Carex rugulosa inhabiting brackish sandbars was also affected. We found four out of six remnant C. rugulosa populations along the Pacific Ocean had become completely extinct. Newly emergent post-tsunami populations, however, had higher allelic numbers than pre-tsunami populations, indicating higher genetic diversity after the tsunami. In addition, genetic differentiation (Fst) between post-tsunami populations was significantly lower than that of pre-tsunami populations. We therefore conclude that the tsunami enhanced gene flow. Seeds of many Carex species persist for long periods in soil, which suggests that seed banks are important genetic resources for post-disturbance recovery of genetic diversity. When its brackish sandbar habitat is no longer subject to disturbance and changes to the land, C. rugulosa is outcompeted by terrestrial plant competitors and eliminated. Disturbance is a driving force for the recovery and maintenance of populations of species such as C. rugulosa—even after near-complete eradication.The magnitude and frequency of disturbances affect species diversity and spatial distributions, but the direct effects of large-scale disturbances on genetic diversity are poorly understood. On March 11, 2011, the Great Tohoku Earthquake in Japan caused a massive tsunami that resulted in substantial alteration of community compositions. Populations of a near-threatened tidal marsh  Because a natural disturbance alters the genetic structure of species11, information of the genetic structures of pre- and post-disturbance populations is required to clarify how the genetic diversity of organisms is affected by disturbance. However, predicting when and where disturbances will occur is generally impossible; thus, samples collected before an extreme disturbance event are rarely available. To understand how disturbances affect genetic diversity, the comparison of pre- and post-disturbance samples is thus valuable and important.The magnitude and frequency of disturbances affect species diversity and spatial distributions13. There have been controversies surrounding the issue of why clonal plants have maintained both vegetative and seed reproduction, despite their fitness being increased by vegetative reproduction14. Silvertown argued that seed reproduction is more favourable than vegetative reproduction under fluctuating environments14; however, this hypothesis has been rarely tested. Seed reproduction enables plants to establish new populations in new habitats via seedling recruitment. Disturbance and seedling recruitment are therefore closely related to spatial and temporal genetic structure. If an undisturbed condition continues following a disturbance, the rate of seedling recruitment within a local population will decline and individuals will expand their habitat via vegetative propagation. Genetic diversity will then decrease with time as a result of clonal propagation or competition between clones in a local population14.Plant life history strategies are partly determined by their tolerance to disturbance and environmental stresses. Resistance properties are classified as competitive, stress-tolerant, and ruderal, based on the intensity of stress and disturbanceC. rugulosa  and ruderal (R) strategies being classified as an SR strategy following Grim13. This species maintains its populations by both sexual (seed) and asexual  reproduction (new shoots budding from rhizomes). The relationship between wave disturbance and genetic diversity is strongly linked to the temporal characteristics of C. rugulosa reproduction: specifically, time to maturity and seed bank longevity17. New shoots budding from rhizomes of C. rugulosa can produce flowering stems within a year18. Carex sp. seeds can persist in soil for long periods of time (from 15 to 130 years)20 and genets are known to be long-lived (over 100 years)21. Therefore, we predict that the above-ground genetic diversity of established populations becomes lower over time in the absence of disturbance because of vegetative propagation by themselves, competition between clones and/or genetic drift14  in Japan, the fourth largest earthquake in the world since 1900, also caused a giant tsunami23 that resulted in substantial alteration of biological community compositions26. In the northeast Honshu coastal region . The 2011 tsunami destroyed approximately 500 km of coastline, from north to south, in the northeast Honshu. In 2008 , populations of C. rugulosa inhabited only six sites along the Pacific Ocean coast of Japan (northeast Honsyu)28. We therefore investigated all these six populations in 2013 or 2014 .The Great Tohoku Earthquake  within and among populations decreased after the tsunami.In this study, we tested the hypothesised relationship between a single, extreme massive wave disturbance and genetic diversity within and among local populations Fig.  using th28, before the tsunami, and after the tsunami in 2013 and 2014  was affected by the earthquake and then extinguished by anthropogenic river improvement. Therefore, neighbouring populations were collected in 2014. A rough estimate of distances between sampling sites of pre-and post-tsunami populations within the same watershed and the tsunami run-up height at each site are given in Supplementary Table Samples were collected in 2008p < 0.01, bootstrap test; Fig. p < 0.001, bootstrap test; Fig. st) within the same watershed were calculated pre- vs. post-tsunami local populations. If genetic composition was unchanged pre- vs. post-tsunami, the Fst of 0 would be obtained. Except for surviving AMG, all populations had Fst values greater than 0.05. Analysis of molecular variance (AMOVA) revealed that the following percentages of total microsatellite variation occurred pre- vs. post-tsunami at each site; AMG 1.8%, KTK 16.3%, HRU 18.2%, USB 25.9%, MTK 7.2%, and THN 6.3%  whose genetic composition was largely similar pre- and post-tsunami samples  than pre-tsunami (Fst = 0.29) populations . As tendencies of THN showed differences from other watersheds, Fst combinations with THN were circled by dashed lines . However, after eliminating THN, post-tsunami Fst decreased significantly (p = 0.039). For HRU vs USB  and the similar effective number of alleles pre- vs. post-tsunami AMG population appears to be due to vegetative propagation over five years, as several genets were shared between 2008 and 2014 samples. Nevertheless, the higher genetic diversity of other populations following the tsunami suggests that new populations were established by floating rhizomes and/or seeds. As the tsunami struck coastlines at the end of the winter, most above-ground plants had already died and some of the rhizomes may have died. We therefore postulate that many populations after tsunami were established by seeds. We also hypothesise that the observed increase in genetic diversity within watersheds was due to soil seed banks32. The increase in the effective number of alleles per locus following the tsunami suggests the introduction of new alleles into populations. Seed banks thus likely played an important role in the restoration of genetic diversity within local populations following the massive tsunami. This suggestion is in agreement with the idea that seed reproduction rather than clonal propagation is enhanced under fluctuating conditions14. The spatial genetic composition of two local populations showed two interesting tendencies; one consisting of only one genotype (a clone), the other consisting of eight genotypes  shows high density, the creation of generational alterations via seed germination is difficult because of the limited light access. Seeds of many Carex species are not only buoyant and suitable for water dispersal, but can also persist in the soil for long periods of time20 and these specific traits of Carex seeds may thus have also promoted extensive gene flow following the tsunami. Although some tidal marsh species such as C. rugulosa are overwhelmed by terrestrial plant competitors under undisturbed conditions18, they can nonetheless maintain themselves and increase genetic diversity via a single disturbance—even if such disturbance almost completely destroys their populations. We acknowledge that different locations pre- and post-tsunami were a necessary limitation because of the destruction of land by the tsunami and that our field surveys were limited to only one pre- and post-tsunami sampling. However, the results of the data showed consistencies in the same general direction; increasing genetic diversity and decreasing fixation indexes. Therefore our findings strongly suggest that the tsunami would have had an important role in the maintenance of the near-threatened salt marsh populations.Even if above-ground populations of C. rugulosa is distributed throughout East Asia33. To clarify this species’ conservation management unit in Japan, we studied 22 populations in 2008 and analysed the genetic structure within and among populations using microsatellite markers34. Of the 22 populations, six were along the Pacific Ocean coast; therefore, the pre- and post-tsunami genetic compositions of those six populations were compared in this study. We visited the Tohoku region in 2013 and 2014. When we found a C. rugulosa population, we roughly estimated its area using a measuring tape to determine its dimensions. For DNA collection, 5-cm-long leaves were collected at about 0.5 m to 1 m intervals along with the longest axis of each population. The number of ramets and flowering stems (May 2013) was counted within 10 plastic quadrats (20 cm × 20 cm). The quadrats were randomly positioned within each population. The total number of ramets per population was roughly estimated using the mean ramet density. Samples were kept at −20 °C until DNA extraction.Plant DNA was extracted from homogenised frozen leaves using a DNeasy Plant Mini kit  following the manufacturer’s instructions.34 with annealing temperatures (Tm) of 57, 57, 62, 57, 62, 57, and 62 °C, respectively. We amplified DNA samples in 10 µL reaction mixtures containing 1 µL template DNA, 1 × PCR buffer for KOD FX NEO, 0.4 mM of each dNTP, 1 U of KOD FX NEO polymerase , 0.5 µM fluorescently tagged forward primer , and 0.5 µM reverse primer. PCR parameters were as follows: a 2-min hot start at 94 °C, followed by 35 cycles of 10 s at 98 °C, 30 s at the appropriate Tm, and 1 min at 68 °C. The amplified DNA was analysed on an ABI 3130xl or ABI 3730xl DNA analyser (Applied Biosystems). Allele sizes were assessed by fragment analysis. To correct for the influence of DNA analyser differences between samples from 2008 and samples from this study, samples from the Ohashi River in Shimane Prefecture previously analysed by Ohbayashi et al.34 were reanalysed and corrected for allele size to enable direct comparison of allele sizes between the previous (pre-tsunami) samples and those in this study .Seven microsatellite loci were assessed, namely CR5, 7, 10, 21, 27, 28, and 39, under PCR conditions slightly modified from previous reports28 and post-tsunami populations along the Pacific Ocean in northeast Honshu were used. Pre-tsunami data from INS, THNa, and THNb populations, which were distributed within the Tone River watershed, were pooled and collectively designated as the pre-tsunami THN08 population for comparison with post-tsunami THN14 populations in all analyses. Approximate distances among these three pre-tsunami population were as follows: INS (furthest upstream) to THNa, 30 km; THNa to THNb (furthest downstream), 3 km; INS to THNb, 33 km. We were unable to access the INS population following the tsunami because it is located in a restricted area established by the Ministry of Environment for plant conservation. Because THNa and THNb population size had reduced due to anthropogenic works, we collected two additional neighbouring populations within 5 km distance in the Tone River watershed. All analyses were performed using only a distinct multilocus genotype (a genet) to eliminate the effects of clonal individuals on allele frequency at each locus (a ramet)35. GENECLONE software36 was used to estimate clonal genotypic diversity parameters, namely, number of distinct and identical multilocus genotypes , as well as genotypic diversity  calculated as R = (G − 1)/(N − 1), where G is the number of distinct genotypes and N is the number of genotyped samples. We also calculated observed (Ho) and expected (He) heterozygosities using CERVUS software37 (version 3.0.6). The effective number of