Coturnix japonica) populations, and their genetic relationships, we examined mitochondrial DNA (mtDNA) D-loop sequences and microsatellite markers for 19 Japanese quail populations. The populations included nine laboratory lines established in Japan , six meat-type quail lines reimported from Western countries , one commercial population in Japan, and three wild quail populations collected from three Asian areas. The phylogenetic tree of mtDNA D-loop sequences revealed two distinct haplotype groups, Dloop-Group1 and Dloop-Group2. Dloop-Group1 included a dominant haplotype representing most of the quail populations, including wild quail. Dloop-Group2 was composed of minor haplotypes found in several laboratory lines, two meat-type lines, and a few individuals in commercial and wild quail populations. Taking the breeding histories of domestic populations into consideration, these results suggest that domestic quail populations may have derived from two sources, i.e., domestic populations established before and after World War II in Japan. A discriminant analysis of principal components and a Bayesian clustering analysis with microsatellite markers indicated that the domestic populations are clustered into four genetic groups. The two major groups were Microsat-Group1, which contained WE, and four WE-derived laboratory lines , and Microsat-Group2 consisting of NIES-L, JD, JW, Estonia, NIES-Br, NIES-Fr, NIES-Hn, W, and commercial and wild populations. The remaining two lines (AMRP and rb-TKP) were each clustered into a separate clade. This hierarchical genetic difference between domestic quail populations is attributed to the genetic background derived from two different genetic sources—the pre-war and post-war populations—which is well supported by their breeding histories.To assess the genetic diversity of domestic Japanese quail ( Coturnix japonica, Phasianidae) is an important animal species in poultry production. Owing to their high growth and egg-laying rates, Japanese quail have been preferentially bred worldwide, including selective breeding to improve egg and meat production, since the beginning of the 20th century , followed by three purification steps using phenol, phenol/chloroform/isoamyl alcohol (25:24:1), and chloroform/isoamyl alcohol (24:1). Feather shaft samples of 19 stuffed specimens of wild Japanese quail, consisting of 14 samples from Honshu of the Japanese archipelago  and five samples from the Liaodong Peninsula in China, were provided by three museums, Osaka Museum of Natural History, Gunma Museum of Natural History, and Togakushi Museum of Natural History in Japan. Genomic DNA was extracted from the basal tips of rachises (approximately 1 or 2 mm in length) using commercial DNA extraction kits .Genomic DNA from four laboratory lines  and commercial quail was extracted from 20 μl of blood using 300 μl of DNAzol BD reagent . Muscle tissues of two wild Japanese quail collected at Tsushima Island were provided by the Yamashina Institute for Ornithology in Japan. The samples were stored in 70% ethanol. Genomic DNA was extracted from tissue samples of about 5 mmAnimal care and all experimental procedures were approved by the Animal Experiment Committee, Nagoya University , and were conducted according to the Regulations on Animal Experiments in Nagoya University.Genomic DNA of historical stuffed specimens is generally degraded and fragmented, and these properties make successful PCR amplification difficult, resulting in the detection of false alleles and/or allelic dropout and leading to genotyping errors –28. To d5′-CCTAACTCCCCTACTTAGTGTACC-3′) and Quail_D-loop-R (5′-TCTCGTGAGGTGTACGATCAAT-3′). Amplification was performed in a 15-μl reaction mix containing 50 ng of genomic DNA, 10 pmol each primer, and 7.5 μl of 2 × Phusion HF Master Mix . The cycling conditions for PCR were as follows: initial denaturation at 98°C for 30 s, followed by 40 cycles at 98°C for 10 s, 60°C for 15 s, and 72°C for 20 s, and a final extension for 5 min at 72°C. PCR products were purified using the 20% polyethylene glycol/2.5 M NaCl precipitation method .DNA sequences were aligned using ClustalW  implemenanalyses ,19 were analyses , F1W1  per locus and expected heterozygosity (He) were calculated using ARLEQUIN 3.5.1.2 [AR) was calculated using MICROSATELLITE ANALYSER 4.05 [Ne, the number of alleles weighted by the square of the allele frequency) was obtained using GENALEX 6.5 [HWE) and calculation of a fixation index (F) were carried out for each line and population using GENALEX 6.5 [FST) were calculated using MICROSATELLITE ANALYSER 4.05 [Based on microsatellite marker data, the mean number of alleles  were divided into two groups . The maxC. coturnix was positioned between Dloop-Group1 and Dloop-Group2; however, the bootstrap support was very low (<50%). In the network of haplotypes (C. coturnix (KJ623812).Dloop-Group2 included four haplotypes, JqD2, JqD3, JqD5, and JqD6, for five laboratory lines , two meat-type quail lines (Estonia and NIES-Fr), and commercial and wild quail, as well as F5 (KF410834), found for one individual in a farm in Spain. Three unique haplotypes of wild quail (JqW1‒JqW3) and H63 (DQ087958) were contained in Dloop-Group2. In the maximum-likelihood tree based on the GTR+G+F model , C. cotuplotypes , JqD1 waF values close to zero (MNA) ranged from 1.48 to 5.70 for 15 laboratory lines and commercial and wild populations , ranging from 1.48 to 1.65, whereas it was higher in NIES-Br, NIES-Hn, W, and commercial and wild (Honshu) populations, ranging from 2.83 to 5.70. Five lines  exhibited low expected heterozygosity (He) values (0.15 to 0.22); in contrast, high He values (0.46 to 0.58) were observed in NIES-Br, W, and commercial and wild (Tsushima Is. and Honshu) populations. The allelic richness (AR) was relatively higher for WE, NIES-Br, W, and commercial and wild (Tsushima Is. and Honshu) populations (from 1.68 for WE to 2.03 for the wild population from Honshu) and lower for AMRP, RWN, rb-TKP, Estonia, and NIES-Fr lines . The ranking of these 17 quail populations based on the numbers of effective alleles (Ne) was quite similar to the rankings based on MNA, He, and AR. Pairwise FST was 0.40, on average, ranging from -0.02 (Honshu and the Liaodong peninsula) to 0.73 (rb-TKP and NIES-Fr), and all FST values were significantly greater than zero , whereas RWN, AMRP and rb-TKP exhibited larger genetic distances to the wild Japanese quail . The FST distances between the populations were shown as a neighbor-joining tree using MEGA 6.0 (The numbers of microsatellite markers amplified from each stuffed specimen of wild quail ranged from 5 (A1798 from Honshu) to 20 (A0279 from Honshu) . The tot to zero , suggestulations . MNA wasMEGA 6.0 .In the DAPC plot, a laboratory line, rb-TKP, was placed as a separate cluster independently from the other 18 populations . To defiThe Bayesian clustering analysis suggested that the optimal number of clusters was four  for our dataset of 19 populations . Five laThe molecular phylogenetic tree and network of mtDNA D-loop sequences revealed the presence of two clades, Dloop-Group1 and Dloop-Group2, within domestic Japanese quail populations. The wide distribution of JqD1 of Dloop-Group1 suggests that the haplotype recently spread among domestic quail populations around the world. JqD1 was also observed for wild Japanese quail from Honshu in Japan and Liaodong Peninsula in China. In addition, our results suggest that Dloop-Group2 is older than Dloop-Group1 because two haplotypes, F5 and H63, in Dloop-Group2 are considered older than other haplotypes of Japanese quail in European countries ,19. F5 iBamHI-RFLP revealed that the 14 lines were separated into two groups: one that contained Quv and RWN and the other that included LWC, AMRP, and rb-TKP. This was comparable to the results obtained in this study, except for AMRP. Shen et al. [The mtDNA diversity was previously investigated for 14 laboratory lines of Japanese quail, including LWC, AMRP, Quv, RWN, and rb-TKP, which were examined in this study, using a restriction fragment length polymorphism (RFLP) analysis . Among 1n et al.  suggesteGenetic clustering analysis using 23 microsatellite markers revealed that 16 domestic quail populations are subdivided into four clusters of populations. Microsat-Group1 consisted of WE and four WE-derived laboratory lines  ,23,50–52panda” plumage coloration [panda mutation appears in Japanese literature published in 1918, which records that the mutant had been maintained as a pet animal by fanciers [The AMRP line originated from a mutant animal with the “loration ,58,59, wloration . The panfanciers . The oldWe consider the genetic background of WE and four WE-derived laboratory lines to be derived from both the pre-war and post-war populations, because the WE line originated from the “CE” line, which was established at the Nippon Institute for Biological Science in Japan in 1966 by the crossbreeding of Japanese commercial quail (post-war population) with a laboratory line imported from the United States (pre-war population) . The mixture of populations from different origins was found for their D-loop haplotypes: LWC, WE, and AWE lines have the JqD1 haplotype of Dloop-Group1, which is considered to be of post-war origin, while Quv and RWN have the JqD3 haplotype of Dloop-Group2, which may have derived from the pre-war population. Genetic differences between the two groups also become apparent by the DAPC and the Bayesian clustering, and Quv and RWN are more closely related to AMRP. This suggests that the genetic background of the WE and WE-derived lines originated from both post-war and pre-war populations.http://www.agr.nagoya-u.ac.jp/~nbrp/en/index.html). The close genetic relationship among these lines indicates that most of existing domestic populations were recently distributed worldwide from the post-war founder population [FST network and the Bayesian clustering at K = 8. These results suggest that some quail populations clustered into Microsat-Group2, such as Estonia and NIES-Fr, initially retained the genetic background of both pre-war and post-war populations, and the genetic contribution of the pre-war population decreased over time during some selective breeding process, such as backcrossing with quail derived from the post-war population.The meat-type lines clustered together in Microsat-Group2 with NIES-L and commercial quail. The body weights of NIES-Br and NIES-Hn quail are approximately two-fold higher than those of common laboratory and commercial quail. For example, average body weights of adult males and females of NIES-Br are 260.3 g and 289.5 g, respectively, while NIES-L males are 122.9 g and females are 148.0 g  [FST = 0.176−0.302, 0.231 on average, in seven indigenous populations) [FST = 0.03−0.32, 0.180, on average, in six local breeds and four commercial lines) [FST values in the quail populations used in this study seem to be attributed to their breeding history and the characteristics of microsatellite markers used in this study. Nine of the 15 lines  were maintained independently as closed colonies for more than twenty years. In this study, microsatellite markers with high allelic polymorphism were selected from more than 100 markers developed in our previous studies [FST values.The pairwise lations) , China (lations) , and Ital lines) . The hig studies ,25. ThesWe identified two genetic sources of domestic Japanese quail populations with different genetic backgrounds by analyses of mtDNA D-loop sequences and microsatellite markers. One (Dloop-Group2 and Microsat-Group1) may include alleles from the population that was exported to the United States from Japan before World War II (the pre-war population). The other (Dloop-Group1 and Microsat-Group2) was probably derived from limited founder populations that were re-established in Japan after the war (the post-war population), which were subsequently exported from Japan and then rapidly spread around the world. Components of the genetic background of the pre-war population can be found in the present domestic quail population, which is the result of persistent crossbreeding with the post-war population. Further studies with additional samples, including wild Japanese quail, are needed to obtain the detailed genetic characteristics of domestic quail populations in the world.S1 FigPutative pre-war and post-war populations are shown by ellipses and hexagons, respectively. Dotted and solid lines indicate breeding histories before and after establishment (introduction) of quail populations, respectively. Filled diamonds indicate a cross between different populations. Genetic groups based on microsatellite markers are shown on the right side.(PDF)Click here for additional data file.S2 FigTissue samples stored in 70% ethanol are shown by filled orange diamonds and feather shaft samples of stuffed specimens by filled gray diamonds.(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S1 File(XLSX)Click here for additional data file.


