Bipolaris sorokiniana, a key pathogenic fungus in the wheat leaf blight complex, was the subject of research that resulted in the development of fifty-five polymorphic microsatellite markers. These markers were then used to examine genetic diversity and population structure in Indian geographical regions. The simple sequence repeat (SSR) like trinucleotides, dinucleotides, and tetranucleotides accounted for 43.37% (1256), 23.86% (691), and 16.54% (479) of the 2896 microsatellite repeats, respectively. There were 109 alleles produced by these loci overall, averaging 2.36 alleles per microsatellite marker. The average polymorphism information content value was 0.3451, with values ranging from 0.1319 to 0.5932. The loci’s Shannon diversity varied from 0.2712 to 1.2415. These 36 isolates were divided into two main groups using population structure analysis and unweighted neighbour joining. The groupings were not based on where the isolates came from geographically. Only 7% of the overall variation was found to be between populations, according to an analysis of molecular variance. The high amount of gene flow estimate (NM = 3.261 per generation) among populations demonstrated low genetic differentiation in the entire populations (FST = 0.071). The findings indicate that genetic diversity is often minimal. In order to examine the genetic diversity and population structure of the B. sorokiniana populations, the recently produced microsatellite markers will be helpful. This study’s findings may serve as a foundation for developing improved management plans for the leaf blight complex and spot blotch of wheat diseases in India. Bipolaris sorokiniana (Sacc. in Sorok) in warm, humid areas of South Asian nations, Shoem, also known as Helminthosporium sativum, teleomorph (Cochliobolus sativa), is one of the most destructive pathogens ..w/v) agaB. sorokiniana isolates from various agroecological zones in India was examined. GenAlEx version 6.503 [The gels were graded according to whether or not they contained pronounced, repeatable amplicons. Every amplicon was given the designation of a locus with two possible alleles. The SSR amplification data from several isolates were converted into discrete variables in a binary data matrix (0 = absence and 1 = presence). To explore the variation in partitioning between populations, a cluster analysis using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) algorithm was carried out using the NTSYS version 2.1 programme . Using Son 6.503  was usedon 6.503 . AdditioK = 1 to K = 15 with five different runs of 25,000 burn in period length at fixed iterations of 100,000 allowed for the most advantageous number of populations (K) to be determined. The optimum K-value was standardised by following the methodology of [The cumulative allelic diversity (Ht), mean allelic diversity within populations (Hs), percentage of total allelic diversity (Gst), and gene flow (Nm) within populations were all calculated using POPGENE version 1.31 software . Using tology of .B. sorokiniana. The most frequent repeat motifs corresponding to dinucleotide to hexanucleotide repeats were (AC/TG)n, (AAG/TCT)n, (ATAC/TGTA)n, (AAAAG/CTTTT)n, and (ACCAGC/CAGCAC)n. The SSR repeat types varied—trinucleotides, dinucleotides, and tetranucleotides, for instance, accounted for 43.37% (1256), 23.86% (691), and 16.54% (479) of the 2896 SSR repeats, respectively. Pentanucleotide and hexanucleotide repeat motifs took up the remaining space, contributing to 7.80% (226) and 8.42% (244), respectively. When all SSR repeat motifs were considered, it was discovered that trinucleotide repeats made up the bulk of the motifs, while pentanucleotide repeats made up the least amount of them, as shown in A total of 3251 SSR loci were discovered as an outcome of the genome sequence search for B. sorokiniana genome, the frequency of each SSR motif type was also determined. The most frequent dinucleotide motif was AC/TG (15.48%), followed by AC/CA (14.47%), AG/GA (11.43%), and AC/AC (10.13). The CG/GC pattern repetitions, meanwhile, were infrequently seen (0.05%). The trinucleotide predominant motif repeats were AAG/TCT (4.21%), ACG/TGA (3.82%), AAG/AGA (3.58%), and AAG/CTT (3.50%), and cumulatively the other 789 trinucleotides motifs shared 62.81% of dominant SSRs. Additionally, ATAC/TACA (3.34%) were the most frequently discovered tetranucleotide repetitions , but that genetic diversity among populations was minimal (3%). Very small but significant genetic distance values between population 1 (Hills) and population 2 (Plains) were revealed in a pairwise analysis (p < 0.001). Between the populations of the Hill and Plain, an average and consistent level of gene flow (Nm = 3.261) was observed, and a pairwise study revealed a genetic identity of 0.071 levels.AMOVA’s outcome  showed tB. sorokiniana into two different groups  and other major wheat-growing countries such as Canada, the United States, Brazil, and Australia [Curvularia inaequalis and Bipolaris spicifera causing leaf blight of Buffalograss in Nebraska [B. sorokiniana isolates and its SSR marker development, genetic diversity, and population structure study in Indian geographical settings. The number of population genetic studies on pathogenic fungus has expanded as a result of this work. Comparatively few population genetic studies of plant pathogenic fungus have been conducted thus far [During standard phytopathological practices of pathogen isolation from symptomatic leaf tissues, researchers obtain multiple microbes on their culture media but generally they discard the disinterested microorganism and only focus on their key interested pathogen using purification techniques, leading to loss of some valuable information, and possibly there is actually a vital role of the discarded microflora on the disease epidemic. A growing understanding of plant pathogen diversity and prevalence has revealed that many diseases formerly assumed to be caused by a single primary agent are actually the consequence of complex interactions between many taxa and the host. Even when a primary agent is recognised, its action is frequently mediated by additional symbionts. As a result, the paradigm of one pathogen–one disease is giving way to the pathobiome concept . The resof maize ,37. In tustralia ; similarNebraska . The prethus far ,41. Undethus far ,43.Bipolaris sorokiniana isolates. The markers found a variety of polymorphisms, from very informative to almost informative. A marker’s PIC value measures a locus’s ability to discriminate between different genotypes while taking into consideration the number and relative frequency of alleles.In this study, 55 polymorphic markers were developed and applied to assess the genetic diversity of B. sorokiniana population in India. Within populations of B. sorokiniana isolates, variation varied to the maximum extent (90%). The Indian B. sorokiniana populations did exhibit some gene diversity, although it was not very high. According to the high migration rate (NM-3.261) estimations, the comparatively low FST value (0.071) between the B. sorokiniana population analysed in this study suggested little differentiation across the groups that may be attributable to gene flow among regions; therefore, in the context of the current study, migration is more significant than genetic drift. It is also a well-established fact that seedlings or young plants contract an infection at the roots, crown, or other below-ground locations, and that the infection later spreads to the above-ground parts. Conidia then form, and secondary conidial spread occurs with the aid of wind, sprinkling rain, or human interventions. Furthermore, it is possible that environmental factors, geographic location, and wheat cultivar genotypes may have an impact on the genetic variations in B. sorokiniana [The outcomes of AMOVA confirmed the existence of genetic diversity in the value 0.01 betweenokiniana .B. sorokiniana isolates from the Indogangetic plains (IGPs) regions of India are closely related. In addition, the population of isolates were divided into the Hill Regions (HR) and Plain Regions (PR) subpopulations on the basis of population genetic analysis. The unweighted neighbour-joining technique and STRUCTURE analysis all supported and showed evidence of mixing between the two populations. The two distinct sub-groups within B. sorokiniana isolates from Plains and the low level of genetic differentiation they exhibited were the results of an intense occurrence of genetic discrimination that presumably occurred at a low level due to migration. The findings are consistent with other studies by [B. sorokiniana isolates probably moved from plain regions to parts of the hilly regions through anthropogenic activities associated with the production and distribution of wheat seed. The limited and sympatric distribution of two discrete clusters (Hills and Plains) in the wheat sampling areas and the fact that two different wheat-growing terrains were clustered in the same lineages were strong arguments in favour of this assertion. Therefore, the plain indogangetic regions population was most likely migrated into the hill regions of Uttrakhand and Himachal Pradesh in recent times. It is not supported by evidence of admixture between isolates from the various locations to divide isolates into clearly distinct subpopulations. The PhiPT value (0.072) among the B. sorokiniana populations examined in this study showed moderate differentiation among the groupings, which may be related to gene flow between locations. The moderate level of variability in populations of B. sorokiniana seen in this study might be explained by the long-distance conidial dispersal that might facilitate pathogen dissemination in wheat-growing regions of India. This dissemination could be connected to the trade of wheat germplasm between farmers, the seed business, and scientists.Understanding of the pathogen’s biology, evolution, and potentially adaptive genotypic diversity in the species is improved by information on the population structure of Bipolaris populations from various territories . The subudies by ,43,44 (HB. sorokiniana isolates from India’s wheat-growing regions. Despite geographical differences, population-observed genetic diversity was lower than predicted, pointing to regional planting material trades and inoculum distribution between the regions. This research produced data that can be used to better understand the biology of the pathogen and its evolutionary potential, as well as to lay the groundwork for future research on disease development, host–pathogen interactions, and the creation and application of disease-resistant wheat varieties. Additionally, the knowledge gained from this study can be used to create new, precise primers for the identification and detection of B. sorokiniana from the wheat leaf blight complex. The number of population genetic studies on pathogenic fungi will be increased as a result of this research. Comparatively few population genetic studies of pathogenic fungus have been conducted thus far [The SSR markers developed in this study were employed to analyse the genetic diversity and population structure of thus far . Understthus far ,47,48,49