alleles per locus (ne) was also calculated. Heterozygote excess or deficiency within a population was tested using a Markov-chain algorithm developed by Guo and Thompson38 and implemented in the software program GENEPOP39 version 4.2. The inbreeding coefficient (Fis) and pairwise population differentiation among populations (Fst) were also calculated and an unbiased estimate of the P-value of a log likelihood ratio (G)-based exact test was performed on Fst using GENEPOP. Although the pre-tsunami USB population consisted of only four genets which were an insufficient sample size for Fst calculation, we included pre-tsunami USB in our Fst calculations to know rough tendencies in this study. Bonferroni corrections were applied to all significant pairwise-test results to adjust for multiple comparisons. The probability of identity (PI)—which is the probability that two individuals obtained at random from a population would have the same multilocus genotype40 —was also calculated.Data from six pre-tsunami41 were used to estimate pre- vs. post-tsunami population genetic structures . We selected the admixture model and the F model  without a priori information. STRUCTURE uses a Bayesian clustering method to assign each multilocus genotype to one of K probable cluster populations. For each K value from 1 to 10, we performed 20 independent runs of 1 × 105 iterations after a burn-in period of 1 × 105 iterations. To detect the true number of clusters, we then calculated ΔK, which is based on the rate of change in posterior probability values for K  between successive K values42. The 20 runs performed with the selected K-value were retained. Replications of STRUCTURE runs were combined using the software programs STRUCTURE HARVESTER43 and CLUMPP44. For K = 2, the FullSearch algorithm in CLUMPP was used to align runs. For K = 3 and 4, we used the Large K-Greedy algorithm, with 1 × 106 random input orders. Results were visualised using the software program DISTRUCT45.Individual-based Bayesian clustering methods implemented in STRUCTUREst differences among populations pre- vs. post-tsunami samples were analysed using the two-tailed paired-sample t-test46 and the binomial test46 in R version 3.3.1. For other genetic parameters , the number of effective alleles (ne)), bootstrap analyses47 were performed to test the differences  for each parameter pre- vs. post-tsunami samples. For this calculation, the AMG population was excluded since the population survived after the tsunami and shared genets pre- and post-tsunami. Mean pairwise differences between the same population in T pre- and post-tsunami sites (where T is the number of observed sampling sites) formed by random sampling from pooled observed data were used as the test statistic. This generated a probability distribution of pre- and post-tsunami population differences under the null hypothesis that there was no mean difference pre- vs. post-tsunami values. The bootstrap test was performed on each parameter until T random combinations of 9999 sets were achieved, following which the observed mean pairwise difference was placed against the probability distribution. If the observed value fell within the upper or lower 250 ranks of the 10000 sets in the parameter (0.025%), we then confirmed that there was a significant pre- vs. post-tsunami difference using a two-tailed test. To estimate genetic variation and genetic differentiation (fixation index) pre- vs. post-tsunami populations, AMOVA was performed using Arlequin48 version 3.5. The null hypothesis was no genetic differentiation among populations exists (Fst = 0), while the working hypothesis was that Fst values are significantly larger than 0 (Fst ≠ 0).The FSupplementary figures and tables


Diatraea Guilding is polyphagous insect pest of many important crops such as corn, sorghum and sugarcane. Losses arising from the attack of Diatraea species have been a serious problem, which may cause loss in sugarcane production around 0.25% in sugar, 0.20% in alcohol and 0.77% of body weight for every 1% infestation and up to 21% in corn production fields. In Brazil, the most commonly reported species are Diatraea saccharalis  and Diatraea impersonatella  (= D. flavipennella). However, multiple other species of Diatraea have been identified in Brazil according to the literature. Currently, little information exists on the presence of the other species causing injury to sugarcane and corn. The objectives of this study were to improve the accuracy of species assignment, evaluate the population genetic structure, and address many of the outstanding questions of systematics and evolution of Brazilian populations of D. saccharalis. To address these main questions, classical taxonomic methods were used, focused on morphological characterization of the reproductive organs, especially the male genitalia. In addition, genetic studies were performed using simple sequence repeats (SSR) and a fragment of cytochrome C oxidase subunit I (COI) gene. The data and findings from this research will contribute to the understanding of evolutionary aspects of insect pests in order to develop more effective and sustainable population management practices.The sugarcane borer or corn stalk borer,  Diatraea Guilding is a genus that is composed of a significant number of species, which contains some of the most important lepidopteran pests of crops, including corn, sorghum, and sugarcane Diatraea n Brazil  reportedn Brazil  narrowedla. Cruz  publisheDiatraea based on the morphology of the male and female genitalia. Expanding this study, Solis and Metz [Diatraea species. Based on the variability of South American species, they synonymized the name Diatraea flavipennella  with D. impersonatella, and we follow the change herein.Box  and Blesand Metz , provideThe collection and observation of adult Lepidoptera is often preferred given the relative ease of identification. The internal morphological characterization of the anatomy of the reproductive organs, especially the male genitalia, has provided excellent results because they have important and conserved characteristics within species , 26, 27.In addition to morphological characterization, molecular analyses may be used to increase the knowledge of a particular group of organisms . SeveralDiatraea species have been published in recent years. Some examples showed that Brazilian populations of D. saccharalis have a high level of polymorphism and genetic structure within the crop production regions [D. saccharalis [D. saccharalis collected in the southern United States and identified two genetically distinct clusters using amplified fragment length polymorphism (AFLP) and COI sequencing. These authors suggested that Florida’s D. saccharalis population could represent a distinct species. However, other studies suggest samples in South America form a separate cluster from those in Central America and the Southern United Stated of America [Very little research on the genetic diversity and population structure of  regions , 45. Stucharalis , 46. Pascharalis  compared America , 47.Diatraea specimens collected in sugarcane and corn plants in the same production regions of Brazil. Our objectives were (a) to improve the accuracy of species identification through morphological characterization and with the polymorphism of sequence of cytochrome C oxidase subunit I (COI) gene (b) to establish a systematics and evolutionary comparison with Diatraea species and (c) to evaluate many of the outstanding questions about population structure with microsatellite (SSRs) fine-scale gene characterizations. In this study, we used these methods to assess the genetic variability of populations collected in the main crop production regions in Brazil. Furthermore, we used DNA barcodes of several species of Diatraea to evaluate the evolutionary relationships within this genus. The findings of this study may help to refine the understanding of evolutionary aspects of insect pests in order to develop more effective and sustainable population management practices. In addition, we adopt the use of D. impersonatella [D. flavipennella [Discrepancies among studies may be associated with collection sites, individuals that were collected in light trap or in a host, development stage (adult or larvae) used for DNA analyses, and the lack of precision with species level identification. We addressed these disagreements with a sequence of methods that valid each other. This study aimed to fill a gap, building a comprehensive study of onatella  instead pennella  as propopennella .Diatraea were collected in the main corn and sugarcane production regions of Brazil. The larvae were collected in equidistant points inside the corn or sugarcane fields. In this study, we defined the term "population” as the city where collection was performed, and the host from where the insect was isolated. Thus, each population was defined as Piracicaba_Sugarcane, Piracicaba_Corn, Jaboticabal_Sugarcane, Morrinhos_Sugarcane, Morrinhos_Corn and Maceio_Sugarcane , and no specific permits were required for these locations/activities because it did not involve endangered or protected species.Diatraea morphology provided by Bleszynski [All moths that emerged were dissected and identified based on the morphological characteristics of the genitalia according to Robinson . The teceszynski  and Solieszynski .The same specimens that were dissected were also used for DNA extractions. The male and female DNA was extracted from the thoracic tissues following the CTAB protocol described by Doyle and Doyle  with sli5’—GGT CAA CAA ATC ATA AAG ATA TTG G– 3’) and HCO 2198 (R) (5’—TAA ACT TCA GGG TGA CCA AAA AAT CA– 3’) [A fragment of the COI mitochondrial gene was amplified by polymerase chain reaction (PCR) with the primers LCO 1490 (F) ( CA– 3’) . The seqhttp://www.technelysium.com.au/chromas.html).Multiple alignment of the sequences .The COI haplotypes found for The microsatellite loci used in the study were provided by the Laboratory of Conservation Genetics and Genomics, Agribusiness Technological Development of São Paulo, Brazil and were developed by Pavinato . DetailsDiatraea populations were genotyped with at least 10 highly polymorphic loci , assuming random model, were estimated using the hierfstat package [poppr package [poppr package [All individuals from  package . Cluster package  and the  package . The sta package . We appl package .D. saccharalis  showed preponderance over D. saccharalis (10.20%) in some areas of the state of Alagoas.The results clearly demonstrate that all specimens of  Freitas  also notD. saccharalis according to keys in Bleszynki [vinculum was smooth, U-shaped, and broadly rounded anteriorly. The uncus narrowed into a beak-like apex, and the gnathos were smooth except for approximately half of the dorsal surface that was densely covered with short teeth. The lateral lobe of the tegumen was rounded, as long as wide, and square in appearance. The basal costal lobe was present with the vertex slightly flattened. The apex of juxta arms were with a single point or rounded with a small, subapical tooth, but never bidentate  gene. The COI sequence polymorphisms allowed the estimation of the genetic relationships among individuals. Aligned sequences of DNA consisted of 666 base pairs for the COI mitochondrial gene for both species. The average frequency of A, C, G and T was 37.8%, 15%, 16.1% and 31.1%, respectively. The strong AT bias (68.9%) is typical of insect mitochondrial genomes , 63. Neisistency . This re species , 65.D. impersonatella specimens and four were present in D. saccharalis specimens  and low nucleotide diversity (0.00316)  and (3/13), respectively. Four haplotypes  occurred in only one individual. In addition, 0.00316)  indicati species . Current species  observedD. saccharalis haplotype diversity and nucleotide diversity were 0.14 and 0.00032, respectively  that was represented in all collection sites. The second most common haplotype was H8 (4/82), being that one individual was collected in Jaboticabal in the sugarcane crop and three in Piracicaba, one was from corn and two from sugarcane. The H9 and H10 haplotypes were represented by unique specimens, and both were collected in corn fields from Morrinhos and Piracicaba. Low genetic variation in the COI sequences was detected in D. saccharalis. The main production regions where the insects were collected and where they shared the same haplotypes, were located within the approximately 200 km between Jaboticabal