Pinctada margaritifera provide the second largest economic income for the country after tourism. This industry entirely relies on the collection of natural spats from few highly recruiting lagoons. In recent years, pearl oyster producers have experienced variable success rates in spat collection, with significant spatial and temporal variability in spat supply, driving uncertainty in the future of pearl production. This study combines, for the first time in a farmed lagoon, genetic (SNPs), demographic , and biophysical data  to shed new light on population dynamics, connectivity, and spat recruitment in Ahe Atoll, a well-studied pearl farming site. Our results indicate that the geographical structuring of the natural populations and the contribution of both natural and exploited stocks to the production of spats result from the interaction of hydrodynamic features, life history traits and demographic parameters: the northeastern natural populations are older, not well connected to the southwestern natural populations and are not replenished by larvae produced by adjacent exploited populations. Moreover, we observe that the exploited populations did not contribute to larval production during our experiment, despite a sampling period set during the most productive season for spat collection. This is likely the result of a strong male bias in the exploited populations, coupled with a sweepstakes reproductive strategy of the species. Our results warrant further investigations over the future of the northeastern older natural populations and a reflection on the current perliculture techniques. In French Polynesia, the production and exportation of black pearls through the aquaculture of the black-lip pearl oyster  Pinctada margaritifera provides the second largest economic income for the country after tourism. The process of pearl making consists in inserting a piece of mantle tissue from a giver oyster alongside a nucleus into the pearl sac of a receiving oyster. In order to obtain both giver and receiving oysters, the pearl industry entirely relies, so far, on the collection of natural spats (oyster larvae that underwent metamorphosis and started their sessile life stage) on plastic structures called collectors placed in the water column in highly reproductive lagoons. The collected spats are then translocated in pearl farms for subsequent grafting and growth within lagoon waters. Spat production and collection is known to be stochastic in space and time [Aquaculture of marine species usually relies on the production of hatchery individuals or on catches from local natural populations that are then transferred into closed structures at sea for a period of growth. In French Polynesia, the production and exportation of pearls through the aquaculture of the black-lip pearl oyster and time ,2 but inand time . As a reand time ,5 and quSpat production and collection declines have already been empirically observed in various lagoons (Direction des Ressources Marines. pers. obs.), including in Ahe, a well-studied and historically highly successful collecting lagoon. There, larval dispersal has previously been modeled in an attempt to identify dispersal routes and their correlation with various environmental drivers ,7,8. ThiP. margaritifera is a protandrous hermaphrodite, all oysters are male during the first two years of their life and slowly transition to the female sex, from which they will be able to go back and forth  according to environmental conditions [Recently, concerns have been raised about common pearl farming practices that affect the global sex ratio of the exploited population ,9. P. manditions . Globallnditions . For peanditions  revealednditions . This apP. margaritifera recently confirmed the level of details that can be obtained with genome-wide markers [Nowadays, multiple techniques can be used to characterize the geographical and demographical origin of individuals: direct tagging of individuals using fluorochromes, dispersal modelling, ,14,15,16 markers .Our goal is to shed new light on population dynamics and spat recruitment in Ahe by combining, for the first time in pearl farmed lagoons, genetic, demographic, and biophysical data to attempt to (i) provide baseline and verify estimates of the demographic parameters of the natural and exploited populations of Ahe lagoon, (ii) assess the genetic diversity and heterogeneity of the natural and exploited stock for each sample collected, (iii) assess the potential contribution of each stock to spat recruitment through larval production and dispersion modelling and finally (iv) confront simulations on dispersal and recruitment from the numerical dispersal model with direct observation of spat collection and parentage analysis through genetic markers. P. margaritifera sized between 6 and 20 cm dorso-ventral were collected in February 2017 all around the Ahe lagoon, distributed in 7 and 6 geographical sites respectively  were placed in the lagoon in the second week of February 2017 in three geographic locations commonly used for collection by professionals (South West lagoon) and in three additional locations to cover the whole study area. Assuming a mean pelagic larval duration of 25 days ,20,21, tP. margaritifera wild populations are protected in French Polynesia, we obtained the permission from the government (Direction des Ressources Marines. DRM) to perform non-destructive sampling of the populations. Sampling was done by SCUBA: the individuals were first extracted from their substrate and brought to the surface. They were anesthetized on the boat with benzocaine (ChemLab) in a seawater bath of 20 L . After 15 min of incubation, individuals were sampled by cutting piece of 0.5cm3 of mantle tissue and oysters were then placed in a second bath containing lagoon water until awakening . Finally, oysters were repositioned on the substrate they were taken from. Shell size was measured using a flat iron ruler and a caliper and sex was also recorded by non-lethal gonadal biopsy and visual observation of the gametes. Exploited individuals were directly bought from the professionals in pearl farms. Since −1) and placed at room temperature for 24 h to allow proper infiltration of the solution in the tissue. They were then placed at −20 °C while in the field and at −80 °C once in the lab, before extraction. For spats, we used the entire individual that we placed in individual 1.5 mL Eppendorf tubes and delicately cracked the shell using a clean pestle before adding 200 µL of RNALater (Sigma Aldrich).All tissue samples were preserved in RNALater (Qiagen)  following the manufacturer protocol. DNA quality and integrity were assessed with Nanodrop 2000 and gel electrophoresis. A total of 262 individuals met the DNA quality requirements for next generation sequencing  and were used for Genotyping by sequencing (GBS) and 500 ng of their DNA was sent to the Plateforme d’analyses génomiques at Université Laval sequencing services (Québec City. Canada) for library preparation. DNA was digested using restriction enzymes MspI and PstI. Individuals were then randomly divided into three libraries of 88 and sequenced on 12 chips (four chips per library) on Ion Proton. P. margaritifera genome using BWA program with default options [All bioinformatic analyses were performed on the DATARMOR cluster of Ifremer. GC content, duplicated and overrepresented sequences and quality score of raw reads obtained were evaluated using FastQC. Trimmomatic  was used options . Aligned options  and unma options  to perfo options , BCFtool options  and VCFlP. margaritifera genome and assembled into 453.322 loci. After filtering based on type of feature, minimum depth of sequencing per locus per sample and minimum allele frequency, 13.408 loci were retained for population genetic analysis containing 4.2% of missing genotypes.The sequencing produced a total of 976 million reads. After adaptor and quality trimming 875.729.388 million reads were retained (89.7%). After demultiplexing, 836.267.245 reads (95.4%) were kept. These reads were mapped on the As inferences of population genetics parameters can be influenced by loci not following a neutral model of selection, we investigated for the presence of putative loci under selection in our dataset. We used BayescanV2.1  with a pradiator R package using the “on-the-fly” random forest method. In order to avoid biases in the estimation of the genotypes we first investigated population pairwise genetic differentiation using the neutral set of loci: we estimated FST coefficients using the assigner R package [Some analyses cannot handle the presence of missing data (4.57% missing genotypes in our dataset). We performed genotypic imputation using the  package . Signifidunn.test and FSA R packages to identify outlier groups. Pairwise FST coefficients were obtained with the assigner R package. We calculated inbreeding coefficients of each individual with the related R package [guellergt method and obtained 95% confidence intervals with 1000 bootstraps.All of the following analyses were carried out on the neutral imputed and on the neutral non-imputed datasets in order to observe any discrepancies. We used VCFtools to extract relative heterozygosity levels for each individual (number of heterozygous sites divided by the number of polymorphic sites listed in the VCF) and estimate levels of relatedness using the method of Yang et al. . Specifi package  using thAdegenet R package [xval function to investigate the optimum number of principal component axes to retain for the analysis (here 80). We then performed a DAPC according to the grouping defined by the find.cluster algorithm using all discriminant axes available (number of groups k minus one), followed by a composition plot (structure-like) showing the assignation of each individual to the k clusters. To assess the number of putative genetic clusters in the dataset, we first performed an unsupervised clustering using the find cluster function of Adegenet and selected the optimum k groups using the Aikake Index Criterion . Then, using the k clusters as grouping variable, we performed a Discriminant Analysis of Principal Components (DAPC) with the  package : first, All R codes and datasets used in this study are available in a Github repository see .P. margaritifera is a broadcast spawner that releases gametes in the water column, which upon fertilization will produce a D larva in the first 24 h post fertilization. The pelagic larval duration (PLD) of P. margaritifera was estimated around 21 to 25 days in hatchery conditions [nditions , though nditions .Larval dispersal modeling was carried out using a revised version of the Ahe lagoon MARS3D hydrodynamic model previously described in Dumas et al. . EnhanceThe simulations were set up to match the field sampling for the exact same period  and between 5 and 10 m from the surface. Fifteen different cohorts have thus been released weekly from the 25/01/2017 to the 03/05/2017. Larval behavior was implemented including nychthemeral variations, the same way as in Thomas et al.  who compDespite our best efforts to obtain a homogeneous distribution of individuals’ lengths across the different sampled populations, the size variation across the exploited samples was higher than that of the natural samples A. The naThe sex ratio of the natural and exploited samples also differed drastically, with more females being observed in the natural samples .Bayescan results indicated that 8 loci were putatively under selection . These lp-value < 2 × 10−16. Both the imputed and non-imputed