Pinus nigra) forests were investigated in the Amyntaio – Ptolemais – Kozani Basin, which has been for decades the largest lignite mining and burning center of Greece, with a total installed generating capacity of about 4.5 GW, operating for more than 70 years and resulting in large amounts of primary air pollutant emissions, mainly SO2, NOx and PM10. P. nigra, a biomarker for air pollution and a keystone species of affected natural ecosystems, was examined in terms of phenology (cone and seed parameters), genetics (283 AFLP loci) and epigenetics (606 MSAP epiloci), using two populations (exposed to pollution and control) of the current (mature trees) and future (embryos) stand. It was found that cone, seed, as well as genetic diversity parameters, did not show statistically significant differences between the exposed population and the control. Nevertheless, statistically significant differences were detected at the population epigenetic level. Moreover, there was a further differentiation regarding the intergenerational comparison: while the epigenetic diversity does not substantially change in the two generations assessed in the control population, epigenetic diversity is significantly higher in the embryo population compared to the parental stand in the exposed population. This study sheds a light to genome dynamics in a forest tree population exposed to long term atmospheric pollution burden and stresses the importance of assessing both genetics and epigenetics in biomonitoring applications.Forest species in the course of their evolution have experienced several environmental challenges, which since historic times include anthropogenic pollution. The effects of pollution on the genetic and epigenetic diversity in black pine ( In recent times, the scientific community has focused on the humanly induced environmental changes that strongly affect forest species, especially keystone species that are the main determinants of sensitive natural ecosystems. Indeed, the health of natural forest tree populations are threatened not only by biotic (insects and diseases), but by abiotic  factors as well . Air polPinus nigra J.F. Arnold) forests are the most prevalent in the area and were found to present a higher capacity of absorbing and accumulating heavy metals  than other tree species in the APKB (Air pollution is quite significant in the region of Western Macedonia and in particular in the Amyntaio-Ptolemaida-Kozani basin (APKB), where the largest lignite deposits of Greece is concentrated. Close to 50-70% of Greece’s total electricity output has been produced in this region since 1950 . The emithe APKB .Field measurements of air pollutant concentrations are used to identify and quantify the impact of air pollutants on human health, agriculture and natural ecosystems. However, for a large-scale comprehensive assessment, modelling of pollutant transport is needed. Relevant models use mathematical and numerical techniques to simulate the physical and chemical processes that affect air pollutants as they disperse and react in the atmosphere. They are useful for investigating air pollutant transport, dispersion, deposition and finally, their temporal and spatial distribution . The advAir pollution exerts significant effects on the biome. While there is clear evidence that morphological features are very sensitive to selective pressures from the local environment , the cleRapid adaptation to environmental stress is also facilitated by epigenetic changes, which form a crucial biological mechanism that interconnects the environment, the genotype and the phenotype. Insofar, DNA methylation has been the most-studied epigenetic mechanism in ecology and population epigenetics . There i2, NOx and PM10 (particulate matter). We selected P. nigra as a biomarker in order to assess quantitative morphological parameters, as well as population genetic and epigenetic diversity using molecular markers, and evaluate possible changes in the black pine forest stands triggered by accumulated pollution stress lasting for the past 70 years. Furthermore, we also studied the epigenetic profile of the next generation (embryos), to investigate if DNA methylation changes were transmitted and/or persisted.Herein, we selected the Amyntaio-Ptolemaida-Kozani Basin as our study area, due to its high levels of air pollution for a prolonged period of time. We used a prognostic air pollution model (TAPM) for investigating the footprint of SO22.12, NOx and PM10 on the P. nigra natural populations in the APKB. In total, eight locations that correspond to the natural distribution of P. nigra in the APKB area were investigated , were selected, following a methodology adapted from The air pollution model TAPM  was usedstigated Table 1.stigated . The mod2.2Sampled trees from APKB were chosen to be at least 30 m apart in order to avoid sampling from related individuals, while isolated trees (no other tree in a radius of 25 m) were excluded from sampling. All sampled individuals were mature trees (average tree age: 80 years) of the dominant class having similar DBH and tree height. A total of 24 trees were sampled per population in terms of needles (10 per tree) and cones (three per tree) for seed extraction.For the genetic and epigenetic analyses, a total of N=48 samples per population were used. Each population sample comprised of 24 samples from needle and embryo tissue (the latter excised from seeds), respectively.2.3The following parameters were assessed: (1) number of scales, (2) percentage of full seeds (out of 1000 seeds) and (3) weight of 1000 seeds (taking into account filled seeds only).2.4In total, 70 soil samples were collected per soil layer per population, 35 from the top 5 cm of soil and 35 corresponding to a 5 -10 cm depth. A total of 48 soil samples were collected at a 3 m distance from the trunk of each sampled tree while 11 additional samples per population were randomly collected from the two sites. The samples were oven dried at 85°C and then homogenized and pulverized using different diameter sieves. The concentrations of Ca, Mg, K and Na were implemented using sequential leaching and those of Cu, Mn, Zn, and Fe by the DTPA method . The con2.5Total genomic DNA of each individual was extracted and isolated from needles and embryos using the Dellaporta protocol . For a g2.6AFLP analysis was performed according to 2.7The detection of methylation-sensitive amplified polymorphisms was based on 2.8t-tests for independent samples, while percentages were evaluated using two sample t-test between percentages. The StatPac software (http://www.statpac.com) was employed.Descriptive statistics of soil related variables, as well as of cone and seed parameters, were calculated. Population differences were assessed using PT with 9,999 permutations, a measure suitable when comparing different data types  and (iii) mean Shannon’s information index (Iepi). GenAlEx 6 , was evaluated to locate the population most exposed to air pollution and the one with minimum exposure (control) .The mean annual concentrations of PM10 (particles with aerodynamic diameter less than 10 microns), the mean annual concentrations of SO2, the dry deposition of NOx and the wet deposition of SO2, as predicted by the TAMP model, are presented in P. nigra in the APKB. Although the values as predicted by the model were generally not high, they differ notably in these areas, for instance the SO2 concentration is 4.2x higher in the former. The two areas present the same average altitude of 1500 m, and prevailing climatic conditions were similar  and the two populations appear to have practically zero genetic distance. The orientation of the populations is presented by a PCoA in The AFLP analysis revealed a total number of 283 amplified fragments, out of which 208 (73.5%) showed polymorphism in the exposed population and 212 (74.9%) were polymorphic in the control population. The slight decrease in the percentage of population polymorphism that was found in the exposed population was insignificant. The values of the basic population genetic parameters are reported in 3.5epi=57.1%) and the control population 413 (Pepi=68.15%). Total methylation polymorphisms that pertain to semimethylated (h) and fully methylated (m) epiloci amounted to Pepi=57.06% for the exposed population and Pepi=68.02% for the control populations, respectively (epi) showed statistically significant differences in diversity between the exposed and the control populations when assessing the mature stand, as shown by a t-test . Differentiation was also studied in the subepiloci level. AMOVA and ΦPT results for n-subepiloci, h-subepiloci and m-subepiloci , showed that the differentiation between the exposed and control mature populations was small as well  and the control population 291 (Pepi = 60.88%). Total methylation polymorphisms, i.e., semi methylated (h) and fully methylated (m) epiloci polymorphism amounted to P=72.02% for the exposed population and P=60.9% for the control population (epi) showed statistically significant differences in diversity between the exposed and the control embryo populations as well  for embryo tissue, which implies weak, but significant differentiation. AMOVA results and ΦPT for n-subepiloci, h- subepiloci and m- subepiloci , showed that there was some differentiation between the two embryo populations , but this is likely the result of the geological background of each region, a finding consistent with pertinent literature .4.2The number of fertile cone scales is an indication of a population’s fecundity and indirectly of the (absence of) inbreeding . The per4.3Pinus pinaster  was lower than the values of The values of population genetic parameters were generally lower than those observed in other studies. For instance, the values of the effective number of alleles were lower than those observed by P. sylvestris. Gene flow between populations was very high. Gene flow is generally very high in forest trees over long distances , by pollution from lignite power stations and mines that has been ongoing for about the past 70 years. It is unclear if the detection of rather lower neutral diversity values in the exposed population can be considered as an early sign of a reaction to selective pressures from the gaseous pollutants in the region, in the presence of some genetic hitchhiking event.In populations that grow in similar altitude and climatic conditions and present strong gene flow, absence of significant differences in their neutral genetic diversity can be expected, except of course for genes under differential selection pressure . The 4.4epi) significantly different between the exposed and the control populations. Total epigenetic differentiation between the exposed and control populations was low but significant, a result that was also found at the subepiloci level. While a higher percentage of the epi-variation resided within populations, this value was less than that found in the respective genetic analysis, where almost all variation resided within populations  between populations of Poa annua, while ST=0.50-0.80) on natural populations of Fallopia japonica grown in different environments , was significantly different between the exposed and control populations. More importantly, while the epigenetic diversity does not substantially change in the two generations assessed in the control population , this is not the case in the exposed population. In the latter, epigenetic diversity increases considerably in the embryo population , showing a significant potential increase in the forthcoming generation.MSAP analysis on embryos was performed in order to assess the epigenetic status of the next generation. Epigenetic diversity . The results agree with Quercus ilex subject to drought and found a reduction of polymorphism in epiloci, but not in genetic loci. Epigenetic variation on a total population basis , was also assessed by the Shannon index. Epigenetic diversity was found to be numerically lower than genetic diversity , may be an indicator of the next-generation adaptability to current local environmental conditions. In this respect, our study may reveal a case where the evolutionary dynamics are first driven by spontaneous epigenetic modifications which arise at faster rates than genetic changes . Our resPrevious studies have suggested that methylation polymorphism in plant genomes is a confounding result of adaptation due to environmental stress . DiversiIn view of the upcoming climate change, epigenetic processes can provide a quick response machinery, allowing evolutionary mechanisms to lead to rapid micro-evolution and adaptation of populations . AccumulP. nigra population and the control population.The role of the interaction between genetic and epigenetic diversity in the process of evolution of natural populations, is beginning to be understood  and moreThe original contributions presented in the study are included in the article/FAA conserved and designed the study. EK has carried out sampling, experimental and data analyses. EA and IG participated in the analyses. FAA, EK and EB evaluated the results. EK and FAA drafted and refined the manuscript. EA, IG and AD, critically read the manuscript. All of the authors have read and approved the manuscript.