SNP) assays for two species of anadromous alosine fishes, alewife and blueback herring (collectively known as river herring), that are native to the Atlantic coast of North America. We used data from high‐throughput DNA sequencing to discover SNPs and then developed molecular genetic assays for genotyping sets of 96 individual loci in each species. The two sets of assays were validated with multiple populations that encompass both the geographic range and the known regional genetic stocks of both species. The SNP panels developed herein accurately resolved the genetic stock structure for alewife and blueback herring that was previously identified using microsatellites and assigned individuals to regional stock of origin with high accuracy. These genetic markers, which generate data that are easily shared and combined, will greatly facilitate ongoing conservation and management of river herring including genetic assignment of marine caught individuals to stock of origin.Freshwater habitat alteration and marine fisheries can affect anadromous fish species, and populations fluctuating in size elicit conservation concern and coordinated management. We describe the development and characterization of two sets of 96 single nucleotide polymorphism ( Elucidation of population structure and patterns of connectivity are often the first steps in the use of genetic data to understand a species’ biology. In addition, common applications for such data include reconstructing pedigree relationships, inferring historical demography, individual identification for mark/recapture type analyses, and evaluating patterns of natural selection and the identification of individuals to population of origin  and blueback herring —collectively known as “river herring”—are migratory sea‐run  fishes that reproduce in lakes and rivers along the east coast of North America, but typically migrate to the Atlantic Ocean as juveniles to grow and reach sexual maturity before returning to their natal freshwater spawning grounds to reproduce  were nested within three regional genetic stocks , whereas US blueback herring populations (n = 21) were nested within four regional genetic stocks , with similar but not identical boundaries. Hasselman et al.  are bi‐allelic markers, ubiquitous in the genome of most species . To identify potential SNPs for alewife and blueback herring, we used double digest Restriction Associated DNA sequencing (ddRADseq), a genome‐reduction technique that uses two restriction enzymes to create DNA fragments with identical fixed endpoints for annealing sequencing adapters  design. Assays were evaluated for consistency and polymorphism by genotyping 382 alewife samples from eight populations and 474 blueback herring from 10 populations throughout the species ranges  and observed heterozygosity (HO) for each locus were estimated using the Microsatellite Toolkit  in R 3.3.1  of 0.290 in blueback herring versus HO of 0.249 in alewife. Similarly, the mean minor allele frequency across blueback herring populations was 0.230, whereas it was 0.197 in alewife. While still adequate for parentage analyses , and one locus in four populations (Aps_5844) Table . For bluThe majority of both blueback herring and alewife individuals were accurately assigned to their population and reporting group of origin in the self‐assignment analyses. For alewife, 67% of all fish were correctly assigned to population of origin and 93% to reporting group of origin Table a when noFST values ranging from 0.006 to 0.140. The highest FST values were between the southernmost populations (Alligator and Chowan) and populations in the Northern New England reporting group (Penobscot and Androscoggin), with somewhat less differentiation seen between these populations and the northernmost ones in Canada (Waughs and Tusket)  Table a. Pairwi.1 Table . Pairwisstructure for the eight populations of alewife found that, at K = 2, the two southernmost populations, Chowan and Alligator Rivers, cluster together  is in the Gulf of the Saint Lawrence Seaway and the other (Tusket) borders the Bay of Fundy, where strong population structure has been identified in other anadromous species: America shad .Genotype data for all fish with all assays, FASTA files with consensus sequences from all ddRAD loci with at least one SNP, as well as haplotype calls from all combinations of locus and individual are in the DRYAD digital repository (doi: Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Amphiareus obscuriceps  (Hemiptera: Anthocoridae) is an invasive species native to the Sino-Japanese Region (SJR) of East Asia. We tested the hypothesis of the ECS land bridge acting as a dispersal corridor or filter for A. obscuriceps during the glacial period. Specifically, we tested whether and the extent to which dispersal ability and host plant habitat requirement influenced the genetic structure of A. obscuriceps during the exposure of the ECS land bridge.On account of repeated exposure and submergence of the East China Sea (ECS) land bridge, sea level fluctuation played an important role in shaping the population structure of many temperate species across the ECS during the glacial period. The flower bug A. obscuriceps is composed of two major lineages, i.e., China and Japan. Divergence time on both sides of the ECS was estimated to be approximately 1.07 (0.79–1.32) Ma, which was about the same period that the sea level increased. No significant Isolation by Distance (IBD) relationship was found between Фst and Euclidean distances in the Mantel tests, which is consistent with the hypothesis that this species has a good dispersal ability. Our Last Glacial Maximum (LGM) niche modeling of plants that constitute preferred habitats for A. obscuriceps exhibited a similar habitat gap on the exposed ECS continental shelf between China and Japan, but showed a continuous distribution across the Taiwan Strait.Phylogenetic and network analyses indicated that A. obscuriceps in its native area. The host plant habitat requirement could also be a key to the colonization of the A. obscuriceps species during the exposure of the ECS land bridge. Our findings will shed light on the potential role of habitat requirement in the process of biological invasion in future studies.Our results suggest that ecological properties , together with sea level fluctuation during the Pleistocene across the ECS, have shaped the genetic structure and demographic history of The online version of this article (doi:10.1186/s12862-016-0748-3) contains supplementary material, which is available to authorized users. Quercus variabilis Blume, 1850 [Thais clavigera  [Microvelia douglasi douglasi Scott, 1874 [The eastern areas of Sino-Japanese region stretch along more than 1500 km in an east–west direction, ranging from East China to Japan and the Korean Peninsula to Taiwan and the Ryukyu Islands . During Platycrater arguta Siebold & Zucc., 1835 [Acer mono Maxim, 1856 [Acanthurus and the rock-associated lizard, genus Dalmatolacerta, indicate that habitat requirement is the main factor that shaped different phylogeographic patterns in these habitat-associated species [In contrast, many studies provided evidence that the ECS land bridge also served as a dispersal filter that obstructed or even inhibited certain species or populations from migrating through the ECS land bridge during the LGM, as seen for two deciduous trees im, 1856 . This phim, 1856 . Recent im, 1856 . Many sm species , 13.Amphiareus obscuriceps  (Hemiptera: Anthocoridae), a native species across the ECS land bridge [A. obscuriceps were usually collected from the deciduous trees, Castanea mollissima Blume, 1851, Salix babylonica L., 1753, Salix matsudana Koidz, 1915, and the shrub, Sorbaria sorbifolia (L.) A. Braun, 1860 [A. obscuriceps has become an invasive species that is rapidly spreading in North America and Europe [A. obscuriceps provides an ideal model for examining the phylogeographic structure and Pleistocene history associated with sea level fluctuation and ecological property across the ECS during the glacial period. We used a comprehensive sampling of A. obscuriceps in its native area, and applied ecological niche modeling (ENM) and multilocus genetic markers to infer a more robust phylogeographic conclusion of this species across the ECS. Specifically, this study aimed to (1) describe the phylogeographic structure; (2) infer the divergence time and historical demography; (3) test the hypothesis of ECS land bridge as a dispersal corridor or filter for A. obscuriceps in its native area during the last glacial period; and (4) evaluate the role of habitat requirement and dispersal ability in driving the current genetic structure during the exposure of the ECS land bridge in LGM period.Few studies are focused on temperate invertebrate species of comprehensive samples, and the influence of dispersal ability and habitat