dataset gave the same results for the following analyses and as such only the results from the imputed dataset will be discussed here. Levels of heterozygosity among the different sites were highly different  and 84 (Cluster 2) individuals A. When oThe cumulated (sum of 15 cohorts) connectivity matrices corresponding to the modelled trajectories of larvae generated between the 25th January and the 3rd May 2017 are shown in Our study used a combination of life history parameters. Genetic data and modelling to better understand the population dynamics and assess the demographic parameters of the natural and exploited populations of pearl oysters. As well as their interaction and potential respective contribution to larval and spat production in Ahe lagoon.The exploited populations showed heterogeneity in the distribution of individuals’ size across sampled populations. This discrepancy might be due to bias in our sampling material, which was directly bought from the available stock that the professionals used in their farms during our sampling period. However, it could also reflect the two types of aquaculture activities occurring in Ahe. Indeed, the lagoon is exploited by professionals who raise spat collected in the lagoon until the age of grafting , and other professionals who use ready-to-graft oysters to produce cultured pearls. Some professionals do both activities while others specialize in one. With both activities occurring in the lagoon, the bimodal distribution of shell sizes of the exploited stock could be expected.The exploited population showed a strong bias in sex ratio, with most populations being dominated by the male sex. This bias could be further aggravated by the higher number of undetermined individuals in the exploited stock , especially for individuals of size 9–15 cm in E1, E2 and E3. This absence of gametes in those individuals could be due to (i) spawning occurring a few days prior to the biopsy, but since spawning is mostly triggered by environmental conditions, it should also have affected natural populations, or (ii) an interruption of gametogenesis, because of stress caused by aquaculture practices  . Genetically, the exploited samples formed a homogeneous group, with all populations resembling each other, which can be expected since pearl farms do buy their oysters from the same professional in Ahe.The natural populations had a relatively unimodal distribution of shell sizes and individuals were on average larger than the exploited individuals. This larger size could reflect a bias due to our sampling technique (diving) and the difficulty to observe small individuals in the wild , althougWhile no genetic differentiation was present among the exploited samples, we found significant genetic structuring and heterozygosity differences within the natural samples, with the presence of two genetic clusters separating four southwestern and three northeastern samples. The four southwestern samples were genetically similar to the exploited samples. The similarity between the southwestern and the exploited stocks and their differentiation to the northeastern samples is not related to the distance between natural and exploited sites, since pearl farms are also distributed in the northeastern part of the lagoon. As such, another process is at play to explain the peculiar genetic signature of the northeastern natural samples. Our results seem to indicate that the northeastern samples are not well connected to the rest of the lagoon: they are made of larger, older individuals, mostly females up to 86%), all with a specific “northeastern” genetic signature, meaning they are not replenished by larval recruitment from either the exploited stock, or the southwestern natural stock. Previous research using modelling of larval dispersal showed the existence of two hydrodynamic systems in Ahe, separating the northeastern lagoon from the southwestern lagoon . The mod6%, all wThe genetically similar exploited and natural samples of the shallow southwestern lagoon where both spat collectors and rearing lines are abundant confirm that this is a likely an area promoting population mixing and contributing to homogenization. The shallow lagoon and overall balanced sex-ratio when considering both natural and exploited samples likely favor effective reproduction in this area and genetic homogenization. This does not prevent, however, the import of larvae from other sectors as shown by genetic data in this study and by modelling data ,6. HowevIS coefficients found in the collected spats indicate that SRS could apply to P. margaritifera, as it has been now reported in many marine invertebrates. However, SRS cannot be the only explanation for the lack of spats produced from the exploited populations. Indeed, we know that gametogenesis occurred in the exploited populations, since we successfully biopsied individuals. However, the northeastern natural populations do not contain any individuals with the exploited genetic signature, despite their presence in pearl farms in the area. SRS could be an aggravating factor for successful reproduction if coupled with regionalization of sex ratio, as observed here. The exploited stock is male dominated, while the natural stock is female dominated, which is likely to prevent mixing of gametes and successful larval production. This is likely a key factor to explain the slow but steady decrease in spat collecting reported by farmers in Ahe atoll.It is possible that only a handful of individuals reproduced during our experiment, all from the northeastern populations. This phenomenon is called “Sweepstakes Reproductive Success” (SRS). SRS states that the chances of a highly fecund marine animal to contribute offspring in a future pool of reproductively mature adults is a process with a few “winners” and many “losers” , which mP. margaritifera, for which only a handful of individuals do reproduce at a given time. The fact that spat produced during our experiment were only produced by the natural stock also strongly suggests that serious measures have to be rapidly taken for the management and protection of those populations. In addition, a reflection on the pearl farming methods and on the possibility to increase the number of female oysters in exploited populations is to be considered, such as keeping the oldest oysters on the lines instead of killing them or organizing the repopulation of the northeastern lagoon coral pinnacles with young individuals. Indeed, the reproduction potential of P. margaritifera in Ahe is far from optimized and could in time jeopardize the recruitment of spats and the activity around the production of Tahitian black pearls in Ahe. The results presented here show that the geographical structuring of the exploited and natural populations and their contribution to the production of spats is a complex phenomenon resulting from the interaction of hydrodynamic features, life history traits and demographic parameters. The strong geographical regionalization of the sex-ratios in the various populations likely prevent the efficient mixing of gametes, especially for the male dominated exploited populations. Gamete mixing is likely further prevented by a possible sweepstake reproductive strategy of Finally, this study also highlights the utility of combining modelling and genetic data for the study of larval dispersal and recruitment. Here, we confirm that larval dispersion modelling can realistically predict spat collecting patterns in Ahe when populations are producing larvae. The addition of genetics data increased the precision of the model, indicating which individual cohort was the one more likely recruited on the collectors and also highlighted an inequal contribution of the natural and exploited stock to spat production during the time of the experiment, by allowing assignation of the collected spats to their stock of origin.


Avocado is an important cash crop in Tanzania, however its genetic diversity is not thoroughly investigated. This study was undertaken to explore the genetic diversity of avocado in the southern highlands using microsatellite markers. A total of 226 local avocado trees originating from seeds were sampled in eight districts of the Mbeya, Njombe and Songwe regions. Each district was considered as a population. The diversity at 10 microsatellite loci was investigated.ST pairwise comparisons revealed lack of genetic differentiation for the seven of 28 population pairs tested. The principal components analysis (PCA) and hierarchical cluster analysis showed a mixing of avocado trees from different districts. The model-based STRUCTURE subdivided the trees samples into four major genetic clusters.A total of 167 alleles were detected across the 10 loci with an average of 16.7 ± 1.3 alleles per locus. The average expected and observed heterozygosity were 0.84 ± 0.02 and 0.65 ± 0.04, respectively. All but two loci showed a significant deviation from the Hardy-Weinberg principle. Analysis of molecular variance showed that about 6% of the variation was partitioned among the eight geographic populations. Population FHigh diversity detected in the analysed avocado germplasm implies that this germplasm is a potentially valuable source of variable alleles that might be harnessed for genetic improvement of this crop in Tanzania. The mixing of avocado trees from different districts observed in the PCA and dendrogram points to strong gene flow among the avocado populations, which led to population admixture revealed in the STRUCTURE analysis. However, there is still significant differentiation among the tree populations from different districts that can be utilized in the avocado breeding program. Persea americana Mill.) is a highly heterozygous diploid species with 12 pairs of chromosomes [Persea americana is a polymorphic species with numerous taxa that are adapted to different climates and altitudinal ranges. These taxa are considered to be botanical varieties [P. americana var. drymifolia, P. americana var. guatemalensis and P. americana var. americana [Avocado , the Njombe urban population recorded lowest value i.e., 4.20 ± 0.36, whereas both Mbeya rural and Njombe rural populations recorded peak value, i.e., 10.70 ± 2.26 and 10.70 ± 0.70, respectively , was 0.061 .Analysis of molecular variance was employed to detect genetic divergence within and among the eight avocado populations. The analysis partitioned 6.08% of the variation among the populations, 17.04% among individuals within populations, and 76.87% within all individuals Table . The detST comparisons (Table P ≤ 0.05), with their FST values ranging from 0.0111  to 0.1475 (Rungwe and Mbozi). The second highest FST value  was recorded for Njombe urban and Mbozi.The genetic divergence between the eight avocado populations was established by calculating pairwise Fns Table . 