Acacia senegal that are undergoing various population disturbances remain scarce. Understanding patterns of gene dispersal in these ecosystems is important for conservation, landscape restoration and tree improvement. We investigated pollen and seed mediated gene flow in two A. senegal populations of contrasting state (less disturbed and heavily undisturbed) using nine microsatellites and 128 genotyping‐by‐sequencing single nucleotide polymorphism (SNPs) multilocus genotypes of two growth stages (juvenile and adult trees) and their spatial locations. We performed parentage assignments using likelihood approach and undertook spatial genetic structure (SGS) analyses for the two growth stages through correlation among kinship coefficients and geographical distances between pair of individuals. The SNPs showed higher resolving power and assignment rates than microsatellites; however, a combination of the two marker‐types improved the assignment rate and provided robust parentage assessments. We found evidence of long‐distance (up to 210 m) pollination events for both populations; however, the majority of seed dispersal was found closer to the putative maternal parent. On average, parentage analysis showed high amounts of pollen (40%) and seed (20%) immigration in both populations. Significant positive SGS was found only for the adult cohorts in the less disturbed population for distance classes 20 and 40 m, indicating historical short‐distance seed dispersals. Our results suggest long‐distance gene flow within the species and we recommend conservation of remnant and isolated populations or individual trees to promote genetic connectivity.Even though pollen and seed dispersals are some of the important factors that determine tree species survival across landscapes, gene dispersal data of important tropical dryland tree species such as  Acacia senegal is important for conservation, landscape restoration, and tree improvement program. We investigated pollen and seed mediated gene flow in two A. senegal populations of contrasting state (disturbed and undisturbed) using multilocus genotypes of seedlings and potential parent trees and their spatial locations. Our results suggest long‐distance gene flow within the species and recommend conservation of remnant and isolated populations or individual trees to promote genetic connectivity.Understanding patterns of gene dispersal in tropical dryland species such as  Our study hypothesize that the heavily disturbed population of A. senegal will display restricted gene flow pattern characterized by short‐distance pollen and seed dispersal, leading to non‐random distribution of genotypes in space compared to the less disturbed population. We used mating patterns and the SGSs of the pre ‐ and post ‐disturbance gene pools to illustrate the extent of gene flow, and separated them into components of identified local against unidentified putative local or immigrant genes. We achieved the aims of the present study using both microsatellite and single nucleotide polymorphism (SNP) markers. Implications of the study findings for conservation and management of the species are discussed.The aim of the present study was to investigate the patterns of gene flow within two natural populations of 22.1A. senegal within Lake Baringo woodland, Solit (00°14′N and 36°35′E) and Kimalel (00°21′N and 36°03′E), with contrasting anthropogenic disturbance status were studied. We surveyed the two populations prior to the study and classified them as heavily disturbed (Solit) or less disturbed  based on population disturbance index (PDI) and density of adult trees  per population. Mean number of adult trees  per hectare was determined for each population. In this study, a tree was considered to be in disturbed habitat when PDI of the population was greater than 5 (presence of more than 60% of disturbance indicators) and the density of adult tree less than 50 per ha. Population with PDI of 1.141 and high density of adult trees was considered as less disturbed while the population with PDI of 6.52 and low adult tree density was considered as heavily disturbed  in each population to estimate the level of realized gene flow within the two populations. Leaf samples from all adults (>5 cm dbh) and juvenile (<5 cm dbh) individuals were collected from a 100 × 200 m quadrat within each population. All the sampled individuals were georeferenced using global positioning system (GPS) and the distributions were as shown in Figure 2.3A. senegal  method described by Hanaoka et al. . We perf2.4A. senegal samples.We used the Institut de Biologie Intégrative et des Systèmes (IBIS) genotyping by sequencing tool of Université Laval, Quebec, Canada to undertake the genotyping  of 0.3, GC score average > 0.85, and sample call rate > 0.90. In addition, the selected 662 SNPs were further tested for adherence to HWE using a Chi‐square goodness‐of‐fit test in GENEPOP v.4 Rousset, . This te2.5A), mean effective number of alleles (Ae), observed heterozygosity (HO), expected heterozygosity (HE), and inbreeding coefficient (FIS) using GenAlEx 6.503 , polymorphism information content (PIC), and non‐exclusion probability (NEP) were calculated for microsatellite and SNP markers. We also evaluated how many SNP loci would be sufficient to ensure assignment of parent pair with 95% confidence. We selected subsets of 128, 100, 80, 60, 40, and 20 most polymorphic SNP loci based on PIC, and repeated simulations using these subsets.We used the same parentage analysis protocol for both microsatellite and SNP markers using the parentage assignment software, CERVUS version 3.0.7 . We also performed heterogeneity test to assess difference in SGS patterns between the two sites  with 10,000 permutations in SPAGeDi software version 1.5  was found in the juvenile cohort than adults in the heavily disturbed population, while there was lower genetic diversity in juvenile in the less disturbed population. However, fixation index was higher for the juvenile than adult individuals in both populations  for microsatellites and two for SNPs in both populations  and 71.7% (38) of the juveniles in the less disturbed and heavily disturbed populations, respectively, to at least one parent with 95% confidence . In these assignments, we regarded the closest parent assigned to an offspring as the maternal parent. In this case,  m Table . However m Table . In the 3.6p < .05; Figure r) values for the less disturbed population adult cohort ranged from 0.136 to −0.203. We identified similar significant spatial structure with Moran Moran's I in SPAGeDi for the distance classes less than 40 m; however, higher Moran's I of 0.153 was found at 20 m distance class . The heavily disturbed population also showed significant spatial structure in the adult cohort at distance class 200 m , however, no spatial structure was detected in any distance class in this cohort using Moran's I analysis. The correlogram of the spatial structure analysis for all the populations and cohorts based on autocorrelation are as shown in Figure p > .05).To assess the presence of SGS within the cohorts, we divided the spatial distances into 20 m distance classes and a combination of SNP and microsatellite markers were used. Among all the groups analyzed, only the adult cohort of the less disturbed population showed significant spatial structure in all distance classes (p < .05) in spatial population structure  at all distance class sizes for adult cohort . Assuming that intraspecific pollination dominates over interspecific pollination, the random distribution of A. senegal trees in heavily disturbed population, could have contributed to extensive pollen dispersal than the less disturbed population.The relationship between distance and probability of mating was significant in both study populations. The average distance between mates within the same area was different between the two population. Although pollen flow may reveal a certain level of stochasticity, the greater average distance between mates observed in heavily disturbed population can be partly related to a lower population density that is, larger distances among individuals within the same area and an opened population structure allowing for more extensive pollinator movement within the population  have been reported for other tropical trees pollinated by bees and wasps ; data curation ; formal analysis (lead); funding acquisition ; investigation (lead); methodology ; project administration (lead); resources ; software (lead); supervision ; validation ; visualization ; writing – original draft (lead); writing – review and editing . Eunice W. Githae: Conceptualization (supporting); data curation (supporting); formal analysis (supporting); funding acquisition ; investigation (supporting); methodology ; project administration (supporting); resources ; software (supporting); supervision (supporting); validation (supporting); visualization (supporting); writing – original draft ; writing – review and editing . Damase P. Khasa: Conceptualization ; data curation ; formal analysis ; funding acquisition ; investigation (supporting); methodology (supporting); project administration ; resources ; software (supporting); supervision ; validation ; visualization ; writing – original draft ; writing – review and editing .The authors declare that there is no conflict of interest.Benefits from this research accrue from the sharing of our data and results on public databases as described above. A research collaboration was developed with scientists from the three institutions and all collaborators are included as coauthors and the results have been shared broadly, including outside the scientific community. The authors are committed to international scientific partner‐ships, as well as institutional capacity building.Appendix S1Click here for additional data file.


