35 cycles at 95°C for 1 min, annealing at 54°C for 1 min, extension at 72°C for 2 min, and a final extension at 72°C for 5 min. All PCR products were confirmed on 1% agarose gels and purified with Inclone™ Gel & PCR purification kit . Direct sequencing of PCR products was performed at Macrogen . Sequence editing and alignment were conducted using Geneious 10.2.2 [For the selection of cp DNA markers with variations within and between populations, 17 chloroplast non-coding or intergenic spacer regions (f6-psbM)  were tesNa), number of effective alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He), proportion of polymorphic loci (P), and private alleles (Pa), were estimated based on the microsatellite allele frequencies over 18 loci using GenAlEx v. 6.5 [Ar) was calculated based on minimum sample size of 5 individuals in each of the 11 populations using FSTAT 2.9.4. software [FIS andFst) was estimated for all populations, and an analysis of molecular variance (AMOVA) was conducted with 999 permutations at two hierarchical levels (among populations and within populations) using GenAlEx v. 6.5 [5 and runs of 2 x 105 repetitions. Twenty runs were conducted for each value of potential genetic clusters (K), with K ranging from 1 to 15. The best K was identified based on the approach of Evanno et al. [Microchecker 2.2.3  was usedx v. 6.5 . Allelicsoftware . Deviatisoftware . The genx v. 6.5 . Principx v. 6.5 . A Mantex v. 6.5  was condx v. 6.5  with 999x v. 6.5 . The admo et al.  using tho et al. . The grao et al. . Bottleno et al. . HeterozD [Fs test [P. scolopendrifolia were deposited in GenBank: MT333568-MT333587 for rpl20-rps12, MT333588-MT333607 for petA-psbJ, MT333608-MT333627 for accD-psaI, and MT333628-MT333647 for petL-psbE.The four combined cp DNA data were used to construct an unrooted haplotype network using the statistical parsimony approach  as impleD  and Fu’s[Fs test , were co[Fs test  was emplP. scolopendrifolia  = 0.24707–0.34803) after excluding null alleles (ENA) were similar to the Fst values  derived from raw microsatellite data, suggesting that the effect of null alleles on the genetic structure of populations is likely negligible. Twelve loci deviated significantly from Hardy-Weinberg equilibrium  across loci varied from 1.278 (JPN-2) to 4.056 (JD-1). The mean of expected alleles (Ne) was 1.810. While the values of observed heterozygosity (Ho) varied from 0.278 (JPN-2) to 0.778 (HN-2), the values of expected heterozygosity (He) ranged from 0.139 (JPN-2) to 0.479 (JD-1). Ho was greater than He in all populations. Regarding the percentage of polymorphism (P), the smallest value was found in JPN-2 (28%) and the largest value (100%) was confirmed in the population JD-1. All populations other than HN-2 had one or more private alleles. Allele richness across loci showed that JD-1 had the highest mean value of 2.808. Negative FIS values were identified in all populations.Genetic diversity was evaluated for 11 populations of drifolia . At the P. scolopendrifolia populations based on 18 microsatellite markers can be seen in 2 = 0.5286, P = 0.001 using all populations; R2 = 0.1867, P = 0.001 using nine Korean populations) (p < 0.05), whereas nine populations showed shift mode, suggesting the occurrence of a recent bottleneck (PCoA results for lations) . Accordilations) . At K = ttleneck .accD-psbI, petA-psbJ, petL-psbE and rps12-rpl20 with polymorphic sites were successfully amplified and sequenced in 182 individuals from the 11 populations. The total length of the combined alignment was 2,370 bp. A total of 20 chloroplast haplotypes (A-T) were identified from this study   to 0.9091 (JD-2). Nucleotide diversity was highest in JD-1 while MP-2, HN-1, and JPN-2 were the lowest as having the value of 0.0000. Overall, genetic diversities were thoroughly evaluated in the Jindo (JD-1 and JD-2) region given its relatively large population size. Values of genetic diversity were high in Korea compared to those in Japan. Tajima’s  < 0.05) . AMOVA a < 0.05) . Accordi < 0.05) .Pelatantheria scolopendrifolia, one of highly threatened and endangered orchid species in Korea. A total of 15 polymorphic microsatellite markers were successfully amplified in all individuals from 11 populations, showing observed heterozygosity values from 0.156 to 1.000 (mean Ho = 0.481) and expected heterozygosity from 0.119 to 0.527 (mean He = 0.325). Although the values of heterozygosity were similar to or lower than those of other orchid species, for example, Caladenia huegelii (mean Ho = 0.551 and He = 0.690) [Cypripedium kentuckiense (mean Ho = 0.436 and He = 0.448) [Pinus resinosa (Pinaceae) [Allium sativum (Lilliaceae) [Artocarpus altilis (Moraceae) [Prunus mongolica (Rosaceae) [Pelatantheria as well as P. scolopendrifolia specially.This study reported microsatellite markers newly developed in = 0.690)  and Cypr= 0.448) , the preinaceae) , Allium liaceae) , Artocaroraceae) , and Pruosaceae) . TherefoPelatantheria scolopendrifolia, we