Solenopsis invicta and Solenopsis richteri, both native to South America, both inadvertently introduced to North America in the early twentieth century. While the two species are known to admix in the introduced range, in the native range no hybrids were found.A major focus of evolutionary biology is the formation of reproductive barriers leading to divergence and ultimately, speciation. Often, it is not clear whether the separation of populations is complete or if there still is ongoing gene flow in the form of rare cases of admixture, known as isolation with migration. Here, we studied the speciation of two fire ant species, S. invicta populations, we evaluated their divergence times and historic and contemporary population sizes, including the original founder population in North America, which was estimated at 26 (10–93) unrelated singly-mated queens.We conducted a population genomic survey of native and introduced populations of the two species using reduced representation genomic sequencing of 337 samples. Using maximum likelihood analysis over native range samples, we found no evidence of any gene flow between the species since they diverged. We estimated their time of divergence to 190,000  generations ago. Modelling the demographic history of native and introduced We provide evidence for complete genetic isolation maintained between two invasive species in their natïve range, based, for the first time, on large scale genomic data analysis. The results lay the foundations for further studies into different stages in the formation of genetic barriers in dynamic, invasive populations.The online version of this article (10.1186/s12862-019-1437-9) contains supplementary material, which is available to authorized users. The separation of populations and their eventual speciation is a fundamental process and a driving force in evolution. However, a newly formed reproductive barrier is often incomplete. Hybridization and introgression between incipient species, or gene flow from one species’ gene pool into another by backcrossing, may still occur. Many cases of species hybridization, resulting from accidental introduction of foreign species into new environments, have been identified and documented –3. OftenHere, we describe one such case study involving two invasive fire ant species. These species, thought to maintain genetic isolation in their native range –6, freelSolenopsis invicta, and the black fire ant, S. richteri, are closely related species, native to South America. Both species were inadvertently introduced to North America in the early twentieth century [S. invicta subsequently migrating to other countries worldwide [S. invicta, amongst the most studied invasive species, and an excellent subject for demographic history analysis.The red fire ant,  century –11, withorldwide , 13. SinS. invicta and S. richteri’ display social polymorphism, with two distinct social colony structures – the monogyne (single queen) and the polygyne (multiple queens) forms. The social polymorphism is a Mendelian trait determined by a supergene of 13 megabases, and is marked by the gene gp-9 with monogyne queens always having gp-9BB genotypes and polygyne queens always having gp-9Bb genotypes [enotypes –16. The enotypes , 18.S. invicta colonies around the world were used to trace the place of origin of the introduced S. invicta populations to the Formosa region in northern Argentina [S. invicta across the globe originated in the introduced USA population. The population of origin of the introduced S. richteri is still unknown.Population genetics analysis of microsatellite and mitochondrial genotypes of thousands of rgentina , 13. It S. invicta queens that were initially introduced to North America was estimated through the screening of haplotypes in mtDNA sequences, as well as genotypes of allozymes, microsatellites, and the complementary sex determination (CSD) locus in individuals sampled throughout the introduced range [The number of ed range . It was S. invicta and S. richteri is prevalent [In the introduced range, admixture between revalent , 8. A hyrevalent , 20, 21.revalent , or not revalent –6. WhileS. invicta and S. richteri. Our data include samples from both species in both native and introduced ranges. The hybrid zone was not included in the sampling because the admixture in the introduced range has been well established in previous studies [S. invicta and S. richteri in their native range to determine if these species had indeed maintained genetic isolation since their separation. We also provide estimates for speciation times, population divergence times and historic and contemporary effective population sizes, including the founding S. invicta population in North America. This is the first demographic history study of these species that uses thousands of genomic markers, which allow population genetic model inference at high accuracy and provide the necessary statistical power to test for gene flow.We used large scale population genomic data to study the demographic history of  studies , 8 . TheS. invicta and S. richteri from nine localities in their native range in Argentina and their introduced range in the USA  were assigned to a fourth cluster. Individuals of the third native S. invicta population, sampled in Herradura, were partially assigned to the cluster of the introduced S. invicta populations and partially to a separate, fifth cluster.TRUCTURE , 23 is aS. invicta is a subset of the genetic variants found in the native Herradura samples, making the latter a possible population of origin of the former. This result is in line with the previous studies that identified the Formosa region, which includes Herradura, as the likely source of the introduced S. invicta [S. richteri populations share a cluster with the introduced populations, indicating that these are not the source population of the introduced S. richteri.The allocation of populations into clusters suggests that the polymorphism in the introduced  invicta , 13. UnlFST. The results can be found in Additional file We calculated the genetic distances between each pair of populations, using the genetic differentiation measure of S. invicta and S. richteri populations by comparing between two competing demographic history models. To this end, we used a full maximum likelihood analysis of coalescent models [3s software [We tested for historic and contemporary gene flow between the native t models , 25 as isoftware . Based oΔlnL = 0.06; p > 0.9). Consequently, the null model could not be rejected, indicating that there was no evidence of gene flow between the native S. invicta and S. richteri populations since their divergence from a common ancestral population.This analysis concluded with an insignificant difference between the likelihood scores of the two scenarios  that was estimated for the honey bee [S. invicta and S. richteri happened 1.9*105 (1*105–3.5*105) generations ago and the speciation of S. fugax from the lineage leading to S. invicta and S. richteri took place 2.5*106 (1.6*106–4*106) generations ago. See Fig. es Table . Using toney bee , 29, we S. invicta and S. richteri, while the ancestral population of these two populations was found to be slightly smaller. The effective size of the ancestral population of the three Solenopsis species was found to be larger than the other populations by an order of magnitude, indicating a very diverse ancestral population. See Fig. The estimates for the effective population sizes were similar between present-day populations of S. invicta populations using Approximate Bayesian Computation (ABC) [DIYABC v2.0.4 [S. richteri populations (poor goodness of fit). This may be because we did not sample the source population of the S. richteri introduction.We estimated the demographic and temporal parameters that define the history of on (ABC) , as implC v2.0.4 , 32. UnfS. invicta are already established, an alternative scenario is unnecessary. Rather, we concentrated on estimating the model parameters of population sizes and time of demographic events as depicted in Fig. An ABC analysis is often used to compare between multiple competing historic demographic models that differ in their scenarios. As many details in the history of S. invicta (TB) is minimal and stands at 2 generations for the maximum posterior estimate, with 95% credible interval ranging between 0 and 16 generations. Combined with the small size of the founder population, this suggests a rapid increase in population size immediately following the introduction. It is in line with historic records in which the presence of the newly introduced fire ants was initially reported as a mere curiosity and quickly changed into a growing concern and even panic, as ant numbers exploded [With a maximum posterior of 39 , the effective size of the founding population is quite low. The length of the bottleneck period of the introduced exploded .NN1) is approximately two orders of magnitude larger than the second native population cluster (NN2) estimated at 1.2*107 and 2.5*105, respectively .The effective population size of the Herradura population  indicated that genetic isolation may be incomplete in the native range . By analdmixture . Our conWolbachia – an endosymbiotic bacterium that infects high proportion of insects [Drosophila [Wolbachia was detected in native populations of S. invicta and S. richteri, but not in the introduced populations [Reproductive isolation between related species is maintained by endogenous (genetic) and/or exogenous  factors . One exp insects , and manosophila . As Wolbulations  it is a ulations . Anotherulations . In factulations –40. Moreulations , 42.S. invicta and S. richteri combined with overlapping generations and multiple nuptial flights throughout the year, make the length of a generation highly variable and difficult to pinpoint. Ross and Shoemaker (2008) have given an estimate of three to six years, based on the relative number of alates produced in different developmental stages of a colony [S. invicta and S. richteri can be estimated to be 1.1 (0.6–2.1) million years ago (MYA) and the speciation time of S. fugax and S. invicta and S. richteri to 15 (9.6–24) MYA. The high end of this estimate is just under the 25 million years  inferred for the divergence of these lineages, based on Bayesian phylogenetic analysis and divergence dating using 27 fossil calibration points [3s, is for the separation of the species (i.e. the formation of genetic isolation). By definition, the divergence between the representative individuals must predate the formation of reproductive barriers that led to speciation.We gave estimates of the number of generations since the two speciation events, which can be converted to years by multiplying them with the average generation time. The social polymorphism of a colony . They len points . It shouDIYABC and 3s, which is the native S. invicta populations cluster, that of Alejandra and El Recreo. Both methods gave similar estimates for it (2.5*105 and 4*105 respectively). It is approximately two orders of magnitude smaller than DIYABC ‘s estimate of 1.2*107 given for the other native population cluster, the Herradura population. This suggests a much larger and more diverse population around Herradura in comparison to the southern population sampled at El Recreo and Alejandra. Of those three locations, Herradura is closest to Corrientes, which was highlighted as the most genetically diverse among the populations that