Moehringia tommasinii (Caryophyllaceae), a narrow endemic plant inhabiting rock crevices in the northwestern Adriatic, and some populations of co-occurring and widespread M. muscosa, an ecologically divergent relative with an overlapping flowering period. We performed reciprocal crosses within and between taxa and used molecular markers to assess the extent of gene flow within and between populations and taxa. Using coefficient of inbreeding, population size, seed weight, pollen-to-ovule ratio, and flower display size, we also looked for evidence of a selfing syndrome.Mating system is one of the major determinants of intra- and interspecific genetic structure, but may vary within and between plant populations. Our study model included all known populations of M. tommasinii. These populations exhibited genetic structuring, with their size positively correlated with both seed weight and pollen production. Although a selfing syndrome could not be confirmed as the majority of selfing resulted from allogamous treatments, the occurrence of selfing was notable. In the presence of M. muscosa, at a site where both species coexist closely, a distinct pattern of fruit production was observed in M. tommasinii following various pollination treatments. Molecular and morphometric data provided evidence of hybridization followed by local extinction at this site.A surprisingly high variation in mating systems was observed among populations of M. tommasinii. Lighter seeds and lower pollen production observed in populations with pronounced selfing do not provide enough evidence for the selfing syndrome. Detected gene flow between M. tommasinii and the sympatric M. muscosa suggested weak reproductive barriers between the taxa, which could pose a conservation problems for the former species. Hybridization leading to local extinction may also resulted in floral polymorphism and disruption of mating patterns of M. tommasinii.Population size proved to be the most important factor affecting the mating system in genetically structured populations of The online version contains supplementary material available at 10.1186/s12870-023-04384-8. Theoretical and empirical studies agree that mating systems are primary determinants of the extent and organization of intra- and interspecific genetic structure within and among plant populations –4. The lGenetic variation within and among populations is also influenced by a range of ecological factors , 12, incOne of the most widely accepted ecological scenarios for the evolutionary transition from outcrossing to selfing is a state of limited pollen/pollinator availability or mate availability, e.g., in case of low plant population size and/or density , 20–24. The change from allogamy (geitonogamy and xenogamy) to autogamous self-fertilisation is usually associated with a selfing syndrome – a characteristic set of changes in floral morphology and function that promote autonomous selfing , 29. As Considerable variation in mating systems has been observed among populations, both spatially and temporally –43, due However, in fragmented and genetically depauperate populations that have evolved differently in contrasting environmental conditions, among-population crosses may result in outbreeding depression by thwarting co-adapted gene complexes  or disruMoehringia tommasinii March., a narrowly distributed chasmophyte inhabiting a preserved, until rather recently human-free, and homogeneous, albeit extreme, habitat as a model system to answer the following questions: (a) What is the extent of variation in mating systems among populations? (b) How do mating systems and population size affect genetic parameters within and among populations? (c) Is there evidence of a selfing syndrome as a result of reduced population size and/or outbreeding depression? Finally, (d) does outcrossing with an evolutionarily closely related but ecologically divergent M. muscosa affects the mating system of our model plant?Assuming that the mating system is a primary, yet not the only, force regulating patterns of genetic variation both within and among populations, and to reduce confounding effects of a variety of ecological, historical, and evolutionary factors, operating within or among populations of phylogenetically closely related taxa , 4, 15, Moehringia tommasinii  Gren. (incl. M. insubrica Degen), M. papulosa Bertol., M. provincialis Merxm. & Grau, and M. dielsiana Mattf. , for each value of K, as well as by calculating an ad hoc statistic ΔK based on the rate of change in the log probability of data between successive K values [K) was made. Runs were clustered and averaged using CLUMPAK [p value was obtained by 9,999 randomizations, with the null hypothesis of no relationship between the two matrices being true.For each microsatellite locus, basic population-genetic parameters  were calculated using Cervus 3.0.7 software . The obt “poppr”  was used / 4 Fst . To asse / 4 Fst . The sam / 4 Fst , 129 gen / 4 Fst . Bootstr / 4 Fst . Fifteen v0.6.92 . was useK values , the cho CLUMPAK . The cor CLUMPAK ; a p valFis), floral display size , seed weight, fruit production by outcrossing (xenogamy), and population size on the one hand, and the within-population fixation index, outcrossing, and population size as factors on the other, were analysed separately for all populations using the same procedure. Response variables  were adjusted to a normal distribution with identity link function using algorithms implemented in R package ‘stat’ to model responses. The significance of slope was estimated using the G–statistics, an equivalent to χ2, with one degree of freedom.The relationships between the within-population fixation index (Additional file 1.Additional file 2.Additional file 3.Additional file 4.Additional file 5.Additional file 6.Additional file 7.Additional file 8.Additional file 9.


Hexaplex trunculus, is a marine gastropod whose reproductive fitness can be severely affected by very low concentrations of antifouling compound tributyltin (TBT). TBT has strong xenoandrogen impacts on snails, causing the development of imposex , thereby affecting the fitness of entire populations. TBT is also known as a DNA-demethylating agent and an obesogenic factor. The aim of this study was to unravel the interactions between TBT bioaccumulation, phenotypic responses, and epigenetic and genetic endpoints in native populations of H. trunculus. Seven populations inhabiting environments along the pollution gradient were sampled in the coastal eastern Adriatic. These included sites of intense marine traffic and boat maintenance activity and sites with low anthropogenic impact. Populations inhabiting intermediately and highly polluted sites exhibited higher TBT burdens, higher incidences of imposex, and higher wet masses of snails than populations in lowly polluted sites. Other morphometric traits and cellular biomarker responses did not show clear differentiation among populations in relation to marine traffic/pollution intensity. An analysis of methylation sensitive amplification polymorphism (MSAP) revealed environmentally driven population differentiation and higher epigenetics than genetic within-population diversity. Moreover, decreases in genome-wide DNA methylation coincided with the imposex level and snail mass, suggesting an epigenetic background of the animal phenotypic response. Banded murex,  Anthropogenic disturbances are progressively changing environmental conditions in all ecosystems. In response to altered environments, organisms can adapt, acclimatise, migrate, or die out. Emerging evidence suggests that in addition to genetic variation, epigenetic variation plays an important role in species’ ability to cope with environmental change . EpigeneNucella lapillus, TBT-induced imposex is known to have caused a complete population collapse [One of the most striking examples of anthropogenically induced compounds affecting the fitness of natural populations is the case of tributyltin (TBT). Since their discovery in 1960s, organotin compounds (OTCs) (particularly TBT) have been widely used as the main ingredients of antifouling paints for ships and fishing nets. However, it quickly became evident that TBT is a strong endocrine disruptor whose effects extend to untargeted species, causing severe outcomes for marine wildlife ,8. Low icollapse ,13,14,15collapse ,16,17. Tcollapse . TBT is collapse ,21,22, acollapse . Due to collapse .Hexaplex trunculus) is a common Mediterranean gastropod. Due to its benthic lifestyle, limited mobility, crawling larvae , and ability to develop imposex at TBT concentrations as low as 1 ng Sn g−1, it has been described as the most reliable indicator of TBT pollution [H. trunculus has been extensively studied for decades. However, the link between environmental exposure to TBT, DNA methylation changes, and organism phenotypic responses has not been examined to date. Therefore, the aim of this study was to unravel the relationships among TBT burden, epigenetic and genetic patterns, and phenotypic responses in natural populations of H. trunculus.Environmental epigenetics using marine invertebrates as suitable sentinel organisms represents an innovative and ground-breaking approach for improving the management and conservation of natural marine resources . Banded ollution ,27. The In this study, the effects of TBT on genome-wide DNA methylation were examined with the aim to provide important insights into the DNA methylation effects in native murex populations upon environmental TBT exposure and their link to reproductive fitness. This study also questioned if and how environmental TBT exposure affects epigenetic population differentiation and diversity. The environment usually impacts the overall genetic makeup of affected populations over the course of generations through the forces of natural selection , altered mutation rates, genetic drift, and altered gene flow. However, in terms of epigenetic responses, one would expect immediate, within-generation local effects. In addition to examining population reproductive fitness, further phenotypic characteristics that might show responses toward TBT load  were also studied to broaden the comprehension of TBT’s overall biological impact on the phenotypes in natural populations.Hexaplex trunculus) specimens were collected in April and May 2014 at seven locations along the pollution gradient in the mid and south Adriatic , is referred to as an intermediately polluted site. Sites within ports and marinas with intense marine traffic activities—Trogir, Vranjic, Split-Špinut, and Split-harbour—are referred to as highly polluted sites. This classification was confirmed by TBT concentrations in the sediment. Sediment samples collected in 2015 revealed a TBT concentration of around 40 ng Sn gon limit . The amoectively . Low, inAt each site, between 48 and 88  adult mu4  and hexane via mechanical shaking at 350 rpm for 30 min. Analyses were carried out on a gas chromatograph  with a pulsed flame photometric detector . Quality control was performed by analysing the certified reference materials (CRM) for mussels . Measurements were performed in two technical replicates, and the mean value of two replicates was used for further analyses. Data are presented as mean ± SD across populations. The Mann–Whitney U test was used to perform statistical comparisons due to non-normally distributed data (level of significance: p ≤ 0.05).The measured organotin compounds (OTCs) included TBT and its degradation derivative dibutyltin (DBT). OTCs in whole murex tissue were determined in 8–10 females per population following the previously described analytical procedure [H. trunculus individuals [Imposex was determined in 215 females , following the previously described recommendations ,29, as dividuals . Each imp ≤ 0.05. The use of ordered logit regression was proposed to model ordinal variables in environmental monitoring studies [In order to test whether the imposex level  could be predicted by either TBT tissue accumulation or DNA methylation (predictors), an ordered logit regression model was applied using the “polr” function available in the “MASS” R package (R studio version 1.0.136). The model was considered significant at  studies .p ≤ 0.05).Biomarker measurements  were conducted on the digestive gland tissue of 6–10 individuals per population in accordance with well-established protocols for marine invertebrates, with slight modifications ,35,36,37For each individual, several morphometric measures were recorded using a digital calliper with a precision of 0.1 mm: shell length (TL), shell breadth (TB), shell opening breadth (AW), and shell opening length (AL). Total mass (TM) was recorded using a digital balance with a precision of 0.1 g. Upon the careful removal of the murex shell, the total mass of the soft tissue (WM) and the mass of the gonads and hepatopancreas (GHM) were recorded. Murex snails were dissected in order to determine their sex and imposex level in females by making a longitudinal cut along the hypobranchial gland. Males were distinguished by a penis located behind the right ocular tentacle, the presence of vas deferens, and  the absence of a capsular gland and vulva, which are typical female characteristics ,38. In op ≤ 0.05). The means of each morphometric measure per population for males and females were separately transformed in a population pairwise proximity matrix with Ward’s method based on squared Euclidean distances using cluster analysis in SPSS Statistics 23.0 (IBM). Proximity matrices were correlated with a matrix of geographic distances between populations and matrices of population pairwise FST values for genetic and epigenetic data using the Mantel test. The test was performed using the “mantel.rtest” function in the “ade4” R package.All morphometric measurements were log-transformed and normalised to the total length. PCA was separately performed on male and female individuals in R studio 1.0.136. An ANOVA and Tukey’s post hoc test were applied to the PC1 and PC2 scores in order to define significance in population separation using the “aov” and “TukeyHSD” functions, respectively, from the “stats” R package (p ≤ 0.05). In order to test whether the WM (response variable) could be predicted by TBT tissue accumulation or DNA methylation (predictors), a linear regression model was applied. The effect of imposex