DcAOX1 was found in populations of wild carrot sampled across a West-European environmental gradient. Even though no direct relation was found with the analyzed climatic conditions or with physical distance, population differentiation exists and results mainly from the polymorphisms associated with DcAOX1 exon 1 and intron 1. The relatively high number of amino acid changes and the identification of several unusually variable positions (through a likelihood ratio test), suggests that DcAOX1 gene might be under positive selection. However, if positive selection is considered, it only acts on some specific populations (i.e. is in the form of adaptive differences in different population locations) given the observed high genetic diversity. We were able to identify two populations with higher levels of differentiation which are promising as hot spots of specific functional diversity.By definition, the domestication process leads to an overall reduction of crop genetic diversity. This lead to the current search of genomic regions in wild crop relatives (CWR), an important task for modern carrot breeding. Nowadays massive sequencing possibilities can allow for discovery of novel genetic resources in wild populations, but this quest could be aided by the use of a surrogate gene (to first identify and prioritize novel wild populations for increased sequencing effort). Alternative oxidase (AOX) gene family seems to be linked to all kinds of abiotic and biotic stress reactions in various organisms and thus have the potential to be used in the identification of CWR hotspots of environment-adapted diversity. High variability of  Crop plants typically include only a portion of the genetic diversity of their wild relatives. Since genetic variation is the raw material of evolution, low genetic diversity has as direct consequence a reduction on the ability of the species to evolve in response to changes in its environment. If until recently we have perceived plant breeding as overall reducing crop genetic diversity, recent assessments have shown spatial and temporal patterns of genetic diversity losses e.g. ,2). Part. Part2])Daucus carota L.), the domestication process rendered more modest changes when compared to their progenitors—and they can even revert to the wild or become self-propagating weeds )allenged –30 the callenged  showed ty exon 1 .DcAOX1 in populations of carrot CWR in Europe, subjected to different climatic stress and thus putatively adapted to different environments. By scanning wild crop relatives with a stress related gene, we expect to highlight hot spots of specific functional diversity.In this study we look into the variability of Sampling was performed following an environmental gradient across Europe. This gradient accommodated sampling points with deviating climatic conditions, such as in Sierra de Guadarrama  or in central Pyrenees and the French Massif Central . In general, the sampling was made on easily accessible non-cultivated fields (thus close to a road), with altitudes ranging from the referred 1100 m to sea level. In total, 13 sites were sampled  by collehttp://globalweather.tamu.edu/). Month averages were obtained to have a workable characterization of the long-term climatic conditions at the sampling locations. A Principal Component Analysis (PCA) was used to define patterns of climatic conditions of the sampling locations. Data was grouped into clusters for data summarization, through an Agglomerative Hierarchical Clustering . These analyses were performed using XLSTAT-ecology, an add-on to MS Excel®.The available climatic data of the last 15 years was collected for the closest by station of the sampling points (DcAOX1 _24Fw or DcAOX1 _94Fw) as forward and at the end of exon 3 as reverse , transformed into bacterial strainJM109  and bacterial colonies were tested using T7 and SP6 primers. Sequence was done from the PCR product, in sense and antisense strands. Because carrot is an outcrossing species, a minimum of four plants per population and two clones per plant were sequenced . A nested PCR approach was selected. For the first reaction, the primers used were located at the beginning of exon 1 (tails in ). A stanequenced . For theSequence visualization was performed in CLC Main Workbench vs 6.8.1 software and, for AOX1 amplicon, exons and intron regions were identified on the sequences and aligned separately. The alignment of segments of the exon regions was relatively straightforward, with few insertions/deletions needing to be inferred. For the intron region an iterative refinement method, which accounts for larger gaps in the sequences (E-INS-i) as implemented in the program MAFFT , was useBayesian inference was conducted using MrBayes version 3.0 ,37. The 2, [DcAOX1, tests for positive selection were performed using the maximum likelihood methods implemented in the CODEML program of PAML [The AOX1 sequences alignment was used to locate polymorphic positions and ambiguities encountered were, whenever possible, resolved by resequencing. Summary statistics and tests of neutrality were calculated with DnaSPv.4.0  on the b2, ) on the 2, . In addi of PAML . The dN/D. carota populations using Arlequin3.5.22 [We determined pairwise FST values among wild in3.5.22 ; levels Daucus carota. Within D. carota the phylogeny is highly unresolved  which shows levels of similarity between themselves higher than 90%;population 3–3 of the 8 haplotypes have here an insertion, being one of them equivalent to I1a (average similarity higher than 90%) whereas the two other haplotypes present a different insertion (I1b) but similar between themselves (99%);population 14—only 1 of the 8 haplotypes present an insertion with 96% similarity with I1a.Rhizophagus intraradices clone JGIBTPH-93C11  or Daucus syrticus (KJ519808.1 and KJ519807.1).I1a blasts primarily with  samples  albeit n samples . This inFrom exon1 the dataset is not complete and from the expected fragment of 432 sites, only 205 sites have no alignment gaps or missing data . Data on the exons  is summarized in Population genetics inferences are primarily based on two sources of information: the site frequency spectrum of mutations (SFS) -being Tajima’s D one of the most popular summary statistics- and the statistical association among those, that is, linkage disequilibrium (LD). Considering an intermediate level of recombination (gene recombination parameter R determined to be 18.60) the observed Tajima´s D value (-1.32) was significant . The observed ZnS value (0.08) was not significant .Likelihood ratio tests (LRTs) revealed that PAML models that allowed for adaptive positive selection fitted the exon 1 sequence data better than those which did not; this was, however, not true for exon 2  althoughST computed between pairs of populations . It shows that variability at exon 1  barriers for interbreeding among either CWR or domesticated forms. The reconstructed phylogeny of carrot CWR (based on the conserved orthologue marker D28n) suggests a Eurosiberian –Mediterranean division, with the exception of the Miranda del Ebro population (population 3). This population, even though still in a Mediterranean climate , grows in an environment close to an oceanic climate zone (Cfb) much more similar, in overall weather conditions, to other more central European locations. The reconstructed phylogeny based on the analyzed DcAOX1 gene fragment does not seem to identify any clear clade that could directly be linked to the population origin. The observed structure in the reconstructed tree is mainly due to the intron region, as the one obtained when using the exons only is generally unresolved (resolved .DcAOX1 genetic pool within the Iberian Peninsula (DcAOX gene sequences. The outcrossing nature of carrot (contrasting with the selfing habits of lettuce) increases the probability of long-distance gene flow, and hence the absence of a distance effect. The level of genetic differentiation depends on gene flow and genetic drift, so the lack of genetic differentiation among Iberian populations likely results from high rates of historical gene flow between populations and/or large effective population sizes. The intron 1 of DcAOX1 is a lengthy variable region, including even a hyper-variable region of simple sequence repeats (SSRs) [There is a lack of structure among the eninsula , and thes (SSRs) , and shoDaucus sahariensis and Daucus syrticus. Arbizu and co-workers [Daucus syrticus, and D. sahariensis together with D. gracilis, are probably the most closely related plants to D. carota [D. carota and D. sahariensis or D. syrticus . Remains to be tested whether the high variation at the intron, has an effect on gene expression and on the functionality of the encoded alternative oxidase protein. Introns can have large influence in the control of gene expression in plants  showed an insertion of 308 bp at the end of intron 1 with high identity with clones of -workers  found th. carota . This ints e.g. ,48). Par. ParDaucts , no linkage disequilibrium was found and the significant negative value of Tajima’s D suggests either a recent selective sweep (or linkage to it) or a recent population expansion following a bottleneck .Nucleotide diversity at the DcAOX1 gene might be under positive selection. However, the high genetic diversity, including several indels, suggests that, if there is positive selection, it only acts on some specific populations (i.e. is in the form of adaptive differences in different population locations). The hypothesis that balanced selection is driving the high genetic diversity at DcAOX1 gene cannot be excluded.The likelihood ratio test identified several positions at exon 1 which are unusually variable (indication of positive selection), and a relatively high number of amino acid changes is observed, together indicating that DcAOX1 gene is not present in a single copy. The adaptive role of copy number variation (CNV) is suspected to be of high relevance, and for specific genes has been linked to important traits such as flowering time, plant height and resistance to biotic and abiotic stress . KnowleDcAOX1 gene seems to result from a fusion of two exons  and thi intron; . Nonethe intron; .a priori knowledge of the AOX1 gene involvement in homeostasis under stress conditions, calls for further characterization.It has been proposed that positive selection promotes the functional divergence of gene family members encoding enzymes involved in secondary metabolism as its products are thought to be a response to challenges imposed by the environment . If, with the present data, AOX does not seem to work as a surrogate for diversity directly linked to the climatic conditions analyzed, we were able to clearly identify two populations with higher levels of differentiation which are promising as hot spots of specific functional diversity. These two populations are thus good targets for a wider approach, either considering several candidate genes or a genome-wide approach, towards the identification of novel genetic resources relevant for modern carrot breeding. Enlarging this study to a wider geo-climatic region, including the center of domestication of carrot, has the potential of identifying further genetic diversity hotspots, particularly if combined with a more fine scale environmental analysis.S1 FigThe phylogeny corresponds to the majority rule consensus tree of trees sampled in a Bayesian analysis. Two insertions at the intron were removed. Arabidopsis thaliana was used as outgroup. The numbers above the branches refer to the Bayesian posterior probability of the nodes (more than 50%) derived from 19500 Markov chain Monte Carlo-sampled trees.