used two types of molecular markers, 18 microsatellite markers and four non-coding cpDNA markers. In this study, we found that the values of observed heterozygosity based on microsatellite genotyping dataset ranged from 0.278 to 0.778 with an average value of 0.529. The values of expected heterozygosity (mean Ho = 0.356) were all lower than those of observed heterozygosity, suggesting heterozygosity excess. The mean He and Ho values observed in P. scolopendrifolia are lower when compared to other endangered or endemic orchids. For example, Caladenia huegelii had a mean He = 0.69 and mean Ho = 0.551 [Cymbidium tortisepalum had a mean He = 0.653 and mean Ho = 0.619 [P. scolopendrifolia were similar to Gastrodia elata which was evaluated as having low genetic variation [P. scolopendrifolia is lower to the mean He and Ho values reported for narrow and regional ranged species [P. scolopendrifolia populations. Historical demographic events, such as founder effects, bottlenecking, population expansion and contraction, and habitat fragmentation, can all impact genetic diversity and structure [P. scolopendrifolia using allozymes suggested that bottleneck and founder effects are possible causes for extremely low levels of allozyme variation [D and Fu’s Fs test to determine the presence of the population expansion, six populations showed negative values on Tajima’s D and Fu’s Fs, but only NJ was significant (p < 0.05), corroborating the aforementioned result. The star-like shape of the haplotype network also supported this assumption. Therefore, this study suggests that demographic events including bottlenecks and population expansion have played important roles in reducing genetic diversity of P. scolopendrifolia. Reproductive pattern may also contribute to this low genetic diversity. Mating system can be very influential, as reproduction through outcrossing is known to lead to a high level of genetic diversity [P. scolopendrifolia is self-compatible, while Son et al. [Megachile yasumatsui) and fruit set. The diversity of the populations MP-2, HN-2, and WD confirmed pollinator visits were not significantly higher than the populations without a pollinator. Furthermore, gene diversity, nucleotide diversity, and haplotype diversity based on cpDNA sequences were unable to reveal any relationship with the presence or absence of a pollinator. These results are contrary to the population genetic theory which generally holds that the genetic diversity of outcrossing species tends to be higher [P. scolopendrifolia can be reproduced through clonal ramets in addition to outcrossing. Vegetative reproduction is one of the causes low genetically variation [P. scolopendrifolia demonstrate that genetic diversity tends to be higher in larger population sizes due to the higher likelihood of maintaining a robust genetic pool. The gene flow is another factor to explain low genetic diversity. In general, pollen and seed dispersal are important for maintaining gene flow. Gene flow between populations prevent from differentiation and increase genetic diversity between populations. Despite the presence of pollinators, the reason for low genetic diversity may be related to the behavior of the pollinator itself. It is not known how far Megachile bees fly, but male bees typically carry pollen farther distances than female bees [P. scolopendrifolia with and without pollinators is approximately 30 km. Therefore, it is probably that little to no pollinator activity occurs between populations of P. scolopendrifolia. Based on these facts, it is inferred that gene flow by pollen dispersal is weak. This is consistent with PCoA and STRUCTURE results indicating the population differentiation  based on allozymes [Fst of P. scolopendrifolia is higher than that of long-lived perennial, narrow-ranged, and outcrossing species  [Fst values, further supported by genetically similarity based on their haplotype distributions  . This reibutions . Althougibutions . The admibutions . Althougibutions  and popuibutions . NotablyP. scolopendrifolia. This raises the possibility that the influx of P. scolopendrifolia to Korea was not only from China, but also from Japan. Three major haplotypes  in the haplotype network support this notion. In addition, founder events may also have influenced the haplotype distribution. After the influx of P. scolopendrifolia from China or Japan, genetic drift related to founder effects or bottlenecks in the process of dispersal from southern to northern populations would have contributed to the low level of genetic diversity and simple population structure. In this process, the Jindo populations (JD-1 and JD-2) with relatively high genetic variation might be situated as a geographic center of seed dispersal. However, after colonization, factors such as excessive collection, asexual reproduction, geographic isolation, and the loss of genetic diversity due to low gene flow provide strong reasons for the absence of a specific pattern in the