21 of tPrincipal components analysis (PCA) was used to study the genetic relationships among the 226 avocado trees Fig. . While tThe genetic distance matrix of the 226 avocado tree samples was used to study the genetic relationships among the eight populations through hierarchical clustering. The UPGMA-based dendrogram produced three major groups, each containing samples from different districts and regions  can be used to assess these within- and among-population components of genetic variation. FIT measures the excess  or deficit (FIT < 0: heterozygosity excess) of homozygotes at the global level [IT), when AMOVA was calculated without considering regions, was 0.231  or deficit (FIS < 0: heterozygosity excess) of homozygotes within a subpopulation [IS) was 0.181 (P < 0.0001), which indicates that within subpopulations, the observed homozygotes exceeded the expected value by about 18%. In other words, there was a reduction by 18% of observed heterozygotes relative to the expected ones within subpopulations. At the global level, this implies that about 78%  of the global heterozygosity deficit was due to the within population deficit. FST is the fixation index that measures differentiation between subpopulations and range from 0 to 1. A value of 0 indicates that the populations under consideration are interbreeding freely (complete panmixis), while a value close to 0 indicates an unstructured population [ST) for the eight avocado geographical populations was 0.061 , P. nubigena (2 samples) and P. kruguii (1 sample). On the other hand, Guzmán et al. [P. americana collected in one country comprising local selections, root stocks and commercial cultivars. Similarly, the 315 samples analysed by Gross-German and Viruel [P. longipes, P. nubigens and P. schiedeana. However, the average FST in the present study is comparable to the 0.05 reported by Cañas-Gutiérrez et al. [Most of the loci in the present study showed significance deviation from the Hardy-Weinberg equilibrium Table . Geograpal level . The glopulation . The avepulation . A valuepulation . In the a et al. , Guzmán a et al.  studied Pisum sativam L.) populations (2%) compared to that of region-based groups (8%).Analysis of molecular variance showed that 6.08% of the total genetic variation was partitioned among the 8 districts Table . When thST that revealed absence of differentiation between pairs of populations, such as Rungwe versus Njombe urban and Buokelo versus Njombe urban. Similar results were obtained when these avocado trees were characterised with morphological markers [In the present study, principal components analysis (PCA) and hierarchical cluster analysis were used to study the genetic relationships among the sampled trees. Neither the PCA nor the dendrogram separated these trees according to their districts or regions. This was in line with AMOVA findings Table  which sh markers . GeneticST values . The bags were then put into plastic bags followed by addition of silica gel to dry the leaves. When needed, we replaced the worn-out silica gel and continued doing so until the leaf samples were completely dry. Complete dryness was determined as when added silica gel retained its original colour. We extracted DNA from the dried young avocado leaf samples using the Thermo Scientific Genomic DNA Purification Kit following the manufacturer’s instructions with minor modifications. DNA integrity was checked in a 1.2% agarose gel electrophoresis and its quality and quantity was assessed with a NanoDrop Spectrophotometer.2, 0.3 μl of 25 mM dNTPs, 0.75 μl of 10 μM of each forward and reverse primer, 0.2 μl of 5 U/μl Taq polymerase and 25 ng genomic DNA. PCR reactions were run in a S1000™ thermal cycler  using a program of initial denaturation at 94 °C for 1 min, 35 cycles of 1 min denaturation at 94 °C, 30 s annealing depending on the specific primers’ annealing temperature and 1 min extension at 72 °C, followed by a 10 min final extension at 72 °C. The size of the PCR products were determined using an Applied Biosystems 3500 Genetic Analyzer .Forty microsatellite markers were selected among those developed by Sharon et al.  and GrosStandard diversity indices for markers; i.e., observed and effective number of alleles, observed Nei’s  and expeST, FIT, FIS, FCT and FSC) and pairwise comparisons between the eight geographical populations.Analysis of molecular variance (AMOVA) was carried out in Arlequin 3.5.2.2 under 1000 permutations, 100,000 steps in Markov chain and 10,000 Dememorisation Steps. Hierarchical global AMOVA was conducted by grouping individual samples according to districts, regions and elevation ranges. Arlequin 3.5.2.2 was used to compute fixation indices  to display the genetic relationships among the eight avocado geographical populations (districts). Allele composition for each tree was computed in the adegenet R package  and then


Habenaria radiata (syn. Pecteilis radiata) is a terrestrial orchid living in wetlands in Japan, Russia, South Korea, and China. The habitats of this plant in Japan have been reduced because of environmental destruction and overexploitation, and thus it is on the Red List of Japan as a Near Threatened species. One of the three petals of the H. radiata flower is called a lip or labellum, which resembles a flying white bird, egret, or white heron, with its proposed function being to attract pollinators. To understand the diversity of H. radiata plants in different areas, we examined the lip morphology and phylogeny of populations from eight habitats in the Kinki area, Japan. The complex shapes of the lips were quantified and presented as a radar chart, enabling characterization of the morphological difference among populations. Phylogenetic analysis with microsatellite markers that we generated showed the variation of genetic diversity among populations, suggesting the different degrees of inbreeding, outbreeding, and vegetative propagation. Our approach offers a basic method to characterize the morphological and genetic diversity in natural populations.Floral organs have evolved from leaves for reproduction, and the morphological analyses help to understand the plant diversity and evolution.  Floral organs have evolved from leaves to have various shapes that facilitate reproduction. The petals in many plants develop to have unique shapes to attract pollinators. The flower shapes in orchids especially vary widely among species, and have established close relations with pollinators; thus, the petal morphology in orchids is critically important for the reproduction.Habenaria is a large genus of orchids with more than 850 species living in the tropical and subtropical regions [Habenaria radiata (Thunb.) Spreng., Syn. Pecteilis radiata (Thunb.) Raf. is a terrestrial orchid living in the wetlands in Honshu, Kyushu, and Shikoku in Japan, the Far East of Russia, South Korea, and China . In recent years, the habitats of H. radiata have been reduced because of environmental destruction and overexploitation, and thus this species is registered as a Near Threatened species in the Red List in Japan .Orchidaceae is one of the largest families of angiosperms, containing more than 28,000 species . Habenar regions . HabenarH. radiata flowers consist of three sepals, two lateral petals, one lip (or labellum), and one column  markers [Molecular markers are powerful tools used to investigate the genetic variation in populations. Microsatellites or simple sequence repeat (SSR) markers are relatively easy to develop and useful for characterizing genetic differences based on genomic DNA sequences. An SSR marker is a short tandem repeat consisting of 1 to 6 nucleotides in the genome, whose repeat numbers can differ among individuals. Molecular markers raised by the SSRs have been widely used for genetic breeding, genome mapping, population genetics, and taxonomic and phylogenetic studies ,9. In Or markers .H. radiata, we examined the morphology and phylogeny of populations from eight habitats in the Kinki area, Japan. Quantification of lip shape by eight indices showed the differences in flower morphology among the populations. We developed novel SSR markers and analyzed phylogenetic relationships, and showed the genetic diversity of H. radiata populations. Our results offer basic methods to characterize the morphological and genetic diversity in natural populations.To understand the diversity of H. radiata plants were collected from eight populations in the Kinki area. During collection, we noticed that the morphologies of the flowers, especially the lips, showed variation among populations. Since the lip shapes are complex, we quantified them by measuring several values and showed these with radar charts, as well as graphs showing each data point . Based on this, we designed 82 SSR primers, and after elimination of unsuccessful primers, 19 SSR primers detecting 21 loci were selected (He) and observed (Ho) heterozygosities were in the ranges of 0.2362 to 0.6410 and 0.3187 to 0.6139, respectively (F) values in Ueno were significantly higher than the HWE expectation, suggesting a high frequency of inbreeding within the population. On the other hand, those in Himeji East–a, South Kyoto, and Yamatokoriyama were significantly low, suggesting excess heterozygosity in these populations  markers. The SSR-enriched libraries from selected . Five maectively . The fixulations . Since HH. radiata populations were classified into three groups: Yamatokoriyama, Himeji, and Ueno and South Kyoto  values was 2 and the populations were classified into two genetic pools  and one hawkmoth (Theretra japonica) visit H. radiata flowers and transfer pollinia by attaching them on their head [Theretra oldenlandiae) visited several flowers and seemed to suck nectar from spurs while hovering, without landing on the flowers  values, representing the complexity of the lip shape, are different between the Himeji region and the others. The former’s flowers have a more simple shape (DI: ~0.04), whereas the latter display values of 0.6~0.8 . This may be because of the reduction of wetland habitats due to excessive land development and overexploitation. Our methods can be used to characterize the population, and thus may help to maintain the local population in natural habitats. This work was limited to the populations in the Kinki area, and we need to examine the other populations left in nature. We also need to think about other natural species living in wetlands to conserve the natural conditions for our future.H. radiata plants were sampled from eight habitats with permission: five from Himeji , and the others from Yamatokoriyama (Nara), South Kyoto, and Ueno Forest Park (Mie). The authors apologize that the details of the habitats are withheld to avoid overexploitation. The information is available upon request, although we may not be able to respond depending on the request content. Note that gathering of any plants and animals is prohibited in these area and parks without permission. For the recording of flower visitors, bulbs were sown on cultivation soil  covered with wet sphagnum and grown outside at the Seika campus, Kyoto Prefectural University, Seika, Kyoto.To quantify the complex shapes of the lips, we defined the middle part of a lip as the body and the two lobes as wings, then measured the dissection index (perimeter/√area) values of the left wing, body length, body width, wing length, angle between body and left wing, and serration number of the left wing . The ratH. radiata genomic DNA for the Korean population [H. radiata genomic DNA as reported previously [Genomic DNA samples were extracted from young leaves at approximately 100 mg using a DNeasy Plant Mini Kit . pulation  was usedeviously . PCR priHe), observed heterozygosities (Ho), and fixation index (F) values were calculated using Genepop version 4.2 [K) were set from 1 to 8 and each K was run 10 times. Each run was carried out with a burn-in period of 50,000 and 1,000,000 MCMC replications. The online STRUCTURE HARVESTER software was used to estimate optimal K values [The genetic parameters of the expected heterozygosities  in the morning or evening on 18 days from 9 July to 3 August 2018. The total recording time was 85 h, 56 min, 35 s. Flowers and petals were captured with a digital camera  and quantified with ImageJ software .