(TIF)Click here for additional data file.S2 FigThe phylogeny corresponds to the majority rule consensus tree of trees sampled in a Bayesian analysis. Only fragments in exons were considered. Arabidopsis thaliana was used as outgroup. The numbers above the branches refer to the Bayesian posterior probability of the nodes (more than 50%) derived from 19500 Markov chain Monte Carlo-sampled trees.(TIF)Click here for additional data file.S3 FigThe phylogeny corresponds to the majority rule consensus tree of trees sampled in a Bayesian analysis. Only intron 1 was considered. Arabidopsis thaliana was used as outgroup. The numbers above the branches refer to the Bayesian posterior probability of the nodes (more than 50%) derived from 19500 Markov chain Monte Carlo-sampled trees.(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


Immunoassays are antibody-based analytical methods for quantitative/qualitative analysis. Since the principle of immunoassays is based on specific antigen–antibody reaction, the assays have been utilized worldwide for diagnosis, pharmacokinetic studies by drug monitoring, and the quality control of commercially available products. Berson and Yalow were the first to develop an immunoassay, known as radioimmunoassay (RIA), for detecting endogenous plasma insulin , a devel Since the development of radioimmunoassay (RIA) in 1960, there has been a rapid increase in immunoassay techniques using radioactive labels . CurrentPlant secondary metabolites are plant-produced organic compounds that play an important role in the defense of plants against herbivores, pests, and pathogens, as well as in their adaptation to the environment, although they are not directly involved in the growth and development of organisms , 4. BecaThe basic facts about ELISA and its practical use for measuring plant secondary metabolites are described in this review.p-nitrophenyl phosphate to produce p-nitrophenol, which can be detected at 405 nm (yellow color), and HRP catalyzes the conversion of chromogenic substrates, e.g., 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, 3,3′,5,5′-tetramethylbenzidine, and o-phenylenediamine into colored products. By using chemiluminescent substrates such as chloro-5-substituted adamantyl-1,2-dioxetane phosphate and luminol for ALP and HRP, respectively, and fluorogenic substrates such as 4-methylumbelliferyl galactoside and nitrophenyl galactoside for β-galactosidase, even more sensitive detection can be achieved. These enzyme–substrate reactions are typically completed within 30–60 min, and the reaction stops with the addition of an appropriate solution, e.g., sodium hydroxide, hydrochloric acid, sulfuric acid, sodium carbonate, and sodium azide, for individual reactions , 1817, 1Indirect competitive ELISA (icELISA) involves the combination of indirect ELISA and competitive ELISA Fig. . The tarThe use of enzyme-labeled secondary antibodies in indirect methods  exhibit advantages over direct methods (direct and competitive ELISA) with respect to sensitivity and versatility . PolycloGambierdiscus toxicus, are the major causative toxins of ciguatera seafood poisoning. Ciguatoxins are structurally classified as ladder-like polyethers with a molecular weight of 1111 Da. Oguri et al. divided these polyethers into two parts and prepared different monoclonal antibodies (MAbs) to individually recognize each part for constructing a sandwich ELISA system to measure ciguatoxins . Sa. Sa16]. uatoxins . More reuatoxins . The sanA highly specific assay can be obtained via a sandwich system because of the use of two antibodies. However, it is an expensive and labor-intensive process to prepare two antibodies. In addition, one more step is required in the sandwich system because immobilization is necessary for capture antibody, which increases the assay time Table .H) and light (VL) chains, which are binding regions for antigens E. coli tlumbagin , and paclumbagin . Similarmination , 67. Themination  and G-Remination . In thesTo date, various methods for the quantitative or qualitative analysis of plant secondary metabolites have been developed because a lot of marketed drugs are generated from plant secondary metabolites, such as morphine , vinblastine (antineoplastic drug), paclitaxel (antineoplastic drug), quinine , digitoxin (cardiotonic drug), and so on, and the accurate, sensitive, and selective evaluation of these drugs leads to safe clinical and general usages.In this review, ELISA has been discussed in detail; it is representative of various analytical methods because of its several advantages over other analytical methods in terms of simplicity, cost efficiency, and selectivity. However, all types of ELISA exhibit more or less advantages and disadvantages. A barrier for further development of ELISA is the preparation of specific antibodies against the target hapten. Even in this advanced era, there are many important plant secondary metabolites for which antibodies are not available. ELISA would be more familiar to us if the antibody or antibody-mimicking probes that are alternatively used in ELISA could be obtained more easily.


