Orchis mascula L. spanning a large-scale climatic gradient in northern Iran were used to investigate patterns of genetic diversity and plant functional traits. Populations were sampled from three land cover types . Plant height, stem length, number of flowers, bulb fresh and dry weight, glucomannan, and starch concentrations showed high variation among populations and were significantly related to land cover type. In general, genetic diversity was high, particularly in those from eastern Hyrcanian; additionally, populations showed a high level of genetic differentiation (G'st = 0.35) with low gene flow (Nm = 0.46). The majority of genetic differentiation occurred within populations (49%) and land cover types (20%). The population structural analysis using the AFLP marker data in K = 4 showed a high geographical affinity for 198 O. mascula genotypes, with some genotypes having mixed ancestry. Temperature and precipitation were found to shape genetic and phenotypic variation profoundly. Significant isolation by the environment was observed, confirming the strong effect of environmental variables on phenotypic and genetic variation. Marker-trait association studies based on MLM1 and MLM2 models revealed significant associations of P-TGG + M-CTT-33 and E-AGG + M-CGT-22 markers with plant height and glucomannan content. Overall, a combination of large-scale climatic variables and land cover types significantly shaped genetic diversity and functional trait variation in O. mascula populations.Understanding how environmental factors shape patterns of genetic and phenotypic variations in a species is necessary for conservation and plant breeding. However, these factors have not yet been completely understood in tuberous orchid species used to make ‘Salep’, an important ingredient in traditional medicine and beverages in middle eastern countries and India. In many areas, increasing demand has pushed species to the brink of extinction. In this study, 198 genotypes from 18 populations of the endangered species  They often display plasticity and genetic variation along environmental gradients, as these gradients can affect gene flow among populations or create extreme abiotic conditions. Interactions between the environment, phenotypic and genetic diversity have been studied in different plant species at the population level, emphasizing the effect of climate gradients 3. Genetic diversity is crucial since it can act as a buffer against abiotic or biotic stress, anthropological disturbance, inbreeding depression, and interaction with invasive species4. Populations with a history of overexploitation and fragmentation can face genetic depletion that can severely impact the survival and evolution of the species by negatively affecting fitness characteristics7. Under extreme conditions such as prolonged drought or high temperature, high genetic diversity is a significant advantage given the higher possibility of having genotypes/alleles that vary in their response to extremes8, enabling adaptation to environmental changes9. A species' capability to occupy new niches can also be affected by the loss of genetic diversity as well as damaging population growth and breeding12. A population's response to the environment  can additionally be examined using functional traits, traits that are thought to have a profound influence on plant fitness15. The plasticity of these traits under various environmental conditions is a fundamental mechanism that plants rely on to react to environmental changes. The convoluted relationship between genetic diversity, functional traits, and environmental heterogeneity of the locations at the population level has been the subject of quite a few studies17 and deserves more attention.Environmental factors play a key role in shaping patterns of genetic diversity and phenotypic variation in plant species18, which traditionally has been an essential ingredient of confectioneries19. In addition to conferring aromatic and flavor to formulations, it also is a thickening and stabilizing chemical agent20. Salep is integral to several medicines, beverages, and ice-creams21. Its critical rheological properties are due to the presence of polysaccharides, especially glucomannan 18, that is, natural fibers with high water solubility. These also have specific medicinal benefits such as normalizing blood sugar, inhibiting liver irregularities, and alleviating pancreas stress22. In recent years the harvest of tuberous orchids has increased due to high prices fueled by international demand, mainly from Iran's neighboring countries such as India, Turkey, and Pakistan23. Increasing public awareness of salep's benefits and growing demand is a dangerous combination that may jeopardize the long-term viability of natural populations of these orchids.Tuberous orchids have a rich history of traditional use and have suffered from overcollection. The dried tubers of specific wild terrestrial orchids are milled to make a flour known as 'salep'25. Agricultural expansion, urbanization, and silviculture have exacerbated the loss of genetic diversity in orchid populations27. Such problematic circumstances have already taken their toll on orchids in Iran, and some orchid species, including Orchis mascula L., have been classified as endangered26. Therefore, conservation strategies are needed to maintain the genetic diversity in the remaining populations and assure the species' long-term viability29. However, the absence of reliable information on the orchid's population status or the ecological consequences of severe over-collection prevents the implementation of robust conservation plans.Besides the ongoing over-exploitation of the wild population of tuberous orchids, climate change may also hasten the extinction of orchid species27. Recently, numerous small-scale regional studies on the population genetic status of tuberous orchids in the west of Iran have been reported32. However, the data required to develop conservation programs to protect genetic diversity or to develop the germplasm stocks in breeding programs can only be gained through sufficiently large-scale analyses, preferably at the landscape level34. O. mascula is a relatively small perennial orchid widely distributed across Europe, western Asia, and northern Africa. In Iran, this species is distributed primarily in the north (Alborz Mountains) and the west (the Zagros Mountains)36. It is one of several widely collected terrestrial orchids with numerous traditional medicinal applications. Overcollection of tubers has reduced O. mascula population sizes to the point that this species is now endangered; thus, fundamental large-scale studies of the functional traits and genetic diversity responses are needed. This study aimed to sample a high number of individuals from 18 populations of the terrestrial orchid O. mascula in northern Iran to address the following questions: (1) does phenotypic plasticity in functional traits exist in response to environmental heterogeneity? (2) how is genetic diversity and population structure affected by the land cover pattern and climatic variables? (3) do any population(s) have superior biochemical and phenotypic traits to be utilized in breeding programs?Although in Iran, as in other countries, legislators have enacted detailed regulations in favor of germplasm protection. Unfortunately, these regulations have not been enforced properly, and current measures are not limiting the collection or marketing of natural resources, particularly orchid tubersp ≤ 0.05 and p ≤ 0.01); the same applies to the population effect , Ziarat (ZIR), Azarshahr (AZS), and Maraveh Tapeh (MVT) populations overall had the highest values in general with the last population MVT, holds the most significant values. When it comes to floral traits, the variation across the populations is almost intangible Table . The MVTThe HCPC provides information on the factor map resulting from principal component analysis based on phenotypic and biochemical traits of the populations with a three-dimensional hierarchical tree, thereby providing a clearer view of the clustering pattern Fig. . DimensiTwo-way cluster analysis (heatmap) identified two main groups for phenotypic and biochemical traits Fig. , column.P < 0.01), the same applies to GLN  and weakly with longitude. BWF and BDW weakly and negatively correlated to land cover (r =  − 0.4), but there was no correlation between land cover and other phenotypic traits  and weakly with annual precipitation (r = 0.5). Also, it was negatively correlated with longitude (r =  − 0.4) and showed no correlation with annual mean temperature. However, the GLN content significantly decreased with the increase in geographical altitude, annual precipitation, and land cover , respectively. A seesaw correlation was unveiled between GLN and starch , as observed earlier in Table Correlation analyses revealed that the phenotypic traits negatively correlated with the annual precipitation and geographical region altitude and positively correlated with longitude. The important traits, PLH, number of flower (NF) and NLP responded positively to annual mean temperature , while AZS had the lowest value (1.65) among populations with an average of 1.89. The highest effective alleles (Ne = 1.64) were related to the TAS population, and the lowest Ne (1.21) belonged to the ZIR population with an average of 1.46 (Table P%) was 31.14%, while MVT from Golestan had the highest (65.63%), and PAJ was the lowest (18.75%). With an average of 0.35, the highest value of Shannon's information index (I) was observed in the JAD population (0.49), while MSL and PBD populations (0.25) had the lowest level. An average of 0.42 expected heterozygosity (He) and 0.30 for Nei's gene diversity index (H) were observed. The parameters of He and H were the highest in the KAH population , and the MSL population  showed the lowest. The average of the populations' total genetic diversity (Ht) was 0.37, and the genetic diversity within populations (Hs) was 0.24.At the population level, TAS, MSL, and CHP had the highest observed alleles (46 Table . Among tG'st) was 0.35, indicating that 35% of the genetic variation was among the populations, and 65% was within the populations. The G'st was higher than average, considering that G'st between 0.05 and 0.15 is defined as moderate and values over 0.30 as high37. Thus, G'st reflected the significant genetic differentiation among the populations. These findings were consistent with the relatively low gene flow value (Nm = 0.46) observed among populations  with a significant share of total genetic diversity (48%), whereas a larger proportion of variation (52%) existed within populations .The coefficient of genetic differentiation among populations  . The population Nei’s genetic diversity demonstrated a negative correlation to annual precipitation , and positively to longitude . This parameter also potently and negatively correlated to land cover , precipitation in June (r =  − 0.4) and geographical altitude (r =  − 0.5). The population expected heterozygosity also significantly correlated to annual precipitation , negatively correlated to geographical altitude (r =  − 0.5), precipitation in June (r =  − 0.3), land cover (r =  − 0.4), positively with longitude (r = 0.5), annual mean temperature (r = 0.4), mean temperature in May (r = 0.4) and mean temperature in June (r = 0.4)  Fig. , which s.4) Fig. .Figure 8In testing for isolation by distance (IBD), the relationship between geographical and genetic distance and geographic and