Elymus breviaristatus, an ecologically important rare grass species with strong resistance, is restricted to a limited area of the QTP. In this study, we investigated the phylogeography of E. breviaristatus using five chloroplast genes and spacer regions in natural populations distributed along the eastern QTP. We identified a total of 25 haplotypes among 216 individuals from 18 E. breviaristatus populations, which were further classified into four haplogroups based on geographical distribution and haplotype network analysis. Notably, we did not observe any signs of population expansion. High genetic diversity was exhibited at both species and population levels, with precipitation being the main limiting factor for population genetic diversity levels. Higher genetic diversity was exhibited by populations located near the Mekong–Salween Divide genetic barrier, suggesting that they may have served as a glacial refuge. The significant pattern of genetic differentiation by environmental isolation highlights the influence of heterogeneous environments on the genetic structure of E. breviaristatus populations. Additionally, the results of ecological niche models indicated that the geographic distribution of E. breviaristatus populations has decreased rapidly since the Last Glacial Maximum but is not threatened by future global warming.Past climatic and topographic variations have created strong biogeographic barriers for alpine species and are key drivers of the distribution of genetic variation and population dynamics of species on the Qinghai-Tibet Plateau (QTP). Therefore, to better conserve and use germplasm resources, it is crucial to understand the distribution and differentiation of genetic variation within species.  The spatial distribution of genetic variations in populations has been shaped by population expansion and contraction triggered by geological events or climatic fluctuation ,2. SpeciThe Qinghai-Tibet Plateau (QTP) is located in the southwestern region of China and is a geographically remarkable area that is the highest and largest plateau on the planet, with an average elevation of over 4000 m . During Chloroplast DNA (cpDNA) is a valuable tool for investigating phylogeography, primarily due to its maternal inheritance in most angiosperms and its limited genetic recombination . These cElymus breviaristatus (Keng) Keng f. (Poaceae: Triticeae), a self-pollinating grass species endemic to the QTP region that is at risk of extinction due to habitat fragmentation [Elymus, characterized by a complex taxonomic history shaped primarily by hybridization processes [E. breviaristatus, the focus of our study, is a hexaploid species with a genome comprising St, H, and Y genomes, exhibiting rich genetic diversity and a high frequency of gene exchange. Given its rarity, short life cycle, and sensitivity to environmental changes, E. breviaristatus represents an excellent model for studying the evolution and genetic variation in alpine plants [E. breviaristatus, we employed a combination of cpDNA analysis and ENM, as well as the geological and climatic changes that may have influenced its distribution shift and genetic diversity. The findings of this study can contribute to the identification of factors contributing to species’ endangerment, germplasm resource assessment, and the development of effective conservation strategies.In this study, we used both environmental data and phylogeography to investigate the factors affecting genetic diversity in entation . This sprocesses . Specifie plants . Furthere plants , emphasie plants . To expltrnH-psbA, trnL-trnF, matK, rbcL, and trnY_GUA-trnD_GUC regions were 667, 1052, 1538, 1432, and 384 bp, respectively. A total of 36 variable sites were detected, comprising one singleton variable site and 35 parsimony informative sites. As cpDNA regions are uniparentally inherited markers, we used these five chloroplast fragments in our subsequent population genetics analysis. A total of 25 haplotypes (H1–H25) were identified among 216 individuals of E. breviaristatus sampled from 18 populations  of 0.825 and a nucleotide diversity (π × 10−3) of 1.14. At the population level, Hd ranged from 0 to 0.818 and π × 10−3 varied from 0 to 2.3. Population QD1 had the highest haplotype and nucleotide diversity, with six haplotypes, while population NC1 had only one haplotype and no nucleotide diversity. The effects of geographical and climatic conditions on haplotype and nucleotide diversity were assessed using Pearson correlation  but decreased with Bio15 . Haplotype diversity was significantly affected by Bio17  and Bio19 . Additionally, the AMOVA indicated considerable genetic variations among and within populations  indicated that the MaxEnt model had a good predictive capacity. Through an analysis of the contribution rates of seven environmental variables to the distribution of Projecting the model into geographical space predicted the potential distribution of the species from the LGM to the present. The species’ potentially suitable habitats have undergone a rapid contraction since the LGM. The current potential distribution areas included the known distribution of species in the southeast of the QTP, involving some adjacent areas of Tibet, Qinghai, Gansu, Sichuan, and Yunnan. In addition, the predicted future distribution range of the species showed different ranges of expansion under different scenario models. Of note, the distribution area will migrate to the northwest in the future .E. breviaristatus is endemic to the QTP and has a narrow distribution, it was initially expected to have lower genetic diversity compared with other Elymus species. However, the present study revealed a high Hd (0.825) and π (0.00114), which was comparable to the genetic diversity of the widespread species of Elymus sibiricus in the QTP region detected based on cpDNA  and even higher than the genetic diversity of E. sibiricus in Xinjiang and northern China  [E. breviaristatus could be due in part to the large number of samples in the study. However, this is also consistent with previous studies that have found high levels of genetic diversity in plant species on the QTP [E. breviaristatus may have accumulated a higher genetic variation in its chloroplast genome during its lengthy evolution and polyploidization history than the tetraploid E. sibiricus.The limited distribution of a species can lead to reduced gene flow and increased genetic drift, which can result in lower genetic diversity. Since ctively) . The hig the QTP ,34. The  the QTP . The hexE. breviaristatus may decrease in the future due to increased precipitation instability.Considerable variation in genetic diversity was observed among the populations studied, with the population from the eastern part of the study area QD1 exhibiting high levels of haplotype and nucleotide diversity, while NC1 from the western part showed no variation. Although the correlation between genetic variation and geographical features, such as altitude and latitude, has been highlighted in previous studies ,37, our Koenigia forrestii and Roscoea spp. [Marmoritis complanatum and Sinopodophyllum hexandrum [E. breviaristatus in the QTP region and shed light on why the eastern populations exhibit a higher genetic diversity. This could be attributed to their proximity to the MSD, which may serve as a hybridization zone for different species or populations and represent a transition zone between distinct environmental conditions [Gene exchange plays a critical role in plant evolution and population structure . Our AMOoea spp. ,24. Anotexandrum ,42. Our nditions .E. nutans. Nonetheless, IBE patterns were detected in all populations. By combining the autocorrelation between environmental distance and geographic distance, we infer that the genetic differentiation pattern of E. breviaristatus is dominated by IBE, which is consistent with our previous microsatellite-based findings [E. breviaristatus populations to heterogeneous environments are a more plausible explanation for the observed differential selection than the neutral process of IBD.The presence of geographic barriers can limit gene flow between populations, leading to the rapid accumulation of genetic differentiation, while also constraining the effective distance of gene transmission, ultimately affecting the differentiation of IBD patterns . Howeverfindings . The patfindings . In concD and Fu’s Fs values were higher than 0 for most populations in the neutrality tests. This suggests that some degree of population contraction may have occurred [E. breviaristatus has experienced a significant decline since the LGM, with a contraction to higher elevations in the mountains. This decline in distribution range may have been caused by past selection pressure or the neutral drift experienced by these populations [Although neutrality tests and mismatch analyses did not indicate a significant influence of selective events on all of our populations and haplogroups, it is important to note that Tajima’s occurred ,48, albeulations .E. breviaristatus will increase and shift to higher elevations in the central QTP, and this trend will be even more pronounced with increasing warming. Previous research by Elsen and Tingley [E. breviaristatus is not at a high risk of range contraction or extinction due to climate warming in the future.In contrast to previous studies that suggested that montane species face range contraction or extinction as they shift upward in response to global warming , our stu Tingley  predicte Tingley . Our finE. breviaristatus is not currently affected by climate change, habitat fragmentation resulting from anthropogenic disturbances remains a major concern for endangered species [E. breviaristatus [The highly endemic montane plants are known to be particularly vulnerable , and des species . The lon species . Additioristatus .E. breviaristatus in the QTP, including E. nutans and E. sibiricus, we recommend incorporating hybridization between different populations or species in the conservation process. This promotes the extensive exchange of genetic information and further enhances the adaptive capacity of the species [Therefore, our recommendation is to prioritize the conservation of natural populations that are distributed near the MSD due to their high haplotype diversity and potential as glacial refuges. These populations possess abundant genetic diversity, which provides them with increased adaptability and resistance to selection pressures . Additio species .E. breviaristatus individuals across the QTP. The seeds of these specimens were planted at the experimental field of the Sichuan Academy of Grassland Science, Hongyuan, China. These comprised 10 from Qamdo (QD1–QD10), 4 from Nagqu (NQ1–NQ4), 2 from Nyingchi (NC1 and NC2), and 2 from Lhasa (LS1 and LS2). Within each population, 12 individuals were sampled at least 5 m apart. A total of 216 silica-dried leaf samples were collected from 18 populations of E. breviaristatus using 10 cpDNA regions identified 5 markers , haplotype diversity (Hd), nucleotide diversity (π), and population genetic distance based on concatenated sequences. A haplotype network was inferred using the median-joining method in PopART version 1.7 [Elymus nutans and Elymus repens as outgroups. DnaSP version 5.0 was used to calculate Tajima’s D [Fs [DnaSP version 5.0  was usedsion 1.7 . To conssion 1.7  was usedjima’s D  and Fu’s’s D [Fs  to detec’s D [Fs  was used’s D [Fs .https://www.worldclim.org/) were used to compute environmental Euclidean distances. We evaluated IBD and IBE by regressing genetic distance against geographical distance and environmental Euclidean distance, respectively.Genetic drift typically results in greater differentiation among populations at larger geographical distances, assuming no dispersal or selection. This phenomenon is known as isolation by distance  (IBD). HE. breviaristatus in the QTP, we used a maximum entropy model (MaxEnt) [To predict the ecological niche distribution of (MaxEnt) . In addiE. breviaristatus, revealing that the species has not undergone significant population expansion. Our results indicate that precipitation under extreme climatic conditions is the primary limiting factor for genetic diversity. We observed that populations located near the MSD exhibited higher genetic diversity, suggesting that these regions may have served as refugia. Population differentiation was predominantly determined using the IBE model, with some evidence supporting the IBD model on a smaller scale due to geographical barriers. While our ecological niche analysis provides a more optimistic outlook for the future distribution of this species, we recommend that appropriate conservation measures be implemented to safeguard the population from potential anthropogenic threats.In this study, we present the first phylogeographic analysis of


