Porphyra sensu lato , an important seaweed grown in aquaculture, is the most genetically diverse group of the Class Bangiophyceae, but has poorly understood genetic variability linked to complex evolutionary processes. Genetic studies in the last decades have largely focused on resolving gene phylogenies; however, there is little information on historical population biogeography, structure and gene flow in the Bangiaceae, probably due to their cryptic nature, chimerism and polyploidy, which render analyses challenging. This study aims to understand biogeographic population structure in the two abundant Porphyra species in the Northeast Atlantic: Porphyra dioica  and Porphyra linearis , occupying distinct niches . Here, we present a large-scale biogeographic genetic analysis across their distribution in the Northeast Atlantic, using 10 microsatellites and cpDNA as genetic markers and integrating chimerism and polyploidy, including simulations considering alleles derived from different ploidy levels and/or from different genotypes within the chimeric blade. For P. linearis, both markers revealed strong genetic differentiation of north-central eastern Atlantic populations (from Iceland to the Basque region of Northeast Iberia) vs. southern populations , with higher genetic diversity in the south vs. a northern homogenous low diversity. For. P. dioica, microsatellite analyses also revealed two genetic regions, but with weaker differentiation, and cpDNA revealed little structure with all the haplotypes mixed across its distribution. The southern cluster in P. linearis also included introgressed individuals with cpDNA from P. dioica and a winter form of P. dioica occurred spatially intermixed with P. linearis. This third entity had a similar morphology and seasonality as P. linearis but genomes (either nuclear or chloroplast) from P. dioica. We hypothesize a northward colonization from southern Europe . In P. linearis recently established populations colonized the north resulting in homogeneous low diversity, whereas for P. dioica the signature of this colonization is not as obvious due to hypothetical higher gene flow among populations, possibly linked to its reproductive biology and annual life history.The genus  Porphyra umbilicalis Kützing  from the Northwest Atlantic has been proposed as originating from Northeast Atlantic refugia by post-glacial colonization  is a chimeric system in multiple ways. When the sporophyte produces a conchospore, meiosis forms a tetrad consisting of the four meiotic products. These four cells develop together into a single leafy gametophytic thallus through successive mitotic divisions. Therefore, each gametophytic blade is composed of cells with different genetic compositions  with contrasting mating systems and seasonality. Porphyra dioica J. Brodie and L. M. Irvine is dioecious, present all year on the coast and endemic to the Northeast Atlantic , reproduction, habitat, and seasonality . Binning and allele rounding were checked with GENEMAPPER v.4.1 and TANDEM  and HS, the expected heterozygosity using the Hardy-Weinberg equilibrium  . When individuals had more unique alleles than possible under these ploidy-scenarios, a random subsample of the alleles was drawn. Since we did not know the exact ploidy of the samples and we did not have any information of the dosage of the alleles, we were not able to calculate the inbreeding coefficient FIS.Two summary statistics of nuclear genetic diversity were calculated for every population in each species using IVE 3.04 : EN, theversity) . Each inversity)  implemenTRUCTURE v. 2.3.2.1 (TRUCTURE was run with the admixture model with the assumed number of clusters (K) ranging from 1 to 25. For each value of K, ten replicate runs were carried out consisting of 100,000 Markov Chain Monte Carlo (MCMC) iterations after a burn-in period of 10,000, without any prior information on the species or population of the sampled individuals. The optimal number of clusters was determined using the ΔK statistic  based on the allelic variation across all genotyped individuals using GENODIVE 3.04  rather than the morphological species identification, with populations grouped within species. We then proceeded to quantify the differentiation within each species separately, based on the main within-species clusters. For these latter analyses, we removed three individuals that were identified genetically as P. dioica in populations where all other individuals were identified genetically as P. linearis. No significance test was performed for these AMOVAs as that would lead to circularity in reasoning . The resulting sequences were edited to eliminate ambiguities and aligned with GENEIOUS 6.1.6  and MEGA X  and winter seasonality of P. linearis but P. dioica genotypes . This new cryptic morphotype was found either as a few individuals intermixed within P. linearis populations (Artabra and Cabo Raso) or as full populations (Patos and Amado) .Sriterion , at whicd Amado) –6 but thd Amado) , showed TRUCTURE and the PCA showed the presence of several admixed genotypes that are presumably hybrids between the two species. All above results were the same when considering species as diploid .Inferring sub-group genetic structure using STRUCTURE cluster  showed a higher number of alleles, and higher gene diversity  was highest in the North of Portugal. For P. dioica there was less spread in the genetic diversity and the most diverse population differed, depending on the summary statistic and the assumed ploidy level.For both species, populations from the South Europe Siversity , 3 and wiversity . For P. FCT statistic that quantifies the differentiation among the two species had values of 0.26, 0.29, and 0.31, for the assumed ploidy levels of 2×, 4×, and 8×, respectively  between the clusters was stronger for P. linearis than for P. dioica, irrespective of the assumed ploidy level (see FSC) was also stronger in P. linearis. When P. dioica was split into two clusters representing the two morphotypes , no differentiation was found according to FCT with values of −0.01, 0.00, and 0.01 for the assumed ploidy levels of 2×, 4×, and 8×.In both species, Sevel see . InteresP. linearis and P. dioica. Within each species, the haplotypes were generally closely related, with only a few mutations steps between haplotypes, with the exception of one divergent haplotype (Haplotype D) found within the P. linearis samples. This could be the case of a recombinant haplotype, or a lineage from an unknown cryptic species, at a introgressed or heteroplasmic individual but further research is needed to clarity this aspect. Haplotype F was widespread in P. dioica but was also found in two P. linearis individuals from one site (Cabo Raso) in the South Europe cluster, where the microsatellites had shown admixture for those two exact individuals. The winter form of P. dioica only displayed this haplotype F, agreeing with the results from the microsatellites  and the winter form of P. dioica matched remarkably well with the clusters observed for the microsatellites, as illustrated in the PCA plot  was found, and a southern area with 6 haplotypes and evidence of introgression. For P. dioica  we also found six haplotypes, all very closely related (only one or two mutation steps difference among them), but there was no clear geographical structure in their distribution. However, also here, the South Europe cluster, with 5 haplotypes, showed more diversity than the North Central cluster, with only 3 haplotypes.The cpDNA phylogroups observed for PCA plot . In P. lP. dioica, presented two morpho/ecotypes, an annual form and winter form indistinguishable from P. linearis. This winter form was found to co-exist with individuals of P. linearis but also to be the only form present at one site. No genetic differentiation was found between the winter form and the annual form of P. dioica, judging from the FCT values between these two groups that were very close to zero. This suggests that the striking morphological differences between the forms could be the result of phenotypic plasticity, which is a genotype’s ability to produce variable phenotypes in response to environmental conditions  showed that the main divergence was at K = 2 for the distinction between the two species, there was also evidence of subclustering within each species (K = 3 and K = 4). For both Porphyra species, the microsatellite and chloroplast markers showed the presence of two main biogeographical areas within the Northeast Atlantic: North-Central Europe  and South Europe . The degree of divergence between the northern and southern clusters, as measured using FCT, was stronger for P. linearis than for P. dioica, independent of the assumed ploidy level. For P. linearis, the southern regions hosted the maximum genetic variability and contained all cpDNA haplotypes including some unique to these areas and the Northern-central regions have fewer and non-unique haplotypes. For P. dioica, all the diversity seemed to be more mixed across its distribution with no clear pattern, though it was also lower in the north compared with the south in general. Additionally, at the more extreme south, introgressed P. linearis individuals and mixed populations with the winter form of P. dioica start to appear.Though both clustering analyses  Guiry; P. dioica, more recent historical events might hypothetically have led to admixture and connectivity among population, in addition to a possible role of different mating systems and all year presence on the shore (vs. only winter/spring presence in P. linearis).Combined, these results point to a postglacial recolonization from southern to northern Europe after the LGM , where n Iberia  harborinPorphyra sensu lato, the phenomena of hybridization and introgression have previously been observed  as P. linearis. We observed such diversity only within the southern cluster of P. linearis, but never in the northern cluster nor in any population of P. dioica.The transfer of genetic material across species by hybridization and/or introgression has a considerable impact on the genetic make-up and evolution of species e.g., . For othed e.g., ; howeverP. linearis reported in the literature [P. umbilicalis Kützing and P. linearis Greville . As the sample is used destructively for DNA extraction, the exact same slice cannot be used for measuring the ploidy level with flow cytometry. The most feasible way to analyze the data of a mixoploid plant was to resample the data sets several times. In that way we could look at the population differentiation simulating each ploidy level separately, and then compare their strength. Though the resulting values of the summary statistics will of necessity be imprecise, this method does allow us to judge whether the assumption of a specific ploidy level would have any impact on the results  – has been challenging. Since these genotypes and ploidy levels can be either distributed across separate sections of the blade or distributed haphazardly see , it is n results . Our anaPorphyra sensu lato taxa or other intertidal red algae considering these facts.In red algae, the number, size and position of nuclei in cells as well as their DNA content (ploidy) vary considerably across taxa. DNA measurements have revealed that nuclei within a single individual can become polygenomic (either polyploid or polytene) and can also represent different stages of the life cycle   or reprePorphyra with higher genetic diversity and evidence of introgression and cryptic plasticity in the southern populations. The southern cluster in P. linearis revealed introgressed individuals that had cpDNA-haplotypes of P. dioica, as well as a winter form of P. dioica that grows intermixed with and is morphologically indistinguishable from P. linearis. For the latter species, we hypothesized that a northward colonization from southern Europe after the LGM has led to more homogeneous and lower genetic diversity in populations toward the north. For P. dioica, the signature of such recolonization is less obvious, possibly due to a higher connectivity and gene flow among populations resulting from its reproductive mode and annual seasonality. We hope the results of this study may provide a starting point showing how to tackle methodological challenges for other Bangiaceae and other intertidal red