haplotype distribution map .According to Qian and Ricklefs , during P. scolopendrifolia is categorized as an endangered species according to IUCN Red List criteria: B2bc. The species is also listed as vulnerable in Japan following ‘Japanese Red Lists’ . Therefore, the development of policies for the conservation of P. scolopendrifolia is essential. Fortunately, since 1998, it has been legally protecting P. scolopendrifolia in Korea. Information on genetic diversity and population genetic structure, as well as ecological knowledge, should be considered when developing and designing conservation strategies for species or populations. Subsequently, this study provides several suggestions. First, population size must be stabilized. Most of the populations were not large, and MP-2, HN-1, JD-1, and JD-2 are easily accessible along the trail, leaving them vulnerable to illegal collection. Therefore, periodic monitoring and strict prohibition of illegal collection seem necessary. Second, the establishment of more efficient conservation strategies will require more extensive research on P. scolopendrifolia ecology. Han [Ramalina intestiniformis was commonly found in association with all populations of P. scolopendrifolia. Though this relationship is not well understood, it appears related to strategies for preserving or obtaining moisture. Factors such as these can be used to create more preferable environments for P. scolopendrifolia reintroduction, thus increasing the success rate of settlement. Lastly, genetic diversity and structure should be carefully considered. Genetic diversity was evaluated as relatively low in this study as a result of low diversity in allozyme data [ogy. Han  reportedyme data , 35 confP. scolopendrifolia and provided information related to genetic diversity and population structure based on microsatellite and cpDNA data. Low levels of genetic diversity and population differentiation were observed. In addition, the identification of the private alleles and population-specific haplotypes, further enhances the need for conservation. However, it was not possible to find a connection between genetic structure or haplotype distribution and specific factors such as the presence of pollinator. Various causes such as small population size, demographic events, clonal reproduction, and low gene flow all likely have role in shaping the observed genetic patterns of P. scolopendrifolia. Lastly, taking these patterns into account informed several suggestions for the development of future conservation strategies.In this study, we newly developed 15 polymorphic microsatellite markers of S1 Tablep < 0.05.+ and − indicate significant and non-significant level at (DOCX)Click here for additional data file.S2 TableProbability values are all under the 0.005.(DOCX)Click here for additional data file.S3 TableQuestion mark means missing data. * indicates the sequencing modification. T and A on 633 site were originally TCTTAATAT and 9 bp deletion, respectively; On 665 site, 1 bp deletion character was coded as A; T and C on 775 site stand for TAAGG and CCTTA, respectively.(DOCX)Click here for additional data file.S4 Table(a) Result based on microsatellite data. (b) Result based on chloroplast DNA data. For this analysis, three population types: coastal, inland, and island, were set as region. Regions: inland , coastal , and island .(DOCX)Click here for additional data file.S1 FigP = 0.001 for all correlations.(a) all populations. (b) Korean populations. The results of the Mantel test indicate the positive correlation between genetic and geographic distances was significant. Note: (TIF)Click here for additional data file.


Odontarrhena are most promising candidates for research on metal tolerance and Ni-agromining, but their genetic structure remains unknown. We investigated phylogenetic relationships and genetic differentiation in relation to distribution and ploidy of the taxa, anthropic site disturbance, elevation, soil type, and trace metals at each population site. After performing DNA sequencing of selected accessions, we applied DNA-fingerprinting to analyze the genetic structure of 32 populations from ultramafic and non-ultramafic outcrops across Albania. Low sequence divergence resulted in poorly resolved phylograms, but supported affinity between the two diploid serpentine endemics O. moravensis and O. rigida. Analysis of molecular variance (AMOVA) revealed significant population differentiation, but no isolation by distance. Among-population variation was higher in polyploids than in diploids, in which genetic distances were lower. Genetic admixing at population and individual level occurred especially in the polyploids O. chalcidica, O. decipiens, and O. smolikana. Admixing increased with site disturbance. Outlier loci were higher in serpentine populations but decreased along altitude with lower drought and heat stress. Genetic variability gained by gene flow and hybridization at contact zones with “resident” species