Jubaea chilensis (Molina) Baill., also named Chilean palm, is an endemic species found in the coastal area of Mediterranean sclerophyllous forest in Chile. It has a highly restricted and fragmented distribution along the coast, being under intense exploitation and anthropogenic impact. Based on 1038 SNP markers, we evaluated the genetic diversity and population structure among six J. chilensis natural groups encompassing 96% of the species distribution. We observed low levels of genetic diversity, a deficit of heterozygotes , and high levels of inbreeding (mean ISF = 0.424). The fixation index (STF) and Nei’s genetic distance pairwise comparisons indicated low to moderate structuring among populations. There was no evidence of isolation by distance . In the cluster analysis, we observed a closer relationship among Culimo, Cocalán, and Candelaria populations. Migration rates among populations were low, except for some populations with moderate values. The K value that best represented the spatial distribution of genetic diversity was ∆K = 3. Habitat fragmentation, deterioration of the sclerophyllous forest, lack of long-distance dispersers, and a natural regeneration deficit may have driven inbreeding and low levels of genetic diversity in the palm groves of J. chilensis. Although extant populations are not at imminent risk of extinction, the rate of inbreeding could increase and migration could decrease if the effects of climate change and human impact become more acute. Understanding the extent to which human activities affect the distribution of the genetic diversity of plant species is mandatory for geneticists and ecologists interested in the conservation of biodiversity . High gePalms (Arecaceae) were among the first plant groups to receive attention regarding the risks of becoming endangered , but despalmar, or palm forest, is an overlooked component of the sclerophyllous forest, which occurs in patches with individuals of the long-lived Jubaea chilensis (Molina) Baill., the Chilean palm, a tertiary-relict endemic species ), Cocalán Culimo (0.280 [0.238–0.322]), Cocalán Petorca (0.266 [0.215–0.317]), and Viña del mar-Valparaiso Ocoa (0.268 [0.222–0.314])  . The log–0.314]) .Based on the maximum value of ΔK, the estimated number of optimal groups was K = 3 . IndividJ. chilensis present low genetic diversity and high inbreeding. Given their biallelic heritage, the highest value of HE expected for SNPs is 0.5. However, the highest value observed for J. chilensis was less than a third of that value, indicating that these populations likely suffer from loss of genetic diversity. Null alleles are an issue for many marker types and could also result in a downward bias in estimated heterozygosity. Unfortunately, we are not able to estimate null alleles’ frequency and SNP null alleles have not been described in this species. The inbreeding coefficient was significant and high for all populations, even though the species is diclino-monoescious, which favored crossing [E and the highest value of ISF. All of these results are indicative that J. chilensis may be suffering from genetic erosion. Other palm species have also shown high levels of inbreeding, and it was usually associated with the mating system, anthropic effects, and low abundance of populations [Pinanga. Cibrián-Jaramillo et al. [Chamaedorea ernesti-augusti, and declare that the high inbreeding is a result of the anthropic effect. Santos et al. [Attalea vitrivir individuals. Other studies using microsatellites confirmed the prevalence of inbreeding in Acrocomia aculeata populations, product of crosses between very close relatives, or even full siblings [Syagrus coronata, for example, the authors take into account the high-level of inbreeding to conduct conservation measures [J. chilensis has been highly exploited and approximately only 121,284 specimens are found in Central Chile , being distributed among highly fragmented populations [Our results show that all studied populations of crossing . Interesulations ,46,47,48ulations  found hio et al.  found his et al. , using nmeasures . Given tulations ,50,51, cJ. chilensis being genetically similar, and no evidence of isolation by distance. These results are unlikely to reflect the current seed dispersal pattern of the species. Currently, seeds of J. chilensis are dispersed close to mother plants, as rodents  and introduced domestic species are the main seed dispersers. The frequency of rodent–seed interaction is low (<25%) and transport usually occurs over short distances , indicaes , leading to inbreeding depression and possibly leading to extinction of the local palm tree [Astrocaryum aculetassimum (Schott) Burret, an endemic Atlantic Forest palm species, it has been reported that the species populations are highly affected by the loss of seed dispersers as a result of fragmentation and hunting [Astrocaryum mexicanum Liebm. ex Mart. species inhabiting Los Tuxtlas (State of Veracruz/México), the abundance of coleopterans and pollinating beetles varies according to the size of the fragment, making reproduction doubly susceptible to the effects of fragmentation [Ceroxylon echinulatum Galeano and Attalea colenda (O.F. Cook) Balslev and A.J. Hend. have failed to survive as a result of deforestation [Phoenix dactylifera L. populations occurring in natural oases in Tunisia also presented HE values higher than HO values with low genetic structure [Nowadays, the geographical distribution of palm groves is highly scattered in a series of small, isolated patches, consisting of small independent local populations at a geographic level, as depicted in alm tree . For the hunting . Moreoveentation . In Ecuaestation ,58,59. Aestation . Similartructure .J. chilensis is a palm tree with a significantly long life expectancy, with 1000 years being the estimated age for the oldest individual [J. chilensis were consumed and dispersed by the extinct megafauna species belonging to the following families: Gomphotheriidae, Camelidae, Equidae, Notohippidae, Homalodotheriidae, Toxodontidae, Astrapotheriidae, Macraucheniidae, Mylodontidae, and Megatheriidae [It is important to highlight that genetic structure analysis may reflect both contemporary and historical processes such as past gene flow and population fluctuation , and thadividual ,62. Therheriidae ,31. Simuheriidae . Cocalánheriidae  and highheriidae .J. chilensis have been carried out [J. chilensis via sowing seedlings has been carried out without any consideration of genetic or biogeographic origin. Understanding the distribution of genetic variability can be especially important for species facing extinction risk, especially when translocations for restoration, genetic rescue, or assisted gene flow are fundamental aspects for the successful long-term population [J. chilensis capacity [Germination and establishment studies of ried out ,66,67,68ried out . As a repulation . Currentcapacity ,60,61. Acapacity ,54.J. chilensis studied have low genetic diversity, high inbreeding, and no evidence of isolation by distance. Habitat fragmentation, deterioration of the sclerophyllous forest, lack of long-distance dispersers, and natural regeneration deficit may have driven inbreeding and low levels of genetic diversity in the palm groves of J. chilensis. Although extant populations are not at imminent risk of extinction, the rate of inbreeding could increase, and the migration effect might be overwhelmed by the effects of climate change and human impact. Thus, considering our findings plus the natural history and ecological context wherein J. chilensis lives, we suggest that this monospecific, relict, and endemic palm species should be considered as critically endangered by international conservation organizations.All populations of Jubaea chilensis is an arborescent and woody monocot, with a bare and cylindrical stipe narrower towards the top, reaching up to 30 m in height and from 0.80 to 1.10 m in diameter. It is a diclino-monoecious plant with cross pollination [J. chilensis’ natural populations are distributed from La Serena (29°54′ S–71°15′ W) in the Coquimbo Region to Tapihue-Pencague (35°15′ S–71°47′ W) in the Maule Region. It inhabits warm climate areas with dry summers and coastal areas with coastal fog influence [lination  and the lination ,28,29,30nfluence , thus tonfluence ,63. The nfluence . The disnfluence , especianfluence .J. chilensis to be sampled, we considered four parameters: (i) populations with more than 50 individuals; (ii) a sampling scheme that covers the entire species’ geographical range; (iii) the exclusion of specimens belonging to plantations; and (iv) a maximum of 30 individuals per population was analysed. We sampled the following populations groups: (a) Culimo (CUL), (b) Petorca (PET), (c) Ocoa (OCO), (d) Viña del Mar/Valparaíso (VAV), (e) Cocalán (COC), and (f) Candelaria (CAN)  , Table 1ia (CAN) , Table 4J. chilensis chosen at random, which included the entire distribution range and with a minimum separation between individuals of 150 m, between February and December 2015 ; only high-quality paired-end sequences (with average PhredScore over 24 and over 65 bp) were used for further analysis. The single nucleotide polymorphisms’ (SNPs) prospection was performed using the software pipeline PyRAD v1.2 [https://github.com/nspope/r2vcftools, accessed on 1 January 2021)—a wrapper for VCFtools [2 < 0.8) [p > 0.0001), and loci with less than 20% missing data.The Genotyping-by-Sequencing (GBS) library was constructed using the standard protocol described by Elshire et al.  and empRAD v1.2 . Reads wRAD v1.2  using thRAD v1.2  and lateVCFtools —was used2 < 0.8) , Hardy–WISF) was calculated based on the variance found in the allelic frequencies [https://github.com/rojaff/LanGen_pipeline, [STF calculations between populations were performed using the dartR package v.183 [STF, using the vegan package [http://beast.community/tracer, accessed on 1 January 2022). We used the Bayesian deviance as calculated by Meirmans . Genetige v.183 . Additioge v.183 ). Mantel package . A princ package ,83. We a package  of the s package  to deters  to sears (2014, . A roughPopulational structure and admixture degree were inferred using the Bayesian grouping method implemented in the STRUCTURE v 2.3.4 program ,89,90. P