To preserve genetic diversity in threatened populations of domestic and wild animals, cryopreservation combined with reproductive technologies are useful. Here, we present a reliable and efficient protocol enabling preservation of epididymal sperm using domestic cattle as a model species. We propose that this technique has great potential in conservation of threatened species because it can be used to rescue genetic variation in the event of unexpected death, castration or in situations where obtaining an ejaculate is not feasible. in vitro fertilization, using bulls of two different age classes. Testicles from 26  and 19  Danish Holstein bulls were collected after slaughter and stored at 5°C. After 0, 24 or 48 h, epididymides were isolated and spermatozoa collected. Assessments included spermatozoal motility, viability and morphology before and after cryopreservation and in vitro embryo production. Results showed that live spermatozoa can be collected from epididymides of bulls after their death. Storage of the testicles at 5°C for 24 h followed by cryopreservation of recovered epididymal spermatozoa resulted in 21% (group 1) and 31% (group 2) blastocysts produced in vitro. These results illustrate that epididymal spermatozoa recovered from testicles kept in specific conditions can be used to preserve genetic material from endangered and threatened species or populations in nature as well as in domestic and zoo animals.The aim of this study was to establish and validate a reliable and efficient protocol for the recovery and cryopreservation of epididymal spermatozoa used for  The ability to preserve genetic material is an important tool in conserving genetic variation in endangered populations and species. Biobanks play an integral part in worldwide conservation efforts, in both domesticated and wild species, to counteract the loss of genetic diversity. Furthermore, material in animal biobanks is useful for various types of research, such as cryobiology, reduction of inbreeding, genomic selection studies, assessment of genetic distances and disease genetics , which is relevant for preservation of genetic material and for evaluation of the fertilizing capacity of spermatozoa after in vitro handling. In contast, such studies have been carried out in bovids, but they are few and have resulted in fairly low in vitro embryo blastocyst rates  and overlaid with 400 µl oil (M5310). Immature cumulus–oocyte complexes were incubated for 23–25 h at 38.5°C in humidified air supplemented with 5% CO2.Ovaries were collected at the Danish Crown slaughterhouse and transported to the laboratory within 2–4 h in 0.9% NaCl solution  at 32–36°C. Cumulus–oocyte complexes were aspirated from 2–6 mm follicles with a 19 gauge needle. The cumulus–oocyte complexes were collected and washed once in Hepes-buffered Medium 199 (M0650) supplemented with 30 IU/ml heparin , 10 µl/ml amphotericin (A2942) and 2% cattle serum , 10 µl/ml amphotericin (A2942) and 2% cattle serum . Cumulus–oocyte complexes with a minimum of three to four cumulus cell layers were selected for g. The supernatant was removed and the pellet resuspended in 2 ml Sperm-TALP. This procedure was repeated twice. After the third spin, the stock volume was reduced to 100 µl in all samples.Owing to limited resources, only bulls at T24 were used for IVF . Straws from each bull were thawed in warm water (35–37°C) for 2 min, and the spermatozoa were washed with 2 ml Sperm-TALP by centrifugation for 10 min at 277in vitro maturation medium to the IVF wells of four-well dishes containing 400 µl IVF medium . The zygotes were recovered and washed in in vitro culture medium . Embryos were evaluated on Days 2 and 7 for cleavage and blastocyst rates, respectively. As a control for each experimental round, IVF was performed using cryopreserved ejaculated spermatozoa from an 18-month-old bull with known in vitro fertility.Presumptive zygotes were vortexed at 250Statistical analyses were carried out using JMP 11 , using both parametric and non-parametric analyses because of the small sample size, where a normal distribution can be difficult to achieve. The Shapiro–Wilk test for normal distribution indicated that some of the analysed variables were normally distributed, whereas others were not. Transformation did not increase normality or homogeneity of variances for those traits that did not fulfil these criteria, and for this reason untransformed data were analysed.A two-way analysis of variance (ANOVA) was used to analyse the effect of storage time , the age of the bull (A = group 1 or group 2) and their interaction (S × A) on pre-freeze and post-thaw bull epididymal spermatozoa samples. The significance of any differences was analysed by the Wilcoxon test, which compares the median of the distributions .6 [95% CI: 469.1 × 106 to 1392.9 × 106 (group 1)] and 13 960 × 106 .Epididymal spermatozoa were successfully collected from all 90 testicles with the retrograde flushing method. The total number of spermatozoa was 9306 × 10In general, motility and viability tended to increase from 0 to 24 h storage, followed by a decrease after 48 h storage  and 21.0 , respectively] compared with group 2 . Group 1 had significantly lower (P < 0.05) cleavage and blastocyst rates than the reference bull , whereas only the cleavage rate in group 2 was significantly different from the reference bull (P < 0.05).The frozen–thawed spermatozoa from testicles stored for 24 h produced cleavage and blastocyst rates that tended to be lower for group 1 [40.5  had lower spermatozoal quality at recovery. This is in agreement with studies of changes in semen characteristics around puberty (39–41 weeks of age, for Holstein bulls; in vitro with satisfying rates, especially from group 2 bulls. Other studies have demonstrated lower developmental rates (Our results showed that refrigeration followed by cryopreservation of epididymal spermatozoa from both group 1 and group 2 bulls allowed production of embryos al rates , differeIn conclusion, cooling of testicles shortly after recovery is important to obtain the best-quality spermatozoa. For preservation of the spermatozoa, storage should preferably be performed for no longer than 24 h, although there is a reasonable flexibility up to 48 h after the testicles are obtained. The younger bulls showed less robust semen, giving less flexibility in the practical work, and emphasizing the importance of working faster with younger males. When done, the embryo production rate after IVF is acceptable, and the method can be used for producing embryos for transfer. We expect the protocol to be fairly simple to adjust to other species, producing a practical tool to save genetic material from various endangered populations and species. The genetic material can thereby be reintroduced into various populations that suffer from genetic consequences of small population size, such as inbreeding depression and loss of genetic variation owing to genetic drift (


