Anopheles nuneztovari and Anopheles albimanus in endemic localities of northwestern Colombia. Genetic and phenetic structures were evaluated using microsatellites markers and wing geometric morphometrics, respectively. In addition, entomological indices of importance in transmission were calculated. Results showed that the main biting peaks of Anopheles nuneztovari were between 20:00 and 22:00, whereas Anopheles albimanus exhibited more variation in biting times among localities. Infection in An. nuneztovari by Plasmodium spp. (IR: 4.35%) and the annual entomological inoculation rate (30.31), indicated high vector exposure and local transmission risk. We did not detect Plasmodium-infected An. albimanus in this study. In general, low genetic and phenetic subdivision among the populations of both vectors was detected using a combination of phenotypic, genetic and environmental data. The results indicated high regional gene flow, although local environmental characteristics may be influencing the wing conformation differentiation and behavioral variation observed in An. albimanus. Furthermore, the population subdivision detected by microsatellite markers for both species by Bayesian genetic analysis provides a more accurate picture of the current genetic structure in comparison to previous studies. Finally, the biting behavior variation observed for both vectors among localities suggests the need for continuous malaria vector surveys covering the endemic region to implement the most effective integrated local control interventions.Population subdivision among several neotropical malaria vectors has been widely evaluated; however, few studies have analyzed population variation at a microgeographic scale, wherein local environmental variables may lead to population differentiation. The aim of the present study was to evaluate the genetic and geometric morphometric structure of  Plasmodium parasites that cause the disease are transmitted to humans by the female mosquitoes of approximately 40 species of Anopheles [Anopheles darlingi, Anopheles nuneztovari and Anopheles albimanus [Anopheles species and the appearance of insecticide resistant species, which affect malaria risk parameters [Malaria remains one of the most important public health problems worldwide. The nopheles . Colombinopheles , and thenopheles –7. Currenopheles . During nopheles , 9. The lbimanus , 11. Vecrameters –14. Hencrameters ; such inAn. nuneztovari and An. albimanus have identified population subdivision across their distributions [An. nuneztovari, several lineages were identified among South American populations [An. nuneztovari populations from the west-northwest and the east-northeast, attributed to physical barriers, geographic distance and ecological variation on both sides of the Andes [COI and the white gene found high gene flow and the existence of a single taxon in An. nuneztovari from UCS, northwest Colombia [An. albimanus as a single taxon in Central and South America [An. albimanus is mainly distributed on the Atlantic and Pacific coasts [An. albimanus as a metapopulation [Plasmodium spp., An. nuneztovari in UCS-northwest and in PAC-west Colombia, and An. albimanus in PAC [Population structure studies of the neotropical malaria vectors ibutions , 17. Regulations , and a nulations . In Colohe Andes . HoweverColombia . Various America , 23, and America . In Coloc coasts , 25, whec coasts , 27. Thepulation . Both sps in PAC , 29.An. albimanus and An. nuneztovari. These activities promote landscapes that impact key mosquito parameters, such as mosquito species composition, vector abundance, biting behavior and natural parasite infection, which determine malaria transmission risk at the local and regional levels [An. nuneztovari and An. albimanus at a microgeographic scale in the UCS region of northwest Colombia. These analyses may provide important information on the factors determining mosquito population variation that can influence vector transmission dynamics.Previous studies in the Colombian malaria endemic UCS suggest that anthropogenic activities such as mining, livestock production, timber extraction, small scale agriculture and wood exploitation favor the presence of the main vectors l levels , 30, 31.Anopheles specimens were collected from two localities in each of five municipalities, six sites per locality, in the malaria endemic region Urabá-Bajo Cauca and Alto Sinú (UCS) in northwest Colombia  loci were analyzed  [An. albimanus  [K) which is pre-determined [K value with the maximum posterior probability Pr (X|K) was retained and assumed to be the most probable number of clusters in that population. Twenty independent runs were performed for each value of K (K = 1 to 10) with a burnin period of 500,000 iterations and 2 million steps for Monte Carlo Markov Chain (MCMC) replications run using Structure Harvester v.0.56.3 [K were averaged using the Large K Greedy method implemented in Clumpp v. 1.1.2 [Given the geographical proximity among localities from the same municipality, specimens were grouped and analyzed by municipality for the genetic analysis. Inter and intra-population genetic diversity was estimated for 139 lbimanus . For An.6, Anu8) ,    . Genetictermined . The K vv.0.56.3 . A totalv. 1.1.2 . A Cavalv. 1.1.2  with 100v. 1.1.2 . Populatv. 1.1.2 .Na), expected heterozygosity (He), observed heterozygosity (Ho), allele richness (Rs), number of migrants per generation (Nm), inbreeding index (FIS), fixation index (FST) and balance of Hardy-Weinberg (HWE) were calculated for each MS loci using Arlequin software v. 3.5 [The number of alleles (e v. 3.5 . The nume v. 3.5 . Molecule v. 3.5 .An. nuneztovari specimens (27–30 per population) and 146 An. albimanus (26–30 per population)  , were moWing conformation among populations was compared by means of a disparity analysis, using 10,000 random permutation tests. To calculate the differences in wing conformation among populations, allometry-free variables were used as input for a Canonical Variates Analysis (CVA). To obtain Procrustes and Mahalanobis distances, the CVA analysis was used to determine whether the five geographic populations of each species could be statistically distinguished based on the relative deformation matrix , 52. MeaAn. nuneztovari and An. albimanus occurrence records throughout the endemic region, to generate an environmental resistance matrix in Circuitscape v. 3.5.8 [FST), phenotypic differentiation , environmental or cost distances and geographical distances were compared using R v. 3.3.2.Population structure based on allele frequency variation of georeferenced individual genotypes and inference of the number of populations and spatial location of genetic discontinuities between those populations was evaluated by a Bayesian clustering algorithm in Geneland 4.0.3 , implemev. 3.5.8 . Finallyv. 3.5.8 . The matPlasmodium falciparum, Plasmodium vivax VK247 and VK210. The positive pools were confirmed by a second ELISA and a nested PCR performed with DNA extracted from the mosquito abdomen and Plasmodium specific primers [Anopheles females collected per hour and per average number of collectors, expressed as the number of bites per person, per night (b.p.n) [Plasmodium positive mosquitoes out of the total number of mosquitoes analyzed, by species and locality. The annual entomological inoculation rate (EIR) per site that corresponds to the number of infective bites that a person may receive in one year [Mosquito parasite infection was tested in the pools of five mosquito heads and thoraxes by an enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies against  primers , 59. The (b.p.n) . Infectione year .An. nuneztovari showed that the ANU11 locus was not polymorphic and was thus excluded from population analyses. For An. albimanus all loci were polymorphic. In An. nuneztovari, the expected mean loci heterozygosity (He) ranged from 0.613 in El Bagre to 0.674 in Mutatá and for An. albimanus from 0.802 in Turbo to 0.840 in Arboletes  (K = 3). There was no particular association among these subpopulations and geographic location  (K = 2); however, there was no association among these subpopulations and geographic location  . Bayesialocation . Similar (526.4) . The genlocation .An. nuneztovari wings showed good repeatability in the x, y coordinates (R = 0.953), centroid size (R = 0.899) and relative deformations (R = 0.937). The Mann-Whitney test revealed significant differences for various centroid sizes in the paired comparisons. The analysis of 60 An. albimanus wings also showed good repeatability in the x, y coordinates (R = 0.981), centroid size (R = 0.992) and relative deformations (R = 0.907)  . Paired = 0.907) , mainly An. nuneztovari wing conformation showed significant differences between the following comparisons: Cáceres and Mutatá (p = 0.0145), Cáceres vs El Bagre (p = 0.0007), Tierralta and Mutatá (p = 0.0433), Tierralta and El Bagre (p = 0.0140) and Turbo and El Bagre (p = 0.0152). Discrimination among populations in the morphospace showed that the first two CV axes represented 76% of the total variation of the data  h in Bahia Cispatá and Puerto Nuevo and another from 20:00–23:00 h in Camerún-Turbo, Broqueles-Moñitos, La Arenosa and the main settlement of Arboletes  and in La Arenosa-Arboletes for An. albimanus  for An. albimanus. The infection rate was of 4.35%, given the detection of one An. nuneztovari specimen infected by Plasmodium spp., in Puerto Anchica-Montelibano, for an annual EIR of 30.31 infective bites per year (The human biting rates (HBR) in most localities were relatively homogeneous with rates up to 6 bites per person per night (b.p.n) for lbimanus . The higper year .An. nuneztovari and An. albimanus at a microgeographic scale in the UCS malaria endemic region. A previous study of An. nuneztovari in Colombia showed that this species is unique, with a population subdivision between the west-northwest and the east-northeast, which suggested high genetic differentiation and reduction of gene flow among populations caused by distance, ecological differences and the presence of geographic barriers  [An. nuneztovari subpopulations were detected that presented