algal species considering mixoploidy and chimerism.Two biogeographical areas in the Northeast Atlantic have been found for chimeric blades of the genus The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/EV-Á conceived the initial idea following discussion with all authors. EV-Á, ES, and MG performed the field collections. EV-Á performed the lab work and wrote the first draft of the manuscript. EV-Á and PM analyzed the data. PM designed the customized R-Scripts used in this study. All authors assisted substantially with the manuscript development and also approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Microtus ochrogaster; N = 231) and meadow vole  within five tall-grass prairie restoration sites embedded within the agricultural matrix of midwestern North America. We predicted that vole dispersal would be constrained by the extent of agricultural land surrounding restored habitat patches, spatially isolating vole populations and resulting in significant genetic structure. We first employed genetic assignment tests based on 15 microsatellite DNA loci to validate field-derived species-designations, then tested reclassified samples with multivariate and Bayesian clustering to assay for spatial and temporal genetic structure. Population connectivity was further evaluated by calculating pairwise FST, then potential demographic effects explored by computing migration rates, effective population size (Ne), and average relatedness (r). Genetic species assignments reclassified 25% of initial field identifications . In M. ochrogaster population connectivity was high across the study area, reflected in little to no spatial or temporal genetic structure. In M. pennsylvanicus genetic structure was detected, but relatedness estimates identified it as kin-clustering instead, underscoring social behavior among populations rather than spatial isolation as the cause. Estimates of Ne and r were stable across years, reflecting high dispersal and demographic resilience. Combined, these metrics suggest the agricultural matrix is highly permeable for voles and does not impede dispersal. High connectivity observed confirms that the restored landscape is productive and permeable for specific management targets such as voles and also demonstrates population genetic assays as a tool to statistically evaluate effectiveness of conservation initiatives.Ecological restoration can promote biodiversity conservation in anthropogenically fragmented habitats, but effectiveness of these management efforts need to be statistically validated to determine ’success.’ One such approach is to gauge the extent of recolonization as a measure of landscape permeability and, in turn, population connectivity. In this context, we estimated dispersal and population connectivity in prairie vole ( The goal of SAFE is to promote wildlife through habitat restoration, with the primary target being high priority species designated by U.S. Fish and Wildlife Service as threatened and endangered (T&E), but with other species benefitting as well. The program is voluntary, thus specific locations and numbers of sites may fluctuate over time. In Illinois, SAFE sites are selected in proximity to permanent prairie habitats, with each site comprised of several restoration patches varying in size and time since initial restoration.Habitat restoration can reduce or even reverse the detrimental consequences of these anthropogenic impacts. In the prairie landscape of North America, numerous conservation initiatives have been implemented to improve remnant grassland parcels and increase population numbers of targeted wildlife. Earliest prairie restoration efforts date as far back as the 1930s  and now Microtus ochrogaster) and the meadow vole (M. pennsylvanicus), as a means of quantifying the effectiveness of prairie restoration efforts within a large-scale agroecosystem. Voles can subsist in small home ranges (100m2) within prairie fragments, and are easily captured via live trapping, a technique that yields reliable abundance and movement estimates . No other population or family structure was concordant among both Structure and Colony outputs. Scant evidence of spatial or temporal structure was apparent for either species.Assignment tests failed to recover distinct gene pools for of K = 2  by the Δtatistic . Rather,tructure . In contlvanicus  was reprlvanicus  or four M. ochrogaster, despite an apparent lack of structure in the empirical data. However, M. pennsylvanicus shows sensitivity to divergence time in that a minimum of 15 simulated generations was required for significant population differences in >90% of replicates. This is likely a consequence of low sample size (N = 6) associated with the Livingston site.Power analysis indicated adequate power to discern population clusters in both species . This waStructure v2.3.4). Colors reflect distinct gene pools and horizontal bars within plots represent individuals. The proportion of each color within an individual bar reflects the probability of ancestry. Samples are grouped by site and year (separated by black bars). Sample sizes are in Plots are based on Bayesian clustering (r) values >0.25. Average within-patch r-values ranged from 0.009–0.106, with higher values generally seen among individuals at the southernmost SAFE site , whereas those for the other patch (PRTO) did not . An insufficient sample size prevented such a comparison for the third patch.We removed 19 individuals from our analyses because of relatedness (P = 0.027). However, relatedness versus distance was non-significant in 2012 .Average pairwise relatedness and geographic distance were significantly but inversely related across patches and sites in 2010, with relatedness diminishing as distances increased  indicated contemporary movements of individuals among SAFE sites  were identified as potential migrants based on an assignment probability threshold <0.01. No potential migrants were identified for M. pennsylvanicus, but it was only captured at two patches, with only six individuals sampled from Livingston and the remaining 77 from Saybrook.Estimates of migration , with a negative but non-significant relationship between geographic and genetic distances , despite a much smaller maximum observed distance  at Prairie Ridge from 148 in 2010 (95% CI = 83–479) to 58 in 2012 . At Montgomery, M. ochrogaster populations also displayed a reduced Ne estimate, with values declining from 579 (95% CI = 67—∞) in 2011 to 169 (95% CI = 67—∞) in 2012 . A test of significance could not be performed due to confidence intervals trending to infinity. Comparison of each confidence interval demonstrated a lack of significance, as they overlapped reciprocally with Ne estimates for each year  and vole (Microtus sp.) being both ubiquitous and linked to complex trophic interactions [Spatially-structured habitats such as the agroecosystems dominating the midwestern plains of North America are characterized by heterogeneity in patch quality, and this serves to modulate demographic processes such as population density and dispersal rates . Small mractions . Howeverractions .M. ochrogaster [M. ochrogaster [versus distance was interpreted as a balance between costs associated with habitat loss versus higher predation risk via distances traveled.Experimental manipulations of patch quality demonstrated that food availability and predation risk indeed affected dispersal in rogaster , as wellrogaster ), but norogaster , 72. Habrogaster  showed lM. ochrogaster we detected neither significant structure .‘Border habitats’ such as fencerows, roadside ditches, and waterways can also promote the recolonization of restored grasslands , and mayDispersal in voles is modulated spatially and temporally  by landsM. ochrogaster  and a substantial drought (2012). Most individuals (47%) were collected in 2012, fewer in 2010 (30%), and least in 2011 23%), with but a few samples (N<5) available for three sites in 2011, and none at two sites in 2010 . Populat%, with bM. ochrogaster socially monogamous with strong pair bonds and an elevated degree of philopatry and sociality [M. pennsylvanicus is promiscuous [At more local scales, genetic structure in voles can be attributed to spatial clustering of kin , or reflociality . Prefereociality . Conversmiscuous  and malemiscuous , with remiscuous , and formiscuous  being faM. pennsylvanicus in our data is attributed to the aggregation of related individuals  are essential to sustain genetically diverse populations , and thuNe, higher relatedness) found in smaller habitat patches  , 96 In tBorrelia burgdorferi) is carried by a vector tick that relies upon vertebrate hosts (including voles) for dispersal [vice-versa [In the context of grassland restoration, Lyme disease is of potential concern , in thatispersal . Some stce-versa . In thisce-versa . Also, ice-versa  and coorce-versa .Restoring agricultural plots in midwestern North America to endemic tall grass prairie is an ongoing process implemented by federal and state agencies, but often with success difficult to quantify. Molecular approaches can provide metrics to gauge if specific restoration goals have been successfully achieved, and were applied in this study to quantify connectivity among vole populations in rehabilitated prairie patches in Illinois.Molecular approaches can complement the ecological assessment of restoration success in multiple ways, and in addition, provide insights not obtainable otherwise. As demonstrated in this study, key aspects include genetic clarification of taxonomic uncertainty, and metrics to quantify population connectivity over larger spatial and temporal scales.Ne and relatedness values, despite a regional population decline in 2011. Despite this, a level of caution is warranted such that genetic data is not over-interpreted in the context of ecological inferences. Genetic connectivity, as examined in this study, does not necessarily equate to demographic connectivity [Voles in this study reflected a certain degree of demographic resilience , 75 as iectivity .Advances in molecular technologies and analyses have expanded the capacity of genetic/genomic data to address questions previously refractive. Genetic data can reveal how species respond to specific management actions , or tesM. pennsylvanicus was a response to social behavior (clustering of kin), and not isolation (reduced geneflow), thus mirroring observations on an iconic prairie species, the Greater Prairie Chicken [Collaborations between molecular and restoration ecologists can invoke a more nuanced perspective of ’restoration success.’ Genetic patterns emerge as a response to different processes, and data must be carefully evaluated . In our  Chicken . EcologyS1 FigField-identification based on morphology was verified by genetic clustering, with 25% of samples reclassified.(PDF)Click here for additional data file.S2 FigGenetic clusters detected in each species at Illinois SAFE sites based on PCA applied to 15 microsatellite loci.(PDF)Click here for additional data file.S3 Fig‘Optimal’ number of retained principal component (PC) axes (X-axis) based on minimum of root-mean-square-error (RMSE) assignment (Y-axis) of 20% of randomly selected samples.(PDF)Click here for additional data file.S4 FigK values 1–10 based on the ’ad hoc statistic’ ΔK and the probability of K.Evaluation of 10 replicates at (PDF)Click here for additional data file.S5 FigAbility to resolve population structure as evaluated with two statistical measures (χ2 and Fisher exact tests).(PDF)Click here for additional data file.S6 FigNe estimates and 95% jackknife confidence intervals between years and two SAFE sites.Comparison of (PDF)Click here for additional data file.S1 TableDetails on geographic location, patch area, acronyms and sample sizes based on phenotypic and genetic identifications.(PDF)Click here for additional data file.S2 TableDetails on primers used to amplify 23 microsatellite loci.(PDF)Click here for additional data file.S3 TableValues calculated for 2010 and 2012 between restoration patches.(PDF)Click here for additional data file.S4 TableEstimates of within-patch average and variance by year.(PDF)Click here for additional data file.S5 TableComparison of population genetic and life history parameters for two vole and three bird species.(PDF)Click here for additional data file.