level (predictor) on WM (response) was tested using a one-way ANOVA. We employed the same method to test whether individual- or population-level TBT burdens were good predictors of genome-wide DNA methylation. Both the linear regression model and the one-way ANOVA were applied using the “lm” R function. The model was considered significant at p ≤ 0.05.Data on the total soft tissue mass (WM) are presented as mean ± SD. Statistical comparisons were performed using an ANOVA  followed by Tukey’s post hoc test using the “aov” and “TukeyHSD” functions, respectively, in R studio . After 1 min at 95 °C, DNA fragments were amplified for 20 cycles under the following conditions: 20 s at 94 °C, 30 s at 56 °C, and 2 min at 72 °C. After the final elongation step for 30 min at 60 °C, the pre-amplification product was diluted to 1:20 with nuclease-free water and used for selective amplification. This procedure was performed using the Eco-AAG, Mse/Hpa-TCC, Mse/Hpa-TAG, Eco-ACT, Mse/Hpa-TAC, Mse/Hpa-TAG, Eco-ACG, Mse/Hpa-TCC, and Mse/Hpa-TAG EcoRI-MspI/HpaII primer pairs. The Msp/Hpa primers were end-labelled with Hex or 6-Fam . The final amplification mixture (20 μL) contained 3 μL of the diluted pre-amplification mixture, 0.2 mM of dNTPs , 2.5 mM of MgCl2, 0.05 μM of the EcoRI primer, 0.25 μM of the MspI/HpaII-labelled primer, 1X a Hot Start DNA polymerase buffer, and 0.5 U of Hot Start DNA Polymerase . After 2 min at 94 °C, the DNA fragments were amplified for 30 cycles under the following conditions: 20 s at 94 °C, 30 s at 66 °C for the first cycle followed by a decrease of 1 °C per cycle for 10 cycles, 56 °C for the remaining 20 cycles, and 2 min at 72 °C. A final extension step was performed for 30 min at 60 °C. The DNA fragments were detected using an automatic GeneScan ABI3130 apparatus from Macrogen Inc. (Korea). The presence or absence of fragments was scored on chromatograms using GeneMapper Genotyping Software 1.5. All fragments between 50 and 400 bp and passing the threshold of 100 RFU were scored. Rare fragments, e.g., those differing only in one individual, were deleted from the dataset. Furthermore, scored fragments were filtered in order to ensure reproducibility between runs. Fragments differing in average frequency for more than 0.15 between samples of the same population run in two different plates were eliminated from further analyses. To check for the error rate between runs, 24 samples were analysed twice using the Eco-ACT, Msp/Hpa-TAC, and Msp/Hpa-TAG primer combinations. The error rate was calculated for both the EM and EH sets as [MSAP was performed on 30 individuals per population. Genomic DNA from 150 mg of murex foot tissue was extracted using the GenElute Mammalian DNA miniprep kit  according to the manufacturer’s instructions. The standard protocol for MSAP was followed  with sliThe error rate calculated using all of the scored fragments was 11.14% for the EcoRI-HpaII fragment set and 10.97% for the EcoRI-MspI fragment set. Using only the fragments retained after fragment-filtering the error rates were 4.03% and 4.75% for the EcoRI-HpaII and EcoRI-MspII fragment sets, respectively. MSAP fragments were analysed using the “msap” R package . Every fp ≤ 0.05), and the results are presented as mean ± SD. For MSL fragments, the frequency of all methylation states across all MSL was calculated for each individual. Populations were statistically compared using an ANOVA followed by Tukey’s post hoc test using the “stats” R package . The GenAlEx 6.5 software was used to perform a principal coordinate analysis (PCoA) using the MSL fragments. PCoA graphs were generated using the epigenetic distance between individuals. An ANOVA and Tukey’s post hoc test were applied to the PCo1 and PCo2 scores in order to define significance in population separation using the “aov” and “TukeyHSD” functions, respectively, in R  using the Arlequin 3.5.2.2., Mcheza , and BayUsing the population pairwise FST values obtained through Arlequin and geographic distances between populations, the Mantel test was performed using the “mantel.rtest” function in the “ade4” R package to check for isolation by distance (IBD).−1, respectively. At the intermediately polluted site of Marina, the mean TBT concentration was 10.7 ng Sn g−1, and at the four highly polluted sites, the mean TBT concentrations ranged from 21.4 to 43.1 ng Sn g−1. The amount of accumulated TBT in specimens with low imposex levels (1–4) was significantly lower in comparison to the amount recorded in snails with higher imposex levels (5–8)  B. The meAt the sites with a low pollution intensity, Čiovo and Ston, the majority of females  had imposex levels in the lower range (1–4) and no individuals with an imposex level of 8 were recorded. At the intermediately polluted site of Marina, 33.3% of females had lower imposex levels (1–4) and 38.9% of females had the highest imposex level (8). At the four highly polluted sites, none of the examined females had the lowest imposex level (1) and between 80.6% and 97% of females had higher imposex levels (5–8) .Both TBT and DNA methylation were found to be significant predictors of imposex, as shown using the ordinal logit regression model . Based oAlthough the biomarker results showed some population-specific responses, they did not indicate the separation of the sites according to the TBT pollution level. The PCA only revealed the significant separation of the intermediately polluted site, Marina, from the lowly and highly polluted sites in PC1 . AltogetH. trunculus morphometric measures showed the significant separation of female snails originating from highly polluted sites, Trogir and Split-Špinut, from the other sites in PC1 . Using linear regression, both TBT accumulation in snail tissue and DNA methylation were significant predictors of snail mass . In general, the increase in snail mass was linked to a higher imposex level, higher TBT accumulation, and decrease in DNA methylation.The total mass of the soft tissue (WM) was significantly different between sites of low pollution intensity, Čiovo and Ston, and intermediately and highly polluted sites, apart from Vranjic . MoreoveAfter fragment-filtering, 568 fragments were retained. We determined 355 polymorphic MSL and 43 polymorphic NML using the “msap” function in R. Considering all methylation states , individuals from the clean site of Čiovo had significantly higher levels of DNA methylation than individuals from the polluted sites of Trogir, Vranjic and Split-Špinut C. Overalp = 0.16) or genetic  differentiation. Neither polymorphic MSL nor NML fragments revealed any obvious epigenetic or genetic structures among the analysed populations using the Structure software. The results of the Mantel test confirmed a lack of isolation-by-distance pattern in either epigenetic  and considerably stronger than the genetic differentiation   . The varp-value of 0.1253). However, the mean population TBT burden was found to be a significant predictor of mean population DNA methylation .The population genetic diversity was observed to be lower than the population epigenetic diversity , as showThe principal coordinate analysis (PCoA) based on individual pairwise genetic distances using the MSL dataset explained 5.65% of variation in first two coordinates and showed a significant separation of sites based on pollution intensity. Sites with a low pollution intensity were significantly separated along the PCo1 coordinate from the intermediately and highly polluted sites . This paFor epigenetic (MSL) fragments, the pairwise FST differentiation between populations ranged from 0.002 to 0.61 and was significant between almost all population pairs except between the high-pollution sites of Split-harbour and Trogir and between Split-harbour and Vranjic . For genUsing the MSL dataset, seven outliers were detected . A total of two outliers overlapped between Arlequin-and Mcheza-detected outliers . Using tOverall, analyses showed higher degrees of epigenomic than genomic within-population diversity and among-population differentiation. No evidence was found that population differentiation stemmed from geographic distances, and PCoA indicated some effect of pollution status on epigenetic divergence.The results of imposex occurrence in murex populations at sites of high marine traffic activities along the Croatian coast presented here are rather alarming, as we recorded that around 50% or more of female snails inhabiting highly polluted sites exhibited the highest imposex level, indicating possible sterility. This is in agreement with other recent data that point to the persistence and constant input of TBT in ports and marinas along the Croatian coast, as well as its notable impact on the reproductive fitness of murex populations ,27. Odontocymbiola magellanica marine gastropod [The TBT-related phenotypic response was further evident in terms of increases in the soft tissue mass (WM) of murex snails, which followed increases in imposex level and bioaccumulated TBT in females. TBT is a known obesogenic factor that can modulate changes in the type and number of generated adipocyte cells . Both inastropod .Epigenetic modifications, such as DNA methylation, have been argued as important players in an organism’s response to altered environmental conditions. In our study, higher incidences of imposex and accumulated TBT in individuals inhabiting ports and marinas were accompanied by decreases in genome-wide DNA methylation. We observed significant effects of both TBT accumulation and DNA methylation on imposex advancement. Furthermore, population-level, genome-wide DNA demethylation was correlated with TBT tissue burden, indicating undirected and stochastic TBT epigenetic alterations that are likely to drive maladaptive biological consequences, such as the observed decrease in reproductive fitness.Sebastiscus marmoratus) and in gastropods (Tritia mutabilis) exposed during embryonic development [T. mutabilis, a simultaneous decrease in DNA-methyltransferase I (DNMT1) expression pointed towards global hypomethylation underlying altered RXR expression. However, we cannot fully confirm the causality of DNA methylation in the cascade of TBT-induced phenotypic responses in the studied imposex-affected murex. The link between epigenetic change and specific phenotypes might indicate that these changes are simply correlated and do not necessarily mean that the epigenetic variant is causative of the phenotype [A decrease in the level of DNA methylation as a result of TBT exposure has also been previously reported in laboratories at both the genome and gene-specific levels. DNA demethylation was observed following the in vitro TBT exposure of preadipocyte mouse3T3-L1 cells . In stroelopment . Moreovehenotype . To fullhenotype . Therefohenotype ,52. Prevhenotype ,53,54. Hhenotype ,55,56,57henotype . It has henotype ,58. ThusDespite the lack of isolation by distance, showing no significant influence of geographic distance on genetic or epigenetic divergence, the studied murex populations were significantly genetically and epigenetically differentiated. Notably, the extent of epigenetic differentiation observed among the snail populations was considerably stronger than the genetic differentiation. A low genetic differentiation implies that at this geographic scale the gene flow among populations is strong enough to prevent substantial genetic divergence. The relatively low resolution of genetic markers used in this study prevented us from conducting the deeper evaluation of population genetic adaptation. Talorchestia capensis exhibited greater epigenetic than genetic variability, and this was attributed to the role of epigenetics in response to local environmental conditions [Potamopyrgus antipodarum for which epigenetic divergence associated with adaptive phenotypes in contrasting environments was observed [H. trunculus stems from differences in local environmental conditions, of which pollution is one major factor. Ecological divergence is increasingly recognised as a major driver of misalignment between patterns of epigenetic and genetic population differentiation . Numerounditions . The effnditions , and in observed . Given TThe higher epigenetic differentiation in comparison to genetic differentiation in our study was further supported by the detected outlier loci in our datasets. Whereas only one outlier was recorded in the genetic loci, seven outliers were defined using the genome scan approach in the epigenetic dataset. The detected outliers remained anonymous, and to this end, their potential role in organism adaptation to local environmental conditions cannot be discussed. The detection of candidate loci that are potentially subject to selection is a widely applied approach using genome-wide genetic data, and it is being increasingly applied for epigenetic markers as well ,63,64. IBuccinanops globulosus gastropod with high imposex occurrence inhabiting harbours has been attributed to TBT and other contaminants such as trace metals and polyaromatic hydrocarbons [Despite the significant differences in TBT accumulation and imposex levels between the sites of low and high marine activity, no clear differentiation in snail biomarker responses that could be linked to the impact of TBT  was recorded in our study. Previous laboratory exposure experiments indicated that TBT acts as an inducer of oxidative stress in gastropods, fish and mammals ,71,72. Tocarbons . Howeverocarbons ,75. The lack of correlation of morphological distances with either geographic or genetic ones indicates the effect of local environmental conditions on morphological differentiation and the probable involvement of phenotypic plasticity. For instance, females from highly polluted sites, Trogir and Split-Špinut, were significantly separated from other sites in PCA. Female snails from those sites also had the highest level of accumulated TBT in their tissue. Several studies previously reported that TBT exposure can affect shell shape in gastropods ,76,77. WIn conclusion, the results of the present study highlight the potential of epigenetic studies to unravel pollution-induced alterations in wild populations. DNA methylation might be crucial for explaining specific phenotypic developmental alterations such as imposex; however, further studies are necessary to determine the exact causative epigenetic mechanisms.