Salmo trutta) in order to monitor the genomic effects of population translocations. In 1979, fish from each of two genetically (FST = 0.16) and ecologically separate populations were simultaneously released, at one point in time, to a lake system previously void of brown trout. Here, whole‐genome sequencing of pooled DNA (Pool‐seq) is used to characterize diversity within and divergence between the introduced populations and fish inhabiting two lakes downstream of the release sites, sampled 30 years later (c. 5 generations). Present results suggest that while extensive hybridization has occurred, the two introduced populations are unequally represented in the lakes downstream of the release sites. One population, which is ecologically resident in its original habitat, mainly contributes to the lake closest to the release site. The other population, migratory in its natal habitat, is genetically more represented in the lake further downstream. Genomic regions putatively under directional selection in the new habitat are identified, where allele frequencies in both established populations are more similar to the introduced population stemming from a resident population than the migratory one. Results suggest that the microevolutionary consequences of population translocations, for example, hybridization and adaptation, can be rapid and that Pool‐seq can be used as an initial tool to monitor genome‐wide effects.Population translocations occur for a variety of reasons, from displacement due to climate change to human‐induced transfers. Such actions have adverse effects on genetic variation and understanding their microevolutionary consequences requires monitoring. Here, we return to an experimental release of brown trout . Two genetically distinct populations released to the same environment are unequally represented in descendant populations, with one introduced population mainly contributing to the lake closest to the release site and the other dominating waters further downstream. Furthermore, this species has a large genome (c. 2.4 Gb) characterized by an ancient whole‐genome duplication c. 90 million years ago  to monitor genome‐wide diversity and divergence of brown trout following introduction into a novel environment. Two genetically and ecologically distinct populations were simultaneously released, at one point in time (in 1979), to a natural lake system void of brown trout prior to the release. Fish from two introduced populations were genetically distinguishable at a few allozyme loci and differed in traits, for example, age at maturity, reproductive and migratory behavior, and body size  and sampled 30 years (c. 5 trout generations) later?Are there any signs of selection in the new environment, that is, any adaptive differences between the released fish compared to fish inhabiting two lakes downstream of the release site c. 30 years later?The following questions are addressed:22.1We studied brown trout originating from two genetically and ecologically distinct populations released to the same novel lake system, as well as fish established in the wild. Established fish were collected over 30 years later  which have remained since.The released fish originated from two populations with diverse ecological features separated by more than 500 km waterway and likely isolated from each other since the last glaciation  lake located 100 km west of the study area. The wild fish that once inhabited this lake were large, slow‐growing, piscivorous, and with the capacity for long‐distance migration  and closely connected lakes, Lake Fälpfjälltjärnarna, located c. 10 km north of the study area with similar ecological characteristics as the presently studied lake system. The fish here are non‐migratory and ecologically typical of populations inhabiting small mountain lakes in northern Scandinavia: with small body size, early maturation, and a primarily insect‐based diet  or the F1 generation for which population assignment was possible using the allozyme marker (no hybrids) are included. Fish established in Lakes LB and HV were caught in 2011.We studied four groups of fish, that is, released fish representing each of the two distinct populations (introduced population A and introduced population B), and fish established in the wild in lakes Lilla Bävervattnet (established population LB) and Haravattnet  using a KingFisher cell and tissue DNA kit (Thermo Scientific) including RNase A treatment. High‐molecular‐weight DNA from each individual was combined at equal concentrations for each population in order to create pools of individuals corresponding to each population to be sequenced. Additional details about DNA extraction are provided in Appendix 2.3Samples were sent to the National Genomics Infrastructure (NGI) at the Science of Life Laboratory (SciLifeLab), Stockholm, Sweden, for the preparation of PCR‐free paired‐end libraries and sequencing (Illumina HiSeq 2000). Additional details about library construction and sequencing are provided in Appendix 2.4http://sourceforge.net/projects/bbmap/). Trimmed reads were mapped per lane to the brown trout assembly  using BWA mem v.0.7.17  using BBDuk implemented in BBTools v.37.31  across all populations using a custom script (available upon request). Pooled heterozygosity score (HP) was calculated according to Rubin et al. , only retaining windows with at least 33 variant sites (the average number of variant sites found per window in PoPoolation2).Allele frequencies were calculated per variant site (SNP) using the “snp‐frequency‐diff.pl” script in PoPoolation2 v1.201  were, therefore, included  as described above. We did not include any migration edges for these four groups of fish.This program uses as input allele frequencies estimated in PoPoolation 2 v.2.2  as would be expected under directional selection on many independent mutations . M‐values for log2 fold change were retrieved by comparing the observed and expected number of SNPs per category and bin . Thresholds for significant enrichment included M > 0.05 and p < 2.5 × 10−11 , we characterize the functional differences between the two, in an attempt to give a more detailed description of divergence than provided by The same procedure of identifying the functional impact of markedly divergent SNPs was performed for the two populations established populations LB and HV, in order to characterize genomic divergence in the new lake system.2.8ΔAF between them, estimated per variant site (1 bp) and per 5 kb windows . For the SNP‐based approach, analysis was restricted to SNPs of marked ΔAF  exhibiting significant allele frequency difference between introduced populations A and B as tested by Fisher's exact test implemented in PoPoolation2 v.2.2  within both introduced populations  and average π for the same window to be below 0.44 × 10−6 in introduced population A and below 0.90 × 10−6 in introduced population B . These thresholds were chosen in order to capture outliers in both FST and π distributions.Second, estimates of divergence and diversity were combined in order to avoid confounding selection with drift in regions of elevated divergence  were obtained by considering non‐synonymous SNPs found within the candidate windows, only including SNPs exhibiting significant allele frequency difference between introduced populations A and B as tested by Fisher's exact test in Popoolation2 v.2.2  and comparing contrasting population pairs: introduced populations A and B compared to established populations LB and HV. Candidates for directional selection were characterized as 5 kb windows of ZHP below the genome‐wide average within each of established populations LB and HV and ZHP above the genome‐wide average within each of introduced populations A and B. Candidates for relaxed selection are defined to have ZHP below the genome‐wide average within each of the introduced populations A and B and ZHP above the genome‐wide average within each of the established populations LB and HV. ZHP was used since its distribution is characterized by μ = 0 and σ = 1. Any deviation in HP from the pool mean is, thus, equivalent to ZHP above or below 0 , and these are used for estimating various population genomic parameters , pooled heterozygosity score (HP), and Tajima's D (TD), show significant differences between populations  and indications of difference in the degree of genetic contribution from the introduced populations A and B to each of the established populations (Table FST = 0.04) than to HV (FST = 0.09), while introduced population A appears to have contributed more to HV (FST = 0.04) than to established population LB (FST = 0.10). Differentiation between established populations HV and LB averages FST = 0.04. The dendrogram of genetic relationships among all four populations has two major branches with introduced population A and established population HV on one and introduced population B and established population LB on the other  visualized within 5 kb windows show similar genetic relationships as FST. ΔAF between the introduced populations A and B exceeds ΔAF between the established populations LB and HV . Genetic distances based on these windows mirror the dendrogram: established population LB is alike introduced population B, whereas established population HV is more similar to introduced population A. However, introduced population B is more similar to established population LB than introduced population A is to established population HV  and exhibiting a significant difference in allele frequency between introduced populations as tested by Fisher's exact test (significance threshold p < 2.5 × 10−11), the majority are intragenic (mainly within introns), intergenic, or found upstream of a gene  and diversity (π) are combined to identify adaptive divergence between the introduced populations A and B. A total of 403 putatively adaptive windows are identified  are associated with immunity in salmonids  and Haravattnet (HV). This contention is supported by genome‐wide divergence and diversity in the four groups of fish. The genome‐wide divergence between introduced populations A and B exceeds the divergence between fish established in the new lake system, as indicated by T Figure  and a deT Figure . Within‐Hybridization between introduced populations A and B is corroborated by a limited SNP panel (comprising 96 SNPs) employed to study fish populating this general mountain area , as well as by Palm and Ryman  in theirInterestingly, we find that genetic contributions from introduced populations A and B to established populations LB and HV are unequal. Introduced population B, which is non‐migratory in its original habitat (Lake Fälpfjälltjärnarna), has contributed more to established population LB in the lake closest to the release sites. In contrast, introduced population A, which is migratory in its natal environment , has contributed more to the established population HV furthest downstream in the system. This trend is observed across the genome Figures  and 4, bThe unequal contribution of introduced populations to lakes in the new system is mirrored in the 96 SNP panel . Further, Palm and Ryman  studied 4.1The introduced populations A and B were initially chosen to represent different body sizes and life history adaptations, for example, growth rate, and migratory and reproductive behaviors Appendix . In thei4.2W = 1462, p < 2.5 × 10−11 and established population LB: W = 1100, p < 2.5 × 10−11; Figure We identified regions with low heterozygosity scores in established populations LB and HV compared to the introduced populations A and B, suggesting direction selection in the new environments. For these regions, we find that both established populations show greater differentiation to introduced population A than to introduced population B  of marked differentiation between introduced population A and both established populations. These genes are all associated with metabolism, and FST between the two introduced populations A and B is high. This indicates that the metabolic requirements, for example, nutrient availability within the Lakes Lilla Bävervattnet and Haravattnet are more similar to those of the small lakes from which introduced population B originates than those of the larger lake that introduced population A is from. It is also striking to find genes related to metabolism since such intrinsic traits may underly other behaviors, for example, migratory and reproductive , gene flow, and drift . However, the magnitude of this difference, estimated as fold change, is near zero . We use stringent cutoffs in order to represent the extremes of the distribution ; data curation (lead); formal analysis (lead); methodology (lead); resources (supporting); visualization (lead); writing – original draft (lead). Nima Rafati: Formal analysis (supporting); methodology (supporting); supervision (supporting); writing – review and editing . Nils Ryman: Conceptualization (lead); formal analysis (supporting); supervision ; writing – review and editing . Linda Laikre: Conceptualization (lead); formal analysis (supporting); funding acquisition (lead); project administration (lead); resources (lead); supervision ; writing – review and editing .The authors declare no conflict of interest.Supplementary materialClick here for additional data file.