requirement on the population genetic structure across the ECS are poorly understood. In this study, we focus on the flower bug d bridge , 15. Thid bridge . In its un, 1860 , all of un, 1860 . RecentlAmphiareus ruficollaris Yamada & Hirowatari, 2003 was used as the outgroup for our phylogenetic analysis. All samples were preserved in 95 % ethanol and stored in a freezer at −20 °C in the College of Life Sciences at Nankai University . Genomic DNA was extracted from the entire body, excluding the abdomen and genitalia using a General AllGen Kit . All individuals were analyzed by sequencing for the mitochondrial markers, cytochrome c oxidase subunit I (COI), cytochrome c oxidase subunit II (COII), and cytochrome b (CytB) and the nuclear marker internal transcribed spacer 1 (ITS1). PCR primers designed in the present study are listed in Table S1  and Maximum likelihood (ML) methods, and implemented in MrBayes 3.2.5  and raxmS), number of haplotypes (Nhap), haplotype diversity (Hd), and nucleotide diversity (π), which were all calculated in DNASP 4.0 [Genetic diversities of mitochondrial and nuclear data were estimated for all populations and for each population using the number of polymorphic sites  and migration rate (m) using the isolation-with-migration (IM) model implemented in IMa2 [q), 10 for the divergence time (t), and 1 for the migration rate (m). Parameters were estimated based on mutation rates for COI (0.6 × 10−8–1.0× 10−8) and CytB (1.1 × 10−8–1.8× 10−8) per site per generation [We estimated the divergence time  delimited by a bounding box containing all known native range occurrences of A. obscuriceps typically used in former studies [C. mollissima, Sa. babylonica, Sa. matsudana and So. sorbifolia [A. obscuriceps have changed since the Pleistocene glaciation. The host plant coordinates in its native area for niche model building were downloaded from the Global Biodiversity Information Facility online database , which determined 335 localities for C. mollissima, 93 for Sa. babylonica, 59 for Sa. matsudana, and 44 for So. sorbifolia. We chose bioclimatic variables to represent annual trends and extreme or limiting conditions, because many taxa are limited by environmental extremes [A. obscuriceps and related host plants, were selected from the WorldClim database [http://www.worldclim.org/), including mean temperature (BIO1), mean diurnal temperature range (BIO2), maximum temperature of the warmest month (BIO5), minimum temperature of the coldest month (BIO6), annual mean precipitation (BIO12), precipitation of the wettest month (BIO13), and precipitation of the driest month (BIO14). We conducted an autocorrelation test of the 7 bioclimate variables that showed relatively low Spearman’s coefficients (r < 0.80)  , COII (540 bp) and CytB (411 bp) genes. A total of 182 unique haplotypes were derived from tandem sequence (COI + COII + CytB) among all individuals. The 277 polymorphic sites included 107 singleton variables and 170 parsimony informative sites. The nucleotide and haplotype diversities ranged from 0.00168 to 0.01199 (mean 0.01926) and from 0.667 to 1.000 (mean 0.9995) respectively (Table p < 0.01), where approximately 18.75 % of the genetic variation was within populations and only 1.12 % was among populations within the group  with higher levels of reticulation were identified. A similar topological structure was recognized using the single COII dataset to reduce the complexity of reticulation and achieve clear exhibition Fig. . The oveges Fig. , indicatA. obscuriceps experienced population expansion events , 0.52 % among populations within groups, and 15.98 % within populations (Table K = 1)   and from 0 to 0.711 (mean 0.664) respectively  .Multimodal mismatch distributions are assumed to characterize old populations of constant size, whereas expanding populations are considered to be unimodal . Mismatc65) Fig. , b & d. A. obscuriceps populations from Mainland China to Taiwan with m = 0.9995, which was greater than zero and thus indicated that gene flow occurred during or soon after the divergence event. Estimation of the divergence time by BEAST was presented in Figure S5  Ma between the population in China and that in Japan and 0.15 (95 % CI = 0.14–0.24) Ma between that in Mainland China and that in Taiwan. We also detected one statistically significant migration event of A. obscuriceps was shown in Figure S6  Koidz., 1784 [Rhinolophus ferrumequinum  [Adelphocoris suturalis  [A. obscuriceps on both sides of the ECS. The ancestral status of A. obscuriceps was probably extensively distributed in East Asia, including the ECS land bridge in the early Pleistocene. At approximately 0.79–1.32 Ma, lineage differentiation between the population in China and that in Japan was driven by population contraction and extinction on the ECS land bridge because of sea level increase and land bridge submergence. However, we could not verify whether the ancestral status of A. obscuriceps was extensively distributed across East Asia in the early Pleistocene in this scenario. This phenomenon indicated that the cold and arid climates during the Plio-Miocene to early Pleistocene were less extensive than that during the middle and late Pleistocene [A. obscuriceps showed that the potential range of the species contracted moderately in the Chinese region and sharply shrank toward the south coastal zone in the Japanese region. This finding suggested that A. obscuriceps was relatively sensitive to the low temperature in the northern regions during the LGM. Therefore, the Plio-Miocene to early Pleistocene environment might have created more favorable conditions for the ancestral status of A. obscuriceps to occupy relatively extensive and continuous regions in East Asia.The results of mitochondrial and nuclear DNA showed that z., 1784 , the horr, 1774) , and thev, 1882) . Based ov, 1882) . From lav, 1882) . During v, 1882) . Based oistocene . Indeed,A. obscuriceps populations between Mainland China and Taiwan.The results of phylogenetic and haplotype network analyses showed that several individuals from Taiwan were nested structures within the haplogroups of China, which indicated that a relatively shallow divergence occurred between the populations in Mainland China and that in Taiwan. The shared ancestral haplotypes showed that recent gene flow also existed between these two regions. The divergence time  between Mainland China and Taiwan approximated the penultimate glaciation period (0.13–0.30 Ma) , which wA. obscuriceps experienced different demographic histories in Mainland China, Japan, and Taiwan. For the regions in Mainland China and Japan, the observed pairwise differences of mtDNA haplotypes fit the sudden expansion model particularly well  [Hesperotettix viridis  and Ceracris kiangsu Tsai, 1929 [A. obscuriceps: similar unsuitable area gaps existed as the geographical barrier between the ECS continental shelf and Japanese regions  . The ENMeps Fig. , indicateps Fig. ; Table 2hem Fig. . Dependis, 1758)  and two ai, 1929 , 65. Thiana Fig. , d & e, A. obscuriceps in its native range. Molecular data revealed a significant genetic differentiation between the populations in China and Japan. Divergence time approximately coincided with the period of sea level increase, and recent gene flow was not detected by the IMa2 analysis. We determined that the vicariance scenario led to divergent lineages of A. obscuriceps on both sides of the ECS. No significant IBD relationship was observed between Фst and the Euclidean distances in the Mantel tests, which is consistent with the hypothesis that this species has a good dispersal ability. Our LGM niche modeling of the four related host plants exhibited similar habitat gaps on the exposed ECS continental shelf between China and Japan, but showed continuous distribution across the Taiwan Strait. We provided a successful example of uncovering the genetic structure and underlying factors of an invasive insect across the ECS. The findings of our study will also shed light on the potential role of habitat requirement in the process of biological invasion.Phylogeography coupled with ENM revealed the Pleistocene history of the invasive species