Halodule wrightii, which is unique in spanning the entire tropical Atlantic. We tested the hypothesis that genetic differentiation estimated across its large-scale biogeographic range can be predicted by simulated oceanographic transport. The alternative hypothesis posits that dispersal is independent of ocean currents, such as transport by grazers. We compared empirical genetic estimates and modelled predictions of dispersal along the distribution of H. wrightii. We genotyped eight microsatellite loci on 19 populations distributed across Atlantic Africa, Gulf of Mexico, Caribbean, Brazil and developed a biophysical model with high-resolution ocean currents. Genetic data revealed low gene flow and highest differentiation between (1) the Gulf of Mexico and two other regions: (2) Caribbean-Brazil and (3) Atlantic Africa. These two were more genetically similar despite separation by an ocean. The biophysical model indicated low or no probability of passive dispersal among populations and did not match the empirical genetic data. The results support the alternative hypothesis of a role for active dispersal vectors like grazers.The transport of passively dispersed organisms across tropical margins remains poorly understood. Hypotheses of oceanographic transportation potential lack testing with large scale empirical data. To address this gap, we used the seagrass species,  Challenges related with empirically tracking the movement and outcome of passive dispersal units have narrowed our knowledge on population connectivity across most marine biodiversity3. Indeed, most marine species, particularly habitat-forming species , migrate via passively-dispersed propagules5. Thus, these species, are expected to form metapopulations that are passively connected by the transportation of such dispersal stages6, but the processes mediating such transportation are poorly understood. Many such marine species, including seagrasses, have wide geographical distribution ranges, which contrasts with their predicted dispersal capacity or ability to maintain regular long-distance dispersal throughout their range8. Biological features of the dispersal phase, its interaction with a range of abiotic, historical, and biotic factors, determine species range limits and gene flow among populations9, which significantly impact a species’ distribution and persistence10.Long-range dispersal of marine organisms with passively transported propagules is often hypothesized to be mediated by ocean currents, a hypothesis that lacks much empirical testing despite its major ecological and evolutionary implications13 but none have addressed the long distance cross-ocean dispersal in the tropical Atlantic region14. Along this wide geographical region, seagrass populations might be isolated by biogeographic barriers such as vast oceanic distances , lack of suitable habitat , or main oceanographic currents . Leading hypotheses predict that oceanographic currents determine most connectivity of passively dispersed stages, functioning as both barriers and promoters of gene flow. The tropical Atlantic region, rich in seagrass diversity14, has only local-scale assessments focused on seagrass genetic diversity, dispersal and connectivity18. Four seagrass genera are known to dominate the tropical Atlantic—Thalassia, Syringodium, Halophila, and Halodule—which either occur as single species or intermixed19. However, only Halodule wrightii is distributed on both east and west tropical Atlantic coastal shores, serving as an optimal model for range-wide connectivity studies across pan-Atlantic spatial scales.Large-scale studies focused on seagrass population connectivity have been developed21. Seagrasses play a strong role in structuring communities and have received much attention due to their large ecological and social significance22. However, they are under increasing pressure from anthropogenic activities and climate change24. As a result, seagrass habitats are declining globally23, becoming lost or fragmented, and further extinctions are forecasted under future climate change scenarios25.Seagrasses form one of the most productive and important ecological groups along with mangroves and corals. They are flowering marine angiosperms that can form dense meadows in shallow coastal waters around the world26 and each reproductive mode produces different types of propagules with diverse dispersal abilities9. There are several circumstances that can drive seagrass propagule dispersal routes over time. Abiotic factors such as wind, waves, tides or currents can lead seagrass-detached fragments to be easily transported away from the source location27. In contrast, seabirds, fish, sea turtles, and dugongs are examples of biotic vectors that may mediate seagrass migration and gene flow by transporting clonal propagules or dispersing seeds after passing through their digestive system29, increasing the chances, or even boosting, their germination success30. Moreover, propagule features such as seed buoyancy can mediate dispersal scenarios4. These biophysical interactions (between propagule features and transport mechanisms) are predicted to shape the extent of connectivity among seagrass meadows over large geographical scales and to determine population longevity8.Seagrasses can reproduce sexually by seed production and asexually by vegetative clonal developmentHalodule wrightii, the tropical species with the most extensive geographic range across the Eastern and Western Tropical Atlantic Ocean31. As for all in most other seagrass species, H. wrightii reproduces asexually and sexually, and the extent of each reproduction mode influences dispersal, genetic diversity, and biogeography. Despite its widespread distribution, the species produces seeds that are neutrally to negatively buoyant, with an apparent lack of long distance dispersal means32. Seeds of H. wrightii are able to form persistent seed banks that can remain dormant up to 4 years33. Therefore, seeds play an important role in the persistence of the species. H. wrightii can also colonize new areas through by asexual clonal growth of detached fragments that can survive in the water column for up to 1 month34. Previous studies have explored the genetic structure and connectivity of H. wrightii35 but only focused on specific areas of its distribution. To date, there is not a comprehensive study that encompasses this tropical amphi-Atlantic seagrass, in contrast with other seagrass species located on a single side of this ocean, such as Cymodocea nodosa36.To address this knowledge gap, this study aims to compare predictions and empirical data on large scale connectivity for the seagrass species H. wrightii in the Atlantic Ocean by comparing oceanographic predictors with genetic information. This is the first study that considers the distributional range of H. wrightii. The main goal of the present study was to uncover the population genetic structure and connectivity of this habitat-forming seagrass species in the tropical Atlantic region. This species is an ideal model for range-wide connectivity studies due to its vast geographical distribution. Here, we ask if: (1) there is genetic structure among H. wrightii populations across distant biogeographic regions along the tropical Atlantic; (2) levels of genetic and genotypic diversity are similar among populations; (3) the main ocean currents explain the present genetic structure; or (4) if the latter could be explained by biotic transportation. These questions and hypotheses were approached by using microsatellite markers and biophysical modelling based on oceanographic transport to model connectivity along the vast H. wrightii distributional range.Here, we aim to test the hypothesis of ocean currents as the main vectors for dispersal of H. wrightii ramet dataset, we obtained a total of 475 sampling units (ramets) successfully genotyped for eight microsatellite loci across 19 populations. For the genet-level dataset, after the removal of repeated copies of each genet, 176 individual genotypes remained for analysis. Measures of genetic diversity such as allelic richness  and gene diversity, or expected heterozygosity  were generally low for ramet and genet level datasets within all populations  estimates were significantly negative for both datasets  was highly variable among populations on both sides of the Atlantic, ranging across both extremes  between groups showed higher differentiation between West Africa against the other groups but differentiation was lower among West Africa populations Fig. .The factorial correspondence analysis (FCA) revealed a first major genetic differentiation between the Gulf of Mexico and the remaining sites, and these latter also split into east and west, resulting in three genetic clusters: Gulf of Mexico, east Atlantic (West Africa), remaining west Atlantic (Brazil and Caribbean) Fig. . The STR. The particle simulations delivered 668,499 particles over a 10-year period. These revealed a sharp decline in potential connectivity with distance , that did not completely match predictions from oceanographic transport of propagules (seeds or shoots) by currents. These results showed that although ocean circulation is an important factor in marine population structure1, long-distance dispersal is likely a very rare event. The results support the hypothesis that rare events of occasional biotic transportation might have been responsible for connectivity between H. wrightii populations that belong to distinct genetic clusters.The genetic structure of the seagrass H. wrightii. This was also supported by the biophysical modelling that revealed a null or very low probability of connectivity mediated by ocean currents between distant geographic regions  and suggested that most of the gene flow mediated by currents occurs at a regional scale  has been reported for fish, sea turtles and birds51. The dispersal distances of such animals known to feed among seagrass meadows would allow long-ranging dispersal . The reported digestion times for such herbivores, 6–7 days for dugongs52 and 1–2 weeks for green turtles53, are compatible with a moderate scale of long-range transport. Satellite tracking of green turtles in Atlantic Africa shows travel of 40 to > 1000 km between H. wrightii sites54 and genetic data indicate cross Atlantic green turtle migration55. Still, direct cross-Atlantic biotic dispersal seems unlikely or at least uncommon within average digestion time scales of grazers. Migratory routes of green turtles that feed on H. wrightii56 include several mid-Atlantic islands that are nesting grounds55, and could serve as occasional stepping-stones, although seagrass presence is mainly unknown in such islands57. It is relevant that a green turtle found dead in Senegal had been nesting in Trindade Island 5 months before58. All this information and our data indicate that transoceanic dispersal must be a very rare event, because the distances may be too vast to allow continuous gene flow even by biotic vectors, and there is a remote hypothesis that stepping stone islands could facilitate occasional dispersal of viable seeds. However, even if propagule dispersal is successful, post-settlement survival may be low, dependent on ending dispersal in a favorable environment34.Despite its low abiotic dispersal potential, 42. This is suggesting that this genetic differentiation might be due to recolonization events that occurred in the Caribbean and Gulf of Mexico after the last glacial maximum59.On the western Atlantic coastline, the biophysical model revealed high probability of oceanographic connectivity between populations, but the genetic differentiation into distinct clusters showed that gene flow is restricted among them. Genetic data revealed isolation between Gulf of Mexico, Caribbean, and Brazil despite the high probability of transport by oceanographic currents, particularly between the Caribbean and Gulf of Mexico. However, no apparent gene flow barrier exists between the Caribbean and the Gulf of Mexico, in contrast with the genetic structure shown in our findings and in previous seagrass studies using microsatellites60. This barrier, which runs for around 2300 km, separates the Brazilian coast from the Caribbean region and affects the genetic structure and dispersal of different marine animal organisms as fish or coral species62. Yet, this barrier is not matched in our genetic data, that suggests gene flow among these regions. Thus, it is plausible to hypothesize that biotic dispersal might also play a role in increasing H. wrightii dispersal potential between these regions. Sea turtles and manatees are known to feed on H. wrightii meadows in these regions63, and for some species, these areas are part of their migratory pathways64. Moreover, our data also suggests a closer level of similarity between population of these two regions , as reported for other seagrass, mangrove, and coral species. Low diversity at range edges is commonH. wrightii was lower than Enhalus acoroides66, Thalassia testudinum16 and Zostera japonica67, but equivalent to Halophila beccarii68, Cymodocea serrulata11, Syringodium filiforme17 and Zostera marina69. Across the H. wrightii populations, negative inbreeding coefficients (FIS) were obtained, which is common in seagrass species70 and supports the hypothesis of selection favoring heterozygous individuals.In comparison to other tropical seagrass species, the general microsatellite genetic diversity of , H. wrightii has been able to achieve such a vast distribution, its dispersal success alone cannot ensure establishment, because that is also influenced by survival, viability, and growth capacity of plants originated from seeds and fragments. The balance between sexual reproduction and clonal growth can be represented by the genotypic richness. Despite the small sample sizes in some populations, our