Ostrinia furnacalis may influence adult mating generation number (voltinism) across different local environments. The degree to which voltine ecotype, geographic distance, or other ecological factors influence O. furnacalis population genetic structure remains uncertain. Genetic differentiation was estimated between voltine ecotypes collected from 8 locations. Mitochondrial haplotypes were significantly different between historically allopatric univoltine and bivoltine locations, but confounded by a strong correlation with geographic distance. In contrast, single nucleotide polymorphism (SNP) genotypes show low but significant levels of variation and a lack of influence of geographic distance between allopatric voltine locations. Regardless, 11 of 257 SNP loci were predicted to be under selection, suggesting population genetic homogenization except at loci proximal to factors putatively under selection. These findings provide evidence of haplotype divergent voltine ecotypes that may be maintained in allopatric and sympatric areas despite relatively high rates of nuclear gene flow, yet influence of voltinism on maintenance of observed haplotype divergence remains unresolved.Diapause is an adaptive dormancy strategy by which arthropods endure extended periods of adverse climatic conditions. Seasonal variation in larval diapause initiation and duration in  The Asian corn borer (ACB) is a major pest insect of cultivated maize throughout China, including Jilin Province where ecotypes of this species differ in voltine traits. The distribution of significant mitochondrial haplotype and nuclear genetic variation among sample locations suggests geography and voltinism are factors influencing contemporary ACB genetic structure in Jilin Province. Cumulative effects of exposures to light (photoperiod) and heat (degree day accumulation) impact species growth rates and modify seasonal responses  is comprised of at least 20 species, many of which have diverse ecologically adapted and phenotypically diverged subspecies or ecotypes  time among univoltine individuals (>20 days) results in a delay in adult emergence compared to multivoltine individuals , are spatial correlated with variation in voltinism among O. nubilalis populations, where estimated frequencies repeat along a northsouth cline in the United States as populations transition from one, two, and three annual reproductive generations  gene vary significantly between populations differing in Pdd , but it remains unknown whether this basis for voltinism differences are ancestral across all Ostrinia.Additionally, univoltine and multivoltine ecotypes of Showers, . A longeO. furnacalis populations pervade in eastern Jilin Province, China, as evidenced by mostly a single yearly adult flight peak  was PCR amplified from a subsample of 282 individuals and direct Sanger sequenced in both directions using forward and reverse primers on an ABI3700  at Sangon Biotech as described previously  as described previously .A Bayesian information criterion (BIC) model was initially used to select the TPM2 + F + I model of sequence evolution (two‐phase model with a proportion of invariable site), which was subsequently applied for a maximum likelihood (ML)‐based phylogenetic reconstruction with 1,000 iterated ultrafast bootstrap pseudoreplicates using IQtree 1.5.6 , and nucleotide diversity (π) were calculated using DnaSP v 5.10.01 , with geographic distance between all sample location pairs to examine adherence to an isolation‐by‐distance (IBD) model  for adherence to an IBD model were estimated using GenAlex  from 1 to 10, with a burn‐in of 100,000 and 200,000 MCMC iterations. The optimal value of K was inferred from ad hoc posterior probability models of [Pr(X|K)]  and heterozygosity (observed and expected heterozygosity under HW equilibrium) were calculated for SNP data, as well as AMOVA, PCoA, and partitioning of genetic correlation of genetic distance 2.4Nem = (1−FST)/4FST  a classical measure of gene flow, and 2) a MCMC simulation approach. First, population genetic structure‐based gene flow was calculated according to the relationship  Wright, , where Nθ  and xNm, where x is 1 for mitochondrial DNA. A Bayesian approach was then used to estimate bidirectional haplotype exchange (Nm), which was calculated from five independent runs for COI sequence data using the following Markov chain setting: sampling of 10,000 genealogies and 3 long chains with sampling of 500,000 genealogies, with burn‐in of 1,000,000 per chain.Secondly, maximum likelihood estimates of haplotype exchange were calculated using the coalescent‐based MCMC simulation approach with the program MIGRATE 3.2.6  were applied to predict the significance of any allelic frequency differences between O. furnacalis samples with mitochondrial COI haplotypes found only at locations primarily with univoltine  or bivoltine ecotypes , and not within sympatric locations (GZ and YT). For this, an FST outlier approach was implemented using LOSITAN  with a false discovery rate (FDR) of 10%, where FDR is defined as the expected proportion of false positives among outlier loci.SNP loci that passed quality filtering criteria . In contrast, amplified fragments from 4 samples were not digested and remained as intact 188‐bp products , which matched the pattern predictive of O. nubilalis or O. scapulalis . Re‐running of assays for HC09, YT43, YT09, and ZL24 along with appropriate controls confirmed nondigested results (results not shown). A total of 276 of the 282 subsampled COI DNA sequences remained after filtering to remove ambiguous or poor‐quality sequences and were submitted to GenBank (accession numbers MN524233 to MN524512). Clustering of these 276 sequences resulted in 65 unique haplotypes  and OR4 results  and ZL24 (Hap_10) were used to define these samples as putative interspecies hybrids and thus were also excluded from further analyses.es Table . Subsequts Table , these wO. furnacalis COI haplotypes for putative voltinism specificity (based on location history and ignoring presence at mixed (sympatric) locations) identified five putative univoltine‐specific haplotypes  ranged from 0.524 in DH to 0.914 in ZL and YT, and nucleotide diversity (π) ranged from 0.001 in YJ to 0.015 in HC for the sampled COI haplotypes across locations , or a mix of univoltine and multivoltine ecotypes . Haplotype differences accounted for a significant degree of variation between univoltine, mixed, and bivoltine locations , although AMOVA partitioned the greatest proportion of variance among individuals within location  surpassed the Bonferroni adjusted significance threshold (p ≤ .018). Among these significant comparisons, 9 (32.1%) were between univoltine and bivoltine, 6 (21.4%) were between univoltine and mix (sympatric), and two among univoltine locations (Table ϕST) and geographic distance  accounted for 0.7% of total variation (p ≤ .001), and relatively low and significant variation was detected among populations within region . AMOVA similarly showed a relatively low level of variation at SNP loci between univoltine and bivoltine locations (FSR = 0.020 and FRT = 0.002) along with a significant level of inbreeding  among pairs of the eight locations ranged from 0.001 to 0.042, of which 21 of 28 pairwise comparisons (75%) were statistically significant after Bonferroni corrections for multiple testing (Table FST) estimates based on SNP marker data showed no significant association with geographic distance  based on 257 SNP loci  = 4 (ΔK of 8.86 maximized with ln P(K) = −41985.6 ± 28.2) among the 257 SNP genotypes from 380 O. furnacalis  from iterative estimates for each value of K. The proportion of estimated co‐ancestries within Q‐matrices at K = 4 showed that Cluster 4 was more prevalent among individuals within the univoltine locations , but this was not significantly different compared to the mean proportion among individuals at bivoltine  or mixed sympatric locations . The univoltine location Dunhua (DH) contained the greatest mean proportion of Cluster 4 (pink) among individual co‐ancestries. Co‐ancestries among individuals from location Yanji (YJ) were distinct in that 0.258 corresponded to Cluster 2 (red), as compared to ≤0.129 among the two remaining univoltine locations and ≤0.040 among all other locations.A Bayesian clustering model implemented by STRUCTURE predicted an optimal 3.4Nm) using the parameter Nm = [(1/ϕST−1)/4] . Results also indicated that haplotype exchange between univoltine and mixed locations was significantly different than between bivoltine and mixed locations . Analogous estimates for Nm using variation between locations based on SNP genotype data yielded estimates of gene flow (number of migrants) that ranged from 5.7 for between YJ and YT to 245.3 between YJ and BC . BLASTx search results showed significant hits for 8 of the 11 parent sequences (72.7%) to protein models of the draft O. furnacalis genome  based on these 11 SNP loci as a function of geographic distances showed no adherence to an IBD model  to more southern (bivoltine) regions of the Midwest United States  compared to 28 more southerly multivoltine locations in China.The current study detected a greater degree of variation between locations defined by voltine ecotype for mitochondrial haplotypes compared to genomic SNP loci. Specifically, significant differences are observed in estimates of subpopulation divergence averaged across univoltine compared to bivoltine  as putatively being under selection , and the anonymous SNP locus contig05909.680, are associated with differences in generation number among multivoltine O. nubilalis ‐based estimates of genetic exchange between populations may be a consequence of mating period asynchrony or topological barriers present across the geographic ranges within this genus. Regardless of these insights, the factors and interactions culminating in the formation and maintenance of biological variants (voltine ecotypes) within natural populations remain to be completely resolved and will undoubtedly be the focus of future research endeavors.Complex mechanisms and interactions, at both the ecological and genomic scale, appear to shape the contemporary structure among subpopulations within species of None declared.Yangzhou Wang: Conceptualization ; Formal analysis ; Methodology ; Project administration (supporting); Writing‐original draft (supporting); Writing‐review & editing (supporting). Kyung Seok Kim: Formal analysis ; Writing‐review & editing (supporting). Qiyun Li: Funding acquisition ; Project administration . Yunyue Zhang: Investigation (supporting); Resources (supporting). Zhen‐Ying Wang: Funding acquisition (lead); Project administration (lead); Resources ; Writing‐review & editing (supporting). Brad Steven Coates: Conceptualization ; Formal analysis ; Methodology ; Project administration (supporting); Supervision (lead); Writing‐original draft (lead); Writing‐review & editing (lead).Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file.Table S4Click here for additional data file.Table S5Click here for additional data file.Table S6Click here for additional data file.