low genetic differentiation and high gene flow. In contrast, previous studies of An. nuneztovari in UCS using mitochondrial COI and nuclear white gene markers suggested genetic homogeneity in northwestern Colombia [An. nuneztovari, showing higher sensitivity compared with mitochondrial and other nuclear markers. Similarly, a previous study on An. nuneztovari s.l. from the Brazilian Amazon region based on the same microsatellite markers, reported the presence of three genetic lineages [An. nuneztovari in UCS.This study evaluated genetic population structure, geometric morphometrics and relevant entomological parameters for e Andes) . In the Colombia . The preColombia . The prelineages . Given oAnopheles nuneztovari geometric morphometric analysis revealed significant differences in size and wing conformation for several of the paired comparisons between populations. This species has shown high wing morphological variability previously in specimens from northwest Colombia [An. nuneztovari specimens are more likely associated with phenotypic plasticity rather than wing conformation variation as previously suggested for other anophelines [Anopheles cruzi form São Paulo state in Brazil indicated vertical population structuring of wing geometry despite the similarity among landscape microenvironments; wing shapes were distinct between lowland and hilltop populations and the wings of hilltop specimens were larger [Colombia , 63, VenColombia  and otheColombia . We founphelines . It is ae larger .An. albimanus from UCS, Bayesian analysis detected two subpopulations with low genetic differentiation, high gene flow and no evidence of geographic differences. The absence of any obvious physical and biogeographic barriers in the UCS region may explain the low genetic differentiation recorded. Previous studies at a larger scale showed moderate to low genetic differentiation between Colombian Caribbean and Pacific populations of An. albimanus and suggested that this metapopulation was influenced by demographic events during the late Pleistocene [An. albimanus were conducted with four microsatellite markers; in this study 11 microsatellites were evaluated and provided a finer resolution as shown by the Bayesian analysis that was able to identify three genetic subpopulations. We hypothesize that the low genetic differentiation and high gene flow in the present study is influenced by the dispersion capacity of An. albimanus adults, with an average flight range of 32 km [An. albimanus to nearby populations increasing gene flow and decreasing population structure.For istocene , 27. Furistocene , 24. Theistocene  and Gutiof 32 km ; in addiAn. albimanus from Arboletes in the geometric morphometric analysis. Also, the multivariate alar conformation and genetic analysis showed a slight separation of the Arboletes population. A previous study of An. albimanus populations from the Colombia Caribbean and Pacific Coasts suggested size variation occurred as a consequence of environmental change experienced during the immature stages [pH and/or oxygen availability, as detected by Soleimani-Ahmadi et al. [Of note, significant variation in centroid size was observed in all paired comparisons for e stages . We hypoi et al.  in theiri et al. .An. nuneztovari and An. albimanus across their Latin American distributions [An. nuneztovari varied according to the locality; however, its main biting peaks in most localities were between 20:00–22:00 h. In other localities of this region, a similar biting peak for An. nuneztovari was observed [An. albimanus, biting peaks also varied, but the highest was found in Arboletes between 22:00–23:00 h. These values differ from those previously reported, in 2009, for this endemic area  [An. nuneztovari and An. albimanus biting behavior was not associated with genetic population differences, and several studies have demonstrated the effects of plasticity on Anopheles species biting behavior [An. darlingi from the Amazon region found an association of genetic diversity with biting behavior not related to population structure [Plasmodium infected Anopheles when they carry out leisure activities in the open spaces of their houses, at the hours when the vectors show their higher biting activity.In addition to genetic and phenetic evaluations, entomological indices were calculated because these estimates are fundamental tools for the design of malaria vector control strategies ; they pributions , 29, 72.observed , 30, 73.behavior , 75. Simtructure . In additructure . In thisAn. nuneztovari and An. albimanus exhibited variable biting behavior among localities and suggest that the UCS human populations are exposed continuously to malaria vector bites mainly during the early part of the night; therefore, control measures should focus to reduce vector-human contact at this time. In addition, the Plasmodium infection results for An. nuneztovari indicate that even the low human biting rate (< 2 b.p.n.) is sufficient to maintain malaria transmission, and although, An. albimanus is an important malaria vector, it was not detected infected; this can be due to the low infection rates usually detected in endemic regions of Colombia [In conclusion, results of entomological parameter estimations indicated that Colombia . FurtherS1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.


Dasyurus hallucatus), an Endangered marsupial carnivore. We used next generation sequencing methods to genotype 10,191 single nucleotide polymorphisms (SNPs) in 352 individuals from across a 3220‐km length of the continent, investigating patterns of population genomic structure and diversity, and identifying loci showing signals of putative selection. We found strong heterogeneity in the distribution of genomic diversity across the continent, characterized by (i) biogeographical barriers driving hierarchical population structure through long‐term isolation, and (ii) severe reductions in diversity resulting from population declines, exacerbated by the spread of introduced toxic cane toads (Rhinella marina). These results warn of a large ongoing loss of genomic diversity and associated adaptive capacity as mammals decline across northern Australia. Encouragingly, populations of the northern quoll established on toad‐free islands by translocations appear to have maintained most of the initial genomic diversity after 16 years. By mapping patterns of genomic diversity within and among populations, and investigating these patterns in the context of population declines, we can provide conservation managers with data critical to informed decision‐making. This includes the identification of populations that are candidates for genetic management, the importance of remnant island and insurance/translocated populations for the conservation of genetic diversity, and the characterization of putative evolutionarily significant units.Mammal declines across northern Australia are one of the major biodiversity loss events occurring globally. There has been no regional assessment of the implications of these species declines for genomic diversity. To address this, we conducted a species‐wide assessment of genomic diversity in the northern quoll ( The northern quoll is a small (<1500 g) generalist marsupial predator  in Australia , and populations are being actively managed by various jurisdictions. Because the spread of cane toads has so far proved impossible to halt, current management actions are aimed at mitigating the impact of toads. For example, the Northern Territory government established insurance populations on two offshore islands in 2003  as well as loci that are significantly associated with the presence of cane toads. We predict that our genomic analyses will identify (i) population genomic structure resulting from vicariance across biogeographical barriers; (ii) patterns of drift associated with isolated island populations and (iii) large impacts of cane toads, or other drivers of population decline, on population genomic diversity, with population bottlenecks in toad‐exposed populations potentially contributing to a reduction in genetic diversity. Our temporal replication will also enable us to directly observe the effect of establishing insurance populations on offshore islands in the short term (16 years). We suggest that this study will provide a quantitative example of the impacts that invasive species can have on the patterns of genomic diversity in native species, the conclusions of which will benefit attempts to mitigate the impacts of invasive species.22.1We collated tissue samples from across the entire species distribution from multiple personnel who had been involved in mammal trapping between 1993 and 2019, covering the regional jurisdictions of Queensland (QLD), the Northern Territory (NT) and Western Australia (WA) Figure . Four ofFor each sampling locality, we determined whether cane toads were present at the date of collection by cross‐referencing with published timelines of cane toad spread  were prepared and extracted in plate format (Qiagen DNeasy 96 Blood & Tissue Kit) following the standard protocol with an extended lysis (incubation at 56°C for 2 h then reduced to 37°C overnight). Samples consisting of dried blood spots on filter paper were extracted with a slight modification to the lysis step  digestion using PstI and NlaIII, (ii) ligation with one of 48 unique inline barcoded adapters compatible with the restriction site overhang, (iii) manual sample pooling, (iv) DNA purification (QIAquick PCR Purification Kit followed by SPRIselect paramagnetic beads), (v) size‐selection targeting fragments of 280–375 bp in size  and (vi) a PCR (polymerase chain reaction) amplification step where one of two multiplexing index primers was added. Indexed libraries were pooled together and loaded onto flow cells for 150‐bp single‐end sequencing on either an Illumina NextSeq 500 (three plates) or a NovaSeq 6000 (two plates) platform.Double‐stranded DNA concentrations were quantified using a Qubit 3.0 Fluorometer and normalized to 200 ng DNA in 25 μl, and then arranged on 96‐well plates, where each plate contained two within‐plate technical replicates, one technical replicate from