Clupea pallasii), which are foundational to coastal food webs and fisheries. While the importance of phenological diversity is well-known from contemporary studies, the extent to which different populations contribute to fisheries over long time scales is mostly unknown. In this study, we investigated the relative contributions of genetically and phenologically distinct herring populations to Indigenous Peoples’ food systems over multiple centuries, using ancient DNA extracted from archaeological herring bones. These bones were excavated from two Coast Salish archaeological sites (Burton Acres Shell Midden and Bay Street Shell Midden) in the Puget Sound region, USA. Using genetic stock identification from seven nuclear DNA markers, we showed that catches at the two sites in central Puget Sound were dominated by January–February and March–April spawners, which are the contemporary spawning groups in the vicinity of the sites. However, May spawners were detected in the older Burton Acres assemblage , and a mixed stock analysis indicated that catches at this site consisted of multiple populations. These results suggest that Coast Salish ancestors used a portfolio of herring populations and benefited from the ecological resource wave created by different spawning groups of herring. This study of ancient DNA allowed us to glimpse into Indigenous traditional food and management systems, and it enabled us to investigate long-term patterns of biodiversity in an ecologically important forage fish species.Phenological diversity in food resources prolongs foraging opportunities for consumers and buffers them against environmental disturbances. Such diversity is particularly important in forage fish such as Pacific herring ( For example, diverse life-histories in salmonids are associated with asynchronous population dynamics which stabilize overall abundance through portfolio effects3. Meanwhile, intraspecific genetic variation represents the evolutionary potential of populations to adapt to new environmental conditions5 and develop resistance to pathogens7. Despite the recognized value of intraspecific variation, population diversity is eroding at a rapid rate globally due to human activities9. This erosion of diversity has implications for ecosystem health, and in particular for Indigenous Peoples who have relied on this diversity for millennia; for them, the loss of diversity is linked tightly to multiple dimensions of social well-being10.There is growing recognition that phenotypic and genetic diversity contribute to the productivity and resilience of wild populations and the ecosystem goods that they provideClupea pallasii) are an abundant forage fish in coastal waters of the Pacific Northwest and important prey for a variety of organisms, including marine mammals12, salmon13, and sea birds15. The spawning migrations of herring provide an ephemeral pulse of food for coastal marine organisms16, as fish aggregate to nearshore environments to spawn.Population and phenological diversity are particularly important in species central to ecosystem functioning that support human societies. Pacific herring , while herring eggs were harvested using evergreen branches or kelp fronds that were submerged during spawning events . Today, herring eggs harvested in this way are a beloved food item in many communities  and herring harvest is an expression of Indigenous culture and sovereignty , while income generated from herring fisheries supports coastal livelihoods30.The resource wave created by herring may have been important to the livelihoods of human communities on the Pacific coast of North America. Humans harvested herring for millennia33. In the twentieth century, large reduction and sac-roe fisheries harvested thousands of tons of herring yearly, but collapsing herring populations in Washington State led to their closure in the latter part of the century34. Today, Washington State only allows a bait fishery to operate in the Puget Sound17.The contemporary use of herring by Indigenous People may be affected by reduced phenological diversity following overexploitation in industrial fisheries. Industrial commercial fisheries for herring began with the colonization of the Pacific Northwest coast in the late nineteenth century by European settlers, and declining catches were reported as early as 1886 in the Puget Sound35, an approximately 50-year time series of spawning biomass surveys indicates that certain herring populations have not recovered to their former abundance and remain severely depressed17. For example, the May-spawning population from Cherry Point was once the largest stock in Puget Sound, but this population has declined over 96% from the initial biomass estimate made in 197317. It is unclear if the dramatic decline of this population is representative of natural variation in population productivity or if this pattern is unique to recent decades, reflecting anthropogenic impacts.Despite the three decades since the last large commercial herring fisheries operated in Puget Sound36, these data can supply long-term information about ecological conditions38 and patterns of resource variability40. Additional layers of information can be provided by analyzing ancient DNA (aDNA) preserved in archaeological remains or sediments, as these archive the genetic diversity of wild food resources that sustained human communities over long periods of time. For example, studies of aDNA in Pacific salmon have identified the migration phenotypes that supported ancient fisheries42 and quantified reductions in genetic diversity associated with the large-scale habitat degradation of riverine habitats in the twentieth century41.One method to gather ecological data over long time scales is to quantify faunal remains in paleontological or archaeological deposits. Although coarser in temporal resolution than modern ecological studiesIn this study, we estimated the relative contributions of genetically distinct populations of Pacific herring to Indigenous food systems over the last millennium, using aDNA from herring bones preserved in archaeological sites in the Puget Sound region. Specifically, we ask the following questions: (i) Which populations of herring did Coast Salish people in the central Puget Sound harvest? (ii) Did the relative proportions of these herring populations vary through time in the archaeological record? (iii) Did the relative proportions of these herring populations differ between archaeological sites? (iv) How does archaeological herring population diversity compare to contemporary population diversity? Answering these questions allowed us to glimpse into Indigenous traditional food and management systems and long-term connections to place and marine resources. Additionally, it allowed us to investigate long-term patterns of biodiversity in an ecologically important forage fish species.43. Archaeological studies indicate that Indigenous People have lived in the Puget Sound region since at least 12,000 years before present44. Today, there are 23 federally recognized Native American Tribes and six tribes that are not federally recognized and/or seeking federal recognition in the Puget Sound region. Some of these tribes are linguistically related, and based on these linguistic relations they are known collectively as the Coast Salish.The Puget Sound ecosystem is a large fjord estuary fed by freshwater from multiple rivers that flow from the Olympic and Cascade Mountains. The diverse topography of the region supports high levels of biodiversity and productive coastal habitats rich in marine resources, including shellfish, finfish, and marine mammals28, Puyallup28, Klallam45, Twana46, Skokomish46, Suquamish47, and Lummi48. Herring were harvested from shallow water using baskets, mazes, and fish traps, and from deeper water using rakes and dip nets that were deployed from canoes49. Herring roe was considered a delicacy, and it was collected from spawning sites by placing kelp or hemlock branches in the water as herring reproduced45. Herring and roe were eaten both fresh and dried28, and historically these foods were cured for trade with inland Tribes49. Herring were widely used as bait for salmon and also valued as “unharvested bait” that facilitated the harvest of other species—for example, Coast Salish people caught salmon, waterfowl, and seals as they aggregated to prey upon schools of herring49.Various oral histories and ethnographies document the importance of herring to Coast Salish peoples in the early twentieth century. In the Puget Sound region, both herring and herring eggs (roe) were harvested by Coast Salish tribes including the Nisqually51. We received permission from the Puyallup Tribe of Indians to use herring bones retrieved from the Burton Acres Shell Midden (45KI437), and the Suquamish Tribe permitted the use of bones excavated from the Bay Street Shell Midden (45KP115). The bones from 45KI437 are curated at the Burke Museum of Natural History and Culture, and the bones from 45KP115 are curated at the Suquamish Museum. Both archaeological sites are located within protected bays in the Puget Sound that encompass or are close to (within 10 km) contemporary herring spawning grounds  of fish remains in certain layers24, while salmonids are the next most abundant group.The Burton Acres Shell Midden is located along the shoreline of Quartermaster Harbor on Vashon Island 51. People at this site had access to both riparian and marine resources, and eight different fish taxa were identified at this site. Pacific herring account for one-third of identifiable fish remains in the total assemblage, indicating prominence in the diet . When available, we selected prootic bones for aDNA analysis to avoid sampling the same individual herring multiple times.Two chronological units—one early and one late—were established for both archaeological sites to enable a diachronic comparison of herring genetic diversity. Herring bones SI Data  were sel53. We hereinafter refer to this layer as “post-contact”.At the Burton Acres Shell Midden, archaeological herring specimens from the deepest excavated stratum (2F) were associated with charcoal yielding a calibrated radiocarbon age estimate of 910–685 calibrated years before present . Specimens from the stratigraphically more recent Stratum 2B was likely deposited after AD 1890 because this stratum was not intermixed with underlying deposits and it contained metal artifacts (such as a dime minted between AD 1860 and 1890), and an 1890s button51 to provide the greatest diachronic separation of samples. Radiocarbon dates provided an age estimate of 800–550 cal BP for Component 1 and an estimate of 400–100 cal BP for Component 3. Table Archaeological herring specimens from the Bay Street Shell Midden were selected from the oldest (Component 1) and the most recent (Component 3) of the three components defined for the site54. Bones were chemically decontaminated through immersion in 6% sodium hypochlorite solution for 5 min. Samples were subsequently rinsed twice with ultra-pure water and bones were exposed to UV light for 15 min. We extracted DNA from each sample following the protocol described in Yang et al.55. In brief, samples were incubated overnight at 50 °C in 3 mL of lysis buffer , and were subsequently concentrated to approximately 100 µL using Amicon 10kDA centrifugal filter units. DNA extracts were purified using Qiagen MinElute columns following the manufacturer’s instructions, and DNA was eluted in 50 µL of warmed (56 °C) Buffer EB. To test for contamination during DNA extraction, blank controls were included in each extraction and processed during all subsequent steps.All sample preparations and aDNA extractions were conducted in a dedicated aDNA laboratory at Simon Fraser University, Canada, following strict protocols to minimize contamination with modern DNA19. We hereinafter refer to these genetically distinct herring populations as January–February spawners, March–April spawners, and May spawners. One of these loci is within a gene (SYNE2) which influences the development of retinal photoreceptors in vertebrates56 and may play a role in the photoperiodic regulation of reproduction in herring57. Additional information on assay development is described in Chamberlin et al.19.Each archaeological sample was genotyped at seven nuclear SNP loci using custom TaqMan assays. These seven loci had divergent allele frequencies when comparing herring populations that spawn at different times of year in the Puget Sound58. These reactions were set up in a dedicated clean room at the University of Washington. Preamplification reactions were conducted in 24 µL volumes containing Qiagen Multiplex PCR Master Mix, 0.2 µM of each