Unio crassus Philipsson, 1788 is one of the examples that has been reported to show a dramatic decline of populations. Hierarchical organization of riverine systems is supposed to reflect the genetic structure of populations inhabiting them. The main goal of this study was an assessment of the U. crassus genetic diversity in river ecosystems using hierarchical analysis. Different molecular markers, the nuclear ribosomal internal transcribed spacer ITS region, and mitochondrial DNA genes (cox1 and ndh1), were used to examine the distribution of U. crassus among-population genetic variation at multiple spatial scales . We found high genetic structure between both drainages suggesting that in the case of the analyzed U. crassus populations we were dealing with at least two different genetic units. Only about 4% of the mtDNA variation was due to differences among populations within drainages. However, comparison of population differentiation within drainages for mtDNA also showed some genetic structure among populations within the Vistula drainage. Only one haplotype was shared among all Polish populations whereas the remainder were unique for each population despite the hydrological connection. Interestingly, some haplotypes were present in both drainages. In the case of U. crassus populations under study, the Mantel test revealed a relatively strong relationship between genetic and geographical distances. However, in detail, the pattern of genetic diversity seems to be much more complicated. Therefore, we suggest that the observed pattern of U. crassus genetic diversity distribution is shaped by both historical and current factors i.e. different routes of post glacial colonization and history of drainage systems, historical gene flow, and more recent habitat fragmentation due to anthropogenic factors.Mussels of the family Unionidae are important components of freshwater ecosystems. Alarmingly, the International Union for Conservation of Nature and Natural Resources Red List of Threatened Species identifies almost 200 unionid species as extinct, endangered, or threatened. Their decline is the result of human impact on freshwater habitats, and the decrease of host fish populations. The Thick Shelled River Mussel  Mussels of the family Unionidae with 680 described species  are impoIn Europe, the remarkable decline suffered by freshwater mussel populations has attracted the attention of conservation organizations . Over reMuch of the global awareness of freshwater mussel decline stems from North American Unionoidea, which constitutes the continent’s most imperiled fauna, but much more numerous in terms of species than the rest of the continents ,25. OverUnio crassus Philipsson, 1788 is one of the examples that has been reported to show a dramatic decline of populations within the western and central European part of its range and thus has become a major target species for conservation  for U. crassus habitat and population declines, might well shape consequently genetic architecture and distribution of mtDNA haplotypes of this mussel. As stated by Zając (2004) [U. crassus preservation is improper mainly due to small population size and unfavorable status of the mussel habitats. Small populations, in turn, are more susceptible to the effect of inbreeding and genetic drift. In our study we included populations reported as declined due to human activity and/or isolated by polluted fragments of rivers at different time scales. Such a population is Jasiołka (JAS), characterized by the presence of one common mtDNA haplotype (CN1) found also in the remaining Polish populations and one private mtDNA haplotype (CN5)  should be similar genetically and share the same haplotypes of mtDNA whereas the Lithuanian populations should be different from the Polish ones. However, we found some mtDNA haplotypes found in central Poland populations  that clustered with haplotypes from Lithuanian populations  , populatN5) vide . Besidesulations . TherefoU. crassus the observed intermingling of haplotypes may be connected to the ancient connection between Neman and Vistula drainages as indicated by the geological data. During the Vistulian Glaciation the ice sheet blocked the pre-existing drainage system and caused the development of vast ice-dammed lakes including waters of the ancient Neman River as well as the Polish rivers Biebrza, Narew, and Vistula [Unio populations, populations located nearby  should be similar genetically and share haplotypes of mtDNA whereas Lithuanian populations should be different from Polish ones. The genetic diversity and differentiation of U. crassus populations revealed during this study can also be explained by colonization from different glacial refugia or postglacial recolonization. Moreover, the observed pattern of genetic diversity may result from specificity between glochidia and host fish vectors.Pleistocene ice ages have exerted considerable influence on the contemporary genetic diversity patterns of freshwater species. Multiple transgressions and regressions of ice caused the situation that many species had to displace or escape to the ice age refugia. In glacial periods, the northern part of Poland and the area of Lithuania were covered to varying degrees by the glacier , causing the withdrawal of organisms from this region . Glacial Vistula . Pre-exi Vistula . In turnU. crassus include fish species like bullhead , Eurasian Minnow , chub , rudd  Three-spinned Stickleback , and perch   are obligate parasites of freshwater fish, they were able to migrate and recolonize the European areas during warmer periods only along inland waters together with their host fish species. Thus, the gene flow among populations of freshwater mussels was mostly affected by migration patterns of fish host species. Potential hosts for ) e.g., ,107,108),108U. crU. crassus has been the subject of many studies, for example: Squalius cephalus [Perca fluviatilis [Cottus gobio [Gasterosteus aculeatus [U. crassus evolutionary lines. However, the U. crassus expansion pattern has some characteristics of the observed distribution of other species of freshwater mussels. For example, the analysis of DNA microsatellite loci of M. margaritifera from central Europe revealed that the diversity of individual populations is not consistent with the modern hydrological network [M. margaritifera was also not compatible with the distribution of genetic variability of C. gobio, one of the fish-host species of glochidia in the same area [U. pictorum from the Vistula are separated from those inhabiting other rivers of the Baltic Sea catchment and cluster with populations of the Danube. Similarly, geographic isolation was found in the present study between populations belonging to the same river basin, which also indicated the existence of hydrological barriers in migrations of glochidia hosts. Therefore, we suggest that the genetic relationships within U. crassus from Poland and Lithuania reflect paleogeographical relationships between river systems during Pliocene and Pleistocene rather that current gene flow.The Ponto-Caspian region  was the main glacial refugium of the recent fish fauna of both Poland and Lithuania  which cocephalus , Perca fviatilis , Cottus us gobio ,114, Gasculeatus . Neverth network . The gename area . These dame area ). A studame area  indicateame area . Interesame area  presenteU. crassus did not match the present-day drainage systems in the case of the central Poland rivers. Individuals from the southern Poland rivers were localized in “Polish cluster” and did not intermingle with Lithuanian populations. The observed pattern of genetic diversity and differentiation of U. crassus populations revealed during this study can be explained either by historical gene flow or different routes of post glacial colonization. In such a case, the region of central Poland could be a contact zone between two haplotype lineages. Interestingly, in the case of our study the present-day population differentiation of Mustela nivalis Linnaeus, 1758 by McDevitt et al., 2012 [We believe that our results suggest the existence of a secondary contact area in central Poland resulting from the recolonization of a given area by populations from separate glacial refugia . The idel., 2012 ). U. crassus, a number of subspecies with local forms of uncertain taxonomic rank has been widely accepted [It is worth mentioning that for accepted ,127,128.U. crassus we dealt with at least two different genetic units. However, the observed genetic structure does not match the present drainage systems. Therefore, we suggest that the observed genetic relationships within U. crassus from Poland and Lithuania reflect rather paleogeographical relationships between river systems during the Pliocene and Pleistocene than being the result of the current gene flow. The present-day genetic pattern of U. crassus diversity may also be shaped by different routes of post glacial colonization and specificity between glochidia and host fish vectors. More recent habitat fragmentation due to anthropogenic factors may also have contributed to the observed populations structure. In conclusion, the results of our study indicated that in the case of eastern Central European populations of


Anthopleura spp.) and their endosymbiont Breviolum ‘muscatinei’, across an extensive geographical range to identify the spatial scales over which local adaptation is possible. Similar to previously published results, two solitary host species exhibited isolation by distance across hundreds of kilometres. However, symbionts exhibited genetic structure across multiple spatial scales, from geographical location to depth in the intertidal zone, and host species, suggesting that symbiont populations are more likely than their hosts to adaptively mitigate the impact of increasing temperatures.Corals and cnidarians form symbioses with dinoflagellates across a wide range of habitats from the tropics to temperate zones. Notably, these partnerships create the foundation of coral reef ecosystems and are at risk of breaking down due to climate change. This symbiosis couples the fitness of the partners, where adaptations in one species can benefit the holobiont. However, the scales over which each partner can match their current—and future—environment are largely unknown. We investigated population genetic patterns of temperate anemones ( EspecTogether, hosts and symbiont genotypes interact across a landscape that varies in space and time, or a ‘geographical mosaic’ . Local aspecies (previously within-clade) can result in the specialization on varying temperature and light conditions [between genera [Although species in the family Symbiodiniaceae are difficult to delineate, recent studies suggest that symbiont populations can respond to mosaics of environmental conditions across a wider range of spatial scales than their hosts. The Symbiodiniaceae family has recently been described and now formally recognizes as genera what were previously ‘clades’ that were defined by genetic and functional differences . Thus, pnditions , as wellnditions . Almost n genera , which sn genera  to hundrn genera . The fewn genera . No studAnthopleura elegantissima, A. xanthogrammica and A. sola inhabit the highly heterogeneous intertidal zone along the west coast of North America and are the only hosts of the microalgal endosymbiont, Breviolum ‘muscatinei’ [Anthopleura spp. Most notably, temperature and light gradients can greatly vary across latitude and depth in the intertidal zone. All three anemone species are sympatric from Bodega Bay, CA to Pt. Conception, CA and in upwelling zones south of Pt. Conception. Anthopleura sola and A. xanthogrammica are both large, solitary species that live primarily lower in the intertidal zone, extending down to 15 m into the sub-tidal zone [Anthopleura along the Pacific coast are panmictic [A. elegantissima exhibits population structure across its geographical range but high rates of gene flow due to long planktonic larval stages prevent locally beneficial alleles from increasing in frequency across the geographical range at most, but not all, host genomic loci [B. ‘muscatinei’ across smaller spatial scales than their host populations of A. elegantissima [Testing for local adaptation or coevolution across variable environments with high species diversity such as coral reefs is difficult due to the large number of potentially interacting host and symbiont populations. However, three congeneric species of sea anemone, catinei’ ,19. Multdal zone . All thrdal zone . Consistanmictic . Howevermic loci . Previountissima , suggestIn this paper, we use a population genetic approach to identify the spatial scales over which host and symbiont populations could match selectively important environmental variation. First, we examine the degree to which differentiated symbiont populations are associated with varying environmental conditions at the macroscale of the geographical range and the mesoscale of the intertidal zone . Second, we test the hypothesis that microscale host–symbiont interactions shape the genetic structure of the partners, consistent with the idea that host morphology or cellular interactions determine which partnerships are successful. We begin addressing this hypothesis by identifying covarying regions of the host and symbiont genome, combined with analyses designed to identify whether host species-level effects or the genotype of each individual host drives these associations.. 2(a)Anthopleura sp. and B. ‘muscatinei’ in the southern portion of the geographical ranges of these species, from Cape Blanco, OR (USA) to the southern tip of Baja California, Mexico. Additionally, we included populations of A. xanthogrammica from more northerly locations up to Washington State to more fully characterize the population genetic structure of this species through a larger portion of its range. The distributions of the three host species largely overlap across this range , where  Mexico; . We samp(b)We extracted DNA from the preserved host tentacles using a standard CTAB extraction procedure . To increase the representation of symbiont reads in the final library, we enriched samples for symbiont cells using a Cytomation MoFlo high-speed cell sorter (Beckman). Gates for each sort were set to keep cells whose fluorescence corresponded to excitation of chlorophyll generated from 488 nm to 635 nm wavelengths. We prepared uniquely barcoded RAD-seq libraries  for all samples and sequenced them on the Illumina 4000 platform.