were sampled across South America [There is one parameter shared by the two different coalescent-based methods of  America .S. invicta USA founder population (NF) was estimated at 39. While for diploid species the effective population size is Nf and Nm are the number of breeding females and males, respectively), for haplodiploid species, the reduced number of chromosomal copies in males results in Ne = 1.5Nf for species in which the queen mates with a single male, as is the case with S. invicta [S. invicta queens is between 10 and 93.The effective population size of the m2Nf+4Nm , 46. Thi invicta . TherefoWe described a unique study system in which the reproductive barrier between two closely related species is maintained for approximately 200,000 generations in their native range and breached in an introduced range. This setting allows sampling from clearly separate pure populations in the native range and admixing populations in the introduced range. Therefore, this is a powerful system for the study of the molecular basis of reproductive isolation, which is key to the process of speciation.S. invicta and two S. richteri populations in the native range in Northeastern Argentina; two S. invicta and two S. richteri populations in the introduced range in Southeastern USA. The specimens were identified by morphological characteristics by James Pitts as described before [Samples were taken from nine ant populations: three d before . For eacSeven restriction-site associated DNA (RAD) libraries were constructed from the sampled populations, with 31–68 individuals in each library. DNA was extracted from the samples and RAD libraries were constructed based on the protocols of Baird and colleagues  and EtteStacks pipeline [S. invicta reference genome  using B[Bowtie2 . For eaclejandra .Stacks pipeline and created a catalog containing 285,847 SNPs for the combined seven RAD libraries, with measured coverage of X16 on average. These SNPs were used to calculate the FST distances between the populations.We analyzed the mapped sequences for single nucleotide polymorphic sites (SNPs) using the We filtered the SNPs catalog further before its use in the population structure analyses. For each locus, we required genotype calls in at least 80% of the samples in each population (i.e. less than 20% missing data); a minimum of three reads made for a genotype call. Additionally, we required a minimal minor allele frequency (MAF) of 1%. This stringent filtering resulted in a collection of 16,759 high confidence SNPs. Finally, we removed samples with more than 30% missing data , leaving 300 samples in the analysis.STRUCTURE over K (number of expected clusters) values of 2–9, 100 times for each K, with 100 different sets of 1000 SNPs randomly chosen out of the high confidence SNPs collection, a total of 800 separate runs. To avoid linked sites from affecting the analysis, we required a distance of at least 5000 bp between the selected sites. Multiple runs over various data subsets contributed to the robustness of the clustering inference and allowed us to use more SNPs than would be possible in a single run. We ran STRUCTURE for 1,100,000 MCMC repetitions and discarded the first 100,000 (burn-in period). All the other parameters were kept at default values. The results were analyzed using CLUMPAK [S. richteri separated to two distinct clusters.We ran  CLUMPAK . The STR CLUMPAK  to identS. invicta and S. richteri have been maintaining complete genetic isolation in South America since their divergence. The alternative to this scenario is that these incipient species have had some form of gene flow between them, including isolated successful mating events (isolation with migration). We used 3s program, which is a coalescent-based maximum likelihood inference tool. The program analyses aligned sequences of three species, two that are closely related and a third which is used as an out-group, for the genealogical process of coalescence and migration. Based on this the maximum likelihood scores of two possible demographic history models are compared, a null model that does not allow genetic flow between the two closely related species and an alternative model that does.We tested the hypothesis that 3s provides estimates for the times of the two speciation events and for historic and contemporary population effective sizes. Speciation times are inferred from the model parameter τ – the average number of substitutions per site since speciation. Effective population sizes are represented by the θ parameter. In diploid species, θ = 4μNe, where μ is the mutation rate and Ne is the effective population size. For a haplodiploid species, θ = 3μNe because of the ploidy ratio between males and females.In addition, 3s, we investigated the demographic history of the closely related S. invicta and S. richteri species with the thief ant S. fugax as an out-group  mutations per site per generation. This mutation rate was calculated using direct measurement of two generations in three colonies and is similar to the mutation rate inferred for other insects [Using 0000000; ). To giv insects , 57.S. richteri individuals from the populations of Las Flores and Buenos Aires (R1 and R2 respectively) and two S. invicta individuals from the populations of Alejandra and El Recreo (I1 and I2 respectively). All four chosen individuals were of the gp-9BB genotype of the social chromosome. Using Bowtie2, we aligned their RAD reads to the reference genome of S. invicta and to a fully sequenced S. fugax individual, allowing no gaps in the alignments. We retained only RADseq reads with no more than 4 mismatches compared to the S. invicta reference genome and RADseq reads with no more than 10 mismatches compared to the S. fugax genome. The mismatches cutoffs are based on the average of 95% sequence identity between S. invicta and S. fugax [The program requires only a small number of individuals with a large number of loci to represent each species. We arbitrarily chose two S. fugax . We alsoS. fugax and one of following pairs of sequences at random: I1 and I2, R1 and R2 or R1 and I2. As the algorithm assumes no linkage disequilibrium (LD) between sites, we only allowed loci that were at least 2000 nucleotides apart. This produced 15,040 triplets of 96 bases long three-ways alignments, to be used as input for 3s software.We assembled for each locus an alignment of three sequences composed of the sequences of the outgroup of 3s and calculated the maximum likelihood values for the two models using the Gaussian quadrature number of points = 16. To examine the robustness of the estimates we ran each of the models three times, with different seed values at each run. We used the likelihood scores and the parameter values that were obtained for the highest scoring run of each of the models and compared them in a Likelihood Ratio Test (LRT).We ran S. invicta population clusters identified by STRUCTURE, for the demographic history model depicted in Fig. DIYABC uses an approximate approach: it runs a series of coalescent simulations over a demographic history model, which includes an historic scenario and parameter prior distributions. The program estimates the posterior distributions for the model parameters based on how well the simulated data fit the observed genetic data (represented by their summary statistics).Using approximate Bayesian computation (ABC), we estimated divergence times and population sizes of the three STRUCTURE analysis found the two native S. invicta populations of Alejandra and El Recreo indistinguishable in term of their genetic polymorphism and so they were assigned to one population, while Herradura’s population was clustered separately. The introduced populations were also clustered together in one population. We formulated the demographic history model used for the ABC analysis with these three distinct population clusters.Our NN1 and NN2, diverged from each other TD generations ago, prior to the introduction to the USA, which took place TI generations ago. As the introduced populations in the USA were found to originate from the region of Formosa (which includes Herradura), in our scenario the founder population, NF, is directly derived from population NN1, and their divergence time coincides with the time of introduction. The scenario also includes a population bottleneck effect after the introduction, which lasted for TB generations and concluded as the size of the population reached its contemporary size, NI.In the historic scenario, depicted in Fig. DIYABC sizeable sampling range, encompassing all reasonable values of these parameters.Coalescent model parameters are either demographic, reflecting population effective sizes, or temporal, indicating the number of generations that had passed between events. We defined parameter priors of normal distributions with averages based on known life history of the fire ants. Prior distribution widths were set widely to allow S. invicta predates sampling time by at least 80 years. Generation time for the fire ants, especially at the time they were dispersing throughout the newly introduced range, is hard to estimate. We therefore defined a prior of introduction time that ranges between 0 to 180 generations. Based on studies indicating that the original number of introduced S. invicta was small, we defined a prior for the effective population size that ranges between 1 and 500 individuals. The ants’ quick expansion in North America suggests a minimal time span of the population bottleneck. This allowed us to narrow the prior distribution of the bottleneck time between 0 and 40 generations. Not much is known about the effective sizes of the native and introduced populations or of the time of divergence between the native populations, and these parameter priors were kept at a wide distribution, which included many orders of magnitudes.The introduction time of An ABC analysis depends on population summary statistics to reduce the high-dimensional genetic information and to evaluate the simulation results. A varied choice of summary statistics is therefore crucial. However, using too many would increase the dimensionality of the analysis, making the available observed data points too sparse in comparison ; a problNN1 and NN2 populations are presumed stable and well represented by their summary statistics. We therefore used all of the within-population summary statistics offered by DIYABC: the proportion of monomorphic alleles, mean and variance of gene diversity of polymorphic loci [NI, a newly established population, we only used the summary statistics of the proportion of monomorphic alleles and mean gene diversity across all loci.The native hic loci  and meanNN1 & NN2 and NN1 & NI): the mean FST distance, the proportion of null FST distances [FST distances.We included between-populations summary statistics for pairs of populations that directly diverged from each other  removed, leaving 15,539 SNPs in the analysis. Populations as before. Figure S3. Evanno analysis for best K. Table S3. - FST between populations. Table S4. Posterior distributions of S. invicta demographic history model parameters. Figure S4. Goodness of fit analysis result for S. invicta demographic history model. (DOCX 446 kb)