each of the other plates and one negative control (blank) well. Plates were sent for double‐digest restriction‐associated DNA (ddRAD) sequencing at the Australian Genome Research Facility in Melbourne, Victoria. An optimal combination of two restriction enzymes was determined using three establishment samples (broadly representative of the species distribution), with 2.3process_radtags function of stacks  >0.6 , and then pruned SNPs based on linkage disequilibrium via the “SNPRelate” package  the level of missing data; (ii) observed heterozygosity; (iii) minor allele counts and (iv) whether they showed signals of being under selection. We filtered individuals based on (i) the level of missing data and (ii) relatedness to other genotyped individuals within sampled localities. To filter SNPs and samples based on the level of missing data, we used a custom script to iteratively remove individuals and SNPs with decreasing missing data thresholds until no SNP had more than 3% missing data and no individual had more than 20% missing data. We then removed any SNP with a minor allele count <3 . One of each pair was then removed from all further analyses. The data set was then further pruned to remove highly related individuals and SNPs showing signals of putative selection, both of which can bias analyses that were developed using the assumption of unrelated individuals and neutrally evolving loci.To ensure that relationships between individuals could be accurately inferred from the data, we used these SNPs and samples to construct a hierarchical clustering dendrogram based on genetic distance, with visual examination of the dendrogram confirming that all technical replicates paired closely together on long branches Figure . We alsoTo estimate relatedness between individuals, we used the recently developed method‐of‐moments estimate for the kinship coefficient , and identification of SNPs that were significantly associated with the presence of cane toads (via a latent factor mixed model). To identify outlier SNPs that showed signatures of diversifying selection, we first computed an ancestry matrix for each individual using the methods implemented in the LEA package  from the data set that were identified using either method. This produced a selectively neutral data set for population genomic analyses, many of which rely on assumptions of neutrality. While future work may attempt to further elucidate and understand the patterns and genomic architecture of selection in response to cane toad invasion, the set of candidate loci identified here  providesAfter all filtering steps had been conducted, we retained a total of 10,191 SNPs and 352 individuals for the remaining analyses. The overall level of missing data for the final filtered SNP‐by‐individual matrix was 1.2%.2.5A), effective number of alleles (AE), observed heterozygosity (HO), expected heterozygosity (HE), Wright's inbreeding coefficient (FIS) and the locus polymorphic index (PE). The East Alligator site was separated into toad‐naïve (2003) and toad‐exposed (2014 and 2017) subsets prior to calculations being made. Similarly, the historical (2005) samples from Astell Island were separated from the more recent (2016) samples. Calculations were made using the “gstudio” package , and analysing the data set using the core program “Populations” with all missing sites removed. The heterozygosity estimates and standard errors in the subsection of the summary output titled “# All positions (variant and fixed)” were recorded, and Pearson's product moment correlation coefficient was used to determine the strength of the relationship between values of autosomal heterozygosity and SNP heterozygosity.Using the filtered SNP data set, we calculated mean values of standard genomic parameters for each sampling locality within each treatment, including the number of alleles  of ancestral populations, allowing for independent investigation of values for k that have low cross‐entropy metrics  across the landscape. To prevent interpolation of genomic structure beyond the geographical range of the northern quoll, we limited the interpolation surface to the known historical (pre‐European) distribution of the species.To assign individuals to ancestral population genomic clusters, investigate patterns of admixture between populations and assess hierarchical population structure, we used the alternating projected least squares algorithm implemented in the “tess3r” package  and total gene diversity (HT) to improve inference of demographic history among populations . A Mantel test was used to test the significance of geographical distance on standardized genomic differentiation, using 10,000 permutations. Isolation‐by‐distance among all mainland individuals was also investigated using spatial autocorrelation of multilocus genotypes, plotted on a correlogram. Pairwise geographical distances between individuals were calculated from latitude and longitude observations for each individual via the earth.dist function of the “fossil” package  or toad invasion and establishment  had on genomic diversity metrics, we constructed individual generalized linear models for each metric, where loci were samples and the predictor variable was a factor corresponding to the year of the survey.To test the relative influence of vicariance and cane toads on genomic diversity among our natural treatments , we constructed four generalized least square models using the “nlme” package  using three methods, of which one is appropriate for estimating recent  trends and two that are most useful for deeper time  trends.To identify patterns of historical demography at a locality, including the potential influence of cane toads on population size, we investigated past changes in effective population size (Ne), we used the version 1.1 software tool  file output from angsd was split into individual VCF files for each sampling locality, with plink version 1.9  were plotted and the x‐axis scaled to time assuming a mean generation time of 2 years  that estimates demographic histories based on the composite likelihood of a population's folded site frequency spectrum , we constructed a folded site frequency spectrum using genotype likelihoods estimated by the angsd software package. All filtering criteria based on mapping and read qualities were the same as previously described, with the filters for polymorphic loci removed to capture information on all monomorphic and polymorphic nucleic sites. By using likelihoods, the site frequency spectrum could be built from between 121 million and 565 million nucleic sites per locality. The site frequency spectrum was loaded into the folded blueprint file provided with the software, with an appropriate mutation rate per site per generation , whereas others were considerably more diverse    . Autosomameters) .z = 38,952, p < .001). Analysis of spatial autocorrelation of genotypes identified significant values of the autocorrelation coefficient r persisting for distances exceeding 500 km (FST = 0.5–0.9), probably due to its very low level of diversity (FST had a significant (p < .001) level of differentiation, based on 1000 bootstraps across loci, even where the same locality was sampled only a few years apart . The FST values were very similar to the GST″ values .Patterns of pairwise genomic differentiation across the landscape broadly corresponded to the pattern of geographical distance between localities , with thg 500 km . Seawateiversity . All pai″ values , with th3.3HE , AE  and PE  all differed significantly among the three  treatments. There was no significant difference in FIS among treatments . Tukey post hoc tests showed that toad‐naïve localities (mean HE = 0.049) had significantly higher HE than toad‐exposed (mean HE = 0.024) and island (mean HE = 0.019) localities , with no significant difference between island and toad‐exposed localities . Values of HO, AE and PE among treatments showed similar patterns to HE (AE = 1.072) had significantly higher AE than toad‐exposed (mean AE = 1.035) and island (mean AE = 1.03) localities , with no significant difference in AE between island and toad‐exposed localities .We found that three of the analysed population genomic parameters varied significantly among treatments Figure . The gens to HE . For exaFIS value for the SNP data set increased from 0.01 to 0.04 . This is despite only minor changes in HE  and HO  over the same period. At the East Alligator locality between 2003 and 2017, where cane toads invaded between sampling periods, there was a significant reduction in PE  and HE , but no change in HO  or FIS , based on the SNP data. Heterozygosity estimates calculated using the SNP data broadly agreed with heterozygosity estimates calculated from autosomal data . However, the estimates calculated from SNP data showed slightly greater stochasticity than the autosomal estimates, probably due to sample size variation among populations/years. For example, SNP estimates of HE at East Alligator increased slightly from 2003 to 2014, and then decreased from 2014 to 2017, whereas the autosomal estimates show a consistent reduction through time in HE and HO, with both values declining from 7e−5 to 6e−5 between 2003 and 2014, and no change observed between 2014 and 2017. As the autosomal estimates are likely to be a more robust measure of heterozygosity, we suggest this pattern is more realistic.Temporally replicated localities showed some significant changes through time, corresponding with isolation on islands or toad invasion and establishment Table . For exasnep suggested that there have been strong declines in the effective population sizes (Ne) of many northern quoll populations for much of the last century  tended to show smaller but somewhat more consistent population sizes through time than mainland populations. The toad‐exposed QLD localities were characterized by small effective population sizes for much of the past century, with noticeable reductions in Ne after arrival of cane toads into Mareeba, Hope Vale and the combined Hope Vale/Black Mountain localities. However, there did not appear to be enough genomic diversity in the sequence data for the Weipa locality