(c)FST values. For the two host species, we further evaluated geographical deviations in gene flow from IBD patterns using the software eems [We evaluated population genetic patterns in the hosts across their geographical ranges, and symbionts across a set of nested spatial scales . Across the geographical range, we characterized isolation by distance (IBD) in the hosts using pairwise are eems . For theare eems  to ident. 3(a)A. xanthogrammica was rare south of Pt. Conception, but was present at BPE and BER, which are geographical locations dominated by stronger upwelling regimes [All three host species were present and common at geographical locations within their previously described contiguous geographical ranges. As expected,  regimes . None ofπ) are highest for A. sola (0.0095), and are much lower for A. xanthogrammica (0.0025); for reference, in A. elegantissima populations π = 0.0035 [A. sola with low levels of genetic differentiation (FST = 0.021–0.049) between populations. Conversely, A. xanthogrammica populations show IBD, largely due to high levels of differentiation between the three individuals we sampled in Baja California, Mexico and the contiguous populations north of Pt. Conception, CA . The overall slope of the best-fit regression of scaled FST to geographical distances is 1.79 × 10−5 for A. sola (p = 0.0047) and 2.45 × 10−5 for A. xanthogrammica . Range-wide patterns of genetic diversity FST , and ADMIXTURE best supports a model of seven subpopulations of symbionts across the geographical range we sampled (ca 2000 km). Four of these subpopulations correspond to geographical location: Bodega Bay, CA (BOD), Big Sur, CA (GAR), Ensenada, Baja California (BPE) and the southernmost populations  of Baja California . These samples are omitted from further discussion due to a lack of homologous loci between them and all other samples.The 1470 SNPs that were successfully genotyped and passed quality filters show genetic structure within and between host species and geographical sites . After excluding hosts whose symbionts exhibited low genotyping rates, 56 individuals remained in the final dataset. Our initial dataset contained 2192 SNPs, which was reduced to 1470 after pruning linked loci. Populations at each geographical location we sampled (regardless of the host species in which they were found) exhibit high levels of lifornia . HoweverA. sola symbiont populations (BBA and BEB) from all other symbiont populations , followed by host species .The AMOVA shows a hierarchical structure in the genetic differentiation of the symbionts across their geographical range and within host species. Overall, geographical location explains the largest percentage of genetic variation between symbionts  . The results from BEDASSLE revealed an aE : aD  ratio of 0.0015. To contextualize this ratio, mean environmental distances between individuals from the same geographical sampling site with relatively large environmental distances (BOD) and relatively small environmental distances (BER) were 11.54 and 6.78, respectively. These mean benthic community distances result in the same correlations in allele frequencies between the symbionts found within two individuals as approximately 4500–7500 km of geographical separation.The benthic community composition  across the intertidal zone correlates with changes in symbiont genotypes within and between sites. Environmental patterns vary within and between sites: PC1 of the benthic community surrounding each individual is correlated with PC2 of the symbiont genetic dataset ( × 10−5) . PartialFinally, we attempted to identify covarying loci across host and symbiont genomes. Overall, based on an analysis that includes all individuals regardless of host species, the LD between the hosts and their associated symbionts is low and, in most cases, near 0. The patterns of LD decrease towards no covariation when the effect of host species is regressed out of the symbiont allele frequencies , from a . 4Anthopleura spp.–B. ‘muscatinei’ partnership, a combination of low rates of gene flow and strong selective gradients across metres in the intertidal zone as well as across their geographical range shape the genetic structure of the symbiont. The opposite is true in the hosts, where populations of all three species of Anthopleura exhibit high levels of gene flow across their geographical ranges, reducing population genetic signals of local adaptation across any of the spatial scales we examined in this study. These patterns highlight significant disparities between the spatial scales over which populations of Anthopleura sp. and B. ‘muscatinei’ might be able to adapt to local conditions, and suggest that symbiont populations can match variation at smaller spatial scales than their hosts. Patterns of genetic differentiation for A. sola and A. xanthogrammica, like those previously reported in A. elegantissima [Geographical mosaics can impose highly variable selection pressures on host–microbial partnerships; the rate and magnitude of response, in turn, is shaped by transmission mode, gene flow and generation times in both partners ,30. In tntissima , imply t(a)A. xanthogrammica and A. sola, combined with previous results in A. elegantissima [A. elegantissima, both A. sola and A. xanthogrammica exhibit limited gene flow in southern populations relative to expectations based on IBD . However, analysis of the populations in ADMIXTURE and eems fails to reveal any evidence for subpopulation structure based on biotic  aspects of microhabitat, as in coral species whose ranges span depths with dramatically different illuminance [. These results suggest that local adaptation is more likely in southern populations due to lower rates of gene flow, which could allow those populations to specialize on the conditions specific to the southern portions of their geographical ranges.One explanation for the increased IBD signal in al range . Finallyuminance ,32. Thes(b)(i)Anthopleura spp. along the Pacific coast are highly differentiated by geographical location, with some of the greatest differences occurring between neighbouring locations (e.g. BPE and BER). Previous studies of symbionts inhabiting A. elegantissima along the Californian coast detected similar patterns using three genetic loci, with major genetic breaks at Cape Mendocino and Monterey Bay [Breviolum ‘muscatinei’, as is the case in the host A. elegantissima [Previous studies demonstrated that at the inter-genus  level, symbiont lineages may exhibit genetic structure at the level of ocean basins , biogeogerey Bay . Similarerey Bay , the evierey Bay , which serey Bay , does nontissima . SignalsB. ‘muscatinei’ populations draw on the same genetic variation to match environmental variation across the geographical range of Anthopleura spp.Symbiont populations are more genetically similar when they originate from locations sharing comparable benthic communities, a proxy for environmental similarity, even when those locations are thousands of kilometres apart. For example, the Bodega population in northern California and the Ensenada population in Baja California, Mexico share genetically similar symbiont populations and a similar benthic community . Cold, upwelled waters are a dominant abiotic factor in both of these locations and act on the scale of hundreds of kilometres ,39, simi(ii)intra-specific patterns and variation at the level of populations on which selection might act.The intertidal zone is a highly variable environment dominated by environmental stress, competition and predation that collectively structure dramatically different biotic communities over the scale of metres ,42. The This study capitalizes on biotic shifts across geographical sites as well as depth in the intertidal zone to create an environmental proxy, which, in turn, is correlated with major axes of symbiont genetic variation. Environmental variation could favour symbiont strains that specialize on a certain location in the intertidal zone. For instance, variation in light regime leads to shifts in the dominant symbiont genus across scales as small as a single coral head . PreviouFurther evidence from this study for how environmental variation shapes symbiont genetic/population structure comes from BEDASSLE, which controls for spatial autocorrelation in environmental variables. This analysis shows that the average variation within a local environment (across approx. 5 m of depth) results in allele frequency correlations of the same magnitude as those that arise between populations separated by thousands of kilometres. This appears to occur independently at most geographical locations, as the results from the ADMIXTURE analysis suggest that overall rates of gene flow across the geographical ranges of these species are relatively low, a pattern apparent in other studies of symbiont populations separated by hundreds of kilometres to thousands of kilometres of km using neutral microsatellite markers . Local bAnthopleura [A. elegantissima can be reciprocally transplanted across the intertidal zone to assess the rate at which genetically identical hosts acquire or alter symbiont strains that match their environment.The overall pattern of genetic structure across the geographical range we sampled in this study, combined with fine-scale matching of symbiont genetic diversity to a local biotic community suggests that the genetic diversity needed to match environmental variation across depth in the intertidal zone is present in the symbiont population at any one geographical location. Alternatively, symbionts may extensively disperse and come to dominate a single geographical location, although dispersal is unlikely to happen while associated with either sessile host adults or dispersing larvae, as symbionts are horizontally transmitted in hopleura . This exhopleura . This st(iii)A. sola are so distinct that they explain most of the variation apparent from the PC analysis, outweighing other patterns associated with abiotic conditions or geographical distance between populations revealed in the second principle component. Furthermore, the A. elegantissima population at BBA hosted symbionts, but these failed to align to the B. minutum genome (or any other published symbiont genome), suggesting that they are the most genetically distinct population we sampled and likely represent another species, if not genus.Genetic differences that allow symbiont populations to match local conditions can arise from the specific microenvironment a host creates  or from interactions between loci in the host and symbiont genomes. We found that symbiont populations are genetically structured by host species within sites and that inter-species covariation (linkage disequilibrium) is partly explained by allele frequency differences in symbiont populations inhabiting the three different host species . These pA. sola and A. elegantissima could favour symbiont strains that specialize on the morphological attributes of host polyps  or the host species-specific variation in microhabitat occupation . Similar beneficial pairings arise in non-nutritional partnerships where symbionts and hosts facilitate each other's persistence in harsh and stressful conditions [Host-specificity in the southern portion of the geographical range could result from historical or contemporary forces. One explanation for symbiont segregation by host species is historical isolation leading to coevolution, where a single isolated host species at this location tightly coevolved with the local symbiont population, leaving the symbiont partner unable to interact with other host species when the host populations subsequently became sympatric ,51. Howenditions , even ifnditions . A key nAnthopleura sp. and their associated Symbiodiniaceae populations across large and small spatial scales both affects and reflects how natural selection and coevolution shape the performance of the holobiont. Across species within the Symbiodiniaceae family, patterns consistent with local adaptation [between Symbiodiniaceae genera are related to their relative performance across different thermal and irradiance regimes. Within the vertical span of the intertidal zone, Anthopleura and their symbionts can experience greater variation in temperature and light than what most sub-tidal tropical species experience across their entire geographical ranges. The populations of Breviolum ‘muscatinei’ in this study have genetic patterns consistent with the hypothesis that genetic variation within symbiont species (as defined by ITS2 and organellar loci) can allow symbionts to match their local abiotic environment, as well as the intra-cellular host biotic environment. However, analysing ITS2 alone would fail to uncover some of these patterns, notably those across the intertidal zone at a single geographical location as well as covariation between host and symbiont loci. Population genetic patterns in the hosts suggest that gene flow likely swamps any multi-generational selective response to environmental heterogeneity across the intertidal zone. More importantly, adaptive benefits could falsely appear to arise from host populations if studies fail to take the genetic variation of the symbionts into account. Future work should experimentally test whether these genetic patterns actually reflect the enhanced performance of the symbiont (and possibly the holobiont) in its local environmental conditions, given that some symbiont strains could be parasitic in some environmental contexts [The genetic structure of aptation  and withaptation  suggest contexts , and thacontexts . Fully cClick here for additional data file.