Xenochrophis tytleri), an endemic, terrestrial and freshwater snake species in the Andaman archipelago, a continental group of islands in the Bay of Bengal. Data was collected from 86 individuals from seven islands and 78 individuals were sequenced for the gene Nuclear Dehydrogenase subunit 4 to identify the number of populations and distribution of genetic diversity across populations. We found 11 haplotypes on seven islands and observed high genetic differentiation between seven populations defined island-wise (FST = 0.82). We further tested the number of populations by incorporating spatial data into Bayesian Clustering Analysis (GENELAND) and identified six populations of the Andaman keelback. We tested for the influence of Isolation-by-distance on these populations. While the overall trend showed a positive correlation between geographic and genetic distance, a correlogram revealed that the positive correlation disappears beyond ∼20–40 km. We also tested for the presence of geographical barriers to gene flow using Monmonier’s algorithm (SPADS), which identified five barriers to dispersal confirming that there are oceanic barriers to dispersal for some island populations of the Andaman keelback. As the Andaman Islands are arranged almost in a straight line from North to South, our data are insufficient to tease apart the roles of geographical distance and barriers to gene flow. We conclude that salt waters between near islands are weak barriers and as the geographical distance between islands increases, so does the strength of the barrier.Limited gene flow between populations due to geographic distance, presence of barriers or inherent low dispersal ability leads to the formation of genetically structured populations. Strong population structure indicates lowered levels or absence of gene flow which might lead to inbreeding and loss of genetic capacity to recuperate from anthropogenic stress and natural calamities. Terrestrial reptiles are generally known to have low dispersal abilities and few studies have explored drivers of their population structure on continental islands, where both anthropogenic stress and natural calamities are relatively common. We investigated the population structure and drivers of diversification of the Andaman keelback ( Limited gene flow between populations of organisms due to distance, barriers or low dispersal ability is known to result in genetically structured populations. The insularity of islands and variation in biotic and abiotic conditions makes them ideal natural laboratories to test population biology theories. Islands present some of the most diverse outcomes of evolutionary processes and have therefore been instrumental in understanding evolution and speciation . In the To explain speciation and genetic variation in space, the concept of isolation-by-distance (IBD)  and the In general, the dispersal patterns of terrestrial reptiles are poorly understood , despiteThe Andaman Islands in the Bay of Bengal are an extension of the Arakan Yoma mountain range; this chain of submarine mountains extends from Cape Negrais of Myanmar to Achin head in Sumatra . These iXenochrophis tytleri; Xenochrophis across South and South-east Asia. Absence of Andaman keelback in brackish water could be a case of false-negative, however, lack of proof until now forces us to consider this species as exclusively freshwater.An increasing number of studies have focused on the phylogeography and population genetics of reptiles in South and Southeast Asia; however, intraspecific studies in archipelago systems are lacking. The Andaman keelback  identify the number of populations of the Andaman keelback, (ii) estimate the genetic connectivity between populations and (iii) understand the effect of IBD and barriers to geneflow between populations.2 including areas in South and Middle Andaman Islands  location (using a GPS Trimble Juno 3) with an accuracy of 2–5 m. When individuals were found on stream banks and wet soil, we captured them with the help of a small stainless-steel hook and bagged them into breathable cotton bags. When individuals were in water, we lifted them from water with both hands and then used the hook to hold them. Captured snakes were restricted in a transparent restraining tube (both ends open). We ensured that the sample collection took minimum time and always provided body balance to individuals. This handling protocol was approved by the Department of Environment and Forest, Andaman and Nicobar Islands Research Advisory Committee and adheres to international Animal Care and Use guidelines.We determined the sex of each individual using sterilized and lubricated sexing probes . We used sterilized surgical steel scissors to cut the tail tip (up to 15 mm from the tip). The snakes were not anesthetized while the tail tip was removed. We stored the tissue sample in 1.5 ml vials filled with molecular grade ethanol. After collecting the tissue, we sterilized the open wound and allowed the individual to settle for a few minutes before releasing them at the capture site. The time taken in handling individuals did not exceed 10 min. The tissue samples were deposited at the Centre for Ecological Sciences, Indian Institute of Science, Bangalore (CESS 1600-CESS 1686). Funds were not available for post release monitoring of sampled individuals. However, individuals of the Andaman keelback sampled in our research base station, Andaman and Nicobar Environmental Team (ANET), a long-term monitoring site, showed no signs of side effects after sampling. These individuals could be identified by their excised tail tip and individuals were frequently sighted in the same areas they were found in prior to sampling.We extracted genomic DNA from 86 Andaman keelback individuals from 38 locations  using the phenol chloroform method . To testd) in DnaSP 5.10.01  to assess the level of population subdivision and obtained population pair wise d groups . IndividFST and shortest geographical distance between a pair of populations. This was calculated by evaluating Tamura-Nei pairwise genetic distances between all individuals belonging to different populations using MEGA 7.0  from 2 to 14; the number of identified clusters saturated at 6. We then fixed K at 6 to estimate the other parameters included in the model, including number of populations, population membership probability of individuals, map of population membership and posterior density of the model.We assessed geographical structure and the number of populations using GENELAND with the Markov Chain Monte Carlo (MCMC) method . We perfFST generated in Arlequin 3.5.2. Single locus average FST estimator computed using Monmonier’s algorithm in SPADS for both the analyses were visualized in the software Barriers . To test whether areas of cluster transition observed in the map of population membership was an artefact of clustering, we tested for the presence of barriers. We determined the barriers by employing Monmonier’s maximum difference algorithm  in the pBarriers . We repeHd = 0.86) with a total of seven variable positions and six haplotypes from CytB (Hd = 0.75) with a total of nine variable sites , although positive, was statistically not significant. Pairwise genetic distances ranged from 0 to 0.02. The gene ND4 has unique haplotypes in most islands, but there are also common haplotypes shared between islands (We obtained seven haplotypes of 16S (le sites . Pairwisositions . The out islands . Middle FST) and among populations within groups (FSC) were obtained . Individual genetic and geographical distances showed a weak positive correlation   explained almost half the variance (r = −0.4 p = 0.003)  , but thevariance . In the = 0.003) .K = 6), we obtained a map of population membership . We detected six different haplotypes, two of them unique to the island and four others shared with four different islands namely Havelock, Long Island, Baratang and North Andaman Island. South and North Andaman Islands are about the same size and could have more haplotypes which were not detected due to insufficient sampling. This study detected both shared and unique haplotypes in both 16S and CytB markers, but this is based on very few samples, so no inferences can be made.The highest genetic diversity in the ND4 gene was detected on Middle Andaman Island which could be due to its central location and its large area  and among populations within groups (FSC)  . This reps (FSC) . This isps (FSC) .Each sampled population of the Andaman keelback displayed low genetic diversity, and were highly differentiated due to limited gene flow and dispersal. As we detected no selection for alleles of ND4 gene in these populations, the genetic differentiation observed among populations is likely to be purely a result of genetic drift.Observed positive correlations between genetic and geographical distances would appear to indicate that IBD is driving diversity patterns . HoweverPopulations of the Andaman keelback identified using both genetic and spatial data assigned individuals to six population clusters  reflectiInference about gene flow patterns between two populations can be made easily in the presence of sharp geographic boundaries between them . We idenWhile most barriers are intuitive, there are some caveats. The barrier between South Andaman (barrier a) and the rest of the northern islands  could beStudies focusing on LGMs and the Pleistocene period have reconstructed maps to look at the influence of lowered levels of oceans on coastlines and vegetation patterns around the world. These maps clearly show that the Andamans was a continuous landmass during lowered sea levels . All theAs the shared haplotypes between islands do not always concur with their geographical proximity, other factors could have influenced gene flow. The Andaman Islands experience regular cyclones and strong monsoon winds which could facilitate over water dispersal. In addition, we cannot ignore the possibility of human mediated dispersal because there is a considerable amount of boat traffic and, historically, the Andamanese tribe are known to have used canoes to travel between islands . DespiteConsidering the ever-increasing challenges for conserving diversity in the tropics, there is a critical need for studying the genetic diversity of populations and factors influencing the spatial distribution of diversity. This is even more pronounced for reptiles, as 95% of reptile species extinctions have occurred on islands . This stDespite limitations in the number of markers and sampling, our study shows that populations of the Andaman keelback on the Andaman Islands are well differentiated, with reduced gene flow because of both IBD and salt water barriers to gene flow. While this pattern can be extrapolated to other terrestrial/freshwater reptiles in the Andaman Islands, we propose that future phylogeography studies can open avenues to comparative phylogeographic studies which are important to understand the impact of islands’ geological histories on contemporary population structures in combination with species specific traits.10.7717/peerj.5752/supp-1Supplemental Information 1Sheet 1: Sample ID, corresponding gene, Bankit ID and sequence accession numbers; Sheet 2: Sample ID, population membership and population name; Sheet 3: Morphological data used for PCA analysis.Click here for additional data file.10.7717/peerj.5752/supp-2Supplemental Information 2H 1 to H 7: labels of each haplotype. Number of ticks on the network between haplotypes correspond to number of base pair differences between them.Click here for additional data file.10.7717/peerj.5752/supp-3Supplemental Information 3A: Populations identified by Geneland, B: Sex.Click here for additional data file.