phenotypic distance were insignificant Table . These rr2 > 0.40 at P < 0.01, where three of the markers revealed a simultaneous association with several important traits. For example, marker P-CCA + M-AGA-49 showed significant simultaneous associations with quantitative traits PLH, STL, NLP, LL, IL, NF, LLI, LMLL, BFW, and BDW on both MLM1 and MLM2 models  as opposed to the woodlands of Mazandaran and low altitudes of Guilan. The determinative effect of light was studied by41, who found that the overall growth rate was higher in coppiced than undisturbed woodland. Although plant species respond differently to shade, many usually do not succeed in flowering under deep shade42. In addition to light, higher nutrient availability in clear-cuts and pastures43 may have facilitated better growth performance.In this study, we observed a strong pattern of phenotypic response to environmental variables, revealing the importance of land cover type and precipitation. Orchis purpurea Huds.44, and in O. mascula and O. pauciflora Ten.45. The results of the current study were, to a large extent, consistent with these studies. In the comprehensive phenotypic study of Ebrahimi, et al.26. quantitative floral traits showed considerably higher variation than the results reported here; however, the number of flowers had the highest coefficient of variation (41.34%), similar to our results  showed an inverse relationship 22 were notably lower than our observation  at the population level with an average of 0.30 was observed to be higher than the previous studies on O. mascula; for example, Jacquemyn, et al.41 reported an average of 0.16 or Gholami, et al.31 0.10. However, the latter used ISSR markers with a small sample size (5). Strong geographical affinity seemed to be followed by the pattern of genetic diversity among populations, analogous to phenotypic variation, the level of genetic diversity was notably higher in the populations of Golestan, where KLH had the highest H (0.39) and other populations from this province similarly showed high H . Thus, the possibility of pollinators visiting more flowers available before leaving due to the food-deceiving nature of this species is higher; however, the gene flow (Nm) mean was low.In this study, we found that genetic variation among G'st: 0.35, Table 56. Low within-population genetic diversity (52%) and high differentiation (G'st: 0.35) in this study are contrary to previous results reported on O. mascula, showing a high genetic diversity within population (92%) and low G'st (0.125)41. Moreover, the woodland type did not affect genetic partitioning. This is in sharp contrast with the results of this study, where 20% percent of the total genetic diversity was partitioned among three groups of land covers . Differences in scale can explain these contrasting results. Whereas Jacquemyn, et al.57 studied 15 populations in the local landscape with slight variation in climatic conditions, in our study, populations were studied across a much larger scale leading to strong differences in climatic conditions between eastern and western populations and significant IBE. This suggests that populations have genetically adapted to the different precipitation regimes across Hyrcania and that this genetic adaptation has contributed to the observed high genetic differentiation. These results further support the findings of Siepielski, et al.58, who showed that precipitation was a dominant driver of natural selection in plant and animal populations for over 150 species.The genetic structuring of populations was strong in K = 2 and K = 4 Figs.  and 7a, O. mascula population sizes have been reduced beyond recovery in many areas in the west, northwest, and north of Iran. Developing a breeding program could be a sustainable option to counteract the further loss of populations. Selection of the most suitable populations and cultivation of local plant material could represent the first steps in a sustainable management of O. mascula populations in the wild. Our results showed that populations from Golestan possessed several important desirable traits, including a high GLN content and bulb dry weight that meets the high-quality threshold48. Another approach to probe for favorable traits is the percent of variation (%var) populations display59. These traits mentioned above also had the highest variation percentage under the effects of land cover and population  in which access to any wild population requires official permission. Dr. Gholizadeh identified the species identity of the samples, and voucher specimens from the populations were deposited in the University of Guilan Herbarium , available for botanical studies with an official request.In the spring of 2020, eighteen populations of on Table , followiA total of 198 individuals from 18 populations, with at least 20 m distance between individuals in each population, were collected from these populations. Nineteen quantitative phenotypic traits related to the size of the plants and flowers Table  were rechttps://www.esri.com/en-us/arcgis/products/arcgis-desktop/resources), a program used to calculate the areas and percentages of land use and land cover by each category.Young and fresh leaves were collected from each individual, immediately dried with silica gel, and transported to the laboratory for DNA extraction. For each population, the land cover was characterized by assessing the percentage of vegetation . Using Google Earth Pro®, high-resolution aerial images of the locations were acquired. The interpretation of the images was based on visual classification using the ArcGIS 10.4® software . The dried specimens were first cut into small pieces and then powdered into flour by milling. Further analysis was carried out to measure the components of interest in the flour.To produce salep powder from bulbs, a traditional method previously described by Tekinşen and Güner1, A2, A3; for starch ΔA, F) in a UV–Vis Spectrophotometer (Shimadzu – UV Mini 1240) at 340 nm (for GLN) and 510 nm (for starch) and using the following formula 1 and 2, respectively65:The samples were prepared according to procedures specified for GLN and starch using GLN (K-GLUM 10/04) and total starch  assay kits . The GLN and starch contents of the samples were determined by measurement of absorbance values of prepared blind and sample solutions , the pigment was first extracted with 0.5 mL methanol/0.1% HCl for 24 h in the dark. Then the absorbance was read at 510 nm using a UV–Vis spectrophotometer 67. The quality and quantity of extracted DNA were assayed by 1% agarose gel electrophoresis and NanoDrop® spectrophotometer and diluted with sterile distilled water to give a final 100 ng/μL. The method described by Vos, et al.68 was employed with slight modification for AFLP analysis. An amount of 250 ng genomic DNA (per sample) was homogenized with EcoRI and MseI primers without a selective base in pre-amplification and with three selective bases in amplification in 20 µL master mix . The obtained combination of genomic DNA and restriction enzymes were incubated for 12 h at 30 °C. Then double-stranded EcoR I (Eco) and Mse I (Mse) linkers were ligated to the restriction fragments without additional nucleotides and afterward amplified by primers with three additional nucleotides at the 3′-end (Table 69. The banding pattern of the amplified DNA fragments was turned into binary codes as presence (1) or absence (0), and only the consistently repeatable bands were scored. Weak or smeared bands were excluded, and the fragments of the same molecular weight were counted as the same locus.Genomic DNA was extracted from silica gel dried young leaves of the individuals utilizing CTAB procedureA principal component analysis (PCA) was performed on the phenotypic data. The first two principal components were retained as they explained the highest portion of the total variation and were treated like traits and subjected to further analysis. To determine how phenotypic variation is partitioned between different types of land covers (fixed effect), among populations (random effect), and among individuals (error), an unbalanced restricted maximum likelihood (REML) mixed-model analysis of variance (ANOVA) was conducted for the 22 measured traits and PC1 and PC2  using "Factoextra" and "FactoMineR" packages73. The same package was utilized to assess the relationship between genetic diversity indices and environmental factors .A correlation analysis between phenotypic traits was carried out using Pearson's correlation coefficients (P 0.01) using the "CorrPlot" packageNa), the effective number of alleles (Ne), expected heterozygosity (He), Shannon's diversity index (I), Nei's genetic diversity (H), total heterozygosity (Ht), and intra-population genetic diversity (Hs) were computed using POPGENE v3.074  was carried out using the "logisticPCA" package77 in the R software to visualize the spatial structure. Bayesian model-based cluster analysis using STRUCTURE v2.3.478 was also used to infer genetic structure and define the number of clusters in the data set. The correlated allele frequencies and admixed model were applied with 50,000 burn-in and 100,000 MCMC. The assumed number of groups (K) varied from 1 to 10, and 10 runs per K were performed. The STRUCTURE HARVESTER79 determined optimum K based on L(K) and ΔK. The structure result at K = 2 and K = 4 were mapped according to geographical locations using the ArcGIS 10.4® software (URL: https://www.esri.com/en-us/arcgis/products/arcgis-desktop/resources).A Cluster (heatmap) analysis was performed at the population level based on the matrix of Nei's unbiased genetic distance using "ggplot2"80 in the R software. Shannon differentiation coefficient (G'st) was calculated based on the following formula, G'st = (Hsp − Hpop)/Hsp , and the gene flow (Nm) among populations was estimated using POPGENE v3.074.To assess the magnitude of genetic differentiation, the partitioning of total genetic variation at different scales was computed with analysis of molecular variance (AMOVA) in "vegan"81 and phenotypic distance using the "dist" function in R software. Euclidean geographic distances were calculated using the "fossil" package82. For sampling sites, climatic variables were collected from WorldClim version 2.067. Bioclimatic variables (19) were considered as different environmental space vectors and used the Canberra distance to calculate the distance between populations in this vector space. For isolation by distance (IBD) and isolation by environment (IBE), the relationship of genetic and phenotypic distances with geographic and environmental conditions was analyzed, respectively, using simple and partial Mantel tests implemented in the R package "vegan"80. Additionally, all other possible comparisons  were tested.To test the effects of geographic distance and environmental differences on the genetic and phenotypic structure, the genetic distances among populations were calculated using Nei's genetic distance implemented in R package "Poppr"http://www.maizegenetics.net/bioinformat-ics) 83: a Mixed Linear Model (MLM) using the kinship matrix (K) estimated by the STRUCTURE HARVESTER79 as a random effect (MLM1) and an MLM using both Q and K (MLM2). Results were compared to determine a better model. Significance of associations between loci and traits was described as P-values (a probability level of 0.001).Two models were compared to test the marker-trait association between AFLP and phenotypic and chemotypic traits using TASSEL 4.0.1. (Supplementary Information.