ISSR) markers. Samples originating from each lake tended to cluster together. Of the six species, only Nymphoides peltata and Myriophyllum spicatum could be divided into plateau and plain groups, once Taihu Lake individuals were excluded. Genetic similarities between populations connected by the Yangtze River were not consistently higher than unconnected populations. Populations from Taihu Lake and/or Weishanhu Lake were distant from other lake populations for all species except Potamogeton lucens. The Taihu and Weishanhu populations clustered for Ceratophyllum demersum and Typha latifolia. Hydrophilous C. demersum had the lowest gene flow (Nm = 0.913), whereas the entomophilous Hydrocharis dubia (Nm = 2.084) and N. peltata (Nm = 2.204) had the highest gene flow. The genetic relationships among distant populations of aquatic plants reflect the comprehensive effects of environmental selection pressure and biotic and abiotic connectivity. Differences in environmental factors between plateau and plain lakes and long distance hydrochory have limited importance on aquatic plant genetic structures. Among multiple evolutionary forces, gene flow mediated by birds may play the most important role in the formation of genetic patterns in the six species examined. For example, the close genetic relationship between Taihu Lake and Weishanhu Lake populations, each in different river systems and with different climates, may be related to the migration routes of birds. Differences in gene flow among the six aquatic plants may be attributable to different bird‐transport and the fruit traits of each species.Many aquatic plant species are distributed over large areas and diverse environments with populations interconnected by abiotic and biotic mediators. Here, we examined differences and similarities in the population genetic structure of six sympatric and widespread aquatic plant species. We sampled the aquatic species from six Chinese lakes found on plateaus, plains, and different river systems and analyzed them using inter‐simple sequence repeat ( For example, the Yangtze River connects many lakes from western upstream plateaus to eastern midstream and downstream plains  separated by large distances and in different climatic zones in China. Among the focal lakes, one plateau lake and three plain lakes are connected by the Yangtze River, and two of the plain lakes are located on the same avian migratory route. By comparing the genetic relationships among lake populations of different plant species, we hope to further understand environmental effects, hydrologic connectivity, and bird migration on population genetic structures in aquatic species. We hypothesize that (1) population genetic differentiation could arise from divergent environmental pressures or demographic factors related to differential population sizes and isolation of lakes, whereas (2) gene flow mediated by water, fish, and birds homogenizes populations.22.1Caohai Lake and Erhai Lake are located on the Yunnan‐Guizhou Plateau in a subtropical plateau monsoon climate Table . Among t2.2Myriophyllum spicatum L., Ceratophyllum demersum L., and Potamogeton lucens L.), two floating‐leaved (Hydrocharis dubia (Bl.) Backer and Nymphoides peltata (Gmel.) O. Kuntze), and one emergent species (Typha latifolia L.) were included  are larger than those of the other four species (fruit diameter 1–5 mm) . The sample size and number of sampling sites (subpopulations) were 177 and 31, respectively, in five lakes (populations) for M. spicatum; 174 and 30 in six lakes for C. demersum; 117 and 20 in four lakes for P. lucens; 98 and 16 in three lakes for H. dubia; 148 and 23 in five lakes for N. peltata; and 101 and 18 in five lakes for T. latifolia , for each species we selected 9–13 primers amplified polymorphic and reproducible bands (Appendix Taq polymerase (0.1 U/μl), dATP, dCTP, dGTP, dTTP (0.4 mM each) and buffer (Beijing ComWin Biotech), ISSR primer (Beijing Genomics Institute), and genomic DNA. PCRs were performed in a C1000 Thermal Cycler (Bio‐Rad Laboratories). A denaturation period of 4 min at 94°C was followed by 40 cycles of 30 s at 94°C, 30 s at 52°C and 1 min 30 s at 72°C, and then 10 min at 72°C for final extension. PCR products were separated on 2% (w/v) agarose gels and visualized under UV after staining with ethidium bromide.Total genomic DNA was extracted from dry leaf samples following a modified cetyltrimethyl ammonium bromide (CTAB) protocol  or absence (0) of bands. Nei's unbiased genetic distances Nei,  among poK in 10 independent runs from 1 to 6, without using sampling location as a prior to assess convergence of Ln P (D) . The value of Ln P (D) is the posterior probability of the data for a given K. Furthermore, ΔK values based on the rate of change in Ln P (D) between successive K values were calculated according to Evanno, Regnaut, and Goudet  that best explain the data.Bayesian cluster analyses of population structure were performed using the software STRUCTURE 2.3 , mean genetic diversity within lake populations (HS), Nei's genetic differentiation index among populations (GST), and gene flow among populations  were calculated using POPGENE version 1.32  was closer than the distance between other populations only for N. peltata. Genetic similarities between populations connected by the Yangtze River were not consistently higher than unconnected populations; however, Honghu Lake and Liangzihu Lake populations had the closest geographic distance among the six lakes and clustered closely in N. peltata and T. latifolia.According to UPGMA dendrograms of lake populations based on Nei's unbiased genetic similarity coefficients, populations in Taihu Lake and/or Weishanhu Lake were distant from the other populations for all species except s Figure , Table 2P. lucens. In M. spicatum and N. peltata, except for Taihu Lake individuals, all individuals tended to divide into two groups: one included individuals from the plateau Caohai and Erhai Lakes; and the other included individuals from Honghu and Liangzihu Lakes.PCA analyses based on data from individual samples Figure  showed tP. lucens. In STRUCTURE analysis, the optimal division with each of the six species was found at K = 2, as indicated by the data Ln P (D) and the rate of change values (ΔK). Thus, the genetic clusters were assigned to two groups. For all species except P. lucens, one group included populations of Taihu Lake and/or Weishanhu Lake, the other group including other lakes.Bayesian cluster analyses Figure  agreed w3.2HT) and mean within‐population genetic diversity (HS) varied among the six species (Table HT (0.262) and HS (0.211) were observed in H. dubia; the lowest values of HT (0.189) and HS (0.154) were found in N. peltata. For C. demersum, the value of HT (0.262) was similar to that of H. dubia, but the value of HS (0.169) was moderate among the six species.Total genetic diversity  and N. peltata  had the lowest differentiation and the highest gene flow. Ceratophyllum demersum had the greatest genetic differentiation (GST = 0.354) and the lowest gene flow (Nm = 0.913). The other three species  had moderate differentiation and gene flow.In terms of values of Nm Table  among thHydrocharis dubia and N. peltata partitioned relatively less genetic variation among populations than the other four species. In within‐population variability, variance among subpopulations was less than that between individuals within subpopulation for each species.AMOVA analyses showed that in the six species 19.29%–36.73% of genetic variation was found among lake populations Table . Hydroch44.1N. peltata and M. spicatum only, individuals could be generally divided into plateau (Caohai and Erhai Lakes) and plain (Honghu and Liangzihu Lakes) groups  via PCA analysis. Previous studies on these species in China have yielded similar findings  of the six species found in the present work were within the range of the reported values of plant genetic diversity and similar to those of the widespread aquatic species (Chen, Li, Yin, & Li, N. peltata measured in this study was considerably lower than that found by Liao et al. (The values of within‐population genetic diversity (o et al. , which mC. demersum, while the opposite was true for H. dubia and N. peltata. Although gene flow can be realized through pollen and propagule flow, the primary contributor of gene flow between geographically distant populations should be propagules rather than pollens (Chen et al., P. malaianus (Chen et al., C. demersum populations may be explained primarily by low seed production (Les, H. dubia and N. peltata may be related to their large and edible fruits (Flora of China High genetic differentiation commonly indicates low gene flow among populations, as was the case in ion Les,  and the Overall, for sympatric and widespread aquatic species with different biological traits, there may be similar aspects to population genetic structure over a large geographic scale. Differences in environmental factors between plateau and plain lakes and long distance hydrochory have limited importance on aquatic plant genetic structures. However, gene flow mediated by birds may play the most important role in the formation of genetic patterns. The close genetic relationship between Taihu Lake and Weishanhu Lake populations, each in different river systems and with different climates, may be related to the migration routes of birds. Differences in gene flow among the six aquatic plants may be attributable to different bird‐transport and the fruit traits of each species.None declared. Click here for additional data file.