forward and reverse SNP primer, ultra-pure water, and 4 µL of template aDNA. Thermal cycling was performed on a Bio-Rad C1000 Touch , using these conditions: initial denaturation at 95 °C for 15 min, followed by 14 cycles of 94 °C for 30 s, 57 °C for 90 s, 72 °C for 60 s, and a final extension of 72 °C for 10 min. Negative controls were included with each preamplification reaction.To increase the amount of template aDNA available for TaqMan genotyping reactions, we first conducted a preamplification PCR with all primers following the protocol of Smith et al.59, each sample was genotyped twice and negative controls were included with every genotyping reaction. We estimated the genotyping discrepancy rate by dividing the total number of genotype mismatches by the total number of samples. Ancient samples that were missing genotypes at more than one locus were removed from the data set.We diluted these preamplification PCR products 1:3 for use in subsequent TaqMan genotyping reactions. All genotyping reactions took place in 12 µL volumes containing 1X TaqMan Universal PCR Master Mix, 1X TaqMan assay, nuclease-free water, and 2 uL of template DNA. Thermal cycling was performed on an Applied Biosystems 7900HT Fast Real-Time PCR system  as follows: initial denaturation at 95 °C for 10 min, followed by 60 cycles of 95 °C for 15 s and 60 °C for 60 s. Following quality control procedures established in Speller et al.18 as the modern herring data set. These modern herring samples (N = 347) were collected from eight distinct spawning aggregations in the Puget Sound between 2007 and 2016  using exact tests in genepop63.Pairwise 64 and implemented in the R package rubias64. In brief, this approach uses Markov chain Monte Carlo (MCMC) to estimate the proportion of individuals in a mixture originating from different reference populations (or aggregates of reference populations known as reporting groups), given genotypic data in the reference populations. Additional information on how we used simulated and empirical data to assess the predicted accuracy of mixed stock analysis is included in the Supplemental Material. Using the seven nuclear loci and three reporting groups , the predicted accuracy of mixed stock analysis ranged from 85 to 88% using simulated data, and 97–99% using empirical samples from contemporary herring populations  using three reporting groups (January–February spawners vs. March–April spawners vs. May spawners).N = 159) at six or more loci. Negative controls did not amplify in any genotyping reaction, and we did not observe any genotype mismatches between repeated genotyping of ancient samples. The number of genotyped ancient samples at each locus, archaeological site, and archaeological layer ranged from 30 to 46 individuals, while observed heterozygosities ranged from 0 to 0.50 (Supplementary Table N = 4) were observed in samples collected from the older Burton Acres archaeological layer . Overall, six out of 42 (14%) tests for linkage disequilibrium were statistically significant (α = 0.05) and all of these occurred in the Burton Acres site .We were able to successfully genotype 83% of archaeological samples  of herring bones are spatially correlated with modern spawning locations, often co-occurring at distances less than 1 km, while sites with lower abundances of herring bones are more variable in regards to their distance to contemporary spawning locations22. These findings suggest that fishing for herring was concentrated around spawning grounds or nearby ‘pre-spawn holding areas’, locations where herring aggregate in the weeks leading up to reproduction. Additionally, there is ethnographic evidence that some of the herring harvest occurred during the spawning season: “During the spawning season when large schools of herring and smelt crowded in to shore, the fish were dipped out with a loosely twined piece of matting…”28.Regional analyses of archaeological sites in the Pacific Northwest17 show that the only May-spawning population reproduces at Cherry Point, a location that is approximately 170 km to the north of the sampled archaeological sites  geographic and temporal stability in spawning activity and (ii) place-based fishing during the spawning season. To rigorously test this hypothesis, future research efforts should analyze archaeological specimens collected from locations used by contemporary May spawners and compare patterns of resource use over large geographic areas. This archaeological information could yield insights into the homing behavior of herring and help place documented extirpations of contemporary herring populations in the appropriate long-term ecological context.Comparing the archaeological data to contemporary distributions of spawning herring can provide insights into the spatial extent and seasonality of fishing, as well as the long-term distribution of herring populations. Both archaeological sites analyzed here are proximate within 10 km) to contemporary winter and early-spring spawning grounds  this population was more widely distributed in the past or (ii) people traded herring captured from different locations in the Puget Sound.Differences in the seasonal distribution and relative abundance of herring populations also likely contributed to the patterns observed in the archaeological record. In other words, it is possible that Coast Salish people in central Puget Sound primarily harvested January–February and March–April spawners because of these populations’ long residence times in the estuary and their localized abundance. It is important to consider this scenario, as it is documented that Coast Salish people also fished for herring outside of the spawning season71, and these items may have been an especially welcome food source in the winter and early spring when other fresh foods were not easily accessible or in short supply. For example, here is an account of the seasonal importance of herring to the Puyallup and Nisqually people in central Puget Sound: “Herring eggs were, also, eaten fresh with smoked salmon or, if the supply of smoked salmon were exhausted by the time herring eggs were available, they were eaten with sprouts”28. Recent oral histories from the Haida First Nation in British Columbia also document this seasonal importance of herring: “Along with the berries, the herring was the first from the ocean to be harvested. Our women would gather in their boats to harvest the K’aaw [herring roe on kelp] and celebrate another cycle of life”72. Thus, the predominance of January–February and March–April spawners in our archaeological sites could have been driven by people’s foraging preferences, and indicate that herring were especially desirable during the winter and early spring, when other fresh foods were unavailable or difficult to obtain.The populations of herring preserved in the archaeological record could also reflect seasonal variability in the desirability of this resource. Pacific herring and its roe are calorie-rich and nutritious73 and New Zealand Maori74. Bioenergetic models indicate that warm oceanic regimes are associated with low recruitment and biomass in Pacific herring75, and water temperatures are estimated to have been ~ 0.9 °C warmer during the Medieval Warm Period than during the Little Ice Age in the Pacific Ocean76. Despite this potential climatic variability, our data suggest that winter and early spring spawners remained abundant enough that fishing communities in central Puget Sound could access them. This could be an indication that herring population diversity and/or traditional management and harvesting practices were resilient to past climatic oscillations. Stability in marine resource use across this time is also observed in other archaeological sites on the Northwest Coast78.Although the temporal resolution of archaeological sites analyzed in this study is limited to centennial scales, our results demonstrate that human communities in central Puget Sound predominantly harvested winter and early-spring spawners over a period of several hundred years. This consistency in resource use is noteworthy given that our sampling period may have encompassed the transition between the climatic oscillations of the Medieval Warm Period  and Little Ice Age , which are associated with large changes in resource use in other coastal communities such as the Norse GreenlandersBy genotyping a small number of SNPs with divergent allele frequencies in modern herring populations, we were able to assign ancient individuals to contemporary populations and estimate their reproductive timing. This approach allowed us to genotype large numbers of ancient individuals in a cost-effective manner and obtain sample sizes similar to those used in genetic studies of modern populations. The ability to analyze robust sample sizes reduces random sampling error and may lead to more accurate estimates of genetic diversity in ancient food resources.SYNE2) that is strongly correlated with spawn timing in both Atlantic79 and Pacific herring18, suggesting that this genomic region is involved in the regulation of reproduction. In addition, allele frequencies at this locus are very divergent between winter and spring spawning populations across the Pacific Northwest coast but temporally stable within spawning groups18. These lines of evidence suggest that allele frequency differences at this locus are maintained by natural selection and have been present for a long time.A limitation of our study is that we could only distinguish between a small number of reporting groups, rather than individual contemporary spawning populations. Furthermore, because we used a small number of loci isolated from contemporary samples, we may have missed additional layers of genetic diversity such as the presence of distinct winter spawning populations or extinct populations that are not included in our contemporary genetic baseline. Finally, our analyses assume that allele frequencies at loci differentiating contemporary populations are stable over time, and were thus also correlated with spawn timing in ancient populations. One of the SNPs used in our analyses is within a gene (80, and the declining cost of sequencing will make the analysis of large numbers of ancient individuals and loci more common in the future81. This will not only facilitate genomic studies in well-studied charismatic species such as large mammals and hominids but may also allow for population genetic and ecological investigations in species that are currently important to human livelihoods and ecosystem processes.At present, there is a trade-off between analyzing whole genomes for small numbers of samples and analyzing many samples at few loci. Although working with aDNA is technically challenging and ancient samples may be limited or scarce, developments in laboratory techniques, such as hybridization capture21. For example, phenologically diverse herring populations are important to the diets of surf scoters20 and Chinook salmon19, and recent declines in the overall abundance of phenologically distinct herring populations in the Puget Sound have may negatively impacted the growth and survival of marine predators like salmon19.Ecological studies have established that resource waves benefit consumers by extending foraging opportunitiesBy analyzing ancient DNA preserved in archaeological faunal assemblages, we were able to identify population-level genetic diversity in herring that sustained communities of Coast Salish people over a period of multiple centuries. Patterns of genetic diversity observed in the archaeological data are associated with phenologically distinct spawning groups in contemporary herring which form the basis of an ecological resource wave. Our results indicate that phenologically diverse herring stocks were important to the food systems of Coast Salish people in the Puget Sound, and that intraspecific biodiversity underpins the ecosystem services provided by healthy forage fish populations.82.Our experimental approach is applicable to investigations of resource use in other systems, and it can be used to assess long-term trends in population diversity and establish ecological baselines for conservation. This type of information will help reveal interactions between human cultures and the ecological services provided by phenologically diverse resources. Such studies may also help identify the seasonality of archaeological site occupation with more precision. Furthermore, the long-term temporal context provided by archaeological data is especially valuable as climate change and other human activities lead to the synchronization of resource phenology and the disruption of food web dynamicsSupplementary Information 1.Supplementary Information 2.


