Peucaea aestivalis) is a fire-dependent species that has undergone range-wide population declines in recent decades. We examined genetic diversity in Bachman's Sparrows to determine whether natural barriers have led to distinct population units and to assess the effect of anthropogenic habitat loss and fragmentation. Genetic diversity was examined across the geographic range by genotyping 226 individuals at 18 microsatellite loci and sequencing 48 individuals at mitochondrial and nuclear genes. Multiple analyses consistently demonstrated little genetic structure and high levels of genetic variation, suggesting that populations are panmictic. Based on these genetic data, separate management units/subspecies designations or translocations to promote gene flow among fragmented populations do not appear to be necessary. Panmixia in Bachman's Sparrow may be a consequence of an historical range expansion and retraction. Alternatively, high vagility in Bachman's Sparrow may be an adaptation to the ephemeral, fire-mediated habitat that this species prefers. In recent times, high vagility also appears to have offset inbreeding and loss of genetic diversity in highly fragmented habitat.Bachman's Sparrow ( Genetic structure in wildlife populations is typically assessed with respect to natural barriers or anthropogenic habitat loss and fragmentation. Fragmented habitats created by natural barriers, such as rivers, oceans, deserts and mountain ranges, have documented major effects on population differentiation In addition to natural habitat fragmentation, recent anthropogenic habitat fragmentation, degradation and loss also have the potential to disrupt gene flow among populations Although natural and anthropogenic fragmentation can shape genetic structure of populations, other underlying natural processes may also influence structure significantly. In particular, species that specialize in ephemeral or disturbed habitat may have dispersal strategies or adaptations that are distinct from or absent in species found in more stable habitats Pinus palustris) forests are a fire-mediated ecosystem with several closely associated plant and animal species. Population structure in one species, the Bachman's Sparrow , is potentially complex because it is influenced by natural and anthropogenic fragmentation as well as ephemeral, fire-mediated habitat preferences. The sparrow currently consists of three subspecies P. a. illinoensis occupies the northern and westernmost areas of Bachman's Sparrow range including Texas, Louisiana, Indiana, Illinois and Missouri; P. a. aestivalis occupies areas east into Florida, Georgia and South Carolina; and P. a. bachmani occupies North Carolina and Virginia In the southeastern US, longleaf pine  and fragmentation of longleaf pine habitat Bachman's Sparrow is listed as a species of conservation concern both internationally (IUCN) as well as within every state in which it breeds The objectives of this study are to: 1) examine genetic structure and diversity in a species adapted to natural disturbances caused by fire; 2) examine Bachman's Sparrow population differentiation across its range to evaluate whether current subspecies designations are valid; and 3) evaluate gene flow among and genetic diversity within habitat fragments to identify areas of restricted gene flow and populations with inbreeding and low levels of genetic diversity. The results of this study should help to ensure that populations of high genetic value are conserved, that genetic variation is maintained and inbreeding depression is reduced in remnant populations, and finally, provide a better understanding of the effect of ephemeral habitat on gene flow.This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Protocols were approved by the Institutional Animal Care and Use Committee of the Louisiana State University AgCenter (Permit Numbers: AE2011-04 and A2012-05) and Tall Timbers Research Station (Permit Number: VE-2002-01). Birds were banded and bled under Federal Bird Banding Permits 07732, 22648 & 24466, State Permits FFWCC LSSC-05-0205 & 29-wmb-02-143 (Florida) and LNHP-11-062 & LNHP-12-023 (Louisiana), Wildlife Management Area Permit WL-Research-2011-03 (Louisiana), and US Department of Agriculture Forest Service Permit 2610 .Sampling in Louisiana was conducted in areas with recent Ebird records and separated by the Mississippi River, a potentially important geographic barrier to dispersal. We sampled four sites on the west and three sites on the east side of the Mississippi River . Western2, 0.8 mM dNTPs, 0.10 µM each of forward and reverse primers, 0.5 µl of 100% dimethyl sulfoxide (DMSO), 1 M betaine, 0.03 µM M13 fluorescent tag, 2.0 units Taq DNA polymerase , and nanopure water to a final volume of 10 µl. PCR amplification conditions were 95°C for 30 seconds followed by 34 cycles of 95°C for 1 minute; 48–60°C  using DNeasy Blood and Tissue kits . Samples were amplified using polymerase chain reaction (PCR) with an Eppendorf Mastercycler pro S thermal cycler. Nuclear microsatellite primer pairs (n = 23) developed in other avian species were tested, and 19 amplified successfully . PCRs co60°C see  for 1 mi2, 0.8 mM of dNTPs, 1.25 µM of each forward and reverse primers, 2.5 units Taq DNA polymerase , and nanopure water for a final volume of 25 µl. PCR amplification conditions were as follows: 95°C for 30 seconds followed by 34 cycles of 94°C for 30 seconds, 50°C (ND2)/60°C (TGFβ2) for 30 seconds, 72°C for 1 minute, and a final extension step of 72°C for 7 minutes. PCR products were sent to Beckman Coulter Laboratories  for Sanger single-pass sequencing. Forward and reverse strands were aligned for each sample and corrected using Sequencher v. 5.0 (Gene Codes Corp.).Sequence data were obtained for one mitochondrial locus, the nicotinamide adenine dinucleotide dehydrogenase subunit 2 (ND2) using primer L5215 from icrocheckerv. 2.2.3 enepopv. 4.1.4 Microsatellite data were checked for genotyping errors using Menetixv. 4.03 statv. 2.9.3 enepop was used to calculate ISF, the inbreeding coefficient Population genetic variation was measured as average observed and expected heterozygosity, average number of alleles per locus, and allelic richness with GnaSP v. 5.10.1 naSP. Estimates of sequence divergence among populations were also calculated using DnaSP, which included the number of net nucleotide substitutions per site among populations (aD) and the average number of nucleotide substitutions per site among populations (xyD).For ND2, a 1038 base pair sequence was obtained for 47 individuals, and for TGFβ2 a 570 base pair sequence was obtained for 43 individuals. Some TGFβ2 sequences were heterozygous, therefore, prior to analyzing sequence data for molecular variation, Bayesian computational inference of TGFβ2 gametic phase was performed using the PHASE module in Denepop with microsatellite data using global STF (θ) as well as pairwise STFSTR (ρ) tructurev. 2.3.2 enelandv. 4.0 enetixv. 4.05.Genetic differentiation among the five regions was calculated in Gtructure assesses whether sampled genotypes are substructured into multiple (K>1) clusters or constitute a single population (K = 1). We implemented Structure with and without the LocPrior clustering algorithm, which accounts for sampling locations and assumes that assignment probability varies among locations. The LocPrior method is appropriate for detecting weak genetic structure K ranging from 1–11. Each run had a burn-in of 150,000 followed by 150,000 iterations K parental populations tructure, the best estimate of the number of clusters (K) was determined using log-likelihood ratios from Structure following Evanno et al. K based on changes in the log probability for successive values of K. The most likely K suggested by initial runs was reassessed in Structure for an additional 25 runs. Averaged results were then calculated to produce a parameter (r) that estimates the information on ancestry provided by sampling location in the LocPrior model. Values of r≤1 indicate that the inclusion of sampling locations is informative, whereas values of r>>1 imply that location data is uninformative Seneland was implemented in R (v. 3.0). Geneland detects population subdivision and barriers to gene flow using a spatially explicit model that incorporates geographic barriers and boundaries among populations into the analysis of genetic structure tructure, all clustering solutions are not equally probable in Geneland. Instead, spatial distributions are used to infer the number of subpopulations, K. Initial runs allowed K to vary under the following conditions; 10,000 stored iterations of the Markov chain, maximum rate of Poisson process set at the default value of 100, minimum population number set to a minimum of 1 and a maximum of 11, and the number of thinnings set to 10. The uncertainty of the coordinates was set to zero because GPS coordinates were available for each sample. A Correlated Allele Frequency model, a true Spatial model and a false Null Allele model were used in the analysis. Five independent runs of these three parameters were run for each potential K.Genetic structure as calculated by Genetix to assess population structure among sampling locations using scores derived from two axes. Isolation by distance (IBD) was tested with IBDWS v. 3.23, which examines the correlation between genetic FCA was run in GSTF using an analysis of molecular variance (AMOVA) implemented in Arlequinv. 3.11 Genetic structure in mitochondrial and nuclear DNA sequence data was examined by calculating estimates of global and pairwise egav. 5 To investigate phylogeographic structuring, relationships among mitochondrial and nuclear DNA haplotypes were constructed using statistical parsimony ottleneck v. 1.2.02 eH) assuming HWE and (2) the number of alleles and sample size (eqH) assuming mutation-drift equilibrium. Both estimates should be similar at equilibrium, but eqH will decrease faster than eH if a population experiences a bottleneck. On the other hand, population expansion would be expected if eH decreased faster than eqH. Estimates of heterozygosity were calculated using a two-phase model that requires two parameters: (1) the percentage of mutations that follow a strict stepwise mutational process, and; (2) the variance in size of multistep mutations Evidence for recent population bottlenecks was evaluated with B0F) immigrants from unsampled populations, we used the Bayesian assignment procedure of Rannala and Mountain eneclass v. 2.0 Lh/Lmax likelihood test statistic to identify migrants with an alpha level of 0.01 To examine whether gene flow may be caused by first generation  from 11 different sampling sites were genotyped at 19 microsatellite loci . One locpulation .DNA sequence analysis produced 19 haplotypes at ND2 and 27 haplotypes at TGFβ2 after data were phased . OverallSTF was 0.012 (±0.002) for microsatellite data, indicating slight genetic structure. Small but significant differences in pairwise STF were detected for approximately half of the sampled populations, with values ranging from 0.0001 to 0.0574  or mitochondrial sequences  where estimates for K = 1 cannot be calculated.Seneland and FCA results suggested a single population. FCA analysis explained only 2.80% of the variation among individuals and produced no discernible separation among geographic areas (eneland results suggested a single population with no barriers to gene flow as given by a map of posterior probability (not shown). Finally, the isolation-by-distance analysis showed no significant relationship between geographic distance and genetic distance  and there was no relationship between geographic distance and genetic distance matrices based on the Mantel test .Gic areas . GenelanSequence data suggested that several populations had unique haplotypes . ND2 seqeneclass, we detected 15 first generation (0F) migrants that were assigned to areas other than their sampling location , North Carolina (p = 0.025), Talisheek Pine Wetlands Preserve (p = 0.049), and Camp Whispering Pines (p = 0.030), but small samples for Talisheek Pine Wetlands Preserve and North Carolina could produce false positives. False positives can also be observed in populations experiencing high rates of migration , which may be relevant here. Using Glocation ; howeverWe examined genetic structure and diversity in Bachman's Sparrow to assess the potential effects of large natural barriers, such as the Mississippi River, and recent habitat loss and fragmentation. Most of our analyses showed high genetic diversity , little Troglodytes aedon), which have lower genetic diversity and less population structure than northern populations with seasonal north-south migrations Spizella breweri) Emberiza schoeniculus) Setophaga cerulea) Our sampling areas overlapped broadly with the distribution of non-migratory populations in the southern half of the species' range STF values were low and non-significant for the most distant populations sampled on Fort Polk, Louisiana and North Carolina , located at the western and eastern extremes of the range  Stipiturus mallee) High vagility as an adaptation to ephemeral habitat is consistent with the lack of genetic structure observed in our study. Bachman's Sparrow habitat suitability is closely linked to ground-cover conditions, and individuals typically abandon areas that have not been burned every 2–3 years Although high vagility as an adaptation to ephemeral habitat may explain weak population structure on a local scale, it is still surprising to see weak population structure among distant populations with different subspecific designations: Bachman's Sparrows probably do not need to travel thousands of km to find suitable habitat. Accordingly, weak population structure between distant populations may be the product of range expansion and retraction. During the early 1900s, Bachman's Sparrow moved northward and occupied suitable habitat on abandoned farms and fallow pastures from Pennsylvania to Illinois, which mimicked the savannah-like understory of southern pine forests Low genetic differentiation among Bachman's Sparrow populations suggests that neither natural barriers nor anthropogenic fragmentation has caused population differentiation, loss of genetic diversity, or inbreeding. The current lack of differentiation across the species' geographic range means that recognition of distinctive subspecies may not be necessary for management purposes. However, an examination of historical genetic variation may be necessary to confirm this conclusion because any genetic structure that was formerly present may have been weakened by range expansion and contraction. Furthermore, distinct populations identified by plumage differences as described in Sibley (2000) and the AOU Table S1Characteristics of 23 microsatellite loci screened in Bachman's Sparrows.(DOCX)Click here for additional data file.