and Piracicaba, the 500 km between Jaboticabal to Morrinhos, and the 650 km between Piracicaba to Morrinhos. Collection sites located in the Piracicaba region had a distance of approximately 30 km between each location, and shared the same haplotypes in different hosts. In Morrinhos city the specimens were collected on the same farm, but from different hosts. Thus, the spatial distribution of haplotypes revealed no major groupings of D. saccharalis haplotypes according to either host plant or geographical location. Each haplotype, when present in two or more individuals, had a wide geographic distribution.ectively , and theD. saccharalis, and -0.25752  for D. impersonatella. These results show that the mutations found in the D. saccharalis sequences probably occurred due a genetic drift, not selective pressure. While for D. impersonatella, the negative Tajima’s D was not statistically significant, and therefore the hypothesis of neutral evolution was rejected for this species. The results of Fu's FS test, which is based on the distribution of haplotypes, also had negative values for all D. saccharalis populations, confirming an excess of rare haplotypes over what would be expected under neutrality. Just as for Tajima’s D, Fu’s FS was not significant for D. impersonatella.When genetic structure has been influenced by rapid range expansion, the Tajima’s D value is expected to be negative, indicating an excess of rare nucleotide variants compared to the expected under a neutral model of evolution . In thisDiatraea. The AMOVA results showed a high percentage of variation between species 99.21% (FST = 0.99). Moreover, low genetic variations were observed among populations within species and within species, -0.02% and 0.81%, respectively  was performed to verify how the genetic variability was distributed among and within the two species collected. Consistent with the other analyses presented here, the AMOVA also shows the separation of the samples into two species of ectively . An AMOVen sites .D. saccharalis and D. impersonatella is also evident by the FST estimates among their populations. The pairwise FST estimates between D. saccharalis populations ranged from -0.33172 to 0.05546. The highest pairwise FST estimate was observed between populations from different hosts, Jaboticabal_Sugarcane and Morrinhos_Corn (FST = 0.05546), however this genetic divergence was not significant , and was often found in association with insect damage in that region. The expansion of Diatraea species to other parts of Brazil is unknown. We can speculate that these pests were introduced into Brazil in the 16th century or later as stalks of sugarcane varieties or native plants from the Amazon basin, were transported throughout the region by colonists.The relationship between these two species of a. Myers , 69 condonatella , 6, 24 tDiatraea species (obtained from GenBank and BOLD) showed that these species cluster together. Other clusters contained species originating from other localities and hosts , possibly the consequence of using two molecular makers instead of one. The length of COI and nad6 sequences used in that study were long, at 1,429 and 497 bases, respectively. The majority of studies involving COI sequences have used a short barcode section of mitochondrial DNA, the first approximate 650 bases of the 5’-end of this gene [D. saccharalis, and the haplotype diversity was lower than expected, in contrast to published records for this species [In contrast to this observations, Silva-Brandão et al. , in a sthis gene , 73, 74.his gene  was iden species , 46.Contrasting results may have emerged due to different factors, such as sampling and the methods employed. For example, light traps may attract insects with different hosts, which may increase the diversity sampled. Other important point is the definition of development stage (moth or larvae) used for DNA extraction. Even though the use of larvae is allowed by International Barcode of Life project (iBOL), bacteria present in the larvae body may be a source of contaminant DNA. Thus, sampling of moths is an advantage with respect to the quantity and quality of DNA isolated , 76. MorD. saccharalis can be explained as a consequence of low mutation rates and/or stochastic processes that resulted in severe bottleneck of the population sizes, as described by negative values of Tajima’s D. According to Myers [D. saccharalis are aquatic and semi-aquatic grasses in the Orinoco Delta River. Therefore, we may speculate that when sugarcane was introduced in the New World by the sixteenth century, D. saccharalis migrated, colonized, and adapted to the new hosts and environments. The bottleneck signal observed suggest that during the colonization a relatively small number of individuals founded the new populations [D. saccharalis [Based on these premises and on examination of almost 100 individuals we can show that low values of haplotype and nucleotide diversity and the higher frequency of the same haplotype is a realistic scenario. The homogeneity observed in COI among widely dispersed and geographically isolated Brazilian populations of to Myers , 69 the ulations , 78. Thicharalis , 81, 82.charalis , 87, 88.charalis . Accordicharalis , the meacharalis .D. saccharalis and D. impersonatella. In relation to intraspecific studies within D. saccharalis, COI analysis did not show genetic structure for populations sampled in this study. The use of combined morphological and molecular approaches, including both mtDNA and a nuclear DNA, is proposed to evaluate relationships that persist uncertain.In this study, we showed that the analysis of polymorphism of oxidase cytochrome C oxidase subunit I (COI) mitochondrial gene is a powerful and accurate species discriminator for D. saccharalis, we performed microsatellite loci characterization on 80 of the 82 individuals collected , Morrinhos_Corn (0.1429), and Piracicaba_Sugarcane (0.1323), reflecting an excess of homozygotes in these populations, while Piracicaba_Corn was the only population with negative value of FIS. These differences among the observed and expected heterozygosities can be attributed to non-random mating among the individuals and inbreeding within populations with positive values of FIS. While the negative inbreeding coefficient observed in Piracicaba_Corn suggests that these processes are not occurring in this populations  per population was 29.83 and ranged from 32.4 to 25.19. The population Piracicaba_Sugarcane showed the highest value of allelic richness. The average values of observed and expected heterozygosity per population were 0.42 and 0.46, respectively. The highest heterozygosity was observed in Morrinhos_Corn (0.44), and the lowest in Jaboticabal_Sugarcane (0.4). For the expected heterozygosity, the highest value was 0.51 for Morrinhos_Corn, and the lowest was 0.39 for Piracicaba_Corn. The expected heterozygosity values were higher than observed heterozygosities for all populations, except for Piracicaba_Corn. The average coefficient of inbreeding was 0.10, ranging from -0.074 to 0.179. The estimate values of Fulations .We identified 12 private alleles, 50% were found in Morrinhos_Corn, and the Piracicaba_Corn population had no private alleles. The molecular marker, which identified the largest number of private alleles, was Dsc11, with 5 alleles distributed in Morrinhos_Corn, Jaboticabal_Sugarcane and Piracicaba_Sugarcane populations .ST indicated genetic differentiation among populations, and ranged from 0.0835 to 0.1812  estimates among sampling sites.In the DAPC, 72% of the total genetic variation was captured by components of PCA and these were used as input to capture two DA functions. This analysis separated the samples in four major clusters that correspond to both geographic locations and hosts . EspeciaIn Brazil, sugarcane and corn are cultivated in a large diversity of environments, which differ in soil conditions, climate, availability and susceptibility of the variety and management control system. It is characteristic of inconstant environments to exhibit a significant loss and fragmentation of natural ecosystems .D. saccharalis populations. The development of new varieties of sugarcane allowed the cultivation in a system ranging from 12 to 18 months. This sequential production system permits D. saccharalis to have host plants yearlong and limits the necessity of migration. In corn production systems, the introduction of Bt technology in the mid-1990s created an agricultural system less dependent on insecticides, but selectively eliminates certain species from the insect populations. Additionally, the Bt technology might provide the primary ecological opportunities needed for the first host shift and encourage adaptation to digest novel plant defensive proteins [D. saccharalis are low, apparently as result of home-range behavior. Over 45% of the adults were recaptured around 50 meters of the release site. Extended dispersal was observed when the moths followed the wind, which increased their dispersal in around 800 meters [Furthermore, these diverse cultivation ecosystems provide habitats for a wide range of pests. Insects demonstrate a high ability of local adaptation and acceleration of the evolutionary process , 94. Theproteins , 99, 100proteins . Results0 meters , 103.D. saccharalis with the major hosts and suggest that the current genetic divergence and inbreeding result from limited gene flow and natural barriers. Evidence of fragmentation influence on genetic diversity was found in other studies with D. saccharalis populations [D. saccharalis collected in corn and sugarcane, just as the results found in our study. However, Nascimento [D. saccharalis in sugarcane. This lack of divergence observed between populations collected in sugarcane may be related with the proximity of samples sites, since all populations belonged to the same state in Brazil.Limited flight ability, genotype–environment interactions, and host year-availability affect the relationship of ulations , 105. Paulations  observedD. saccharalis, such as the DAPC and pairwise FST results. Pavinato [D. sacharalis populations from corn and sugarcane. Subpopulations of the same host (sugarcane or corn) tend to be more similar to each other [Another important factor for the population divergence observed in our study may involve different selective pressures across environments. In addition, low rates of gene flow between populations increase the likelihood of host-associated genomic differentiation. We found strong evidence of host-associated genetic divergence across the range of Pavinato  support ch other .D. saccharalis collected in Brazil, which suggested the presence of genetic groups correspondent with their geographic sampling locations. It is possible that these populations have evolved some degree of adaptation to local environmental conditions.In conclusion, microsatellite loci were polymorphic and highly informative, allowing the study of genetic variability of D. saccharalis and D. impersonatella that included characteristics from genitalia anatomy, mtDNA, and nuclear molecular markers. Our results clarified some outstanding questions about Diatraea populations in the Brazilian territory. In this study, we observed, through taxonomic methods and COI sequencing, that D. saccharalis and D. impersonatella are the two species responsible for attacking sugarcane and corn in Brazilian crop fields. Sequencing of COI revealed to be an accurate species discriminator for this genus. Microsatellite analyses revealed host-plant preference in populations of D. saccharalis. Moreover, genetic structure showed little connection among populations. There are preliminary indications that low interactions can relate to the fragmentation process between crop productions regions and may affect gene flow. In summary, we have demonstrated that microsatellite polymorphisms provide a valuable tool for population genetic analysis of D. saccharalis. Additionally, we strongly recommend the adoption of the name D. impersonatella as a nomenclatural change from D. flavipennella as proposed in Solis and Metz [Diatraea populations.We conducted a systematic study of and Metz . Our finS1 TableF = forward primer sequences; R = reverse primer sequences; Ta = annealing temperature.(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S6 Table(PDF)Click here for additional data file.S1 Dataset(TXT)Click here for additional data file.S2 Dataset(XLSX)Click here for additional data file.