to provide snep with the information content required to see past the arrival of cane toads to the area.Investigation of historical demographics using y Figure . LocalitNe of island populations around the time of isolation due to sea‐level rise after the end of the last glacial maximum . The EBSPs showed considerably less certainty with estimated population sizes, highlighted by the large confidence intervals for all localities that were analysed and mediocre convergence (posterior effective sample size) in some localities . Results suggested recent population declines in most populations, consistent with the outputs from snep and Stairway Plot 2 . This was most pronounced for populations in the highly remote and rugged areas of the western Kimberley, where threats may have developed more recently.Stairway plots showed expected fluctuations in m Figure , suggesty Plot 2 , with thy Plot 2 . While w4While biodiversity declines are well recognized as a global problem, the consequences of these declines for patterns of genomic diversity are often poorly known. Using genomic data to infer recent and long‐term historical demographic trends can help identify the extent to which loss of genomic diversity itself is threatening species' persistence. Northern Australia's vast savannas are the location of some of the world's most dramatic recent declines of mammals   in toad‐exposed populations, it is worth noting that this metric does not provide information on the cumulative level of inbreeding in a population, but rather detects nonrandom mating in the most recent generation  could help quantify the relative importance of recent (<50 years) changes in landscape management as drivers of decline. For example, while feral cats have probably been present for ~150 years , Groote Eylandt (NT), eastern Kimberley (WA) and Cape York (QLD), respectively  that biogeographical barriers separate subregions each containing substantial amounts of unique genomic diversity; (ii) that remnant island populations harbour only a subset of the diversity present within each subregion; (iii) that large insurance populations on islands or in intensively managed/fenced safe havens can maintain high levels of genomic diversity  and (iv) that declines induced by cane toads are dramatic, but play out against broader declines resulting from other anthropogenic stressors and threats. With the exception of cane toad impacts, these considerations are likely to be similar for many of the widespread faunal species within the Australian monsoonal tropics . These challenges span agriculture, biomedicine, the environment and industry, as well as extending to relevant international endeavours.A research collaboration was developed with researchers around Australia, who provided tissue samples for genetic analysis. All collaborators were offered the opportunity to contribute to the manuscript as co‐authors, and the results of research are available for the broader scientific community through online databases and open access publishing. The research addresses a priority concern, in this case the conservation genetics and management of the threatened northern quoll, and findings will be incorporated into management actions for the species. Bioplatforms Australia is a nonprofit organization that supports Australian Life science research by investing in state‐of‐the‐art infrastructure and expertise in genomics, proteomics, metabolomics and bioinformatics. Investment funding for Bioplatforms Australia is provided by the Commonwealth Government National Collaborative Research Infrastructure Strategy. The Bioplatforms Australia Oz Mammal Genomics Initiative is one of many national collaborative projects that generate high‐impact data and knowledge resources to support some of Australia's biggest scientific challenges (Appendix S1Click here for additional data file.


Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae), is a globally distributed agricultural and horticultural insect pest. Despite the economic importance of this insect in Ethiopia, its genetic diversity and demographic history are poorly understood. We examined the nucleotide variation of the mitochondrial cytochrome c oxidase subunit I (COI) gene fragment of 74 H. armigera individuals from six collection sites in Ethiopia. We recorded 15 COI haplotypes in H. armigera, ten globally shared and five exclusive to Ethiopia . Haplotype HaET1 was the most widely geographically distributed and frequent (71.62%). Analysis of molecular variance (AMOVA) revealed a high and significant level of variation within H. armigera populations (STθ = −0.0135). Negative values of the neutrality test and nonsignificant index of mismatch distribution supported the demographic expansion of H. armigera populations in Ethiopia; furthermore, this was also supported by the nonsignificant values of the sum of squared deviations (SSD) and raggedness index (r). The high genetic variation and population expansion of H. armigera have immense implications for devising locally adapted management strategies in area‐wide integrated pest management IPM programs. However, a comprehensive study of H. armigera genetic diversity and population structure using various molecular markers is needed for future confirmation.The Old World bollworm,  H. armigera haplotypes based on COI gene region showing novel Ethiopian haplotypes (a) and its relation with other countries haplotypes (b)The TCS of  Then, they were preserved in absolute ethanol and labeled with their host plants, site, date of collection, and GPS coordinates and stored at −20°C until required for DNA extraction.2.2Genomic DNA was extracted using the established protocols described by Behere et al.  with mod2.3® Blend Master Mix (Solis BioDyne), 0.5 μl of each 10 µM primer  , and the alignments were verified visually. A TCS network  GenBank (www.ncbi.nlm.nih.gov/genbank/) repository by blasting the gene sequences for TCS network construction. These sequences were obtained from 14 countries: one each from the Dominican Republic , haplotype diversity (dH), and nucleotide diversity (pi) were used to estimate genetic variability between haplotypes using DnaSP v. 5.10 program .The number of haplotypes  (Table We generated 402‐bp mtDNA COI gene sequences of 74 dH), which could be attributed to its low sample size, followed by PJR  ranged from 0.00083 to 0.00329 with an average of 0.00242  polymorphic sites, followed by the ChAD and TTK populations Table . The seqJR Table . The Hd3.2H. armigera haplotypes were identified from the alignment of all 74 COI gene sequences from the six populations, of which five haplotypes were HaET4 from the ChAD population, HaET7 from the NJR population, HaET10 from the PJR population, HaET14 from the SDD population and HaET15 from the TTK populations, which were represented by a single individual  negative, with values of −1.220 , indicating deviation from evolutionary neutrality, and −1.102 , indicating an excess of rare haplotypes in the population compared to what is expected under a neutral model of evolution  values of SSD and raggedness index for all (except the TKC population) of the populations, indicating the presence of nonequilibrium and a population expansion event, and the data have a relatively good fit to a model of population expansion in H. armigera , which occurs in many areas of Africa, including Kenya, Cameroon, South Africa, Tanzania, and Uganda; the H. fletcheri (Hardwick) (Lepidoptera: Noctuidae), which was reported from Africa, including Sudan  (Lepidoptera: Noctuidae), which is also present in most of Africa, such as Eritrea, Egypt, Sudan, Algeria, and Chad  and nucleotide diversity (pi) values of 0.821 and 0.0028, respectively, were detected by Leite et al. . Recently, Arnemann et al. . The low haplotype diversity and nucleotide diversity observed among H. armigera populations in the present study can be explained by the dispersal capacity  between countries when using similar molecular markers, which they stated could be due to the high mobility in H. armigera. Additionally, the low haplotype diversity in our findings also reflects the small number of H. armigera specimens and populations used.Our data indicated lower haplotype diversity and higher nucleotide diversity when compared to several previous reports, reflecting the presence of few segregating sites across different haplotypes of the populations. In populations of e et al.  in Brazig et al.  in Chinan et al.  reported4.2H. armigera populations using COI gene sequences. The majority of the haplotypes were randomly distributed throughout the six populations. The presence of unique haplotypes across the populations may be due to the small sample size used in this study. Leite et al.   programs in the region.The results of this study indicate a relatively low genetic diversity and demographic expansion of 4.5H. armigera populations is critically important for designing effective and sustainable H. armigera management programs. Our results revealed a total of 15 H. armigera haplotypes for the COI gene sequences of 74 individuals from six populations with lower haplotype diversity and higher nucleotide diversity. Demographic analysis using neutrality tests and a mismatch distribution analysis with COI mtDNA markers revealed expansion of H. armigera within the Ethiopian territory. However, a comprehensive study on H. armigera genetic diversity and population structure using various molecular markers is needed for future confirmation.Understanding the genetic diversity and demographic history of Tarekegn Fite: Conceptualization ; Data curation ; Formal analysis ; Investigation ; Methodology ; Software ; Validation ; Visualization ; Writing – original draft ; Writing – review & editing . Tadele Tefera: Conceptualization ; Funding acquisition (lead); Project administration ; Resources ; Supervision ; Validation ; Writing – review & editing . Muluken Goftishu: Data curation ; Methodology ; Supervision ; Writing – review & editing . Tebekew Damte: Conceptualization ; Data curation ; Supervision ; Visualization ; Writing – original draft ; Writing – review & editing .The authors declare no conflicts of interest.