Admixture mapping has been enormously resourceful in identifying genetic variations linked to phenotypes, adaptation, and diseases. In this study through analysis of copy number variable regions (CNVRs), we report extensive restructuring in the genomes of the recently admixed African-Indian population (OG-W-IP) that inhabits a highly saline environment in Western India. The study included subjects from OG-W-IP (OG), five different Indian and three HapMap populations that were genotyped using Affymetrix version 6.0 arrays. Copy number variations (CNVs) detected using Birdsuite were used to define CNVRs. Population structure with respect to CNVRs was delineated using random forest approach. OG genomes have a surprising excess of CNVs in comparison to other studied populations. Individual ancestry proportions computed using STRUCTURE also reveals a unique genetic component in OGs. Population structure analysis with CNV genotypes indicates OG to be distant from both the African and Indian ancestral populations. Interestingly, it shows genetic proximity with respect to CNVs to only one Indian population IE-W-LP4, which also happens to reside in the same geographical region. We also observe a significant enrichment of molecular processes related to ion binding and receptor activity in genes encompassing OG-specific CNVRs. Our results suggest that retention of CNVRs from ancestral natives and de novo acquisition of CNVRs could accelerate the process of adaptation especially in an extreme environment. Additionally, this population would be enormously useful for dissecting genes and delineating the involvement of CNVs in salt adaptation. AMY1) has been linked to high starch diet preference  range in size from 1 kb to several megabases and include deletions, duplications, and large insertions–deletions (indels) . CNVs oceference  and indetibility . Earliertibility . The strtibility .Genomic dissection of admixed populations offers excellent opportunities for mapping disease loci and signature of selection events especially if the admixture is recent, and the parent populations have distinct ancestral history . Many geOur study reveals a surprising excess of CNVs in the admixed population when compared with the ancestral Indian and African as well as the HapMap populations. Population structure analysis indicate that the admixed population has evolved its own genomic structure through copy number variable regions (CNVRs) that are unique and common in this population as well as retained from ancestral population that resides in the same habitat. The CNVRs span the entire genome and are significantly enriched in processes related to ion channels and receptor activity. Our results substantiate the hypothesis that CNVRs especially in admixed populations could accelerate the process of adaptation as in this case the admixed population resides in an extremely saline environment, a habitat known to be nonconducive especially for African natives from equatorial regions. Additionally, this population would be enormously useful for dissecting genes and delineating the involvement of CNVs in salt adaptation.supplementary table S1, Supplementary Material online.In this study, a total of 152 healthy and unrelated subjects from 6 different Indian populations and 3 populations of HapMap Project  were useGenotyping of Indian samples were carried out using genome-wide Affymetrix 6.0 SNP array . In brief, 250 ng of DNA samples were processed for restriction digestion with Sty1 and Nsp1 separately, following the manufacturer’s recommended protocols. After amplification, both the set of amplified products were pooled and further processed for hybridization and scanning. Genotyping quality was assessed using Affymetrix Genotyping Console Software, and samples having contrast quality control > 0.4 were further used for CNV analysis. HapMap samples genotyped on Affymetrix 6.0 platform  were conwww.Affymetrix.com) to check the concordance of Birdseye’s CNV calls.BirdSuite software (v1.5.5)  was usedThe following filtering criteria were applied for calling CNVs from Birdseye data: 1) only autosomes were considered, 2) number of contiguous probes ≥5, 3) probes less than 10 kb apart, 4) LOD score ≥10, and 5) CNV events of size <1 Mb. Further, in-house Perl script was used for construction of CNVRs by merging overlapping Birdseye’s CNV segments with any base overlap criteria. Additionally, we applied sample frequency threshold of 5% to avoid singletons/rare calls. Frequency matrix of deletion and duplication calls for CNVRs was also generated. Circos  was usedCNPs and birdseed genotype calls obtained from Birdsuite were also filtered. Genotypes with confidence value <0.1 were retained, and values >0.1 were treated as missing data.Supervised Random Forest algorithm  impleme was usedFST) analysis. Population clustering was performed using STRUCTURE 2.3.4 , normal copy (2), and duplications (3 and 4) were converted to allelic states—0/0 (zero copy), 0/1 (one copy), 1/1 (two copies), 1/2 (three copies), and 2/2 (four copies). Biallelic data format was used for population clustering and population differentiation  close to Indian ancestor(s), 2) close to African ancestor(s), and 3) unique. Further, functional annotation was done for ancestry informative CNVRs (AICs).CNVRs were divided into three subsets: deletions, duplications, and gain–loss on the basis of CNV states in four populations . Duplication and deletion subset included 24 and 483 CNVRs, respectively; whereas gain–loss data set had 58 CNVRs. Deletion and duplication CNVR markers were used for further analysis to avoid ambiguity. Ancestry informativeness was computed using frequency of CNVRs in OG and assigned hypothetical Indian (IE-W-LP4) and African putative ancestors , as described in our earlier article . Method P value ≤ 0.05 with Bonferroni correction for enrichment analysis.CNVR data sets were annotated using RefSeq database downloaded from University of California, Santa Cruz table browser. We considered coordinates of longest transcript of a gene if it has multiple isoforms for annotation. Functional enrichment analysis was performed using ToppFun module of ToppGene suite that uses background information from multiple resources . We usedsupplementary fig. S1, Supplementary Material online). Average number of CNV calls in OG was significantly higher than observed in Indian and HapMap populations . Frequency distribution of CNV events binned into different segment sizes ranging from 1 kb to ≥500 kb revealed CNVs of smaller size to be more frequent . Compared with other populations, the segment size of CNVs in OG was much larger . OG population encompasses larger number of CNV events compared with the other populations; especially deletion CNV events are overrepresented . As discussed in the Materials and Methods section, we carried out a random forests approach to compute distance matrix using CNVR data followed by MDS to visualize population stratification. The predicted accuracy of the random forest model is about 81%, and error rate of the model was described by confusion matrix, where comparison between actual and predicted population labels was made . MDS analysis reveals that OG individuals cluster separately from African and Indian populations and is close to one of the Indian populations (IE-W-LP4) residing in same geographical region . To understand the potential effect of CNV markers on SNPs, 22,472 SNP markers within CNV boundaries were also used for population stratification analysis. Population stratification using SNPs within CNV markers also had different spectrum with respect to genome-wide markers but similar to what we observed using CNVRs . We checked the consistency of the population structure and clustering revealed by SNPs within CNV boundaries by randomly selecting the SNP markers of approximately same data size. Population structure using random data sets was same as reflected by genome-wide marker data sets .Using a set of 872,188 autosomal SNPs from Affymetrix 6.0 platform, we revalidated our earlier observation of distinctness of OG population as it comes in a perfect cline between Indian (Large populations of Northern and Western India) and African  populations, and there is almost no contribution from European ancestry . However, at k = 3, we observed separation of ancestral populations as expected and a third component contributes maximally (76%) to the ancestry of admixed OG individuals . To quantify and analyze the effect of CNVs on admixed populations at fine resolution, we have used SNP markers within CNVs to compute admixture proportions. At k = 2, ancestry contribution from Indian populations was higher (0.61%) in comparison to African populations (0.38%) . This finding was contrasting with our previous study using genome-wide SNPs, where proportion of ancestry estimated for African and Indian populations was approximately 60% and approximately 40%, respectively. However, at k = 3, contribution of third ancestry component (0.46%) was higher for OG when SNPs within CNVs were used  and African (YRI) populations were chosen for clustering. At ere used b. This iAs OGs have very recently settled in the Indian subcontinent from Africa, deviation in population structure and excess of unique ancestry proportions in OG individuals through CNVs could be indicative of their role in selection .FST distances were computed using CNVRs between OG and ancestral populations. It was observed that FST between OG and IE-W-LP4 was minimum (0.16) compared with other studied Indian and African populations . These CNVRs were considered as OG specific. These regions had enrichment of molecular processes from genes related to trace-amine receptor activity and calcium ion binding activity for OG-specific CNVRs . For example, association of variations in TTN with cardiomyopathy , which are a family of G protein-coupled receptors known to bind endogenous biogenic amines and affect behavioral phenotypes . It also complements the observations made by SNP in the earlier study. Interestingly, some of these CNV regions were also shared with the parental population residing in same region. Though in terms of SNPs, the parental population is closer to the other IE population, a distinct set of CNVRs, which it shares with OG seem to differentiate it from IE populations of other geographical regions.Irrespective of previous ancestries, populations residing in the same geographical region are exposed to similar evolutionary pressures and hence might share genomic regions selected for adaptation. One such example is of salt retention in African populations, an adaptive trait against heat stress . Variatimyopathy , ITPR2 warcinoma , etc. Inenotypes . Genes wTo successfully inhabit a contrasting location, OG seems to have shaped its own genome through extensive CNVs. Although a large number of CNVs have been acquired denovo and are present in multiple individuals in the OG population, some CNVs have been retained from the ancestral Indian population, which together might have allowed the population to adapt to its new habitat. This provides an important genetic resource for mapping/identification of genes that could be involved in salt stress adaptation/maladaptation relevant in many phenotypes and diseases.Supplementary text S1, tables S1–S7, and figures S1–S9 are available at Genome Biology and Evolution online .