Branchinecta lynchi, is one of several endemic and federally listed species being targeted for translocations. To guide reintroduction and conservation, detailed information on range-wide population structure and diversity is needed. We collected genetic data from two mitochondrial genes throughout the known extant range of B. lynchi to elucidate population structure and diversity of the species. We found support for phylogeographic structure throughout the range of B. lynch associated with isolated watersheds and vernal pool regions previously identified in the recovery plan for the species. The underlying mechanisms responsible for this broad pattern of genetic structure have yet to be identified. However, the evidence of only a few haplotypes being shared across the species range and patterns of isolation by distance within vernal pool regions suggests dispersal limitation may play a role. These results stress that conservation programs, at a minimum, should consider using individuals from regional populations as sources for reintroductions to maintain historical patterns of genetic differentiation. Additionally, because genetic structure is associated with vernal pool regions which are based on local hydrology and geology, translocations should proceed considering the distance between donor and recipient sites.Wetland habitats across the world are experiencing rapid modification and loss due to accelerating habitat conversion. Impacts to wetland habitats are particularly acute in California where up to 90% of wetland habitats have been modified or lost. Vernal pool ecosystems have therefore undergone a dramatic loss in habitat and along with them an entire endemic fauna is under threat of extinction. Recent efforts to conserve vernal pool habitat and associated species have involved restoration and creation of vernal pools as well as translocations of threatened species. The vernal pool fairy shrimp,  Wetland ecosystems in America have experienced greater than 50% declines in extent since the 1800s –4. An asCalifornia’s vernal pool wetlands have been grouped into 17 distinct geographic regions based on species endemic to each region as well as geomorphology and underlying soils . These vBranchinecta lynchi  [B. lynchi has the largest current distribution of any of the listed Branchiopods ranging from Jackson County in southern Oregon south to Riverside County in southern California; however, within this range B. lynchi occurs locally in a small proportion of the vernal pools [B. lynchi is particularly susceptible to loss of habitat and demographic disruption from landscape fragmentation and would therefore benefit from targeted reintroductions [B. lynchi occurs in vernal pools that vary in size (from 0.02 to 10 hectares), elevation , temperatures (4.5 to 23°C), and are rarely found to co-occur with other fairy shrimp species [Lepidurus packardi) and the diet of vernal pool fairy shrimp largely consists of algae, bacteria, protozoa, rotifers and detritus [ shrimp)  is one oal pools  and is cductions . B. lync species . It is pdetritus . Thus, tdetritus . Given tdetritus , 15.B. lynchi, additional information describing phylogenetic relationships among vernal pool regions and distribution of genetic variation throughout the species’ range is needed. Although B. lynchi is limited in mobility, several dispersal mechanisms, including waterfowl, wind, and flood events can likely result in long-distance movements of resting branchiopod eggs [Limnadopsis species display no population structure across large portions of central Australia, possibly due to dispersal by large populations of water birds [To guide ongoing reintroduction efforts and ensure preservation of extant genetic diversity of pod eggs –18. Becapod eggs , 20. Thipod eggs , 20, 21.pod eggs , 23. In er birds , 25.B. lynchi, Aguilar [B. lynchi. In this study, we examined nearly 400 individuals and sequenced two mitochondrial (mtDNA) genes, cytochrome c oxidase I (COI) and the ribosomal RNA gene 16S to test for regional population structure and potential management units across the species range, as well as identify if long distance dispersal is common or uncommon across the range.Within  Aguilar  found li Aguilar , 28 precB. lynchi is listed on the by the federal government as a threatened species and is of conservation concern, we sought to minimize new collection of samples from extant populations and, to the extent possible, we utilized museum collections for the sampling throughout the species’ range. There was no ethics review required for use of specimens previously collected from museums and all collections from new individuals was conducted by individuals possessing permits issued by the Fish and Wildlife Service and were collected in strict accordance to the requirements under the federal Endangered Species Act. Permit numbers and individual are listed in the acknowledgments section.Because B. lynchi individuals . From the collection, we were given permission to destructively subsample 483 specimens for genetic analysis  we designed based on an alignment of sequences that had worked with the other primer sets above. We paired this newly developed primer with the previously designed universal primer for Branchinecta for amplification (BrUniR-2042) [5’- AGGGCCGTGGTATTTTAACC-3’ and 16S-RintE, 5’- ATCCCTGAACCAACATCGAG—3’) using Primer 3 with default settings in version 4.0.0 [DNA from the right ventral phyllopods of museum specimens or freshly collected specimens N = 696,  was extriR-2042) . We desion 4.0.0 , 34.m of the two primers and then subtracting 5°C. PCR success was assessed on a 1.2% agarose gel stained with Gelstar . PCR products were purified using the ExoSap-it kit  and then sequenced bi-directionally on an ABI 3130/3730 sequencer using di-deoxy chain termination chemistry with BIG Dye v3.1  following recommended Applied Biosystems protocols. Sequences from forward and reverse primers were aligned and edited with SEQUENCHER v4.9 . A global alignment for each gene was exported from SEQUENCHER in a nexus format and used for phylogenetic analysis. Sequences generated for both loci have been deposited in GenBank  , BSA (1x), magnesium chloride (2mMol), dNTPs (0.20μMol), 0.05 units Faststart TAQ , 1 μL of DNA template (10 ng/μL), forward and reverse primers (0.50 μMol), and molecular grade water in a total reaction volume of 10 μL. Reported concentrations are in the final volume for the PCR. PCR for all primer sets involved