Tursiops truncatus) is one such species, exhibiting complex patterns of genetic structure associated with local habitat dependence in various geographic regions. Here we studied bottlenose dolphin populations in the Gulf of California and Pacific Ocean off Baja California where habitat is highly structured to test associations between ecology, habitat dependence and genetic differentiation. We investigated population structure at a fine geographic scale using both stable isotope analysis (to assess feeding ecology) and molecular genetic markers (to assess population structure). Our results show that there are at least two factors affecting population structure for both genetics and feeding ecology (as indicated by stable isotope profiles). On the one hand there is a signal for the differentiation of individuals by ecotype, one foraging more offshore than the other. At the same time, there is differentiation between the Gulf of California and the west coast of Baja California, meaning that for example, nearshore ecotypes were both genetically and isotopically differentiated either side of the peninsula. We discuss these data in the context of similar studies showing fine-scale population structure for delphinid species in coastal waters, and consider possible evolutionary mechanisms.For highly mobile species that nevertheless show fine-scale patterns of population genetic structure, the relevant evolutionary mechanisms determining structure remain poorly understood. The bottlenose dolphin (The online version of this article (10.1007/s11692-018-9445-z) contains supplementary material, which is available to authorized users. Despite high dispersal potential, many highly mobile marine species show significant population structuring  has been recently proposed  and Southern California bottlenose dolphins have also shown evidence of phenotypic, ecological and genetic differentiation, which supports the recognition of “coastal” and “offshore” ecotypes  for a highly mobile species in a heterogeneous environment. Specifically, we test the hypothesis that local nearshore and offshore populations either side of the Baja California peninsula will show differentiation for both genetic markers and stable isotopes, indicative of an association between prey choice and population genetic structure. We further test for evidence of structure between ecologically distinct northern and southern portions of the GC and outer Baja California coastal range see Fig. , and theA total of N = 281 tissue samples were analysed in this study, comprised of biopsy samples, material from stranded and captive animals and DNA from samples used in an earlier study. Skin biopsy samples (N = 175) were collected from regions within the Gulf of California and along the western coast of Baja California and South California Bight Fig. . SamplesWhen possible, source individuals were categorised as ‘offshore’ or ‘nearshore’ ecotypes using physical characteristics : 5′ CCT CCC TAA GAC TCA AGG AAG 3′ and H16343 (D): 5′ CCT GAA GTA AGA ACC AGA TG 3′ , nucleotide diversity (π), fixation indices (Fst and ϕst), tests for neutrality (Tajima’s D and Fu’s Fs) and mismatch distributions were estimated using ARLEQUIN 3.5 (Excoffier and Lischer −7 substitutions/year) for the mtDNA control region HVR1, after Ho et al.  to verify base calling and aligned with CLUSTAL X  and expected heterozygosity (He), regional differences in frequencies, deviation from Hardy–Weinberg equilibrium, and Fst were all computed in ARLEQUIN 3.5 , in units per mil  space and significant differences tested using the MANOVA statistic.A subsample of 60 skin biopsies could be analysed for A total of 34 new mtDNA control region haplotypes were identified among new samples (accession numbers HE617258–HE617297) and pooled with 32 haplotypes from a previous study by Segura et al.  . There was no consistent pattern, but one locus (TexVet7) was out of HWE in three of seven putative populations. However, analyses run with and without TexVet7 showed no difference in pattern or significance (data not shown), and so all eight loci were retained.The clustering analysis performed in STRUCTURE showed a plateau of similar likelihood values between K = 2 and 5, while ∆K was 2 (see figure S1). Assignment histograms for K = 2, 3 and 5 are shown in Fig. ST = 0.098), followed by differentiation among the two ecotypes (FCT = 0.068) and then differentiation among populations within ecotypes (FSC = 0.032), and all were highly significant  have been previously distinguished within the GC and PO by means of morphological, ecological and mtDNA molecular data  affinity within bottlenose dolphin populations. Sampling is unfortunately incomplete, however it is sufficient to show a distinction between profiles for nearshore populations either side of the Baja California peninsula, together with a clear distinction between nearshore and offshore forms within the gulf. There was greater similarity among offshore populations within GC, while the single western offshore sample fell out of that cluster . Oceanographic conditions such as dynamic eddies around Punta Eugenia  was small and not clearly differentiated from any of the other gulf populations in our sample set, given the level of resolution obtained. In fact, genetic assignment analyses suggest they may represent a mixture of GN and GCo individuals see Fig. . PossiblT. truncatus preferably inhabits coastal shallow waters of 15–21 °C SST, with high turbidity. T. truncatus is also the only cetacean known to venture into the Colorado River; while common dolphins remain in deeper, less turbid waters  and all other putative populations, including the nearshore population within the GC. The WN population is also the strongest candidate for having a founder expansion origin  population in the Gulf of California  the confidence limits on tau were too broad for useful expansion time estimates Table . Howeverons Fig. .Tursiops truncatus in the GC and the Pacific Ocean off Baja California, and suggests possible mechanisms for the evolution of this structure. The GC has been recognized as a priority for conservation and management actions, given the outstanding levels of biodiversity present in this marginal sea. It is also a region that has seen recent differentiation and incipient speciation for a diversity of taxa  and totoaba  are endangered endemic species currently under management actions for population recovery


Astyanax mexicanus are outstanding models to study the tempo and mode of adaptation to a radical environmental change. They are currently assigned to two main groups, the so-called “old” and “new” lineages, which would have populated several caves independently and at different times. However, we do not have yet accurate estimations of the time frames of evolution of these populations.Cavefish populations belonging to the Mexican tetra species We reanalyzed the geographic distribution of mitochondrial and nuclear DNA polymorphisms and we found that these data do not support the existence of two cavefish lineages. Using IMa2, a program that allows dating population divergence in addition to demographic parameters, we found that microsatellite polymorphism strongly supports a very recent origin of cave populations . We identified a large number of single-nucleotide polymorphisms (SNPs) in transcript sequences of pools of embryos (Pool-seq) belonging to Pachón cave population and a surface population from Texas. Based on summary statistics that can be computed with this SNP data set together with simulations of evolution of SNP polymorphisms in two recently isolated populations, we looked for sets of demographic parameters that allow the computation of summary statistics with simulated populations that are similar to the ones with the sampled populations. In most simulations for which we could find a good fit between the summary statistics of observed and simulated data, the best fit occurred when the divergence between simulated populations was less than 30,000 years.Although it is often assumed that some cave populations have a very ancient origin, a recent origin of these populations is strongly supported by our analyses of independent sets of nuclear DNA polymorphism. Moreover, the observation of two divergent haplogroups of mitochondrial and nuclear genes with different geographic distributions support a recent admixture of two divergent surface populations, before the isolation of cave populations. If cave populations are indeed only several thousand years old, many phenotypic changes observed in cavefish would thus have mainly involved the fixation of genetic variants present in surface fish populations and within a very short period of time.The online version of this article (10.1186/s12862-018-1156-7) contains supplementary material, which is available to authorized users. Astyanax mexicanus. Twenty-nine cavefish populations have been discovered so far in limestone caves in the Sierra de El Abra region of northeastern Mexico  and 10,750  for Pachón cave population and surface fish population, respectively. These estimations make sense as it is obvious after several trips in Sierra de El Abra that the census population size (Nc) of surface fish is much higher than the census population size of the cavefish. Nc has been estimated for Pachón cavefish   suggests that the Pachón population is much younger than usually thought. Indeed, and without any complex computations, a simple glance at the microsatellite data set  showed unambiguously that these surface fish are closely related to the surface fish sampled in the Sierra de El Abra region. Despite this evidence and the absence of a high structuration of the genetic diversity of the surface fish in the Sierra de El Abra region, if there is a genetically closer surface population living near the Pachón cave, the straightforward conclusion would be that the result we obtained is an overestimate of the age of the Pachón cave population.Even though the recent evolution of Of note, the Texas surface fish shared about 7% of their SNPs with Pachón cavefish. As shared polymorphism is expected to decrease quickly if at least one population has a low effective population size and the two populations are completely isolated, it suggests that the divergence is recent or the migration rate is high.In order to make estimations of the model parameters  that could explain the distribution of SNPs within and between populations, we ran simulations of the evolution of the SNPs forward in time in two populations, allowing migration from the surface to the cave. Running these simulations with different sets of parameters allowed finding simulations for which, after a given number of years of divergence, the distribution of the SNPs within and among simulated populations was very similar to the distribution observed within and among the real populations. The time of divergence was taken as an estimation of the age of the cave population. The use of simulations in order to estimate unknown values of model parameters is common in population genetics, for example in Approximate Bayesian Computation methods .The rationale of this analysis is that when a population is divided into two populations, ancestral polymorphism is shared by the daughter populations for a period of time, even if the populations are totally isolated. As divergence proceeds, each descendant population experience fixation and loss of alleles at loci that were polymorphic, and this random sorting of alleles is part of the way populations diverge. The divergence of the populations also increases through time because new alleles appear at new and different polymorphic sites in both populations. However, migration allows sharing of the ancestral and new polymorphism and can restrict the divergence of the populations. As already discussed it is thus challenging to estimate if shared polymorphism is due to a recent split, gene flow or both . HoweverN is the population size, p is the allele frequency) [We found 2.34 times more derived alleles that reached fixation (substitutions) at synonymous polymorphic sites in the cavefish population than in the surface fish population. An excess of non-synonymous and non-coding substitutions were also observed. We searched for an explanation for these observations that could appear at first glance unexpected, in particular for synonymous substitutions which are for most of them probably neutral or nearly neutral. It is well known that if two populations have diverged for a long time and if the mutation rate is the same in both populations, the neutral substitution rates