Fenneropenaeus chinensis, is an important species in both aquaculture and fishery activities in China. To investigate inbreeding depression in natural populations, four Fenneropenaeus chinensis natural populations  were collected from the Bohai and Yellow seas. Microsatellite markers were used to evaluate individual inbreeding coefficients (F) of all samples. Furthermore, the effects of inbreeding on growth traits were investigated. The results showed marker-based F was continuous and ranged from 0 to 0.585, with an average of 0.191 ± 0.127, and there was no significant difference among the average F of the four populations. Regression analysis using the four populations showed inbreeding had a very significant (p < 0.01) effect on body weight. When analyzing a single population, regression coefficients were also all negative and those in Huanghua and in Qingdao were significant at the level of p < 0.05 and < 0.01, respectively. Inbreeding depressions, expressed as the percent change in body weight per 10% increase in F, were 2.75% in Huanghua, 2.22% in Qingdao, and 3.69% in all samples. This study provided a piece of rare evidence of inbreeding depression in natural populations and also guidance toward the conservation of wild Fenneropenaeus chinensis resources.Understanding inbreeding depressions (IBDs), the effect on the phenotypic performance of inbreeding, is of major importance for evolution and conservation genetics. Inbreeding depressions in aquatic animals were well documented in a domestic or captive population, while there is less evidence of inbreeding depression in natural populations. Chinese shrimp,  Inbreeding is defined as the mating of individuals that are related by ancestry and results in the reduction of heterozygosity . The inbF. chinensis, is an important species in both aquaculture and fishery activities in China. Over the past few decades, the natural population of F. chinensis was largely reliant on released shrimps to maintain its size, and their contribution to the total landings has been consistently > 90% , observed heterozygosity (Ho), expected heterozygosity (He), and polymorphism information content (PIC) at 11 genotyping microsatellite loci were obtained using CERVUS software . A fixatsoftware . There asoftware , and a tsoftware  was adopsoftware . The trisoftware  to obtaiy is the measured value for body weight; a is the y-intercept; b is the regression coefficient of F; and e is the residual term. The residual term in this model includes individual genetic effects, day-age effects, and error terms, all of which are unrelated to inbreeding. In particular, since the interaction effect between sex and F was not significant (p > 0.05), the analysis of sex effects in Huanghua and Qinhuangdao populations were also subsumed into the residual term.A linear-regression model was used to evaluate the effects of F on body weight:a and b are y-intercept from the regression of y on F and the regression coefficient of F, respectively (see Eq.  see Eq. .N was 25.18 ± 14.68. The p < 0.05). The marker-based F was continuous and ranged from 0 to 0.585, with an average of 0.191 ± 0.127. The average F of Qingdao and Haiyang were 0.180 and 0.185, respectively, lower than those of Qinhuangdao and Huanghua, 0.202 and 0.194, respectively  effect on body weight. When analyzing a single population, regression coefficients were also all negative and those in Huanghua and Qingdao were significant at the level of p < 0.05 and < 0.01, respectively. However, those in Qinhuangdao and Haiyang were not significantly different from zero due to their high standard errors. In particular, when regression analysis was based on four populations, the interaction effect between the population and F was not significant. For analyses with significant regression coefficients, estimates of IBD on body weight were further calculated. Those in Huanghua and Qingdao were 2.75% and 2.22%, respectively, and when all individuals were fitted, the estimate of IBD was 3.39% . This suggested that there was no or very little genetic differentiation among these populations, which was consistent with the expected average F. Most previous studies thought of F. chinensis distributed along northern China as one population and suggested a lack of genetic structure in this species . In addition, when the samples were divided into four populations according to where they were collected, regression analysis results in two of them supporting this conclusion. Insignificant regression coefficients in another two can likely be attributed to sampling errors which are associated with both a small sample size and a small number of microsatellite loci. Moreover, the interaction effect between the population and F was not significant, which also suggested that IBD should be universal in different populations. Similar to microsatellites, single nucleotide polymorphism (SNP) is another available molecular marker to estimate individual F  in a breeding population. Some research has indicated fast inbreeding  caused more harm than slow inbreeding  , there wbreeding . This viF. chinensis for the first time (to the best of our knowledge). Although a study has demonstrated the existence of IBD in F. chinensis breeding population (F. chinensis in China was largely reliant on released shrimps to maintain its size (F. chinensis natural population (F. chinensis.In this study, we demonstrated the phenomenon of IBD in a natural population of pulation , the finpulation , and IBDpulation . Over thits size . Due to pulation . Therefo


Plantago maxima Juss. ex Jacq. is a perennial species distributed along the Eurasian steppe. The westernmost range edge of the species’ distribution is located in the Pannonian basin, in Hungary where it is represented by a few, fragmented and highly endangered populations. We studied population diversity of all Hungarian range edge, natural populations, and one established ex situ population. One population from the centre of distribution (Kazakhstan) was implemented in the cpDNA haplotype study to compare the peripheral vs. central populations. We performed morphometric trait-based analysis, chromosome studies  and genetic diversity evaluations using inter simple sequence repeats (ISSR) and cpDNA trnL-trnF to evaluate differences between the in situ and ex situ populations as well as central vs. peripheral populations.The analysis of genetic diversity of protected plant species can greatly support conservation efforts. n = 2x = 12). The karyogram consists of 5 metacentric and 1 acrocentric chromosome pair. Plantago maxima has one 35S and two 5S rDNA loci, located on the acrocentric chromosome pair. Eight variable ISSR primers yielded 100 fragments, of which 74.6% were polymorphic (mean He = 0.220). A high level of genetic variation within population was observed (92%) while the genetic differentiation among the populations was only 8%. STRUCTURE analysis revealed that the largest Kunpeszér population separated from the rest of the Hungarian populations, indicating a high rate of admixture among the other ones. Based on the trnL-trnF sequence analysis the Hungarian populations represent a single haplotype, which can indicate a reduced diversity due to isolation and recent population decline. By contrast, Kazakh population represents a distinct haplotype compared to the Hungarian samples.Our results showed no obvious morphological differences among the in situ and ex situ populations in the period between 2018 and 2020. One ex situ subpopulation develops flowers three years in a row from 2019, which is a favourable indicator of the introduction success. Hungarian populations are exclusively diploids (2Plantago maxima populations from the westernmost range edge of the species’ distribution.The present study draws the attention to the high conservation value of the The online version contains supplementary material available at 10.1186/s40529-022-00365-6. It has been emphasized since long time that preserving genetic diversity of endangered species can significantly affect the long-term survival and evolution in changing environments Frankham . In situPlantago genus (Plantaginaceae) is 5–18.5 Million years old . Plantago maxima is the member of subg. Plantago and sect. Lamprosantha  for Plantago maxima which emphasizes the need to evaluate the chromosome number of the living Hungarian populations.Basic chromosome number of Magulaev . In contMagulaev  and RahnMagulaev  reportedPlantago genus  polymorphism requires no prior knowledge of the DNA sequence and evolve rapidly enough to exhibit variation, therefore ISSRs are universally applicable as dominant markers even for exploring new species  markers are widely used for taxonomic and phylogenetic comparisons  evaluate the possible haplotype differences using a cpDNA marker between a central vs edge populations.The goal of this study was to (1) evaluate the morphological trait variation of the Hungarian populations and the genetic representativeness of the ex situ stock; (2) give detailed information about the chromosome number and rDNA localisation; (3) assess the level of genetic diversity of both in situ and ex situ Hungarian P. maxima were included in the analysis from Hungary . Morphometric measurements were conducted between 2018 and 2020 at the time of main flowering. The seed and leaf