Spodoptera frugiperda, the fall armyworm (FAW), and the role of host-plant association as a source of genetic diversification. By strain-genotyping all field-collected individuals we found that populations collected from corn were composed primarily of corn-strain individuals, while the population collected from rice was composed almost entirely of rice-strain individuals. Outlier analyses indicated 1,184 loci putatively under selection  related to 194 different Gene Ontologies (GOs); the most numerous GOs were nucleotide binding, ATP binding, metal-ion binding and nucleic-acid binding. The association analyses indicated 326 loci associated with the host plant, and 216 loci associated with the individual strain, including functions related to Bacillus thuringiensis and insecticide resistance. The genetic-structure analyses indicated a moderate level of differentiation among all populations, and lower genetic structure among populations collected exclusively from corn, which suggests that the population collected from rice has a strong influence on the overall genetic structure. Populations of S. frugiperda are structured partially due to the host plant, and pairs of populations using the same host plant are more genetically similar than pairs using different hosts. Loci putatively under selection are the main factors responsible for the genetic structure of these populations, which indicates that adaptive selection on important traits, including the response to control tactics, is acting in the genetic differentiation of FAW populations in Brazil.We applied the ddRAD genotyping-by-sequencing technique to investigate the genetic distinctiveness of Brazilian populations of the noctuid moth  Rhagoletis pomonella Walsh  [Studies of ecological speciation , 2 comprritidae) –11, ecolritidae) –15.If divergence among populations is due to genetic drift and host-independent selection, gene flow among populations would be estimated by genetic distance in neutral loci . On the Spodoptera frugiperda (J. E. Smith), the fall armyworm (FAW), throughout its distribution in Brazil, and to investigate the role of host-plant association as source of genetic structure of field populations of FAW. Genotyping-by-sequencing has been widely applied in population genetics studies of insects in recent years [The emerging area of population genomics aims to identify these genes, and the use of Next Generation Sequencing (NGS) techniques has made possible studies of population genomics with data comprising all the genome information of organisms , 21–23. nt years –34. The Spodoptera frugiperda is the most important pest of corn (Zea mays L.) in South America [Oryza sativa L.), and cotton (Gossypium hirsutum L.). Pest control of FAW is made by using both Bacillus thuringiensis genetically modified corn (Bt-corn) and insecticides [ America , and is cticides .Bt toxins [The FAW is differentiated into host plant-related strains with their own ecological, genetic and physiological features . One strt toxins  and to ct toxins , 47. Rect toxins . All thet toxins . In soutt toxins , and a lt toxins . Genetict toxins . Signifit toxins .S. frugiperda [Taken together, these studies point to a speciation process in populations of ugiperda , 54. By S. frugiperda were collected on non-Bt-corn (refuge areas) and rice in 11 localities throughout Brazil, separated by distances between 151 and 1,957 Km  to confirm sample quality. The DNA concentration and 280/260 and 260/230 nm ratios were estimated by UV absorption in a NanoDrop spectrophotometer . Next, the final concentration was estimated in a Qubit® 2.0 , and the amount of DNA per sample was normalized to 20 ng/μL.Total genomic DNA was extracted from thoracic tissue using the CTAB protocol . ThoraciMspI site in the mitochondrial gene cytochrome c oxidase subunit I (COI). The ca. 569-bp fragment of COI was amplified using the primers JM76 and JM77 [2, 40 μM dNTPs, 0.2 μM of both forward and reverse primers, 1U GoTaq DNA Polymerase , 10% volume of 10X Taq buffer and 10% volume of 5% dimethyl sulfoxide (DMSO). The PCR program included an initial incubation at 94 °C (2 min), followed by 38 cycles of 94 °C (45 s), 56 °C (45 s), 72 °C (1 min), and a final incubation of 72 °C for 5 min. After amplification, 1.0 μL of FastDigest MspI (Thermo Scientific) was added to 10 μL of each reaction, incubated at 37 °C for 10 min, and the complete volume was loaded in a 2% agarose gel in TAE buffer .The strain of each field-collected individual was identified using the strain-specific and JM77 . ReactioPlateforme d’Analyses Génomiques of the Institut de Biologie Intégrative et des Systèmes . Total genomic DNA (200 ng) was simultaneously digested with both high-fidelity PstI  and MspI (New England Biolabs). One of 96 barcoded adapters was ligated on the PstI cut site for each individual sample, and a common adapter (adapter 2) was ligated onto the MspI cut site of all samples with T4 ligase (New England Biolabs). All 96 samples (same volume) were pooled and size-selected using a 2% agarose gel cassette on a BluePippin instrument  with the elution time set from 50 to 65 min. Eluted fragments were used for multiplexed PCRs, using standard forward primer A and reverse primer C [Genotyping-by-sequencing libraries were constructed using standard protocols , with miprimer C . Final lhttps://github.com/bioinfo-guy/Stacks_GBS_pipeline.Samples demultiplexing and SNP calling were performed using a pipeline with specific parameters on the software Stacks . A custopopulation  and all possible output files were generated for downstream analyses. When necessary, the data-conversion tool PGDSpider [First, raw sequence reads were demultiplexed and cleaned (process_radtags). Next, the data from each sample were grouped into loci, and polymorphic nucleotide sites were identified, using the package ustacks with no-reference genome . Cstacks was used to group loci across samples and to create a catalogue . Sstacks was then applied to match each sample against the catalogue in order to define the allelic state at each locus . Finally, the allelic states were subjected to population-genetics statistics using the package GDSpider  was usedST values for all loci in relation to He (expected heterozygosity). We ran the program three times: the first run included all loci under an attempted neutral mean FST, with 50000 simulations, 99% confidence interval, infinite alleles mutation model, and false discovery rate of 0.1%. After the first run, all loci outside the confidence interval were removed and the mean neutral FST was recalculated to reduce the bias in the estimation of the mean neutral FST by eliminating extreme loci from the computation [ST, with all other parameters maintained. This procedure was repeated three times, and loci recovered as outliers in all three replications were inferred to be under selection.Lositan  was usedputation . Only thhost plant where the individual was collected, and its strain. We applied a standard case/control association analysis available in the package Plink v. 1.9 [population. As we consider that the two features are not independent, since the preference for a host plant is mostly due to the individual strain, we constructed a Venn diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/) using a list of loci independently associated with the two features, to permit us to visualize both the shared loci and the exclusive loci associated with each feature. We used the same approach to sort loci that are simultaneously associated with the two features and are putatively under selection.To investigate the mechanisms involved in the relationships among the populations of the FAW, we tested the association between loci and two individual features, k v. 1.9 , using BAll loci putatively under selection and feature-associated loci were mapped on the transcriptome proposed for the FAW  , using b−5 and HSP similarity threshold of 80%. Patterns of RNA families were indicated by hmmscan [https://transdecoder.github.io/).Previously assembled contigs  had thei hmmscan  using RF hmmscan . Pattern hmmscan  were pro–10 and 90%, respectively) sorted by the latter; RNA and protein families from hmmscan were filtered by the Expectation Value (1 e–10) on the full sequence column from the resulting analyses. The gene ontology-controlled vocabulary terms were assigned to all sequences by a custom perl script using all search results, following the thresholds described above and removing all obsolete terms.The sequence description was achieved by integrating all database searches. Blast best hit results, from all databases previously described, were designated by a restrictive e-value and HSP similarity cutoff  and insecticides [Bt and insecticide resistance [Because cticides , we manusistance –74.https://github.com/vcflib/vcflib). The .xml files with neutral loci and loci putatively under-selection were created on the BEAUti interface (comprised in the package Beast v. 2.4.5) using the GTR model of nucleotide substitution, clock rate = 1.0 and Yule process of speciation. One MCMC analysis included 50 mi generations (with a pre-burn-in of 20%), storing parameters every 1000 steps. Tracer v. 1.5 (Drummond & Rambaut 2007) was used to examine the ESS of the different parameters and to define the ‘burn in’. TreeAnnotator v. 2.4.5 was used to conduct a 20% ‘burn in’ and to generate a maximum clade credibility topology of all the sampled trees rescaled to match posterior median estimates. Finally, the software FigTree v. 1.3.1 was used to visualize the topology of the Bayesian trees.We expected that loci under-selection could be more differentiated among populations of FAW than neutral loci, if the ecological speciation model was at least partially responsible for their discrimination in the field. To test the ability of neutral loci and loci under-selection to resolve the relationships among all collected individuals of FAW, we used a Bayesian approach available in Beast v. 2.4.5 . We firsMspI site in COI. Non-hierarchical analyses were first computed using all loci and then estimated using neutral loci or loci under-selection separately. Hierarchical AMOVA was conducted for two grouping configurations using combined neutral loci and loci under-selection: (1) among all populations collected from the two host plants, corn and rice; and (2) among populations from Rio Grande do Sul (RS) collected from the two host plants. Genetic structure was interpreted from the Φ statistics associated with the different hierarchical levels in which variation is distributed [ST values was evaluated using 16000 permutations, computed distance matrix using pairwise difference, and gamma a value = 0. Pairwise genetic distances were estimated using Slatkin’s method [Genetic structure of FAW populations was estimated by non-hierarchical locus by locus Analysis of Molecular Variance (AMOVA), using the software Arlequin v. 3.5 , and partributed . The sigs method  in Arleqpopulation, as before. First, a genetic-distance matrix of all collected individuals, including strain information and all loci, was computed with Plink v. 1.9 [k) was set to 10, and a k = 40 was also used to test for large-scale genetic structure.The population structure was also estimated using a network-based approach in the R package Netview P v. 1.0 . Input fk v. 1.9 . NetviewA Discriminant Analysis of Principal Components  was alsMspI site in COI, and ranged from 69–97% of the individuals within localities. All but one individual collected from rice (R-RS) were characterized as rice strain (RS) (The corn strain (CS) was more common in all populations collected from corn, according to the strain-specific ain (RS) .The number of reads for each run per lane of HiSeq2000 ranged from 142 to 196 million, with 96 samples per library. Each library was run in two lanes. From a total of 1.418.091.728 sequence reads, 87.1% was retained for Stacks analyses, generating a catalog containing 3.073.340 loci. Ustacks presented 3.8464,70 as coverage depth mean among all populations included on this study, with standard deviation of 193.082,3.The population genetic statistics calculated in Stacks recovered 7,664 SNPs. The population from São Paulo (SP-17) was removed from downstream analyses due to a lack of data, which could increase the bias in posterior analyses, and the final matrix had 269 individuals.Bacillus thuringiensis (Bt) resistance included zinc carboxypeptidases and ABC transporters (loci 12150 and 19063). The two contigs where these ABC loci were mapped . The functional annotation gathered 194 different Gene Ontologies (GOs) using the FAW transcriptome as reference. The most represented GO was nucleotide binding, functionally described as elongation factor Tu GTP binding domain, multidrug resistance-associated protein 1, ABC transporter transmembrane region, and insulin receptor, among other descriptions . Other cctively, ) showed a exigua , 84, andarmigera , and ABC tremula , includi tremula  included tremula ; diamide tremula . Three uugiperda : ecdysonS. frugiperda as reference  , and a predicted Helicoverpa armigera cadherin-related tumor suppressor (LOC110371604) . The same contig showed