Taint in grapes and wine following vineyard exposure to bushfire smoke continues to challenge the financial viability of grape and wine producers worldwide. In response, researchers are studying the chemical, sensory and physiological consequences of grapevine smoke exposure. However, studies involving winemaking trials are often limited by the availability of suitable quantities of smoke-affected grapes, either from vineyards exposed to smoke or from field trials involving the application of smoke to grapevines. This study compared the accumulation of volatile phenol glycosides  in Viognier and Cabernet Sauvignon grapes exposed to smoke pre- vs. post-harvest, and found post-harvest smoke exposure of fruit gave similar levels of volatile phenol glycosides to fruit exposed to smoke pre-harvest. Furthermore, wines made from smoke-affected fruit contained similar levels of smoke-derived volatile phenols and their glycosides, irrespective of whether smoke exposure occurred pre- vs. post-harvest. Post-harvest smoke exposure therefore provides a valid approach to generating smoke-affected grapes in the quantities needed for winemaking trials and/or trials that employ both chemical and sensory analysis of wine. Howeverof smoke  and are of smoke . Glycosiof smoke ,22,23, wSmoke-derived volatile phenols and their glycosides have become important chemical markers of grapevine smoke exposure. Several studies have evaluated direct and/or indirect methods for determining free and bound volatile phenol concentrations in grapes and/or wine ,26,27,28It is difficult for researchers to study the direct effects of grapevine exposure to bushfire smoke, given the occurrence of bushfires cannot be reliably predicted, and the inherent occupational health and safety risks associated with fires. While some studies have made use of fruit and/or wine samples arising from vineyards that have been exposed to bushfire smoke  generates fruit suitable for research purposes. Kennison and colleagues found post-harvest smoke exposure of grapes yielded wines containing elevated levels of smoke-derived volatile phenols , but untA range of analyses were applied to grapes, wine and/or acid hydrolysates of wine to determine the effects of bunch excision and smoke exposure. Berry weight was monitored following the application of experimental treatments and with the exception of excised Viognier bunches at t = 1, the mass of smoke-affected grapes was found to be significantly higher than for the corresponding control grapes, at each time-point . This wao-cresol was detected in Cabernet Sauvignon grapes following pre-harvest smoke exposure, but only at t = 1. These results are consistent with findings from recent studies [The volatile phenol levels of control and smoke-exposed grapes were also measured . Low lev studies ,17,19 an studies ,17.Background  levels of volatile phenol glycosides were detected in both Viognier and Cabernet Sauvignon grapes. Total glycoside levels ranged from 45 to 53 µg/kg for control Viognier grapes and from 11 to 27 µg/kg for control Cabernet Sauvignon grapes , with siSmoke exposure resulted in approximately two to three-fold higher levels of volatile phenol glycosides in Viognier grapes and wines (compared with corresponding controls), i.e., 128–165 µg/kg and 134–142 µg/L, respectively . In contThe most abundant volatile phenol glycosides detected in smoke-exposed Viognier grapes were pentose-glucosides of guaiacol, cresol and phenol, and the glucose-glucoside (gentiobioside) of syringol , in agreA recent study reported a delay in the accumulation of volatile phenol glycosides in Cabernet Sauvignon grapes following grapevine exposure to smoke . VolatilSignificant differences were observed between the glycoside profiles of wines made from control and smoke-exposed grapes . Backgroo-cresol (at 1 µg/L each) in wines made from smoke-exposed Cabernet Sauvignon grapes provided compositional evidence of smoke exposure (Guaiacol was the only volatile phenol detected (at ~1 µg/L) in control Viognier wines, while syringol was present (at 5 µg/L) in control Cabernet Sauvignon wines ; backgroexposure . Sensoryexposure . Signifiexposure a, with sexposure . Fewer sexposure b, again exposure , but theEvidence of smoke exposure was more apparent when the volatile phenol concentrations of wines were compared after acid hydrolysis . Acid hyo- and m-cresol, syringol and 4-methylsyringol), for 60 h. Low levels (up to 2.0 µg/kg) of guaiacol and o-cresol were detected in control grapes  forms and from 17 to 2114 µg/kg in glycosylated forms) in treated grapes  was undertaken. Grape bunches were placed in a 156 L glass chamber, together with an aqueous volatile phenol solution  at considerably higher concentrations than syringol and 4-methylsyringol. This likely reflects relative differences in volatile phenol concentrations in the gas phase, as a consequence of their different physical properties, i.e., a combination of differences in molecular weight, water solubility, boiling point and vapor pressure. Significant variation was also observed in both volatile phenol and volatile phenol glycoside levels between the two grape varieties , enabling both chemical and sensory analysis of wine; especially in years where commercial fruit or wine affected by bushfire smoke is not available. The density and/or duration of smoke exposure could be increased to obtain more heavily tainted grapes and wine, with higher levels of volatile phenols and their glycoconjugates, and more intense smoke-related sensory attributes. The only real limitation of this approach will be the need to apply smoke close to maturity, given grapes are non-climacteric and do not continue to ripen once removed from the grapevine. Model experiments involving exposure of excised bunches to one or more volatile phenols (in gaseous form) in lieu of smoke could also be undertaken. Again, the concentration of volatile phenols and duration of exposure can be manipulated to influence to what extent grapes are tainted.In the current study, grape bunches were exposed to smoke immediately after harvest, while exposure to volatile phenols commenced within ~6 h of harvest, because grape metabolic processes, including glycosylation by glucosyltransferase enzymes, were expected to decline progressively following harvest. However, results from a recent study suggest glycosylation may continue for some time post-harvest. Bound volatile phenols were observed in table grapes 24 h after they were exposed to smoke , despited3-guaiacol, d3-4-methylguaiacol, d7-o-cresol, d3-syringol and d3-syringol gentiobioside) were synthesized in house, as previously reported [Chemicals  were purchased from Sigma Aldrich  and solvents (HPLC grade) were purchased from Merck . Deuterium-labelled internal standards . Grapevines were: planted (in 1998) in north-south aligned rows on their own roots; trained to a bilateral cordon, vertical shoot positioned trellis system; hand-pruned to a two-node spur system; and drip irrigated. Three adjacent grapevines were exposed to straw-derived smoke (for 1 h), 7 days before maturity, using a purpose-built smoke tent (6.0 × 2.5 × 2.0 m) and experimental conditions described previously [Field trials were conducted in a vineyard located at the University of Adelaide’s Waite Campus in Urrbrae, South Australia  using Viognier and Cabernet Sauvignon grapevines  were co2 were adjusted to 3.5 and 30 mg/L respectively, before bottling (under screw cap closures). Bottles were stored at 15 °C for six months prior to chemical analysis.Bunches were de-stemmed by hand and 1 kg of fruit (per replicate per treatment) crushed with the addition of 50 mg/L sulfur dioxide (added as a 10% solution of potassium metabisulphite (PMS)). Tartaric acid was added to adjust the pH of must to 3.0 and 3.5 for Viognier and Cabernet Sauvignon, respectively, prior to inoculation with 300 ppm of PDM yeast  and addition of diammonium phosphate (150 mg/L). White and red musts were fermented on skins (to maximize extraction of volatile phenol glycoconjugates) at ambient temperature (22–23 °C) for 7 days, with the cap plunged twice daily. Wines were then pressed and held at 20 °C until completion of fermentation , after which they racked from gross lees and cold stabilized (at 0 °C for 1 week). No wines underwent malolactic fermentation. Wine pH and free SOWines were subjected to strong acid hydrolysis according to methodology described previously ,21. Brieo- and m-cresol, syringol and 4-methylsyringol). After 60 h of treatment (which commenced within 6 h of harvest), the grapes from each cluster were rinsed (with water), dried, weighed and homogenized (as above), prior to chemical analysis. Bunch weights were obtained before and after treatment; berry numbers were also counted prior to homogenization. Control grapes (3 bunches per variety) were similarly harvested, weighed and homogenized for chemical analysis. The volume of aqueous volatile phenol solution remaining after treatment was measured and ranged from 3.6 to 7.4 mL per dish .An additional trial involving post-harvest