sampling for chromosome and genetic diversity studies was carried out in 2019 and 2020. The four in situ populations are located in Molinia meadows  were measured with a measuring tape. The observed traits were as follows: biggest leaf length, biggest leaf width, number of leaves. The generative parameters were not assessed, since the first flowers appeared in the ex situ stock in 2019, but in statistically non-significant quantities. However, the flowering individuals were monitored during the years to evaluate the background factors necessary for the development of generative organs.Statistical analysis was carried out using IBM SPSS Statistics 27 software. Multivariate ANOVA (MANOVA) model was used to evaluate statistical differences between populations. Morphological traits as factors and years were treated as blocks in the model. Wilk’s lambda as unexplained variance rate was tested. Normality of the residuals was proved according to their skewness and kurtosis  and stored at 4 °C. Photographs were taken with an Olympus BX51 microscope, equipped with a highly sensitive digital camera (Olympus DP70).Seeds were collected from the four Hungarian in situ populations in 2019. Ploidy level from pooled seed samples, from 9 to 23 individuals per population were determined. Chromosome numbers were counted from mitotic metaphases from root tips using classical squash methods according to Mlinarec et al.  with somCucurbita pepo, cloned into pUC19 . The preparations were mounted in antifade buffer Vectashield  containing DAPI counterstain (2 ug ml−1) and stored at 4 °C until use. Signals were visualised and photographs captured with an Olympus BX51 microscope, equipped with a highly sensitive digital camera (Olympus DP70). Images were merged and contrasted using Adobe Photoshop 22.5.4. An average of 10 well-spread metaphases were analysed for each individual.Chromosome preparations for fluorescent in situ hybridization (FISH) were conducted according to Mlinarec et al. . BrieflyLeaves were collected and stored in silica gel until use. DNA was extracted with E.Z.N.A.® SP Plant DNA kit  following the manufacturer’s recommendations. DNA concentration and quality was assessed using NanoDrop .http://www.michaelsmith.ubc.ca).We performed 15.5 μL PCR reactions with 1 μL of genomic DNA, 10.72 μL of Milli-Q ultrapure water , 1.5 μL of (10x) DreamTaq Green PCR buffer , 0.3 μL of (10 mmol·L–1) dNTP mix , 0.5 μL of (2.5 mmol·L–1) MgCl2, 0.5–0.5 μL (10 mmol·L–1) of each primer, and 0.03 μL of DreamTaq Green DNA polymerase , 0.15 μL BSA (1%) (ThermoFisher) and 0.3 μL DMSO (2%). ISSR primers . The PCR products were applied on a 1.5% (w/v) ethidium bromide stained agarose gel in 1xTBE buffer. PCR products were separated for 60–150 min at 110 V. Size comparison with external standards (Thermo Scientific GeneRuler 100 bp Plus DNA Ladder) was evaluated using GelAnalyzer 19.1 software  clustering was employed using unweighted pair group method with arithmetic averages (UPGMA) method. Cluster analysis based on Nei’s genetic distances calculated in GenAlex was also carried out using the (UPGMA) method. Dendrograms were plotted using NTSYS-pc 2.10 software Rohlf . InterpoPlantago L. (Plantaginaceae) , with significant intercept , and significant difference between the studied years . In case of the biggest leaf length  = 36.32;p < 0.001) and biggest leaf width  = 45,10;p < 0.001) there were significant differences between the populations  and (Kunpeszér). At least five metaphases were investigated per individual. One 35S and two 5S rDNA loci were positioned on the acrocentric chromosome 6. 35S rDNA was located subterminally on the short arm, while one 5S rDNA loci were located proximally to the centromere and the other subterminally on the long arm. 35S rDNA locus was heteromorphic. 5S rDNA locus positioned proximally to the centromere was at the edge of visibility. The mitotic metaphase, FISH and idiogram is presented in Figs. The karyotype formula of The amplification of the ISSR fragments in the 100 individuals, analysed with eight primers, found variable after a primary test, yielded 100 unambiguous and reproducible electrophoretic bands , while this value was the highest in Kunpeszér (He = 0.257). The values of Shannon’s information index reflect similar results, the lowest in ex situ collection, the highest 0.396 in Kunpeszér population . Between the Hungarian and Kazakhstan samples the difference occurred in two positions: mononucleotide (A) microsatellite in 226–236 bp position, and also a mononucleotide (T) microsatellite in 589–597 bp region. The Kazakhstan population comprised two different haplotypes (H2-H3) based on the two microsatellite regions length. The Plantago maxima sample from NCBI (of unknown origin) represented another haplotype with two SNP in the 38 and 247 bp positions (H4).The total length of the pes Fig. . H5-H8 hPlantago maxima is one of the remaining relict steppe plants in the Hungarian flora. The present study is the first to examine the genetic structure and population variability of in situ and established ex situ giant plantain populations. To plan future conservation actions, it is important to analyse the current state of the populations and to compare the naturally occurring diversity to the established ex situ collection.The Pannonian Basin holds a great botanical value providing primary and secondary refugia for European steppe species  as Soó  as Soó  reportedPlantago maxima reaches a relatively high genetic diversity , in comparison to other endangered and endemic Plantago species where He = 0.1965–0.2309 was reported using ISSR markers  C. Lewis and Zona  was similar in the ex situ collection and wild populations. Also a great proportion of genetic variability was preserved in the ex situ population of Sinocalycanthus chinensis W. C. Cheng and S. Y. Chang  -trnF(GAA) region was formerly successfully used in a molecular phylogenetic study of Poa badensis  from the ex situ population (n = 92), therefore introduction of new individuals is required to strengthen the botanical garden population. Kunpeszér population bears the most elevated genetic diversity, therefore the establishment of new ex situ collection with propagules originating from that population is highly recommended. The lower number of individuals in Táborfalva and Tatárszentgyörgy preserving similar genetic diversity in these two localities also requires the need of establishing new ex situ gene stocks.Additional file 1: Fig. S1. ISSR banding profile of Plantago maxima individuals with UBC 808 primer. Fig. S2. ISSR banding profile of Plantago maxima individuals with UBC 818 primer. Fig. S3. Cluster analysis among the individuals of Plantago maxima based on ISSR molecular marker.


Ne) using whole genomes make it feasible to examine how temporal dynamics influence diversity across populations. We investigate Ne variation and climate‐associated range shifts to examine the origins of a previously observed latitudinal heterozygosity gradient in the bumble bee Bombus vancouverensis Cresson (Hymenoptera: Apidae: Bombus Latreille) in western North America. We analyze whole genomes from a latitude‐elevation cline using sequentially Markovian coalescent models of Ne through time to test whether relatively low diversity in southern high‐elevation populations is a result of long‐term differences in Ne. We use Maxent models of the species range over the last 130,000 years to evaluate range shifts and stability. Ne fluctuates with climate across populations, but more genetically diverse northern populations have maintained greater Ne over the late Pleistocene and experienced larger expansions with climatically favorable time periods. Northern populations also experienced larger bottlenecks during the last glacial period, which matched the loss of range area near these sites; however, bottlenecks were not sufficient to erode diversity maintained during periods of large Ne. A genome sampled from an island population indicated a severe postglacial bottleneck, indicating that large recent postglacial declines are detectable if they have occurred. Genetic diversity was not related to niche stability or glacial‐period bottleneck size. Instead, spatial expansions and increased connectivity during favorable climates likely maintain diversity in the north while restriction to high elevations maintains relatively low diversity despite greater stability in southern regions. Results suggest genetic diversity gradients reflect long‐term differences in Ne dynamics and also emphasize the unique effects of isolation on insular habitats for bumble bees. Patterns are discussed in the context of conservation under climate change.Understanding historical range shifts and population size variation provides an important context for interpreting contemporary genetic diversity. Methods to predict changes in species distributions and model changes in effective population size ( We use whole genome demographic modeling to demonstrate that existing patterns of genetic diversity across latitudes in a montane bumble bee are shaped by historical differences in population size dynamics. High‐diversity populations have a history of larger effective population sizes through time, even with larger losses in habitat suitability during glacial periods resulting in larger Pleistocene