should be equal and independent of the population sizes . Neverthequency) . The strequency) . In otheWe defined summary statistics describing the polymorphism and the divergence of two populations that could be accurately estimated using pooled RNA-seq . The evoAstyanax species [In our simulations, we set the generation time to two and five years for the surface and cave populations, respectively. This surface fish generation time is twice the estimations obtained for other  species  and the  species . This es species . As the We also took into account that genetic drift occurred during several generations in the lab. In order to estimate the effect of genetic drift in the lab and the accuracy of alleles frequency estimations using pooled RNA-seq, we compared the frequencies of two alleles (Pro106Leu) of MAO gene estimated using a sample of wild caught Pachón fish, a sample of adult fish maintained in the lab with frequencies estimated using pooled RNA-seq of lab embryos. Although it has been published that Pachón population is not polymorphic at this locus , we founOf note whereas the estimation of the divergence time depends on the generation time and the mutation rate with IMa2, our estimation depends only on the generation time. This is due to the fact that the summary statistics we used, i.e. SNPs class frequencies, to compare simulated and real populations, do not depend on the mutation rate. We set a mutation rate allowing a number of SNPs within and between simulated populations large enough to have accurate estimations of the summary statistics.Without migration, shared polymorphisms were quickly lost and the best fit of the model to the data was obtained when the cavefish population size (1250) was smaller than the surface fish population  and the age of the cavefish population was 20,600 years. When migration was included, good fit with the data also implied large differences in population sizes, a low migration rate and low numbers of migrants. The very best fit was observed for a surface fish population size of 20,000, a cave population size of 1250, and a cave population age of 54,200 years. Nevertheless, most very good fits were obtained with a divergence time in the range of 20,000 to 30,000 years.As expected no fit was found when the surface and cave populations had similar sizes. The best fit was observed when the ancestral population size was similar to the surface population size. It is at odds with the estimation of a much larger ancestral population size estimated with IMa2. We do not have a clear explanation for this discrepancy, but it could be the consequence of the admixture of two divergent populations at the origin of the ancestral population. Such admixture could have increased the number of alleles at each microsatellite loci, and IMa2 thus inferred a larger ancestral population size. For SNPs, admixture of divergent populations results in more polymorphic sites, but the number of alleles at a given locus is not increased as the probability that two parallel mutations occurred independently at the same locus in two populations is extremely low.In a recent analysis of the expression of 14 crystallin genes in the Pachón cavefish, 4 genes are not expressed or expressed at a very low level, but no stop codon or frameshift could be identified . This reSecond, a recent study has shown that the heat shock protein 90 (HSP90) could restrict the expression of eye-size polymorphism in surface populations . Indeed,A recent origin of the so-called “old” Pachón population can solve a conundrum put forward by previous and the present analyses. First, at the SNP and microsatellite level, the diversity is not that low in Pachón cave when compared with surface populations, i.e. about one third. If the populations are at migration/drift equilibrium, it means that the effective population size of Pachón cavefish is about one third of the surface populations, and this is at odds with the likely large difference in census population sizes , 33. Of The new time frame we propose for the evolution of the cavefish populations would not allow enough time for the fixation of many de novo mutations and most derived alleles that reached fixation in caves were probably already present in the ancestral population. This was also suggested by a recent population genomic study . This maAlthough we estimated that all cavefish populations are probably recent, less than 20,000 years old, the number of independent and approximatively simultaneous adaptations to cave and evolution of cave phenotype is still an open question. The evolution in a short period of time of the phenotype of individuals belonging to a population adapting to a new environment, is actually not that unexpected and has already been observed in other fish species such as the stickleback , dwarf wMexican cavefish could thus be another and striking illustration that many phenotypic changes can accumulate in parallel and in a short period of time thanks to standing genetic variation . The relWith a multilocus microsatellite polymorphism dataset  and the Astyanax mexicanus cavefish and surface fish, founded with fish collected respectively in the Pachón cave  and at the San Solomon Spring   Fig. , and obtSA) Fig. . Both obHyphessobrycon anisitsi fish were purchased from Chalet-Hérault Aquariophilie .In 2012, thirty In order to identify polymorphisms at the population level based on a Pool-seq approach , for eacAstyanax mexicanus transcriptome was assembled with Newbler ver. 2.8 (Roche 454) sequence analysis software using 454 sequences (2.106 reads) of both the Pachón cave and surface fish pooled embryos. We obtained 33,400 contigs (mean contig length = 824 bp). We also tried to generate a transcriptome assembly using the Illumina sequences, but whereas this resulted in more contigs  than the 454 sequences, many of them were concatenations of different transcripts and in some cases the same transcript was found in more than one contig. We therefore mapped the Illumina sequences onto the 454 contigs to identify and annotate SNPs. Putative coding sequences in each contig were identified using the zebrafish (Zv9) proteome available at EnsEMBL 73 as a reference [− 5. We found 13,240 protein coding contigs (contig mean length = 530 bp). We identified contigs containing domains that matched different zebrafish proteins and which were most likely chimeric contigs. These contigs were removed . In total, we analyzed 12,871 putative protein coding contigs.The eference . A contiHyphessobrycon anisitsi sequences were aligned to Astyanax contigs using a lower maximum edit distance (n = 0.001).Illumina sequences were aligned to contigs with BWA  using thSNPs calling was performed using GATK UnifiedGenotyper v2.4.9 . BecauseHyphessobrycon anisitsi , otherwise only the sequence matching the coding sequence of the zebrafish was annotated as coding and the flanking sequences were not annotated. The 55,950 SNPs in the coding sequences were annotated as synonymous or non-synonymous, according to which amino acid was coded for by the alternative codons resulting from the SNP. The ancestral allele and the derived allele were inferred according to the allele found in the outgroup Astyanax mexicanus SF and CF were classified into eight classes , we would expect that the eighth class (divergent polymorphism) of SNPs would be a very rare outcome because it is the result of two independent mutations at the same site, either in the ancestral population or in the CF and SF populations. We found only one SNPs in this class (Table The populations being closely related (they belong to the same species) and the mutation rate for a SNP origin being very low . When the minimal number of reads per population at a SNP site was set to 100 or higher, the relative frequencies of the eight SNP classes were very stable, indicating that 100 was a good compromise between the stability of the distribution of the SNPs into different classes and the number of SNPs discarded  with a small window size. When the threshold was set to > 50 bp on each side of the SNP, the distribution was stable  in the lab populations should be set around 5% because the effective population size in the lab is low. All the above thresholds, apart from that for MAF, are trade-offs between quality and quantity of the data. The lowest MAF value possible in the pooled embryo samples depends on the unknown number of parents of the embryos, and the MAF threshold of > 5% could therefore be considered arbitrary. Nevertheless, using MAF thresholds of 1%, 5% and 10% we obtained similar SNP class frequencies .In order to estimate the age of the Pachón cave population, we compared the distribution of SNPs into seven classes (the divergent polymorphism class was empty and thus excluded) defined above with the distribution obtained in simulations of the evolutionary process Fig. . More deAdditional file 1:Supplementary figures and tables. (PDF 1092 kb)Additional file 2:Four taxon unrooted nuclear gene phylogenies. (PDF 101 kb)Additional file 3:Allele frequency distributions at 25 microsatellite loci. (PDF 2321 kb)Additional file 4:Rationale of dating with pool-seq SNPs. (PDF 420 kb)


We found that populations of the threatened plant Arnica montana have conserved a considerable amount of genetic diversity, even when they have become fragmented. However, populations in the Vosges mountains differed strongly in their genetic makeup from populations at lower elevations. It has been suggested to reinforce lowland populations of rare plants with seeds from the uplands, but in view of our results this does not seem to be a good idea for A. montana. Because they are genetically different, plants from the mountains could be less able to cope with conditions in the lowlands than lowland plants.  Arnica montana in western central Europe using AFLP markers. Our results indicate that in contrast to our expectation even strongly fragmented colline populations of A. montana have conserved a considerable amount of genetic diversity. However, mean seed mass increased with the proportion of polymorphic loci, suggesting inbreeding effects in low diversity populations. At a similar small geographical scale, there was a clear IBD pattern for the montane Vosges but not for the colline populations. However, there was a strong IBD-pattern for the colline populations at a large geographical scale suggesting that this pattern is a legacy of historical gene flow, as most of the colline populations are today strongly isolated from each other. Genetic differentiation between colline and montane Vosges populations was strong. Moreover, results of a genome scan study indicated differences in loci under selection, suggesting that plants from montane Vosges populations might be maladapted to conditions at colline sites. Our results suggest caution in using material from montane populations of rare plants for the reinforcement of small genetically depauperate lowland populations.Due to land-use intensification, lowland and colline populations of many plants of nutrient-poor grasslands have been strongly fragmented in the last decades, with potentially negative consequences for their genetic diversity and persistence. Populations in mountains might represent a genetic reservoir for grassland plants, because they have been less affected by land-use changes. We studied the genetic structure and diversity of colline and montane Vosges populations of the threatened perennial plant  Nutrient-poor grasslands in lowland areas have been strongly fragmented during the last decades due to changes in land-use, nutrient enrichment through fertilizers or the cessation of traditional agricultural practices . As a coTo enhance the chances of survival of small and isolated populations, it has been suggested to artificially augment threatened populations by introducing seeds from extant large populations to increase their size and genetic diversity  impose sArnica montana, a characteristic species of acid nutrient-poor grasslands in Central Europe, in the colline Ardennes-Eifel and Hunsrück regions and the nearest montane region, the Vosges mountains. A. montana has strongly declined in lowland and colline regions and is now considered to be endangered in many parts of Europe  Are the colline populations genetically depauperate? (3) Are there differences between colline and Vosges populations in AFLP loci?We asked the following questions: (1) Do the genetic diversity and genetic structure of the colline Ardennes-Eifel and Hunsrück populations and montane Vosges populations of Arnica montana (Asteraceae) is a long-lived