Malus species. We used genotyping-by-sequencing (GBS) technology to analyze samples of 112 individuals belonging to 18 populations of wild Malus spp. Using GBS, we identified thousands of single nucleotide polymorphisms in the species analyzed. M. baccata and M. toringo, two dominant mainland species of the Korean Peninsula, were distinguishable based on their genetic structure. However, M. toringo collected from Jeju Island exhibited a different genetic profile than that from the mainland. We identified M. cf. micromalus as a hybrid resulting from the Jeju Island M. toringo (pollen donor) and the mainland M. baccata, (pollen recipient). Putative M. mandshurica distributed on the Korean Peninsula showed a high structural and genetic similarity with M. baccata, indicating that it might be an ecotype. Overall, this study contributes to the understanding of the population history and genetic structure of Malus in the Korean Peninsula.This study was to understand the genetic structure and diversity of the Korean  Malus Mill.  is an economically important genus comprising 25–55 taxa and is widely distributed in the temperate regions of the northern hemisphere3. Malus domestica Borkh., a representative crop of apples, underwent hybridization and gene introgression with various wild Malus species over decades4. Interspecific hybridization in Malus is well documented9. Although several taxonomists proposed reasonable classification systems based on its morphology19, reticulated processes of Malus have hindered the accuracy of its phylogenetic inferences7. The concept of hybridization leading to the formation of new species has been proposed in the 1950s20. Hybrids usually possess an intermediate phenotype of the two parental species; describing their morphology can be subjective and difficult to predict owing to the combined expression of parental genes21. Some hybrids are identified based on their morphological properties, but their origins remain unknown . Similarly, distinguishing interspecific hybrids of Malus among species of the Korean Peninsula can be difficult. Since the identification of two subspecies by Nakai10  Kom. ex Skvortsov]), up to eight taxa including cultivar have been reported in the Korean Peninsula over the last 20 years  and has been widely used as a rootstock for breeding because it is disease-free and cold-resistant24.rs Table . M. baccM. baccata and M. toringo (Siebold) Siebold ex de Vriese are the most widely distributed representative taxa in the Korean Peninsula19. M. baccata is predominantly distributed along the main mountain-range connecting the north and the south  up to the central region and is vertically distributed from the lowlands to about 1500 m above sea level , petiole (M. baccata: 2–5 cm and M. toringo: 1.5–2.5 cm), pedicel length (M. baccata: 4–7 cm and M. toringo: 1.2–4 cm), bud characteristics (M. baccata lineage: convoluted bud and M. toringo lineage: folded bud), and fruit size (M. baccata: 6–9 mm and M. toringo: 4–6 mm)26. However, individuals with intermediate morphology between M. baccata and M. toringo possess the following characteristics27: (1) ovate leaf but slightly lobed, short petiole and pedicel; (2) slightly lobed leaf, long petiole and pedicel; and (3) variations in fruit sizes.M. baccata and M. toringo are distinguished based on their morphological characters29, maximum likelihood (ML) tree constructed using 79 protein-coding chloroplast genes clustered the sequences based on geographic distribution, and not the species monophyletic group30. In addition, the unweighted pair group method with arithmetic mean (UPGMA), based on morphological characters and random amplified polymorphic DNA (RAPD) analysis, revealed that the individuals formed two distinct clusters based on species data32. Intermediate morphological features27 and inconsistent molecular evidence30 have raised doubts over the natural hybridization of the two species as suggested by Chang et al.14; however, no assessment has been conducted at the population level.Previous research has not deduced consistent results with regard to the relationship between the two species. Although phylogenetic studies performed using sequences of partial chloroplast and nuclear regions have confirmed that M. micromalus Makino is another putative hybrid species, which was first described by Makino in 1908, and introduced in South Korea from China33. This species is presumed to have resulted from a cross between M. baccata and M. spectabilis (Sol.) Borkh34. M. micromalus is distributed at high elevations in areas such as the Hallasan Mountain (> 1500 m) on Jeju Island, which is approximately 140 km south of the Korean Peninsula mainland33. The species has the following morphological characteristics: tomentose in the midrib, pubescent petiole when young, pedicels tomentose-pubescent, and persistent calyx at pomes33. Although this taxon has been described in various studies, there are discrepancies in its classification13. Moreover, literature on whether calyx is caducous or persistent is inconsistent33, making it difficult to identify distinct morphological differences between M. micromalus and other related species.M. mandshurica (Maxim.) Kom. ex Juz., which is distributed in China, Russia, Japan, North Korea, and South Korea25. Although M. mandshurica possess similar characteristics as M. baccata, it exhibits sparse pubescence on the petiole and abaxial pubescence on leaves, or subglabrous along midvein and lateral veins26. However, preliminary investigation of specimen and image data of both herbariums  revealed that it was difficult to differentiate M. mandshurica from M. baccata. Moreover, there have been discrepancies in the ranking of this taxon, with some studies classifying it as a separate species and others classifying it as a variety of M. baccata26. Additionally, M. mandshurica has also been classified as an ecotype and treated as a synonym of M. baccata35. Previous studies that performed simple sequence repeat (SSR) analyses using several markers could not differentiate the two species because the genetic distance within M. baccata taxa was scattered across other Malus species36. Additionally, the low resolution of a phylogenetic approach cannot efficiently represent the relationship between the two species37. Species boundaries are often difficult to identify morphologically; therefore, integrated taxonomy which includes additional information, such as molecular data, biogeography, and ecology is required39. When several lines of evidence  independently indicate a certain species boundary, discretely evolving lineages can be identified42.Another relevant species is Malus45. Molecular diagnosis of genetic diversity within the nuclear genome using GBS has the potential to examine hybrid signals, introgressions, and the population–species boundary48. This is the first study to explore the possibility of hybridization and evolutionary relationship of Malus across the Korean Peninsula. The aim of this study was to identify Malus-specific SNPs in the Korean Peninsula and establish their phylogenetic relationships by GBS approaches. The objectives of this study were to (1) verify whether there is a hybrid or genetic exchange between M. baccata and M. toringo; (2) identify the entity of M. cf. micromalus distributed in Jeju Island; (3) compare the genetic structure of M. toringo distributed in the mainland and Jeju Island; and (4) verify the entity of M. cf. madshurica collected in the Korean Peninsula.Recently, next-generation sequencing (NGS) has identified sufficient single nucleotide polymorphisms (SNPs) from population genetic studies to facilitate the study of genetic diversity in M. baccata, three accessions of M. toringo, four accessions of M. mandshurica, four accessions of M. micromlaus, and three accessions of M. spectabilis) were obtained from a previously conducted GBS study49. During the field survey, young leaves were collected from 112 individuals belonging to 18 populations  in South Korea  from natural habitats Table  as well 50, with minor modifications. Briefly, DNA samples were digested with ApeKI . Adapters were subsequently ligated to the sticky ends by adding T4 DNA ligase  to each well. Digested DNA samples, each with a different barcode adapter, were combined and purified using a commercial kit , according to the manufacturer’s instructions. The libraries were sequenced using Illumina NextSeq500 for 95 samples and Hi-seqX for 20 samples.The GBS libraries of 112 individuals were constructed as previously described51. Reads were trimmed, and adaptors were removed using cutadapt 3.552. In this study, two data matrices were used: (1) data set A: SRA data (25 accessions) + 112 individuals of Malus species collected from the Korean Peninsula; (2) data set B: 112 individuals of Malus species. A total of 137 individual reads were aligned to the Malus domestica chromosome sequence retrieved from NCBI (ASM211411v1) to generate the BAM files using BWA v0.7.17 and SAMtools v1.9 with the default parameters55  which were subsequently input into the “Populations” program of STACKS v2.60 that utilize SNP calling. The ‘-p’ option, which indicates the minimum number of populations required to process a locus, was set to 23 for 137 accessions (data set A) and 18 for 112 accessions (data set B). The designation of parameters 23 and 18 in two data sets for SNPs calling is a strict strategy to extract only the SNPs observed in all groups. The minimum percentage of individuals in a population required to process a locus for that population (-r) was set to 1 for 137 accessions of data set A because for the maximum value (1) is to minimize the error of SNPs generated due to external data. And we set the 0.8 for 112 accessions of data set B. The options listed were later equally applied to both datasets. The minimum percentage of individuals in/across a population required to process a locus for that population were increased . Additionally, we set the minimum minor allele frequency to 0.05 (--min_maf 0.05), maximum observed heterozygosity to 0.95 (--max_obs_het 0.95) and restricted the study to only the first SNP per locus (--write-single-snp).https://qgis.org/en/site/) was used for visualizing the heat map and the source of GPS coordinates was collected from 833 specimens deposited in KH and KB herbariums.Qgis v3.26.1 desktop application (https://github.com/edgardomortiz/vcf2phylip.git), with the value for minimum samples per locus (MIN_SAMPLES_LOCUS) set to 137 in data set A and 112 in data set B. The network was created and visualized using SplitsTree4 software56.To explore the genetic distance of data set A and B, we constructed a SplitsTree network using strictly filtered 563 and 613 SNPs, respectively. The vcf file generated from “populations” in STACKS v2.60 was converted into the FASTA format from the vcf2phylip  of K = 1–10 values, using admixture_ linux-1.3.060. The graphical display of the population structure was generated using DISTRUCT61.To investigate the population structure, we used VCF file generated using the “populations” function in STACKS v2.60. To illustrate the relationship among various individuals, principal component analysis (PCA) was conducted based on 563 SNPs (137 accession) in data set