of primary ultramafic habitats promoted expansion of the tetraploid O. chalcidica across anthropogenic sites.Albanian taxa and populations of the genus  Bornmuellera and Odontarrhena  to: (1) compare the levels of genetic diversity and divergence within and between species and populations, (2) assess the possible effects of site conditions such as elevation, soil type and metal concentration , and (3) test the hypothesis that human disturbance could have contributed to the shaping of genetic structure in the group by promoting hybridization. AFLPs were used also to identify deviant loci potentially involved in adaptation to serpentine soil, trace metal concentration, and elevation.Odontarrhena species.Elucidating the genetic structure in this group also pointed to the potential risk of using it for agromining applications on ultramafic outcrops outside its native range and inhabited by native Odontarrhena accessions), only 50 positions (7.9%) were phylogenetically informative. The mean genetic distance among ingroup accessions was 0.015. The Bayesian consensus phylogram  internal transcribed spacer region (ITS) alignment consisted of 629 bp (including 22 coded gaps 608-629), of which 479 conserved, 145 variable but phylogenetically non-informative and 99 (15.7%) informative; considering only the ingroup , as well as the three accessions of typical O. muralis from outside Albania. Small terminal clades with PP > 0.90 were formed by other Balkan species but these did not include Albanian accessions.All Albanian taxa and accessions were included in a large unresolved group. However, the two diploids Alyssum representatives (outgroup) 565 sites were conserved, 16 were variable but non-informative, and only five were (0.68%) phylogenetically informative. Mean genetic distance in the ingroup was 0.003. The IGS sequences therefore provided even less phylogenetic signal than did ITS. The resulting Bayesian tree was completely unresolved and is, therefore, not shown.The chloroplast DNA trnL-trnF IGS (trnL-F) alignment was 733 bp long, (gaps in pos. 712–733). By excluding the p < 0.001) and a genetic structure dominated by within-population variation . Among-population variation was considerably higher in tetraploid than in diploid accessions  was deviating in having four genetic groups instead of two as in pops. 19 and 27  showed a significantly lower admixing than tetraploid populations (p = 0.00443).Genetically admixed populations (with at least two groups both of which with ≥10% posterior probability of belonging into that genetic group) were the large majority (29 = 91%); only three populations consisted of only one group . HoweverO. albiflora and O. rigida included no admixed individuals, whereas the great majority of the O. chalcidica accessions (14 out of 17) and all those of its hybrid O. decipiens included a variable proportion of admixed individuals. The two O. moravensis accessions no. 19 and no. 27 were formed by non-admixed individuals, while population no. 20 included five admixed individuals. When excluding the latter, the diploid profiles showed a significantly lower admixing than the tetraploids (p = 0.00338).At the individual level, 75 samples (20%) included more than one genetic group ; O. albip = 0.0147).Based on multifactorial analysis B the numO. albiflora and population 14 of O. rigida. Most individuals of O. chalcidica and O. decipiens were mixed with those of other species.Neighbor-Net analysis  confirmeO. moravensis from the type locality (no. 27), whilst the highest (87.6%) was in those of O. decipiens and O. chalcidica (O. smolikana (73.5%) and lowest in O. albiflora (35.8%), though no significant differences existed between taxa. Diploid and tetraploid populations/taxa also did not differ significantly, but diploids had on average a lower polymorphism than tetraploids (57.9% vs. 66.8%). Similarly, polymorphism was higher in serpentine vs. non-serpentine populations (66.2% vs. 52.8%), though differences were not significant because of large variability in O. chalcidica. Heterozygosity was lowest in O. albiflora and highest in O. smolikana, O. decipiens and O. chalcidica  was in the population of alcidica . The latalcidica . The latalcidica B, while O. chalcidica accessions . The NJ tree of the populations from the type localities  ranged from 0.002 to 0.956 and were not related to geographic distances  and tetraploids ; the second group was formed only by tetraploids, with O. smolikana subsp. glabra distantly connected to one accession of O. decipiens and three of O. chalcidica.In the first one, there were both diploids (O. smolikana subsp. glabra (0.302) from distant massifs, whereas it was lowest in O. decipiens . Odontarrhena smolikana was more strongly connected to O. chalcidica (Nm = 4.2) than to their hybrid O. decipiens (Nm = 2.3). Gene flow between diploids was not significant (Nm = 1.98).Within single species, mean inter-population distance was highest in subalpine accessions of s 