Plantago lagopus populations along the drove road with populations in the surrounding agricultural matrix, at varying distances from human settlements. We observed significant differences in coefficients of inbreeding between the drove road and the agricultural matrix, as well as significant trends indicative of higher genetic diversity and population nestedness around human settlements. Trends for higher genetic diversity along drove roads may be present, although they were only marginally significant due to the available sample size. Our results illustrate a functional landscape with human settlements as dispersal hotspots, while the findings along the drove road confirm its role as a pollinator reservoir observed in other studies. Drove roads may possibly also function as linear structures that facilitate long-distance dispersal across the agricultural matrix, while local P. lagopus populations depend rather on short-distance seed dispersal. These results highlight the role of herbivore corridors for conserving the migration capacity of plants, and contribute towards understanding the role of seed dispersal and the spread of invasive species related to human activities.Habitat fragmentation is one of the greatest threats to biodiversity conservation and ecosystem productivity mediated by direct human impact. Its consequences include genetic depauperation, comprising phenomena such as inbreeding depression or reduction in genetic diversity. While the capacity of wild and domestic herbivores to sustain long-distance seed dispersal has been proven, the impact of herbivore corridors in plant population genetics remains to be observed. We conducted this study in the Conquense Drove Road in Spain, where sustained use by livestock over centuries has involved transhumant herds passing twice a year en route to winter and summer pastures. We compared genetic diversity and inbreeding coefficients of  Increased habitat fragmentation has been perceived as a major worldwide threat for ecosystems and biodiversity for the last decades . FragmenDispersal is a central life-history trait that structures the properties of ecosystems . ParticuDrove roads—routes traditionally used for mobile pastoralist livestock in many regions of the world—become particularly interesting to promote functional connectivity between isolated grasslands. Historic ones are presumed to often derive from ancient migratory routes of wildlife  Drove roAnthyllis vulneraria fragments historically subjected to rotational grazing (Bromus erectus). More recently, however, rotational grazing livestock has been observed to genetically link island grassland communities of Campanula rotundifolia that are otherwise isolated  Is the CDR activity influencing gene flow among populations? (ii) Do the populations in the CDR have similar genetic diversity and/or inbreeding values than those located in the agricultural matrix? (iii) Can other landscape factors  modify the genetic structure of the P. lagopus populations?In this study, we aimed to check whether dispersal processes in drove roads subjected to active use influence the population genetic structure of oad CDR;  in Spainoad CDR; . The CDRPlantago lagopus L. (Plantaginaceae) is a diploid polycarpic annual or biennial forb, abundant in grazed annual-dominated pastures of Central Spain . Populations located in the agricultural matrix were located at least five km from the CDR. Each population consisted of a group of plants not further than 100 m from each other, and all populations were at least five km apart from each other .In winter 2013 we selected 13 populations of ch other ; Table 1l fields . AuthoriTo test for the potential effect of other landscape factors on genetic descriptors, we measured the Euclidean distance from the sampling points to the nearest human settlement using Google Maps. At the agricultural matrix points, we also checked the time since last plowing by inspecting historic aerial photographs from the area —the oldest of which being 70-years-old. The CDR, as in the case of all other major drove roads in Spain, has been protected from plowing for the last centuries .P. lagopus in collaboration with the Unidad de Genómica, Parque Científico de Madrid (http://fpcm.es/en/servicios-cientificos/). Briefly, the library was developed from DNA of five individuals of P. lagopus extracted using QIAGEN plant Extraction Kit, using manufacturer’s protocol. DNA was fragmented with ultrasounds using Bioruptor  and size fragment was selected for an approximated size of 1.2 kb. DNA sequencing was performed using a 454-FLX-Plus Roche sequencer. We obtained approximately 185,000 sequences. We used Pal_Finder to select those sequences with more than five repetition motifs and a size that ranged from 100 to 300 bp. We used Primer3 (We developed specific microsatellite markers for  Primer3  to confiHO and HE) using INEST 2.1 (AP) using GeneAlEx 6.5  equilibrium with GeneAlEx 6.5  using Bottleneck 1.2.02  with 1,000 permutations. To assess the presence of isolation-by-distance (IBD) patterns, we performed a Mantel test with 999 permutations using genetic differentiation  and Euclidean geographical distance between populations with Vegan package in R , ranging from 1 to 15. Each run comprises a burning period of 100,000 permutations, followed by 1,000,000 Markov Chain Monte Carlo iterations. We assumed correlated allele frequencies and genetic admixture. To assess the most plausible number of clusters (K), we used the STRUCTURESelector module (K. We considered the Evanno method (K and identified the maximum ΔK when plotted against K. MedMeaK (median of means), MedMedK (median of medians), MaxMeaK (maximum of means) and MaxMedK (maximum of medians) following We assessed genetic structure using the Bayesian clustering method STRUCTURE v. 2.3.4  with prir module  to obtaio method  with theFIS and rA  to examine the relationship between genetic diversity metrics with the population location , the distance to nearby human settlements (in km) and the interaction location × distance.To assess whether movement of transhumant herds along the drove road leaves a footprint, we selected the genetic descriptors that have most commonly been used previously in similar studies, i.e., S and Ar . We builFIS the amount of grasslands in 500-m radius circles centered on sampling points  was also included. The landscape descriptors (distance and percentage of grasslands) were standardized and centered to interpret model coefficients as standardized effects. Spatial autocorrelation was taken into account by building the GLS models with three alternative spatial covariance structures most commonly used to describe spatial correlation . The presence of nearby grasslands was included for the analysis of FIS, for this genetic descriptor is considered to be particularly influenced by pollinator activity due to its effect over selfing and/or kinship crosses  and for nestedness, we did not try to simplify the models but to identify the relevant predictors with p-values based on nested log-likelihood ratio tests. The genetic descriptors we used as response variables in the models were the inbreeding coefficient (FIS), the genetic diversity statistic (rA) and the nestedness estimates. We confirmed nearby grasslands to be only a relevant factor for FIS by comparing models of the three metrics with and without the grassland factor via AICc. Additionally, we tested the correlation of the genetic descriptors with the plowing history (years since last plowing) with base R and Anova function, car package and just for the populations in the agricultural matrix—the drove road having been protected from plowing for the last centuries.For the three genetic diversity metrics  were similar between populations in the agricultural matrix and populations in the drove road, except for AP, which was greater in the populations of the agricultural matrix, due to the large NA found in LMA08 (HE was 0.88 (0.02) in drove roads populations and 0.89 (0.03) in matrix populations. However, matrix populations showed greater variability in HE, with values ranging from 0.92 in LMA13 to 0.84 in LMA08. The same pattern (similar average but different variability) was found in the other genetic diversity indexes  varied greatly among populations and between matrix and drove road locations. Drove road populations showed a mean value of 0.08 (ranging from 0.01 in LCR04 to 0.17 in LCR01), while matrix populations showed a mean value of 0.11 ranging from 0.06 in LMA05 to 0.17 in LMA14 . Bottleneck signals were found in some populations under SMM model , but the results were not significant in all genetic models and when applying multi-comparisons corrections (The inbreeding coefficient (in LMA14 . Inbreedrections . LinkageFST) between populations were low  and a moderate one between populations within each category (FSC = 0.11). Both values are significant. Mantel test  did not detect the presence of an IBD pattern, but results were significant when only analyzing the six populations included in the drove road . The genetic structure analyzed with a Bayesian clustering approach  for LMA14 population, another cluster (purple) for LMA16, LMA13 and LCR10 populations and a third cluster (orange). The other populations are assigned to the last cluster, with admixture of the others. The K = 12 distribution showed an important population admixture and one cluster assigned to LMA14 population .The distance to the nearest settlement had an impact on both genetic descriptors analyzed . This reFIS; FIS increased with distance from human settlements in the agricultural matrix, but followed the opposite trend in drove road populations , the distance to settlements had a consistent negative and significant effect, while the effect for population position and its interaction with distance was only marginally significant . None of the geographic parameters, however, significantly affected the rank (Significant nestedness was found among the the rank .P. lagopus populations in the study area. This assessment of the population genetics of P. lagopus in the study area indicates relevant differences both in terms of coefficient of inbreeding and of genetic diversity between populations and between matrix and drove roads positions. Furthermore, proximity to human settlements appeared to drive the hotspots for genetic diversity in this landscape. Drove roads were able to invert the inbreeding that otherwise appears in distant populations from the agricultural matrix. The loss in genetic diversity with increasing distance to human settlements seems weaker at drove roads, although such effect is only marginally significant with the sample size and the molecular markers used in this study. Such a trend would, however, be in line with the one that was observed by other studies involving livestock being moved on hoof (P. lagopus is capable.Drove roads have a significant capacity to shape some genetic features of  on hoof , as well on hoof . The higP. lagopus, population inbreeding is mainly determined by the ratio between selfing and outcrossing, where outcrossing is enhanced by pollinator availability. Thus, the smaller selfing coefficient and the reduced influence over FIS observed along the drove road indicates a relevant role played by them in providing pollination services. This relationship is even clearer after controlling for landscape effects, namely the presence of grassland patches in the vicinity of the studied plant populations that could harbor further pollinators. In fact, we found the same potential negative relationship between the presence of such patches and the pollinators, mirroring the results of In a self-compatible plant species pollinated both by wind and by insects, such as P. lagopus populations on the drove road don’t show to be affected by these types of dispersal, likely because their human use is much diluted and human activities are comparatively simplified and less dense, consisting just in accompanying or herding livestock. They also show to be pollinator reserves with a higher relevance than peri-urban grassland patches. Such important role played by human settlements for seed dispersal is not surprising, given that human-mediated dispersal adds to livestock-mediated dispersal mechanisms (Distance to human settlements showed a consistent effect on genetic diversity and inbreeding. The resulting functional landscape can be interpreted as settled, high human density areas being hotspots of dispersal that modulate the genetic patterns of diversity, probably mediated by commercial livestock exchanges but also by other dispersal types, mainly human-mediated . The P. chanisms  which archanisms . Howeverchanisms  may be vThe trend of a higher genetic diversity on drove roads compared to the agricultural matrix , even ifFunctional herbivore corridors would sustain diversity by reducing the risk of local extinctions due to increased inbreeding and loss of genetic variation in small populations . The effP. lagopus, in spite of the rapid transit of transhumance flocks—ca. 15 km/day or 1.5 km/h when moving (The marginally significant, yet observable trend of drove roads in reducing genetic diversity loss of n moving  is very n moving , as trann moving  and thern moving  where thMobile pastoralism is globally threatened by non-conductive policies , weakeniOur results are relevant for shaping conservation policies that take into account the role of herbivores, be them wild or domestic. We confirm the relevance that herbivore corridors and mobile pastoralism have in reducing inbreeding levels in plant populations, as well as their potential, within the possibilities of our limited data, in facilitating long-distance plant migration that has been envisioned in seed dispersal studies . Such co10.7717/peerj.7311/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.7311/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.7311/supp-3Supplemental Information 3Click here for additional data file.