perennial plant that produces large rosettes from a rhizome. The species is restricted to Europe , which are wind-dispersed. Although the seeds of A. montana are small (mass c. 1.3 mg) and possess a pappus, their dispersal is very limited  The colline region of the Ardennes (Belgium), the Oesling (Luxembourg) and the Eifel (Germany), (2) the colline region of the Hunsrück (Germany) with the neighbouring Pays de Bitche region (France) and (3) the montane belt of the French Vosges mountains, which is the mountain range nearest to the colline study area (To study the genetic structure of udy area . Apart fA. montana (The studied 20 colline populations (281–633 m a.s.l.) represent a large part of the extant populations in the area. Although some large populations still occur in the colline region of the Ardennes-Eifel, many of the extant populations are small due to small habitat size and low habitat quality, in particular high nutrient levels which are known to be detrimental to A. montana. The geographical distance between montane populations was much smaller than that between colline populations .Ten montane populations were sampled in the French Vosges mountains  per population as it is not possible to distinguish individual genets in the field due to the clonal growth of A. montana is a clonal species, the minimum distance between the sampled rosettes was 1 m to avoid sampling of the same genetic individual twice. In very small populations, the number of sampled rosettes was less than 20  was performed on 10 mg of dried leaf tissue for 494 samples after grinding . Extracted DNA was purified from secondary metabolites with a ChargeSwitch® gDNA Plant Kit (Invitrogen®). A further purification step was done by electrophoresis on a 2 % agarose gel  in 10X TBE buffer UltraPure . Extraction of the samples from the gel was done using a QIAquick® 96 PCR Purification Kit (Qiagen®).EcoRI and MseI , 0.65 µL of 10X REact® 1 Buffer  and 0.65 µL of 10X REact® 3 Buffer  in a final volume of 10 µL. Endonucleases were inactivated by 15 min at 70 °C. Adaptor ligation was achieved by adding 12.48 µL of Adapter/Ligation Solution  and 0.52 µL of T4 DNA ligase (1 U/µL) before incubation for 2 h at 20 °C. Ligation solution was diluted to 1:6 and 4 µL were used to perform the pre-amplification by adding 16 µL of pre-amp primer mix (Invitrogen®), 2 µL of 10X pfu buffer with MgSO4 (Fermentas®) and 1 U of pfu DNA polymerase (Fermentas®) in a final volume of 24 µL.DNA (0.1 µg) was digested at 37 °C for 2 h using pfu buffer with MgSO4, 0.4 U of pfu DNA polymerase, 1 µL of EcoRI primers (100 mM) and 1 µL MseI primer (50 mM) to a total volume of 20 µL. Amplifications were programmed for 1 cycle at 94 °C for 2 min, 10 cycles consisting of 20 s at 94 °C and 30 s at 66 °C and 2 min at 72 °C. The 66 °C annealing temperature of the 10 cycles was subsequently reduced by 1 °C every cycle, and continued at 56 °C for the remaining 20 cycles, with a final hold at 60 °C for 30 min.Polymerase chain reaction was performed using a thermocycler . A first cycle was performed at 94 °C for 3 min, then 20 cycles were performed at 94 °C for 30 s, 56 °C for 60 s and 72 °C for 60 s, with a final cycle of 5 min at 72 °C. Three primer combinations with distinct polymorphic loci were selected for the selective amplification: E-CTA/M-ACA, E-CTA/M-AAC and E-CAA/M-ACA (Invitrogen). To assess reproducibility of the primer combinations, three different and independent individuals were repeated three times from the same DNA extraction for each combination. The mean reproducibility values for the three combinations were reasonably high (86.1–93.0 %). Amplifications were performed using 5 µL of 1:6 diluted pre-amplification reaction, adding 0.4 µL of dNTPs (10 mM), 2 µL of TM 500, GE Healthcare). Samples were prepared for analysis by diluting the final amplified product to 1:10. All samples included 1 µL of MegaBACE ET550-R DNA size standard  diluted at 1:6. Samples were run for 75 min using GT Dye Set 2 .Capillary electrophoresis of all samples was performed with the selective amplification products of AFLP on an automated 48-capillary DNA sequencer (MegaBACEHe) that averages expected heterozygosity of the marker loci  and manually scored as either present (1) or absent (0). Fragments with lengths between 60 and 500 base pairs were included in the analysis. Estimates of allelic frequencies were computed using the square root method of the null homozygote frequency assuming Hardy–Weinberg equilibrium, as implemented in the program AFLP-SURV V1.0  between 1 and 30 in order to quantify the amount of variation of the likelihood of each K. We found that a length of the burn-in and Markov chain Monte Carlo (MCMC) of 10 000 each was sufficient. Longer burn-in or MCMC did not significantly change the results. The model choice criterion implemented in STRUCTURE to detect the K most appropriate to describe the data is an estimate of the posterior probability of the data for a given K, Pr(X|K) (D)’ in STRUCTURE, which we refer to as L(K) afterwards. An ad hoc quantity based on the second-order rate of change of the likelihood function with respect to K (ΔK) did show a clear peak at the true value of K (K  =  m(|L(K + 1) −2L(K) + L(K−1)|)/SD[L(K)] where m is the mean and SD the standard deviation. The best estimate of K was defined by the model giving the highest probability of the data, with a peak in the ΔK graph, and which also gave consistent results over multiple runs. Finally, the runs of the STRUCTURE simulation were aligned using the FullSearch option with the cluster matching and permutation program CLUMPP V1.1.2  was used to partition the genetic variability among colline and montane Vosges populations, populations within population groups and individuals as implemented in GenAlEX 6.41 : annual mean temperature, temperature seasonality, temperature annual range, temperature of driest quarter, and annual precipitation. Because these variables were intercorrelated, we identified two principal components (PCs) by PCA with varimax rotation (SPSS 19.0). PC ALTI explained 45.6 % of the variation and was highly correlated with annual precipitation (r  =  0.97) and mean annual temperature (r  =  −0.96), indicating that PC ALTI corresponded to a climatic gradient related to altitude. PC CONTI explained a further 33.5 % of the variation and was highly correlated with temperature seasonality (r  =  0.98) and temperature annual range (r  =  0.96); indicating that PC CONTI corresponded to a gradient in continentality.We obtained the following bioclimatic variables for each study site (representative of 1950–2000) in a grid-size of about one square kilometre (30 arc seconds) from the Worldclim database version 1.4.  all pairs of populations, (2) separately for the colline and the montane Vosges populations and (3) for a subset of the colline populations whose geographical distances were similar to those of the montane Vosges populations were related to geographical distances and the significance of the relationships tested with a Mantel–Test implemented in GenAlEX (1000 permutations).The clusters identified by the PCA-analysis of the bioclimatic variables corresponded well to the three geographical regions . A firstF2,27  =  14.55, P < 0.001) and was higher in the Ardennes–Eifel region (75.1 %) than in the Hunsrück (59.2 %) and Vosges region (62.4 %). The proportion of polymorphic loci in the montane Vosges populations was lower than in the colline populations .Using three primer combinations, 399 AFLP bands were scored with no private bands specific to a population. All 494 individuals had a unique multilocus genotype. The proportion of polymorphic loci (PPL) in the 30 populations ranged from 46.6 to 86.7 % . The proe) assuming Hardy–Weinberg equilibrium was 0.210. Genetic diversity was 10.2 % lower in montane Vosges (He  =  0.195) than in colline populations , and differed significantly among the three geographical regions Ardennes–Eifel, Hunsrück and the Vosges mountains . Among the colline regions, mean genetic diversity of the populations in the Hunsrück region was significantly lower than that of the populations of the Ardennes–Eifel region . In multiple regressions relating the measures of genetic diversity to altitude and (log)population size, separately for the three regions, Nei’s gene diversity was not significantly related to the two explanatory variables in any of the regions. However, adjusted for the effects of altitude, the number of polymorphic loci significantly increased with population size in the Ardennes-Eifel region . Seed mass increased with the proportion of polymorphic loci .The mean value for Nei’s genetic diversity within populations  allowed us to identify with STRUCTURE several groups corresponding to the uppermost hierarchical level of partitioning between populations. The highest modal value of ΔK was at K  =  3, corresponding to the number of geographical regions. There was a nearly complete correspondence between the clusters identified by STRUCTURE and the three geographical regions  than the value obtained by the AMOVA (ФST  =  0.159). Results of the AMOVA showed that there was a significant genetic differentiation between the Vosges and the colline region (8.2 %) and among the populations within the groups , although the largest part of the total genetic variation was due to differences between plants within populations  was much higher than that among Vosges populations (FST  =  0.004). Overall genetic differentiation between all 30 populations (pairwise ФST) was related to their geographic distance . This isolation by distance pattern was much stronger for the colline populations .A separate analysis with AFLP-SURV for the colline and the montane Vosges populations indicated that the genetic differentiation among colline populations  were identified as outliers with FDR values below 0.001. Divergence of 49 loci (77.8 %) was higher and that of 14 (22.2 %) significantly lower than under a neutral expectation indicating that directional selection occurred at a higher frequency than stabilizing selection.P <0.05) relationship with one or two of the principal components derived from a PCA of bioclimatic variables. After correcting for spatial autocorrelation and for the genetic structure of neutral loci two loci were significantly related to climatic PCs in analyses of deviance, suggesting that these loci may be adaptive and their frequency related to climatic conditions . This was also the case when the variation was partitioned among the colline and the montane Vosges populations (ΦST  =  0.06 vs. ΦST  =  0.08), suggesting that not only genetic drift but also divergent selection has influenced the genetic differentiation among the colline and montane Vosges populations.A second AMOVA, using a reduced data set with the 63 non-neutral molecular markers removed, resulted in lower ΦA. montana populations still exist at both altitudinal levels. Although colline populations are much more isolated than the montane Vosges populations, even most small colline populations have conserved a considerable amount of genetic diversity. Due to their isolation, current gene flow among most colline populations is probably very low, but the effects of genetic drift in small populations are not yet very pronounced, as there was no clear relationship between genetic diversity and population size. However, we only could estimate the number of rosettes in the populations, and the relationship between the number of genets and rosettes in this clonal plant is not known and might vary strongly among populations. A prevailing assumption has long been that sexual recruitment is rare in clonal plants implying low genetic diversity, but an increasing number of studies indicate that populations of clonal plants may maintain considerable amounts of genetic diversity  in comparison to that of other species studied using dominant markers  and a gradient in continentality , which suggests that these molecular markers may be under directional selection. These results could indicate that populations of A. montana are adapted to the local climatic conditions. Although this is only correlative evidence and the observed pattern could also be due other environmental factors, local adaptation in response to local climatic conditions has frequently been found in plants  and by the Musée National d’Histoire Naturelle, Luxembourg.The project was supported by T.M., G.C., D.M. and S.M. designed the study. T.M. carried out the practical work. T.M., G.C. and D.M. analysed the data, T.M., G.C., D.M. and S.M. wrote the manuscript.None declared.