Pinus densata, a pine of hybrid origin on the southeastern Tibetan Plateau, to gain an understanding of the contemporary dynamics of species barriers. We analyzed genetic diversity in a range-wide collection of P. densata and representative populations of its progenitors, Pinus tabuliformis and Pinus yunnanensis, using exome capture sequencing. We detected four distinct genetic groups within P. densata that reflect its migration history and major gene-flow barriers across the landscape. The demographies of these genetic groups in the Pleistocene were associated with regional glaciation histories. Interestingly, population sizes rebounded rapidly during interglacial periods, suggesting persistence and resilience of the species during the Quaternary ice age. In the contact zone between P. densata and P. yunnanensis, 3.36% of the analyzed loci (57 849) showed exceptional patterns of introgression, suggesting their potential roles in either adaptive introgression or reproductive isolation. These outliers showed strong clines along critical climate gradients and enrichment in a number of biological processes relevant to high-altitude adaptation. This indicates that ecological selection played an important role in generating genomic heterogeneity and a genetic barrier across a zone of species transition. Our study highlights the forces that operate to maintain species boundaries and promote speciation in the Qinghai-Tibetan Plateau and other mountain systems.Most species have clearly defined distribution ranges and ecological niches. The genetic and ecological causes of species differentiation and the mechanisms that maintain species boundaries between newly evolved taxa and their progenitors are, however, less clearly defined. This study investigated the genetic structure and clines in  Guo et al. explore the evolutionary mechanisms that contribute to strong genetic structure and species barriers in outcrossing conifers. They show genomic evidence of ecological adaptation and reproductive isolation across a species transition zone, indicating that both intrinsic genetic incompatibility and extrinsic ecological selection play a role in the maintenance of species boundaries. Mating barriers and adaptive divergence are major drivers of speciation . KnowledGymnosperms, especially conifers, are mostly outcrossing and show weak genetic incompatibilities even among taxa that diversified more than 10 million years ago . This crPinus densata forms extensive forests in the southeastern Tibetan Plateau  and highly repetitive genome of pine (P. densata and P. yunnanensis to examine genomic patterns of introgression. We aimed to (1) characterize genetic variation over the range of P. densata to discern the clinal genetic and gene flow patterns across the landscape, (2) reconstruct the demographic histories of P. densata to understand the impact of climate oscillation on population persistence, and (3) quantify patterns of genomic clines across a species transition zone to define the role of selection in maintaining species barriers. Our results show that introgression, geographical barriers to gene flow, persistence during glacial cycles, and ecological selection over heterogeneous landscapes have contributed to the strong population structure in P. densata and the buildup of intrinsic barriers with its progenitor. This pattern may be generally applicable to plant speciation and diversification on the Qinghai-Tibetan Plateau (QTP), one of the most prominent biodiversity hotspots on Earth.To explore these questions, we performed genome-wide population genetic analyses using exome capture sequencing with 40 000 exome probes in a range-wide collection of  of pine , which cP. densata, two representative populations of P. tabuliformis, and nine populations of P. yunnanensis in this study  <0.05 filtering reduced the final dataset to 79 028 SNPs. Exclusion of Pinus sylvestris (outgroup) samples yielded 77 368 SNPs (MAF >0.05) in the 260 samples of P. tabuliformis, P. yunnanensis, and P. densata.We included 24 populations of is study A. Exome K = 5 to be an optimal number of clusters to explain the genetic structure in the P. densata species complex , southwest (SW), central (C), and east (E). The eastern range of P. densata was dominated by P. tabuliformis ancestry  was significant, with FST between the groups ranging from 0.083 to 0.294 with an average value of 0.176, which was 5-fold higher than the average FST (0.036) between populations within each group  in each group of P. densata decreased gradually from east (π = 0.0040) to west (π = 0.0023)  E. Tajimaent past .Table 1GFST values among geographical regions motivated us to generate an overview of gene flow across the landscape. The estimated effective migration surface (EEMS) highlighted scattered barriers to gene flow  of the migration parameter  over time (Ne of ∼1.60 × 105. Group C experienced a steep decline at ∼0.5 Mya followed by a quick recovery to the previous Ne of 0.81 × 105 and has remained relatively stable since. Group SW experienced a contraction at about 0.4 Mya followed by a recovery and then remained at a stable Ne of 0.32 × 105. Group W had a weak expansion around 0.3 Mya and then maintained a low Ne (∼0.23 × 105) over the past 0.3 million years. To validate these results, we used fastsimcoal2  was two, under which the model explained 99.97% of the variation in the data  was between group E and the ancestor of groups SW and W  analysis on 57 849 SNPs in the 17 populations to identify loci with exceptional introgression in the admixed populations. We found 1941 outliers, including 640 positive outliers . The 640p < 0.05), including 77 biological process, 32 molecular function, and 14 cellular component terms. In the biological process category, we identified, in addition to growth and material transport and metabolism, a number of terms related to high-elevation adaptation. These included terms related to DNA repair , carbohydrate metabolism (GO:0005351 and GO:0005402), and flavonoid biosynthesis (GO:0009962 and GO:0009963); flavonoids act as sunscreens and antioxidants to prevent damage and/or help plants adapt to a wide range of stress conditions in alpine environments  overrepresented pathways, including ribosome biogenesis in eukaryotes (ko03008), Fanconi anemia pathway (ko03460), circadian rhythm-plant (ko04712), longevity regulating pathway (ko04211), FoxO signaling pathway (ko04068), and glycosaminoglycan degradation (ko00531) (Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed six significantly (ko00531) . The GO p = 0.001, adjusted R2 = 26.9%) on allele frequencies among populations  that explained 8.40% and 1.92% of the total variance. The first axis was primarily driven by bio1, whereas the second was associated with bio15 and WET.We performed redundancy analyses (RDAs) as another test for signatures of genetic adaptation. Forward selection retained three environmental variables  and one distance-based Moran’s eigenvector map (dbMEM). Their variance inflation factors (VIFs) were all below 10, suggesting that multicollinearity among these predictors was properly controlled. In the partial RDA that controlled for the spatial component, environment had a large and significant effect (ulations A. When cSPL8) that regulates flowering, growth and development, and drought tolerance (GolS4) whose encoded enzyme synthesizes galactinol and protects plant cells from oxidative damage (LUT1) required for carotenoid epsilon-ring hydroxylation activity  in the nuclear genome is only 3%  the populations in this zone are distributed along an elevation gradient that runs north to south, with a contact zone and admixed populations in the middle; (2) they are distributed along major river valleys that provide corridors for gene flow. Thus, possibilities for gene exchange among populations along valleys are not hindered by major geographical barriers, and an isolation-by-distance model provides a valid neutral expectation.Zones of hybridization and introgression are good settings for understanding genetic differences that are potentially associated with adaptation and reproductive isolation between diverging species . We seleP. densata ancestry and one-third showed excess P. yunnanensis ancestry. This could be the result of either divergent selection operating on different loci within the contact zone or genetic drift. Among the 1941 BGC outlier loci, 14 showed more rapid than expected introgression rates (negative β), suggestive of adaptive introgression, and 64 showed slower than expected introgression (positive β), suggestive of loci associated with reproductive barriers  of the 57 849 loci analyzed in the admixed populations. Approximately two-thirds of these outlier loci showed excess barriers . In the ted taxa . We prev as 0.51 . Thus, d as 0.51 . The potβ outliers, whereas weakly selected loci will be shown as having excess ancestry  exhibit heterogeneous maternal lineages as shown by a mix of mtDNA haplotypes found in the two parent species . We performed probe capture sequencing using the same set of 40 000 exome probes as in UniGenes . The majUniGenes . Libraryhttp://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Adapter sequences and low-quality bases  were removed using Trimmomatic . Reads around indels were locally realigned using RealignerTargetCreator and IndelRealigner in GATK. Variant calling was performed using HaplotypeCaller. GenotypeGVCFs was then used to perform multisample joint aggregation and genotype likelihood correction with the parameter “includeNonVariantSites.”To reduce the computation time of the Genome Analysis Toolkit (GATK) pipeline , we prepP. taeda genome v.1.01) and with mapping quality <40 were removed, genotypes with genotype quality <20 or read depth <3 were masked as missing, and SNPs that met any of the following criteria were removed: missing rate >20%, MAF < 5%, heterozygosity >70%, or allele number >2. The remaining SNPs were used for population genomic analyses, with the exception of site frequency spectrum (SFS) and nucleotide diversity-based analyses, for which no MAF filtering was applied.Several filtering steps were performed to minimize genotyping errors: indels were removed, SNPs located in repetitive regions (with reference to the K) required to explain the structure in the dataset set to 1–16. The best K value that maximized the marginal likelihood of clustering in the data was identified by the script “chooseK.py”. The graphical illustration of ancestry coefficients was plotted using “distruct.py”. Differentiation (FST) between genetic clusters was estimated using the R package HIERFSTAT (π)   and TajiT (π) (D  of each T (π) (D .m) were generated and plotted using the rEEMSplot package (https://github.com/dipetkov/eems).To visualize spatial patterns of gene flow, we used EEMS . EEMS usP. densata (see Results). We inferred the demographic histories of these groups  using the flexible multi-epoch coalescent approach implemented in Stairway Plot 2 (https://github.com/isaacovercast/easySFS). The final folded SFS for P. densata contained 7 544 066 sites. We set the mutation rate to 7 × 10−10 per site per year and the generation time to 50 years (Ne) over time. We also used another coalescent simulation-based