Sciurus vulgaris) is an emblematic species for conservation, and its decline in the British Isles exemplifies the impact that alien introductions can have on native ecosystems. Indeed, red squirrels in this region have declined dramatically over the last 60 years due to the spread of squirrelpox virus following the introduction of the gray squirrel (Sciurus carolinensis). Currently, red squirrel populations in Britain are fragmented and need to be closely monitored in order to assess their viability and the effectiveness of conservation efforts. The situation is even more dramatic in the South of England, where S. vulgaris survives only on islands . Using the D‐loop, we investigated the genetic diversity and putative ancestry of the squirrels from Southern England and compared them to a European dataset composed of 1,016 samples from 54 populations. We found that our three populations were more closely related to other squirrels from the British Isles than squirrels from Europe, showed low genetic diversity, and also harbored several private haplotypes. Our study demonstrates how genetically unique the Southern English populations are in comparison with squirrels from the continental European range. We report the presence of four private haplotypes, suggesting that these populations may potentially harbor distinct genetic lineages. Our results emphasize the importance of preserving these isolated red squirrel populations for the conservation of the species.The Eurasian red squirrel ( While this squirrel is common in much of its broad geographic range, which extends from Ireland across Eurasia to Japan  in the late 19th century  was developed to provide a rational basis for prioritizing conservation effort and defined as unique, population(s) that evolved independently Moritz, . This asS. vulgaris may be lower in UK populations, compared with European congeners, with potential implications for their susceptibility to disease  in this process, is a well‐known example of disease‐mediated invasion  and SQR SQR (5′‐CTTACTTGACCAATCCCTCACT‐3′) from Trizio et al. . The PCR2.3FST and AMOVA calculations were performed using Arlequin ver. 3.5.2.2.  was used as an out‐group. The substitution model HKY + G was chosen using jModelTest .A phylogenetic tree was generated with MrBayes . Only two haplotypes were present on Brownsea and Furzey . Fu and Li's D were also calculated and found to be −1.286 on the Isle of Wight and 1.296 on Brownsea Island, again both values were nonsignificant. Fu and Li's D was statistically significant on Furzey Island with a value of −1.827 (p < 0.05) which might indicate a possible population extension on Furzey.Genetic diversity for Brownsea Island, Furzey Island, and the Isle of Wight was low . A Mantel test was also performed on the British populations alone, and no correlation between the FST and the geographical distances was found . Interestingly, the correlation was higher between genetic and geographical distances when calculated in Europe without the British Isles .Pairwise Fct value was obtained when 14 groups were tested, where 44.49% of the molecular variation was attributed to among groups variation (Table Fct was found when four populations were pooled (Northern East England and Dorset as well as Northern West England and Argyll), and all the rest were assigned to a single group. Several AMOVAs were also tested for continental Europe, and the highest Fct value was found when the dataset was divided into 18 groups. AMOVA indicated that 40.49% of the molecular variation was attributed to among‐group variation  as an out‐group. A total of 216 haplotypes was found in the dataset  than on Brownsea (<300) and Furzey (~30). Furthermore, many studies highlight the positive correlation between island area and genetic diversity (Cheylan, Granjon, Granjon, & Britton‐Davidian, S. vulgaris, despite being an “urban island,” was viable and self‐sustaining. However, it was also found that migration of red squirrels to the park was possible via ecological corridors and forested habitats in the urban environment (Rézouki et al., S. vulgaris on the Isle of Wight is not documented.The Isle of Wight is home to the largest remaining population of the red squirrels in southern England. The population has been estimated as 3,300 squirrels (Pope & Grogan, 4.4S. vulgaris throughout its range, island populations are likely to become increasingly important. This may include populations in “urban islands” such as urban parks (Rézouki et al., S. vulgaris in Wales included donor individuals from populations outside of the region in order to maximize genetic heterogeneity in the founders (Ogden et al., With the growing threat to 5S. vulgaris populations in the UK and represent the only remnant populations of Southern England. Our analysis confirmed a British origin of these populations as well as lineages of S. vulgaris that appear to be unique to the islands and, therefore, reinforces the importance of preserving these S. vulgaris populations for the conservation of the species.The preservation of island population genetic diversity may be crucial for the conservation of the locally adapted individuals. The three islands studied are more than 250 km away from the main None declared.KHH, AKS, AM, PL, HB, and REK conducted the fieldwork for this investigation. EAH, OGR, EC, WJL, and JF conducted the laboratory work. EAH and MBS conducted the data analysis. EAH and KHH conceived the study and wrote the final manuscript. All authors were involved in writing and data interpretation and read and approved the final manuscript. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Isoetes yunguiensis Q. F. Wang & W. C. Taylor is a lycophyte of an ancient genus, and it is endemic to China. It is a first-class protected plant in China. This living fossil is used in paleoecology and studies on the evolution of Lycophytes in the Yunnan-Guizhou Plateau. In recent years, human activities have caused the disappearance of several wild populations, and the number of plants in the existing populations is low. Study of the genetic structure, distribution pattern, and historical dynamics of I. yunguiensis in all areas of its distribution is of guiding significance for its rational and effective protection.I. yunguiensis, and noncoding chloroplast DNA (cpDNA) sequences were used to study the pedigree, population dynamics history, and glacial shelter of I. yunguiensis. A maximum entropy model was used to predict the past, present, and future distribution patterns of I. yunguiensis.Expressed sequence tag-simple sequence repeat (EST-SSR) markers were used to study the genetic diversity and structure of I. yunguiensis showed high genetic diversity and that genetic variation was significantly higher within populations than between populations. Based on cpDNA data, it was concluded that there was no significant geographic pedigree in the whole area of I. yunguiensis distribution ; 21 haplotypes were detected using DnaSP v5. Neutral test and LAMARC simulation showed that I. yunguiensis has experienced rapid expansion in recent years. The maximum entropy model predicted that the potential distribution area of I. yunguiensis in the last glacial maximum period has increased significantly compared with the present distribution area, but the future distribution area did not show substantial changes.Analysis with EST-SSR markers revealed that  It is divided into the Yunnan and Guizhou Plateaus, with the Wumeng Mountains as its boundary. The Yunnan Plateau is located in the higher elevation area in the west, and the Guizhou Plateau is located in the lower elevation area , and it is the first plant to receive national level I protection  as well as in Xindan Tiansheng Bridge and Pingba (Guizhou Province). With climatic and environmental changes, wild I. yunguiensis populations are gradually declining, and several historical populations in Yunnan have already disappeared. Therefore, protection of the existing populations is paramount. I. yunguiensis is quite similar in appearance to Isoetes japonica A. Braun and was considered as I. japonica for a long period. Renchang Qin, for the first time, distinguished I. yunguiensis from I. japonica and described it as I. yunkweiensis Ching. Owing to the lack of complete information, this finding was never published . Recent studies on I. yunguiensis have mainly focused on its morphology  markers and chloroplast DNA (cpDNA) were used to determine the genetic diversity and structure of I. yunguiensis populations. The distribution and evolutionary patterns of genetically distinct I. yunguiensis groups were revealed, and the causes of the present distribution patterns of I. yunguiensis were discussed, which may provide scientific evidence for the protection of I. yunguiensis.otection . In receublished . Subsequublished . The speguiensis . In a surphology , palynolrphology , and molrphology . HoweverAfter obtaining approval from the Guizhou Province Wild Flora and Fauna Management Station, a total of 167 samples were collected from 14 populations in Yunnan and Guizhou Provinces from October 2018 to January 2019 . BrieflyMN463102 –MN463268 for psbC-trnS, MN463269 –MN463435 for psbD-trnT, and MN463436 –MN463602 for trnG-trnS.DNA was extracted from dried leaves using the TIANGEN Plant Genomic DNA Extraction Kit . PCR analyses were performed using the EST-SSR primers SJSSR5, SJSSR12, SJSSR14, SJSSR45, and SJSSR53 and the cpDNA primers trnS-trnG, psbC-trnS, and psbD-trnT . PCR reaπ) of cpDNA