Quercus castanea, to infer the historical population dynamics of this oak distributed in the Mexican highlands. A total of 36 populations of Q. castanea were genotyped with seven chloroplast microsatellite loci in four recognized biogeographic provinces of Mexico: the Sierra Madre Occidental (western mountain range), the Central Plateau, the Trans‐Mexican Volcanic Belt  and the Sierra Madre del Sur . We obtained standard statistics of genetic diversity and structure and tested for signals of historical demographic expansions. A total of 90 haplotypes were identified, and 29 of these haplotypes were restricted to single populations. The within‐population genetic diversity was high (mean hS = 0.72), and among‐population genetic differentiation showed a strong phylogeographic structure . Signals of demographic expansion were identified in the TMVB and the SMS. The ecological niche models suggested a considerable percentage of stable distribution area for the species during the LGM and connectivity between the TMVB and the SMS. High genetic diversity, strong phylogeographic structure, and ecological niche models suggest in situ permanence of Q. castanea populations with large effective population sizes. The complex geological and climatic histories of the TMVB help to explain the origin and maintenance of a large proportion of the genetic diversity in this oak species.The Mexican highlands are areas of high biological complexity where taxa of Nearctic and Neotropical origin and different population histories are found. To gain a more detailed view of the evolution of the biota in these regions, it is necessary to evaluate the effects of historical tectonic and climate events on species. Here, we analyzed the phylogeographic structure, historical demographic processes, and the contemporary period, Last Glacial Maximum (LGM) and Last Interglacial (LIG) ecological niche models of  High genetic diversity in Mexican oaks. High phylogeographic structure in a widespread oak species. Biogeographically, the Mexican Highlands comprise most of the Mexican Transition Zone which also includes the Central American mountains forming the Chiapas Highlands province Morrone, . In the The Mexican Transition Zone is characterized by an impressive biological and physical complexity in terms of biogeographical and macroecological patterns Morrone, , geologiThe most important facts that phylogeographic studies have shown for species in the Mexican Transition Zone include pre‐Quaternary divergence times, east–west phylogeographical breaks in the Trans‐Mexican Volcanic Belt that are incongruent with geographic distance or topography, and north–south phylogeographical breaks within the Sierra Madre Occidental  have been frequently used to study the geographic distribution of genetic diversity from a historical point of view in the Nearctic and Neotropical regions  as a focal species. This species has high ecological importance and has been intensively studied for gene flow estimation in fragmented landscapes , (b) test for signals of historical demographic expansions and to determine the timing of the population expansion, (c) contrast the conclusions derived from genetic data with models of the contemporary period, LGM and LIG ecological niche of Q. castanea, and (d) contrast the phylogeographic patterns revealed in this study with the regionalization of the biogeographic provinces based on the physiographic features.Specifically, the objectives of the present study were to (a) determine the patterns of genetic diversity and differentiation of populations of 22.1Quercus castanea belongs to the section Lobatae . Fragments were analyzed and sized with the Peak Scanner program 1.0 (Applied Biosystems).Genomic DNA was extracted from 100 mg of leaf material using the modified CTAB protocol of Lefort and Douglas . Seven c2.3Sh) were calculated using SPAGeDi 1.1   and RST . We also performed a hierarchical grouping to obtain the variance components of genetic variation among groups, among populations within groups, and within populations. The categories were defined by the four above‐mentioned biogeographic provinces. The significance of partitions was tested using 104 permutations.Tests of population genetic structure were performed using analyses of molecular variance (AMOVA) with ARLEQUIN 3.5  in SPAGeDi 1.1  ranging from two to 35. The analyses were set up considering ten independent runs, correlated allele frequencies, one million generations with a thinning value of 100 and a burn‐in value of 1,000. Finally, the distribution of GENELAND spatial clusters was mapped using ArcMap 10.3 (ESRI Inc.).We analyzed the spatial distribution of genetic variation and identified genetic discontinuities between 2.5FS  and suggest population expansion or purifying selection. In turn, positive SF values indicate a recent population bottleneck or the action of balancing selection. Simulations have shown that SF is a more precise indicator of population historical demography than Tajima's D , while in stationary populations, the distribution is predicted to be ragged and multimodal  and the initial and present effective population sizes scaled by mutation rate (Θ0 = 2µN0 and Θ1 = 2µN1), where µ represents the mutation rate, t is the number of generations since the population expansion occurred, l is the number of loci evaluated, and 0N and 1N are the previous effective population sizes and the sizes after the expansion, respectively , the TROPICOS database , and the Global Biodiversity Information Facility database . Records that did not match the natural distribution of the species reported by Valencia‐Á  with a 30 arc seconds resolution. The LGM climate layers were obtained from two past climate scenarios developed by the Coupled Model Intercomparison Project Phase 5 .Contemporary climate layers (average of the 1960–1990 period) were downloaded from the WorldClim 1.4 database . In addition to elevation, the bioclimatic variables retained represent annual trends , seasonality , and extreme or limiting environmental factors (precipitation of the driest month).To avoid redundancy among variables, the initial set of 20 variables was reduced to a group of seven variables using a correlation matrix implemented with the Q. castanea records were used as training data, and the remaining 30% occurrences were used to validate it. As a threshold‐independent method for model validation, we used the area under the receiver operating characteristic curve (AUC) , binomial outputs calculated using the above‐mentioned threshold rule were summed using the raster calculator tool in ARCMAP 10.4 (ESRI Inc.) and the extent of the stable areas was compared to the entire area of the contemporary distribution model.To evaluate whether the models suggest changes in the distribution of 33.1Q. castanea (Table hS) were higher in the Trans‐Mexican Volcanic Belt  and the Sierra Madre del Sur  than in the Central Plateau  and the Sierra Madre Occidental   and longitude  and decreased with latitude .A total of 90 haplotypes were found in the 36 sampled populations of ea Table  with 29 hS) was 0.725 (0.022), and the total diversity (hT) was 0.989 (0.003). The analysis of molecular variance (AMOVA) showed that most of the variation occurred among populations under the IAM and SMM . The hierarchical AMOVA showed that when distances among haplotypes are not taken into account (IAM), most of the differentiation resides among populations within regions (35.39%), followed by among the four regions (35.13%), and within populations  was 0.630 (0.089) and was 0.266 (0.022) for unordered alleles (GST). The permutation test for the comparison of the two differentiation values was significant (p < .001), indicating phylogeographic structure in the populations of Q. castanea. When phylogeographic structure was analyzed considering the distance between oak populations, there were significant differences observed between NST and GST even at the lower distance intervals. However, it was also observed that phylogeographic structure expressed as NST > GST (p < .001) increased constantly as higher geographic distance intervals were considered  within Q. castanea after 10 independent runs (ln Pr(K = 14)= 1,152.97)  for both regions were 8.34 for the Sierra Madre del Sur and 8.49 for the Trans‐Mexican Volcanic Belt. This finding signifies that the demographic expansion could have occurred in the Sierra Madre del Sur between 5.9 Ma  and 151 ka . Similarly, the most recent expansion in the Trans‐Mexican Volcanic Belt would be dated between 6 Ma and 151 ka.We obtained nonsignificant negative and positive el Table . Howeverur Table . Values 3.4Q. castanea suggests that 19% of the species suitable habitat has remained stable from the LIG to the contemporary period  for the training and test data for the Maxent modeling were 0.971 and 0.963, indicating an appropriate model performance. The jackknife analysis indicated that the climatic variables with the highest relative contributions to the contemporary distribution model were related to elevation and temperature seasonality. The estimation of the distribution extent of  Figures . Such st4The analysis of the evolutionary patterns reported for the Mexican Highlands  is frequently high and has been explained as a product of complex interactions between geographical and environmental factors in several North American and Central American species, such as Q. castanea and other sympatric oak species . Older dates for the demographic expansion seem more credible than younger ones considering the expected long periods needed to allow the accumulation of cpDNA genetic variation. The ENM analyses also support the idea of a stable enough distribution for Q. castanea to maintain large populations, at least since the LIG with moderate range expansions and contractions during the last glacial cycle. Therefore, it is plausible that in response to climate variation, populations of this species mostly experienced altitudinal displacements and a series of expansions and contractions rather than large latitudinal migrations, favoring continuous levels of gene flow and promoting a species distribution rearrangement as it has been proposed for Neotropical species (Bennett, Bhagwat, & Willis, Besides the evidence that geological events predating the early Pleistocene and a complex physiographic landscape explain the phylogeographic structure observed in Q. castanea have occurred within the Trans‐Mexican Volcanic Belt, which is also a hotspot for oak species diversity and has acted as a natural bridge between other biogeographic provinces (Bryson et al., Finally, our results indicate a similar pattern to previous studies in the Mexican highlands that have reported pre‐Quaternary divergence times (Bryson et al., 5Q. castanea, as has been found for other species in the Mexican highlands (Mastretta‐Yanes et al., Our study suggests that together tectonics and paleoclimate have shaped the genetic structure of Authors have no competing interests to declare.JMPR and KO conceived and designed the study. JMPR and VRR performed field and laboratory work. JMPR, HRC, AGR, and KO analyzed and interpreted the data. JMPR, HRC, and KO drafted the article. All authors contributed equally to subsequent drafts and gave final approval for publication.