0.224; . The ests 0.224;  showed sOdontarrhena could not be satisfactorily resolved due to the low rates of variation in both ITS and trnL-F regions, in line with previous studies that suggested rapid and recent diversification as a possible cause [O. muralis from outside Albania, including one from the type locality in Romania, are clustered in a separate clade, unlike all others from this country. Most of the previous records of this important model taxon for studies on Ni-accumulation physiology and agromining applications, as well as the numerous Ni-accumulation reports from the Balkan ultramafics  and diploid chromosome complement. When looking at population genetic data, the two species showed a lower mean population heterozygosity (He) and were characterized by a lower number of genetic groups (KPop) compared with tetraploids. Remarkably, population 10 of O. rigida and 27 of O. moravensis showed an identical composition of genetic groups, accounting for their close relationship in neighbor-joining analysis and supporting their phylogenetic relationship.Relationships between Albanian and other taxa of le cause ,30,31. Ps , shouldOdontarrhena, such as Italian O. bertolonii [O. lesbiaca [O. serpyllifolia [Odontarrhena was dominated by within-population variation, likely due to the prevalent outbreeding in the species of this genus [O. bertolonii and the facultative O. serpyllifolia mentioned above. In the latter species, distance was even more important than soil type in explaining differentiation [AFLP-markers were more successful in detecting significant overall differentiation between the 32 populations analyzed, as expected from their mostly different species identity, edaphism, ploidy level, and geographic origin. Similar levels of differentiation were found in other Mediterranean endemics of rtolonii , Aegean lesbiaca , and Ibellifolia . As in tis genus . Consistntiation , and serntiation . In the ntiation .O. decipiens. As in other plant groups [O. decipiens, O. smolikana, and O. chalcidica. Introgressive hybridization especially between the former two taxa is supported by the high level of estimated gene flow between them (Nm = 8.9) and is related to their wide, mostly sympatric distribution in Northern and Central Albania, their phenotypic plasticity [O. smolikana, O. moravensis, O. rigida, and O. albiflora, and the ‘novel’ habitat is that created by all those human land-use activities that have shaped, and still shape, large patches of the Albanian territory. Multifactorial analysis showed significantly higher levels of genetic admixing in populations and individuals of O. chalcidica and O. decipiens from anthropogenic sites, whereas narrow-ranged endemics from primary habitats such as O. albiflora, O. rigida, and O. moravensis (when excluding population no. 20) were much less variable in terms of genetic group composition, in line with their low phenotypic variability. As predicted by the ‘disturbance hypothesis’ [O. chalcidica has likely acquired adaptive genetic variability by crossing with locally sympatric ‘resident’ taxa along contact zones, widened its niche in a more generalist sense (no longer restricted to serpentine), and spread in the many non-natural sites across the country to reach an almost continuous distribution and large population size. Reduction of geographic isolation has further promoted introgressive backcrossing and the genetic swamping of the group, a process that leads to the rarefaction or extinction of pure parental genotypes though not of their alleles [A second relevant finding was that grouping of populations by species identity could not explain overall genetic variation in our sample, and that most populations consisted of two or more of these groups in variable proportions. In addition, a significant percentage of individual genetic profiles were also admixed, especially in the morphologically intermediate tetraploid hybrid t groups ,36 admixt groups . In our asticity  and, posasticity . The rolasticity ,39,40, aasticity . In our othesis’ ,42, O. c alleles . Anthrop alleles ,44,45.O. chalcidica and diploid O. moravensis, suggested by the significant admixing detected in a population (no. 20) of the latter species. This population lies at the foot of Mt. Moravë in Eastern Albania, at the contact zone between the species core range on ultramafic rocks and a plain agricultural area where O. chalcidica is abundant. Moreover, Cecchi et al. [O. elatior (F.K. Mey.) Španiel et al., described from this area [O. moravensis and the three other O. chalcidica accessions shown in the NJ tree (Arabidopsis lyrata (Brassicaceae), where introgression with interploidal flow of adaptive genes can take place from diploid to tetraploid populations [Further investigation should address possible hybridization events between tetraploid i et al.  suggestehis area , could b NJ tree . Formatiulations .Odontarrhena in Albania and the likely mechanisms behind it, our findings apparently provided