Amaranthus palmeri) is a major weed in United States cotton and soybean production systems. Originally native to the Southwest, the species has spread throughout the country. In 2004 a population of A. palmeri was identified with resistance to glyphosate, a herbicide heavily relied on in modern no-tillage and transgenic glyphosate-resistant (GR) crop systems. This project aims to determine the degree of genetic relatedness among eight different populations of GR and glyphosate-susceptible (GS) A. palmeri from various geographic regions in the United States by analyzing patterns of phylogeography and diversity to ascertain whether resistance evolved independently or spread from outside to an Arizona locality (AZ-R). Shikimic acid accumulation and EPSPS genomic copy assays confirmed resistance or susceptibility. With a set of 1,351 single nucleotide polymorphisms (SNPs), discovered by genotyping-by-sequencing (GBS), UPGMA phylogenetic analysis, principal component analysis, Bayesian model-based clustering, and pairwise comparisons of genetic distances were conducted. A GR population from Tennessee and two GS populations from Georgia and Arizona were identified as genetically distinct while the remaining GS populations from Kansas, Arizona, and Nebraska clustered together with two GR populations from Arizona and Georgia. Within the latter group, AZ-R was most closely related to the GS populations from Kansas and Arizona followed by the GR population from Georgia. GR populations from Georgia and Tennessee were genetically distinct from each other. No isolation by distance was detected and A. palmeri was revealed to be a species with high genetic diversity. The data suggest the following two possible scenarios: either glyphosate resistance was introduced to the Arizona locality from the east, or resistance evolved independently in Arizona. Glyphosate resistance in the Georgia and Tennessee localities most likely evolved separately. Thus, modern farmers need to continue to diversify weed management practices and prevent seed dispersal to mitigate herbicide resistance evolution in A. palmeri.Palmer amaranth ( Amaranthus palmeri S. Wats.)  , which i. Wats.) .Amaranthus palmeri is a dioecious, annual species with prolific seed production, pollen-mediated gene flow due to obligate outcrossing, and high genetic variability  which produces increased EPSPS transcription and protein activity  generated by genotyping-by-sequencing (GBS) to identify numerous sequence differences at presumably random parts of the genome , United States. Glyphosate was used as the sole weed management technique for the cotton portion of the production cycle for more than 10 year before glyphosate resistance was first suspected in 2012, 8 years after the first report in the species. The objective was to determine whether GR e genome , were usA. palmeri individuals  from each of eight different locations in the United States were used for the analyses (Table 1), except for AZ-S2 for which only eleven individuals were used to leave a blank on the plate. Locations AZ-S1, AZ-S2, KS-S, GA-S and NE-S were verified as glyphosate-susceptible (GS) and locations AZ-R  were used. DNA extraction was performed following a modified cetyltrimethylammonium bromide (CTAB) extraction protocol (HindIII (Thermo Scientific) were performed on all or 10% of the samples, respectively, to confirm DNA quality and normalization.For DNA extraction, young leaf tissue was collected, immediately frozen in liquid nitrogen, and stored at -80°C. For GR samples only individuals that survived 800 g a.e. haprotocol  and quanin vivo shikimate accumulation assay with excised leaf tissue . A shikimate standard curve was used to calculate the ng shikimate μl-1 accumulation above the background level. Each biological sample was run in three technical replicates for each dose.To confirm glyphosate resistance and susceptibility for the individuals used for GBS, an f tissue  was condEPSPS gene copy number determination, DNA concentrations were adjusted to 5 ng μl-1 and primer sets  and qPCR conditions were used as previously described . EPSPS gene copy number relative to ALS was determined using the 2ΔCT method where ΔCT = CT(ALS) - CT(EPSPS). Each biological sample was run in three technical replicates.For escribed . Quantit-1 pyrithiobac-sodium  with 0.25% v/v non-ionic surfactant . Plants were sprayed at the six-leaf stage using a CO2 pressurized backpack sprayer equipped with a three nozzle (TeeJet XR8001VS) boom delivering a carrier volume of 112 L ha-1 at 172 kPa at 4 km h-1. The experimental design was random with five replications per dose. Above-ground biomass was harvested 27 days after treatment (DAT), dried at 60°C and dry weight was measured.Greenhouse dose response studies were conducted to confirm pyrithiobac-sodium [acetolactate synthase (ALS inhibitor)] resistance in AZ-R with AZ-S1 as a susceptible control. The experiments took place at the University of Arizona Campus Agricultural Center in Tucson, AZ, United States. Seeds were planted in artificial soil mix in 10 cm pots and after emergence seedlings were thinned, fertilized, and irrigated as needed. ALS-inhibitor treatments included 0, 0.0001, 0.0005, 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, and 1 kg a.i. haALS gene was sequenced from three individuals each of the AZ-R, GA-R, and TN-R populations using the same DNA used for the EPSPS copy number test and SNP calling. ALS gene sequencing was conducted as previously described  and the mitochondrial genome of sugar beet (Beta vulgaris) to identify SNPs specific to the cytoplasmic regions.e genome . They caassette’ . Becausesequence  and the cassette . The 1,3K in the dataset was determined by the averaged likelihood at each K [In Pr (X|K) or In (Kn)] and the variance between replicates was determined by running a continuous series of K = 1–15 to determine the optimal number of populations present within the 95 individuals. The analysis was carried out using a burn-in of 30,000 iterations and a run length of 100,000 Markov Chain Monte Carlo (MCMC) replications in ten independent runs. Prior knowledge about the number of populations was not included. The optimum number of clusters was predicted following the ad hoc statistic ΔK  and allelic richness (AR) per population were calculated using the package ‘PopGenReport.’ Observed (HO) and expected heterozygosity (HE) were calculated with ‘adegenet’ (FIS) was calculated following the formula 1 - (Ho/HE). Principal component analysis (PCA) was conducted using ‘SNPRelate’ and ‘gdsfmt’ (DST) (FST) among populations were performed with 1,000 bootstrap replications using ‘StAMPP’   and pair‘StAMPP’ . The ana‘StAMPP’  and ‘ade‘StAMPP’ , respectA. palmeri populations showed higher shikimate accumulation (11.8–146.3 ng μl-1 at 500 μm glyphosate) than GR populations (0–3.8 ng μl-1)  while GR populations showed higher genomic EPSPS copy number  than GS populations  . Thus, the mechanism of glyphosate resistance was determined to be EPSPS gene duplication in all the sampled GR populations . The 500 μm glyphosate concentration was a clear discriminating dose between GR and GS individuals.Glyphosate-susceptible ulations . The aveALS gene was conducted. The ED50 values for dry weight (pyrithiobac-sodium dose causing 50% reduction in dry weight) were 6.9 and 1.3 g a.i. ha-1 for AZ-R and AZ-S1, respectively (P = 0.027) . A different AZ plant was heterozygous for a AGC → AAC mutation resulting in a change from serine to asparagine at position 653 (S653N). No individual carried both mutations within the same allele. The remaining individuals tested showed no mutations at these positions , whereas the S653N mutation confers resistance to imidazolinones and the pyrimidinylthio-benzoates only  is not expected to influence the analysis of genetic relatedness in this case.The AR) ranged from 1.445 (AZ-S2) to 1.654 (KS-S) with an average of 1.560. The observed (HO) and expected heterozygosity (HE) values ranged from 0.161 (AZ-S1) to 0.219 (TN-R) and from 0.163 (AZ-S2) to 0.211 (KS-S/GA-R), respectively, with an average of 0.193. Low values for HO indicate small effective population sizes or population bottlenecks. The HO values in most populations were less than the HE values  for these three populations was negative. AZ-R was the population with the highest FIS value (0.121) (Table 2).The 1,351 loci used for this study had an average percentage of missing data of 1.07% and an average MAF of 0.159. A high degree of polymorphism (MAF ≥ 0.30) was found in 14.41% of the dataset. The proportion of MAF < 0.1 was 45.37%. One AZ-S2 individual was removed from all future analysis because it was an extreme outlier. The observed number of alleles within a population ranged from 2,017 (AZ-S2) to 2,395 (KS-S), with an average of 2,217. Levels of heterogeneity were compared among populations to examine genetic variability within populations. Allelic richness , The long branch lengths for the individuals indicate high within-individual genetic variability.The consensus tree separated GA-S, TN-R, NE-S, and AZ-S2 with over 86% certainty with GAS, TN-R, and AZ-S2 being the most divergent populations. AZ-S1, AZ-R, and KS-S clustered together. Except for KS-S and AZ-R, all individuals clustered within their sampling location . After removing GA-S, TN-R, and AZ-S2, AZ-R did not separate from the cluster with KS-S and AZ-S1, while GA-R and NE-S clustered distinctively according to PC 2, supporting the UPGMA consensus tree. PC 1 in the second PCA on the subset of populations accounted for 6.87% of the variation in the dataset and again roughly separated GR from GS individuals. Individuals from the same population occupied different areas of the cluster, which indicates a population substructure .To confirm this clustering, a similar pattern of differentiation among populations was constructed using PCA which is used to bring out strong patterns in the dataset based on their variance. The first two principal component (PC) axes cumulatively accounted for 16.69% of the total variation. PCA showed that all individuals clustered according to their collection site. Three distinct outgroups  emerged while the remaining individuals from the other five populations clustered into one group. The first dimension (PC 1) accounted for 8.91% of the variation and roughly separated GR from GS individuals (K) in the dataset required to explain the total sum of genetic variation observed was determined. Evanno’s test  while the remaining populations contained unique alleles. This is supported by a high number of shared alleles among these three populations at K = 8 where KS-S displayed a high degree of admixture with AZ-R and AZ-S1. Although less than with KS-S and AZ-S1, AZ-R still shared alleles with GA-R while AZ-S1 and GA-R shared none . Investigating the dataset without the three outgroups GA-S, TN-R, and AZ-S2 at K = 5 supports that AZ-R shares alleles with KS-S, AZ-S1, and GA-R and very few with NE-S   while the genetic distance was lower among AZ-R, AZ-S1, and KS-S. Thus, AZ-R was most closely related to AZ-S1  and KS-S  and most distantly related to the three outgroups GA-S , TN-R , and AZ-S2 . This was further visualized by a heatmap in Supplementary Information Figure FST values indicated that all populations were significantly different from each other, except for AZ-R and KS-S, where only 5% of the genetic differences between populations were attributable to their geographic origin (Table 3).As expected, very high genetic distances (DST)  and FST P < 0.001) of the total genetic variation was among populations, 4.87% was among individuals within a population (P < 0.01) and the remaining 77.35% (P < 0.001) of the genetic variation was within individuals  indicate that genetic differentiation between populations was high.An AMOVA revealed that 17.78% . The observed correlation of 0.076 further suggests that the two distances are not associated . Structure analysis with the identified sub-populations of K = 4 and K = 5 . At K = 8 AZ-R was closest related to AZ-S1  , while the remaining populations AZ-R, AZ-S1, GA-R, and KS-S clustered together  leaving K = 5 identifiable clusters among the eight populations   from California had multiple independent origins within the Central Valley and evolved many years before its first detection. From there it spread, possibly due to increased selection by the herbicide  populations (Ipomoea purpurea)   , and GR L. Pers)  found muA. palmeri with glyphosate resistance due to EPSPS gene amplification. An investigation using four genomic loci as markers of GR A. palmeri from several sampling sites within North Carolina suggested that adaptation to glyphosate application took place in parallel. The authors based this conclusion on the fact that four out of five identified population clusters were statistically associated with increased glyphosate resistance , it may be difficult to identify a parallel adaptation event. The nativity of A. palmeri to the southwestern United States and adaptation to local and heterogenous environments  and do not necessarily reflect the view of the National Institute of Food and Agriculture (NIFA) or the United States Department of Agriculture (USDA).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer MJG and handling Editor declared their shared affiliation.