a 4-minute denaturing at 94°C before thermocycling for 35 cycles. The thermocycling profile was 94°C denaturing for 30 s, annealing for 30 s, and 72°C extension for 1 minute, followed by a final 7-minute extension at 72°C. Annealing temperatures for COI primers sets were: 48°C for primer set LCO1490 and HCO2198 and primer set CO1-FS-intF and BrUniR-2042 and 50°C for primers JyothiF-1489 for the forward and BrUniR-2042 or LYR2198 for the reverse . The annB. lynchi for phylogeographic analysis, we defined success for PCR amplification as a PCR product of expected size visible on a gel. Sequencing success was defined as the generation of high quality sequences for both the forward and reverse directions from the PCR product, but did not necessarily take the final length of sequence into account.In order to determine the utility of museum specimens of Branchinecta lindahli) as the outgroup for phylogenetic analysis with the COI gene and B. coloradensis, B. oriena, B. constricta, and B. paludosa for rooting the 16S gene phylogeny. Support for phylogenetically distinct groups within the species was assessed by bootstrap values greater than 65% and/or a posterior probability of 95 or greater for the Bayesian analysis.Only specimens that produced the full length sequence for the standard barcode region of COI (658 bp of the 5’ end of COI)  were incs), transversions (nv), and substitutions (nd) as implemented in Arlequin v3.5. Mean pairwise differences among haplotypes (π) and haplotype diversity (h) within vernal pool regions were calculated using DNAsp v 5.10.01 [st among vernal pool regions using Arlequin v3.5 [st estimates use haplotype frequencies for mtDNA and there were only two instances of a shared haplotype between vernal pool regions, we therefore estimated Фst with the Kimura 2-parameter (K2P) genetic distance as implemented in Arlequin v3.5. This estimate of Фst takes sequence differences among haplotypes into account [Due to a lack of phylogenetic signal in the 16S gene, all remaining phylogeographic analyses were performed using only COI haplotypes except for the network analysis. COI haplotypes within vernal pool regions were compared to estimate the number of transitions . Likewise, significant and negative Tajima’s D values are evidence of low frequency alleles relative to expectations and can be indicative of recent population expansion, but can also be purifying selection. Significant (p < 0.05) positive values can be interpreted as recent population bottlenecks or balancing selection. We additionally performed mismatch distribution analysis [S and Tajima’s D and multimodal mismatch distributions) or populations under demographic or range expansion .Qualitatively we evaluated patterns of phylogenetic clustering among  4.2.0.1 . In thes2.0.1 [S  and Taji2.0.1 [S  as impleanalysis  that comAn analysis of molecular variance (AMOVA) was performed with only COI mtDNA pairwise estimates of Фst in Arlequin v3.5  to test st because so few haplotypes were shared between vernal pool regions. Significance of isolation by distance correlations were determined by a Mantel test with 1,000 permutations as implemented in Arlequin v3.5 [To test for dispersal limitation, we conducted an isolation by distance analysis within and between vernal pool regions. We estimated the pairwise geographic distance between sample locations in kilometers using QGIS v2.6  and corruin v3.5 .mtDNA due to the age of a specimen, of the 696 samples analyzed for COI, only 224 samples successfully produced COI sequences of 658 bp and only 386 samples produced 16S sequences of 358 bp for a total of 399 specimens producing sequence data for at least one gene  and because our samples are from post habitat reduction; thus, not allowing a full analysis across the historic species range. Unfortunately, due to preservation techniques , and as our data indicate  , 53. ThiB. lynchi populations sampled here, we observed potential long-distance dispersal only once across the species range. This, in combination with the phylogeographic signal recovered among vernal pool regions, suggests that long-distance dispersal of B. lynchi by birds, or another mechanism, is not likely to be a common event.We found evidence of a potential long distance dispersal event between the SSFH and KMVP regions. While the haplotype sampled in KMVP region is similar to those sampled in SSFH region, it was not identical, indicating that it has diverged since dispersal occurred, or that the haplotype found in KMVP was not sampled from SSFH in our study. This long distance dispersal event was also detected in a previous study ; howeverBranchinecta species used as outgroups, supports the monophyly of the species throughout its patchy range. The 16S gene is known to evolve at a slower rate compared to COI in crustaceans [Branchinecta species in California.The reduction in informative sites by using the partial barcode for COI likely led to the previous conclusion of weak phylogeographic structure for this species ; in contstaceans  and is aB. lynchi, found that use of non-denatured ethanol likely contributed to the decreased value of museum collections for future DNA studies [Use of historical samples from museum specimens proved quite challenging in this study. Unfortunately for many specimens collected prior to 2000 the original fixative was not recorded and they are now only stored at 75% ETOH in the museum collection. We suspect any future studies using museum specimens from before 2000 will require significant time and investment to yield useful genetic data. A recent study testing known preservation methods used for Anostracan species, including  studies . Given tThe molecular evidence supports, at a minimum, considering each vernal pool region as a separate genetic management unit . PreviouS1 FigThe geographic sample information for each number associated with each haplotype is given in (PDF)Click here for additional data file.S2 FigThe geographic sample information for each number associated with each haplotype is given in (PDF)Click here for additional data file.S3 FigSolid line depicts expected pattern under a constant population size and the dashed line indicates observed values. Samples from the San Joaquin Valley (SJVP) vernal pool region was excluded because the sample size was to small for estimating the distribution.(PDF)Click here for additional data file.S4 FigOpen circles in network are hypothesized, but not sampled haplotypes. Abbreviations are as in (TIF)Click here for additional data file.S1 TableTable includes the geographic location in latitude and longitude, the vernal pool region for each site sampled, the haplotype name as given in (XLSX)Click here for additional data file.