poor molecular support to the taxa delimited based on correlation between morphology, karyology, phenology, distribution, and ecology of natural populations [O. albiflora, in fact, there was only partial correspondence between these taxa and the detected genetic groups, though their discrete status was already described by specialized students of this plant group and of the Albanian flora [O. chalcidica and O. decipiens) compared with less variable and geographically more restricted diploids from primary serpentine habitats (O. rigida and O. moravensis). However, the partial genetic-taxonomic discrepancy can be explained by the process of diversifying selection which allows maintenance of species  boundaries despite ongoing gene flow between them. This mechanism was recently documented in other Mediterranean species complexes with strong attitude to introgressive hybridization, such as those in the genus Senecio L., and is probably an underestimated speciation pathway across angiosperms [Odontarrhena populations and on the role of diversifying selection along the elevational gradient and/or levels of metal concentration in the soil or site disturbance. The persistence of morphologically distinct taxa along these gradients, as in the case of O. chalcidica and O. smolikana from low altitude to mountain slopes, could in fact be possible because this mechanism acts on only a few loci that drive adaptive speciation in spite of very low levels of genome-wide differentiation [In revealing the complex genetic structure of ulations . With than flora ,24. Thisiosperms . This pontiation .Odontarrhena examined here. Likewise, no evidence for ecotypic differentiation was found within O. chalcidica, the only facultative serpentine species in our group. Reduced heterozygosity observed in some accessions of this species from severely disturbed sites  is likely due to recent founder effect events, which are known to result in the genetic impoverishment of recently established populations [Although heterozygosity and polymorphism were slightly higher in serpentine populations, there were no significant deviations in the levels of genetic variability and differentiation between the accessions and taxa of ulations . Other sulations ,52,53,54O. serpyllifolia showed no differences between accessions from ultramafic and non-ultramafic sites across the Iberian Peninsula [Senecio on Mt. Etna, Chapman et al. [S. chrysanthemifolius to hotter, more arid conditions at the base of the volcano. Remarkably, Etna’s basaltic lava and serpentine soils share a very dark color, which greatly enhances heat and drought stress during spring and summer. Reduced severity of these limiting factors may therefore provide a working hypothesis to explain the decreased incidence of outliers in Albanian populations of Odontarrhena from higher elevations.In our study, additional insights came from the analysis of outlier loci, which represented a considerable proportion (11.7%). Although the statistical procedure adopted here cannot be excluded to have caused false positives, we are confident that the proven robustness to this problem of AFLP data treated with Bayesian methods ,56,57 haeninsula . Moreoveeninsula . Adaptiveninsula ,67,68,69n et al.  found anOdontarrhena across its distribution range. The conservation of metallophyte biodiversity and the identification of threats are indeed major issues that require increasing attention and ad hoc research [O. muralis s.l. populations are used in several nickel agromining experiments on ultramafic outcrops across Central and Southern Europe [O. muralis s.l. is likely to be capable of crossing with adjacent congeneric populations of distinct taxa, if existing, and cause the formation of hybrids with enhanced expansion ability. We, therefore, advocate caution before introducing these populations in ultramafic areas hosting endemic Odontarrhena species.Our findings have relevant implications for the conservation of native taxa, ecotypes, and genotypes of research . At presn Europe  and thisOdontarrhena are small chamaephytes or long-lived caespitose herbs with branched inflorescences of numerous bright yellow flowers that are visited by a variety of pollinating insects including bees, hover flies, flies, wasps and small beetles. Allogamy is the prevailing breeding system [Alyssum [Species of g system , though [Alyssum .Odontarrhena are given in Cecchi et al. [Detailed information on the morphology, distribution, ecology and chromosome features in the six Albanian taxa of i et al.  and summO. albiflora is a tetraploid endemic known from a single site in Eastern Albania, on calcareous cliffs at 900–1300 m a.s.l.; O. smolikana subsp. glabra is a tetraploid obligate serpentine endemic, occurring in rocky subalpine slopes at 1200–2200 m a.s.l.; O. rigida is a diploid serpentine endemic of Central Albania at 250–1200 m a.s.l., in mostly undisturbed sites; O. moravensis is also diploid and restricted to undisturbed serpentine