Sorbus domestica at its northern range margin, in western Switzerland. New microsatellite data were combined with published data from across the European distribution of the species. Analyses revealed the presence of mainly one of two species-wide ancestral clusters, i.e., the western European cluster, with evidence that it consists of two cryptic sub-clusters. Average pairwise FST of 0.118 was low across the range, and only allelic richness was reduced in the northern margin compared to more southern and southeastern areas of Europe. Based on our finding of considerable genetic diversity of the species in western and northern Switzerland, we suggest that a national propagation program should focus on collecting seeds from natural, high-density tree stands and propagate locally. More generally, our study shows that rare tree species in marginal areas of their distributions do not necessarily have low genetic diversity or heightened levels of inbreeding, and in those cases probably need no assisted migration in efforts to propagate them.Climate change has aroused interest in planting warm- and drought-adapted trees in managed forests and urban areas. An option is to focus on tree species that occur naturally, but have centers of distribution in warmer and drier areas. However, in order to protect the species pool of genetic diversity, efforts of planting and promotion should be informed by knowledge on the local genetic diversity. Here, we studied the macro- and micro-scale population genetic structure of the rare European fruit tree  The service tree is a Mediterranean species occurring from Spain to northwestern Turkey, reaching its northern distribution limit in central Europe  and an eastern (Austria) cluster  and northwestern Switzerland (N = 73) were collected in 2020 (2) in liquid nitrogen, followed by immediate DNA extraction using the DNeasy Plant Mini Kit . We genotyped all individuals at the seven microsatellite loci used by i.e., MSS5, MSS16, CH01h10, CH01h01, CH02c09, BGT23b and MS14H03  among all European localities with 10’000 permutations. We estimated allelic richness (AR) by rarefaction for each locality with at least 10 individuals with FSTAT 2.9.4 (Ho) and expected heterozygosity (He), the number of alleles, the inbreeding coefficient (FIS) and the number of private alleles by using GenAlEx. Finally, we tested for isolation-by-distance by performing a Mantel test between pairwise geographic and Euclidean genetic distances among individuals within Switzerland. Testing was performed using the package vegan  as the best supported scenario for the complete dataset , separat as the bThe two leading principal component (PC) axes for the complete dataset accounted for 13.0% and 9.0% of the total variation, respectively. While populations generally sorted out by longitude along PC1, there was substantial variation within populations . NeverthFST) being 0.118 , consistent with the presence of both genetic clusters in this region  and localities of the southern and southeastern species range (N = 10), with northern localities having lower allelic richness . The number of alleles identified across the seven loci was 88  are highlighted in bold. The full names of localities are presented in Table 2.Significant Click here for additional data file.10.7717/peerj.14397/supp-5Figure S1A—Allele frequencies for all newly genotyped individuals from CH NW. B—Allele frequencies of the three re-genotyped individuals used by Click here for additional data file.10.7717/peerj.14397/supp-6Figure S2A—Optimum at K=2 for the dataset including all European samples . B—Optimum at K=2 for the dataset including samples from Switzerland and surrounding areas . C—Optimum at K=2 for the dataset including Swiss samples only .Click here for additional data file.


Dactylorhiza hatagirea (D. Don) Soo is medicinally important herb, which is widely used in ayurveda, unani, and folk/traditional medicine system to cure diseases. Due to its immense ethno-botanical properties, the trade of D. hatagirea is estimated to be USD 1 billion/year in India. Unfortunately, due to overexploitation of the herb from the wild, has resulted in dwindling of its populations in their natural habitats, which has led to its critically endangered status. Molecular genetic studies are still scarce in D. hatagirea, therefore, in current study, genetic diversity and population structure analysis was carried out of 10 populations  collected from three cold desert regions (2527 m–3533 m amsl) of Himachal Pradesh. Mean observed heterozygosity (Ho) and expected heterozygosity (He) was recorded 0.185 and 0.158. The maximum values for Fst (fixation index) and Nm (gene flow) were recorded 0.945 at locus KSSR14 and 1.547 at locus KSSR 4 respectively. Mean genetic differentiation (Fst) coefficient was estimated to 0.542. Overall, low levels of genetic diversity was recorded in the populations of D. hatagirea, might be due to habitat specificity , restricted distribution and high anthropogenic activities. However, two populations viz., Bathad and Rangrik were recorded with high diversity and largest number of private alleles, stipulates that these populations might have high evolutionary significance and response to selection. Dendrogram analysis revealed that the populations of D. hatagirea were clustered into four major clusters, which was supported by Bayesian based STRUCTURE predictions. Clustering pattern of majority individuals of different populations revealed consistency with their geographic origin. Outcomes of current study reveals the status of genetic diversity and population structure of endangered D. hatagirea, which can be futuristically utilised for appropriate planning of conservation strategies. This region is the longest bioclimatic gradient which covers approximately 2800 km long area, and is around 220 km–300 km wide along with altitude ranges from 200 to 8000 m2. The Indian Himalayan region biodiversity, represents plentiful of angiosperms  and gymnosperms 3. Abundant flora of the region also acts as “Carbon Sink” which plays an important role in reducing the impact of global warming. Number of plants from IHR has been reported to have immense medicinal and therapeutic values and the Himalayan plants have been valued since Vedic period (Rig Ved 4500-1600BC). It is estimated that, ~ 90% of the medicinally important plant are extracted from wild and 69% of the plant material is collected using destructive harvesting4. Increased anthropogenic activities in the Himalayas is considered one of the critical factor responsible for decline in the availability of medicinally important plants and their populations.Indian Himalayan Region (IHR) is a hotspot for biodiversity due to tremendous variation in climate and altitudinal rangesDactylorhiza hatagirea (D. Don) Soo belongs to Orchidaceae family, is native to India, Pakistan, Bhutan, Afghanistan, Nepal, and Tibet, is of high value due to its medicinal and ornamental properties. The plant is found at an altitudinal range of 2500–5000 masl in alpine meadows and grassy slopes5. It is a monocotyledonous, terrestrial, perennial herb with erect hollow stem and bears purple flowers with green bracts. It is a slow growing, habitat specific species and regenerate poorly due to its requirement of association with mycorrhizae fungi for germination6. Bioactive compounds of D. hatagirea, which are of medicinal importance are dactyloses A, B, and dactylorhins A to E. Bulbous roots of the plant is used to prepare “Salep” which is utilized to cure wounds, diarrhoea, dysentery, cough, fever, burns and fractures7. The plant has high annual demand (5000 tonnes) and price of dried rhizome is ~ 72 US $ per kg, which has resulted in its overexploitation from its natural/wild habitat4. Moreover, it has also been observed that D. hatagirea in wild shares habitat with highly traded species like Ophiocordyceps sinensis and with expanding species like Polygonum polystachyum, which is also affecting negatively the number of individuals in a population of D. hatagirea8. Biodiversity loss is among the most disastrous environmental problem and world is now facing the challenges required to reduce the rate of loss of biodiversity9. Moreover, due to the increase in the demand of natural compound in the healthcare system, natural populations of medicinally important plants like D. hatagirea are under tremendous pressure due to overexploitation, indiscriminate extraction and unsustainable management.D. hatagirea is very challenging due to the non-availability of genetic resources. Till date, only 17 nucleotide sequences10 and one transcriptome study has been reported for D. hatagirea11. Few genetic diversity studies using RAPD and ISSR markers from Ladakh region recorded moderate level of diversity among natural populations13. Variability among natural populations of D. hatagirea from Uttarakhand was also analysed using morphological, biochemical and isoenzymes analysis14. However, no population structure and genetic diversity study has been reported from the cold desert regions of Kullu, Kinnaur, Lahaul and Spiti valley of Himachal Pradesh, where D. hatagirea is among the most exploited medicinally important plant for domestic usage and is under