findings revealed that genotypic richness of H. wrightii is very variable among locations. Higher genotypic richness values were found in populations located at the center of the distribution. Higher genotypic diversity  may enhance productivity and community recovery72. Seagrass genotypic diversity can range from near monoclonal36 to very high73.Although74. Indeed, clonal propagation was the dominant reproductive mode prevalent in southern marginal populations across east and west Atlantic, showing edge-of-range reduced sexual reproduction relative to populations at center of the species’ range (geographic parthenogenesis)76. Such a pattern could be attributed to limited population sizes, geographical isolation, climate oscillations, and poor seed dispersal from other source populations. However, the northern marginal population in West Africa , revealed a much higher genotypic richness compared to other marginal populations. This may be due to the Banc d'Arguin’s habitat suitability for seagrass species, as opposed to what would be predicted for a marginal range. The Banc d'Arguin is huge, protected, shallow, and nutrient-rich, with inputs of desert dust from the east and coastal upwelling from the northwest77. Furthermore, this tropical seagrass species is projected to expand there with climate change78.Reduced genotypic richness can be a common scenario for coastal populations found at the species’ range edgeH. wrightii relies primarily on clonal propagation throughout the Caribbean region. Seagrasses reproduce both sexually and asexually, and their relative proportions may depend on several factors, including the level of disturbance79. Specifically, events that destroy seagrass meadows create opportunities for rapid colonization and growth by new shoots or clonal fragments, faster than through seed production81. There are reports of fast establishment of H. wrightii after a hurricane (e.g. hurricane Wilma in 200582). Thus, we hypothesize that the low genotypic richness found among Caribbean populations of H. wrightii could be due to past disturbances followed by fast colonization83 through asexual reproduction of previously established or selected genotypes.Our findings showed that , H. wrightii showed a low mean of genotypic richness (R = 0.38), lower when compared to the mean genotypic richness found for other Atlantic seagrass species such as Zostera noltii (R = 0.85)84, Zostera marina (R = 0.61)85 or Thalassia testudinum (R = 0.55)73, but comparable to Syringodium filiforme (R = 0.3717). However, seagrass populations can show a wide range of genotypic richness, from high rates of sexual reproduction86, to high levels of clonality73 such as in Z. noltei87 and also H. wrightii35.OverallWe found discordance between genetic differentiation and predicted ocean connectivity patterns, suggesting a role for other means of dispersal, such as the hypothesis of grazers mediating transport. This study underlines the importance of taking multi-pronged approaches to understand meta-population dynamics and connectivity. Using genetic data and modelling predictions enabled us to study the dispersal and connectivity patterns at different spatial scales while testing hypotheses of ocean currents mediating connectivity.Genetic differentiation requires time for evolutionary processes to accumulate population differences. Therefore, although differentiation requires the lack of homogenizing gene flow, the absence of differentiation can be caused by either gene flow or insufficient time for differentiation among recently isolated populations, also designated as shared ancestral polymorphism. We used genetic methods as an empirical indicator of gene flow patterns, and biophysical modeling through propagule dispersion simulations to complement our findings. All these methods, like any other ways of assessing connectivity, have inherent drawbacks that should be highlighted. Genetic differentiation is affected by historical factors, such as founder events, bottlenecks, and variable population sizes. While this offers a significant advantage in discussing such processes, it also poses a limitation in addressing dispersal as the single cause of the patterns, given the simultaneous influence of these other processes. Additionally, the possibility of remaining ancestral polymorphism could create the false impression of contemporary gene flow even where populations are presently isolated. These complex demographic processes like population bottlenecks, founder events, or changes in population size over time can affect the genetic structure of populations in addition to gene flow. In our data interpretation, we therefore considered that there are other ecological factors besides ocean currents, such as habitat continuity and biological dispersal features, that may have an impact on gene flow, raising the hypotheses of influences of large flow of major rivers in breaking habitat continuity, and grazer-mediated transport.H. wrightii can be used to highlight areas where local protection is necessary or where populations could be managed in a metapopulation strategy. It could also be used as a baseline for future comparisons of the genetic diversity and differentiation of this species, motivating further research. The study also comprises a valuable and unique species distribution baseline confirmed by genetic tools, which may be particularly useful in future studies for example of cases where the validity of putative sister taxa to H. wrightii may be unclear.Our results also shed light on the population genetic variability of an important tropical seagrass across its entire range. This new information for H. wrightii along the east and west Atlantic coastlines  were performed in a total volume of 15 μL, containing 1× Colorless GoTaq" Flexi Buffer , 2 mM MgCl2 (2.5 mM), 10 mM forward and reverse primers, 0.2 mM of dNTP’s, 1 U GoTaq G2 Flexi DNA Polymerase  and 5 mL of diluted template DNA . PCR conditions included an initial denaturation step at 95ºC for 5 min, followed by 30 cycles of 95ºC for 30 s, Ta for 30 s, extension at 72 °C for 30 s and a final extension at 72 °C for 10 min. Amplified fragments were separated on an ABI3130 XL automated DNA sequencer , with 0.25 mL GeneScanTM 500" LIZ Size Standard  plus 9.75 mL of Hi-Di formamide, after denaturation at 95 °C for 5 min. Alleles were manually scored using STRAND , and binned using the R package ‘MsatAllele’89. To minimize all ambiguities, a manual review of microsatellite amplification and scoring was conducted, and the final genotyping alleles for each sample were obtained after a double-check reading process to reduce scoring errors.Genomic DNA was extracted using the NucleoSpin Plant II Kit  following the protocol from the supplier. Individuals were genotyped for eight microsatellite markers Table  develope90 was used to check for null alleles to avoid bias in estimating genetic parameters. The package ‘RClone’91 of R software v.3.6.292 was used to assess clonality for all individuals, because seagrass can proliferate vegetatively and the number of shoots can represent duplicate genotypes from the same clone. To verify if individuals with the same number of multi-locus genotype (MLG) were true clones, Psex was calculated, i.e., the probability of finding identical MLGs resulting from distinct sexual reproductive events93. When Psex threshold was below 0.01, the identical MLGs were considered the same clone . Following previous theoretical assumptions94 we estivated population genetic metrics with our dataset spited into (1) a ramet level dataset, that includes genetic information for all genotyped individuals, and (2) a genet level dataset, where only the genets were kept (after Psex estimates). For the second set of data  found among the individuals sampled (N) was used to estimate genotypic diversity in each population, using the clonal richness (R) index, (R = (G − 1)/(N − 1), ranging from 0 (one single clone) to 1 .The software Micro-Checker 2.2.3E), observed heterozygosity (HO) and inbreeding coefficients (FIS), using GENETIX 4.0595. We have interpreted our results in the context of previous theoretical studies, with a particular focus on the distribution of FIS values among loci and populations for both datasets. By comparing both, we aim to assess whether the clonal rates in populations result in differences in FIS values97. Also, by examining the distribution of FIS values per locus per site, it may be possible to understand the extent to which asexual and sexual reproduction may be involved at a particular location. A factorial correspondence analysis (FCA) was performed to determine the genetic relationship between populations using GENETIX 4.05. To estimate the distribution of genets among genetic groups that minimize Hardy–Weinberg and linkage disequilibrium, a Bayesian assignment of genotypes to K groups was made using the program STRUCTURE98. The value K was estimated from the mean log-likelihood for each K value and the ΔK statistic99 to identify the optimal number of populations groups. This was analyzed for K ranging from 2 to 23 with ten replicates per value and a 50,000 burn-in followed by 500,000 MCMC replicates per iteration. All runs were performed in parallel on multiple cores using the R package ‘ParallelStructure’100. A discriminant analysis of principal components (DAPC) and a Principal component analysis (PCA) implemented in the R package adegenet 2.1.10101 were used to complement structure analysis. Genetic differentiation was estimated between sites (FST).Microsatellite genetic diversity was quantified for each population by estimating allele frequencies, mean standardized allelic richness (Â) adjusted for the minimum genet sample size, standardized number of private alleles (PÂ), Nei’s gene diversity (HH. wrightii. The simulations used daily data of ocean currents assembled from the Hybrid Coordinate Ocean Model (HYCOM), a hindcast of high-resolution three-dimensional ocean velocity fields . This model can resolve key oceanographic processes such as oceanic fronts, eddies, meandering currents, and filaments because it integrates the effect of precipitation, wind stress, wind speed and heat. When hindcast data on ocean current direction and intensity is combined with biological features of pelagic viable time in biophysical modelling, the component of connectivity that is exclusively mediated by ocean currents can be predicted. This modelling approach was previously validated using demographic and genetic data for macroalgae, seagrasses, limpets, mussels, fish, echinoderms, and crustaceans104.Biophysical modeling based on simulations of propagule dispersion coupled with network analysis was used to estimate the connectivity potential of H. wrightii rafts strictly transported on the ocean surface, and as in other studies at large spatial scales104 no shape or density of rafting fragments is considered. Occurrence records describing the species distribution were compiled from the literature and biodiversity information facilities (OBIS and GBIF). The simulation encompassed locations distributed along the east and west Atlantic coastal shores. On the east Atlantic, the simulation included the west coast of Africa from Angola to Mauritania (~ 6250 km of coastlines from -10.0° to 21.5° latitude) and on the west Atlantic, from Florida to south Brazil . A high-resolution polygon was used to define landmasses105. Every hour of simulation, the model determined the position of all drifting rafts while fitting a bilinear interpolation estimate over the velocity fields to smooth ongoing trajectories. Rafts were permitted to float for up to 60 days (extreme propagule duration estimate). Rafts that arrived at a coastline or got lost in the open ocean (outside of the model domain) were excluded from the simulation. Trajectories were aggregated to build asymmetric matrices of pairwise probability of connectivity between source/sink sites, by dividing the number of virtual rafts released from site i that reached site j, by the total number of rafts released from site i. For the ten-year period 2008–2017, interannual variability was analysed by running individual simulations per year. The overall approach of the biophysical modelling is documented in106 and the source code is openly available at: http://github.com/jorgeassis/biophysicalModelling.Individual virtual particles were released on a daily basis  from source / sink sites, where the species is known to occur, spaced 1 km apart  over the course of a year. These particles simulate 107. Graph analyses were performed in R92, using the package igraph.Graph theory was used to generate networks that allow the visualization of connectivity patterns. The graph nodes  and the strength of edges (probability of connectivity) were structured using a connectivity matrix based on a 10-year average of individual simulations. Stepping-stone probability estimates were determined using the graph, by using a product function over the probabilities of connectivity along the shortest paths between all pairs of sites, as found with Floyd–Warshall’s algorithm, which minimises the sum of log‐transformed probabilities. This approach aimed to capture all multigenerational potential connectivity across the study region, beyond the time frame considered in the biophysical simulationsSupplementary Information.