Drosophila suzukii has recently become an invasive pest insect of significant economic impact in Europe and the USA. In contrast to other Drosophila species, D. suzukii is able to infest intact fruit by means of a saw-like ovipositor, which allows females to deposit eggs beneath the skin of the fruit. Classical biological control using the parasitoid wasp Ganaspis cf. brasiliensis is currently being researched as an environmentally sustainable option for the control of D. suzukii. In particular, the host specificity of this parasitoid has been assessed for populations from different regions in China and Japan. In order to study the relationship between the differences in specificity and molecular variations, we have adapted a matrix-assisted laser-desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based method, originally developed for use with plant material, to discriminate between example populations of G. cf. brasiliensis. We have employed a combination of principal component analysis and blind-tested comparison between reference sample MALDI-TOF MS spectra and test sample spectra to discriminate, on the basis of the acid-soluble insect protein spectra generated, between four populations of G. cf. brasiliensis . MALDI-TOF MS analysis is able to discriminate with 100% accuracy between populations G. cf. brasiliensis. The Chinese populations were observed to be similar, but the Tokyo population is slightly different and the Hasuike population is significantly different from the other populations. The Tokyo population appears more closely related to the Chinese populations than the Hasuike population, even though both originate from Japan.The fruit fly  CHROMASOLVTM LC–MS-grade water was purchased from Fluka .Ethanol ≥99.8%, ≥98% (TLC-grade) α-cyano-4-hydroxycinnamic acid (HCCA) matrix, LC–MS-grade acetonitrile, 99% ReagentPlusG. cf. brasiliensis parasitoids were collected into ethanol and placed on roughly 12  × 4 mm strips of Whatman filter paper, Grade 3. The paper was then folded lengthways, wrapped in cling film and gently tapped over the whole surface using a hammer against a solid surface to transfer insect proteins to the paper. The cling film was then removed, and the paper was unfolded and allowed to air dry for 1 h. The dried and protein-impregnated paper was placed inside a capped 1.5 ml Eppendorf tube, which was stored at 4°C until protein extraction. For Method 1, proteins were extracted by immersing the paper in 200 µl of  (referred to as Solution 1 in the following), capping the tube, briefly vortexing, soaking for 10 min and vortexing again. One microlitre of the supernatant was then pipetted onto the Bruker sample plate, air dried and loaded into the spectrometer. For Method 2, 10 µl of the protein-containing Solution 1 from Method 1 was diluted with 40 µl of Solution 1. One microlitre of this dilution was pipetted onto the Bruker sample plate and air dried, followed by application of a further 1 µl of this dilution, air drying and loading into the spectrometer. For each of the two above-described methods, eight paper-only negative controls were carried out using 6-mm diameter Whatman filter paper, grade 3 discs.Single adult male Eshcerichia coli proteins supplemented with ribonuclease A and myoglobin), using peaks with masses at 3637.8; 5096.8; 5381.4; 6255.4; 7274.5; 10 300.2; 13 683.2 and 16 952.3 for calibration according to the manufacturer’s instructions. Spectra were acquired using MALDI Biotyper RTC Version 4.0 (Build 19) using the manufacturer’s standard settings . Database entries were made as single-spectra Main Spectra (MSPs) using the Bruker Online Client software suite , again using the manufacturer’s standard settings. For spectral comparisons, Bruker identification scores were derived using the standard Bruker algorithm. This first converts raw mass spectra into peak lists, which are then compared between spectra. Three separate values are computed: the number of peaks in the reference spectrum that have a closely matching partner in the test spectrum , the number of peaks in the test spectrum that have a closely matching partner in the reference spectrum  and the peak height symmetry of the matching peaks . The above three values are multiplied together and normalized to 1000, and the base-10 logarithm is then taken to give the final Bruker score (range 0–3). Bruker scores of scores between 2.3 and 3.0 indicate very close relatedness, scores between 2.0 and 2.3 indicate close relatedness and scores <1.7 indicate low relatedness.MS covering the range from 2  to 20 kDa was carried out using a Bruker Microflex LT linear-mode instrument running the MALDI Biotyper 4.0 applications , using a 60 Hz frequency and 3 ns pulse duration nitrogen laser , with a wavelength of 337 nm and spot size of 100 µm, with 240 laser shots per sample. The laser settings were Global Attenuator Offset (0%), Attenuator Offset (25%) and Attenuator Range (30%), and the ion source voltage was 19.98 kV. Bruker MBT Biotarget 96 plates (Bruker ref. 1840375) were used for all samples in this study. Calibration was carried out using the manufacturer’s ‘BTS’ controls  were carried out using the Bruker On-line Client software. PC1 versus PC2 ordination plot were generated, PCA was unsupervised, and all peaks were weighted equally. Differences in Bruker scores between test spectra were analysed for each reference spectrum of a parasitoid population separately using one-way analyses of variance, with the scores as dependent and parasitoid population as independent variable. In all cases, the residuals sufficed the assumptions of normal distribution and homogeneity of variance, as assessed graphically.Original spectral data held on the Bruker Microflex PC is available on request.All eight paper-only controls for sample preparation Method 1 (described in the Methods section) gave no peaks , Fig. S1Using Method 1, the overall accuracy in blind testing was 19.5 correct identifications from 24 tests 81%, , which iThe average Bruker scores obtained from Method 2 resulted in an overall repeat-testing accuracy of 24 correct identifications from 24 tests 100%, , which iIn order to investigate the relatedness between the various samples, the spectra used to derive F3,20 = 342.29; P < 0.0001), Hasuike , Tokyo  and Ximing . A significant effect of the affiliation to different parasitoid populations on the Bruker score was found for all reference spectra: Dali  for hig  differentiation between all populations, which at least indicates the existence of regional biotypes. These tentative conclusions must, however, be treated with caution because, in contrast to DNA sequencing-based approaches, MALDI-TOF MS analysis is not a comparably linear technique because unit changes in DNA sequence  do not necessarily lead to unit changes in protein molecular weight. By referring to the genetic code [We observed clearly separated PCA grouping of the spectra for the Hasuike population and separated grouping of the spectra for the Tokyo population, with the spectra for the Dali and Ximing (Chinese) populations overlapping. While the Hasuike population is well separated from all other populations in the ordination plot, the Tokyo population  appears to be more closely related to the Chinese populations than the Hasuike population. This conclusion is also supported by the spectral comparison data. Interestingly, from the four tested populations, Hasuike is the only one that originates from a site in a temperate climate zone (a ski resort in the Nagano Prefecture), while the other three are all from a subtropical climate zone . The avetic code , at one G. cf. brasiliensis , for which MALDI-TOF MS analysis is able to discriminate with 100% accuracy between populations. The Chinese populations were observed to be very similar, but significant differences were found between the Hasuike population and all others, as well as, in some cases, between the Chinese populations and the Tokyo population. The Tokyo population appears more closely related to the Chinese populations than the Hasuike population even though both originate from Japan.In conclusion, we have successfully adapted the method of Reeve and Buddie, originally developed using plant material, for the analysis of insects. We have employed a combination of PCA and blind-tested comparison between reference sample MALDI-TOF MS spectra and test sample spectra to discriminate, on the basis of the acid-soluble insect-protein spectra generated, between four populations of the potential biological control agent Supplementary DataClick here for additional data file.