bottlenecks. Results suggest that southern high‐elevation restricted populations have a reduced maximum population size through time than northern low‐elevation populations despite greater niche stability, which has implications for conserving genetic diversity in these more isolated regions under climate change. The impact of Quaternary climate variation on genetic diversity is well‐recognized , SMC models are a powerful tool for examining how climate fluctuations may influence genetic variation  are abundant insects in terrestrial biomes, where they provide important pollination services  but exhibit a clear latitude‐altitude gradient in genetic diversity, with diversity lower in southern high‐elevation populations while northern populations appear more well‐connected with higher heterozygosity  If current genetic diversity differences observed among populations can be explained by long‐term Quaternary Ne differences, Ne in less diverse southern populations will be persistently smaller than in northern populations through time. (2) If more recent Ne declines can be detected with SMC models, insular genome data should demonstrate strong contemporary bottlenecks. (3) If bumble bee populations can rapidly respond to expansions and contractions of the geographic range, temporal fluctuations in population size in individual genomes should be mirrored by variation in the spatial extent of the species' niche.In this study, we take advantage of existing whole genome data from prior studies  and mapped to the B. vancouverensis genome (NCBI RefSeq GCF_011952275.1) with bwa‐mem v0.7.15‐r1140  was used to remove PCR duplicates and sort and index BAM files. We created a BED file to only retain positions on large scaffolds , which can be beneficial for SMC methods  followed by ‘makeMappabilityMask.py’ from msmc tools (https://github.com/stschiff/msmc‐tools/blob/master/README.md) . We used the SFS for all samples in each population group for diversity calculations, but for plotting single population SFS, we also performed calculations for nine randomly selected individuals per region so that the SFS would have the same bin sizes. We determined the two estimates of θ  and Tajima's D for each scaffold  from each sample . For each region pair, we also estimated the folded 2D SFS and weighted FST (not performed for San Juan Island genome with only one sample). Visualization of population structure was performed using PCA with pcangsd v1.03  < 20, within 10 bp of an insertion–deletion, and below or above coverage thresholds  as missing data. Samples were sequenced to similar coverage so we specified a minimum depth of d = 10 and a maximum depth of D = 50 .The Pairwise Sequentially Markovian Coalescent (PSMC) model  with the ‐R option and plotted using geom_step of ‘ggplot2’ 3.3.5 . Data files were prepared with minor modifications of scripts from J Rick (https://github.com/jessicarick/msmc2_scripts). MSMC2 also required the mappability mask. Separate VCF files for each scaffold and individual were created using samtools and bcftools as above and excluding sites with <10x coverage. Mask BED files representing sequenced sites for each sample and scaffold required for MSMC2 were created using ‘bamcaller.py’ in the MSMC tools repository. Input files for populations were prepared using VCFs, individual masks, and the masked reference genome using “generate_multihetsep.py” in MSMC tools. MSMC2 was run setting the ‐I parameter to only evaluate coalescence between haplotypes within individuals for each population . Such spikes are a typical sign of model overfitting , using records from the USDA‐ARS Pollinating Insect‐Biology, Management, Systematics Research Collection, the National Museum of Natural History Collection, the Smithsonian Institution Collection, the Canadian Biodiversity Information Facility Collection, the Illinois Natural History Survey Collection, and the Yale University Peabody Museum Collection  and 20% testing (N = 97) points. Although the data remain more abundant in the contiguous USA and southern Canada, filtered occurrence records were much more even and largely represent the known distribution of the species  and determining the 33.12 = 261.7, d.f. = 2, p < .001; delta‐AIC = −257.7), being highest in northern populations (θπ = 0.0032 per site), intermediate in the mid‐range region (θπ = 0.0030 per site) and lowest in southern populations (θπ = 0.0029 per site), consistent with previous observations  coverage genomes, suggesting that populations across the Sierra‐Cascades region have been affected by similar species‐wide events Figure . Nonethep Figure . Followis Figure .Inferences for the high coverage genomes from northern and southern regions were essentially the same as moderate coverage samples Figure , suggest3.2.2Ne and late Pleistocene bottleneck size were larger in northern populations, with the magnitudes of both declining toward the south. Population Ne trajectories generally matched the individual‐genome PSMC plots, both for estimates of Ne and the timing of size changes , and extending north through western Canada and into Alaska. At the LGM, the predicted range contracted everywhere but with most of the northern range becoming unsuitable owing to glacial advance and populations largely restricted to south of present‐day Canada. Exceptions for our northern region include some Oregon locations and regions around Vancouver, BC that maintained a moderate degree of environmental suitability but were isolated. In montane areas, populations would have occupied lower elevations. Mid‐range populations  were largely stable, and although the highest elevation southern region sites would have been unsuitable, extensive areas with highly suitable climates would have been available in adjacent lower elevations throughout California. LIG estimates were similar to the present day, consistent with the period of Ne expansion.The final Maxent model had a training and test AUC = 0.86 Figure , and botIn contrast to expectations for a stability‐diversity hypothesis, heterozygosity was inversely related to the difference in raster suitability between time points Figure , suggest4B. vancouverensis. Our objective was to better understand north‐to‐south gradients in genetic diversity throughout the Sierra‐Cascade Mountain region of the western USA and to test whether such differences may be explained by long‐term evolutionary processes or might require a more recent demographic explanation. Analyses suggest that all populations experienced fluctuations in Ne alongside major range shifts. However, variation in genetic diversity can be explained by divergent population histories impacting the degree of these fluctuations, with more diverse contemporary populations exhibiting signatures of elevated Ne throughout the late Pleistocene, particularly during periods of expansion when the species range was more continuous.Combining temporal coalescent analyses from modest genome resequencing with paleo‐SDMs proved useful for understanding variation in contemporary genetic diversity in B. vancouverensis populations fluctuate at the same temporal scales throughout the latitude‐altitude cline examined in this study. It is unsurprising that Quaternary climate dynamics drive concurrent demographic and genetic dynamics , while southern populations remain relatively isolated at high elevations even during favorable periods. Although most populations occupy areas that would have been unsuitable during the LGM, southern populations would have had less genetic variation to lose at this time owing to the weaker interglacial expansion. Furthermore, the southern B. vancouverensis range did not suffer a regional collapse of suitable habitats at the LGM; high‐elevation populations would have only had to shift to nearby lower elevations via short distance dispersal to stay within their niche, contributing to greater stability and more tempered bottleneck signatures. By contrast, the northern range largely disappeared due to the presence of ice sheets  processes. The correlations with predicted habitable area from SDMs also provide support for the reliability of the SMC models; however, because we expect changes in population structure over time, some inferred differences could be due to migration rates rather than a simple reflection of the numbers of individuals alone. Another potential concern is that, while temporal SDMs have been commonly used for understanding patterns of genetic diversity, these models assume that a species niche is conserved through time, with no temporal adaptation to changing conditions that might allow persistence in regions with low predicted suitability based on contemporary occurrences. Local adaptation may be present across the B. vancouverensis range ; data curation (lead); formal analysis ; funding acquisition ; investigation ; methodology ; project administration (lead); visualization (lead); writing – original draft (lead). Jamie Strange: Funding acquisition ; investigation ; resources ; writing – review and editing . Sam D Heraghty: Data curation ; formal analysis ; investigation ; writing – review and editing .US National Science Foundation DEB 1457645, URoL 1921585.Neither I nor my co‐authors have a conflict of interest. No materials should require permission to reproduce.Appendix S1Click here for additional data file.