A and 8426 SNPs (112 accession) in data set B, using graph Laplacian PCA (gLPCA)Ho and He), nucleotide diversity (π), inbreeding coefficient (FIS), and population differentiation (pairwise FST), were calculated using the “populations” function in STACKS v2.60.Common measures of genetic diversity, including private allele number (AP), percentage of polymorphic loci (%Poly), observed and expected heterozygosity  all samples ; (2) M. toringo, including C3 and C5 ; (3) M. baccata (C1 and C2) and M. mandshurica ; (4) M. baccata (C1 and C2), M. mandshurica (C7), and M. micromalus , as well as M. toringo (C3 and C5) and M. micromalus .An analysis of molecular variance (AMOVA) was performed to estimate genetic variation among and within populations, using the adegenet66. Structure file produced from the “populations” program of STACKS v2.60 was converted into the SNP model (heat map data) using the “vcfR2migrate” plug in vcfR package67. We redefined groups based on the genetic clusters inferred from STRUCTURE , with the mutation rate as a constant68. A more permissive acceptance criteria was applied (“heating”) with four chains set at different temperatures . The run used 100 long-samples with an increment of 10,000  after a burn-in of 100,000. The number of migrants per generation (Nm) was calculated using the following equation: Nm = [(θ x * M y → x)/4])68.To investigate the ancient gene flow, we utilized a coalescent approach implemented in MIGRATE- N 3.6.11 to calculate the migration rates between groups representing populationsThe materials used in this study are not included IUCN red list. Sample collections was conducted in compliance with the regulations of the Act on the creation and furtherance of arboretums and gardens.Malus domestica reference genome. Approximately 83.2% of the 112 accessions and 92.3% of the 25 SRA data were mapped to the genome  were generated from data set B Table . After qme Table . Subsequme Table  and S3. Malus and its relatives , M. mandshurica (including M. cf. mandshurica in Mand_GW population), and M. spectabilis. Within cluster I, comparisons between collected samples and SRA data formed a subgroup based on their original collection site. Particularly, four SRA-M. baccata  were closely related to the Korean population  were found to be of a different lineage, a long branch that was separately from the Korean population. Nevertheless, no clear distinction at the species level could be observed in the SRA branch. Putative M. micromalus collected from Jeju Island  from M. toringo, M. baccata, M. cf. mandshurica, and M. cf. micromalus. PC2 separated M. toringo into two groups, according to the geographic distribution. Three populations of cultivar were located in the center of M. toringo in PC2  formed a group which was distinct from the other accessions in quadrant 4  and the other with two populations (T_JJ2 and T_JJ3). The distinct groups remained consistent across the entire range of K values  population collected from Jeju Island shared both M. toringo and M. baccata genetic composition. Specifically, the M. baccata genotype was mixed with geographically more distant populations  than closer populations . The highly polymorphic and diverse JEJUtype population exhibited mixed components of M. baccata  and M. toringo (T_JJ2 and T_JJ3).Population genetic structure analysis (PCA and STRUCTURE) was conducted based on 8426 SNPs, using the STACKS v.2.60 software. The eigenvalues of PC1 (47.7%) and PC2 11.35%) explained approximately 59.1% of the total variance  of the 18 Malus populations varied from 0.074 in T_GW to 0.203 in the island JEJUtype. The observed heterozygosity (Ho) of the 18 Malus populations varied from 0.074 in T_GW to 0.260 in B_GN. Wright’s F-statistic (FIS) of the 18 Malus populations ranged from -0.170 in B_JB to 0.042 in Mand_GW  groups, which were geographically distributed close to the M. toringo population  between populations ranged from 0.011–0.47  with similar geographical distributions. The lowest FST value was identified among the three M. baccata populations . These findings indicated that the genetic difference within populations was greater than those between populations as well as the occurrence of gene flow between populations. Most FST values between the M. baccata and M. mandshurica groups as well as M. toringo were more than 0.3 , and three M. toringo populations  were extremely low. Additionally, the FST values between M. mandsurica and M. baccata (0.074–0.181) were lower or similar to the FST values measured between M. baccata populations, indicating the occurrence of gene flow between populations70.Pairwise 47 Table ; a simil47 Table  and 6. TM. toringo; M. baccata, and M. mandshurica; and M. baccata, M. mandshurica, and M. micromalus) was extremely high . Our analysis further revealed that between the two groups of M. toringo and M. micromalus, 52% of the total variations occurred within samples, while the remaining 37.8% and 19.2% of variations occurred between groups and between populations/within group, respectively. The results of the migration rate analysis revealed that the Nm between all populations without model specification was 0.373, with the highest value obtained (0.748) at M3 → 5 78. However, this discrepancy could be attributed to differences in research methodology79. Moreover, the findings of the present study are in agreement with those of previous GBS-based studies80. However, the low diversity observed in most Korean Malus populations is unusual for Malus  and Zone IV (Miratic zone 2)91. C2 belonged to the boundary between Zone II and Zone III in the low-altitude regions of the Korean Peninsula91, which is similar to the biogeographic regional boundary proposed by Lee and Yim90. Nevertheless, the possibility that the distance between populations, altitude, and climatic zone changes affect the genetic composition of Malus cannot be ruled out  collected from Jeju Island did not exhibit a close relationship with SRA-M. micromalus, nor did it show a close relationship with M. spectabilis , which has been the center of debate for nearly 100 years since Makino described a new species  in 190833, is a different entity from M. micromalus. To better understand the evolutionary history of this hybrid, extensive sampling is necessary to identify genetic clues in their natural habitat and assess their phenetic characters, particularly in the species-rich neighboring countries (China and Japan).The present study indicated that the JEJUtype did not cluster with previously reported targets  analyzed in this study exhibited similar morphological characteristics with previously identified M. mandshurica, such as tomentose hair on petiole and adaxial leaf pubescence26. However, SplitsTree analysis indicated that there was no close relationship between M. cf. mandshurica accessions (population: Mand_GW) and SRA-M. mandshurica, which is a remarkable finding  using the reference mapping method. Notably, majority of the Malus populations distributed in the Korean Peninsula form geographically distinct groups that coincide with those in the floristic zones91. Contrary to our prediction that hybridization would occur between M. baccata and M. toringo, we observed that the two species were genetically differentiated. Putative M. micromalus from the Jeju Island might represent a new hybrid. Our findings provide valuable insights into the genetic profile of Korean Malus. However, thorough morphological studies with extensive sampling are needed to clarify these species attributes.The current study primarily explored the genetic structure and geneflow of Supplementary Tables.


Festuca species are an important source of feed for animals due to their high tolerance to low temperatures and drought. Using simple sequence repeat (SSR) markers developed from expressed sequence tags (ESTs), the genetic diversity of 43 populations of Festuca species from Oruro, La Paz, Potosi and Cochabamba departments was evaluated for the purpose of providing information for effective conservation and breeding. In total, 64 alleles were detected across the 43 populations. SSR locus NFA 142  had the highest number of detected alleles, while locus FES 13  had the highest polymorphism information content (PIC) at 0.55. Based on Nei’s genetic distance between populations, the unweighted pair group method with arithmetic mean (UPGMA) cluster analysis revealed two major clusters, each consisting of populations from the four departments. However, the analysis of molecular variance (AMOVA) revealed that only 5% of the total variation separated these two groups, indicating low genetic differentiation between the populations. It was also found that there was a low but significant differentiation (0.08%) between the population groups of the four departments (p = 0.01). The newly developed EST-SSR markers are highly valuable for evaluating the genetic diversity of Bolivian fescues and other related species.In the highlands of Bolivia, native  Festuca L. belongs to the Poaceae family and is, along with the genus Poa L, the largest genus of the tribe Poeae. Festuca species are geographically distributed all over the world with a chromosome number ranging from diploid (2 n = 2 x = 14) to dodecaploid (2 n = 12 x = 84) [Festuca by conventional plant breeding is slow since many species are predominantly, if not completely, allogamous wind-pollinated grasses [Festuca is centered in the Holarctic zone of Eurasia and North America, approximately 140 species of this genus are found in South America [Festuca described in the Catalog of Vascular Plants of Bolivia [The genus  x = 84) ,4 and in grasses . Althoug America , where t Bolivia , 17 are  Bolivia ,11,12,13Parastrephia-Baccharis grassland, Páramo rangelands and Bofedals) [Festuca species are an important source of feed for animals inhabiting the highlands of Bolivia due to their high tolerance to low temperatures and drought. However, overgrazing, climate change, and the advance of the agricultural frontier have resulted in grassland reduction [The most important feed sources for both camelids and ruminant species in the highlands are native grasslands, which are composed of different combinations of plant species  . Naturaleduction .Festuca dolichophylla J. Presl (known as “chilliwa”) and Festuca orthophylla Pilg. (known as “iru ichu”) are the two most important forage species of this genus in the highlands of Bolivia [Festuca species, such as Festuca rigescens J. Presl Kunth, Festuca boliviana E.B. Alexeev, and Festuca humilior Nees and Meyen, have also been reported as forage sources in the region [ Bolivia ,17. The  Bolivia ,19,20, w Bolivia ,22. OtheFestuca species worldwide, especially in the two most economically important species, the hexaploid tall fescue (Festuca arundinacea) and the diploid meadow fescue (Festuca pratensis) [Extensive research related to genetic diversity using different molecular markers such as simple sequence repeats (SSRs), amplified fragment length polymorphism (AFLP) and inter simple sequence repeats (ISSRs) has been conducted on atensis) ,24,25,26atensis) ,29,30,31atensis) ,32,33,34atensis) . Even thAssessment of genetic diversity using molecular markers provides highly valuable basic information that can be utilized