Mytilus coruscus) is widespread in the temperate coastal areas of the northwest Pacific and holds a significant position in the shellfish aquaculture market in China. However, the natural resources of this species have been declining, and population genetic studies of M. coruscus are also lacking. In this study, we conducted whole-genome resequencing (WGR) of M. coruscus from eight different latitudes along the Chinese coast and identified a total of 25,859,986 single nucleotide polymorphism (SNP) markers. Our findings indicated that the genetic diversity of M. coruscus from the Zhoushan region was lower compared with populations from other regions. Furthermore, we observed that the evolutionary tree clustered into two primary branches, and the Zhangzhou (ZZ) population was in a separate branch. The ZZ population was partly isolated from populations in other regions, but the distribution of branches was not geographically homogeneous, and a nested pattern emerged, consistent with the population differentiation index (FST) results. To investigate the selection characteristics, we utilized the northern M. coruscus populations  and the central populations (Zhoushan and Xiangshan) as reference populations and the southern ZZ population as the target population. Our selection scan analysis identified several genes associated with thermal responses, including Hsp70 and CYP450. These genes may play important roles in the adaptation of M. coruscus to different living environments. Overall, our study provides a comprehensive understanding of the genomic diversity of coastal M. coruscus in China and is a valuable resource for future studies on genetic breeding and the evolutionary adaptation of this species.The hard-shell mussel ( Mytilus coruscus, commonly known as the hard-shelled mussel, has a wide distribution area in the northwest Pacific Ocean, including Hokkaido, Japan; Jeju Island, Korea; the Yellow Sea; the Bohai Sea; the East China Sea; and other regions [M. coruscus is the primary mussel species cultured in Zhejiang province and is a crucial species for marine aquaculture in China. In 2021, the mussel culture area in Zhejiang was 1837 hectares, with a production of 227,700 tons, ranking first in the country. Shengsi Islands, located in Zhoushan City, are the main production area for M. coruscus in Zhejiang (source: https://www.zj.gov.cn/ (accessed on 1 March 2023)). In recent decades, there has been a decline in the natural juvenile stocks of M. coruscus. Surprisingly, despite this decline, the number of mussel farms has increased, with a dependency on natural populations. Furthermore, the rapid depletion of the M. coruscus resource can be attributed to the overexploitation of fishery resources, severe pollution in offshore areas, and ecological degradation [M. coruscus and to protect the resources of this species. regions ,2. Not o regions ,4. M. coradation ,6. ModerM. coruscus, focusing on its physiological and biochemical functions, biomaterials, adaptation to climate change, bacterial-host interactions, and biofouling and antifouling properties. It serves as a model organism for these research areas and plays a crucial role in the fields of economy, ecology, and biology [M. coruscus in the East China Sea and mitochondrial 16SrRNAs of M. coruscus from four regions along the southeast coast [M. coruscus species in Korea [M. coruscus through WGR is deemed to provide more comprehensive and in-depth information on the population genetic relationships of M. coruscus.To date, several studies have been conducted on  biology ,13,14,15st coast ,18,19. Sin Korea . These sCrassostrea ariakensis and sequenced the genomes of 261 individuals from three typical habitats in China to examine the phylogenetic relationships among the three geographic populations and to detect selective traits of temperature and salinity adaptation in the southern and northern populations [Mizuhopecten yessoensis from Dalian Zhangzi Island by analyzing whole genome resequencing data, and identified candidate genes associated with the phenotype of the dominant population, which laid the foundation for exploring scallop breeding [Mytilus galloprovincialis, M. edulis, M. californianus, M. chilensis, and Mytilisepta virgata. Lallias et al. (2007) initially constructed a linkage map for M. edulis [Mytilus genus [M. coruscus [M. galloprovincialis and M. edulis and M. coruscus, as well as the latter two species, especially between M. galloprovincialis and M. edulis [M. coruscus and M. galloprovincialis for genome assessment, completed the assembly of the M. edulis genome, and performed a positive selection gene identification and functional analysis by calculating the evolutionary rate of three species in the family Mytilidae  for the identification and functional analysis of positively selected genes [In recent years, next-generation sequencing (NGS) technologies have facilitated the discovery and acquisition of SNPs, which has enabled more accurate and efficient population genetic studies at the genomic level ,22. Wholulations . Lv et abreeding . In the us genus ,28; inveus genus ; and recus genus . Additiocoruscus . This fu. edulis . Corroched genes .M. coruscus genetics by re-sequencing the entire genomes of 80 mussels from eight distinct regions along the Chinese coast. Our objectives were to analyze the genetic diversity and population structure of M. coruscus from different regions, identify the genomic selection signatures among these groups, and investigate their habitat adaptation. Our findings will supplement the current M. coruscus genomic resources and provide a foundation for future conservation of genetic diversity and the improvement of cultured M. coruscus species.In this study, we aimed to deepen the understanding of M. coruscus individuals from eight sites and obtained a total of 6.96 billion high-quality paired-end reads, with an average data size of 56 GB per sample and an average sequencing depth of 14×  on the chromosomes and 3,025,618 (11.7%) on unplaced scaffolds. The majority of SNPs were evenly distributed across the 14 chromosomes of M. coruscus, with roughly 13 SNPs per Kb  of SNPs were located in intergenic, upstream, and downstream regions of the genes, while the other 47.9%  were distributed within genes, including different types of exons, introns, Stopgain, Stoploss, unknown, and SNV. Of all the genetic region variant loci, most were distributed in the intron region, accounting for 95.7%  of the total number of SNPs. Only 4.2%  of the SNPs were located in exonic regions, with synonymous substitutions and nonsynonymous substitutions accounting for 1.94%  and 1.77%  of the total SNPs, respectively c.M. coruscus populations in eight regions, we calculated several metrics including genome-wide nucleotide diversity (π), inbreeding coefficient (FIS), observed heterozygosity (Ho), and expected heterozygosity (He). Our analysis revealed that the TS and ZZ populations had the highest genetic diversity based on observed heterozygosity  and expected heterozygosity . All of the populations showed moderately high nucleotide diversity, with the TS, XS, and ZZ populations exhibiting the highest nucleotide diversity , and the ZS population showing the lowest nucleotide diversity (π = 3.213 × 10−3). It is worth noting that nucleotide diversity is a reflection of genetic diversity, to a certain extent. Inbreeding levels varied among the populations, with the YH and DL populations displaying higher levels compared with the other populations. All populations passed the HWE test, and the mean p-values of HWE ranged from 0.810 to 0.843  showed that individuals from each group were closely clustered together, but they were not clearly separated from other populations. Only a few samples from the ZZ region and the LYG region showed significant deviation from other district groups, indicating a high degree of homogeneity among ulations a. The NJulations b.To further investigate the relatedness between populations, we performed an unsupervised clustering analysis with ADMIXTURE. The cross-validation (CV) error was at its lowest at k = 1, indicating the presence of one ancestral cluster. At k = 2, a red cluster with a score above 0.75 consisted of 15 individuals, 8 from ZZ, 3 from LYG, 2 from Zhoushan, and 1 each from YH and TS. Among the 80 individuals, 53 showed mixed ancestry. Under K = 3 and 4, all eight populations were a mixture of the same three genetic components, suggesting a common ancestral origin. However, the LYG and ZZ populations appeared to show weak differentiation .M. coruscus population was in a state of population contraction in all of them. Additionally, the results of SMC++ analysis revealed that the effective populations of M. coruscus in all eight regions have undergone a continuous decline for millions of years. Specifically, the Ne of the eight populations began to diverge at around 0.6 Ma, and then stabilized at 0.05 Ma  values to be in the selection. We then combined overlapping windows for further analysis. Our results showed significant selection signals in the genes from the overlapping regions identified by both techniques. We also examined the degree of genome-wide population differentiation through Manhattan fixation index plots (FST) and found that chromosomes 1, 2, and 4 of the ZZ population exhibited more significant differentiation features. This indicates that these regions experienced stronger selection, with 36.9% and 37.8% of the selection signatures coming from these chromosomes for the northern vs. southern and central vs. southern groups, respectively  were found to be shared in the comparison with both groups , while 432 genes (approximately 48.2%) were found only in the comparison with one group  .LPGAT1). Using GO enrichment analysis, we identified 143 genes that were significantly enriched in 29 GO terms . The most enriched GO terms were involved in the establishment or maintenance of apical/basal cell polarity (GO:0061245) and in the establishment or maintenance of bipolar cell polarity (GO:0061245) in the biological processes subclass. The most abundant cellular component categories were the apical part of the cell (GO:0045177), synaptic membrane (GO:0061245), and bipolar cell polarity (GO:0061245). The main molecular functions were ubiquitin-like protein transferase activity (GO:0019787) and catalytic activity, acting on RNA (GO:0140098)  b,c.Hsp70), phosphoenolpyruvate carboxykinase (PEPCK), toll-like receptor 2 (TLR2), and ubiquitin carboxyl-terminal hydrolase 19 (USP19). Our GO enrichment analysis revealed that 41 genes were significantly enriched in 25 GO terms , with the most enriched GO terms involved in the positive regulation of the pattern recognition receptor signaling pathway for immune system development (GO:0062208) and the positive regulation of interleukin-8 production (GO: 0032757), among others. The major molecular function represented was the extracellular matrix structural constituent (GO:0005201)  b,c. ThesM. coruscus has higher nucleotide diversity at the whole-genome level than M. yessoensis and C. ariakensis and lower nucleotide diversity than C. gigas and Semibalanus balanoides, so we believe that M. coruscus may have a moderately high nucleotide