exposure of grapes to volatile phenols (in gaseous form) was also performed. Three bunches of Viognier grapes and three bunches of Cabernet Sauvignon grapes  were placed in a 156 L glass chamber (91 × 38 × 45 cm), together with six glass dishes each containing 20 mL of an aqueous solution of volatile phenols  were measured in grapes, wine and acid hydrolysates of wine, by the Australian Wine Research Institute’s (AWRI) Commercial Services Laboratory . Volatile phenols were measured using an Agilent 6890 gas chromatograph coupled to a 5973 mass selective detector , using stable isotope dilution analysis (SIDA) methods described previously [TM 4500 tandem mass spectrometer, with a Turbo VTM ion source . Data acquisition and processing were performed using Analyst software (version 1.7 AB SCIEX). The preparation of isotopically labelled internal standards, method validation and instrumental operating conditions were as previously reported [The concentrations of volatile phenols . Prior to wine evaluation, panelists completed a brief induction, during which they were introduced to both the RATA procedure and a list of attributes adapted from previous studies [The replicate wines from each treatment were evaluated (by two sensory experts with >10 years of experience working with smoke tainted wines) to ensure there were no obvious faults or sensory differences amongst the replicates. The wine replicates from each treatment were then blended and their sensory profiles determined  method ) with a  studies ,23. RATAp < 0.05. Sensory data were analyzed by two-way ANOVA using participants as a random factor and wines as a fixed factor, with Fischer’s LSD post-hoc test (p ≤ 0.05) to determine significant differences between the treatments at p < 0.05, using XLSTAT .Chemical data were analyzed by analysis of variance (ANOVA) using GenStat . Mean comparisons were performed by least significant difference (LSD) multiple comparison test at


In the context of climate change, in situ conservation of plant genetic resources is becoming increasingly important as it supports natural  adaptations of plants to the changing environment. The aim of this study was to synthesize a comprehensive general methodology for the maintenance of genetic reserve sites of medicinal plants based on the analysis of relevant legal documents, literature sources, databases, and authors’ own experience. A methodology was developed for the application of various maintenance measures for target species populations in genetic reserve sites to ensure their long-term sustainability. It uses a systematic approach to the intervention measures by grouping them into habitat-specific and factor-specific ones, and follows the specific principles of biodiversity conservation, such as the concept of ecosystem, priority of in situ conservation, caution and validity of decision-making, and regional approach. An extensive discussion on major intervention measures is provided. The methodology is intended to be used as a tool for the preparation and implementation of management plans of individual medicinal plant genetic reserve sites by the responsible agencies and protected area managers and is directly linked to the implementation of the EU (European Union) Biodiversity Strategy to 2030 at the national level. Medicinal and aromatic plant species, including crops and their wild relatives, make up 77% of the total species number in the Euro-Mediterranean region . The intIn Lithuania, 34 genetic reserve sites of medicinal plants have been established accommodating more than 120 medicinal plant species. The purpose of establishing genetic reserve sites, as defined by Article 16 of the Law on Protected Areas of the Republic of Lithuania, is to “preserve the resources of genetic material necessary for activities” . The susThe aim of this study was to synthesize a comprehensive general methodology for the maintenance of genetic reserve sites of medicinal plants. The methodology is intended to be used as a tool for the preparation and implementation of management plans for the individual genetic reserve sites by the responsible agencies and protected area managers and is directly linked to the EU Biodiversity Strategy to 2030 implementation at the national level .Maintenance of genetic reserve sites presents itself a system of various interventions focused on the long-term sustainability of target species populations, the main object of conservation. It is envisioned to be achieved by maintaining the existing populations and their habitats as close to their natural conditions as possible to make use of ecological and evolutionary adaptations to an ever-changing environment. In general, all maintenance interventions can be grouped into habitat-specific interventions and factor-specific interventions. Habitat-specific interventions are direct object-focused  support and maintenance measures, the degree of applicability of which is predefined by the type of habitat. Here we distinguish three main groups of habitat types: (1) meadow/grassland including heaths and shrub formations, (2) forest, and (3) mire/wetland including bogs, fens, and transition mires. Factor-specific interventions are reason-focused prevention and control measures predefined by the type of factors in effect: biotic, abiotic, or anthropogenic. Naturally, both groups of interventions are interrelated. Finally, an assessment of maintenance interventions is set up with some stipulations on the use and supervision of genetic reserve sites. A schematic illustration of the entire process is presented in Mowing is carried out at least every two years and not more than once a year. Mowing begins no earlier than July 15 by cutting the grass at a height of 3–5 cm. Cut grass must be removed from the site within two weeks. Mowing machinery should be adequate to the site conditions to avoid soil compression and scraping.Nardus grasslands , 6270 * Fennoscandian lowland species-rich dry to mesic grasslands, 5130 Juniperus communis formations on heaths or calcareous grasslands, and where no risk of habitat trampling or erosion is posed due to higher soil moisture or surface inclination, grazing may be applied. Grazing intensity, on average during grazing season (from May 1 to October 30), must not exceed one livestock unit (LU) per two hectares (or 0.5 LU/ha). One LU is one animal of cattle or horses or 14 animals of sheep or goats. The lower limit of effective grazing should not be below 0.3 LU/ha. The remainders of ungrazed grass must be mowed by October 15 and removed from the site within two weeks.On sites where plant communities have been formed through regular grazing, such as in EU habitat types 6230 * Species-rich Mowing under appropriate conditions (as described above) can be combined with grazing, e.g., mowing in one year and grazing in the other.When no mowing and no grazing has been applied for more than two or three years, tree and shrub cutting should be considered along with grass mowing or grazing.Assisted propagation of target species can be applied with the propagation material collected from the same genetic reserve site.To support target species pollination, beehives can be placed in the genetic reserve sites or nearby during flowering time.Special, i.e., biodiversity maintenance and other special forest cuttings, as defined by the Regulations on forest cuttings , are appIn forests on moist or wet substrates, like in habitat types 91D0 * Bog woodland and 9080 * Fennoscandian deciduous swamp woods, the maintenance of the soil moisture regime is of crucial importance. Thus, any drainage attempts should be timely observed and prevented.Assisted propagation of target species can be carried out with the propagation material collected from the same genetic reserve site.Genetic reserve sites occurring in EU habitat type 9070 Fennoscandian wooded pastures and alike are maintained by grazing and/or mowing in accordance with the recommendations provided for meadow-type habitats.Tilia cordata Mill., Malus sylvestris Mill., Prunus spp., etc.), to support pollination beehives can be placed in the genetic reserve sites or nearby during their flowering time.For some target tree species  occurring in EU habitat types 7140 Transition mires and quaking bogs and 7230 Alkaline fens, mowing is possible following the recommendations provided for meadow-type habitats.Menyanthes trifoliata L., Rubus chamaemorus L., Vaccinium spp., etc.), to support pollination beehives can be placed in the genetic reserve sites or nearby during their flowering time.For some target species (Heracleum sosnowskyi Manden.). In Lithuania, among other control measures, herbicide encapsulations and inoculations are recommended for such woody invasives as Acer negundo L., Amelanchier spicata (Lam.) K. Koch, Padus serotina (Ehrh.) Borkh. and Robinia pseudoacacia L. [Invasive non-native species. To avoid this problem, preventive measures must be applied in the first place: in forest habitats, forest litter must be protected; in meadow habitats, mechanical damages to the sward layer must be avoided, and mire sites should be maintained as natural as they normally are. No cut grass or woody plant residues can be left as well as no fertilizers can be applied in the sites. Plants of invasive species are eradicated, and their populations are controlled by mechanical , biological  and, in exceptional cases, chemical  means and combinations thereof. Herbicides can only be used against particularly aggressive species, such as Sosnowsky’s hogweed  Roth) is one of the most problematic species, spreading intensively in unmanaged or improperly managed (mowed grass is not removed) grassland communities. Some less problematic species are common reed  Trin. ex Steud.), cow parsley (Anthriscus sylvestris (L.) Hoffm.) and some other nitrophilous plants, which compete strongly with herbaceous and young woody plants. A more frequent selective mowing should be used to control populations of these species, especially when plants are young, with the cut grass removed after each mowing.Problematic native species, also called expansive