method, fastsimcoal2 version 2.7  the levels of gene flow between groups, (2) the mode of population size change after splitting from the ancestral population, and (3) whether and when strong bottlenecks occurred. Detailed descriptions of fastsimcoal model testing are presented in the supplemental information.Population genetic analyses identified four distinct groups in y Plot 2 . We cons50 years . Two hunsion 2.7 , to infer2 threshold of 0.05) along windows of 50 SNPs and a step size of five SNPs using PLINK  statistical tests with Dsuite version 0.4r43 to detect introgression (P. sylvestris) were tested . We selected eight populations from P. densata group C and nine populations of P. yunnanensis that formed a transition from pure P. yunnanensis to admixed populations with P. densata to pure P. densata. Additionally, they also formed a geographical gradient along the major river valleys of the region that run north to south following elevations ; the probability of being inherited from reference population 2 (P2) is therefore 1 − φ. BGC summarizes introgression using a locus-specific α that quantifies the change in ancestry relative to a null expectation based on the genome-wide hybrid index and a β that specifies the slope of the cline  or adaptive introgression (negative β) (ABBA-BABA tests detected introgression between evations B. We inving BGCs . BGC ideing BGCs . The hybhe cline . Loci idozygotes . Negativative β) . For botP. yunnanensis populations (P1), four pure P. densata populations (P2), and six admixed populations (α or β = 0) or their median of posterior distribution surpassed the (1−0.975)/2 and 0.975/2 quantiles of the probability distribution  as the cutoff for outliers. Each SNP was then associated with all environmental predictors using Pearson’s correlation coefficient (r)  , and thoP. taeda v1.01 assembly were determined using eggNOG-mapper v2 (Outlier SNPs were annotated and categorized using SnpEff v4.3t  on the bapper v2  with parapper v2  to reducapper v2 .10.13039/501100004359Swedish Research Council (VR 2017-04686) and T4F program, Sweden.This study was supported by grants from the X.-R.W. and W.Z. designed the study. W.Z. and J.-F.M. conducted field sampling. J.-F.G., W.Z., and B.A. analyzed the data. J.-F.G., W.Z., and X.-R.W. wrote the manuscript draft. All authors contributed to the revision of the manuscript.


Zostera marina L.), the most widely distributed marine flowering plant or seagrass from its origin in the Northwest Pacific, based on nuclear and chloroplast genomes. We identified two divergent Pacific clades with evidence for admixture along the East Pacific coast. Two west-to-east (trans-Pacific) colonization events support the key role of the North Pacific Current. Time-calibrated nuclear and chloroplast phylogenies yielded concordant estimates of the arrival of Z. marina in the Atlantic through the Canadian Arctic, suggesting that eelgrass-based ecosystems, hotspots of biodiversity and carbon sequestration, have only been present there for ~243 ky (thousand years). Mediterranean populations were founded ~44 kya, while extant distributions along western and eastern Atlantic shores were founded at the end of the Last Glacial Maximum (~19 kya), with at least one major refuge being the North Carolina region. The recent colonization and five- to sevenfold lower genomic diversity of the Atlantic compared to the Pacific populations raises concern and opportunity about how Atlantic eelgrass might respond to rapidly warming coastal oceans.Currents are unique drivers of oceanic phylogeography and thus determine the distribution of marine coastal species, along with past glaciations and sea-level changes. Here we reconstruct the worldwide colonization history of eelgrass ( Zostera marina L.) is used to trace its colonization history from its origin in the Northwest Pacific.Ocean currents play a crucial role in the distribution of marine coastal species. Here the nuclear and chloroplast genomes of this eelgrass ( Seagrasses are foundation species of entire ecosystems thriving in all shallow coastal areas of the global ocean except Antarctica2. By far the most geographically widespread species is eelgrass (Zostera marina), occurring in Pacific and Atlantic areas of the Northern Hemisphere from warm temperate to Arctic environments3, spanning 40° of latitude and a range of ~18 °C in average annual temperatures . Three independent lineages descended from freshwater ancestors that lived ~114 mya  as applied at the population level10, making it possible to construct a time-calibrated phylogenetic tree from SNP (single-nucleotide polymorphism) data11. Our data set comprised 190 individuals from 16 worldwide locations that were subjected to comprehensive whole-genome resequencing (nuclear and chloroplast).One major objective of the present study was to provide time estimates of major colonization events. We asked how evolutionary contingency—specifically the timing of large-scale dispersal events—may have affected the timing of arrival of eelgrass on East Pacific and North Atlantic coastlines12. Such local extinctions and subsequent recolonizations from refugial populations are expected to leave their genomic footprint in extant marine populations15 and may restrict their potential to rapidly adapt to current environmental change17. Hence, we were also interested in how glaciations—in particular the Last Glacial Maximum ; ref. 18)—have affected the population-wide genomic diversity of Z. marina and which glacial refugia permitted eelgrass to survive this period.Superimposed on the general eastward colonization are Pleistocene cycles of glacial and interglacial periods that resulted in frequent latitudinal expansions and contractions of available habitat for both terrestrial and marine biotaZ. marina specimens collected from 16 geographic locations . To avoid reference-related bias, owing to the large Pacific–Atlantic genomic divergence, and to facilitate phylogenetic reconstruction within a conserved set of genes20, we focused on core genes—the set of genes shared by most individuals. From a total of 21,483 genes, we identified 18,717 core genes that were on average observed in 97% of the samples, containing 763,580 SNPs , comprising 54 haplotypes.A complete chloroplast genome of 143,968 bp was reconstructed from the reference sampleπ) and genome-wide heterozygosity (Hobs) - to 6.6 (Hobs)-fold higher genetic diversity compared to the Atlantic ones  followed by Japan-North (JN). Alaska-Izembek (ALI) and Alaska-Safety Lagoon (ASL) showed approximately a third  of the diversity in the more southern Pacific sites (average of San Diego (SD), Bodega Bay, California (BB) and Washington State (WAS)). In the Atlantic, a comparable loss of diversity along a south–north gradient was observed. Quebec (QU) showed 42% (π) and 47% (Hobs) of the diversity of North Carolina (NC) and Massachusetts (MA), while the diversity values in Northern Norway (NN) was 31% and 43% of averaged values of Sweden (SW) and Wales, respectively.As measures of genetic diversity, we assessed nucleotide diversity (bs) Fig. . Consistbs) Fig. , PacificTo reveal the large-scale population genetic structure, we performed a principal component analysis (PCA) based on the most comprehensive SNP selection  randomly selected from the ZM_Core_SNPs. The most likely number of genetic clusters was determined using a combination of the Delta-K method23 and other metrics introduced by ref. 24  haplotype is shared by both MA and NC, with nine private NC haplotypes. A single mutation separates both MA and QU, as well as MA and Wales-North. Also extending from the central haplotype were SW and NN Fig. . Togethe26 using all ZM_Core_SNPs. Pacific populations were connected in a web-like fashion , suggesting substantial admixture. For the Atlantic side, D values indicated recent or ongoing connection between the Atlantic and Mediterranean Sea, consistent with the admixture signal detected by STRUCTURE . The resulting clock rate for fourfold degenerative transversions of paralogous gene sequences yielded a divergence time estimate of 9.86–12.67 mya between Z. marina and Z. japonica . Further support for JN being a dispersal hub is its smallest pairwise FST with all Atlantic populations . The Northwest and Northeast Atlantic populations also diverged from each other very recently at ~18.8 kya  and shared a common ancestor during the LGM, indicating that they were partially derived from the same glacial refugium in the Northwest Atlantic (likely at or near NC). Some admixture found in the SW population stemming from the Mediterranean gene pool Fig.  likely e10, we used alignments of 617 core genes across all samples . This approach resulted in a topology consistent with the one depicted in Fig. In a second coalescent approach−9 per site per year, this genetic distance corresponds to a divergence time of 392 kya  haplotypes as an alternative dating method. SD and BB along the Pacific East coast showed very different haplotypes, separated by about 30 mutations from the other Pacific and the Atlantic clades. Assuming a synonymous cpDNA mutation rate of 2 × 1033 to infer past effective population size Ne  varied. Given a range of plausible generation times under a mix of clonal and sexual reproduction, it is likely that an Ne minimum shown by several locations coincides with the LGM, which in turn can be used to estimate the long-term generation time. For example, a local minimal Ne at 5,000 generations ago, at locations JS, WAS, BB, SD and MA would translate to 3 year × generation−1 × 5,000 generations = 15 kya, just after the LGM. In general, lower Ne values were related to lower clonal diversity at sites in northern (NN) and southern Europe . As for the Atlantic side, the Northwest Atlantic populations NC and MA and the southern European populations PO and CZ (and to a lesser extent Mediterranean FR) showed little evidence for bottlenecks , suggesting that these localities were refugia during the LGM  Ne Fig. . We hereFor the Atlantic, we determined the most likely post-LGM recolonization through approximate Bayesian computations , as the major dispersal gateway. It bifurcates north into the Alaska Current and south into the California Current . Subsequent trans-Pacific events that headed south at the gateway eventually resulted in an admixture zone involving WAS and BB.We identified the North Pacific Current, which began to intensify ∼1 mya , our analyses only support a single colonization event into the Atlantic, in contrast to findings for other amphi-Arctic and boreal marine invertebrates41 and seaweeds42. Genomic variation characteristic of extant Alaskan populations was not detected in any North Atlantic populations, in line with earlier microsatellite data40, corroborating that the Atlantic was only colonized once. While we cannot rule out an earlier colonization, this would require that Z. marina became extinct without leaving any trace in nuclear genomes or cpDNA haplotypes, which we consider unlikely.Although the Bering Strait may have opened as early as 5.5–4.8 mya , including the species Z. marina some 5–1.62 mya (ref. 5) in the Northwest Pacific . These are common patterns of many terrestrialfic Fig.  and 6b, 47) has been attributed to