in breeding programs with the aim of developing suitable cultivars adapted to different environments and utilization systems . MicrosaFestuca species using EST-SSR markers. The results are important for the effective management of native fescues in Bolivia, comprising both the development of conservation strategies and the future breeding of this forage grass.The main objective of this study was to evaluate the genetic diversity of 43 Bolivian populations of Festuca spp. were collected from 43 populations in the highlands of the departments of Oruro, La Paz, Potosi and Cochabamba from March to May 2015  were added, and then the samples were homogenized in a Mixer Mill MM400  for 1 min at 30 Hz. A total of 400 µL of lysis buffer  was added, and the tube was then centrifuged at 13,000 rpm for 10 min. DNA was isolated from 200 µL of lysate by a QIAcube HT extraction robot using the QIAamp DNA mini kit  based on the standard protocol provided by the supplier with the addition of RNAse A having a final concentration of 0.1 mg/mL in the elution buffer. The quality and quantity of the isolated DNA were evaluated by agarose gel (1.5%) electrophoresis and spectrophotometry .http://bioinfo.inf.ufg.br/websat/; accessed on 01 November 2022).Based on the EST sequences of tall fescue available at the National Centre for Biotechnology Information (NCBI), sequences containing SSRs with two to six repeat motifs were screened using WebSat , a web shttp://bioinfo.ut.ee/primer3-0.4.0/; accessed on 01 November 2022).Once redundant, overlapping and very short sequences were excluded, 28 sequences containing SSRs were chosen as candidates for designing primer pairs using Primer3 primer designing program  Initial denaturation step of 3 min at 95 °C, 38 cycles of 30 s denaturation at 94 °C, 30 s primer annealing at 60 °C, 45 s primer extension at 72 °C, and a final primer extension at 72 °C for 20 min (for NFA036).(2)Initial denaturation step of 3 min at 95 °C followed by nine touchdown cycles of 30 s denaturation at 94 °C, 30 s primer annealing at 58 °C reduced by 1 °C every cycle and 45 s extension at 72 °C. This was followed by 29 cycles of 30 s denaturation at 94 °C, 30 s annealing at 48 °C and 45 s primer extension at 72 °C with a 20 min final extension step at 72 °C .(3)Initial denaturation step of 3 min at 95 °C followed by nine touchdown cycles of 30 s denaturation at 94 °C, 30 s annealing at 59 °C reduced by 1 °C every cycle and 45 s primer extension at 72 °C. This was followed by 29 cycles of 30 s denaturation at 94 °C, 30 s annealing at 49 °C and 45 s primer extension at 72 °C with a 20 min final extension step at 72 °C (for FES04 and NFA126).(4)Initial denaturation step of 3 min at 95 °C followed by nine touchdown cycles of 30 s denaturation at 94 °C, 30 s annealing at 60 °C reduced by 1 °C every cycle and 45 s primer extension at 72 °C. This was followed by 29 cycles of 30 s denaturation at 94 °C, 30 s annealing at 50 °C and 45 s primer extension at 72 °C with a 20 min final extension step at 72 °C .PCR was performed in an S1000TM Thermal Cycler  in a reaction containing 2.5 µL PCR buffer , 0.3 mM of each dNTP, 0.3 mM of each forward and reverse primer, 1 U (0.04 U/µL) Dream Taq DNA Polymerase and 10 ng/µL of template DNA. Sterile Millipore water replacing DNA was used as a negative control. A 50 bp ladder  was used to estimate the size of the amplified products. PCR products were analyzed using electrophoresis with 1.5% agarose gels containing GelRed™ and visualized using a UVP M-26XV benchtop UV transilluminator. Fragments were amplified using one of the following temperature profiles:Amplified products were kept at 4 °C until electrophoresis. The PCR products of the 12 EST-SSR loci were multiplexed into five panels based on their differences in fragment sizes or fluorescent labels of the forward primers, with each panel containing PCR products of two or three loci. For multiplexing the PCR products labeled with the same fluorescent dye, a minimum expected fragment size difference of 54 bp was considered to avoid overlapping. The multiplexed PCR products were diluted 25× using Millipore water, and the fragment analysis was performed on a 3500 Genetic analyzer .® V2.7.0 software  was used for peak identification and allele size determination. Each peak was considered as an allele, and the genotype of each individual at each locus was determined and exported to Excel for statistical analysis. Observed number of alleles (Na), effective number of alleles (Ne), Nei’s gene diversity of each locus (H) [GeneMarkerocus (H) , Shannonocus (H) .To examine the genetic relationship among the 43 populations, Nei’s measure of genetic distance was calculated, and a dendrogram was constructed using the unweighted pair group method with arithmetic mean (UPGMA) using MEGA7 software ,55. To eOf the 12 EST-SSRs, five were newly developed, and seven were previously published for tall fescue . Their rIn total, 64 alleles were recorded in the 10 polymorphic loci, with the observed number of alleles (Na) per locus ranging from three (NFA036 and NFA147) to twelve (NFA142), and an average of 6.4. The effective number of alleles (Ne) at each locus varied from 1.03 (NFA036) to 3.29 (NFA142) with an average of 2.0 . The NeiAllele frequencies for the 10 polymorphic loci ranged from 0.001  to 0.987 (NFA036) .The percentage of polymorphic loci (PPL) in each population ranged from 40 to 90%, with a mean of 61.2% . The higp = 1; p = 0.24). When populations were grouped according to departments, AMOVA allocated 0.08% of the total variation among groups indicating a very low level of genetic differentiation  using present/absent allele data of the 43 populations revealed no significant genetic variation among populations p = 1; . Similarp = 0.88). Therefore, the wide range of altitudes of the sample collecting sites (3217–4325 masl) did not influence the genetic variation among populations , no significant differentiation was found (ulations .Nei’s genetic distance between populations ranged from zero (between populations 18 and 19 from La Paz) to 0.863 (between population 4 from Oruro and 36 from Cochabamba). The second lowest genetic distance (0.002) was recorded between population 8 from Cochabamba and 21 from La Paz. The second highest pairwise genetic distance (0.845) was observed between populations 4 and 38, followed by 0.831 (population 4 vs. 37) and 0.805 (population 26 vs. 36) .Populations 36, 38 and 37 were the most distantly related populations, with mean genetic distances of 0.48, 0.47 and 0.46 from the other populations. The overall mean genetic distance between the populations was 0.31.p < 0.05) between the two clusters, with 5% of the total variation differentiating them.A UPGMA clustering with a bootstrap test was conducted based on Nei’s genetic distances at the population level. The 43 populations were grouped into two major clusters (I and II) supported by a moderate bootstrap value of 83% . ClusterFestuca species. These results are consistent with previous reports, which showed that EST-SSR markers have a high transferability rate among species of the same genus [Festuca species). The average number of alleles per locus in the present study was 6.4, which is less than the value reported by Mian [Analysis of genetic diversity is not only important for crop improvement but also for efficient management and protection of the available genetic resources. EST-SSR markers have the advantage of facilitating population genetic analysis and are interesting because of their capacity to amplify conserved homologous sequences across different grass species , where sme genus ,43,58,59me genus . The locme genus ,58 of otAmong the five EST-SSR loci developed in the present study, FES09 was monomorphic, whereas FES04, FES13 and FES24 showed a relatively high level of polymorphism with PIC of 0.22, 0.30 and 0.55 and gene diversity (H) of 0.53, 0.65 and 0.66, respectively. Interestingly, the mean PIC of the newly developed polymorphic EST-SSRs (0.29) was higher than the mean PIC (0.12) of those polymorphic loci selected from Saha et al.  suggestip = 0.01) was obtained among accession groups that were grouped according to their departments , although it was very low. Among the four departments, the average gene diversity of populations from Cochabamba (H = 0.27) was slightly higher than that of the other departments (H = 0.23 to 0.24). A rare allele (at locus NFA-142) unique to Cochabamba and shared by only two populations (7 and 40) was also identified (Festuca species [Festuca diversity.When the 43 populations were grouped according to their biogeographic provinces (xerophytic and mesophytic Puna), there was no significant differentiation among the groups indicating similar genetic diversity of Bolivian fescues across the two biogeographic provinces. The estimates of the within-population genetic diversity varied about twofold in both provinces, although the mean gene diversity (H) of xerophytic Puna (0.23) was slightly lower than that of mesophytic Puna (0.25). Both provinces also have a similar number of population-specific alleles . The resentified . The res species  or other species ,62. A lo species , who indFestuca species, which will allow effective conservation and genetic improvement in breeding programs.The absence of significant differentiation among populations, as revealed by AMOVA, strongly suggests the outcrossing nature of Bolivian Fescues that led to high gene flow between the populations. This is in line with a lack of a clear pattern of clustering of populations according to their geographic regions. For example, populations from Cochabamba were not clustered separately from those from La Paz, Oruro and Potosi, which can be attributable to a high gene flow between the regions. The fact that some populations from Cochabamba were closely clustered together with those from La Paz or Oruro or Potosi indicates a lack of significant correlation between genetic distance and geographical distance between the populations. Considering the absence of previous studies on native Bolivian fescues using molecular markers, the present results are important for the development of effective management strategies for native and endangered SSR loci FES04, FES13, FES24 and NFA142 can be considered suitable targets for future population genetic analysis in fescues or other Poaceae species owing to their high genetic polymorphism. In this study, no significant differentiation among populations was found, indicating a lack of clear population structure caused by factors such as high gene flow. The up twofold differences in genetic variation within populations obtained in this study have direct implications for setting conservation strategies, which include identifying genetic diversity hotspots for Bolivian fescues for in situ conservation. Although no clear genetic diversity hotspot was identified in the present study, the results suggest Cochabamba is a better-suited department for in situ conservation of Bolivian fescues. The fact that a significant differentiation  was found among population groups at a department level suggests the need for further studies that cover wider geographic areas to shed more light on the genetic diversity of Bolivian fescues and beyond.