sequences, and neutral tests and mismatch distribution analyses of cpDNA fragments were performed at the species level. LAMARC 2.1.8 was used to further verify the group expansion events for I. yunguiensis, and Permut 2.0 , effective allele number (Ne), observed heterozygosity (Ho), and expected heterozygosity (He) were calculated, and principal coordinate analyses (PCoA) and Mantel tests  were perrmut 2.0  was usedrmut 2.0  and IQ-Trmut 2.0 . A mediarmut 2.0 .I. yunguiensis during the last interglacial (LIG) period and last glacial maximum (LGM) as well as in the present and future. Coordinates of I. yunguiensis populations were determined using field records from the China Digital Herbarium (http://www.cvh.ac.cn/), as described previously (http://www.webmap.cn). Climatic data were downloaded from the World Climate Data website . Correlations among 19 bioclimatic variables were calculated with the Raster Correlations and Summary Statistics tool in the SDMtoolbox. The environmental and extreme climatic factors with a correlation coefficient of —r— ≤ 0.8 were selected. Finally, eight environmental variables were selected as the prediction variables of the distribution of I. yunguiensis: average daily range (bio 2), temperature seasonality (bio 4), maximum temperature of the warmest month (bio 5), average temperature of the wettest quarter (bio 8), average temperature of the coldest quarter (bio 11), precipitation of the wettest month (bio 13), precipitation of the driest month (bio 14), and precipitation of the warmest quarter (bio 18). Briefly, environmental data and spatial coordinates were imported into MaxEnt 3.4, parameters were set to default values, and distribution ranges during various periods were predicted. Subsequently, ArcGis 10.2 was used to generate ASCII raster layers and simulated distribution maps.MaxEnt 3.4  was usedeviously . Maps weI. yunguiensis populations is presented in The genetic diversity of EST-SSR loci in The genetic diversity of different populations is summarized in I. yunguiensis populations and up to 77.75% was present within populations  cluster analyses were performed based on Nei’s genetic distances using MEGA 7.0, and 14 UPGMA phylogenetic trees of structed . With a structed  were conI. yunguiensis populations. At K = 2, ΔK reached the maximum value ; therefore, I. yunguiensis exhibited no distinct phylogenetic structure in the studied areas. Consistently, AMOVA . The genetic variation in I. yunguiensis was predominately higher within populations. Mean Nm was 2.18, which indicated substantial gene flow between I. yunguiensis populations.The total genetic diversity (Ht) was 0.631, mean genetic diversity (Hs) was 0.515, and the genetic differentiation coefficients between (GST) and within (NST) populations were 0.183 and 0.344, respectively. The haplotype variation structure of the 14 y, AMOVA  showed tI. yunguiensis populations. Although the mismatch distribution was bimodal (p < 0.001), Fu & Li’s D = −8.03665 (p < 0.02), and Fu & Li’s F = −6.94682 (p < 0.02) were significantly negative. The population growth index obtained by LAMARC simulation was 921.590 (G > 200). Taken together, these results indicated that the I. yunguiensis populations have experienced expansion events.DnaSP v5 was used to examine the combined cpDNA sequence of all the individuals in the 14 sampled  bimodal , Tajima’I. yunguiensis haplotypes  network diagram and phylogenetic tree based on the maximum likelihood method were constructed using Network 5.0 . BecauseI. yunguiensis are located in the central Guizhou Plateau and northwestern and eastern Yunnan Plateau and that the suitable areas of distribution are mainly located in the central Guizhou Plateau. The future distribution pattern was similar to the present distribution pattern. The distribution range of I. yunguiensis during the LIG period was significantly reduced compared with the present distribution range, and the distribution ranges in the Yunnan–Guizhou Plateau were clearly separated. The distribution range during LGM was slightly expanded in the Yunnan Plateau and reduced in the Guizhou Plateau compared with the future distribution range.MaxEnt 3.4 was used to predict distribution ranges during the LIG period  and LGM I. yunguiensis showed high genetic diversity compared with other rare, endemic, and endangered plants, such as Isoetes sinensis Palmer , Dalbergia odorifera T. Chen , indicating that I. yunguiensis shows no distinct biogeographical patterns. I. yunguiensis is mainly dispersed through spores released in flowing water  have summarized the genetic structures of 36 species of alpine plants from the Qinghai–Tibet Plateau and surrounding areas. Some hardy plants did not migrate to lower altitudes during the glacial episodes; however, plants on the plateau surface experienced small-scale expansions of ranges following LGM. The genetic structures of species are often characterized by high variations or specific haplotypes, with population distributions in the highland mesa of one or more isolated areas. In addition, some species occupied narrow ranges during the glacial period, leading to small areas of expansion following LGM. These populations were characterized by high genetic diversity and unique haplotypes distributed evenly throughout the distribution range. The present study demonstrated that although I. yunguiensis occupied specific areas of the Guizhou and Yunnan Plateaus during the glacial period. Because the ancestral population from the Yunnan Plateau is extinct, only one population was noted in the Yunnan Plateau in the present study, and the specific location of the extinct population in this plateau remains unknown.I. yunguiensis plants, a provincial nature reserve has been established; the DPJ population used in the present study was sampled from this reserve. However, it has been difficult to protect this population. The SCZ population in the present study was sampled from a farm where sewage is directly discharged into the habitat. The DJC population was sampled from the middle of Longli Forest Farm; seven populations were sampled from this farm, accounting for half of the surveyed populations. However, although these populations were not under the threat of sewerage exposure, limited attention and protection have led to a state of low survival and even extinction. Therefore, the establishment of a protected area dedicated to I. yunguiensis in Longli Forest Farm and training of staff and residents of the surrounding areas regarding protective measures for I. yunguiensis are warranted. In a previous study, populations with high genetic diversity were selected from similar environments for transplantation (I. yunguiensis habitats should be maintained to avoid contamination, and the growth of companion species should be controlled. Improved knowledge and awareness regarding the areas of I. yunguiensis distribution are paramount. To strengthen the conservation strategies for endangered species, further studies are required to identify threats. Moreover, considering that the number of wild I. yunguiensis individuals included in this survey was <10,000, the populations are declining further.The present study demonstrated that the BSH, CX, DJC, DPJ, GJS, SG, and PB populations showed high genetic diversity. It is important to understand genetic diversity and population structure to establish scientific and effective protection measures. Therefore, to protect the endangered antation . AccordiI. yunguiensis exhibited higher genetic diversity than other rare and endangered plants, and its genetic structure demonstrated is similar to that previously reported. Genetic variation was significantly higher within populations than between populations. Based on the data of cpDNA, although no distinct pedigree was detected in the entire area of I. yunguiensis distribution, there has been rapid expansion in recent years. The distribution pattern simulated using the MaxEnt model showed that the past and future distribution patterns of I. yunguiensis are not considerably different from its present distribution pattern. EST-SSR marker and cpDNA data support that the 14 populations examined in the present study can be divided into two groups: those in the Yunnan Plateau and those in the Guizhou Plateau. This indicated that there were at least two refuges for I. yunguiensis during the glacial period. The Wumeng Mountains between the two plateaus have a substantial influence on gene exchange in I. yunguiensis. Based on the high genetic diversity and ancient haplotypes of the populations associated with the refuges, it can be concluded that the CX population from the Yunnan Plateau and the BSH, HFH, DJC, SG, DPJ, GJS, and PB populations from the Guizhou Plateau should be primarily protected. These plateaus are ice-age refuges where these populations should be protected. In the present study, only one population was identified in the Yunnan Plateau. Further investigation is required to identify the new population and discuss the area of refuge. To date, the cause of I. yunguiensis endangerment remains unclear, and related research is urgently warranted.In the present study, 10.7717/peerj.8270/supp-1Supplemental Information 1Raw data from the EST-SSR applied for data analyses and preparation for Click here for additional data file.10.7717/peerj.8270/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.8270/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.8270/supp-4Supplemental Information 4Click here for additional data file.