diversity among bivalve shellfish [M. coruscus among bivalve species is also likely to be intermediate. However, there was not much variation in genetic diversity between regions, except for ZS, where genetic diversity was low  and they are able to attach to natural and artificial substrate rafts, which allows them to reach their final settlement site even in the late larval stages [Some organisms in the ocean release planktonic larvae that disperse using ocean currents over days to months, exhibiting effective long-distance larval dispersal and thus becoming the main source of their dispersal ability, such as yriaster ,53,54. Ll stages ,56. ThusM. coruscus in China, rich in natural germplasm resources, and many larvae are dispersed in the region during the annual winter breeding season  annotations to gain insight into their biological functions and their potential roles in adapting to the southern environment. As expected, many genes known to be associated with heat response were identified in the southern population that were not found in comparison with the central population, such as heat shock protein 70 kDa (Hsp70), cytochrome P450 protein (CYP450), and E3 ubiquitin-protein ligase  [CYP450) belong to evolutionarily conserved antioxidant proteins and are considered to be another general molecular chaperone [HSPs and CYP450 genes is significantly induced by heat stress in intertidal species [HSPs and CYP450 genes are thought to be important for the survival of the snail, Echinolittorina radiata, in the harsh thermal environment of rocky shores [Heliocidaris erythrogramma exhibit significantly higher thermal tolerance than southern (cooler) populations, making it possible for northern thermotolerant populations to migrate poleward with the East Australian Current [Mytilus edulis and Littorina littorea [M. coruscus are better adapted to thermal environments than northern individuals.To investigate the genetic differentiation of  RNF1_2) ,68,69,70 RNF1_2) ,71. As p RNF1_2) ,73. Cytohaperone ,75. The  species ,77. For y shores ,79,80. Fy shores ,82,83. Flittorea ,85. TherM. coruscus at different latitudes, which provides important information for further investigation of the functional significance of these genes in M. coruscus and their role in influencing temperature adaptation. However, the estimated genetic diversity may be biased due to the small sample size, which may also result in a less comprehensive and uncertain identification of genomic variation. Therefore, future studies should use larger sample sizes to obtain more precise information on genetic variation and to determine the causal relationships between these selected genes and the environment.Our study has provided a rich genomic resource of selection signatures related to temperature adaptation in M. coruscus were collected from eight sites in Dalian (DL), Qingdao (QD), Lianyungang (LYG), Zhoushan (ZS), Xiangshan (XS), Yuhuan (YH), Taishan (TS), and Zhangzhou (ZZ), China, from September 2020 to March 2021 , the purified PCR product was cyclized using the ligated oligonucleotide sequence to yield single-stranded circular DNA (ssCir DNA). This ssCir DNA constituted the final library and was rigorously characterized by quality control procedures. Libraries meeting quality standards were sequenced using the MGISEQ-2000 sequencer. ssCir DNA molecules were circularly replicated to form a DNA nanoball (DNB) containing more than 300 replicate copies. These DNPs were then loaded onto patterned nano-arrays using advanced high-density DNA nano-chip technology. Ultimately, 150 base pair reads were generated from the other end of the ssCir DNA using the combinatorial probe anchor synthesis (cPAS) method.http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ (accessed on 11 May 2022)).The raw data generated were filtered using the Trimmomatic tool v0.39 . The rawM. coruscus reference genome (unpublished data) using the BWA-MEM algorithm of the Burrows−Wheeler Aligner software v0.7.17-r1188 [http://broadinstitute.github.io/picard/ (accessed on 1 June 2022)) to sort, organize, and eliminate PCR duplicates, and to generate an index of the BAM files. For variant calling, we employed GATK software v4.1.9.0 [The sequencing results were quality controlled in the above steps and the reads were aligned to the 17-r1188  to convev4.1.9.0  and utilv4.1.9.0 . VariantM. coruscus to classify all SNPs into exons, introns, upstream, downstream, and splice sites.To annotate the location and other attributes of the variants, this study used ANNOVAR v2.4  to deterFST) values for all SNPs were calculated using the -fst option of the PLINK software. The Hardy−Weinberg balance was detected using the “-hew” option of PLINK. Calculation of the inbreeding number (FIS) in the population was performed using Genepop software v4.7 [https://github.com/BGI-shenzhen/PopLDdecay (accessed on 3 July 2022)) [2, the higher the genetic diversity of the population [The genetic diversity and population structure among varieties and cultivars, as well as the heterogeneity of each genetic parameter , minor allele frequencies (MAF), and nucleotide diversity (θπ) were estimated using PLINK (v1.9)  and VCFtare v4.7 . In addiy 2022))  was usedpulation .M. coruscus into plink files. These files were then filtered based on a minimum allele frequency (MAF) of >0.01 to ensure that all populations contained at least three allelic mutations per locus. The genomic data were further processed using PLINK software to remove linked loci with the parameter “-indep-pairwise 50 5 0.2”. After filtering, a total of 2,883,362 SNPs were used for the principal component analysis (PCA), phylogenetic tree construction, and population structure analysis to illustrate the genetic evolutionary relationships among M. coruscus populations. Neighbor-Joining (NJ) trees were constructed by calculating the genetic distance matrix through PHYLIP v3.69 [The VCFtools software was utilized to convert the VCF files of 80 individual t IP v3.69  softwareIP v3.69 . The popIP v3.69 . The numM. coruscus populations, resulting in a total of 32 samples. The SMC++ model v1.15.4 (https://github.com/popgenmethods/smcpp (accessed on 11 July 2022)) [M. coruscus or closely related species, we used a mutation transformation matrix based on oyster data [−9 were used [−9 -sp cubic.Two strategies were used to extrapolate the historical demography. First, Tajima’s D  was calcy 2022))  was thenter data . A generere used ,104. TheFST) and nucleotide diversity (π) metrics to detect possible selection traits [FST value to the ratio of π values [To compare population differences across geographic regions, we used a combination of the coefficient of fixation  enrichment analysis using the R package clusterprofiler . The falM. coruscus from eight regions along the Chinese coast to investigate the genetic diversity and population structure. The findings revealed that the ZS region had a lower genetic diversity than the other regions, while the remaining seven regions showed no significant differences. The genetic structure of M. coruscus showed a trend of weakening, with partial differentiation observed in the ZZ region. Conversely, other regions did not show significant genetic differentiation, and there was no correlation between phylogenetic relationships and geographic distribution. Additionally, we assessed temperature adaptation in the southern and northern populations. These findings provide valuable insights into the genetic distribution of this marine organism and serve as a reference for genetic breeding and studies on the environmental adaptability of intertidal bivalves.In this study, we conducted whole-genome resequencing of


Mnemiopsis leidyi in native habitats. Hybrids are present at the distribution border of two lineages, especially in highly fluctuating environments including very low salinities, while hybrids occur at lower frequency in stable high-saline habitats. Analyses of hybridization status suggest that hybrids thriving in variable environments are selected for, while they are selected against in stable habitats. Translocation of hybrids might accelerate invasion success in non-native habitats. This could be especially relevant for M. leidyi as low salinity limits its invasion range in western Eurasia. Although hybridization status is currently disregarded, it could determine high-risk areas where ballast water exchange should be prevented.Hybridization of distinct populations or species is an important evolutionary driving force. For invasive species, hybridization can enhance their competitive advantage as a source of adaptive novelty by introgression of selectively favored alleles. Using single-nucleotide polymorphism (SNP) microarrays we assess genetic diversity and population structure in the invasive ctenophore   •M. leidyi hybrids in native habitats including low saline areasSNP-chip detects •Hybrid analysis suggests selection against admixed individuals in stable habitats•Hybrid prevalence in fluctuating, low saline areas suggests a selective advantage•Hybrid translocation from low saline areas could facilitate successful invasions Environmental science; Nature conservation; Evolutionary biology The geographic extent of hybrid zones is normally narrow and maintained by balancing dispersal, selection, hybrid fitness, and ecological conditions.Mnemiopsis leidyi provides a case to test the role of hybridization and introgression as a potential source of adaptive novelty in the context of biological invasion. The species is native to the Atlantic coasts of North and South America.,b and six nuclear microsatellite loci, Bayha et al.The ctenophore M. leidyi lineagesM. leidyi in its native habitat along the US Atlantic coast. Estuaries in the northwestern Atlantic experience differing salinity conditions, especially Chesapeake Bay, the largest estuary in the US, which has a large spatial and temporal variation in environmental conditions including extremely low salinity levels.Here, we use a diagnostic single-nucleotide polymorphism (SNP) microarray developed for the northern and southern M. leidyi individuals from eight locations in two regions (New England and mid-Atlantic Coast) was conducted , Woods Hole, , Fort Adams , and Fort Wetherill . Therefore, temporal samples within locations were pooled for further analyses.No temporal genetic differentiation was detected at the four locations, where samples were collected in both 2018 and 2020: Sandwich  indicated three clusters (A highly significant genetic differentiation was detected between the two Virginia locations (Wachapreague on the Altantic coast and Gloucester Point inside Chesapeake Bay) and all other samples, with no genetic structure detected for samples collected North of Virginia along the cost and estuaries of New England . In accoclusters . One groM. leidyi have also been confirmed from large salinity ranges, including very low salinities  as the most likely , which clinities .Figure 3A detailed classification of the hybrids identified in STRUCTURE was conducted in NEWHYBRIDS , with po2 hybrids, 22.7% first-generation backcrosses, and 9.1% second-generation backcrosses. All first-generation backcrosses were classified as pure northern × F1 hybrid