Brassica rapa plants from two locations that rapidly evolved changes in multiple phenotypic traits, including flowering time, following a multiyear late‐season drought in California. These ancestor‐descendant comparisons revealed evolutionary shifts in allele frequencies in many genes. Some genes showing evolutionary shifts have functions related to drought stress and flowering time, consistent with an adaptive response to selection. Loci differentiated between ancestors and descendants (FST outliers) were generally different from those showing signatures of selection based on site frequency spectrum analysis (Tajima's D), indicating that the loci that evolved in response to the recent drought and those under historical selection were generally distinct. Very few genes showed similar evolutionary responses between two geographically distinct populations, suggesting independent genetic trajectories of evolution yielding parallel phenotypic changes. The results show that selection can result in rapid genome‐wide evolutionary shifts in allele frequencies in natural populations, and highlight the usefulness of combining resurrection experiments in natural populations with genomics for studying the genetic basis of adaptive evolution.There is increasing evidence that evolution can occur rapidly in response to selection. Recent advances in sequencing suggest the possibility of documenting genetic changes as they occur in populations, thus uncovering the genetic basis of evolution, particularly if samples are available from both before and after selection. Here, we had a unique opportunity to directly assess genetic changes in natural populations following an evolutionary response to a fluctuation in climate. We analysed genome‐wide differences between ancestors and descendants of natural populations of  see also the Perspective by Hancock Arabidopsis lyrata  that are the subject of this study were previously shown to have undergone adaptive evolution in response to selection. These populations, located in southern California, experienced a series of wet years leading up to 1997 and then a series of dry years, in which drought was particularly severe late in the growing season, leading up to 2004  ancestral and postdrought (2004) descendant gene pools in two southern California populations of Brassica rapa L. (Brassicaceae), commonly known as field mustard, is an annual, obligate outcrossing, herbaceous plant introduced into the United States. Although considered a mesopolyploid, it behaves functionally as a diploid, and has been fully sequenced , ensuring the plants represent an unbiased sample of the gene pools of the original populations. Our set of samples subjected to sequencing contained 205 accessions derived from the predrought (1997) and postdrought (2004) seed samples from both the Arb (50 pre‐ and 74 postdrought) and BB populations (22 pre‐ and 59 postdrought).We collected seeds from thousands of individuals from each population before (1997) and after (2004) the drought. From each of these collections, 500 seeds were randomly selected and grown under greenhouse conditions in 2006. Based on the large number of plants that served as the source of the collection, and the large number of seeds used, we do not expect that this procedure would have caused a strong population bottleneck. Plants were crossed randomly within each group (population and generation), and seeds from these intrapopulation crosses were collected to generate ancestral and descendant lines  leaves collected after 10–14 days of growth were lysed  followed by genomic DNA extraction . We then normalized DNA concentrations among samples because we wanted to make sure that each individual contributed equally to the pooled sample for each library. We normalized using quantitative PCR of the GAPDH gene, which is commonly used in qPCR studies. This gene was chosen because it is a housekeeping gene with important function, and thus is unlikely to display polymorphism (Wedel & Soll B. rapa reference genome (version 1.18) (http://brassicadb.org/brad/). Default parameters were used with the exception that q  was changed from 0 to 20 to reduce read trimming (in addition to read trimming performed above). We used samtools  were calculated using the packages popoolation ‐corrected q‐values with fdrtool . We tested 29 samples from each population (BB and Arb) for each time period (ancestors and descendants) for a total of 116 samples. These 116 samples were chosen randomly from among those for which we had sufficient quantities of DNA.To confirm our pooled results at the individual level, we performed Kompetitive Allele‐Specific PCR genotyping . We chose 10 SNPs from well‐annotated loci with an r = 0.83, P < 0.001; Fig. S1, Supporting information), indicating that our next‐generation sequencing of pooled DNA from multiple individuals in each population provides equivalent estimates of allele frequencies in those populations to estimates that are based on individual sample genotyping.Nine of our ten chosen SNPs amplified consistently and were used for the validation analysis. We found high concordance in allele frequencies obtained from KASP genotyping compared to pooled Illumina sequencing  analysis was performed using ErmineJ, with annotations from Our low‐coverage (~25× per pooled library) whole‐genome shotgun sequencing identified 5 812 602 sites with SNPs. Within regions annotated as coding sequence, we found 42 360 high‐quality, biallelic SNPs segregating at moderate to high frequency  in these populations. Average overall nucleotide diversity per site (π) was 0.00957.FST) analysis  between ancestors and descendants in both populations . Thus, during the course of the drought there were rapid changes in allele frequencies in multiple genes in both populations. The average genome‐wide (genic and nongenic) between‐year FST was 0.035 (0.034–0.035) for Arb and 0.032 (0.031–0.033) for BB (95% bootstrapped CIs in parentheses). These values, while very low, were significantly greater than zero, suggesting that rapid evolutionary shifts occurred at both coding and noncoding loci. For comparison, genome‐wide FST between the Arb and BB populations (which are separated over space rather than over time) was 0.044 (0.043–0.045) in 1997 and 0.047 (0.045–0.048) in 2004.To identify genes showing evolutionary shifts in the Arb and BB populations, we conducted outlier fixation index . Tajima's D values were strongly correlated before and after the drought for both Arb  and BB , indicating a lack of a genome‐wide effect of this recent evolutionary event on Tajima's D. In addition, the loci identified by Tajima's D and outlier FST analyses tended to be distinct . Many of the outlier genes we detected have annotations related to drought response traits  in descendants as compared to ancestors  and high divergence (FST) are focused on a relatively narrow region within SOC1, with both 2004 populations showing an elimination of variation (He = 0) at many SNPs. This result suggests strong recent selection acted on variation within this gene. Other outlier genes are related to flowering time and hormones that influence flowering time, such as gibberellin, as well as drought and osmotic stress tolerance  and protein phosphatase type 1  in the Arb population [which showed a much greater shift in flowering time than the BB population  and BB (433 genes), only 11 (0.025%) are found in both populations  with 4 biosamples: SAMN03397805: Back Bay 1997 (TaxId: 3711); SAMN03397806: Back Bay 2004 (TaxId: 3711); SAMN03397807: Arboretum 1997 (TaxId: 3711); and SAMN03397808: Arboretum 2004 (TaxId: 3711).All sequences have been deposited in the Short Read Archive: PRJNA277879: Rapid genome‐wide evolution in N.B.O. conducted the laboratory work; N.C.K., J.S.R. and S.J.F. designed the experiments; all authors contributed to writing and editing the manuscript; and all authors contributed to data analyses.Fig. S1. High correlation between KASP and Illumina next‐generation determined allele frequencies in Brassica rapa populations.Click here for additional data file.Fig. S2. Tajima's D, calculated using a 100‐kb sliding window, shown across the genome for ancestral (blue) and descendant (red) populations with trend lines added using a local regression smoothing with a span of 0.05.Click here for additional data file.Table S1. SNPs chosen for KASP validation of Brassica rapa samples.Click here for additional data file.Table S2. Genes showing evolutionary shifts in both populations.Click here for additional data file.Data S1. All FST outlier genes, which evolved during the course of a drought.Click here for additional data file.Data S2. Methods.Click here for additional data file.