the highest identity with the scaffold 9577 (96%) when blasted against the corn variant assembly 3.1 of the genome of S. frugiperda (https://bipaa.genouest.org/is/lepidodb/spodoptera_frugiperda/), and with the SFRU RICE 028070 when blasted against the rice variant assembly 1.0 (96% identity). The alignment of the contig 63619 with several cadherin sequences available did not permit to determine if it is in any important region related to Bt resistance  loci were exclusively associated with host plant, 33 with strain, and 183 were simultaneously associated with both features . One huneference . One locsistance . Locus 2Many loci significantly associated with the host plant have one allele fixed in individuals collected from rice , while tThe tree topology recovered with loci under-selection was better resolved than the topology recovered with neutral loci . It is pST = 0.056. When only neutral loci were considered, FST value was reduced by half (0.028). Conversely, when we considered only loci under-selection, the value of FST increased 10× in relation to neutral loci (0.287) (ST = 0.096), but the structure decreased when we considered only neutral loci, and was strongly increased when only loci under-selection were considered. Genetic structure was the lowest when we consider individual strains of FAW  . For pops of FAW .ST were similar for the two sets of populations  and all other populations, including the other two populations collected in Rio Grande do Sul from corn . Pairwise structure was low among all other populations  was connected with the individuals from R-RS. Sixteen individuals from PR-51  formed a second isolated network  . This locus was mapped to the contig 54090 in the transcriptome of S. frugiperda, a contig with 351 amino acids. When contig 54090 was blasted against the NCBI data bank, two sequences were indicated with the highest similarity, a predicted Spodoptera litura cadherin-related tumor suppressor (LOC111348058) , and a predicted Helicoverpa armigera cadherin-related tumor suppressor (LOC110371604) . This contig showed the highest similarity (98%) with the scaffold 46658 when blasted against the corn variant assembly 3.1 of the genome of S. frugiperda, and with the SFRU RICE 028070 when blasted against the rice variant assembly 1.0 (99%). The alignment of the contig 54090 with several cadherin available showed a high similarity to the cadherin repeats 10–12 involved in the interaction with Cry proteins in other lepidopterans  hydroxamic acid, DIMBOA , which confers resistance to herbivorous insects and pathogens because of its antifeeding, insecticidal, antimicrobial and allelopathic properties –101. Remtrations , 103. Thtrations , that isOther plant secondary compounds may also be responsible for the FAW differential use of host plants, such as phytoecdysteroids. Phytoecdysteroids are steroidal compounds synthesized by plants that interfere with insect ecdysis and development , 106. ThOn the other hand, female oviposition preference was suggested for FAW based on host plants choice trials at laboratory conditions –113. A mS. frugiperda larvae, such as variation in the competitive abilities of the two strains, behavioral isolation through sexual communication mediated by different pheromone blends, and differential timing of reproduction. In conclusion, each barrier could theoretically contribute individually and simultaneously to the host-plant preference in FAW larvae [Several possible pre- and poszygotic isolation barriers have been postulated to explain the asymmetric use of host plants by W larvae .MspI site in the mtDNA COI region to characterize strain genotypes of FAW [Tpi) are capable to distinguish between the two FAW strains, and to indicate corn-rice hybrids [Tpi here, and we did not find it in the loci obtained by genotyping-by-sequencing technique, so we do not have the hybrid information for the populations of FAW we studied.The main question, however, is whether individuals of the two strains are reproductively isolated in the field, and the answer we are able to provide at this moment is "partially". Most of the rice-strain individuals collected from rice were genetically isolated from corn-strain individuals in general, even if they were geographically close, but the rice-strain individuals collected from corn belonged to the same genetic cluster as their corn-strain co-host fellows. The marker that we used to identify FAW strains may be responsible for this pattern. Here we applied the most often used strain-specific s of FAW , and we s of FAW , 54, 115The fact that several loci responsible for the genetic cluster configuration in the DAPC analysis are fixed in the rice cluster (cluster 3) consists an additional evidence that these rice-strain individuals are genetically isolated from the other Brazilian populations of FAW. As these loci include molecular functions such as the carbohydrate metabolic process and oxidoreductase activity, both functions related to digestion in insects, they also suggest the existence of adaptive differences in the rice-strain individuals that allow them to feed on rice.S. frugiperda collected from rice is more isolated from all other populations collected from corn, even from geographically close populations; and 2) all populations collected from corn show low genetic structure compared to each other. Some individuals collected from rice, however, are connected to individuals from corn, which indicates current gene flow among individuals of FAW feeding on these different host plants. Our results coincide with studies that found the host-plant association as the main reason for the genetic differentiation within and among populations of S. frugiperda [Pairs of populations of polyphagous herbivorous insects using different host plants are expected to be more reproductively isolated than pairs using the same host plant if they are under selective pressure due to ecological-speciation mechanisms. Here we were able to show two important results related to this expectation: 1) the only population of ugiperda , 51, butugiperda , 116.Bt-resistance in Lepidoptera [Helicoverpa armigera [Ostrinia nubilalis fed on transgenic Cry1Ab or non-transgenic corn for 6 hours [Bt-corn seeds with Cry toxins were introduced to reduce FAW infestations in Brazil in 2008 [Bt-crops. It is interesting that larvae found in this study feeding on non-Bt-corn and on non-transgenic rice show different allele frequencies at this locus, which indicates that these individuals are under divergent selective pressures on this trait.If genetic structure is at least partially due to host-plant association, corn and rice populations could be under different selective pressures. Irrespective of the strain to which an individual belongs, if it is feeding on corn it is adapted to this host plant. So, how do these individuals differ from individuals that feed on rice in the field? Our association analyses indicated that loci significantly associated with the host plant where the individual was collected included unigenes related to digestion in insects, and to resistance to insecticides and genetically modified plants. Zinc carboxypeptidases, for instance, are related to idoptera –73, and armigera . Zinc ca 6 hours . Bt-corn in 2008 , and in Bt-crops [Cry1A toxin-binding in lepidopteran insects [Bt Cry toxins, due to changes in receptor binding, as reported for H. armigera [Bt-corn and non-transgenic rice on this trait. The locus 14975, also annotated as a cadherin domain, is a locus that contributed to the arrangement of individuals in three clusters. This locus showed a high similarity with sequences of cadherin well characterized in other pest species of Lepidoptera, such as Helicoverpa armigera [Manduca sexta [S. frugiperda is also shaped by the response of its populations to the methods of control used at field conditions.Cadherin, also associated with resistance to Bt-crops –73, is r insects  and is tarmigera . The locarmigera  and Mandca sexta , includiTwo other loci were significantly associated with both host plant and strain in the FAW populations: a locus annotated as glutathione transferase showed a large difference in allele frequency in corn and rice plants and strains. Glutathione transferases are closely associated with insecticide resistance in insects, including pyrethroids , 123, inAnother locus associated with both features was annotated as insulin receptor, which was shown to play an essential role in feeding behavior in insects, as a key metabolic hormone related to carbohydrate and lipid metabolism . In the Bt-crops and insecticides. In addition to the unigene annotated as zinc carboxypeptidase, we found two loci annotated as ABC transporter, another mechanism related to Bt-resistance [Bt resistance in other species of Lepidoptera. Furthermore, we found several unigenes annotated as cytochrome P450 and carboxylesterases that, together with glutathione transferase, are related to increased detoxification of insecticides in insects [All those annotated loci simultaneously associated with the host plant and strain are also putatively under selection in the populations of FAW we investigated, but we found other loci under selection that are also related to resistance to sistance –73, and  insects . Our ana insects . As we iA recurrent question related to FAW is its taxonomic status: are the two host-related strains sibling species or races? Genome-wide analyses have indeed found significant genomic differentiation between the two strains . SeveralS. frugiperda are structured according to the host plant where they were collected, although we were able to sample only one population from rice, and pairs of populations using the same host plant are more genetically similar than pairs using different host plants. Populations collected from corn are genetically more similar to other populations collected from the same host plant, which indicates current or historic gene flow among those populations. The only population collected from rice is more isolated from all other populations from corn, even from geographically close populations. Other factors, however, also contribute to the genetic structure of Brazilian populations of FAW (vide population PR-51 isolated from other corn-collected populations). Loci putatively under selection are the main factors responsible for the genetic structure of these populations, which indicates that adaptive selection on important traits, including response to control tactics, is acting in the genetic differentiation of FAW populations in Brazil.Considering our initial questions, there is evidence that Brazilian populations of S1 TableGene Ontology annotation (GO) and description of loci putatively under selection.(PDF)Click here for additional data file.S2 Table1 and A2 = two alleles forms presented in each loci; F_A1 = frequency of allele A1 in rice or corn host plant or strain; Bonf = Bonferroni adjusted significance values of each allele frequency comparison in each feature.Gene Ontology (GO) annotation and molecular function description of loci associated to the host plant where the individuals were collected, to the individual strain or to the two features. All annotated loci simultaneously associated to the two features are also putatively under selection. A(PDF)Click here for additional data file.S3 TableGene Ontology (GO) annotation and description of loci that contributed to the arrangement of individuals in three clusters found in the DAPC analysis. † Alleles fixed in cluster 3, composed of individuals from R-RS. ‡ Aspect: M = Molecular function, B = Biological process, C = Cellular component.(PDF)Click here for additional data file.S1 FigSpodoptera litura ABCC1-like (KM453742) was the best match when BLAST was used ; sequence Spodoptera exigua ABCC2 (KM068116) is discussed in [Spodoptera exigua ABCC3 (KF926101) discussed in [Helicoverpa armigera ABCA2 (KP259911) is annotated as in [Sequence ussed in ; sequencussed in ; sequencussed in ; sequenced as in . TpM = t(PDF)Click here for additional data file.S2 FigSpodoptera litura subfamily F member is a predicted ATP-binding protein (LOC111362986), and was the best match when BLAST was used ; sequence Spodoptera exigua ABCC2 (KM068116) is discussed in [Spodoptera exigua ABCC3 (KF926101) discussed in [Helicoverpa armigera ABCA2 (KP259911) is annotated as in [Sequence ussed in ; sequencussed in ; sequenc(PDF)Click here for additional data file.S3 FigHelicoverpa armigera (LOC 110371604) is a predicted cadherin-related tumor suppressor; sequence Helicoverpa armigera (BtR) (AY647974) is annotated as in [Manduca sexta (BT-R1) (AF319973) is annotated as in [Sequence ed as in , and added as in , 128; seed as in . EC = ca(PDF)Click here for additional data file.S4 FigLoci contributions to clustering pattern above the threshold of 0.0008.(PDF)Click here for additional data file.S5 FigHelicoverpa armigera (LOC 110371604) is a predicted cadherin-related tumor suppressor; sequence Helicoverpa armigera (BtR) (AY647974) is annotated as in [Manduca sexta (BT-R1) (AF319973) is annotated as in [Sequence ed as in , and added as in , 128; seed as in . EC = ca(PDF)Click here for additional data file.