Caenorhabditis elegans populations under partial selfing, exclusive selfing or predominant outcrossing, while they adapt to osmotically challenging conditions. We find that the ancestral genetic load, as measured by the risk of extinction upon inbreeding by selfing, is maintained as long as outcrossing is the main reproductive mode, but becomes reduced otherwise. Analysis of genome-wide single-nucleotide polymorphisms (SNPs) during experimental evolution and among the inbred lines that survived enforced inbreeding indicates that populations with predominant outcrossing or partial selfing maintained more genetic diversity than expected with neutrality or purifying selection. We discuss the conditions under which this could be explained by the presence of recessive deleterious alleles and/or overdominant loci. Taken together, our observations suggest that populations evolving under partial selfing can gain some of the benefits of eliminating unlinked deleterious recessive alleles and also the benefits of maintaining genetic diversity at partially dominant or overdominant loci that become associated due to variance of inbreeding levels.Partial selfing, whereby self- and cross- fertilization occur in populations at intermediate frequencies, is generally thought to be evolutionarily unstable. Yet, it is found in natural populations. This could be explained if populations with partial selfing are able to reduce genetic loads and the possibility for inbreeding depression while keeping genetic diversity that may be important for future adaptation. To address this hypothesis, we compare the experimental evolution of  Classic theoretical models posit a key role for deleterious recessive alleles in the evolution of reproductive mode modifiers , then prcf. Although classic models suggest the evolution of predominant selfing or predominant outcrossing should be common, comparative studies in plants and in animals have found that a number of natural populations show mixed selfing and outcrossing at intermediate frequencies . To explCaenorhabditis elegans has become a good experimental model to study how standing genetic variation for fitness depends on reproductive mode . For these reasons, and because sex segregates in a Mendelian fashion , and also the fertility of surviving inbred lines . The risAll populations in this study are ultimately derived from a hybrid population obtained by a funnel-crossing scheme where 16 parental wild isolates were reciprocally crossed two-by-two, and their hybrid offspring subsequently crossed also in a pairwise fashion, until obtaining the final hybrid population (ion (103 . During ion (103 .xol-1(tm3055)] into A6140, a mutant impairing X-chromosome dosage compensation and leading to the arrest of male development, to derive a monoecious population composed exclusively of hermaphrodites [homozygous for xol-1(tm3055)] that only reproduce by selfing (named M00). In parallel, we mass introgressed an autosomal sex determination recessive mutant allele [fog-2(q71)] into A6140, a mutant that eliminates self-spermatogenesis in hermaphrodites, to derive a trioecious population composed of males and females [homozygous for fog-2(q71)], and hermaphrodites [homozygous for fog-2(wt)] at an initial frequency of The genetic manipulation of sex ratios in A6140 has been detailed in The evolution experiment has also been previously reported in For line derivation, we followed standard protocols similar to mutation accumulation evolution experiments, where population sizes are kept to a minimum to allow fixation of segregating alleles in the population within each line in mostly a neutral fashion  populations so that we could derive inbred lines by selfing from all experimental populations and thus not confound potential selection under biparental and uniparental inbreeding as in Data from the inbred lines in low salt (25 mM NaCl), the environment where previous lab domestication and line derivation was done, was previously reported in Genomic DNA from A6140 and each of the G50 populations was extracted from individuals with the ZyGem prepGEM Insect kit and from pools of individuals from each inbred line using the Qiagen Blood and Tissue kit. A total of 830 SNPs distributed evenly across genetic distance, which was estimated by linear interpolation of the expected F2 map distances from All genotype data for the lines has been previously published in In ancestral and evolved experimental populations, SNP allele diversity was estimated as ozygotes . To avoinlme R package , with the following input formula for the nlme function:Linkage disequilibrium between pairs of SNPs was estimated as  i and j . Comparic, which is the genetic distance between SNPs, and x, which determines the rate of LD decay and can differ between populations . The underlying assumption is that the expected decay of linkage disequilibrium with genetic distance follows the equation given by N refers to the effective population size. Only SNPs segregating at frequencies between 0.05 and 0.95 were used.With this, we model the observed inbreedR . Likelihood ratio tests were employed for model selection. Zero-inflated negative binomial error distributions were chosen for subsequent analyses as they provided the best fit. We then employed generalized mixed effect models separately for 25 mM and 305 mM NaCl conditions, using the function glmmadmb from package glmmADMB (vs. evolved) and the reproductive system at generation 50, with assay blocks and replicate populations as random factors. Differences between experimental populations and the average of inbred lines derived from them was again carried out with the lsmeans and pairs functions in R  mutant allele in the heterozygous state, meaning they will also produce females through selfing, and male mating ability has had the opportunity to improve through sexual selection (As previously noted (P < 0.0001), a result that contrasts with values from androdioecious populations which, together with trioecious populations, only show a small decrease in allele diversity  did not result in a reduction of the ancestral genetic load. Trioecious populations maintained the ancestral risk of extinction . When coWe also attempted to detect genetic loads by comparing fertility between experimental populations and the inbred lines derived from them, given that a difference between the mean values of fitness components in populations and the mean value among the surviving inbred lines would indicate inbreeding (negative difference) or outbreeding (positive difference) depression . MoreoveGiven that inbred line derivation was done at low salt, the observed line extinction differences between reproduction systems might not be representative of fitness differences during experimental evolution. This problem does not appear to be of much concern because we detected a positive genetic correlation in fertility across the two salt environments for the androdioecious populations and no obvious trade-off between high and low salt in the other reproduction systems .SNP and haplotype diversity metrics were calculated among the lines that survived inbreeding by selfing in order to detect selection during inbreeding. In trioecious and androdioecious populations, simulations of genetic drift during inbreeding and sampling predict that SNP diversity among lines is maintained or slightly reduced whereas haplotype diversity is always expected to be reduced . HoweverFurther evidence for strong selection during the inbreeding assay comes from the SNP frequency changes observed during experimental evolution . For trioecious and androdioecious populations, there is a negative correlation in differentiation during experimental evolution and inbreeding, while in monoecious populations only a much weaker relationship could be found.C. elegans reproducing by selfing for 50 generations evolved reduced risk of extinction when maintained at very small sizes and under extreme inbreeding conditions. This result, which is consistent with the reduction of an ancestral genetic load, was likely caused by selection on viability loci given the similar reproductive outputs that were found in populations and among the surviving lines after inbreeding appear to rule out fertility as a major fitness component. Trioecious populations, which reproduced predominantly by outcrossing, had the same risk of extinction upon extreme inbreeding as the ancestral population, suggesting that they maintained the ancestral genetic load. We also found that populations that experienced some degree of outcrossing during experimental evolution (trioecious and androdioecious populations) maintained more genetic diversity than that expected solely under genetic drift, or under genetic drift and directional selection.In this study, we found that genetically diverse populations of i.e., variation in the degree of individual heterozygosity created by variation in past ancestry of selfing and outcrossing, could result in the maintenance of genetic diversity because of selective interference between completely recessive deleterious alleles, or because of the interaction between genetic drift and selection on beneficial alleles  associative overdominance generated by deleterious recessive alleles at high linkage disequilibrium and in repulsion phase or, (2) heterozygote advantage generated by overdominant loci . LinkageIf excess genetic diversity during experimental evolution resulted from overdominant loci and/or deleterious recessives in identity disequilibrium, then androdioecious populations should have maintained some of the ancestral genetic load .C. elegans, albeit always in strong linkage disequilibrium and at close physical distance, e.g., Under partial selfing (androdioecy), heterozygote advantage could be also generated by recessive lethals in repulsion phase, which are effectively overdominant loci despite the potentially large physical distances between them. Distinguishing these alternatives would require mapping the relevant fitness loci in the genome, an endeavor that is notoriously difficult and beyond the scope of the present study. Recessive sub-lethals in repulsion have been described in natural populations of Monoecious populations show an overall reduction in genetic diversity that is in line with neutral expectations under exclusive selfing . It is pPhysa acuta, specifically designed to disentangle sexual selection from selfing in the purging of deleterious recessives, has failed to find a significant role for sexual selection . We have previously shown that in the novel salt environment populations from all three reproduction systems had similar adaptive rates [Figure 4 in Selfing rates under androdioecy were unstable during experimental evolution and, unless male fitness components were to evolve, exclusive selfing would be achieved in less than 100 generations . It is t