increasing demand for export16.Understanding of population structure and genetic diversity pattern is important for sustainable utilisation and planning of conservation strategies for critically endangered plant species. Moreover, maintaining genetic variation in a population is one of the most important objective for conserving critically threatened and endangered species. Whole genome sequencing of critically endangered species to create genomic resources are paramount to understand the genome-wide diversity between and within species, which is beneficial for conservation practitioners. Unfortunately, planning of conservation strategies in critically endangered D. hatagirea, and samples were collected from 10 locations representing three regions  of Himachal Pradesh, India. Fifteen stable and highly polymorphic SSR (Simple Sequence Repeats) markers developed in our previous study were utilised for genetic diversity characterization and population structure analysis of collected natural populations of D. hatagirea10. SSRs were utilised as they are the most ubiquitous and hypervariable type of repeat elements, which are abundant in the plant genomes. Moreover, SSR markers have added advantages for precise estimate of genetic diversity over other markers because of their co-dominant nature, high reproducibility and transferability17. Accordingly, realising the medicinal, ornamental and industrial applications of D. hatagirea along with its critically endangered status in the Himalayas, current study was performed with an aim to understand the diversity pattern and population structure of natural populations of D. hatagirea for their sustainable management, effective conservation strategy planning and genetic resource management for future breeding programmes.In current study, attempts were made to perform extensive geographic sampling of natural populations of Na) varied from minimum 1.1  and maximum of 2.0 (KSSR16), number of effective alleles (Ne) ranges from minimum 1.047 (KSSR14) and maximum of 1.718 (KSSR16), Shannon's information index (I) ranges between 0.05–0.599, observed per locus heterozygosity (Ho) ranges from 0.00 (KSSR10) to 0.623 (KSSR16) with mean of 0.185 and expected heterozygosity (He) varied from 0.032 (KSSR14) to 0.411 (KSSR16) with mean 0.158, respectively. The Polymorphic Information Content (PIC) of 15 markers varied from minimum of 0.0939 (KSSR18), 0.1411 (KSSR37) to maximum of 0.5499 (KSSR12), 0.6279 (KSSR16) with an average of 0.3321 (Table Fst) coefficient and gene flow (Nm) analysis at each locus using F-statistics revealed significant differences. The maximum values for Fst and Nm were recorded 0.945 at locus KSSR14 and 1.547 at locus KSSR 4, respectively. Mean genetic differentiation (Fst) coefficient was estimated to 0.542 for 15 locus ranging from 0.139 (KSSR4) to 0.945 (KSSR14) and mean gene flow recorded was 0.363 ranging from 0.015 (KSSR14) to 1.547 (KSSR4)  from three cold desert regions  of Himachal Pradesh was performed using six di- and nine tri-nucleotide SSR markers. Initially, for screening of SSR markers, two random individuals from each population were utilised. The number of different alleles (D. hatagirea (Table PPL) across 10 populations was found moderate (42.67%) which ranged from 20.00% (Chhitkul) to 66.67% (Mane) with only three population revealing PPL more than 50% . The number of alleles (Na) per population ranges from 1.200 (Chhitkul) to 1.733 (Mane) with an average of 1.453 and number of effective alleles (Ne) varied from 1.139 (Chhitkul) to 1.447 (Tosh) with a mean of 1.274. The observed heterozygosity (Ho) varied from maximum of 0.293 (Mane and Sichling) and minimum of 0.080 (Sangla) and expected heterozygosity varied from (He) 0.235 (Mane) and 0.081 (Chhitkul) with a mean of 0.185 and 0.158 across 10 populations, respectively. Further, the mean value of Shannon's Information Index (I) was 0.239 with a range of minimum of 0.119 (Chhitkul) and maximum 0.363 (Mane). The fixation index (F) with an average of − 0.126 along with range varied from − 0.084 in Rangrik to 0.242 in Sangla was recorded (Table 18.Population level genetic diversity parameters were found considerably different across 10 populations of ea Table . The aveoderate 4.67% whicoderate 4.67% whicP < 0.001) difference was recorded for the genetic differentiation i.e. Fst between the different pairs of 10 populations, which varied from 0.042 between Sichling and Mane to 0.648 between Bathad and Chhitkul. However, gene flow (Nm) values ranges from 0.072 between Chhitkul and Bathad to maximum of 3.071 between Shakoli and Chhitkul  indicating isolation-by-distance phenomena in D. hatagirea populations  population was found intermixed with these two populations. Cluster II represented individuals from Bathad population (Kullu) and cluster III represented majority of individuals from Shakoli , Tosh (Kullu), Sangla and Chittkul (Kinnaur) , Kullu (Tosh), and Lahaul valley (Shakoli) were grouped together in one group and populations from Spiti valley  were grouped together. Further, the maximum likelihood clustering with K = 4 revealed that Bathad population from Kullu region and Rangrik population from Spiti valley belongs to different groups, however, one individual from Mane and Giyu revealed intermixing  Fig. . The Priing Fig. .Figure 219. Orchids are the largest plant families which are commercially traded for various purposes in local, national and international markets. D. hatagirea is among the widely utilised and traded Orchid around the world for its potential medicinal properties and health supplements. It has been estimated that approximately 6200 to 31,000 kgs of D. hatagirea is harvested annually from the North-Eastern Himalayan region of India20. Unfortunately, each kilos of the D. hatagirea comprised of around 100 individuals and much of the procurement, harvesting and trade is done illegally and unsustainably, results in its critically endangered status with huge conservation concerns7. Understanding the genetic diversity of endangered plant species is of utmost importance, because high genetic diversity is crucial for maintaining the fitness and survival of natural populations, which also ensure the presence of pre-adapted genotypes21. Moreover, information related to genetic diversity present within and among populations of endangered plants like D. hatagirea is important to establish the conservation strategies22.The drastic changes in the environmental conditions due to climate change and overexploitation by human’s intervention, the risks of extinction of various endangered plants of Himalayan region has increased many timesD. hatagirea populations from three cold dessert regions of Himachal Pradesh was investigated, because the plant is considered as model to study orchids and their immense pharmacological and therapeutic properties like anticancer, antioxidants and activation of immune system to fight infectious diseases23. Despite the medicinal and economical importance of D. hatagirea along with its critically endangered status, only few molecular studies using ISSR and RAPD markers have been carried out to understand the genetic diversity and population structure pattern of the D. hatagirea populations. However, SSR markers which are widely utilised and recommended for diversity analysis due to their co-dominant and high polymorphic nature has not been used till date to analyse the diversity pattern of D. hatagirea populations in the cold desert regions of North-Western Himalayas24.In current study, genetic diversity of Ho = 0.185 and He = 0.158) was recorded in the populations of D. hatagirea which was also reported in earlier studies where genetic diversity of D. hatagirea from Ladakh region was estimated using ISSR and RAPD markers13. The low genetic diversity in few populations of cold desert regions of Kullu, Kinnaur and Lahaul Spiti valley stipulates that the species is under serious threat of extinction in these regions25. Moreover, the low genetic diversity recorded might be because of the isolated populations, habitat specificity, restricted distribution and high anthropogenic activities26. Moreover, the populations of D. hatagirea in alpine meadows ecosystem, prefers humid laden undulating habitat and its population density in alpine meadows usually ambit between 0.70 and 2.43 individuals/m226. Another possible reason for the low genetic diversity is the small size of the populations and large spatial distance between the populations which hinders the gene flow between the populations, results in self-pollination and inbreeding24 In case of D. hatagirea, selfing and crossing among related individuals has been reported12. All these factors might have resulted into the reduced life span, and health of D. hatagirea individuals with exhausted capacity to grow population size27.In current study, overall low levels of genetic diversity  at population level was recorded uniform for majority of populations and was moderated to high in only three populations viz., Mane, Rangrik, Sichling from Spiti valley and one Tosh from Kullu valley. The genetic diversity pattern in these populations indicates low level of human interferences/disturbance in comparison to other populations. Further, individuals