Minuca rapax among Atlantic  populations , whereas Caribbean populations show significantly restricted gene flow among the constituent islands and towards continental populations. Our data support the hypothesis of the Mona Passage hindering larval exchange. Contrastingly, Caribbean Leptuca leptodactyla populations appear to be devoid of detectable variation, while Atlantic-continental (i.e. Brazilian) populations show much higher haplotype and nucleotide diversities and display slight genetic differentiation among populations within the Atlantic region, though not statistically significant. Both species show a pronounced divergence between regions, supporting the presence of a phylogeographic barrier.Fiddler crabs , like many other marine organisms, disperse via planktonic larvae. A lengthy pelagic larval duration is generally assumed to result in genetic connectivity even among distant populations. However, major river outflows, such as of the Amazon or Orinoco, or strong currents may act as phylogeographic barriers to ongoing gene flow. For example, the Mona Passage, located between Puerto Rico and Hispaniola, has been postulated to impair larval exchange of several species. In this study, Cox1 mtDNA data was used to analyze population genetic structure of two fiddler crab species from the western Atlantic, comparing the continental coastline and Caribbean islands. The results indicate genetic homogeneity in  Ongoing gene flow among widespread populations is essential for genetic homogeneity within a species, while its disruption leads to genetic differentiation and heterogeneity. Being rather limited in spatial expansion as adults, a dispersive planktonic larval stage is part of the reproductive strategy of many coastal marine organisms –3. The lIn marine ecosystems, barriers to dispersal are much less obvious compared to terrestrial ones. Oceanographic features such as major currents, ocean fronts, or eddies can severely impair larval migration , 16, 17.Uca, now represented in several genera,  al al22] albean Sea , but hasbean Sea . MoleculM. rapax and L. leptodactyla. Both species show significant restrictions in genetic exchange between Caribbean and Atlantic populations which may indicate that the Orinoco, possibly enhanced by the Amazon, may function as a biogeographic barrier to dispersal. The Amazon alone, however, seems not to impede larval exchange, as both species exhibit ongoing gene flow. Within the Caribbean, contrasting patterns become obvious. While there is no evidence for genetic structuring in L. leptodactyla in this region, the opposite is true for M. rapax. Not only did we find significantly restricted gene flow among populations in this region, but a severe lack of genetic interchange between Hispaniola and Puerto Rico seems to be the case. This supports the suggestion that the Mona Passage may indeed function as barrier for this fiddler crab.Our data suggest that gene flow is not entirely unimpaired among populations of


Notamacropus eugenii) is one of the most intensively studied of all macropodids and was the first Australasian marsupial to have its genome sequenced. However, comparatively little is known about genetic diversity and differentiation amongst the morphologically distinct allopatric populations of tammar wallabies found in Western (WA) and South Australia (SA). Here we compare autosomal and Y-linked microsatellite genotypes, as well as sequence data (~600 bp) from the mitochondrial DNA (mtDNA) control region (CR) in tammar wallabies from across its distribution. Levels of diversity at autosomal microsatellite loci were typically high in the WA mainland and Kangaroo Island (SA) populations  but significantly reduced in other endemic island populations . Autosomal and Y-linked microsatellite loci revealed a pattern of significant differentiation amongst populations, especially between SA and WA. The Kangaroo Island and introduced New Zealand population showed limited differentiation. Multiple divergent mtDNA CR haplotypes were identified within both SA and WA populations. The CR haplotypes of tammar wallabies from SA and WA show reciprocal monophyly and are highly divergent (14.5%), with levels of sequence divergence more typical of different species. Within WA tammar wallabies, island populations each have unique clusters of highly related CR haplotypes and each is most closely related to different WA mainland haplotypes. Y-linked microsatellite haplotypes show a similar pattern of divergence although levels of diversity are lower. In light of these differences, we suggest that two subspecies of tammar wallaby be recognized; Notamacropus eugenii eugenii in SA and N. eugenii derbianus in WA. The extensive neutral genetic diversity and inter-population differentiation identified within tammar wallabies should further increase the species value and usefulness as a model organism.The tammar wallaby ( Notamacropus eugenii) (see . At. At14]. N. eugenii [During the last 200 years, observed morphological differences amongst tammar wallaby populations has led to the description of 10 distinct species or subspecies , althoug eugenii ,23. Howe eugenii ,24, are Previous genetic comparisons of the Kangaroo Island (SA) and Garden Island (WA) populations, using several techniques, revealed substantial differentiation  with Kangaroo Island . This stCR). This will, for the first time, enable a comprehensive picture of the distribution of diversity within and amongst extant tammar wallaby populations to be compiled.The publication of the tammar wallaby genome  has reinIn 2009 animals from Middle (n = 12) and North Twin Peak (n = 2) Islands were captured in cage traps as previously described . A 2–5 mMacropus giganteus)  [N. eugenii are given in Individuals were genotyped at 16 polymorphic autosomal microsatellite loci derived from the tammar wallaby  and eastern grey kangaroo  –43, as p, G31.1) . The genCR was determined using marsupial-specific primers [equencher v4.8  and aligned using Clustal X [DNA sequence variation in the hypervariable Domain I of the mtDNA  primers  and sing primers ) as prev primers . All unilustal X .enePop v7 [Ho), expected heterozygosity (HE), allelic diversity (A) and effective allelic diversity (Ae: corrected for sample size n = 15) were estimated using Fstat v2.9.3 [rA)  and unique (private) alleles (uA) per locus was also calculated. Differences in diversity indices amongst sampled populations were assessed via a Wilcoxon rank sign test using SYSTAT9. The effective inbreeding coefficient  was calculated from the equation: Fe = 1 –HIS / HM where HIS represents heterozygosity for island populations and HM represents heterozygosity for mainland populations [For the autosomal loci, conformance to Hardy-Weinberg equilibrium was conducted using GnePop v7  via a ManePop v7 . Observet v2.9.3 . The meaulations .A) and haplotypic diversity (h) were calculated using GenAlEx v6.5 [AR) was calculated in FSTAT v2.9.3.For Y-linked microsatellites the average number of alleles per locus  present using values of K between 1 and 12 with 10 replicates of each. The inferred number of populations within the sample was deduced using both maximum posterior probability (L(K) [tructure Harvester 0.6.93 [istruct v1.1 [enAlEx v6.5.For the autosomal microsatellite data, several methods were used to infer population structure. Firstly, differentiation amongst populations was assessed by calculating pairwise FST , using Fe v2.3.4 . Structuty (L(K) ), and maty (L(K) ) implemer 0.6.93 . The resuct v1.1 . ThirdlyϕPT values amongst populations were calculated using GenAlEx v6.5, with significance estimated using 999 permutations. A Y-haplotype network was also constructed in NETWORK v4.6.1.3 [w = 2 and imperfect repeats  weighted w = 5. For the mtDNA CR data pairwise differentiation (ФST) amongst populations was estimated and tested for significance in Arlequin v3.5 [For the Y-linked microsatellite loci pairwise v4.6.1.3 , using tv4.6.1.3 . Loci wiv4.6.1.3 , with peuin v3.5  using 50N. eugenii CR haplotypes were analysed using three methods (maximum parsimony (MP), maximum likelihood (ML), neighbour-joining (NJ)) in Paup* v4b10 [odeltest v3.7 [CR sequences from an eastern grey kangaroo (Macropus giganteus) and western grey kangaroo (M. fuliginosus) , were used as outgroup taxa. For complete sequences of all new CR haplotypes see GenBank accession numbers KY623685-KY623712.Phylogenetic relationships amongst identified p* v4b10 . MP analest v3.7 ), with rn = 40) and Kawau Island, New Zealand (n = 30). From WA two mainland  and six island  populations were sampled.Samples were obtained from 287 tammar wallabies from ten populations throughout the current range, including from Kangaroo Island, SA (p<0.05) deficiencies of heterozygotes were identified.All of the 16 autosomal microsatellite loci were polymorphic in most populations . ExceptiA total of 270 autosomal microsatellite alleles were identified; 174 in SA (33% unique) and 209 in WA (46% unique) . Of the rA was significantly (p<0.05) higher in the Kangaroo Island population  lower in all island populations compared to the Tutanning and Kangaroo Island populations . The introduced North Island population had significantly (p<0.05) lower values of A and Ae than all other populations (p<0.05) lower diversity values