The strawberry tree diverged into two groups during the Quaternary, but before the LGM, surviving in refugia located in the western end of the Mediterranean region and with the Eastern Mediterranean being colonized more recently. This migration was possible because Europe and North Africa were occasionally connected through the straits of Gibraltar and Sicily. Likewise, our evidence supports arrival in Ireland from northern Iberia in post-glacial times. Altogether, our results reveal the considerable ability of the strawberry tree for dispersal, allowing it to migrate over thousands of kilometres and cross stretches of sea, which may be crucial for its future survival. Arbutus unedo), are particularly useful to unravel these patterns. Here, we describe its phylogeographic structure and check for concordance with patterns seen in other Mediterranean plants: longitudinal/latitudinal clines of diversity, evidence for glacial refugia and the role of sea straits in dispersal. We also identify the most likely source for the disjunct Irish population. With this aim, we sequenced four chloroplast non-coding fragments of A. unedo from 23 populations covering its whole distribution. We determined the genetic diversity, population structure, haplotype genealogy and time to the most recent common ancestor. The genealogy revealed two clades that separated during the last 700 ky but before the last glacial maximum. One clade occupies Atlantic Iberia and North Africa, while the other occurs in the Western Mediterranean. The Eastern Mediterranean is inhabited by newer haplotypes derived from both clades, while the Irish population is closely related to Iberian demes. The straits of Sicily and Gibraltar partially restricted the gene flow. We concluded that a vicariance event during the Late Quaternary in the western end of the species' range followed by eastward migration seems a likely explanation for the observed phylogeographic pattern. The role of straits indicates an occasional communication between Europe and North Africa, suggesting that the latter was a novel refugia. The East–West genetic split in Iberia is consistent with the refugia-within-refugia model. Finally, the strawberry tree possibly reached Ireland from Iberia instead of throughout the maritime fringe of France as previously thought.Phylogeographic studies give us the opportunity to reconstruct the historical migrations of species and link them with climatic and geographic variation. They are, therefore, a key tool to understanding the relationships among biology, geology and history. One of the most interesting biogeographical areas of the world is the Mediterranean region. However, in this area, the description of concordant phylogeographic patterns is quite scarce, which limits the understanding of evolutionary patterns related to climate. Species with one-dimensional distribution ranges, such as the strawberry tree ( Phylogeography uses molecular information to infer the historical forces that have shaped the genealogical architecture of populations and closely related species . The typDespite the above, commonalities still exist, even though they frequently show some inconsistencies. Similarly to other areas of the world, there is a south–north decrease in genetic diversity . This laAnother interesting pattern is the finding that genetic diversity increases from west to east along the Mediterranean in many species . FinallyArbutus unedo (strawberry tree), a small tree with a neat circum-Mediterranean distribution (Arbutus as a component of the Madrean–Tethyan flora that existed between western North America and Eurasia until the end of the Oligocene (25 Mya). The divergence between Mediterranean and North American Arbutus was estimated in at least 21.2 Mya  to test whether some patterns detected in other Mediterranean plants also apply to the strawberry tree  and (iii) to identify the likely source for the disjunct Irish population. To our knowledge, this is the first range-wide study of the phylogeographic structure of A. unedo.In this study, we sequenced four chloroplastidial non-coding fragments to address a number of specific issues: (i) to estimate the time to the most recent common ancestor (TMRCA) of Arbutus unedo is a small–medium tree from the family Ericaceae, subfamily Arbutoideae. Genus Arbutus includes several members in USA and four in Europe: A. andrachne, A. pavarii, A. canariensis and A. unedo . At each population, leaves were collected from 12 adult trees separated at least 15 m from each other to minimize the risk of sampling relatives. Leaves were wrapped in Kimtech Science wipes  and stored in silica gel until DNA extraction. DNA was extracted with the Realpure Genomic DNA extraction kit (REAL) according to manufacturer’s instructions except that we extended the incubation times for cell lysis (2 h at 55 °C) and protein precipitation (20 min at −20 °C). Extractions were conducted in batches of 23 samples plus a negative control; each batch combined individuals from at least five populations. The quality of extracted DNA and negative controls was checked on 1.5 % agarose gels.The sampling plan covered the whole range of es Table . Additio−1 of each primer and 1 µL of DNA (diluted 1 : 10). Amplification conditions were 30 s at 98 °C for DNA denaturation; 30 cycles of 10 s at 98 °C, 20 s at the specific annealing temperature of each primer and 30 s at 72 °C; and a final extension of 7 min at 72 °C. Annealing temperatures were 60.8 °C (trnT-trnL), 59.7 °C (trnL-rpl32), 61.8 °C (Intron L) and 66.8 °C (trnH-psbA). Before sequencing, PCR products were checked on 1.5 % agarose gels and purified with 1 µL of Exonuclease I (20 U µL−1) and 2 µL of FastAP (10 U µL−1) . Purified PCR products were sequenced under BigDye Terminator cycling conditions on an Automatic Sequencer 3730XL . The program Geneious R6 v.6.1.4 was used to check the quality of the chromatograms and to perform alignments. Singleton polymorphisms and unique haplotypes  were corroborated with reverse sequencing reactions to discard sequencing artefacts. As the non-recombinant nature of cpDNA makes it equivalent to a single-locus marker, sequences from the four fragments were combined into a single haplotype for every individual. These sequences can be obtained from GenBank using the accession numbers: BankIt 1868954: KU194976–KU195222 (Intron L fragment); BankIt 1874447: KU205359–KU205584 (trnT-tnrL fragment); BankIt 1874452: KU205585–KU205820 (trnL-rpl32 fragment); BankIt 1874463: KU205821–KU206025 (trnH-psbA fragment). It is noteworthy that the identification of each population is represented by the last two letters and two numbers of the sequence definition name. Supporting Information—Table S1 shows the equivalence between the population codes used in this article and the population codes that identify the populations in GenBank. Still, the identity of each fragment was retained when we calculated the TMRCA to allow for differences in the mutation rate of each region.After reviewing previous phylogeographic studies, initial trials were conducted with five non-coding cpDNA regions commonly used with trees and, specifically, in studies of closely related species: trnS(GCU)-trnG(UUC), trnH(GUG)-psbA, trnL(UAG)-rpl32, trnT(UGU)-trnL(UAA) and Intrón L . These iπ) using Arlequin 3.5 (FS test to detect any evidence of demographic expansion (K) using a simulated annealing procedure to maximize the variance (FCT) among those groups. The analysis was performed in SAMOVA 2.0  and 20 for indels. To improve the interpretation of the resulting tree, we removed the resulting loops, but always without modifying the interpretation. Complementarily, we estimated the genealogy using TCS 1.21 , which allowed BEAST to obtain the precise mutation rate. The four cpDNA fragments were designed as unlinked partitions to allow for variable mutation rates. Chain convergence was assessed with Tracer 1.6 (available in the BEAST2 package at http://beast.bio.ed.ac.uk/) and by checking the effective sampling size (ESS) values. Trees were summarized in a maximum clade credibility tree obtained in TreeAnotator 2.1 and visualized in FigTree 1.4.1 .Estimates of the TMRCA were conducted with the 207 The calculation of the TMRCAs for the groups later detected was based on independent running of BEAST, by combining two searches with 10 million MCMC, using only the haplotypes assigned to each group.ftp://ftp.essc.psu.edu/pub/emsei/pollard/Stage3/PLOT/.The climatic reconstruction observed in Fig. A. unedo  were private (Hp); interestingly, five Hp were detected in North Africa (Morocco). The two individuals of A. andrachne produced a single haplotype that differed by 16 new point mutations from the consensus sequence of A. unedo. Haplotype diversity in A. unedo was 0.901 (±0.01) (standard error), while nucleotide diversity (π) (×102) was 1.238 (±0.60). Nonetheless, genetic diversity was unevenly partitioned among populations as Hd and π estimates for the 23 populations ranged 0.000–0.810 and 0.000–0.934, respectively .A total of 28 haplotypes were found 2 = 0.58 ± 0.30) to the Western Mediterranean , and to the Atlantic coast .Total gene diversity (ht) was 0.919 (±0.02), while the average within-population gene diversity (hs) was 0.585 (±0.04). We also calculated the diversity at a regional scale by partitioning the populations into three groups suggested by the phylogeographic analyses (see below): Eastern Mediterranean , Western Mediterranean  and Atlantic coast . Diversity at regional scale increased from the Eastern . NST (0.645 ± 0.067) was significantly higher than GST (0.363 ± 0.040) (P < 0.001) indicating the existence of a strong phylogeographic structure   Table . One gro[seeSupporting Information—Table S1]. The median-joining network , while the other clade was dominated by H4, again surrounded by 9 less-common haplotypes (H4-derived).Haplotypes H2 and H4 dominated the data set (37 %), whereas 12 haplotypes were very rare, as they were detected in just one (10 unique haplotypes) or two individuals (2 haplotypes) [seeSupporting Information—Fig. S2] and BEAST [seeSupporting Information—Fig. S3]. The previous description in two sublineages was corroborated and also the distribution of the main haplotypes. Even if the general patterns were concordant, some exceptions were found. TCS assigned H11 and H10 to the sublineage H2, while H20 was placed into sublineage H4 and it placed H7 and H9 as a different group, although closest to H2 than to H4  (not shown). Likewise, BEAST analysis located H15 into sublineage H2, while H18 and H20 were placed into sublineage H4. Despite these minor modifications, the previous interpretation of the Network tree is still consistent.The partition of haplotypes in two large lineages was remarkably consistent with their geographical location as well as with the clusters of populations identified by SAMOVA Fig. . AccordiFS tests run for the complete data set and for each genetic clade treated separately . Therefore, the final estimates of TMRCA relied on a constant growth model. However, the presence of 11 out of the 14 private haplotypes in the Atlantic clade might indicate a higher ability to diverge and spread. The latter is further supported by the higher number of haplotypes and more extensive geographical distribution of this clade. The Bayesian tree inferred by BEAST reproduced the neat division in two clades (Atlantic and Mediterranean) found in the network analysis [seeSupporting Information—Fig. S2]. The BEAST inference suggested that the split between Atlantic and Mediterranean lineages must have occurred before the LGM, around the Middle/Late Pleistocene boundary. Thus, the TMRCA for A. unedo was 365 ky . The calculation of the TMRCAs for the Atlantic and the Mediterranean lineages was 258 ky  for the Atlantic and 194 ky  for the Mediterranean.Initial trials in BEAST were run using exponential growth as the coalescent tree prior. Regardless of the data set tested , the posterior distribution of the growth rate [95 % highest posterior density (HPD)] always included zero indicating no support for the existence of exponential growth and establishes the plausibility of the constant population growth model, based on our data. The absence of demographic expansion was confirmed by Fu's A. unedo, we infer that the diversification in two lineages also occurred in the late Pleistocene but before the LGM, suggesting that it may have coincided with the hardest glaciations recorded in the Quaternary  a predominance of younger haplotypes in this area (H2-derived and H4-derived) and (ii) the likely unsuitability of the West Mediterranean during glacial maxima. Accordingly, paleoclimatic reconstructions of the LGM indicate that minimum monthly temperatures in the Aegean–Anatolia area were below the 4 °C limit required by the strawberry tree for survival EurophacaA. unedo because only a handful of H2-derived haplotypes managed to reach the Eastern Mediterranean, contrarily to other trees where several haplotypes were common to both sides of the strait  with north-east Iberia (SP7) and the Balearics (SP8). Indeed, north-east Iberia (SP7) is more closely related to French demes than to other Iberian populations. Thus, the strawberry tree adds to the many cases of trees with a clear-cut East–West division in the Iberian Peninsula . Since wA. unedo in Ireland . This ability for long-distance migration could be useful in a scenario of future changes, allowing the species to migrate to new areas such as Northern France, where a drier and warmer climate, with a greater number of extreme weather events, it is expected ; BankIt 1874447: KU205359–KU205584 (trnT-tnrL fragment); BankIt 1874452: KU205585–KU205820 (trnL-rpl32 fragment); BankIt 1874463: KU205821–KU206025 (trnH-psbA fragment).Spanish Ministry of Science and Innovation (research grant CGL2009-11356), the European Regional Development's Fund (ERDF) and also by the Spanish Ministry of Education (FPU fellowship AP-2009-0962 to X.S.).This work was supported by the R.R. and R.B. conceived the ideas; X.S. and R.R. collected the data; X.S. and L.L. performed the work on laboratory; X.S., L.L. and R.B. analysed the data and X.S., L.L., R.B. and R.R. led the writing.None declared.The following additional information is available in the online version of this article –Figure S1. The variable positions in the 28 haplotypes detected in A. unedo. The first 18 haplotypes belong to the Atlantic clade and the last 10 to the Mediterranean one.Figure S2. The genealogy of the haplotypes obtained with FigTree, which visualized the analysis made with BEAST and TreeAnotator. We observed two main clades, the upper one (Atlantic) with 17 haplotypes and the lower one (Mediterranean) with 11 haplotypes.Figure S3. The genealogy of the haplotypes obtained with TCS. They are grouped into three clades, being the ancestral haplotype of each clade represented inside the rectangle.Table S1. The number of individuals per haplotype in 23 populations of A. unedo.