species. In genetic reserve sites of medicinal plants, wood small-reed (Castor fiber L.), it is controlled that these animals do not dam the streams or drainage ditches in a reserve site or nearby. To protect individual trees from chewing various kinds of fencing and deterring materials can be applied.When there is a risk of site flooding due to activities of the Eurasian beaver (Cervidae) and other large herbivores is achieved by fencing the sites or by isolating individual, more vulnerable trees, or groups of trees with suitable protective materials  or by using repellents.Protection of target species from damage caused by deer  in genetic reserve sites is permitted in accordance with the Regulations on the use of wild plant resources . Some liNo feeders and luring spots for ungulates can be established in genetic reserve sites .It must be prohibited to build roads, parking lots, forest warehouses, recreational facilities, hunting installations  as well as to change natural ground-water regime  or perform other activities in the areas of genetic reserve sites, which may adversely affect the populations of the target species and their natural recovery.The purpose of a genetic reserve site inventory is to identify and assess changes in the qualitative and quantitative indicators of the populations and habitats of the target species and the factors causing them, to properly plan and take the necessary measures in a timely manner.The inventory includes the estimation of target species cover-abundance, identification of actual and potential threats, assessment of the effectiveness of intervention measures, collection of seed samples for ex situ conservation and general assessment of site condition according to the methodology developed for this purpose .Periodicity of inventory. In genetic reserve sites where the main target species are herbaceous plants, the inventory is carried out every 4 years. In the case of forest communities, this period may be longer, but not more than 8 years. When actual or potential threats are identified, inventories are performed more frequently.The inventory data are used to update passport data of the genetic reserve site and, if necessary, the cartographic material, and are stored in the database of a Coordinating Centre for Medicinal Plants as well as in the Central Information System of the National Plant Genetic Resources. Feedback is provided regarding maintenance interventions to the site managers.Monitoring of genetic reserve sites is carried out on a selective basis by ecogeographical areas and habitat types using methodologies specifically developed for that purpose. Feedback is provided to the site managers.Collecting of plant genetic resources  maintained in genetic reserve sites and use for research, breeding, propagation, and cultivation purposes are permitted in accordance with bona fide principles, without depleting their populations. Users are encouraged to provide feedback to site managers regarding intervention measures needed based on visual site assessment.Use of state-protected species is permitted only with the authorization of the Environmental Protection Agency as detailed in the Description of the procedure for the use of protected species . Users aProtection, use, and restoration of genetic reserve sites are supervised by the Lithuanian State Forest Service of the Ministry of Environment, which is responsible for the management of the Central Information System of National Plant Genetic Resources. The Lithuanian State Forest Service organizes and coordinates the periodic inventories of plant genetic resources in cooperation with the Nature Research Centre and other research institutes and universities.To facilitate practical implementation, the interventions are estimated by their applicability from very common (5 points) to very rarely used (1 point) or not applicable (0) in genetic reserve site management by habitat type and supporting references are provided .Lithuania belongs to the Boreal biogeographic region where the final stage of vegetation succession is forest . Therefo−1) grazing was the most appropriate treatment since it maintained species richness and typical floristic composition of the pastures [A condition in favor of livestock grazing application is compensations paid to the farmers participating in the activity “Extensive grassland management by grazing livestock” of the Lithuanian Rural Development Program’s 2014–2020 measure “Agri-Environment Protection and Climate” . In any pastures . This ispastures . The abapastures  is reasoIn the boreal biogeographical region, with Lithuania being part of it, the prevailing vegetation type is forests, so trees and shrubs naturally occupy the largest part of the zone. Therefore, cutting of trees and shrubs as an intervention measure is nearly equally expected in all three types of habitats. However, the need for cutting application in meadows and mires indicates that: grassland was abandoned of haymaking or grazing, and wetland was drained. Two types of cuttings are used in genetic reserve sites according to the Regulations on forest cuttings : cuttingArnica montana L. in north-eastern Lithuania by using seed from southern Lithuanian populations [Tilia cordata Mill. carried out by De Jaegere et al. revealed that in Europe, the genetic diversity of T. cordata tends to decrease as latitude increases [This kind of intervention is better established in forest tree genetic reserve sites when the self-regeneration of trees is insufficient for normal stand recovery or age structure of stands needs to be diversified to ensure stand sustainability. Assisted propagation may also be applied with rare and threatened herbaceous species. For the recovery of genetic stands, there is a provision in Regulations for natural and complex reserves  to use pulations . A reviencreases .Vaccinium myrtillus L., V. vitis-idaea L., V. uliginosum L. and V. oxycoccos L., which grow on mires. Thus, supportive pollination should be considered for all major habitat types by placing beehives in genetic reserves.Polination is an increasingly important element of plant conservation strategies. IPBES  report  lists thPinus sylvestris L. after cutting of pinewoods in dry habitats, like 91T0 Central European lichen Scots pine forests [There are some other population-focused types of intervention, for example, controlled burning. Suggestions exist on the use of controlled burning to support the regeneration of some species, namely,  forests . Control forests . HoweverArnica montana can be often found on mineralized belts and forest block lines [Phragmites australis (Cav.) Trin. ex Steud.) as stated in the nature management plan of the lake Žuvintas and its shores [Triggered by climate change, invasive species is one of the most challenging issues. Currently, 18 species of vascular plants are considered invasive in Lithuania . Basicalck lines . This cock lines . One morck lines . In exces shores . Detailes shores . Recommes shores .The most damage caused by Eurasian beavers is due to flooding of sites. Large populations of beavers can also cause significant damage to tree species as they chew and cut down even large trees. There are various tools invented to prevent rivers and streams from beaver dam construction . MultiplPopulus tremula L., Quercus robur L., and Fraxinus excelsior L., as well as shrub species Sorbus aucuparia L. and Frangula alnus Mill. are foraged by cervids mostly, while European bison (Bison bonasus L.), reintroduced in Lithuania in early 1970s, prefer soft deciduous tree species  Moench) [A review of the research on ungulate herbivory in forests confirmed that manipulation of such herbivory is often highly influential on tree regeneration and on the abundance, diversity and composition of understory vegetation, although it found few studies of boreal areas and long-term herbivory effects . Data fr Moench) . Most re Moench) .It should be noted that chemical plant protection products are primarily used in the agricultural sector, but some of them may also be used in forestry. A useful source of information, in this regard, is the EU Pesticides Database . HoweverAn adequate water regime is one of the most important abiotic factors for ecosystem sustainability. Management interventions related to floods are relevant at the riverine sites mostly. Flood water can make damage when sites remain flooded for an unusually long time. Some proposals exist to solve or to mitigate the problem. One idea is to modify the water regime by controlling the flow in the links connecting the floodplain depressions to the river and to each other .One of the most reasonable solutions to mitigate drought impact is canceling of mowing of grass and livestock grazing. In extreme cases, watering in some areas can be considered. For that purpose, natural small water retention measures which combine drought mitigation, flood protection, and biodiversity conservation, are very relevant . AccordiPinus mugo Turra) in 1800s [Natural, or non-anthropogenic, soil erosion is not a considerable threat to genetic reserve sites in countries with flat topography, like in the case of Lithuania and the other Baltic States. In the past, wind-induced erosion was a problem on open sandy soils in the coastal region of the Baltic sea until it has been controlled by introducing mountain pine , cowberries (V. vitis-idaea L.), cranberries (V. oxycoccos L.), wild raspberries (Rubus idaeus L.), and wild strawberries (Fragaria vesca L.). The total amount of purchased wild berries in 2012 was 1,637,371 kg, with 91% of bilberries. Meanwhile, medicinal plant raw materials totaled 8294 kg [Locations of genetic reserve sites may often coincide with the sites rich in harvestable plant resources. Then the potential conflict issues may arise between the conservation of plant genetic resources and the use of their physical resources. Although the Regulations on the use of wild plant resources  cover ti 8294 kg . No offiField inventories include botanical and ecological site inventories. Botanical inventory aims at identifying changes in the target