I) ranged from 0.25 in control to 0.98 in Sabah population. The mean numbers of effective alleles and Shannon's information index were 0.36 and 64.90%, respectively. Similarly, the allelic diversity was very high with mean expected heterozygosity (He) of 0.60 and mean Nei's gene diversity index of 0.36. The dendrogram based on UPGMA and Nei's genetic distance classified the 53 rice accessions into 10 clusters. Analysis of molecular variance (AMOVA) revealed that 89% of the total variation observed in this germplasm came from within the populations, while 11% of the variation emanated among the populations. These results reflect the high genetic differentiation existing in this aromatic rice germplasm. Using all these criteria and indices, seven accessions  from three populations have been identified and selected for further evaluation before introgression into the existing breeding program and for future aromatic rice varietal development.Aromatic rice cultivars constitute a small but special group of rice and are considered the best in terms of quality and aroma. Aroma is one of the most significant quality traits of rice, and variety with aroma has a higher price in the market. This research was carried out to study the genetic diversity among the 50 aromatic rice accessions from three regions  with 3 released varieties as a control using the 32 simple sequence repeat (SSR) markers. The objectives of this research were to quantify the genetic divergence of aromatic rice accessions using SSR markers and to identify the potential accessions for introgression into the existing rice breeding program. Genetic diversity index among the three populations such as Shannon information index ( Oryza sativa L.) is one of the most important food crops in the world. Approximately three billion people around the world consume rice as a basic food which provides about 50 to 80% of their daily calories. Aromatic rice is preferred over nonaromatic rice during special occasions and for export, and thus it commands a higher market price. One of the major features of this kind of rice is its special aroma that is appreciated by many people and represents a high value-added trait [Rice , thus making environmental factors mask their true phenotypic performance , 10. PheFor marker-assisted selection as well as gene tagging, rice microsatellites had shown their utility , 17. The indica type. The experiment was conducted inside the net house at the experimental field of Universiti Putra Malaysia. The sprouted seeds of the 53 rice accessions were sown in the different pots using randomized complete block design (RCBD) with three replications.A total of 53 rice accessions including three local check varieties  were used in this study as shown in A total of 147 SSR markers were selected for diversity analysis, out of which 32 primers showed clear, distinct polymorphic bands among the 53 aromatic rice accessions selected for the analysis as shown in The DNA was extracted from 21-day-old seedlings leaves of aromatic rice genotypes using hexadecyltrimethylammonium bromide (CTAB) method . The quaμl and this included 1 μl of about 50 ng DNA template, 7.0 μl DreamTaq PCR master mix (Thermos Scientific Inc.), 1 μl of each primer (forward and reverse primer), and 5.0 μl nuclease free water. Touch-down PCR protocol was followed [Polymorphic thirty-two (32) SSR markers were used for genotyping the entire 53 rice accessions. Total PCR reaction was optimized to be 15 followed . The banNe), gene diversity (h), Shannon's information index (I), gene frequency, and gene flow (Nm) were computed [N is the effective population size and m is the fraction of individuals in a population. If Nm is <1, this indicates that populations tend to differentiate, and when Nm ≥ 1, there is little differentiation among populations. Polymorphism information content (PIC) was computed from the formula given below:Pij is the frequency of the jth allele for the ith marker and summed over n alleles.Genetic diversity parameters such as percentage polymorphic loci (PPL), effective allele number  was conducted to assess the genetic structure of the populations  using ArThe present study evaluated the genetic diversity of 50 aromatic rice accessions and 3 check varieties as a control. These accessions were obtained from three different regions, namely, Sarawak, Peninsular Malaysia, and Sabah. The study of genetic diversity in any breeding population is essential as it constitutes the backbone of any breeding and improvement program. It helps in the development of crop that is suitable and adaptable to rapid climate change through the introduction of foreign genes , 24. ThuFrom 147 SSR markers screened, 32 markers (21.77%), which displayed clear and repeatable polymorphic bands, were selected for analysis as shown in Ne) varied from 0.10 (RM16655) to 2.12 (RM3134) with an average of 1.48. Nei's gene diversity (h) ranged from 0.05 (RM172) to 0.98 (RM1), with a mean of 0.36. Shannon's information index ranged from 0.22 in RM460 to 0.91 in RM195; the average was 0.58 as shown in PIC was observed to differs significantly, from 0.25 (RM3872) to 0.98 (RM321). The effective number of alleles  varied from 0.80 (control) to 1.98 (Sabah). The effective number of alleles per locus (Ne) was small compared to the number of alleles per locus and it ranged from 0.16 (control) to 0.54 (Sabah) with the mean number of 0.36. Shannon's information index (I) was very high and varied from 0.25 (control) to 0.98 (Sabah) with 0.58 as mean as shown in The average number of alleles per locus  have been identified as diverse accessions and suitable as a parent in the future breeding program.