Sander lucioperca  is an important fish species in the development of European aquaculture. The aquaculture of a fish species can be facilitated by knowing the genetic variability within and among populations. Here, we assessed the genetic background of 8 wild populations along with 13 broodstocks  of pikeperch through a combination of genetic markers. We underlined that current broodstocks have a genetic diversity similar to wild populations. When focusing on genetic differentiation, we highlight that European pikeperch populations are divided into two groups: one predominantly present in Northern Europe and around the Baltic Sea and another mainly in Central Europe. Broodstocks appear to contain fish of a single origin with only a few exceptions. Ultimately, we have proposed baseline information about genetic diversity of pikeperch along with a genetic tool that can help pikeperch producers manage and improve their fish stock.The pikeperch b gene sequences were also used to infer phylogeographic relationships. Results show that on average, the domesticated populations do not exhibit significantly lower levels of genetic diversity compared to the wild ones and do not suffer from inbreeding. Nuclear data provide evidence that pikeperch populations in Europe belong to at least two genetically differentiated groups: the first one is predominantly present in Northern Europe and around the Baltic Sea, while the second one comprises populations from Central Europe. In this second group, Hungarian origin populations constitute a differentiated stock that needs special consideration. Aquaculture broodstocks analyzed appear to contain fish of a single origin with only a few exceptions.The pikeperch is a freshwater/brackish water fish species with growing interest for European aquaculture. Wild populations show signs of decline in many areas of the species natural range due to human activities. The comparative evaluation of genetic status in wild and domesticated populations is extremely useful for the future establishment of genetic breeding programs. The main objective of the present study was to assess and compare the genetic variability of 13 domesticated populations from commercial farms and 8 wild populations, developing an efficient microsatellite multiplex tool for genotyping. Partial cytochrome  Sander lucioperca) is a temperate predatory freshwater fish species that tolerates brackish waters )For the studied domesticated pikeperch populations, our results show that current fish stocks do not seem to suffer from inbreeding nor strong genetic diversity loss. This means that (i) current broodstock management methods seem to allow maintaining genetic diversity within stocks, and (ii) current domesticated fish stocks could provide a solid basis for future selective breeding programs.The present overview of genetic background in wild and domesticated pikeperch populations could be further supported by complementary analyses, including higher sample sizes and stocks originating from a broader distribution area as well as a higher number of genetic markers (such as SNPs) coming from whole genome or reduced representation sequencing methodologies.Current results already provide valuable insight for fish farmers. First, knowing genetic divergences between domesticated fish stocks paves the way (i) to develop accurate pedigree assignment  and . Nevertheless, this can result in outbreeding depression . IndeedThird, considering genetic divergences between wild and domesticated populations can potentially improve the prevention of environmental risk near farming locations. Special attention should be paid to the risk of escapees in areas where farmed stocks and wild neighboring populations are genetically distinct to avoid nuisances observed in other fish species e.g., ,60). Wit. Wit60])


DMY gene is deleted in all males of the Sanya population (SY-medaka) of the Hainan medaka, Oryzias curvinotus, as recently reported by us. However, due to limited knowledge regarding their population genetic background, it is difficult to explore the possible evolutionary pathway. Herein, we resequenced the whole genome of four populations, including SY-medaka. A total of 56 mitogenomes and 32,826,105 SNPs were identified. We found that the genetic differentiation is highest between SY-medaka and the other populations. The results of the population history of the O. curvinotus suggest that the SY-medaka has been in a bottleneck period recently. Further analysis shows that SY-medaka are the most strongly affected by environmental selection. Moreover, we screened some potential genomic regions, and the genes contained in these regions may explain the potential mechanism of the selection process of the SY-medaka. In conclusion, our study can provide new clues for the adaptation process of medaka in the new environment of Sanya.The  Oryzias has always been a species of interest for genetics and evolutionary biology, and research on the genetic diversity of Oryzias has been reported continuously. In terms of morphology and physiology, when the medaka was first studied, it was found that there were significant differences in the average number of anal fins of Oryzias latipes in different regions   . In addi-medaka)  (Supplem-medaka) . Obviousferences . Thus, iPrevious studies of genetic diversity and population structure have often been based on mitochondrial genome (mtDNA) . But mtDO. curvinotus from four geographic groups at different latitudes with the aim of obtaining a high density of SNPs and also analyzed the population structure and evolutionary history of O. curvinotus at the nuclear gene level.Here, we performed a batch of whole-genome re-sequencing of the wild All the 56 wild individuals sequenced in this study were collected from four geographical locations  along thAfter collection of each population, all the samples were euthanized and stored in absolute ethanol. Genomic DNA was extracted using the phenol-chloroform method. The fragment size of the sequencing library was 350 bp, and sequencing was conducted on the Illumina HiSeq Xten platform using terminal pairing (PE) 150 bp reads. The sequencing depth of each sample was ×10. The resequencing raw data of all samples in this study have been uploaded to the NCBI Sequence Reading Archive (SRA) with storage number SRR17331594-SRR17331649.O. curvinotus, and GATK4 (MarkDuplicates) software was used to remove PCR duplication  was used to compare sequencing results to the reference genome  of lication .The standard process of GATK4 software was used to identify variation and extract SNPs. High quality SNP data was filtered using GATK4  and VCFtools  .O. curvinotus genome using the package ANNOVAR (Gene-based SNP annotation was performed according to the annotation of the  ANNOVAR . Based ohttps://github.com/BGI-shenzhen/VCF2Dis) and the NJ-tree was built with 1000 bootstrap by PHYLIP 3.69 (http://evolution.genetics.washington.edu/phylip.html).The P-distance matrix was calculated based on the all SNPs data by VCF2Dis  values between any two SNPs within 100 kb were computed by using the software PopLDdecay  of each population, the squared correlation coefficient  and maximum θπ ratio  were identified as selected regions. We used clusterProfiler to preform KEGG enrichment analysis on the candidate selective gene located in selected regions.O. curvinotus from the four geographical groups are abbreviated as ST-medaka, JWM-medaka, GQ-medaka, and SY-medaka. Genome mapping of all individuals across the O. curvinotus’s 800 Mb genome resulted in an average of 98.21% sequencing coverage  that was different from the NJ tree based on SNPs, all other individuals formed clusters with other local individuals. This demonstrates that the genetic structure analysis based on SNPs is reliable. In addition, the result of ML tree indicate that O. curvinotus first differentiated into two branche. One branch includes ST-medaka and JWM-medaka, and the other branch includes GQ-medaka and SY-medaka.The VCF2Dis software was used to calculate the genetic distance between all individuals, and the neighbor-joining (NJ) method was used to construct the evolutionary tree. The unrooted tree  demonstrThe first three principal components, namely first principal component (PC1), second principal component (PC2), third principal component (PC3) were extracted and plotted. In particular, the PC1 (variance explained = 48.44%) separated the SY-medaka from the other populations, the PC2 (variance explained = 11.93%) separated the GQ-medaka from the other populations . The PC3O. curvinotus are similar, and some poor results only appear for more extreme parameter values. Taking the estimate of g = 0.67, μ = 2.5 × 10−8 as an example, the effective population sizes of all subgroups of medaka were basically the same until about 100,000 years ago, when the common ancestor of them had not yet diverged. Therefore, the effective population sizes dating back to this period are basically the same. During the period from about 40,000 to 100,000 years ago, the population size changes showed two trends, one is an upward trend containing SY-medaka and GQ-medaka, and the other is a downward trend containing JWM-medaka and ST-medaka, suggesting that two branches of the medaka diverged during this period. While the trends in effective population sizes of WM-medaka and ST-medaka remained consistent and the population sizes were very similar from approximately 10,000 to 40,000 years ago. In contrast, the trends of the other two populations were clearly distinguished, with GQ-medaka showing an upward and then a downward trend, while SY-medaka showed a sharp decline. All four populations of the O. curvinotus live along the coast of the South China Sea, but the demographic changes of several of them are very different, which may be due to the different effects of geological events and climatic environmental changes on them.In O. curvinotus population is very fast and stabilized at an attenuation distance of about 20 kb. In addition, the decay rates of the ST-medaka, JWM-medaka, and GQ-medaka are similar, but significantly lower than that of SY-medaka, indicating that SY-medaka may be under stronger selection pressure. It is worth noting that the LD value of the final convergence of the four groups is correlated with the latitude. At lower latitudes, the stable LD value of each group was higher.The LD attenuation diagram  shows thFST were calculated between the populations , 65 genes were obtained for JWM-SY, 303 genes were obtained for GQ-SY, and 39 candidate genes were obtained by taking the intersection of the three groups of genes . The same situation exists in O. latipes  arose in 27.3 Mya and that O. latipes formed in 9.2 Mya, on the basis of which the species formation and population divergence of the O. curvinotus would have occurred within 9.2 million years , Gaoqiao, and Sanya within 1 year (“Weather O. curvinotus occurs.In terms of possible environmental factors acting as selective pressures on SY-medaka, we only considered temperature and ultraviolet light. Because “Weather .. While “Weather , especiaO. curvinotus have adapted to the new environment of Sanya. Among them, the circadian rhythm pathway may provide possible clues for environmental stress factors. In mammals, heat stress can regulate circadian rhythm through a series of physiological reactions (O. curvinotus also responds to environmental