and other species cover and abundance as well as health and general condition of the target species populations. Ecological site inventory aims at detecting changes in habitat conditions, and the factors behind, as well as identifying actual and potential threats. A systematic and explicit list of threats is provided at the Reference Portal for Natura 2000 . The assAs stated by the Law on Wild Vegetation , to moniAs there are no specific regulations on collecting plant genetic resources in genetic reserve sites, from the formal point of view one should follow the Regulations on the Use of Wild Plant Resources . HoweverIn Lithuania, plant genetic resources are under the domain of the Ministry of Environment. Therefore, the protection, use, and restoration of genetic reserve sites is supervised by the Lithuanian State Forest Service of the Ministry of Environment, which is responsible for the management of the Central Information System of National Plant Genetic Resources. The State Forest Service organizes and coordinates periodic inventories of plant genetic resources and cooperates with the Nature Research Centre and other state research institutes and universities.This methodology was based on analysis of relevant legal documents, literature sources and databases as well as authors’ own experience and historical land use that has formed the habitats in Lithuania. It systematically presents measures for the maintenance and protection of genetic reserve sites, aimed at the long-term sustainability of target species populations of medicinal plants. It complies with the Law on Protected Areas , Law on A long-term experience on the development of Lithuanian national network of genetic reserve sites for in situ conservation of medicinal and aromatic plant genetic resources acquired by the authors , as wellSpecific principles of biodiversity conservation, such as the concept of ecosystem, priority of in situ conservation, caution and validity of decision-making, regional approach , were foFor general provisions on management interventions in genetic reserves, Maxted et al. were consulted . SpecifiThe results are presented in numbered subsections following the structural scheme of maintenance interventions presented in The systematic approach to genetic reserve site maintenance presented above contributes to the planning and application of intervention measures in genetic reserve sites by the better perception of the entire process centered on conservation object (populations of target species) and factors  affecting it. Nevertheless, the recommended measures should be treated in a flexible, pragmatic manner, considering different target species requirements, local conditions, and potential impacts on the environment by preparing individual site-specific management plans. The individual plans, in turn, should be revised and updated according to the results of periodic inventories and monitoring as well as feedback from the genetic resource users and protected area managers.It is notorious that anthropogenic factors, including indirect human impacts, are the most critical in ecosystem conservation. The exploitation of natural resources, particularly when seeking the most profitable ones, is among that kind of anthropogenic factor. Meanwhile, wild medicinal plants usually coexist with more profitable natural resources, such as wood and peat, and human interests often focus on the exploitation of the latter, while ignoring medicinal plant conservation efforts. Thus, success of the conservation is predefined much by human attitude towards natural resources. In any case, sites that are out of interest to the other natural resource users or are protected by nature itself  can be successfully maintained with much less effort. This way a significant part of the resources can be lost. Therefore, it is of critical importance to cooperate with the European ecological network NATURA 2000. It provides several advantages, including better protection of sites and compensation mechanisms to landowners. A standardized habitat classification system and explicit listing of threats, pressures and activities related to their maintenance are additional benefits contributing much to the development of protected site management plans including medicinal plants’ genetic reserve sites.


Lucia and I.insularisinsularis from the Grenadines. Its former description is completed by the use of 38 new samples for genetic and morphological analysis. Seventeen microsatellites were used to estimate genetic diversity, population structure and the level of introgression with other Iguana species over nearly the whole range of the species. ND4 and PAC sequences were also used to better characterize hybridization and to complete the description of this lineage. The I.insularis population of St. Vincent shows a high level of introgression from I.iguana whereas in the Grenadines, most islands present pure insularis populations but several show evidence of introgressions. Of the two remaining populations of I.insularissanctaluciae, only one is still purebred. The recent identification of this and other distinct insular species and subspecies in the eastern Caribbean, and evaluation of where hybridization has occurred, are timely and important because the native iguanas are in urgent need of conservation action. Among the greatest threats is the ongoing human-mediated spread of invasive iguanas from Central and South America, which are destroying the endemic insular lineages through multiple diachronic introgression events.The newly described horned  Iguana which was long thought to be represented by only two species (I.delicatissima (Laurenti 1768), endemic to the Lesser Antilles, and the invasive common or green iguana (Iguanaiguana) [I.delicatissima hybridizes with invasive I.iguana in the Lesser Antilles, resulting in the progressive elimination of I.delicatissima by genetic swamping.Resolving taxonomic or phylogenetic uncertainties and delineating management units according to the genetic characteristics of populations are important in conservation biology . This is species : the Lesaiguana) , with a Iguanaiguana complex sensu 2 .The iguanas used by naiguana , 2020 deI.iguana endemic to north South America, east of the Andes, I.rhinolopha endemic to Central America, and, in the eastern Caribbean, I.delicatissima in the northern Lesser Antilles, I.melanoderma endemic to Saba and Montserrat, and I.insularis  becomes totally black with age and the body barred with broad black bands, whereas the dewlap of the Grenadines pink rhino iguana (I.insularisinsularis) is typically light cream to cream, its bands are narrower, and, in old individuals, markings fade until the animal is almost uniform light cream to nearly white. The genetic analysis was based on a relatively large number of individuals from St. Lucia but only four individuals from two islands in the Grenadines  and Mustique were visited between 10–15 September 2019  and photographs were taken. Tissue samples (tail tip or shed skin) were collected and preserved in 70% ethanol. The procedure was done as quickly as possible, and the iguanas were released back in their habitat. Photographs were also taken of individuals that evaded capture. Iguanas observed and/or caught in St. Vincent and the Grenadines were identified by using the suite of morphological traits recognized as diagnostic by For this genetic study, we took biopsies from 34 iguanas from 15 Grenadine Islands and four iguanas from St. Vincent. These 38 samples were genotyped using 17 microsatellite markers amplified as described by Ar), expected heterozygosity (He) and Fis using FSTAT ver. 2.9.3.2  in 2018, 17 iguanas from northeast St. Lucia corresponding to I.insularissanctaluciae, seven I.rhinolopha collected from southwest St. Lucia , seven I.iguana from French Guiana  values between groups of individuals  in the adegenet package (PCA) to transform the raw genetic data retaining all principal components to maximize the variation of the original data. The best number of clusters K was estimated using the function find.clusters that implemented a K-means clustering minimizing the variation within clusters and a Bayesian Information Criterion (BIC) approach. We assumed a maximum number of 10 clusters and ran the K-means algorithm with 1,000 random starting values and 108 iterations to ensure convergence. A Discriminant Analysis (DA) was then applied with the DAPC function using 30 principal components explaining more than 95% of the total variance of the data and retaining two discriminant functions that carried most information.We conducted a Discriminant Analysis of Principal Components ( package  for R veK) of genetic cluster(s) and the admixture coefficient of individuals to be assigned to the inferred clusters. We selected the admixture model and the option of correlated allele frequencies among populations. As recommended by K (with K varying from 1 to 10) with a total of 100,000 burn-in and 100,000 recorded iterations. To determine the number of genetic clusters from structure analyses, we used the STRUCTURE HARVESTER program  to compare the mean likelihood computed from the 20 independent runs.At the individual level, we also accessed the genetic structure using the Bayesian approach implemented by the software STRUCTURE . This clThe best number of clusters was determined using the hierarchical approach delta K method . In a fiIguanainsularissanctaluciae in St. Lucia  on the following Grenadine Islands (listed from north to south): Bequia, Petit Nevis, Isle à Quatre, Pigeon Island, Battowia, Baliceaux, Mustique, Petit Canouan, Canouan, L’Islet, Catholic Island, Mayreau, Baradal, Jamesby, Petit Bateau, Petit Rameau, Union Island, Palm Island, Frigate Rock, and Petit Saint Vincent  were photographed Fig.  and did 141 Fig.  was a feP = 0.0005). Only eight of the 105 groups of individuals/locus combinations deviated significantly from Hardy-Weinberg expectations . These deviations occurred only for populations of I.insularis and likely resulted from a Walhund effect because the individuals came from