Potamogeton pectinatus L.  giving structure to various aquatic habitats. We compared the level of genetic diversity in a heterogeneous series of aquatic habitats across Iran and tested their differentiation over distances and across mountain ranges (Alborz and Zagros) and desert zones (Kavir), with values obtained from temperate region populations. The diversity of aquatic ecosystems across and along large geographic barriers provided a unique ecological situation within Iran. P. pectinatus were considered from thirty-six sites across Iran at direct flight distances ranging from 20 to 1,200 km. Nine microsatellite loci revealed a very high number of alleles over all sites. A PCoA, NJT clustering and STRUCTURE analysis revealed a separate grouping of individuals of southeastern Iranian sites and was confirmed by their different nuclear ITS and cpDNA haplotypes thereby indicating an evolutionary significant unit (ESU). At the level of populations, a positive correlation between allelic differentiation Dest with geographic distance was found. Individual-based STRUCTURE analysis over 36 sites showed 7 genetic clusters. FST and RST values for ten populations reached 0.343 and 0.521, respectively thereby indicating that allele length differences are more important and contain evolutionary information. Overall, higher levels of diversity and a stronger differentiation was revealed among Iranian P. pectinatus than previously observed for temperate European regions, due to regional differences across mountain ranges over long distances.Biogeographic barriers for freshwater biota can be effective at various spatial scales. At the largest spatial scale, freshwater organisms can become genetically isolated by their high mountain ranges, vast deserts, and inability to cross oceans. Isolation by distance of aquatic plants is expected to be stronger across than alongside mountain ridges whereas the heterogeneity of habitats among populations and temporary droughts may influence connectivity and hamper dispersal. Suitable aquatic plant habitats became reduced, even for the widespread submerged  Biogeographic barriers for freshwater biota can be considered at three spatial scales, namely inter and within basin and continental. At the largest spatial scale, freshwater organisms are isolated by their high mountain ranges, inability to cross oceans and vast deserts . Gene flBesides merely distance as a limit to connectivity, especially large deserts and mountain ranges act as additional natural barriers. The flora of the mountains provides many examples of subspecies with populations and very closely related species that show restricted gene flow due to these inherent geographic barriers , 8. IsolDrought as a climatic event is one of the most threatening challenges that occur due to climatic change and global warming . The hetPotamogeton pectinatus L. is one of most diverse submerged aquatic species [The importance of submerged vegetation in aquatic ecosystems management and biodiversity has been highlighted , 18, 19. species , 28. Thi species , and uti species . These u species , 32. Pla species .P. pectinatus reproduces both sexually and vegetatively through tubers emerging from the rhizomes and acting as propagules. Asexual reproduction is thought to be responsible for short distance dispersal while sexual reproduction with seeds is more important to ensure long distance dispersal and long-term survival [P. pectinatus. Hettiarachchi & Triest [et al. [P. pectinatus. Hangelbroek et al [et al. [et al. [et al. [P. pectinatus increased with geographic distance. Following the development of nine polymorphic microsatellite loci for P. pectinatus [P. pectinatus but also other submerged plant taxa was found between coastal brackish water and freshwater inland habitats in lowland regiosn of W. Europe by Triest et al. [P. pectinatus in river populations and managed pond [et al. [P. pectinatus maintained a high level of variation within and between two contrasting and distant lakes in China. Those previous studies over the last decades confirmed that P. pectinatus has a mixed reproduction system, an overall high level of gene diversity and an increased differentiation over long distances of hundreds of kilometers. All previous studies were done in temperate and temperate-cold areas having many permanent water bodies and often at close vicinity. Therefore, it can be hypothesized that P. pectinatus populations might exhibit a stronger genetic differentiation over similar distances across mountain ranges and in a drier climate zone with often temporary habitats of very isolated wetlands areas and rivers habitats that show strong fluctuations due to high evaporation levels and use for irrigation in agriculture.survival , 29. The& Triest  used isoek et al  used RAP [et al. , 34, 35. [et al.  and Kingctinatus  an evoluctinatus  were shot et al. . They alged pond  revealed [et al.  using AFThe specific biogeographic location of Iran  with itsP. pectinatus populations over a long gradient of regions across mountain ranges as potential barriers, whether in such habitatsOur aim was to test, by considering sites with P. pectinatus can maintain its usually high levels of allele and gene diversity.Differentiation over distances and across mountain ranges and around deserts would be more pronounced and structured than in temperate regions.Therefore we considered a sampling design with a large number of sites across the various biogeographic regions and used the same microsatellite loci as a previous European studies, following a preliminary taxon verification with nuclear ITS and maternal chloroplast marker genes.P. pectinatus L. —as in Triest and Sierens [Samples were dried on silica gel prior to extraction and a voucher specimen for each population was deposited at HUI (Herbarium of the University of Isfahan). One subset  of the collection was used for cpDNA haplotypes and ITS (accession number: DQ840279.1) characterization (results not shown in this paper). Identification of  Sierens . A more For each specimen we measured 29 morphological characters. Among these, the leaf apex and leaf width (36 measurements per site) could differentiate morphologically the specimens characterized for a nuclear ITS-A or B. B. Mean rank for Krustal-Wallis was between 1.12–3.11 mm for the group of specimens sharing ITS-B. Morphological measurements were tested for significant differences with one-way ANOVA  and pairwise Mann-Whitney U test.Genomic DNA was extracted from leaf tissue using CTAB by Gawel and Jarret  with somP. pectinatus is hexaploid, the identified primer pairs amplify microsatellite loci that are ‘‘diploids”, confirming the observations of Nies & Reusch [FIS), total inbreeding (FIT), subpopulation differentiation (FST) and gene flow (Nm) using GenAlex. Weir & Cockerham [FIT (CapF), FIS  and FST (Theta) following Rousset [Potpect24, Potpect26, Potpect28, Potpect32, Potpect34, Potpect37, Potpect39, Potpect40, and Potpect42 . Amplifi& Reusch , 37. To & Reusch  and GenA& Reusch . At locu& Reusch  with inbockerham  estimati Rousset  were calDest [et al. [ST was tested by calculating multilocus estimates of kinship coefficient (Fij) between all pairs of individuals implemented in the software SPAGeDi v1.4 [We tested for recent bottlenecks in each population under the two-phase model (TPM) with 95% single-step mutations and 5% multiple-step mutations (Wilcoxon’s test 1-tailed) using bottleneck 1.2.02 . GeneticDest  was calcDest  and usedDest  was carr [et al. . For tes [et al.  softwareeDi v1.4 .The haplotypes obtained from amplified product lengths of TrnH-PsbA showed congruent differences for Aligoodarz (1), Borujen (5), Googhar (15) and Yaschaman (35) when compared to all other populations. Morphological measurements between this group (mean rank of 1.12–3.11 mm) and specimens from other sites (mean rank of 0.25–1.27 mm) were significant with one-way ANOVA  (P = 0.036) and pairwise Mann-Whitney U test (P = 0.012). The lowest morphological value was 0.25 mm (2-Amirkelaieh) whereas the highest value 3.11 mm (15-Googhar).Potamogeton pectinatus populations, we tested these cross-amplified loci with microchecker, but there was no evidence for null alleles, large allele dropout or scoring error due to stuttering. Over all sites and loci, the nine microsatellite loci revealed a total of 130 alleles. The mean number of alleles was highest for potpec 39 (3.3) and lowest for potpec24 (1.9) with an overall observed heterozygosity (Ho = 0.2–0.7) nearly similar to the overall expected heterozygosity (He = 0.3–0.7) (As the primers were previously developed and used on European 0.3–0.7) .FIS was estimated -0.077–0.846. The populations of Azbaran (3), Vimcheh (34) and Izeh (19) showed evidence of recent bottleneck events  were obtained with re-encounters only occurring within a same site k events .From the 10 considered populations, the percent of individuals assigned to the same population was higher (76%) than those assigned to other populations (24%). The populations of Amirkelaieh (2), Azbaran (3) and Kaniborazan (21) did not share any allele.P. pectinatus identity of nuclear ITS and trnH-psbA (380 bp length) for the samples from Hamzeali (16) Borujen (5), Googhar (15), and Yaschaman (36) (A Neighbor Joining tree of microsatellite gene diversity was congruent with the man (36) .A PCoA at the level of all individuals revealed a gradient along a first axis that explained 28.9% of all variation but where several individuals were clearly separated as a group Figs  and 4. MPercentage of variation explained by the first three axes in PCoA at 10 populations is 28.80% and 17.94% for first and second axes, respectively.FST was between 0.156–0.595 among the ten populations. The lowest FST was between Izeh (19) and Amirkelaieh (2) and the highest was between Azbaran (3) and Yaschaman (35) (The range of man (35) .Dest values were between from 0.001 to 0.925. There was no relationship at all between Fst and Dest (Pearson correlation = 0.007) indicating that allelic differentiation can be more important as an explanatory variable of IBD. There was a positive relationship between FST and geographic distance (R2 = 0.35 and P = 0.01) but there was an improved positive correlation between Dest and Ln (1+Km)  (The range of  = 0.56) .An AMOVA showed that the percentage of molecular variance among regions and populations were 15% and 18%, respectively .Potamogeton pectinatus in Iran showed 6 genetic clusters . In the largest distance class, the RST value (0.698) and FST value (0.496) were highest. Also in all distance classes, except second distance class, the values for RST were significantly higher than for FST. FST for all loci P between neighboring populations of Izeh and Shushtar was not significant. Genetic analyses at the individual level in 5 distance classes showed that pairwise correlation coefficients of allele size are significant for upper distance classes (beyond 610 km).The higher values for P. pectinatus and previous genetic studies on submerged aquatic plants based on dominant markers [P. pectinatus L. is one of most diverse submerged aquatic species [et al [et al [et al [In spite of preliminary studies on the vegetative mode of reproduction of  markers , 33, 2,  markers , 39 a hi markers , 24, 25  markers . Among a species , 28. Acrs [et al  observedl [et al  in genetl [et al , Jackrell [et al  used lowFST value in this study was 0.5 that was relatively high due to spatial differences of studied populations and stochastic founder events as in Triest et al [Dest) was much more pronounced because of the large amount of alleles detected across the different areas. Also the He value in our study is higher than in central or western Europe [P. pectinatus due to sexual reproduction is most likely explaining the observed high genetic diversity [P. pectinatus has an important effect on its genetic structure [P. pectinatus is a polymorphic species with high phenotypic plasticity [Under an Island Migration Model, increasing geographical distance among populations is expected to lead to enhanced genetic isolation, which essentially corresponds to a stepping-stone population structure and requires that dispersal is primarily local rather than long-distance , 3,4,5,6st et al  and Niesst et al . Howevern Europe , 39. Geniversity , 23. Thetructure  and we aasticity . As a phasticity ,70.Potamogeton pectinatus. Mountains act as strong genetic barriers but river valleys recognized as corridors for gene flow [Potamogeton seems to be linked to mountain regions as indicated by the STRUCTURE results (for 10 populations)  showed very low or absent connectivity across mountains north (Albroz) and south (Zagros/Kerman).With regard to our results, we found strong genetic differentiation between Iranian sites of ene flow . The dislations)  and by tlations) . We obtaP. pectinatus in Iran. We found a strong positive correlation between Dest and geographic distances  (In spite of short (direct flight) distances between some populations, a high level of differentiation occured because of dispersal barriers such as Alborz mountain (Amirkelaieh (2), Azbaran(3)) or (Kaniborazan (21); Miandoab (24)). The highest differentiation was between Azbaran and Yaschaman (= 0.925) . The comRhodiola dumulosa (Crassulaceae) between rivers in Mountains ridges also showed strong differentiation [There is however some connectivity between both sides of Zagros mountains namely between SW Zagros 19, 25, 27, 30) and N Zagros . This area in Iran was reported as a route for bird migration . It thus, 27, 30 Potamogeton represents the largest genus in Potamogetonaceae, including about 100 species and 50 interspecific hybrids worldwide. Hybridization therefore can be another complexity that is thought to be relatively frequent, because complex series of polyploidy and aneuploidy are present within the genus [P. pectinatus having 78 chromosomes could form hybrids with other linear leafed taxa such as P. filiformis and P. amblyphyllus [Potamogeton populations. In our study we have detected only P. pectinatus haplotypes in correlation with specific microsatellite alleles for each site. No chloroplast capture was found. Consequently, we assume the absence of hybrid specimen in our samples.The most common haplotype (cp 378) can be observed in most regions but cp 379 in only two regions  despite their large allelic differentiation of microsats. Thus the mutation of the chloroplast mononucleotide repeat (1 base only) is most likely independent as event in both regions and not an indication of long distance dispersal. Another shorter variant (cp377) also occurs in only two regions . The morphological characters such as leaf sheath, width of leaf and tip of leaf were different from all other populations. These populations clearly belong to an evolutionary lineage within P. pectinatus and do not represent another species (e.g. P. amblyphyllus) because their ITS in a NJtree was separated (97% bootstrap) from P. amblyphyllus (S. amblyophylla) of NCBI (next paper under submission).It is proven that habitat diversity often makes ecological barriers against gene flow that leads to interpopulation genetic separation ,74. DiffP. pectinatus populations over a long gradient of regions across mountain ranges and around deserts in Iran, their genetic differentiation over distances was more pronounced and structured than for temperate regions. However, across such habitats P. pectinatus maintained an even higher level of allele and gene diversity than in temperate regions.As a conclusion, when considering


Culex pipiens and Culex torrentium. Due to their morphological similarity, ecology, distribution and vector abilities, knowledge about these species' population structure is essential. Culicidae from 25 different sampling sites were collected from March till October 2012. All analyses were performed with aligned cox1 sequences with a total length of 658 bp. Population structure as well as distribution patterns of both species were analysed using molecular methods and different statistical tests like distance based redundancy analysis (dbDRA), analysis of molecular variances (AMOVA) or McDonald & Kreitman test and Tajima's D. Within both species, we could show a genetic variability among the cox1 fragment. The construction of haplotype networks revealed one dominating haplotype for Cx. pipiens, widely distributed within Germany and a more homogeneous pattern for Cx. torrentium. The low genetic differences within Cx. pipiens could be a result of an infection with Wolbachia which can induce a sweep through populations by passively taking the also maternally inherited mtDNA through the population, thereby reducing the mitochondrial diversity as an outcome of reproductive incompatibility. Pairwise population genetic differentiation (FST) ranged significantly from moderate to very great between populations of Cx. pipiens and Cx. torrentium. Analyses of molecular variances revealed for both species that the main genetic variability exists within the populations . Based on a distance based redundancy analysis geographical origin explained a small but significant part of the species' genetic variation. Overall, the results confirm that Cx. pipiens and Cx. torrentium underlie different factors regarding their mitochondrial differentiation, which could be a result of endosymbiosis, dispersal between nearly located populations or human introduction.Nowadays a number of endemic mosquito species are known to possess vector abilities for various diseases, as e.g. the sibling species  Aedes aegypti, Aedes albopictus or Ochlerotatus japonicus, which have been introduced and established in different countries. However, numerous indigenous mosquito species are known to be potential carriers of diseases such as Sindbis virus, Ockelbo virus, Usutu virus, Batai virus, West-Nile virus or even malaria Since the late 19th century, mosquitoes are known as vectors for various diseases as malaria, dengue, yellow or Chikungunya fever Culex with more than 750 described species worldwide Culex, the subgenus Culex includes seven species in Europe Cx. pipiens being one of the most common and widespread holarctic species. Together with its palaearctic biotypes Cx. pipiens pipiens and Cx. pipiens molestus, Cx. pipiens belongs to the Culex pipiens complex which also includes the non-european species Cx. quinquefasciatus, Cx. australicus as well as Cx. globocoxitusCx. pipiens pipiens are known to be ornithophilic, but several studies also mention a potential anthropophilic diet  in order to distinguish this type from Cx. torrentium. Recently a multiplex real-time PCR for simultaneous detection and differentiation of Cx. pipiens biotypes and Cx. torrentium was established Cx. pipiens and Cx. torrentium within the Frankfurt/Rhine-Main Metropolitan Region showed a genetic variability within both species Cx. pipiens and Cx. torrentium and to investigate whether there are differences in their genetic composition and patterns of distribution. Here, we present first-time population comparisons for both species in Germany.A former study about 2 and/or BG Lure as an attractant as well as EVS-traps with dry ice. Caught specimens were stored at −20°C. Larvae were collected from natural as well as artificial water pools using hand nets or ovitraps, fixed and preserved in 70% ethanol or kept alive to raise them to adults. For morphological identification (based on Culicidae from 25 different sampling sites in 22 German cities see  were colbased on  of larvabased on . No endaGGTCAACAAATCATAAAGATATTGG 3′) and HCO 2198 (5′ TAAACTTCAGGGTGACCAAAAAATCA 3′) 2, 1U Taq polymerase  as well as varying concentrations of DNA and Millipore water, in a total volume of 30 µl. The cycle parameters were the following: 1 cycle of initial denaturation at 94°C, 60 s; 6 cycles of 94°C, 40 s; 45°C, 40 s; 72°C, 60 s; 36 cycles of 94°C, 40 s; 51°C, 40 s; 72°C, 60 s and for terminal extension 1 cycle 72°C, 5 min; with a final ramping to 8°C. The yield and quality of DNA was analysed with SYBR-Green (Life Technologies GmbH) staining and agarose gel-electrophoresis. Sequencing and sequence analysis were carried out as previously described The DNA extraction was carried out with glass fiber plates  following a former described protocol Cx. pipiens and Cx. torrentium and to analyse the processes that might have shaped the present day distribution, we used the cox1 barcoding fragment, which can distinguish between both species. The McDonald & Kreitman Test was calculated to show neutral evolution or selection among the analysed mitochondrial DNA. In addition we used Tajima's D to test recent demographic or range expansion. We also calculated pairwise FST's to show differences between haplotype compositions of sampling points and analysed the molecular variances as well as genetic distances and genetic variability depending on geographical origin. All analyses were performed with aligned cox1 sequences with a length of 658 bp where no frame shifts or stop codons were found. The genealogical relationship between haplotypes of Cx. pipiens and Cx. torrentium was analysed by reconstructing phylogenetic networks for each species. We used the method of statistical parsimony as described by Templeton et al. To infer the population structure of CT, FSC, and FST, which refer to distance among groups, among populations within groups and within populations . We tested whether the cox1 sequences evolved neutrally with the McDonald & Kreitman Cx. modestus as outgroup taxa. We also analysed Tajima's D ST's, to determine significance by permuting genotypes among populations (1023 permutations), using Arlequin 3.5.1.2. Additionally we tested whether there was a recent range expansion, a bottleneck or a selective sweep within the two species and their populations For population analyses, models of sequence evolution for the population genetic analysis were chosen according to MODELTEST Cx. pipiens  and Cx. torrentium  from 25 different localities within Germany were sequenced and compared with sequences deposited in the GenBank using the BLAST algorithm Cx. pipiens was much more abundant than Cx. torrentium and could be detected at 21 out of 25 sampling sites  was used as the reference (H1) for other haplotypes. For Cx. torrentium only one to three different haplotypes were observed in 12 out of 15 localities (Cx. pipiens the highest haplotype diversity was observed in Heldenbergen and Dresden (both n = 6). A haplotype diversity with more than