for all parameters than the Kangaroo Island population. The Perup population had significantly lower A, He and rA than Tutanning, although this most likely reflects the large difference in sample size , as well as between Kangaroo and Kawau Islands (H2)  .CR haplotypes from SA and WA ; although more modest divergence amongst haplotypes within each region: range of 0.17–3.5% within SA and 0.17–6.4% within WA. Mean divergence within the Kangaroo Island population (1.4 ± 1.4%) was greater than that found between the Kangaroo and Kawau Island populations (0.89 ± 1.2%). Within WA, mean sequence divergence amongst haplotypes within endemic island populations was low (range 0.4–1.0%), but higher mean divergence was evident between island and WA mainland haplotypes (range 3.3–4.9%). WA mainland haplotypes differed by 0.2–5.5%, with a mean of 3.8 ± 1.0% separating the two sampled populations.There was substantial sequence divergence between FST and ΦST) was detected amongst all adequately sampled populations , North and West Wallabi Island populations , as well as between Kangaroo and Kawau Islands   and each other  .Significant genetic differentiation  . The WA  = 0.86) . The SA tructure indicated that either seven (maximum L(K)) or eight (maximum ΔK) populations were present in the data  . The PCAΦPT = 0.15), and between Kangaroo and Kawau Islands (ΦPT = 0.04) (ΦPT = 0.86) and the WA mainland (mean ΦPT = 0.82). The SA and WA populations were also well differentiated (mean ΦPT = 0.72) . Distinct eastern (SA) and western (WA) clusters were also apparent in the Y haplotype network  . The WA  = 0.72) . An AMOV network .CR revealed two well supported clusters of highly divergent haplotypes, showing complete reciprocal monophyly and corresponding to the sampled SA and WA populations  and western (WA) populations, with each region being characterised by a high proportion of unique and divergent alleles/haplotypes. This pattern of divergence is consistent with the long-term isolation of SA and WA N. eugenii populations and the lack of recent gene flow between them, allowing each to evolve independently. A similar pattern of genetic divergence is also present between eastern and western populations (or species pairs) in many taxa across southern Australia, including grey kangaroos [Both our mtDNA and microsatellite analyses have revealed significant differentiation amongst angaroos , dasyuriangaroos ,68, bandangaroos , rodentsangaroos , birds [angaroos –73 and rangaroos . This wiangaroos ,76. Althangaroos  suggesteangaroos .CR sequence (14.5%) detected here between eastern and western tammar wallabies is similar to that reported between eastern and western grey kangaroos (14%) [Petrogale spp. 10–17% [N. eugenii, largely reflecting distinct eastern and western groupings, although with alterative clustering for some southern island populations [The divergence in os (14%) , and amo. 10–17% ,80). In uence 14.% detecte. 10–17% . Morpholulations ,24.N. eugenii) continue to be recognised. There is however, the need for further research, since a reduced breeding efficiency when producing Garden Island by Kangaroo Island F1 hybrids in captivity has been reported [Since the allopatric populations of SA and WA tammar wallabies are genetically highly differentiated, with levels of divergence typical of different species, there would be some justification in recognising each as a separate species, for consistency with data from other macropodid species. However, despite their genetic divergence at neutral loci, SA and WA tammar wallabies are known to be fully inter-fertile (in captivity), with F1 and back-cross hybrids of both sexes showing normal fertility . This isreported . It is treported  may be rreported ,85.sensu [N. eugenii populations (SA) be known as N. eugenii eugenii, and western (WA) populations as N. eugenii derbianus  (sensu ). We wousensu ,90. Neveerbianus  as recenPetrogale lateralis pearsoni) also found in the Investigator Group, SA is most closely related to the black-footed rock-wallaby  from southwest WA [Notechis scutatus) [Isoodon obesulus) [The western affinity of some SA animal populations is not unprecedented; for example, the Pearson Island rock-wallaby . Howcutatus)  and soutbesulus)  from theCR sequence divergence between WA island and mainland populations ranged from 3.3–4.9%, but up to 5.5% divergence was found between haplotypes within the Tutanning population alone. Similarly, although the East and West Wallabi Islands populations appear highly divergent based on autosomal microsatellite data  [Although we recommend that a single subspecies be recognised in WA we note that substantial differentiation in microsatellite loci also occurs among many of the sampled WA populations, some of which have historically been proposed as separate taxa . Howeverite data , Fig 4 tite data  indicatiite data . These rite data , togethelucatus) .n = 2). However, since the populations do not share CR haplotypes and are morphologically distinct (unpublished data) the later hypothesis seems more likely.An exception is the North Twin Peak Island tammar wallaby population, which was found to share all of its autosomal microsatellite alleles with the nearby (31 km west) Middle Island population. Whether this similarity reflects recent gene flow or the preferential retention in both populations of the higher frequency alleles present in the common ancestral population, must await more comprehensive sampling of the North Twin Peak population (currently n = 6), unique CR and Y haplotypes were detected and 55% of autosomal microsatellite alleles were not shared with the much better sampled Tutanning (n = 50) population located 200 km to the northeast. These preliminary data suggest that mainland WA populations are also structured with limited gene flow by both sexes between sites. More comprehensive sampling of remaining tammar wallaby populations throughout southwest WA is required to confirm these findings. However, if these data are typical then considerable unique diversity may exist within each remaining mainland WA population. In this context, it would also be important to examine the pattern of male and female mediated gene flow and extent of population genetic structure throughout the abundant Kangaroo Island tammar population. Since Kangaroo Island is over 150 km long, tammar wallaby populations may show significant genetic structure across the island.On the WA mainland some genetic differentiation is also apparent between the two sampled southwest WA populations (Tutanning and Perup). Although only a limited sample was available from Perup  to haplotypes recorded in Kangaroo Island and so may represent recent mutations in the Kawau Island population or be as yet unsampled in the Kangaroo Island population. In light of this uncertainty, definitive conclusions as to the origins of the Kawau Island population and the distinction of the SA mainland and Kangaroo Island populations remain elusive. To further resolve this matter would require not only genetic data from definitive historic SA mainland tammar wallabies , but also a better understanding of the distribution of genetic diversity throughout Kangaroo Island, since the population we sampled is from the western end of the island and the degree of population structure across the island remains unknown. The similarity between the Kangaroo Island and Kawau Island tammar populations detected in this study should not impact the ongoing re-introduction of Kawau-derived tammar wallabies to the Yorke Peninsula on the mainland SA, since returning SA tammar wallabies to the mainland is a worthy endeavour for biodiversity conservation and restoring ecosystem function. However the recent use of eugenii eugenii to refer to the extinct SA mainland tammar wallaby population as distinct from eugenii decres on Kangaroo Island  and no scientific name has yet been specifically associated with the SA mainland population  that bepulation .A, He) are amongst the highest yet reported in marsupials [Ne since isolation from the mainland population and so reduce the impact of genetic drift [Although island populations typically have reduced diversity compared to mainland populations ,94, a rersupials . A remarrsupials ), comparrsupials ) and manrsupials . These hic drift . Some maic drift ,99, althic drift ,100. Theic drift . Similaric drift ,95,100. Notamacropus rufogriseus), employs different strategies at different latitudes [To date, most studies of tammar wallaby physiology, reproduction, genetics and development have utilised the Kangaroo Island population, and they are now amongst the best known of marsupials ,11. We hatitudes . The extatitudes  and advaS1 Table(XLSX)Click here for additional data file.S2 TableKI = Kangaroo Island; KwI = Kawau Island, New Zealand; Tut = Tutanning; Per = Perup; GI = Garden Island; EWI = East Wallabi Island; WWI = West Wallabi Island; NI = North Island; MI = Middle Island; NTP = North Twin Peak Island.(DOCX)Click here for additional data file.S3 TableKI = Kangaroo Island; KwI = Kawau Island, New Zealand; Tut = Tutanning; Per = Perup; GI = Garden Island; EWI = East Wallabi Island; WWI = West Wallabi Island; NI = North Island; MI = Middle Island.(DOCX)Click here for additional data file.S1 Fig(DOCX)Click here for additional data file.