Ulmus lamellosa (one of the ancient species of Ulmus) is an endemic and endangered plant that has undergone climatic oscillations and geographical changes. The elucidation of its demographic history and genetic differentiation is critical for understanding the evolutionary process and ecological adaption to forests in Northern China.U. lamellosa via DNA sequencing. All haplotypes were divided into three phylogeographic clades fundamentally corresponding to their geographical distribution, namely THM (Taihang Mountains), YM (Yinshan Mountains), and YSM (Yanshan Mountains) groups. The YSM group, which is regarded as ancestral, possessed higher genetic diversity and significant genetic variability in contrast to the YSM and YM groups. Meanwhile, the divergence time of intraspecies haplotypes occurred during the Miocene-Pliocene, which was associated with major Tertiary geological and/or climatic events. Different degrees of gene exchanges were identified between the three groups. During glaciation, the YSM and THM regions might have served as refugia for U. lamellosa. Based on ITS data, range expansion was not expected through evolutionary processes, except for the THM group. A series of mountain uplifts  following the Miocene-Pliocene, and subsequently quaternary climatic oscillations in Northern China, further promoted divergence between U. lamellosa populations.Polymorphic haplotypes were detected in most populations of U. lamellosa. These results provide important data and clues that facilitate the demographic study of tree species in Northern China.Geographical topology and climate change in Northern China played a critical role in establishing the current phylogeographic structural patterns of The online version contains supplementary material available at 10.1186/s12870-020-02723-7. Examining how historical and contemporary ecological factors contribute to the demographic history and genetic differentiation of plants is a central question in ecology and evolution. Climatic oscillations have dramatically influenced the demographic history and patterns of genetic diversification in many plant species, particularly during the Pleistocene periods with more frequent glacial–interglacial cycles –8. In AsMeanwhile, the potential role of geology in facilitating the population divergence and species diversification of plants has been revealed , 15, 16.Previous researches have revealed different refugia patterns for forest trees in China, such as single refugium, multiple refugia, microrefugia, and cryptic refugia –33. NeveUlmus lamellosa (Ulmus) is primarily distributed across and endemic in Northern China, which is an important tree in many communities and terrestrial ecosystems. The abundance of decylic acid in the seeds of this species makes it a critical medicinal and light industrial resource   was utilized to estimate the expansions time by using g = a + [S/(1 − S)]  , 74.U. lamellosa haplotypes, the divergence time was calculated using BEAST software with a calibrated molecular clock  was selected to date the demographic events in this study [To relate the historic events and differentiation among ar clock , 75–79. ar clock , 81, andis study .Ulmus  were to be calibrated. The analysis was implemented for 50 million generations, with sampling frequency of once per 1000 generations. The log files were run in TRACER [The fossil records for n TRACER  and the n TRACER , 84.U. lamellose [U. lamellosa populations into three geographical groups . First, seven population divergence scenarios were tested with 7,000,000 coalescent simulations: scenario 1 (Simple split model), scenarios 2–4 , and scenarios 5–7 (Isolation with admixture model). For the common parameters between models, the range was the same and the prior distribution was uniform. Based on the observation data closest to 1% of the simulated data, the posterior probability of each evolutionary event was predicted and the confidence interval up to 95% could be obtained by logistic regression. The optimal evolution scenario was filtered by using the highest a posteriori probability (PP) as the selection criterion.ABC analysis (Approximate Bayesian Computation) , 85–87 wamellose . To simpIn order to verify a goodness fit scenario, the “model checking” function in DIYABC software, namely principal component analysis (PCA), was used to analyze the simulated datasets  in the summary statistics. Based on the pseudo-observed datasets , type I and type II error rates was evaluated using the “evaluate confidence in scenario choice” functional option of DIYABC. According the above indices, the ancestral group of the optimal scenarios was further finally inferred.U. lamellosa groups [M = m/μ, where m = migration rate) among groups and the effective population size  of different groups [Nm = θ ∗ M was employed to compute the number of migration per generation (Nm) of each population of U. lamellosa [The coalescent method, implemented in MIGRATE-N software, was employed to investigate the historical gene flow pattern between the three geographical a groups –92. The t groups , 93. Theamellosa , 93, 94.FST calculations. Each replicate was analyzed with 3 long chains and 10 short chains. The static heating treatment scheme corresponded to four temperature levels  and 1000,000 trees was discarded as burn-in. Then, the average of the results of five independent operations, namely the historical gene flow, would be obtained with a confidence interval of 95%.Furthermore, the gene flow analysis was performed under the HKY mutation model. It was run for five replications with constant mutation rates and starting parameters based on U. lamellosa groups. This analysis was ran for 10,000,000 iterations with a sampling frequency of 1000 following a burn-in of 1000,000. The delta values for a , m (migration), and f (inbreeding) was adjusted to 0.5 for guaranteeing the fixed numbers of changes were limited within the range from 20 to 60% of the total number of iterations. The data with the lowest deviance in 20 runs were selected in the further analysis. And the 95% confidence interval was calculated by mc ±1.96 × SD [BAYESASS software was used to calculate the contemporary gene flow , 92 betw.96 × SD .U. lamellosa [To assess the influences of geographic isolation (IBD) and / or environmental conditions (IBE) on the observed genetic differentiation of amellosa , 95, 96,FST estimated from the ITS / Aat sequences was used as the genetic distances matrix. And the geographic distances between populations was calculated by using the GENALEX software [Pairwise software . While tsoftware .2) and their significance were determined based on 9999 random permutations.In addition, a multiple matrix regression with randomization (MMRR) was also performed to test whether the genetic distance responded to variations in the geographic and/or environmental distances via using the R package ‘PopGenReport’ , 100. CoAat sequences from 232 U. lamellosa individuals produced a consensus sequence of 652 and 392 base pairs, respectively. For the ITS sequences, eighteen haplotypes were defined with 27 polymorphic sites, including 17 parsimonious informative sites  were defined, which contained 33 polymorphic sites, 26 of which were parsimonious informative sites  was 0.424 and 0.941, whereas the nucleotide diversity  was 0.00212 and 0.01601, respectively. For the group level based on ITS data, the highest values of Hd ITS (0.909) and πITS (0.00939) existed in the YSM group, followed by the THM and YM groups. On the contrary, the Hd Aat and πAat values of the YSM group were slightly lower than that of the THM and YM groups via Aat data.A high genetic diversity was detected for d value was detected in the different populations for different data . The highest πITS value also existed in the SFS population (0.00583), and the highest πAat value was found in the SGS and HHG populations  constructed using the Bayesian approach, revealed that all haplotypes were clustered into three major clades I-III Fig. , which wAat data, the genetic differentiation between populations was significantly higher when computed using a distance matrix  than when using haplotype frequencies , which indicated a significant phylogeographic structure within U. lamellosa.No matter the ITS or K supported the existence of three clusters  occurred between the three groups  for all groups based on the ITS sequences. Nevertheless, the unimodal mismatch distribution for the THM group was a perfect fit to the expected expansion distribution  of Tajima’s D statistics for the three groups .When oup Fig. . The hapU. lamellosa, all haplotypes could be roughly classified into three lineages, and the divergence times from clade I to clade III were 4.56 Ma (95% HPD: 1.62–8.34 Ma), 2.89 Ma (95% HPD: 1.24–8.68 Ma), and 9.64 Ma (95% HPD: 5.53–13.78 Ma), respectively.This suggested that the intraspecific divergence of all haplotypes most likely proceeded during the period between the Middle-Miocene to the early stage of the Late Miocene. Within U. lamellosa. Scenario 2  and did not overlap with other scenarios . Consequently, Scenario 2, namely the YSM group, might be a common ancestor of the THM and YM groups, which diverged at time t2. Subsequently, the YM group diverged from the THM group at time t1 , and 8.25 × 106 (Aat: 95% CI: 2.56 × 106 _ 12.73 × 106) years with a generation time of 130 years.An ABC framework permitted an evaluation for population divergence scenarios of o 2 Fig. a, b had 5, 95% CI: 1.88 × 105_ 9.52× 105), followed by the YSM group  and YM group . For Aat, the median values of the effective population size were 2.35 × 105 (95% CI: 4.65 × 104_ 8.70× 105) for the THM group, 2.79 × 105 (95% CI: 4.50 × 104_ 9.21 × 105) for the YSM , and 2.17 × 105 (95% CI: 1.56 × 104_ 9.27 × 105) for the YM group. There was no significant difference in the effective population size between all groups based on Aat data.The effective population size was estimated based on the sequencing data. For the ITS data, the THM group has the largest effective population size   between the three groups ranged from 0.008 to 0.260  based on ITS data between the three groups ranged from 0.008 to 0.279 Fig. b. Simila260 Fig. d. The hiFST values between populations (used as genetic distance) were correlated against the respective pairwise geographic and environmental distances . However, the correlations between genetic distances estimated from the ITS /Aat and environmental distance were non-significant (P > 0.05).Pairwise Aat: MMRR ≈ 3.7 times as much).Multiple matrix regression with randomization (MMRR) analyses also revealed a significant effect of geographical distance on the divergence of this species. Furthermore, IBD explained more genetic variation than IBE , which was similar with other plants, such as Gentiana lawrencei var. farreri , and was consistent with the results of Liu et al. [d = 0.783, π = 0.00891) [U. lamellosa was higher than that of ITS sequences and lower than that of Aat sequences  , the genU. lamellosa time-scales [U. lamellosa was widely distributed in Northern China  over ancestral populations (YSM group). Furthermore, a higher gene flow occurred from THM to YM  and H4 (Aat data) in the populations  likely began during the period between Middle-Miocene and the early stage of the Late Miocene  to the Taihang Mountains .The intraspecific divergence of all ene Fig. . This esU. lamellosa migration, from the northwest and northeast, which is supported by the more frequent gene exchange between the three groups    and 3.25 Ma  uplifted in Northern China once Tertiary and Quaternary climatic oscillations blocked gene exchange and further promoted the divergence of populations.Higher genetic diversity and significant genetic variations occurred in U. lamellosa were suggested based on geological evidence and paleoclimate data. The results in this study may provide insights into how the endangered U. lamellosa species might be protected and utilizing. Further, they may play an important role in the exploration of genetic differentiation patterns and dynamic changes of other tree species, while offering a scientific basis toward understanding the evolutionary history and ecological adaption of forests in Northern China.In summary, the causes of the phylogeography pattern of Additional file 1: Table S1. Locations and sizes of individuals in the sampled populations of Ulmus lamellose.Additional file 2: Figure S1. The maximum likelihood tree of single-copy nuclear gene Aat haplotypes.Additional file 3: Figure S2. Results of Bayesian clustering analysis conducted by STRUCTURE. The clustering patterns of ITS (a) and Aat (b) by three clusters (K = 3). The clustering patterns of Aat (c) by four clusters (K = 4).Additional file 4: Figure S3. Correlation between geographic distance and pairwise FST/environmental distance for Ulmus lamellosa. (a), (b): relationship estimated by ITS and (c), (d): relationship estimated by Aat.