changes through these mechanisms. Other genes may also be related to some potential mechanisms. For example, we found that many genes are closely related to cell growth and death. The expression of stk3, ing3, trit1, and laptm4b may inhibit cell growth and induce apoptosis (cep192 and bub1b may play an important role in mitosis (39 selected genes of SY-medaka participate in multiple biological processes, which may indicate the potential processes through which eactions , to makeeactions . Of courpoptosis , and cep mitosis . FurtherAlthough many genes lack annotation information and related pathway research, we believe that positive selection of these genes is very important for the adaptation of SY-medaka to the new environment. Furthermore, these genes provide us with an important research basis and research direction. We also note that the genome mapping rate of SY-medaka is about 1% lower than that of other populations, suggesting that more genes are selected against and not found in SY-medaka. It is difficult to identify these genes through the GQ-medaka genome. Therefore, we have started an SY-medaka genome project at the time of writing this paper, which is expected to further explore the environmental adaptation process of SY-medaka.O. curvinotus individuals from four geographic locations, identified 32,826,105 SNPs. The population genetic structure analysis showed that the O. curvinotus can be divided into three subgroups. Among them, ST-medaka and JWM-medaka were the most closely related, while the SY-medaka was the most divergent from the other populations. The results of the population history of the O. curvinotus suggest that the SY-medaka has been in a bottleneck period recently and a variety of evidence shows that them were subjected to continuous strong selection pressure. By selective screening, we also identified some potential gene regions that were subject to significant positive selection in the SY-medaka. Candidate genes located in these regions may play important roles in biological processes such as circadian rhythm and cell cycle regulation, which may suggest potential adaptive processes that occurred in the new Sanya environment. Our study provides a genetic basis for the study of environmental adaptation of medaka.In this study, we performed WGS on 56


Bactrocera oleae is the major pest of olive orchards. The damages it causes become in considerable economic losses as well as a decrease in oil quantity and quality. A key question for the success of pest control strategies is the further knowledge about the species, and genetic data becomes essential for this purpose. The present work analyses more than 250 fruit flies from six different Mediterranean countries, showing relevant data about the genetic structure and gene flow of this damaging pest. These findings are helpful to improve the integrated pest management strategies according to the current European Guidelines.The output of olive industry at the Mediterranean Basin, headed by Spain, is huge worldwide. The olive fruit fly Bactrocera oleae, the main pest of olive crops. To improve its control, one of the challenges is the further knowledge of the species and populations dynamics in this area. A phylogeographic work is necessary to further characterise the levels and distribution patterns of genetic diversity of the Spanish populations and their genetic relationships with other Mediterranean populations. A 1151 bp fragment of the mitochondrial cytochrome oxidase subunit I (COI) gene has been analysed in over 250 specimens of the six main Mediterranean countries via sequencing. Genetic diversity parameters were high; 51 new haplotypes have been identified showing a geographical pattern across the Mediterranean area. The data revealed that olive fruit fly populations have been long time established in the Mediterranean Basin with two genetic groups. Gene flow seems to be the main process in shaping this genetic structure as well as fly’s colonisation routes that have paralleled those of the olive tree.Spain is the leading producer of olives and olive oil. Ninety-five percent of world production originate from Spain and other regions of the Mediterranean Basin. However, these olive-growing countries face a major problem, the harmful fly  Olea europaea, (Linnaeus 1753), is an iconic species of the Mediterranean Basin, where it covers more than nine million hectares of groves [The olive tree, Linnaeus 753, is af groves . Concernolf) Bactrocera oleae  is the major damaging pest of the Olea genus. Crop losses are due primarily to larvae feeding habits, which seriously reduces the fruits’ oil content and the organoleptic properties and causes the premature fruit drop. This tephritid is widespread throughout the Mediterranean Basin and the Middle East. It is also found in the South and East of Africa, India, and Pakistan; since the end of the 20th century, the olive fly has been also reported in California and Mexico and very recently (2019) in Hawaii [Besides the economic magnitude, the olive cultivation also has social, territorial and environmental repercussions. Its environmental role is significant, mainly in terms of biodiversity, carbon sequestration, soil conservation and the generation of valuable agricultural landscapes at the current scenario of global change, which benefits pest species from increasing their growth rates . The olin Hawaii . B. oleae populations need to be effectively controlled. The national and European competent institutions  request integrated control strategies that limit the use of phytosanitary products. For this challenge, a better understanding of the species is essential [The ssential , with geolf populations yielded conflicting results on the genetic structure of Mediterranean olf populations. The debate over the number of genetic (sub)groups and the relationships among them was affected by the heterogeneity of sample sizes and the genetic markers used. Results vary from no evidence of genetic differentiation among populations , a tendfragment ,12 and ifragment ) to two fragment ,15,16). B. oleae populations has been limited, since those surveys included few individuals from very restricted locations, despite the large area under olive cultivation in the country. Then, the first objective of this article was to increase the phylogeographic knowledge of this harmful pest in Spain and, secondly, its genetic relationships with other populations. Then, samples from other Mediterranean countries were included as external references to get a comprehensive scene of the genetic structure, demographic history and colonisation routes in this region. This survey has represented the most exhaustive study so far, with representative populations of the entire Spanish geography using the cytochrome oxidase subunit I (COI) mitochondrial marker. This gene is the universal barcode for animal species identification, but its utility goes far beyond taxonomy, given the high informative genetic variation it holds at the intraspecific level [Notwithstanding, the characterisation of the Spanish ic level .Olive flies were sampled from 15 Spanish populations across the whole productive area . When poIndividual genomic DNA was extracted using the DNeasy Blood and Tissue Kit  following the instructions given by the manufacturer with minor modifications. The concentration and purity of the DNA were estimated both electrophoretically and by absorbance at 260–280 nm with a NanoDrop ND-100 . COI was amplified with the C/N 2769 and LCO 1490 primers at a final volume of 12.5 µL [4, 200 µM of each dNTP, 0.65 µM of each primer and 1 U of the high-fidelity Vent® DNA polymerase . The PCR program included an initial denaturation step at 94 °C for 5 min, followed by 35 cycles of 94 °C for 45 s, 60 °C for 1 min and 72 °C for 1 min, plus a final extension step of 72 °C for 10 min. All reactions were performed in a LabCycler apparatus .A 1.75 Kbp fragment of the mitochondrial gene  12.5 µL ,19. PCR TM  . Both strands were sequenced using the BIG Dye® Terminator Cycle Sequencing Ready Reaction Kit  in a 3730 DNA Analyser  at the Genomics Unit of the University Complutense of Madrid, with the same primers employed for amplification. The amplicons were firstly checked on 0.8% agarose gels with TBE buffer , stained with ethidium bromide (0.5 mg/mL). Then, they were purified in a single-step enzymatic clean-up method with enzymes exonuclease I (Exo I) and FastAPCOI sequences were aligned with CLUSTAL W software and edited with BioEdit v.7.0.9.0 [COI sequences were trimmed to a final length of 1151 bp and deposited in GenBank under accession numbers of KC005742-KC005762, KP704354-KP704404 and JX073648.The .7.0.9.0 ,21. To c.7.0.9.0 ,23. AfteHd), the nucleotide diversity (π) and the number of segregating sites (S) were calculated with DNAsp v 5.10.01 program [B. oleae populations, the network was repeated discarding the exclusive haplotypes and incorporating olf COI sequences from GenBank with accession codes GU108459-GU108479 and HQ677155-HQ677162 and Bactrocera dorsalis  DQ845759 as an outgroup [The parameters of the haplotype diversity  [STF ≈ 1/(1 + 2Nm) [To infer the genetic differentiation between populations, Frrection ,31,32. Ts (PCoA) , employis (PCoA) . Gene fl1 + 2Nm) . The analysis of molecular variance (AMOVA) was performed to check the hierarchy of the genetic structure detected at the Mediterranean Basin, using Arlequin software with 10,000 permutations. Additionally, to explore patterns of genetic divergence in more detail, the spatial analysis of molecular variance (SAMOVA) was also applied defining homogeneous groups and maximising the proportion of genetic variance due to differences among groups of populations . These a2 and Tajima’s D neutrality tests were performed with the genetic groups revealed by PCoA and SAMOVA [ut where τ is estimated via mismatch distribution analysis and expressed in units of evolutionary time, t is the time in generations, and u = µk, in which µ is the substitution rate in the specific DNA region, and k the length of the analysed sequence. A generation time of 0.25 years and a substitution rate of 2.3% of divergence per million years for the COI gene were applied [Finally, Fu’s Fs, Rd SAMOVA ,38,39. Sd SAMOVA ,40. Mismd SAMOVA . The sumd SAMOVA . Evoluti applied ,42.B. oleae in Spain has revealed a high diversity at the mitochondrial level. The 1151 bp sequence analysis detected 45 polymorphic sites that defined 49 haplotypes, of which 34 were exclusive, i.e., they were present in a single fly. These haplotypes were separated by 1–3 SNPs (Single Nucleotide Polymorphism), being the most frequent H1 followed by H2 and H15. In the Spanish populations as a whole, the haplotype diversity (Hd) resulted 0.864, and the nucleotide diversity (π) was 0.00151 , and π varied from 0.00176 to 0.00112 (SPA4−SPA7). The SPA5 sample showed very low values in relation to the range found (Hd = 0.644 and π = 0.00089). The study of 15 samples representative of the distribution range of  0.00151 . FocusinCOI haplotypes were found, differing at 1–9 mutational steps, six encompassed 63.63% of the analysed flies, with not only H1, H2, H17 and H15 being the most common as in Spain, but also H21 and H22. The remaining were at very low frequencies, and some were population-specific. Overall, B. oleae showed a Hd value of 0.908 and a π value of 0.00259 , of which 26 were parsimony informative. Although 