backcrosses. Second-generation backcrosses were all classified as backcrosses between first-generation backcrosses (pure northern × F1 hybrid) and F1 individuals.At the Atlantic coast of Virginia (Wachapreague), samples consisted of 63.6% pure northern individuals, 4.6% F2 hybrids, 30% first-generation backcrosses  and 40% second-generation backcrosses. Most individuals were classified with high confidence , where only hybrid individuals were identified, i.e., no pure northern or southern individuals. Individuals from Gloucester Point were classified as 30% FABBA over BABA and an average D = 0.24 (p < 0.001). This suggests recent introgression from Miami into Wachapreague. Similarly, a highly significant amount of shared ancestry was found between Gloucester Point inside Chesapeake Bay and Miami, with a c. 3-fold excess of ABBA over BABA and an average D = 0.42 (p < 0.001).In all comparisons with the locations north of Chesapeake Bay , introgression tests confirmed no significant deviation from the null expectation of D = 0, suggesting no shared ancestry between Miami and the northern locations and no indication of introgression . HoweverM. leidyi in native habitats long the Atlantic coast of the US. We show that hybrids are present at the transition zone between northern and southern lineages, around Cape Hatteras, which represents an oceanographic front where the Gulf Stream begins its arc offshore.M. leidyi is an example of a species with high dispersal capacity facilitated by its holoplanktonic life cycle and self-fertilizing reproduction mode.b, which only detected northern haplotype individuals north of Chesapeake Bay, in an extensive sampling scheme including the Long Island Sound and Rehoboth Bay, DE.The increased resolution of genetic markers sheds new light on the status of hybrids and the nature of introgression between northern and southern lineages of In contrast, two locations just north of Cape Hatteras around/in Chesapeake Bay were highly differentiated from the northern and southern lineages due to the presence of hybrids. In order to understand environmental drivers and document adaptive introgression in hybrid zones, we compare two locations within the hybrid zone with contrasting environments. First, a coastal area along the Atlantic coast characterized by high salinity and relatively stable environmental conditions, and second, Chesapeake Bay, an estuary characterized by high levels of spatial and temporal variability in environmental conditions. The proportion and nature of hybrids found during our investigation were contrasting in the two locations, as outlined in the following sections.M. leidyi population at this sampling site is composed of pure northern individuals plus a few late-generation hybrids. The population structure found during our investigation is consistent with and suggests a hybrid tension zone. In a hybrid tension zone, the population structure is balanced between dispersal of parental individuals and selection against hybrids. This has been suggested to be due to intrinsic fitness differences of hybrid lines.,b as marker gene.The Atlantic coast east of Chesapeake Bay is characterized by relatively stable environmental conditions, which are more similar to other areas along the north-east coast of the USA.,As observed in our data, contact between the northern and southern populations can result not only in hybridization, but also introgression because of backcrossing. However, in a hybrid tension zone, hybrid fitness is expected to be lower relative to the parental forms due to endogenous incompatibilities including the loss of coadapted gene complexes involved in local adaptation due to recombination.M. leidyi is present in a spatially and temporally varying environment, especially when considering salinity, ranging from > 2 to 27.1 PSU  hybrids as well as first- and second-generation backcrosses. This is indicative of a stable hybrid population, actively producing viable offspring and forming a hybrid swarm.,,,,M. leidyi is known to form large populations, including in areas of very low salinity (<5).,M. leidyi in Chesapeake Bay; regions of low salinity act as refugia for M. leidyi to avoid predation by the scyphomedusa Chrysaora quinquecirrha, which is less tolerant to low salinities.,,A contrasting setting was found inside Chesapeake Bay, where  PSU see ;30,37,42Eurytemora affinis.,,One mechanism that might explain the putative fitness advantage of hybrids in the fluctuating environment of Chesapeake Bay is beneficial reversal of dominance, as demonstrated experimentally in the invasive copepod ,,1 hybrids and first-generation backcrosses with pure northern individuals. This indicates that pure northern animals must have been present at low frequencies or originated from nearby coastal areas, with subsequent selection for hybrid lines. Irrespective of the source of the animals observed in Chesapeake Bay, our data suggest the potential presence of a hybrid swarm in this habitat, which is characterized by high environmental variability,M. leidyi has been found  post-introduction hybridization in the recipient habitat; and (2) translocation of hybrids directly from native habitats to the non-native range. It is interesting to note that the northern and southern M. leidyi lineages seem to differ in their salinity tolerance as inferred from their observed distribution range in invasive populations.M. leidyi has not been established, such as the Baltic Sea.M. leidyi in the native range, this study contributes to the general understanding of how hybridization in native populations might contribute to successful invasions in the marine environment. We encourage genomic monitoring of native populations of highly invasive species in order to identify hybrid populations to prevent translocation of admixed individuals from hybrid zones in the native area to new, thus far uninvaded habitats.In the case of M. leidyi using a large number of genetic markers. We acknowledge that our spatial and temporal sampling of Chesapeake Bay is limited. At present, our study does not include the very low saline locations which would be needed to further support a putative advantage of hybrids in Chesapeake Bay, while temporal samples would allow to assess the overall stability of the hybrid zones. However, previous studies using low number of markers,,,Chrysaora quinquecirrha are likely the drivers to maintain hybrids in Chesapeake Bay. Experiments should be conducted to substantiate a hybrid advantage at extreme salinity levels.We are the first to disentangle the status and proportion of admixed individuals and the nature of the hybrid zone in the native habitat of coja@aqua.dtu.dk).Further information and requests should be directed to and will be fulfilled by the lead contact, Cornelia Jaspers , Woods Hole , Fort Adams , Fort Wetherill , Greenwich Cove , Esker Point , Wachapreague  and in Chesapeake Bay at Gloucester Point   are high and >19 even in Narraganset Bay,M. leidyi has been confirmed from salinities as low as >2 (All permissions and regulations to sample the invertebrate comb jelly (ctenophore)  10) see . Salinitow as >2 .,M. leidyi populations for mitochondrial cytochrome b, but not for microsatellite markers.Samples were collected at all locations during late summer/early fall 2018 (N = 102), additional samples were collected at four locations in summer/early fall 2020 (N = 74) to allow for temporal genetic analysis. All samples were collected north of Cape Hatteras and correspond to the northern lineage identified in previous genetic studies.M. leidyi were individually placed on coffee filters and dried for 48 hours at 60°C. DNA was extracted using the DNEasy Blood & Tissue kit (Qiagen) following the manufacturer’s protocol except for the sample processing and the elution steps. A 1 cm x 1 cm piece of dried tissue was cut from coffee filters and placed into a 2 ml microcentrifuge tube. 180 μl ATL buffer and 20 μl proteinase K were added, mixed by vortexing and incubated for 3 hours at 56°C with occasional vortexing in between. After centrifuging at 10,000 rpm for 1 min, 200 μl AL buffer was added, mixed thoroughly by vortexing and incubated at room temperature for 10 min. In the final elution step, after discarding the collection tube and transferring the column into a new 1.5 ml Eppendorf tube, DNA was eluted by adding 50 μl AE buffer (pre-heated to 60°C) to the center of the spin column membrane, incubating for 2 min and centrifuging at 10,000 rpm for 1 min. DNA concentration and purity were measured and afterwards samples were diluted 1:100 for further analyses.All individuals were genotyped at a total of 96 single nucleotide polymorphisms (SNPs) using a high throughput low-density SNP microarray, developed from whole-genome re-sequencing data.ST) was calculated between all sample pairs. Second, a hierarchical AMOVA was conducted partitioning genetic differentiation into a geographical and temporal component.Genetic diversity was estimated using observed (Ho) and expected (He) heterozygosities and standardized allelic richness (AR) per population, calculated in Arlequin v3.5.2.2.All SNP data were used to conduct a Principal Component Analysis (PCA) to visualize population structure using smartPCA from the Eigensoft package,Hybridization patterns were assessed using the framework of Bayesian model-based clustering implemented in NEWHYBRIDS,D statisticD = 0. In our case, we used the baseline population of Miami as P3 in order to infer the amount of shared ancestry between the northern populations and Miami and the direction of introgression. We included the North Sea population from Jaspers et al.Introgression was also investigated by testing for an excess of shared derived alleles using the ABBA-BABA test.1 vs. F2 hybrids was more difficult since both classes shared similar admixture proportions and F1 hybrids presented no exclusive genotypes relative to F2 hybrids. However, F2 hybrids could be distinguished from F1 hybrids by the presence of recombinant genotypes and correctly assigned with a confidence of 94%. Regarding backcrosses, a correct assignment with high confidence was obtained for first-generation (on average 90.4%) and second-generation backcrosses with the same parental type (on average 94.4%). The remaining second-generation backcrosses were correctly assigned with lower confidence (60.1-61.8%). Overall, results suggest that our SNP panel has enough discriminatory power to correctly identify parental northern, parental southern, F2, first-generation and second-generation hybrids. While simulated F1 hybrids could also be assigned as F2 hybrids, they could not be assigned as parental or backcrosses. It should also be noted that we extended the original hybrid classes to include second-generation backcrosses but we did not include later generation hybrids such as F3 or F4 hybrids, which would be not possible to distinguish from F2 hybrids, hence we refer to those hybrids as F2 (or later generation) hybrids in the discussion.To test the power of the markers to classify hybrids, a total of 120,000 individuals were simulated using all SNPs in the dataset for 12 categories including first- and second-generation backcrosses and reassigned blindly.