different Grenadine islands and likely displayed different genetic signatures. All microsatellite loci were polymorphic with an allelic richness (Ar) ranging from 1 to 2.921 and a genetic diversity (He) ranging from 0 to 0.821 across groups of individuals  , native individuals from both Montserrat and Saba (endemic I.melanoderma), individuals from St. Lucia , individuals from the Grenadines (endemic I.insularisinsularis), and French Guiana (I.iguana). It confirmed there is clear genetic differentiation between I.insularisinsularis from the Grenadines and I.insularissanctaluciae from St. Lucia. Moreover, the populations of St. Vincent and Grand Anse (St. Lucia) show mostly hybrids  Table . DAPC  from other taxa . The results also highlighted individuals showing intermediate admixture coefficients on both St. Lucia and St. Vincent, suggesting hybridization  and the Grenadine Islands (n = 11) sequenced for ND4 for this study, the haplotype of St. Lucia AF217782 identified by n = 3), Battowia (n = 3), Tobago Cays (n = 3), Pigeon Island (n = 1), Union Island (n = 1) and Petit St. Vincent (n = 1). Moreover, an insularis haplotype (MK687402-3) previously identified by MN590142) and Mustique (MN590150).Of 14 individuals from St. Vincent , in the one from Petit St. Vincent, and in one of the two iguanas from Union Island. Palm Island has three different haplotypes, but we did not find AF217782 here. Jamesby (Tobago Cays) and Mustique have another haplotype. Thus of the 28 sequences available for Iguanainsularis . insularis haplotypes at the end of a branch.The ND4 haplotype n = 10) had the same endemic haplotype of the PAC gene (JN811117) as the one found in Louvet (St. Lucia) by n = 6. This haplotype differs by a G instead of a A at site 323. Moreover, we also found this haplotype in three hybrids from St. Vincent that were homozygous for this gene. Conversely, the population of Grand Anse (St. Lucia) had two iguanas  with this endemic JN811117 haplotype, whereas two others  are homozygous for a widespread Central American haplotype (JN811107).All the sequenced iguanas from the Grenadines (JN811117) of the PAC gene identified by I.insularis: three from St. Lucia (Louvet), ten from the Grenadines and three hybrid individuals from St. Vincent. Two iguanas from the Grand Anse (St. Lucia) hybrid population out of the four sampled are homozygous for this haplotype while the other two recognized as hybrids (JN811107) present in iguanas from Belize, El Salvador, Honduras, Guatemala, and Nicaragua. For example, the 70 PAC gene sequences obtained by JN811117, found nowhere else, unambiguously identifies Iguanainsularis among all other lineages and thus confirms the complex hybridization history of some populations.The haplotype  to at least as far south as Petit St. Vincent. I.insularisinsularis (Juliana Coffey in litt. to JD). Indeed, the native range of this taxon might extend as far south as the main island of Grenada, as suggested by the pioneering work of I.iguana and I.rhinolopha.While t. Lucia , the newiguana populations in this region and thus it might be supposed that no population has remained untouched. Natural dispersal between nearby islands may also serve to homogenize these populations.One of the challenges to elucidating the natural ranges of iguanas in this region is that they are often transported by people. For example, it is common practice for hunters to collect live iguanas from the Grenadines to sell as bushmeat on St. Vincent and Grenada during the hunting season from October through January . I.insularisinsularis by South American I.iguana. Similar observations were reported by I.insularisinsularis on Union Island that harbored a South American mitochondrial haplotype . Introgression is already a well-known phenomenon in iguana populations, notably between I.iguana and I.delicatissima in Guadeloupe and Anguilla and between I.rhinolopha and I.delicatissima in St. Barthélemy , which supply the pet trade , Petit Canouan, L’Islet, and the Tobago Cays . On Battowia, Union Island, Palm Island, and Petit St. Vincent, on the other hand, the situation is more complex with some individuals being admixed with continental iguana alleles and, on Union and Palm islands, I.insularissanctaluciae alleles.Our samples from Battowia, Baliceaux, and Petit Canouan (Grenadine Islands) showed evidence of introgression of rthélemy  and St. rthélemy . Iguanaret trade . Iguanaih Guiana . In the I.insularisinsularis is considered endemic to the Grenada Bank (which includes the Grenadines) and I.insularissanctaluciae is endemic to St. Lucia, I.iguana whereas their PAC and ND4 sequences are insularis and their microsatellites are a combination of Iguanaiguana, I.rhinolopha, and I.insularisinsularis. We have no data on the morphology of the fourth specimen, IGU132, but its microsatellite genotype corresponded with I.insularisinsularis. It is, however, impossible to establish whether this last specimen represents an endemic iguana from the original population of St. Vincent or is the descendant of released or escaped iguanas from the Grenadines . The homozygous PAC diagnostic locus (JN811117) shows that have been at least backcrosses with I.insularis. The microsatellites also indicate that this population is deeply introgressed. We see on St. Vincent an important discrepancy between the genetic analysis of this hybrid population and its morphology, which seems to indicate a nearly pure Iguanaiguana population.While es e.g., . The ND4I.delicatissima in the iguanas from St. Vincent and the Grenadines. Conversely, in one population sampled on St. Lucia , I.insularissanctaluciae with a delicatissima mitochondrial haplotype (MK687394-95). Moreover, these two individuals are homozygous for a PAC Central American haplotype (JN811107) and present microsatellites typical from I.iguana and I.rhinolopha. The third individual has a delicatissima ND4 like haplotype (MK687392) and is homozygous for the PAC insularis diagnostic haplotype (JN811117), while its microsatellites show introgression with both I.iguana and I.rhinolopha. The fourth individual from this population has ND4 and PAC haplotypes and microsatellites typical from I.insularissanctaluciae. Thus, this St. Lucian population demonstrates the complexity of hybridization in the genus Iguana, where some individuals possess genetic sequences belonging to four phylogenetic species that all have the ability to breed and produce fertile offspring, as demonstrated by the backcrosses. One of us (JD) was told in May 2021 that the Forestry Department of St. Lucia used to take iguanas handed in by the public to release at Grand Anse. This was done at a time where the morphological differences between iguanas were not well understood and these unfortunate translocations may well explain the alien genes in that population.We have not found any genetic or morphological signs of I.iguana and I.rhinolopha) and their fertile hybrids are now widely scattered across the Eastern Caribbean and pose a serious threat to all remaining indigenous populations of I.insularis, I.melanoderma and I.delicatissima  is severely depleted and restricted to northeast St. Lucia, where it is under immense pressure from habitat loss, illegal poaching for bushmeat and the international pet trade, and feral and invasive alien mammals. Furthermore it faces a rising population of invasive alien I.rhinolopha that is spreading from southwest St. Lucia  still occupy at least 21 of the 35 named islands. Unfortunately, most of these sites are very small and unprotected, and there is little to prevent incursions of alien iguanas from St. Vincent or Grenada, especially during the hunting season when live iguanas are openly transported between islands. Evidence of past hybridization with I.iguana was detected on several islands. Other threats observed during our field surveys included invasive alien cats, dogs, and rats (which prey on iguanas and eggs), domestic goats (which destroy vegetation), and bushfires   . Iguanasaribbean . By recoaribbean  and thisinsularis lineage compared to other representative populations in the Iguanaiguana complex. Furthermore, comparison of the two subspecies of insularis, in a broader geographic context , we lean towards splitting this complex into several species, but we know well that further research is needed, especially in South America, to get a consensus for the taxonomy of this iconic lizard.All of these genetic data (unique PAC and ND4 haplotypes) also confirm the uniqueness of the iguana populations of the southern Lesser Antilles, which were first identified by Iguanainsularis includes St. Lucia  and at least 21 islands in the Grenadines (subsp. insularis). The first descriptions of these taxa were informed by genetic analysis of a relatively large number of individuals from St. Lucia but only four from the Grenadines . However, this gene flow is a recent phenomenon due to human activities. These anthropogenic movements of iguanas have disrupted the normal and independent evolution of these different island populations.The current range of the southern Antilles horned iguana enadines . The preIguanainsularis faces multiple threats, including unsustainable hunting, habitat loss and invasive alien species, including alien iguanas. While purebred I.insularisinsularis populations survive on several islands in the Grenadines, our results reveal evidence of hybridization with I.iguana, an invasive alien species from South America, and I.rhinolopha from Central America. The I.insularis population of St. Vincent shows a high level of introgression from I.iguana, while on St. Lucia, a growing population of invasive Central American I.rhinolopha endangers the remnant population of I.insularissanctaluciae. Experiences from other islands suggest that both invasive alien species are capable of driving native iguana to extinction through competition and introgression. Stronger protection of I.insularis is required throughout its range, coupled with concerted efforts to curb the spread of alien iguanas, I.iguana and I.rhinolopha.Despite having only recently been described,