sites of the E Albanian mountains, at 800–1500 m a.s.l.; O. chalcidica is a tetraploid species widely distributed especially in the northern, central, and eastern parts, on serpentine and other soil types , at 50–1300 m a.s.l., usually in anthropogenic habitats; O. decipiens is a putative allotetraploid hybrid between O. smolikana and O. chalcidica, already recognized by Nyárády [Data on Ni levels in soil and plants are also available from Bettarini et al. . In summ Nyárády  and founDuring ad hoc field trips  we sampled a total of 32 populations of the six taxa throughout Albania, in all major outcrops of ultramafic rocks  and in O. albiflora) to 17 . Sampling was more intensive for O. chalcidica and O. decipiens also because these are the two taxa of the O. muralis group more widely used in research on metal uptake and Ni-agromining [The number of sampling sites for each of the six taxa was unavoidably uneven due to their different frequency and range extent, from 1  according to an empirical ordinal scale with three levels : 1 = no Genomic DNA of all 12 individual samples from the 32 populations was extracted using a modified 2xCTAB protocol  and quanO. muralis from type locality in Romania (ITS and trnL-F) and 14 accessions of related but non-Albanian taxa for trnL-F. Additional sequences were retrieved from GenBank, bringing the total number of ingroup accessions to 50 (for ITS) and 23 (trnL-F), plus, respectively, three and two Alyssum outgroups  using thttp://www.hiv.lanl.gov/content/sequence/findmodel/findmodel.html). These were GTR + Γ for ITS, with gamma-distributed rate variation across sites, and GTR + I + Γ for trnL-F.Trees were first obtained using Bayesian inference of phylogeny as implemented in MrBayes 3.1.2 . SelectiThe analyses were performed using four incrementally heated Markov chains  simultaneously started from random trees, and run for one million cycles sampling a tree every ten generations. The stationary phase was reached when the average standard deviation of split frequencies reached 0.01. Trees that preceded the stabilization of the likelihood value (the burn-in) were discarded, and the remaining trees were used to calculate a majority-rule consensus phylogram.The trees were viewed and edited with TreeView , with inFor both markers (ITS and trnL-F), estimates of genetic distances between the in-group accessions were calculated using the maximum complete likelihood model  as impleArabidopsis halleri [O. serpyllifolia [We used amplified fragment length polymorphism (AFLPs), a still widely used tool for inferring genetic structure in plant populations because of reproducibility, robustness, and high resolving power of polymorphism . This DN halleri , serpentllifolia , and genllifolia . Moreovellifolia ,95,96.AFLP-fingerprinting was applied to 384 individual samples following the standard procedure with minor modifications .The quality of AFLP profiles was preliminarily tested on 20 samples randomly selected from the 32 populations . The preliminary AFLP protocol for quality control was performed using six primers pair combinations . The finFollowing the program manual, we adopted a low peak detection threshold setting and a “stutters peak” filter to remove stutter peaks within 2.5 bp of each detected allele peak. After running the data with the size standard and the specific panel, the trace comparison report was analyzed with the duplicated samples, also checking the allele report file, and no mismatches were detected. During this step, the presence, absence, and questionable presence of alleles is shown, and in the case of complete mismatch of the peaks, the sample is automatically removed by the program to eliminate errors.Analysis of molecular variance  was theNext, we used STRUCTURE v.2.3.3  to deterPrincipal component analysis was used to summarize and display the variation in the average proportion of each genetic group across the six groups of conspecific populations (taxa).In addition, a Neighbor-Net analysis was performed on the matrix of uncorrected P-distances using SPLITSTREE v. 4.15.1 .Genetic variation was assessed as the proportion of polymorphic loci in the dataset, and as within-population heterozygosity (He) based on Nei’s metric  as impleThe mean number of outlier loci was determined for each population and the six groups of conspecific populations. After testing normality with the Shapiro–Wilk test, the six taxa were compared using ANOVA followed by a post hoc Tukey test.O. smolikana subsp. glabra, O. rigida, O. moravensis, O. albiflora, and the “type” populations of also taxa recently included in O. chalcidica and O. decipiens as synonyms; Cecchi et al., [Genetic distances among populations were estimated as Slatkin’s linearized pairwise FST values . MEGA X  et al., ; Supplem et al., . The num et al., : FST ≠ 1The relationships between population genetic variables , site variables , and site disturbance were first explored using multiple factor analysis  as impl