frequent intermediate extinctions6. Our data suggest a second plausible process, namely multiple long-distance genetic exchanges within and among ocean basins that may have impeded allopatric speciation . Our range-wide sampling has allowed an overview of evolutionary history in this lineage of seagrass and opens the door for exploration of functional studies across ocean basins and coasts. Future work will explore the pan-genome of Z. marina with the consideration of how the high diversity and robustness of Pacific populations may be able to contribute to management and rescue of populations along rapidly warming Atlantic coastlines.In conclusion, the relatively low number of extant seagrass species (~65 species in six familiesZ. marina L.) is the most widespread seagrass species of the temperate to Arctic Northern Hemisphere3. It is being developed as a model for studying seagrass evolution and genomics49. Z. marina is a foundation species of shallow water ecosystems17 with a number of critical ecological functions including enhancement of fish and crustacean recruitment50, improvement of water quality51 and the sequestration of ‘blue carbon’53.Eelgrass  stemming from a sexually produced individual (=genet or clone) are intermingled to form the seagrass meadow. This also implies that generation times are difficult to estimate or average across populations. Nevertheless, we assumed here based on personal observations that in perennial eelgrass populations, individuals become reproductive in year 2 after germination, while attaining their maximal reproductive output in year 3. Extended clone longevity results in overlapping generations, but not in longer generation times. Additional evidence for an average generation time of 3 years used here for later modelling comes from the historical demographic analysis  and sexual reproduction via seeds, with varying proportions across locationsZ. marina specimens from 16 geographic locations , frozen in liquid nitrogen and stored at −80 °C until DNA extraction.2. Genomic DNA was extracted using the Macherey-Nagel NucleoSpin plant II kit following the manufacturer’s instructions. DNA concentrations were in the range of 50–200 ng µl−1. Quality control was performed following Joint Genome Institute guidelines (https://jgi.doe.gov/wp-content/uploads/2013/11/Genomic-DNA-Sample-QC.pdf). Plate-based DNA library preparation for Illumina sequencing was performed on the PerkinElmer Sciclone NGS robotic liquid handling system using Kapa Biosystems library preparation kit. About 200 ng of sample DNA was sheared to a length of around 600 bp using a Covaris LE220 focused ultrasonicator. Selected fragments were end-repaired, A-tailed and ligated with sequencing adaptors containing a unique molecular index barcode. Libraries were quantified using KAPA Biosystems’ next-generation sequencing library qPCR-kit on a Roche LightCycler 480 real-time PCR instrument. Quantified libraries were then pooled together and prepared for sequencing on the Illumina HiSeq2500 sequencer using TruSeq SBS sequencing kits (v4) following a 2 × 150 bp indexed run recipe to a targeted depth of approximately 40x coverage. The quality of the raw reads was assessed by FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and visualized by MultiQC58. BBDuk (https://jgi.doe.gov/data-and-tools/bbtools/bb-tools-user-guide/bbduk-guide/) was used to remove adapters and for quality filtering, discarding sequence reads (1) with more than one ‘N’ (maxns = 1), (2) shorter than 50 bp after trimming (minlength = 50) and (3) with average quality <10 after trimming (maq = 10). FastQC and MultiQC were used for second round of quality check for the clean reads. Sequencing coverage and mapping rate was calculated for each sample  (ref. 59). To categorize, extract and compare core and variable (shell and cloud) genes, primary transcript sequences  from the Z. marina reference  were aligned using BLAT using default parameters60 to each de novo assembly. Genes were considered present if the transcript aligned with either (1) >60% identity and >60% coverage from a single alignment or (2) >85% identity and >85% coverage split across three or fewer scaffolds. Individual presence–absence-variation calls were combined into a matrix to classify genes into core, cloud and shell categories based on their observation across the population. The total number of genes considered was 20,100. Because identical genotypes and fragmented, low-quality assemblies can bias and skew presence–absence-variation analyses, only 141 single representatives of clones and ramets with greater than 17,500 genes were kept to ensure that only unique, high-quality assemblies were retained. Genes were classified using discriminant analysis of principal components61 into cloud, shell and core gene clusters based on their frequency. Core genes were the largest category, with 18,717 genes that were on average observed in 97% of ramets.To analyse genetic loci present throughout the global distribution range of eelgrass, we focused on identifying core genes that are present in genomes of all individuals. To do so, each of the 190 ramets were de novo assembled using HipMer  was used to identify and tag duplicate reads in the BAM files. The mapping rate for each genotype was calculated using Samtools . To avoid the reference-genome-related biases, due to the large Pacific–Atlantic genomic divergence, we focused on the 18,717 core genes that were on average observed in 97% of ramets. Bedtools66 was used to find overlap between the SNPs and the core genes, and only those SNPs were kept . Genotypes that were outside our custom quality criteria were represented as missing data.BCFtools67). To ensure that all genotypes assessed originated by random mating, ten ramets showing evidence for selfing were excluded. Seventeen multiple sampled clonemates were also excluded  of 24,127 bp each, a large single-copy region of 83,312 bp, and a small single-copy region of 12,402 bp. All regions were equally taken into SNP calling analysis except for 9,818 bp encoding 23S and 16S ribosomal RNAs due to bacterial contamination in some samples. The raw Illumina reads of each individual were aligned by BWA MEM to the assembled chloroplast genome. The alignments were converted to BAM format and then sorted using Samtools62. Genomic sites were called as variable positions when the frequency of variant reads was >50% . For the phylogenetic tree reconstruction, we further selected 108 SNPs that represent parsimony-informative sites .The chloroplast genome was de novo assembled by NOVOPlastyhttp://pcingola.github.io/SnpEff/) was used to annotate each SNP as genic or non-genic, and within the former category as synonymous or non-synonymous. To obtain putatively neutral SNPs, we kept only SNPs annotated as ‘synonymous_variant’ . For the SNPs in ZM_Neutral_SNPs , only SNPs without any missing data were kept, which resulted in 44,865 SNPs, the data set used for calculating π . The package vcfR22. To reduce the run time, we randomly selected 2,353 SNPs from ZM_Core_SNPs (20%) to run STRUCTURE . Ten runs were performed for K values 1–10. StructureSelector25 was used to help determine the optimal number of clusters (K) based on the original Delta-K method23 in conjunction with additional metrics proposed by ref. 24 that give an upper limit to the number of clusters. We considered the hierarchical structure of our data set owing to the marked Pacific–Atlantic divide and always performed a qualitative inspection of alternative major and minor K modes.We used Bayesian clustering implemented in STRUCTURE to study population structure and potential admixture5; number of Markov chain Monte Carlo runs, 2 × 106) as described above, with possible K values 1–7.To detect hidden hierarchical population structure, we further analysed populations from the Atlantic and Pacific alone. Pacific data were extracted from ZM_Neutral_SNPs , excluding monomorphic sites and those with missing data. To obtain putatively independent SNPs, we thinned sites using VCFtools, so that no two sites were within 3,000 bp distance  from one another . Those 12,514 SNPs were subjected to PCA, while a set of randomly selected 6,168 SNPs was used in STRUCTURE to reduce run times . To obtain putatively independent SNPs, we thinned sites using VCFtools according to the above criteria. The resulting 8,552 SNPs were then used to run another separate PCA and STRUCTURE using the parameters above. For STRUCTURE analysis, 73 SNPs. 71 with epsilon 0 and 1 implemented by PopART72, based on 151 polymorphic sites. The topology was additionally confirmed using a maximum-likelihood phylogenetic tree, reconstructed by IQ-TREE v1.5.5 with 1,000 bootstrap replicates32 based on 108 parsimony-informative polymorphic sites  for each population at all synonymous sites using each of the six chromosomes as replicates for 44,685 SNPs without any missing data /) was calculated using a custom Python3 script based on all synonymous SNPs .VCFtools was used to calculate nucleotide diversity   was used to plot a heat map that jointly visualizes both the D value and the associated P value for each comparison of P2 and P3. The colour of the corresponding heat map cell indicates the most significant D value across all possible populations in position P1. Red colours indicate higher D values, and more saturated colours indicate greater significance.Patterson’s 11. We used the software SNAPP9 with an input file prepared by script ‘snapp_prep.rb’ (github.com/mmatschiner/snapp_prep). Two specimens were randomly selected from each of the included populations, and genotype information was extracted from ZMZJ_Neutral_SNPs  data set, excluding monomorphic sites and missing data. We thinned sites again using VCFtools, so that all sites were ≥3,000 bp distance from one another . The crown divergence for all Z. marina populations, estimated in the first SNAPP analysis, was used as calibration point, assuming a lognormal prior distribution .Most of the 6,169 SNPs above represented the genetic differences between D-statistics . Only chromosomal regions that permitted unique mapping of sequencing reads were considered. We generated one mask file for all core genes along each of the six main chromosomes. We generated one ramet-specific mask file based on the BAM format file using bamCaller.py (https://github.com/stschiff/msmc-tools), containing the chromosomal regions with sufficient coverage of any genoytpe, with minDepth = 10. We also generated a ramet-specific VCF file for each of the six main chromosomes based on ZM_HQ_SNPs using a custom Python3 script.The MSMC75 were run to distinguish between alternative models of the recolonization history of Z. marina after the LGM. Considering that the Mediterranean Sea had its own glacial refugium, the ABC modelling was conducted for the Atlantic only. We constructed three recolonization scenarios (Supplementary Fig. Simulations using DIYABC-RFFurther information on research design is available in the Supplementary InformationSupplementary Notes 1–8, Tables 1–6 and Figs. 1–12.Reporting SummarySupplementary DataSupplementary Data Table 1: Sequence coverage. Supplementary Data Table 2: Mapping rate. Supplementary Data Table 3: Accession number of each library.