Tetrix undulata pygmy grasshopper populations in disturbed and stable habitats. Genetic differentiation was evident among the different populations, but there was no genetic separation between stable and disturbed environments. The incidence of long‐winged phenotypes was higher in disturbed habitats, indicating that these populations were recently established by flight‐capable colonizers. Color morph diversity and dispersion of outlier genetic diversity, estimated using AFLP markers, were higher in disturbed than in stable environments, likely reflecting that color polymorphism and variation in other functionally important traits increase establishment success. Neutral genetic diversity estimated using AFLP markers was lower in disturbed habitats, indicating stronger eroding effects on neutral diversity of genetic drift associated with founding events in disturbed compared to stable habitats. Functional diversity and neutral diversity were negatively correlated across populations, highlighting the utility of outlier loci in genetics studies and reinforcing that estimates of genetic diversity based on neutral markers do not infer evolutionary potential and the ability of populations and species to cope with environmental change.Genetic structure among and diversity within natural populations is influenced by a combination of ecological and evolutionary processes. These processes can differently influence neutral and functional genetic diversity and also vary according to environmental settings. To investigate the roles of interacting processes as drivers of population‐level genetic diversity in the wild, we compared neutral and functional structure and diversity between 20  In combination with selection, this molds functional genetic structure and diversity of natural populations. Neutral genetic variation is shaped by historic relationships, random processes such as genetic drift, recombination, and mutations, and gene flow between populations  Orthoptera, Tetrigidae) in disturbed and stable habitats  data  habitats or disturbed (n = 12) habitats   is a measure of the proportion of possible comparisons which are unalike and was calculated based on the equation: u2=1−∑i=1pi2, where p represents the proportion of the population in each of the ith categories of the categorical variable  .To quantify color morph diversity in populations, we used the coefficient of unalikeability   were regarded as the outliers (loci under selection). To obtain a neutral dataset, loci with log10 PO > 1  estimations for a subset of eight populations suggested a range of Fis values (0.06 to 0.68) randomly distributed among the populations without suggesting a plausible Fis > 0 values for all populations. These calculations were performed in I4A  or with differences in elevation have resulted in genetic differentiation among populations. It is also possible that the existence of disturbed and stable environments has favored the evolution of genetically different ecotypes of pygmy grasshoppers. To evaluate these hypotheses, population genetic structure was investigated using estimates of pairwise FST for the nDATA and sDATA datasets. Two separate calculations were performed for each dataset: one with structure and one without structure. For the former, to partition the total genetic variation and test for overall differentiation between grasshopper populations in disturbed and stable environments and according to geographic location or elevation, we performed a nested AMOVA where the populations were nested in either of two disturbance regimes (disturbed or stable). For the latter, all 20 populations were grouped together. The statistical significance of the AMOVA was tested by 10,000 permutations.An analysis of molecular variance AMOVA  on the distribution of the genetic variation among FST) and geographic (log km) distance using a Mantel test  Slatkin, , we estiTo examine whether genetic structure and pairwise divergence among populations in neutral AFLP loci were associated with the divergence in outlier AFLP loci putatively influenced by selection, we used a Mantel test. Mantel tests were performed in the zt software  using correlation analysis. Prior to statistical analyses, the proportion of long‐winged individuals was arcsine‐square root‐transformed, and census population size was log10‐transformed.Analyses of genetic structure uncovered significant differentiation among most study populations and no signature of isolation by distance (see Results), justifying that the different study populations can be considered as independent observations. We tested for differences between populations inhabiting disturbed and stable environments in proportion of long‐winged phenotypes, color morph diversity , neutral and outlier genetic diversity, and census population size using either 33.1Of the 1,419 AFLP loci, 1,277 had dominant allele frequencies >2% and <98%, and they were included into the outlier analyses. According to BayeScan analysis, 28  loci exceeded the threshold for “decisive” evidence of selection with a FDR of ≤0.001043 as the outliers, and they were used for the outlier dataset (sDATA). Alpha values were positive for all 28 loci, suggesting diversifying selection. In addition to these 28 loci, 41 loci that exceeded the threshold of “strong” evidence of selection were excluded from the neutral dataset (nDATA) that included 1,208 loci. These 41 loci also had positive alpha values, except one with negative value suggesting balancing or purifying selection.3.2r = 0.84, n = 20, p < 0.0001). Estimates of Nei's genetic diversity and of mean deviations from centroid for neutral (DevCneu) and for outlier loci (DevCout) are reported for each population in Table r = 0.60, p < 0.0049, n = 20). Estimates of functional AFLP diversity within populations based on sHj and deviations from centroid of outlier loci (DevCout) were negatively correlated . Calculation of Hj is dependent on correct Fis values , it is possible that estimates of sHj might deviate from the actual values. By comparison, multivariate analyses (including PERMDISP) do not require a specific genetic model of populations, and we therefore consider the deviations from the centroid more reliable estimates for quantification and comparisons of within‐population diversity.Analyses of the 1,419 polymorphic AFLP markers (the dataset ALL) revealed high within‐population genetic diversity estimated with proportion polymorphic loci (PPL) and average heterozygosity  across all sampling localities .Estimates of neutral and functional genetic diversity were not strongly or consistently associated with sample sizes , whereas the location effects were strong . The lack of the genetic differentiation between different regimes is also evident in multidimensional scaling ordination plots based on analysis of Jaccard distances for neutral and for outlier loci according to population and environmental state , there was no significant signature of environment .Pairwise genetic differentiation among populations, as estimated by FST values suggested that the genetic divergence between populations in neutral AFLP loci was correlated with the genetic divergence in outlier AFLP loci .Analysis based on pairwise 3.42 = 9.68, df = 1, p = 0.0019, Figure The proportion of long‐winged phenotypes (indicative of recent establishment and immigration) was higher on average in populations in disturbed  than in stable  environments  than in stable (0.79 ± 0.09) environments  was higher in disturbed  was lower in disturbed (0.28 ± 0.018) than in stable 0.31 ± 0.032) environments  did not differ significantly between disturbed (0.33 ± 0.0731) and stable (0.37 ± 0.062) environments  than in stable (0.362 ± 0.0074) environments    in neutral loci was significantly lower in disturbed .There was no difference in census population size between populations in disturbed  and stable  environments  but was not associated with outlier genetic diversity .Color morph diversity decreased with increasing neutral genetic diversity across all populations .Color morph diversity increased with increasing immigration rate .Color morph diversity increased with increasing census population size . Results from comparisons between contrasting environments uncovered that populations in disturbed habitats had a higher incidence of long‐winged phenotypes, as expected if these populations were recently established by flight‐capable colonizers. Intrapopulation functional color morph diversity was also higher in disturbed than in stable environments. This outcome was expected given that color polymorphism increases establishment success Forsman, . Neutral4.1The pattern of genetic structure and intrapopulation diversity seen in these grasshoppers can be accounted for by combined effects of ecological and evolutionary processes associated with environmental change, as summarized in Figure T. undulata is an almost exclusively short‐winged species  might indicate that divergence in functional traits has not only been influenced by selection, but also by gene flow and stochastic processes associated with founder events and drift in small populations , populations inhabiting large (source) as opposed to small (sink) habitat patches, populations in ephemeral or temporal as opposed to permanent habitat patches, or populations at the core of the distribution range as opposed to populations in marginal areas that represent invasion fronts of expanding species  to additive genetic variation and by the physical inhibition of recombination associated with chromosomal rearrangements in small populations (Neiman & Linksvayer, Tetrix japonica—a closely related pygmy grasshopper species (see Figure S1 in Forsman, The color polymorphism in pygmy grasshoppers likely has a polygenic inheritance Fisher, . A transWhen interpreting results from comparisons of neutral and functional genetic diversity in populations between contrasting environments, it is necessary to take into consideration any systematic differences in effective population size. It is not only the eroding effect of genetic drift that depends on effective population size. Evolvability, or the efficiency by which selection can remove deleterious alleles and drive advantageous alleles to fixation, increases with increasing effective population size (Willi et al., 5T. undulata grasshoppers illustrate that genetic diversity within natural populations is determined by a combination of ecological and evolutionary processes, the importance of which are different for neutral and functional genetic diversity and vary between environmental disturbance regimes (Figure Our results for s Figure . A highes Figure , combines Figure . NeutralNone declared.A.F. designed the research. A.F. and J.T. collected animals. J.T. performed laboratory work. J.T. and Y.Y. analyzed molecular data. Y.Y., A.F., and J.T. performed statistical analyses. All authors interpreted the data and contributed to the writing of the paper.Data on sampling locations, sampling year, sex, and AFLP genotypes of individuals used for genetic analyses are available as electronic supplementary material. Click here for additional data file. Click here for additional data file.