73  0.00259 , althougolf populations from Spain, Portugal and Tunisia (TUN1 and TUN3) and also Greece (GRE1 and GRE2). H22 (6.2%) was detected in all eastern olf populations, and H21 (6.5%) was detected in Israel, Greece (GRE1) and two populations from Tunisia (TUN1 and TUN2). The most widely distributed haplotype was H17 (13%), found in some Spanish populations and in all ones from Tunisia, Italy and Greece.This haplotype diversity was represented on a map with pie charts on the coordinates of each population sampled . The hapSTF estimates were practically null when comparing the 15 olf samples, with 0.101 being the highest one population. When comparing the Spanish populations with the others . Regarding the genetic structure of this pest in Spain, the hest one . These l outcome . Spanishe others , low FSTp < 0.0001; STF = 0.649)), whereas the gene flows among population within the two groups Nm were 3.14 and 3.51, respectively. The PCoA provided a two-dimensional graphical view of the relationships among these populations . The X-aThe MJ networks exhibited a star-shape topology with the three haplotypes , from which the other ones were radiated and detected . From thCOI sequences and removing exclusive haplotypes. The network  and Greek (H2 and H17) COI sequences, besides the Algerian (H2) and Moroccan ones (H74), joined the Mediterranean group I. The sequences from Turkish and Israeli flies  were integrated in haplogroup II. The USA flies also carried the haplotype H22, typical of this haplogroup. The haplotype H76 was characteristic of flies from Kenya, South Africa and Turkey; it was placed at one SNP apart from the Mediterranean and the widely distributed H17. Finally, a different haplogroup comprised the COI sequences from Pakistani flies with haplotypes H77, H78 and H79, separated by four or five polymorphisms from the Middle East and African haplotype H76. B. dorsalis rooted this network, allowing designating the Pakistani haplotypes as ancestral. It also confirmed that haplotype 17 was the ancestral of the 73 detected in the Mediterranean populations analysed.For a more complete overview of the genetic structure of the olive fly pest, a second haplotype network was elaborated by including 31 additional  network  accordedp > 0.05; Fs and very low R2, which were statistically significant or close to significance. This expansion might occur about 66,000 years ago. Likewise, haplogroup I might have a demographic expansion under the sudden expansion model  occurring about 49,000 years ago. Haplogroup II was adjusted to a spatial expansion model assuming a constant population size  that might take place about 61,500 years ago. When considering Mediterranean B. oleae samples as a single population, it fitted to a spatial expansion model , estimated about 61,000 years ago. In short, these results pointed to an expansion of the populations of olive fly populations in the Mediterranean dated at the Pleistocene.The Spanish olive flies experienced a demographic expansion that fitted a sudden expansion model . COI marker in some polyphagous Dacini revealed a haplotype (h) and a nucleotide (π) diversity of 0.92075 ± 0.017 and 0.01083 ± 0.00052 in the fruit pest Bactrocera zonata from seven Iranian locations [Bactrocera correcta, parameters were h = 0.9337 and π = 0.0132 from 15 Thai populations and around 0.95 and 0.008 in a compilation of B. dorsalis surveys, both of which were economically important polyphagous fruits pests in Asia [Zeugodacus. Tau complex, h and π estimates were 0.8954 and 0.0033, respectively [B. oleae, of which the larvae are totally dependent on the olive fruit, despite its reduced environmental diversity, the results reflected a high genetic variability, in the similar range as the polyphagous dacines ones. Our results suggested that B. oleae does not conform the expectations of low genetic diversity given its limited environmental diversity [Genetic diversity estimates from the ocations . For Bac in Asia ,47. Likeectively . Howeveriversity . This ouolf populations, where most SNPs were silent transitions, and the presence of exclusive haplotypes may be due to large population sizes. This hypothesis is supported by the high fecundity of the females, which lay more than 200 eggs, with two to five generations per year [olf have already parasitised the wild variety [The considerable neutral genetic variability observed in the Spanish per year . This fa variety ,52. STF, PCoA, AMOVA and SAMOVA analyses), regardless of other variables such as the olive grove management regimes. Therefore, the Spanish and Portuguese B. oleae populations constituted a single genetic meta-population on a country-wide geographical scale, reliably supporting our previous surveys [olf dynamics of decline/recolonisation. Likewise, the adaptive history of the species to long and current local conditions cannot be avoided to improve an efficient pest control [Iberian Peninsula populations were not differentiated . In turn, there was additional regional sub-structure discriminating the populations of Spain and Portugal from those of Tunisia and Italy, which constituted genetically distinct entities, according to our group’s preliminary work [olf sample from Thessaloniki in the analyses (n = 8) revealed its integration into group I [olf samples showed genetic differentiation [COI gene fragment used. The employment of a longer fragment than those usually used, located at the 5’ region of the gene, which is more variable, has demonstrated the suitability of this marker for inferring new phylogeographical features of this damaging pest. Traditionally in dacines and mainly on pest species, much works has utilised universal COI primers for barcoding and genetic diversity assessment [Genetic analyses revealed the Spanish olive fly samples to be genetically very similar not only to Portuguese ones, but also to samples from Italy, Tunisia, particularly TUN3, and part of Greece . All of ary work . It was  group I . None ofntiation ,16. Thessessment ,54,55 anGene flow is the main factor that has shaped the observed genetic structure overall. The intensive cultivation of olive trees in the Mediterranean Basin, accounting for 90% of the world’s olive oil production, favours these genetic diversity patterns. Olive flies have been reported to be able to cover considerable distances, depending on fruit availability; therefore, in cases of shortage of olives, adults may travel several kilometers in a few days ,57,58,59COI sequences joined the Mediterranean group I. Greek (H2 and H17) haplotypes also fell into this genetic group, as well as the Algerian (H2) and Moroccan (H74) ones. The sequences from Turkish and Israeli flies  were integrated in haplogroup II, but also the USA flies, which held H22. This outcome confirmed the recent American infestation from the Eastern Mediterranean area, as suggested using nuclear markers, although not enough time has elapsed to differentiate these olive flies from the Mediterranean ones [The additional haplotype network included 38 sequences from Genbank, in an effort to integrate and compare the results from all studies concurrently. This network corroborated the patterns of distribution of genetic diversity we detected in the Mediterranean, but also enhanced it . The addean ones ,16. B. dorsalis, a phylogenetically very close tephritid species. Then, H76 is the one of the ancestral olf COI haplotypes until now reported. The Mediterranean and widely distributed H17 are the ancestral of the 73 detected in the Mediterranean populations we have analysed . Howeverolf haplotypes, which seemed to indicate an early divergence, as suggested [B. oleae var. asiatica. Thus, in this Asian easternmost group, an early isolation, small population size and the genetic drift shaped the current structure of this population. Notwithstanding, the small sample size stressed the need to analyse a larger one for further inferences.Lastly, the haplogroup of the Pakistan samples  was clearly apart, differing by up to five SNPs from the Middle East and African uggested ,16,27. Iolf populations is another relevant factor responsible for the distribution of genetic diversity revealed by the mitochondrial marker. Some authors suggested a westward expansion of the pest, in parallel with the expansion of olive cultivation, following the trade routes of the Phoenicians, Greeks and Romans [olf already parasitised the wild variety. Then, the genetic structure of B. oleae populations detected had a more ancient component. The olive tree species complex originated in Central−Eastern Africa and diverged into two subspecies, cuspidata, typical from South Africa and Asia and europaea, the Mediterranean subspecies around 4.5 million years ago in the Pliocene [B. oleae is still controversial, this species has probably arisen in sub-Saharan Africa. The genetic results seemed to indicate that the fly’s colonisation routes paralleled those of the olive tree as genetically distinct sub-Saharan, Mediterranean and Pakistani populations have been identified  subsp. europaea diverged between 284,000 and 139,000 years ago, before the last glaciation, as reflects its genetic structure, according to the glacial refugia of the three Mediterranean peninsulas. In this scenario, demographic inferences (B. oleae populations in Spain to occur in the Pleistocene around 66,000 years ago, when climatic fluctuations had a major impact on the distribution and evolution of species in temperate regions [olf to be in Mediterranean olive trees for at least 400,000 years. As the wild olive trees were gradually replaced by cultivated olive ones, the olf moved to host domesticated olive groves, where it found a better habitat.The olive wild trees of ferences  have poiolf, revealing very high values of genetic diversity and unrestricted gene flow in Spanish populations. A marked pattern of geographic distribution in the Mediterranean Basin and two major genetic groups have been identified, with a patent substructure, mainly due to gene flow and colonisation routes. These genetic results seemed to indicate that the fly’s colonisation routes paralleled those of the olive tree, with olf populations being long established in the Mediterranean Basin. The use of this COI genetic marker provides robustness to the results obtained, with a matrilineal inheritance and lack of recombination that provides a more ancient view of the evolutionary process occurring, allowing a sharper knowledge about the olf genetic structure in this area. All the information obtained in this study may be relevant in the major challenges that control programs of B. oleae have to face in the scenario of global trade and global change.In short, the mitochondrial analysis has been extremely useful in the phylogeographic study of the B. oleae analysed displayed high values of genetic diversity and unrestricted gene flow in Spain. The data revealed olf populations have been long time established in the Mediterranean Basin. A marked pattern of geographic distribution in the Mediterranean basin and two major genetic groups has been identified with two genetic groups. Gene flow seems to be the main process in shaping this genetic structure as well as fly’s colonisation routes that have paralleled those of the olive tree. These findings are helpful to improve the integrated pest management strategies according to the current European Guidelines.The populations of