three different haplotypes at a sampling site was identified in Berlin, Lebus, Rietschen and Stralsund.In total, 597 individuals of ng sites . At ten calities  whereas Cx. pipiens resulted in one single network with 24 different haplotypes and no subnetworks. The highest outgroup probability within the network was calculated for H1 which was the most frequent  and observed at all sampling sites  the dataset was partitioned into 27 haplotypes which implies a higher genetic variability within Cx. torrentium compared to Cx. pipiens. The structure of the network was not dominated by one single haplotype. In general the pattern was also rather star like with rare haplotypes (n = 10) that are directly derived from the most frequent one  and only five hypothetical haplotypes are missing. Haplotype three was found 82 times and was present at eight different localities across Germany. Furthermore this haplotype is linked to the second most frequent haplotype (H5) via H24 which differed only by one mutation from both. In contrast to the haplotype network of Cx. pipiens, the most frequent haplotype was not the ancestral one. In Cx. torrentium the most probable ancestral haplotype (H1) is represented by five individuals and was found only at four different localities . It also differed only by one mutation from the two most frequent haplotypes (H3 and H5) and was directly linked to both. Besides the most frequent haplotype H3, additional haplotypes occurred at high frequencies . The second most frequent haplotype, H5, occurred at four different localities and was mainly found in Mönchengladbach. Furthermore this haplotype occurred in central Germany (Frankfurt) and eastern Germany (Dresden). Additionally some of the more frequent haplotypes were exclusively found at single localities like H26, which only occurred at Mönchengladbach. The haplotypes H3 and H5 were much more frequent at central and western localities  than in the eastern parts of Germany  Furthermore, haplotype H8 followed a similar geographical pattern to haplotypes H3 and H5 .The statistical parsimony network calculated with TCS using 198 cox1 sequences of  one H3, . The hapCx. pipiens and 0.03% in Cx. torrentium. The mean genetic distance between both species was 3%. The genetic differentiation based on population pairwise FST is shown in ST values are indicated in ST values into 4 categories Cx. torrentium and Cx. pipiens showed significantly different pairwise FST values. Generally more populations were very greatly significantly differentiated in Cx. torrentium  was highly differentiated from all other populations except for Frankfurt-all with a moderate differentiation. Furthermore, significant genetic differentiation was detected in geographically much closer Cx. torrentium populations in Hesse  derived from the AMOVA calculations where the FST value of Cx. torrentium (0.33) is much higher than the FST value within Cx. pipiens (0.11).The calculated AMOVA revealed that most of the genetic variability within  88.38%, . The gro 88.38%, . Only 3. 88.38%, . The AMO 66.54%, , but in  66.54%, . The higCx. pipiens and also some indications for strong genetic differentiation between populations from the western parts of Germany and central and east-German populations of Cx. torrentium (see above), we tested for isolation by distance using distance based redundancy analysis (dbRDA). However, only a very low proportion of the genetic variation could be significantly explained by geographical distance; the spatial coordinates only explained 2% of the genetic variability in Cx. pipiens and 5% of the genetic variation within Cx. torrentium. The ratio of non-synonymous to synonymous polymorphisms within Cx. pipiens/Cx. torrentium and within the outgroup was not significantly different from the ratio of non-synonymous to synonymous polymorphisms fixed between these groups (Fisher's exact test P = 0.337/0.380). Thus, the McDonald-Kreitman test is consistent with neutral evolution of the cox1 gene and there were no indications that there is selection acting on the studied mitochondrial gene fragment. To reveal signs of population expansion, signs of bottleneck within the population or sudden contractions, we used Tajima's D for which the level of significance was assessed using the permutation test implemented in Arlequin. The overall Tajima's D for Cx. pipiens was negative (−0.75 +/− 0.73) but non-significant (p>0.05). For Cx. torrentium the overall mean Tajima's D was only slightly negative (−0.13+/−1.03) and also non-significant (p>0.05). All calculations for Cx. torrentium populations resulted in non-significant Tajima's D which was either strongly positive or negative. In contrast several populations of Cx. pipiens showed a strongly negative Tajima's D . These highly negative Tajima's D might indicate a recent demographic or range expansions within species As there were indications of genetic differentiation between populations from central and eastern Germany within Cx. torrentium and Cx. pipiens is of particular interest as both species are ornithophilic and potential enzootic vectors for certain arboviruses Culex pipiens could be detected at 21 out of 25 sampling sites whereas Cx. torrentium was only found at 15 out of 25 sampling localities. One explanation for the dominance of Cx. pipiens at several localities could be the different developmental rates of both species which can lead to approximately one generation less per year in Cx. torrentiumCx. pipiens was described to take place between temperature ranges of 8–30°C Cx. torrentium was found in areas with a mean temperature of 10.5°C for May to August 1950–2000 Cx. torrentium was often found in artificial water bodies. Although this has been described previously Cx. torrentium utilizes more natural larval habitats than Cx. pipiensCx. pipiens as well as of Cx. torrentium were found, often sympatrically, in various kinds of habitats. Therefore, it seems unlikely that habitat constitutes an important factor for species composition at the localities, as both species were equally observed in different habitats.Information about the genetic variability of Cx. torrentium and Cx. pipiens provide insights into the genetic structure of these potential disease vectors in Germany. While Cx. pipiens with its bioforms Cx. pipiens pipiens and Cx. pipiens form molestus belongs to the worldwide distributed Cx. pipiens complex, Cx. torrentium is characterized as a sibling species The genetic analyses of populations of Cx. pipiens complex and Cx. torrentium play in enzootic and/or epidemic transmission of arboviruses in Europe. A clear identification of Cx. pipiens form pipiens, Cx. pipiens form molestus and Cx. torrentium is essential but due to the difficulties in morphological identification, molecular methods are necessary to distinguish these forms. Today, commonly used techniques are the amplification of the cox1 gene fragment followed by restriction enzyme digestion of the amplicons Culex pipiens and Cx. torrentium are treated in many studies just as bundles of “Cx. pipiens/torrentium” and have not been separated, leading to the point that not many population structure analyses of these species have been performed in the past for Europe Cx. torrentium showed a higher genetic diversity than Cx. pipiensCx. torrentium, but some haplotypes of Cx. pipiens provide more substitutions within the analysed fragment. The much lower genetic differentiation of Cx. pipiens populations is most likely due to the observed dominant and widespread haplotype H1 which occurred at every sampling locality. The structure of the network with one dominant haplotype and many rare haplotypes  and that these bioforms differ in their relative abundance in different parts of Germany. Former studies Cx. pipiens form molestus in the eastern part of Germany; nevertheless the appearance of this form cannot be excluded and could be an explanation for the observed higher haplotype diversity in eastern populations.It is also obvious that there is a moderate differentiation between western and eastern populations of  Germany . The reaCx. pipiens and Cx. torrentium is the much higher genetic differentiation between populations of Cx. torrentium; the overall FST value was 0.33 for Cx. torrentium compared to a three times lower FST value of Cx. pipiens (FST = 0.11). Furthermore the absence of one single dominant haplotype, the higher haplotype diversity, the stronger genetic differentiation between populations . As the dbRDA is based on pairwise genetic distances between individuals and geographic coordinates, all individuals from every sampling site were included in this analysis.One main difference between the population structure of ions see  and the ions see  reflectsCulex spp. is about 0.2–2.6 km, depending on various factors Cx. erraticus of 0.967 km which is close to the mean range by mark-release-recapture experiments (0.73 km) Cx. erraticus as a stronger flyer than most other mosquito species that disperse only a few hundred meters. In our study, Frankfurt city center with many skyscrapers and very patchy distributed larval habitats seems to serve as a barrier between the localities FFM-Ostend (FZ), FFM-Bornheim (FB) and FFM-Bockenheim (KS). Between these localities and for FFM-Sachsenhausen (FS) the Main River serves as an additional geographic barrier which can limit both geographic as well as genetic expansion. Furthermore, the habitat of FFM-Sachsenhausen differs from the others sampling stations, as it is located in a rural, forested area. As stated by LaPointe Cx. quinquefasciatus undertaken in a forest, dispersal is the result of appetential flights, searching for hosts or oviposition sites. Here, both species were found in municipal and in rural areas, respectively. Within the urban localities the highest numbers of specimens were found at places with a slight suburban character like gardens or patios. This indicates that Cx. pipiens as well as Cx. torrentium both have adapted to a life in human neighborhoods. Suitable conditions for reproduction are met in these environments; artificial containers serve as egg deposits for both species (own observation) and hosts for blood meals are frequent, i.e. humans and synanthropic birds e.g. sparrows, pigeons and blackbirds. Therefore long appentential flights are not necessary in these areas, which might explain the differentiation between the Cx. torrentium populations within Frankfurt. Furthermore, the population structure of Cx. pipiens and the lower genetic differentiation than in Cx. torrentium might be due to the infection with different strains of Wolbachia symbionts. Not all populations are compatible with each other. Crossings of males from a southern German population with females from the North result in fertile descendants whereas reciprocal crosses, on the other hand, result in fertile clutches but no hatchings, indicating a reproduction barrier Wolbachia symbionts. Additionally, several factors such as the Wolbachia strain, amount of infection, host species, temperature and rearing density influence the CI intensity Wolbachia-free females are at a reproductive disadvantage when they copulate with infected males; the spread and fixation of Wolbachia infections is facilitated Wolbachia