Viburnum japonicum is a rare plant species and endemic to the coastal region of Eastern Asia with extremely small populations. Within mainland China, this species can be only found in narrow habitats of the northeast coastal islands of Zhejiang Province. However, there are scarce conservation genetic studies on V. japonicum, which has limited the effective conservation and management of this rare species. Here, 51 individuals in four natural populations covering the Chinese geographic range of the species were sampled to assess the genetic diversity and population structure. A total of 445,060 high-quality single nucleotide polymorphisms (SNPs) were identified using double digest restriction-site associated sequencing (ddRAD-seq). The overall average values of observed heterozygosity (Ho), expected heterozygosity (He), and average nucleotide diversity (π), were 0.2207, 0.2595, and 0.2741, respectively. The DFS-2 population exhibited the highest level of genetic diversity among all the populations. Genetic differentiation between populations was moderate (FST = 0.1425), and there was selfing between populations . Of the total genetic variation, 52.9% was found among populations through AMOVA analysis. The Mantel test  combined with analyses of the Maximum Likelihood (ML) phylogenetic tree, ADMIXTURE, and principal component analysis (PCA), revealed that populations of V. japonicum were genetically segregated and significantly correlated with their geographical distribution. Our study demonstrated that V. japonicum maintained a medium level of genetic diversity and differentiation with a strong population structure, and the results were mainly affected by its island distribution pattern and self-crossing characteristics. These results provide insights into the genetic diversity and population history of V. japonicum, critical information for conserving and sustainably developing its genetic resources. Genetic diversity is a critical component of biodiversity, as it serves as an indicator of a species’ adaptability and changeability for change within its habitat . During Viburnum japonicum (Thunb.) Sprengel, commonly known as Japanese viburnum, is an evergreen broad-leaved shrub in the family Adoxaceae. It can be easily distinguished from other recorded species of Viburnum by having characteristics such as rounded and evergreen shrubs, broad leaves, and glabrous texture. In particular, some studies have reported the leaf extraction of V. japonicum contained rich medical value. This indicates that the species has great potential for ornamental and economic use. In the past, the species was thought to be only naturally distributed on the coast of Japan . In 1994, V. japonicum was reported by Chinese plant taxonomists from the east coast of Zhejiang Province (V. japonicum again occurred in South Korea (Gageo-do Island)  and listed among Zhejiang Province’s Key Protected Wild Plants. Despite its endangered status and anthropogenic disturbance, genetic and genomic resources available for V. japonicum are scarce, compared to other Viburnum species. Previous research mainly focused on phytochemistry, resistance physiology, and breeding techniques. To the best of our knowledge, there was only one recent study relating to V. japonicum genetic diversity that has relied on the traditional molecular marker . However, this traditional marker can provide only a small number of polymorphisms from a large sample collection. Next-generation technologies have revolutionized the depth of information we can get from a species’ genome  The characterization of the pattern of genetic diversity and population structure of the four populations in China, (2) investigation of the gene flow and migration history, and (3) development of appropriate conservation and management strategies for long-term.Province . By 2003 Island) . Based o’ genome . In the V. japonicum samples from nearly all known natural populations known to exist across mainland China. A total of 51 individuals from four natural populations were sampled and all the populations originated from islands within an altitudinal range from 45.84 to 230.81 m . To achieve this, genomic DNA was digested with the suitable restriction enzyme ustacks function in Stacks software v 2.5.5  maximum observed heterozygosity of 0.75; (2) minor allele frequency (MAF) ≥ 0.02; and (3) minimum stack depth of m = 4 and p = 8 . Based on these calculations, a high-quality SNP dataset was obtained for further analysis.The initial check of the raw reads was performed using FastQC v.0.11.9 . To incr v 2.5.5  was emplHo), expected heterozygosity (He), average nucleotide diversity (π), and inbreeding coefficient within individuals (FIS) were calculated using Stacks software v 2.5.5. The selfing rate (s) was calculated from s = 2FIS/(1 + FIS) (FST) were estimated using Arlequin v 3.5.2.2  value was determined by the cross-validation (CV) error. To further investigate the historical migration between four populations, an ML drift tree of populations and residual fitting heat map using TreeMix v1.3.1 (The genetic diversity parameters for each population including the average number of nucleotide differences (Num Indv), observed heterozygosity ( FIS) . To eval 3.5.2.2 . To veri 3.5.2.2  with 9,9ee v 2.1 , and a rv 1.94.1 . The pop v 1.3.0  to clustx v1.3.1  was alsoV. japonicum accessions, we utilized the ddRAD-seq method for SNPs genotyping. A total of 8,611,606 tags were acquired with an average sequence depth reaching 47.05 (range 34.48–64.32) ×. After filtering poor data, 445,060 SNPs were detected across the four populations. Among these, the majority were transitions (Ts) type SNPs , making up 60.04% of the total SNPs, while transversions (Tv) type SNPs  account for only 39.96%. The transitions to transversions ratio (Ts/Tv) was 1.503. Furthermore, heterozygosity SNPs  dominated, accounting for 63.86% of the total SNPs, whereas homozygosity SNPs  represented only 36.14%. Nevertheless, the distribution of SNP mutations was not uniform. The mutation spectrum analysis revealed that genome-wide SNP mutations could be divided into six types, with C:G > T:A and T:A > C:G as the main SNP mutants (To obtain high-resolution genotypic information on the 51  mutants .V. japonicum populations. The highest level of average nucleotide diversity (π), observed heterozygosity (Ho) and expected heterozygosity (He) was recorded in population DFS-2 . By contrast, population TA exhibited the lowest diversity . Additionally, a lower observed heterozygosity (Ho) value was observed in all populations compared to expected heterozygosity (He), and, the FIS values varied from 0.2130 to 0.2627, resulting in a mean selfing rate (s) of 24.52%.At the species level, the AMOVA statistics of the 445,060 SNPs indicated significant genetic variation among (52.90%) and within (47.10%) the population .FST value was 0.1425 (range 0.0431–0.1924). DFS-2 population to TA had the highest level of differentiation compared to the remaining populations  and geographic distance  was detected by the Mantel test  and the second principal component (PC2) in the principal component analysis (PCA) were 21.46% and 5.46%, respectively. Furthermore, the PCA scatter plots of PCA reflected that DFS-1 and DFS-2 individuals were closely clustered together but distinct from individuals from DC and TA .K value was 2  revealed a high level of genetic diversity  between populations with the AMOVA analysis indicating that 52.9% of the genetic variation occurred across geographical regions. These results align well with the study findings of FIS values, and the selfing rate (s) estimated was 24.52%, indicating that inbreeding occurred within all populations. The narrow geographical distribution and distance between the islands where V. japonicum grows can limit gene flow (pollen and seed) between populations, leading to increased genetic drift and inbreeding in the native population. These factors contribute to the increase of genetic differentiation observed among populations.Several factors affect genetic differentiation among plant populations, with geographical isolation being a crucial factor that affects population growth, gene flow, and ultimately species’ persistence . In thisNm ≈ (1-FST)/4FST = 0.046 < 1, indicating that gene flow between the two populations is limited. Nevertheless, field observations showed that V. japonicum has a high flowering capacity, displays bright red fruit, and produces an abundance of seeds. These traits attract local pollinators over short distances and a variety of frugivorous birds that can feed on the fruit and disperse seeds over long distances. Therefore, we hypothesize that sporadic birds dispersing seeds between islands facilitate the maintenance of gene flow between populations of V. japonica.Notably, the distance between the TFS-2 and DC populations examined in this study was approximately 177.751 km, posing a significant geographic barrier hindering gene flow between these populations. However, one gene flow event was observed between the two populations in TreeMix. We calculated that V. japonicum . This finding demonstrates that the geographic distance could be a limiting factor to gene flow between populations of V. japonica. The Mantel’s test results were further solidified through analysis of the ML tree, PCA, and ADMIXTURE.The Mantel test is a frequently used approach to evaluate spatial processes that influence population structure . Our stuV. japonica, further exacerbated by the geographical isolation of islands which restricts the gene exchange between populations and intensifies the genetic differentiation among populations. Therefore, to enhance the protection of V. japonica and its utilization, it is recommended to strengthen the in situ protection of natural populations, regulate economic activities and artificial construction, such as wind turbines and solar power panels, in the vicinity of the populations, and prevent deforestation and reclamation to promote the population renewal and reproduction. Moreover, a germplasm resource bank can be established on Tian’ao Island, which can facilitate the mixing and breeding of individuals from different islands such as Dongfushan Island, Dachen Island, and other islands can be to promote gene exchange between populations and maximize the protection and restoration of the genetic diversity of V. japonica. Considering V. japonica’s specific habitat requirements, it would be beneficial to advance research on artificial breeding techniques to improve survival rates in ex-situ conservation. Additionally, by employing in situ population protection and ex-situ cultivation, excellent varieties can be chosen to support island vegetation restoration and landscaping design.Island plants tend to have lower genetic diversity than terrestrial species, and have a higher extinction risk . Human d