from Spiti valley represents the hub for genetic diversity of D. hatagirea. However, low levels of genetic diversity in other populations might be due to overexploitation, habitat destruction, poor regeneration due to its specific requirement of growth association with mycorrhiza fungi for germination. Fixation index (F) revealed that five populations , majority from Lahaul and Spiti valley have negative values, indicating cross pollination or outcrossing and other populations  from Kinnaur, Kullu and Spiti valley revealed excess of homozygotes associated, which indicates inbreeding24.Current study has reported for the first time the genetic diversity pattern of the 10 populations representing forty-eight individuals from three cold desert regions of Himachal Pradesh. The study reveals the overall variation in the polymorphism level between these populations. Overall, genetic diversity , which indicates that genetic differentiation is due to genetic drift29 and it might be an important factor which is influencing the genetic structure of D. hatagirea populations in these regions of Himachal Pradesh. Moreover, Mantel test analysis revealed weak positive correlation between the genetic diversity and geographic distance between populations of D. hatagirea. Therefore, the differences in the genetic differentiation might be due to few geographic barriers, resulting in isolating populations in different gene pools of D. hatagirea. Further, AMOVA analysis revealed that high molecular variance was recorded among populations and low within populations, which further stipulates habitat fragmentation due to human interventions and anthropogenic activities, leading to low genetic diversity within populations13. Moreover, lower genetic variation within populations might be due to vegetative propagation of D. hatagirea in the local regions of Western Himalayas.Two important parameters viz., the genetic differentiation coefficient and gene flow plays an important role to understand the genetic structure of the populations and these two parameters are negatively correlatedD. hatagirea. The distribution pattern of majority individuals of different populations on the basis of these analysis revealed consistency with their geographic origin. The UPGMA analysis grouped the populations into 4 major clusters wherein, cluster I and IV represented population from Spiti valley; cluster II represented population from Kullu and cluster III revealed grouping of populations form Kinnaur, Kullu and Lahaul valley. Despite the long distance between the populations which were represented in cluster III, stipulates low levels of gene flow (mean Nm = 0.363) among them, might be due to collection of D. hatagirea from one region and its introduction to the other region. Interestingly, two populations viz., Bathad and Rangrik might be of completely different origin as compare to other populations, also possessed large number of private alleles, and might have role in response to selection and of high evolutionary significance18. Structure analysis also clearly suggests that individuals of these populations belong to four major clusters with admixture of few individuals in which populations from Spiti valley seems to be different from rest of the populations. Furthermore, PCoA was also found similar to STRUCTURE results which validate the UPGMA tree. Small admixture retrieved in the structure stipulates the low gene flow (mean Nm = 0.363) among some individuals of 10 populations. Overall, above analysis revealed that it is most likely that majority of the D. hatagirea populations in these regions might have been distributed via anthropogenic method and propagated vegetatively, resulted in low population differentiation, gene flow and are genetically similar  from three regions  of Himachal Pradesh were collected. Collection of plant material was done without disturbing the natural habitat of the plants and according to the institutional and national guidelines. Moreover, the permission was taken from the Department of Forest, Himachal Pradesh, India and all the samples were collected under their guidance. The formal identification of the plant material was done by ethnobotanist Dr P. K. Sharma, working at Chaudhary Sarwan Kumar Himachal Pradesh Krishi Vishvavidyalaya, Palampur, Himachal Pradesh, India, who has vast experience in identification and research in medicinal and aromatic plants of North-Western Himalayas. The specimen of the plant material has been preserved and deposited to the herbarium of IHBT , Palampur, Himachal Pradesh and field book number has been assigned with specimen voucher no. 27. Tender and healthy leaves of plants which were 10 m apart were sampled before flowering season and immediately frozen in liquid nitrogen container for DNA fixing and then shifted to − 80 °C till further DNA isolation. Leaf samples, a total of 5 individuals from Sangla , 4 from Chhitkul , 5 from Rangrik , 4 from Shaloki , 5 from Bathad , 4 from Tosh , 5 from Mane , 5 from Sichling , 4 from Shego , and 7 from Giyu  were collected  and integrity using 1% agarose gel with uncut λ as reference. For PCR amplification, 50 ng of diluted genomic DNA, 0.5 µl of each forward & reverse primers (10 pmol), 0.1 µl of dNTPs (100 µM), 1.5 µl of MgCl2 (15 mM), 2.0 µl of 10 × buffer, 0.17 µl (5U/µl) Taq DNA polymerase (Genei Labs) was used and final reaction volume was made upto 10.0 µl. PCR reaction was performed using thermal cycler  and amplification cycles were used viz., pre-denaturation at 95 °C for 5 min, 35 cycles of denaturation at 95 °C for 1 min, annealing of primers for 1 min followed by extension at 72 °C for 1.30 min and final extension at 72 °C for 7 min. Amplified PCR products were separated on 6% denaturing PAGE (Poly Acrylamide Gel Electrophoresis) gel and were visualised using silver staining method along with size estimation of amplified product against 50 bp ladder (ThermoFisher).Total genomic DNA extraction was performed using CTAB protocol with little modificationsh), shannon’s information index (I), effective number of alleles (Ne), percent polymorphic marker loci (%PPL), analysis of molecular variance (AMOVA), principal coordinate analysis (PCoA), and mantel test of geographic and genetic distance estimation using GenAlEx version 6.531. To understand the genetic relationship among and between populations of D. hatagirea, POPGENE version 1.32 was utilised to calculate the gene flow, and Nei’s coefficient of genetic differentiation among populations32. Further, ARLEQUIN ver 3.5 was used to calculate the Fst and pair-wise Fst34.Stable and highly polymorphic fifteen SSR markers (KSSR) were utilised for genotyping of 48 individuals from 10 wild natural populations belonging to three cold desert regions  of Himachal Pradesh Table . PresencNei’s genetic similarity matrix generated in GenAlex from 15 SSR markers using UPGMA (Unweighted Pair Group Method with Arithmetic mean). The data was further utilised to develop a dendrogram using unpaired group mean arithmetic and Jaccard’s similarity coefficients in DARWIN 6.0 software35. STRUCTURE analysis based on Bayesian clustering was performed using STRUCTURE version 2.3.436. The population genetic structure (k) was classified by six iterations and value of k was set between “1 to 5” with a burn-in phase 100,000, also Markov Chain Monte Carlo (MCMC) repeats was set 100,000 after burn in and final structure determination was performed using Structure Harvester version 0.6.9437.Cluster analysis was performed using D. hatagirea is one of the most important species of Orchidaceae family which is exploited for its bioactive compounds viz., Dactylorhins A-E, and Dactyloses A-B. The species hold immense significance in the clinical and drug discovery research to identify potential bioactive compounds for treatment of diseases associated with respiratory, circulatory and reproductive systems. Unfortunately, due to over-exploitation and unsustainable management, the populations of D. hatagirea are dwindling in their natural habitat and loss of genetic diversity within/among these populations further increases the risk of extinction due to factors like inbreeding depression. Moreover, small size of the population also accumulates deleterious genetic variations/mutations due to genetic drift and results in highly vulnerable populations39. Therefore, increasing the population size is one of the important parameter in conservation of endangered plant species, as effective population size (Ne) have huge evolutionary potential and essential to prevent inbreeding depression40. The population structure and genetic diversity analysis of D. hatagirea revealed low gene flow. Interestingly, populations from two regions viz., Bathad from Kullu and Rangrik from Spiti valley revealed unique and different genetic structure, can be considered as potential populations for conservation strategy planning via introducing them to different regions to induce artificial gene flow41. Therefore, conservation of diverse individuals of each population is important to preserve the genetic diversity of D. hatagirea in the cold desert regions of Himachal Pradesh as diverse gene pools improves the chances of adaptation for future climatic conditions42.