with each generation. As such an infection can sweep through the population leading to reduced mitochondrial diversity Wolbachia strains resulting in CI or a higher degree of uninfected specimens. Two problems which could lead to misinterpretation of the results are pseudogenes and maternally inherited symbionts, like Wolbachia. While the situation for Cx. pipiens has been discussed already, the situation for Cx. torrentium is different, as pseudogenes can be excluded because of the previous testing of the dataset. No Wolbachia infections have been found so far in Cx. torrentiumWolbachia infections occur in German populations of Cx. torrentium, it could mean that there are no reproductive barriers resulting from CI and no mitochondrial sweep through populations, which explains the heterogeneity of most of the analysed populations.Previous studies have shown that the mean dispersal range of Cx. pipiens and Cx. torrentium are synanthropic and share similar ecological habitats, which in some cases contradict the common conception. One example of this is the larval habitat. Culex torrentium was thought to utilize more natural breeding sites, whereas in this study larvae of both species were often found sympatric in artificial water bodies. However, different factors underlie the mitochondrial differentiation in both species. Some studies argued that feral and synanthropic forms seem to be sufficient for gene flow to diminish drift in Cx. pipiensWolbachia or insecticide-resistance due to previous eradication events. Another aspect leading to decreased genetic differentiation may be the dispersal between nearly located populations or mixing by human introduction. In the case of Cx. torrentium these mechanisms seem to act in the opposite direction: dispersal as well as human introduction influences the mitochondrial diversity of populations. In previous works, Wolbachia could not be detected in Cx. torrentiumCx. pipiens has not occurred and the mitochondrial diversity of populations could remain constant or even increase.As shown here, Cx. torrentium with a higher haplotype diversity as well as the absence of one dominant haplotype which was only found for Cx. pipiens. Different species can vary in their insecticide resistance, ecological habits and vector competence. However, whether cryptic species are involved, or Cx. torrentium might be a variable species should be the focus of future studies. Hybrids between the two bioforms pipiens and molestus and between molestus and torrentium are of particular interest, as these represent potentially important bridge vectors for different zoonotic arboviruses possibly having a major impact in the risk assessments for arboviruses in Germany. It has to be noted that due to possible insecticide-resistance or Wolbachia-induced sweeps in populations, mitochondrial markers are not, or just in a limited way, useful for analyses of such infected populations. On the other hand, mitochondrial markers are generally producing reliable results for species without mitochondrial population-sweeps. Another explanation for the low haplotype diversity can be the result of insecticide-induced sweeps after eradication programs, when only a small percentage of the original population remains. Apart from the aforementioned remaining questions, future studies should focus on dispersal for both species in different areas as different dispersal capabilities can result in varying differentiation and possibly even in distinct vector competences for different populations.Nevertheless, the species show a big natural diversification. The very strong differentiation between populations indicates a splitting within Table S1Names and contact details of all private persons can and will be given on request.(DOCX)Click here for additional data file.


Lithobates catesbeianus) is endemic to eastern North America, but now has a global distribution and is considered to be one of the worst invaders in the world. In Montana, several introduced populations have been reported, but little is known of their sources and vectors of introduction and secondary spread. We evaluated the genetic composition of introduced populations at local (Yellowstone River floodplain) and regional (Montana and Wyoming) scales in contrast to native range populations. Our objectives were to (1) estimate the number of introductions, (2) identify probable native sources, (3) evaluate genetic variation relative to sources, and (4) characterize properties of local‐ and regional‐scale spread. We sequenced 937 bp of the mitochondrial cytochrome b locus in 395 tadpoles collected along 100 km of the Yellowstone River, from three additional sites in MT and a proximate site in WY. Pairwise ΦST revealed high divergence among nonnative populations, suggesting at least four independent introductions into MT from diverse sources. Three cyt b haplotypes were identical to native haplotypes distributed across the Midwest and Great Lakes regions, and AMOVA confirmed the western native region as a likely source. While haplotype (Hd = 0.69) and nucleotide diversity (π = 0.005) were low in introduced bullfrogs, the levels of diversity did not differ significantly from source populations. In the Yellowstone, two identified haplotypes implied few introduction vectors and a significant relationship between genetic and river distance was found. Evidence for multiple invasions and lack of subsequent regional spread emphasizes the importance of enforcing legislation prohibiting bullfrog importation and the need for continuing public education to prevent transport of bullfrogs in MT. More broadly, this study demonstrates how genetic approaches can reveal key properties of a biological invasion to inform management strategies.Reconstructing historical colonization pathways of an invasive species is critical for uncovering factors that determine invasion success and for designing management strategies. The American bullfrog ( Retracing historical colonization pathways provides fundamental insights into the origin and genetic composition of species invasions and the factors determining invasion success (Dlugosch and Parker Dreissena spp.) has prompted mandatory boat inspection stations at many water bodies and at jurisdictional borders  have been introduced across the globe and are listed as one of the 100 worst invasive species  identifying the number of introduction events, sources, and genetic diversity of a successful invasion and (2) providing guidance about how to allocate limited management resources between prevention of new bullfrog introductions and control of secondary spread from established invasive populations in the region. In Montana, state border inspection stations and regulations that identify species as prohibited or regulated are the primary tools for preventing the initial entry of invasive species, whereas management approaches to limit the secondary spread of bullfrogs within the state are uncommon.If the genetic data indicate support for within‐state translocation, then control of established populations and outreach education should be prioritized. In contrast, if data suggest that bullfrog populations in Montana are genetically distinct from one another, then efforts to stop bullfrog importation into the state should continue to be prioritized and bolstered. In order to distinguish between the two scenarios, we specifically used mitochondrial sequence data to (1) estimate the number of introductions, (2) identify probable native population sources, (3) determine whether genetic variation is reduced relative to sources, and (4) characterize the properties of local‐ and regional‐scale secondary spread.n = 40); Flathead River (n = 5); Tongue River (n = 15); and Yellowstone River  locus to reconstruct pathways of bullfrog introductions and secondary spread within Montana. We performed polymerase chain reaction (PCR) to amplify a 1047‐base pair (bp) segment of the cyt b gene using the MVZ15‐L , 0.6 U of GoTaq DNA polymerase , 1 μmol/L of each primer, and distilled sterile H2O to volume. The following thermocycling conditions were used: initial denaturation at 94°C for 6 min; 35 cycles of 94°C for 20 s, 52°C for 30 s, and 72°C for 30 s; and final extension at 72°C for 5 min. We included a negative control in all PCR runs. Amplified PCR products were purified using Exo‐SAP‐IT  and cycle‐sequenced in both directions using forward and reverse primers. We submitted purified products to GENEWIZ (http://www.genewiz.com/) for custom sequencing.We sequenced the mitochondrial DNA (mtDNA) cytochrome F‐statistics) in Arlequin 3.5 (Excoffier and Lischer S), allelic diversity (A), haplotype diversity  as outgroups. The best‐fit model of nucleotide substitution was identified using Akaike information criteria ‐based approach. Bayesian inference involved a four‐chain Metropolis‐coupled Markov chain Monte Carlo analysis as implemented in MrBayes ver. 3.2  pairwise tests. A significant pairwise ΦST reflected two populations that were genetically distinct and, following Funk et al.  was determined by 1000 bootstrap replicates and significance assessed with 10,000 permutations. We constructed a Mantel correlogram to explicitly consider the spatial structure at different distance classes spaced at 5‐km intervals. All statistical analyses were conducted using R  from the most upstream site  as the explanatory variable. We specified a binomial error structure and weighted the data by site sample size. In addition, we performed a generalized additive model to examine the relationship between site pairwise age Wood . Variatiage Wood  that tesage Wood . Pairwisb haplotypes in introduced bullfrog populations of Montana and GTNP . All haplotypes were private within populations in the study region, each only found in a single location (Table S3). Three haplotypes were identical to those previously discovered in native range bullfrogs , confirming that the bullfrog invasion in Montana and GTNP most likely originated from this region (Table P = 0.09). In contrast, there was significant among‐group variation between the introduced and the east  or northeast  native bullfrog regions.The three native haplotypes that were identical to haplotypes found in this study  had a non Table . Low, maHd = 0.000, median π = 0.00), relative to the west  and overlap  native range regions. However, Mann–Whitney U‐tests revealed that the levels of genetic diversity were not significantly different than west native source populations . In contrast, Hd was significantly lower in introduced versus potential overlap source populations , whereas differences in π were only marginally significant .Genetic diversity of introduced populations within the introduced range of Montana and GTNP was very low (median P < 0.0001), with the proportion of haplotype MT1 decreasing and haplotype MT2 increasing with distance from the most upstream site  and geographic distance . Inspection of the Mantel correlogram revealed a roughly linear pattern, with an inverse relationship between Mantel's R and distance between sites  proportion and river distance . Interestingly, populations with the highest genetic diversity  are widespread and abundant  as a function of geographic distance.Click here for additional data file.