Peromyscus leucopus) sampled across a purported recent range expansion gradient. We found mixed support for the epigenetic compensation hypothesis and a lack of support for expectations for expansion populations of mice at the range edge, which likely reflects a complex history of expansion in white-footed mice in the Upper Peninsula of Michigan. Specifically, epigenetic diversity was not increased in the population at the purported edge of the range expansion in comparison to the other expansion populations. However, input from an additional ancestral source populations may have increased genetic diversity at this range edge population, counteracting the expected genetic consequences of expansion, as well as reducing the benefit of increased epigenetic diversity at the range edge. Future work will expand the focal populations to include expansion areas with a single founding lineage to test for the robustness of a general trend that supports the hypothesized compensation of reduced genetic diversity by epigenetic variation observed in the expansion population that was founded from a single historical source.Populations at the leading front of a range expansion must rapidly adapt to novel conditions. Increased epigenetic diversity has been hypothesized to facilitate adaptation and population persistence via non-genetic phenotypic variation, especially if there is reduced genetic diversity when populations expand . In this study, we use the spatial distribution of genetic and epigenetic diversity to test this hypothesis in populations of the white-footed mouse ( Populations at the leading front of a range expansion or invasion must rapidly adapt to novel conditions to successfully colonize new environments. Often, such populations also lack genetic variation due to low population size, bottleneck effects, founder effects, limited gene flow, and assortative mating , theoretFallopia spp.; Poa annua; Didemnum vexillum; Passer domesticus) from a recent invasion (<50 years ago) compared to an older invasion (150 years ago) had reduced genetic diversity, but epigenetic diversity was similar in the two populations (Xenostrobus secures) and the Australian tubeworm  methylation, can facilitate colonization of novel habitats and ecological range expansions (reviewed in ulations . In othePeromyscus leucopus noveboracensis). We then test whether spatial patterns in diversity are consistent with the hypothesis that increased epigenetic diversity compensates for reduced genetic diversity at the leading edge of the expansion. We characterize epigenetic variation using DNA methylation, an epigenetic marker associated with transcriptional regulation and modulation of gene expression. Peromyscus leucopus is a native North American rodent that is currently shifting its range northward, likely due to shorter and milder winter conditions (A) where regular small mammal censuses in the region indicated that P. leucopus was absent in the UP prior to 1981 except in the southernmost county of the UP (Menominee), where they were documented in natural history collections that began in 1939 , while epigenetic diversity would be lowest in the core populations and increase along the expansion gradient .Using this background, we sampled white-footed mice collected from four sites in northern Michigan to test the prediction that epigenetic (methylation) diversity compensates for reduced genetic diversity in expansion populations . These sFig. 1In a companion analysis conducted concurrently with the work presented here , we usedB). We also sampled two expansion populations within the UP. The first, UP expansion population 1 , is located approximately 120 km northeastward of UP Core and represents the estimated range edge in 2002. The second, UP expansion population 2 , is located 230 km northeastward of UP Core and represents the range edge in 2016. Data on sample collection, including the site and date of sampling, are shown in We sampled three sites in the UP and one in the Lower Peninsula (LP) of Michigan, which were categorized as either expansion populations  or core populations (populations from the historic range). The two core populations occur at a similar latitude and represent the estimated northern range limit of the species up to 1981  vouchered at the University of Michigan Museum of Zoology or Miami University  in some of our study areas, but species identity can be discerned visually  by inserting a sterile micropick into the nasal cavity to dislodge 5–12 mg of tissue . Prior tPeromyscus . We trimmed all reads to 96 bp, and only reads with Phred score >10 and unambiguous barcodes were retained. We excluded individuals with fewer than 200,000 raw reads, as low-quality specimens can result in assembly problems and excessive filtering of loci.We processed raw sequence reads in STACKS v.2.41 . We checP. leucopus genome (NCBI assembly ID: GCA_004664715.1) using Bowtie2 . We exported the SNP data in Variant Call Format (vcf) and processed in R v. 3.3.2 using the PEGAS package (We extracted the consensus sequence for each assembled locus using  package . We remoprocess_radtags script of STACKS with default settings except a maximum barcode distance of one (–barcode_dist 1) and disabled restriction site check (–disable_rad_check). We trimmed demultiplexed reads for quality and adapter contamination and removed cut site sequences using TrimGalore v.0.6.0 . After quality processing and adapter trimming, we visually assessed the reads for degradation at read ends using Mbias plots. We removed cut site sequences by trimming 5 positions from the 5ʹ end of forward reads (–clip_r1 5) and 4 positions from the 5ʹ end of reverse reads (–clip_r2 4). To remove low quality positions, we further trimmed forward reads by 3 positions at the 5ʹ end and truncated reads to 119 bp at the 3ʹ end (–hardtrim5 118).We restricted our analysis to methylation occurring in the context of CpG dinucleotides; in mammals, DNA methylation occurs almost exclusively in this context . . Although CpG methylation shows tissue-specificity, it is highly variable and even within a single cell type the methylation state of a given CpG position in the genome may vary between individual cells. As a result, methylation at a given position is typically expressed as a percentage ranging from fully methylated (methylated in 100% of reads) to fully unmethylated (methylated in 0% of reads). Usually, a given CpG position sampled within a single tissue type is either fully methylated or fully unmethylated. Less commonly, methylation of a CpG position varies between cells; such positions are called partially methylated  and nucleotide diversity (π) from a dataset of 5937 loci. We calculated genetic diversity measures using the STACKS module populations for loci with a maximum of 25% missing data  in % methylation) using non-parametric Kruskal Wallis tests. We conducted post hoc comparisons using Tukey's Honestly Significant Difference tests.We compared the mean locus diversity values among populations for all four diversity measures  and nucleotide diversity (π), are highly correlated . For both genetic diversity measures, mean diversity differs significantly across populations, and all pairwise comparisons are significant . Genetic diversity is highest in the LP Core population , consistent with our prediction that genetic diversity would be highest in historic populations with larger population sizes, and is higher than in either expansion population: UP Exp1 , and UP Exp2 . The genetic diversity of the UP Core population  is higher than the UP Exp 1, but not the UP Exp2 . The higher genetic diversity of the UP Exp2 population relative to the UP Exp1 population was not what we predicted based on the expansion history informed from field surveys and museum records . Mean epigenetic diversity across loci also differs in both measures, though this difference is only marginally significant for uh when using a p value cutoff of 0.05 . Pairwise comparisons revealed that mean epigenetic diversity does not differ significantly across the three UP populations , but epigenetic diversity is significantly lower in the LP Core population . Low epigenetic diversity in the LP Core population is expected given the corresponding high genetic diversity . However, epigenetic diversity in the UP Core population is similar to the epigenetic diversity in the expansion populations and there is not an increase in epigenetic diversity toward the range edge proposed by Fig. 2C, D). Unsurprisingly then, genetic diversity and epigenetic diversity are not correlated . A partial Mantel test comparing pairwise genetic and epigenetic distance after controlling for the effect of geographic distance also is not significant .The two epigenetic diversity measures, unbiased haplotype diversity (uh) and the standard deviation of % methylation (SD), are significantly positively correlated  from a recent invasion (<50 years old) to an older invasion (150 years old) and found that while genetic diversity was greatly reduced in the recent invasion, epigenetic diversity was similar in both populations. Likewise, X. secures, and Australian tubeworm, F. enigmacticus).In light of this updated expansion (two-source) model, a different sampling design will be required to test our hypothesis in this system; however, we can still test updated predictions about diversity and colonization using the data presented here. First, we can test our original predictions for a single-source model by focusing on the westernmost expansion population (UP Exp 1) and its source population (UP Core). In these populations, genetic diversity is significantly higher in the source population and lower in the expansion population, as we expected. Epigenetic diversity, in contrast, does not differ significantly between the two populations. Taken together, these trends support an epigenetic compensation hypothesis because epigenetic variation relative to genetic variation is higher in the colonist population compared to the source population. Similar trends have been found in other colonist systems. Second, we can make updated predictions about diversity in UP Exp 2, the contact zone between two lineages in the two-source model. Intraspecific admixture of two separate lineages in a colonist population generates novel allelic combinations and counteracts the effects of bottlenecks and inbreeding  can vary at least partially independently of genetic markers (reviewed in Future work can comprehensively test the compensation hypothesis in this system by focusing on western populations in Wisconsin and the western UP, where the expansion appears to be derived from a single, western lineage. Another avenue for future work, whether in this system or others, is to use historic museum specimens to characterize temporal changes in epigenetic and genetic diversity. Measuring diversity in a colonist population over time would be a powerful test of the epigenetic compensation hypothesis. Several studies have successfully characterized cytosine methylation in historic and even ancient tissues, however, obtaining high quality data can be challenging (see obad038_Supplemental_FilesClick here for additional data file.