Cerastium alpinum L. in Poland, an arctic-alpine perennial plant with a wide distribution in North America, northwestern Asia, and Europe. To determine whether the isolated Polish populations are genetically distinct, we have performed an evaluation of C. alpinum from Babia Góra with the use of iPBS markers. A total number of 133 individuals of C. alpinum from seven populations representing four localizations of the species were analyzed, i.e., from Babia Góra (Poland), Alps (Switzerland), Nuolja massif (Sweden), and Kaffiøyra . Genetic analysis of all C. alpinum samples using eight PBS primers identified 262 bands, 79.4% of which were polymorphic. iPBS markers revealed low genetic diversity (average He = 0.085) and high population differentiation (FST = 0.617). AMOVA results confirmed that the majority of the genetic variation (62%) was recorded among populations. The grouping revealed by PCoA showed that C. alpinum from Svalbard is the most diverged population, C. alpinum from Switzerland and Sweden form a pair of similar populations, whereas C. alpinum from the Babia Góra form a heterogeneous group of four populations. Results of isolation by distance analysis suggested that the spatial distance is the most probable cause of the observed differentiation among populations. Although significant traces of a bottleneck effect were noted for all populations of C. alpinum from Babia Góra, the populations still maintain a low but significant level of genetic polymorphism. These results are of great importance for developing conservation strategies for this species in Poland.Babia Góra massif is the only site of occurrence of the The online version contains supplementary material available at 10.1007/s13353-022-00731-x. Cerastium (Caryophyllaceae) consists of over 200 species , the majority of which are spread in the Northern Hemisphere in the temperate climate zone, but only a few of them are cosmopolitan species. Cerastium alpinum is a perennial that grows in rocky, sandy, or gravely substrates and is characterized by its matted growth form, hairy basal leaves, flowering stems, white, five-petalled flowers about 20 mm in diameter, and a branched root system   method described by Kalendar et al.  is basedC. alpinum populations; (2) to estimate genetic differentiation and relationships among studied C. alpinum populations from different geographic regions; and (3) to evaluate the genetic condition of C. alpinum from the Babia Góra massif to provide information essential for the development of effective conservation strategies for the genetic resources of C. alpinum in Babia Góra.The objectives of this study were as follows: (1) to test the suitability of iPBS to detect genetic polymorphism within and among Cerastium alpinum representing seven sampling sites (from now on referred to as populations) from Poland, Switzerland, Sweden, and Svalbard (Norway) . The amount and purity of isolated DNAs were estimated spectrophotometrically (NanoDrop). Additionally, the quality of DNA was verified on 1.5% (w/v) agarose staining with 0.5 µg/ml ethidium bromide.C. alpinum  were screened for Eight PBS primers that gave clearly identifiable and repeatable amplification products (bands) were selected for subsequent genetic diversity studies Table . The iPBNB), percentage of polymorphic bands (P), Shannon’s information index (I), and expected heterozygosity (He).The amplified bands were scored as either 1 (present) or 0 (absent) across genotypes. Based on the obtained binary matrix of amplification products, the following genetic parameters were estimated with the use of GenAlEx 6.5 software , achieving linkage equilibrium within clusters  based on the matrix of Euclidean distances between individuals from all analyzed populations. This analysis was performed in PAST software  was performed on Arlequin 3.5 software  using a Mantel test with 9999 permutations of the relationship between the matrix of pairwise FST/(1 − FST) and that of the logarithm of geographical distance between populations   Table , File S1Of a total of 262 scored bands, 20 7.63%) were represented as private bands that were found only within one population and absent in the others (Table % were reI) and expected heterozygosity (He). Population 3 had the highest values for both parameters, while population 5 had the lowest values.The iPBS markers revealed the presence of genetic polymorphism between individuals within a population and genetic variation among populations Table . The numPCoA indicates that 51.56% of the variation was explained by the first three components . The projection of the analyzed populations on the first two axes is shown in Fig. K has a maximum at K = 7, but a marked secondary peak at K = 5 was also found , in the case of populations 1 and 4, the predicted values of the proportion of membership of these populations to separate, individual clusters were > 0.7. The highest admixture was observed for populations 2 and 3, for which the predicted proportion of membership in the certain cluster was ≤ 0.499. For K = 5, an analogous pattern of population subdivision is observed. Pairwise FST analysis was also performed. The highest FST value (0.839) was observed between populations 5 and 6, whereas the lowest (0.268) was between population 1 and population 3 .The Bayesian clustering analysis performed in Structure and Structure Harvester software revealed that Δund Fig. . Both po= 5 Fig.  and K = = 7 Fig.  reveal aThe results of AMOVA showed that most of the identified genetic variation occurred among populations 61.69%), whereas the remaining 38.31% was attributed to variation within populations , which means that the genetic distance between populations, expressed as pairwise FST/(1 − FST) values, increases with the spatial distance between them , but only for populations 5 and 7 did it significantly deviate from zero  were used to analyze the bottleneck effect in studied populations Table . In eachCerastium alpinum occurs mainly in the Northern Hemisphere, North America (Canada and Greenland), Europe, and Western Siberia  and populations of the species from Switzerland , Sweden , and Norway .a Hultén . Our resCerastium alpinum is a common perennial in Fennoscandia. In alpine regions, it has a continuous distribution on alpine heat and serpentine soils, while in boreal forests, it has a scattered distribution on ultramafic soils and steep slopes . In Poland, C. alpinum occurs only in one place, on the Babia Góra massif in the Babia Góra National Park. This species is critically endangered in Poland due to its small range and geographical isolation  of the detected amplification products in our study on the genetic diversity of C. alpinum using the iPBS method were polymorphic, and the average polymorphism was found to be at 25.35% (across loci and populations). From 13.36% in population 5 to 45.80% in population 3, this parameter’s value was found. There were even higher levels of polymorphism in earlier studies using iPBS markers, such as 82.35% for Nicotiana tabacum . The highest value of that parameter was observed for population 3 (He = 0.162) and the lowest for population 5 (He = 0.051). Several authors found similarly low levels of heterozygosity in other isolated plant populations. The arctic-alpine species Dryas octopetala forms isolated populations in mountainous regions on northern Greece’s southernmost edge of the species’ distribution range. Studies by Varsamis et al. (He = 0.156). In Pilosella alpicola subsp. ullepitschii, an endemic plant in the Carpathians, in the Western Carpathians (Slovakia and Poland), this species occurs at many sites, but only four sites are known in the Eastern and Southern Carpathians (Romania). Studies by Šingliarová et al. (He = 0.134) in isolated populations of the Eastern and Southern Carpathians in comparison to populations from the Western part (He = 0.235). The results suggested that this population experienced a robust genetic bottleneck, probably due to a founder effect. On the other hand, studies of the influence of alpine habitat isolation on the genetic diversity of the rare and isolated population of Campanula thyrsoides showed a large genetic variation , the analyzed populations of C. alpinum were characterized by very high genetic differentiation . The significant pairwise FST values between all pairs of studied populations and the significant results of IBD analysis suggest that this population differentiation could be attributed to limited gene flow between them. This is, however, a surprising observation in the case of C. alpinum populations from the Babia Góra massif, which are not separated by high geographical distances (the pairwise distance between populations from this site ranges from 0.14 to 0.55 km). Therefore, the most likely explanation is the seed dispersal method (gravity) and the fact that the C. alpinum seeds are not morphologically adapted to wind dispersal.The results of our study revealed that despite a low level of polymorphism revealed by iPBS markers  and low genetic diversity  . It is an endemic plant species that is extinct in the wild in Poland and is known only from one transplanted population  with similar habitat conditions to its native site. Some individuals were transplanted to the Botanical Garden of the Polish Academy of Sciences in Warsaw to protect the species from complete extinction. After 18 years of ex situ conservation, Rucińska and Puchalski . Even lower values of He were observed with the application of the same DNA markers (iPBS) for populations of C. quitensis , regular monitoring of the species abundance and population condition should be continued.Considering the low genetic diversity and high population differentiation revealed in this study, we suggest that all Cerastium alpinum populations. The results showed a relatively low level of genetic diversity and high population differentiation. Furthermore, the genetic structure analyses reveal that the C. alpinum population from Babia Góra forms a distinct and heterogeneous group with greater genetic diversity than populations from other regions. Although C. alpinum populations from Babia Góra revealed significant traces of a recent bottleneck, like all other studied populations of the species, they still represent valuable genetic resources deserving protection. This has become especially important in light of the recent observations pointing to the shrinking area of its occurrence. Therefore, immediate action is required to develop an efficient conservation strategy to protect this species’s only Polish location.Currently, we observe the increasing recognition of the value of genetic data to support the decisions associated with developing and managing species conservation strategies. In this paper, the retrotransposon-based DNA markers (iPBS) proved helpful in revealing the genetic variation of Supplementary file1 (DOCX 16 KB)Supplementary file2 (XLSX 117 KB)Supplementary file3 (RAR 72.1 MB)Below is the link to the electronic supplementary material.