Malacosoma , is an important group of forestry pests responsible for large-scale defoliation across much of the Nearctic and Palearctic. The present study uses sequence variation in the mitochondrial cytochrome c oxidase 1 (COI) gene to examine the population genetic structure of the three widespread Malacosoma species . Populations of all three species showed highest diversity in the south, suggesting that modern populations derived from southern refugia with loss of variation as these lineages dispersed northwards. However, despite similar life histories and dispersal abilities, the extent of regional variation varied among the taxa. M. americana, a species restricted to eastern North America, showed much less genetic structure than the western M. californica or the widespread M. disstria. The regional differentiation in the latter reflects the likely derivation of modern lineages from several refugia, as well as taxonomic uncertainty in M. californica. In these respects, the three species of Malacosoma share phylogeographic patterns similar to those detected in vertebrates which are characterised by greater phylogeographic breaks in the western half of the continent and limited structure in the east.While phylogeographic structure has been examined in many North American vertebrate species, insects have received much less attention despite their central ecological roles. The moth genus  The patterns of genetic variation in species and the processes which underlie them are of particular interest to evolutionary biologists. Diverse factors, both historical and contemporary, influence how variation is distributed among populations; these include geological and climatic events, and the presence of physical and behavioural barriers . Past glComparisons of genetic variation spanning multiple species can identify both general and species-specific patterns, revealing how particular life history characteristics impact population structure. Species-specific traits, such as dispersal ability or niche requirements, may affect how a species responds to environmental, climatic, and geological changes. For example, the majority of northern species experienced major population declines reflecting the loss of habitat during the Pleistocene glaciations, while the interglacials favoured range expansion and population growth . In contc oxidase 1 (COI) locus  locus . This stMalacosoma  is a Holarctic genus found across much of North America, Europe, and Asia, with six species native to Canada and the United States  from eastern United States, found limited variation and a lack of regional genetic differentiation , and the forest tent caterpillar M. disstria   . We emplc oxidase I (COI) for all Malacosoma specimens from the United States and Canada were downloaded from the Barcode of Life Data System  was used as the outgroup.Sequences of the 658 bp barcoding region of cytochrome Malacosoma species: M. americana, M. californica,and M. disstria. These species were selected based on distribution and available sample size (Hd) and nucleotide (π) diversity indices, calculated in DNAsp v5.10  were calculated in Arlequin v3.5.1.2  was run in GenAlEx v6.5  which identifies the maximum between group variance with the use of additional geographic information , Saskatchewan (SK), and New Brunswick (NB), as well as those identified as M. californica pluviale, did not group with the majority of M. californica samples. The taxonomic status of M. californica has been widely debated; it is currently viewed as including six largely allopatric subspecies, many of which have previously been considered distinct species, although Franclemont questioned the validity of M. californica pluviale as a subspecies rather than a species  and eastern groups. There were no fixed differences in M. americana. Haplotype and nucleotide diversities were high in all species  were removed from the correlation analysis, coefficients were much stronger for M. americana and M. californica , and slightly weaker for M. disstria (r =  − 0.360).Intraspecific analyses examined 79  samples . For eacdisstria . There wM. americana showed the least population structure , the fewest significant pairwise comparisons : MN, NB, and the separation of the remaining samples into northern  and southern  populations, while the Bayesian clustering analysis separated the samples into three clusters . The BEAST analysis generally supported the BAPS groups with BAPS1 and BAPS2 forming weakly supported clades while BAPS3 contained the remaining samples , and the highest nucleotide diversity (n = 3), and the small geographic distance between cBC and swBC. The greatest differences existed between sCA and all other populations (ΦST = 0.73–0.94), and between AZ and all other populations (ΦST = 0.54–0.79). The SAMOVA analysis identified genetic breaks between three groups: sCA, AZ, and all other populations . Bayesian clustering analysis identified six clusters . When sCA, CA, and AZ were removed from analysis, the distribution was still significantly different than random . The BEAST analysis identified four main clades with numerous subclades within them: two well-supported clades representing the AZ and sCA samples , one clade found in eBC with one CA sample (posterior = 0.75), and one found across BC and WA , and 42 of 55 pairwise comparisons were significant following correction for multiple tests (ST = 0.49–0.98). Diversity within populations was generally high with Hd > 0.7 in 10 of the 16 populations. The lowest values were in SK (Hd = 0) followed by eBC  and TX . Bayesian clustering analysis showed a slightly different pattern with four identified clusters . The BEAST analysis identified four main clades, generally supporting the BAPS groups, although with slightly different membership: two well-supported clades found mostly in the west (posterior probability = 1 and 0.75), and two clades present mostly in the east and southeast , possibly along the Rocky Mountains (clusters : three wn ≥ 10): three found in a single region , one shared by ON and NB/NS, and one found across several regions in the east and south , and identified a general separation of northern and southern populations along coordinate 2 (14.8%). Coordinate 3 explained 12.6% of the variation . The stand south . The BC Malacosoma species showed high levels of variation and diversity, with some highly divergent populations in M. californica. All three species show evidence of persistence in one or more southern refugia with subsequent recolonisation of northern regions. Diversity patterns exhibited the characteristic “southern richness, northern purity” (M. americana where diversity in southern populations was twice that in northern regions (π = 0.0046–0.0061 versus 0.0013–0.0027), and in M. californica where diversity was four-fold higher in Arizona (π = 0.0093) than in Washington  separated by multiple fixed differences which may represent different subspecies or ecotypes. This pattern is common in many southwestern and western species, and is explained by both the current and historical topography of western North America: four major mountain ranges  run along a north-south axis, and large plains and deserts abound, all of which contribute to a complex habitat mosaic. This heterogeneity, coupled with the resulting complex glacial histories of the region, has resulted in extensive structuring in many birds , while M. californica has disjunct populations among many of the southwestern deserts , likely representing the described subspecies M. californica lutescens as a separate species  and distribution of haplotypes (h), haplotype diversity (Hd), and nucleotide diversity (π) among the (A) M. americana, (B) M. californica, and (C) M. disstria sampling locations. Shared haplotypes have been allocated a letter. The allocation of samples into Bayesian clusters (BAPS) is also given.Sample size (Click here for additional data file.10.7717/peerj.4479/supp-3Figure S1Malacosoma. The triangles represent multiple specimens from the same species, with the length of the triangle representative of the sequence variation. Posterior values ¿0.8 are given. The shaded boxes represent the samples used in this study; further detail is given in Bayesian analysis based on 474 COI sequences from five North American species of Click here for additional data file.10.7717/peerj.4479/supp-4Figure S2M. americana, M. californica, and M. disstria. The correlation coefficients were −0.421, −0.284, and −0.508, respectively. All populations in this study above 40°N are in previously glaciated regions.Comparison of diversity relative to latitude for Click here for additional data file.10.7717/peerj.4479/supp-5Figure S3M. americana sequences and rooted with the congeneric M. californica. Posterior values are given for major clades. Tree files are available upon request.Bayesian analysis based on 79 Click here for additional data file.10.7717/peerj.4479/supp-6Figure S4M. californica sequences and rooted with the congeneric M. americana. Posterior values are given for major clades. Tree files are available upon request.Bayesian analysis based on 207 Click here for additional data file.10.7717/peerj.4479/supp-7Figure S5M. disstria sequences and rooted with the congeneric M. constricta. Posterior values are given for major clades. Tree files are available upon request.Bayesian analysis based on139 Click here for additional data file.