Acridotheres tristis) is one of the most invasive bird species in the world, yet its colonisation history is only partly understood. We identified the introduction history and population structure, and quantified the genetic diversity of myna populations from the native range in India and introduced populations in New Zealand, Australia, Fiji, Hawaii, and South Africa, based on thousands of single nucleotide polymorphism markers in 814 individuals. We were able to identify the source population of mynas in several invasive locations: mynas from Fiji and Melbourne, Australia, were likely founded by individuals from a subpopulation in Maharashtra, India, while mynas in Hawaii and South Africa were likely independently founded by individuals from other localities in India. Our findings suggest that New Zealand mynas were founded by individuals from Melbourne, which, in turn, were founded by individuals from Maharashtra. We identified two genetic clusters among New Zealand mynas, divided by New Zealand’s North Island’s axial mountain ranges, confirming previous observations that mountains and thick forests may form barriers to myna dispersal. Our study provides a foundation for other population and invasion genomic studies and provides useful information for the management of this invasive species.The common myna ( Invasive species are organisms which are not native to a particular area that may reproduce and expand demographically and spatially  have established from their native range in Central to Southeast Asia to become a globally invasive species and are one of only three bird species on the IUCN ‘100 of the World’s Worst Invasive Alien Species’ list  and similar genotyping by sequencing (GBS) methods subsample the genome by using restriction enzymes to fragment genomic DNA and then sequence adjacent to the cut sites with next-generation sequencing  markers – identified from RADseq reads mapped against a draft myna genome assembly - from 814 myna individuals from six countries to 1) identify the likely sources of invasive population(s), and more specifically of the New Zealand myna population, 2) clarify our current understanding of the myna’s colonisation history in New Zealand, and 3) identify population structure and genetic diversity in New Zealand mynas. This study provides a foundation for further invasion genomic studies and useful insights for species management.Data filtering and analyses performed in R were conducted in R version 4.1.2 , sample data missingness, locus data missingness, and linkage disequilibrium, and finally filtered to only retain SNPs that are under Hardy-Weinberg equilibrium (HWE). Depending on the analyses performed, the datasets were filtered using different stringencies   was retained at random prior to further SNP filtering. A further three samples  were removed from the ALL and IND dataset due to the samples exhibiting heterozygote excess  were removed from the dataset using VCFtools to remove potential artefactual alleles  and plotted this against pairwise distance using PopLDdecay , require prior knowledge of the population structure. However, incorrect delineation of populations may result in inaccurately clumping genetically distinct populations, or separating genetically homogenous populations.Further downstream analyses, such as population pairwise-Consequently, initial principal component analysis (PCA) and sparse non-negative matrix factorisation (sNMF) population genetic structure analysis were performed on the NZ, IND, and ALL dataset prior to HWE filtering to avoid the inadvertent introduction of a Wahlund effect  as a separate population  from other Maharashtra individuals due to separate clustering. Other populations were also separated based on the location and timing of the sample collection . This delineation of populations will be referred to as ‘popdef1’ hereafter , loci were filtered to remove non-neutral loci by testing for HWE using the Uneven sample sizes across genetic populations have been shown to potentially affect analyses such as principal component analysis (PCA)  were grouped together and populations were delineated as follows for subsampling: IND: Maharashtra subpopulation A, IND: Other, AUS: Melbourne, AUS: Sydney, AUS: Gold Coast, NZ: Napier, NZ: Other, South Africa, Fiji and Hawaii. This delineation of populations will be referred to as ‘popdef2’ hereafter  to ensure consistency of results Appendix  and S8.7FST values calculated using the gl.fst.pop function in the ‘dartR’ R package (100 bootstraps performed) for all population combinations.Inter-population genetic differences with the HWE-filtered dataset were examined across the dataset using pairwise-gl.ibd function in the ‘dartR’ R package to determine whether genetic differentiation between populations within India and within New Zealand can be explained by geographical distance. To do so, we correlated the transformed population pairwise-FST  was performed using the  Rousset . Mantel gl.report.heterozygosity function in the ‘dartR’ R package. The proportion of polymorphic loci was calculated after randomly subsampling the dataset to n = 6 per population, 100 times. This subsampling accounts for the uneven sampling per population, as populations with more samples will likely have more polymorphic loci. Mean allelic richness was calculated using a rarefaction method to account for uneven sampling using the allel.rich function in the ‘PopGenReport’ R package version 3.0.4 , with low but significant differentiation between the ROM (collected in 1984) and modern (collected between 2017–2020) samples  from the other New Zealand populations. Pairwise-FST values among the other New Zealand population are generally very low (pairwise-FST < 0.005) and/or insignificant .The PCA of the New Zealand-only (NZ) dataset shows two clusters on PC1 Fig. : individn ≤ 5) found no significant isolation by distance . The cluster of points with The Mantel test on the populations within NZ: Other , and Maharashtra subpopulation A, a Mantel test on the populations within India found significant isolation by distance .PC1 vs PC2 from the native-range India-only (IND) dataset shows two to four clusters Fig. . The mosFST values suggest low (FST = 0.001–0.03) but significant differentiation between all Indian populations except for Maharashtra subpopulation A which showed moderate levels of genetic differentiation from other Indian populations (FST = 0.042–0.073)  Fig. .The global (ALL) dataset was subsampled due to the large variation in sample sizes from different countries. Across 10 replicates, PCA on this subsampled data showed consistent clustering of the populations (results not shown), with a representative run presented in Fig. The sNMF population structure analysis of the ALL dataset was best explained with K = 8, and supports seven genetic clusters Fig. . The sevFST between all populations showed high genetic differentiation (pairwise-FST > 0.247) among distant bottlenecked populations  and low genetic differentiation (pairwise-FST = 0.005–0.037) between Maharashtra subpopulation A and Melbourne, Napier, and Fiji .Population pairwise-FST < 0.05), comparable to populations from IND: Other , and NZ: Other  . However, both populations showed lower population pairwise-FST with IND: Other (pairwise-FST = 0.105–0.131) than with other populations in this study.Population pairwise-o), mean expected heterozygosity (He), mean allelic richness (AR), proportion of polymorphic loci and mean private allelic richness (PAR)) inferred that myna populations in India were more diverse than the introduced populations  and population structure can be identified and compared to historical records and current observations. The differences in genetic diversity, differentiation, and composition between populations can elucidate the connectivity between populations and population introduction history. Using common myna samples from the native and the invasive range, we were able to utilise genomic analyses to identify the population structure, and evaluate and infer the myna introduction history from India to Australia and then to New Zealand.Our extensive sampling from the native and invasive range has allowed, for the first time, inference of the origin of mynas introduced to New Zealand. Historical records suggests that mynas in New Zealand were founded by individuals from an established population in Australia which in turn was founded by individuals from the native range in India Thomson . The cluTwo genetic clusters in New Zealand were identified that appear to be consistent with the historical record of the species introduction and the distribution in New Zealand . Mynas were only first recorded on GBI in 1960  shows low but significant levels of genetic differentiation from other New Zealand populations . This clustering, and given that Napier has higher genetic diversity than NZ: Other, raises the hypothesis that Napier and NZ: Other were separate introductions from Melbourne, versus NZ: Other being established from Napier birds. Attempts were made to resolve the population history with phylogenetic trees and coalescent simulations using fastsimcoal2 version 2.7.09 , it is most genetically similar to sNMF cluster 2C  and is most genetically similar to Melbourne,  is more diverse and has a smaller distribution. Further analyses can be built upon this study to provide more information for management of the species in New Zealand. Our gene flow analysis was unsuccessful, but further analyses on higher-density sequencing data  may help resolve the very recent establishment of the New Zealand populations. Additional samples from more locations will also help elucidate finer scale population structure, and reduce the chance that key populations are not sampled. For example, samples from the highways crossing the North Island axial mountain range can potentially help deduce if there is admixture between the NZ: Other and NZ: Napier populations, and if this requires any management interventions. Similarly, tracking individuals in key populations may provide an alternative source of information on the species’ dispersal ability and population connectivity, which may further help inform myna management programmes on the feasibility of myna eradication in some locations (e.g. island populations).The identification of Maharashtra subpopulation A as the potential source of the mynas in Australia and Fiji also raised other questions regarding this potential source population. Maharashtra subpopulation A is genetically less diverse than the other Indian populations and has potentially experienced a population bottleneck. These samples were collected in 1975 from Maharashtra, although the exact location is unknown. Other samples from Maharashtra, and the rest of India were also collected from a similar time period  and the modern samples collected in 2014–2020, suggesting that the genetic composition of mynas in these populations may have changed since the collection of the ROM samples. Although there may be some bias from sample size differences, in addition to variability introduced from stochastic sampling effects and differences between sampling locations, it is likely that much of this signal is due to drift and population size changes. Interestingly, unlike Melbourne and Napier where samples from different time periods show similar levels of genetic diversity, modern Sydney samples show higher genetic diversity compared to Sydney ROM samples, and modern Sydney samples are more genetically similar to modern Melbourne (and Melbourne ROM) than Sydney ROM . In all cases, we found low but significant pairwise-ROM Fig. . These rROM Fig. ). AnalysThis study presents the most extensive population genomic studies of the common myna to date, which utilises samples from multiple locations across the world. We were able to identify the source population of mynas in a few invasive locations: populations in Melbourne and Fiji were likely founded by individuals from Maharashtra, and populations in New Zealand were likely founded by individuals from Melbourne. We were able to identify two distinct populations in New Zealand, east and west of North Island’s axial mountain ranges. This observation confirms previous observations that mountain ranges and thick forests may form barriers for myna dispersal and provide useful information for the species management. These findings support the use of thick forests and mountains as barriers of management units and a more localised management strategy of mynas in New Zealand . This also highlights the potential additional benefits of reforestation programmes in New Zealand (e.g. One Billion Trees Programme (Te Uru Rākau Supplementary materialTable S1.2