Bactrocera tryoni (Froggatt), has been subject to significant long‐term quarantine and population reduction control measures in the major horticulture production areas of southeastern Australia, at the species southern range limit. Previous studies have employed microsatellite markers to estimate gene flow between populations across this region. In this study, we used an independent genetic marker, mitochondrial DNA (mtDNA) sequences, to screen genetic variation in established and adjacent outbreak populations in southeastern Australia. During the study period, favorable environmental conditions resulted in multiple outbreaks, which appeared genetically distinctive and relatively geographically localized, implying minimal dispersal between simultaneous outbreaks. Populations in established regions were found to occur over much larger areas. Screening mtDNA  lineages proved to be an effective alternative genetic tool to assist in understanding fruit fly population dynamics and provide another possible molecular method that could now be employed for better understanding of the ecology and evolution of this and other pest species.Understanding the relationship between incursions of insect pests and established populations is critical to implementing effective control. Studies of genetic variation can provide powerful tools to examine potential invasion pathways and longevity of individual pest outbreaks. The major fruit fly pest in eastern Australia, Queensland fruit fly  The other larval samples (n = 13) were collected from infested fruit grown in the GSPFA during outbreaks, from sites that were also sampled contemporaneously for adult B. tryoni  was chosen for this study as in B. tryoni, the amplification conditions have previously been optimized, and it is also known to be highly variable, does not contain indel gaps, or appear to have nuclear copies .DNA was extracted from dry fly samples using 5% Chelex  was also conducted in GenAlEx to examine the partitioning of genetic variation within and between regions and sites.A neighbor‐joining tree of genetic relationships between sites was constructed in Mega 5.1  diversity in samples collected from a limited number of sites in Gippsland and the FFEZ in 2008 and 2010 was examined to test for the persistence of mtDNA lineages through time, that is, temporal variation at these sites.33.1B. tryoni samples in this study (Table A large number of haplotypes n = 153) were detected from the 53 were d3.1.1A comparison of haplotype diversity  compared with site locations was conducted Figure . A stron3.23.2.1p < .05) within each region suggested relationships between groups of sites and genetic variation. In a combined analysis of all of the sites together  between sites sampled in this study Figure  indicate3.2.3n = 5, Haps 102, 106, 115, 116, 145), while the rest represented shared widespread  or localized  haplotypes present at southern B. tryoni sites tested in this study. Interestingly, while the number of Queensland larvae sampled here is low, many of the shared haplotypes occurred at outbreak FFEZ or GSPFA sites  the large geographic scale of widespread established southeastern Australian populations Figure ; 2) the the2; Gi4.2B. tryoni populations suggest a degree of localization of genetic variation within a number of outbreaks (Table B. tryoni lineages (represented here by different mtDNA haplotypes) within each area. These results indicate that gene flow is greatly reduced in GSPFA outbreak populations. The patterns of diversity between sites observed here appear to be at least partially explained by the different status of B. tryoni in each region tested: B. tryoni is established through eastern Australia, including east Gippsland (the most southerly sites sampled here); however, all westerly populations of B. tryoni examined were the result of outbreaks, with the degree of B. tryoni control measures being greatest in the GSPFA.Overall the spatial analyses of southern ks Table a. Examinks Table . The degks Table . This paB. tryoni between sites is described as jump diffusion, that is, the human‐assisted long‐distance movements followed by local diffusion through natural insect flight  between Australia's capital cities of Brisbane in Queensland (where B. tryoni is established) and Melbourne in Victoria (which is B. tryoni free). Genetically, Dubbo appears similar to other nearby towns , as well as distant sites in Victoria  into the eastern FFEZ and subsequently to the GSPFA and South Australia . Interestingly, Wagga Wagga appears more similar to some FFEZ sites, more closely matching sites from the “Central FFEZ”  to Melbourne (B. tryoni free), which appear to match the adjacent “Southern FFEZ” sites  that were not found elsewhere in our study. It would be useful in future studies to sample a greater range of B tryoni genetic diversity from a greater part of the species range, including sites from the northern parts of the species range, for comparison with the data presented here.Ours is the first genetic study to examine the most southerly d Figure , Table 1d Figure . In our d Figure  and 5a. 4.3At the time of our study, outbreaks in some parts of the southeastern FFEZ had been continuous for more than five years. This region appears to show continuous gene flow over relatively large distances Figures , and 5. B. tryoni into the GSPFA from multiple sources. Furthermore, these introductions appeared to show very limited dispersal between GSPFA sites.If there was a widespread low‐level resident population within areas of the GSPFA prior to the study period, gene flow between sites would be expected to result in some shared common haplotypes across the GSPFA. This does not appear to be the case, and overall genetic patterns appear to indicate that over the study period, there were many independent introductions of 4.4B. tryoni freedom. If there is no traveler awareness campaign and no vehicle inspections, about 18% of the traveling public carry fruit , while others appeared to have not yet become widespread and persistent at the time of the study.Overall, it appears that screening mtDNA  lineages does provide an extremely useful alternative tool for helping to understand B. tryoni at the southern extremity of the species range, that could now be built upon, screening additional sites to include a greater part of the range of B. tryoni  as well as examining the same sites over time. It would be particularly beneficial to revisit the sites sampled in our study to observe the genetic changes that have occurred since control measures were reduced in the FFEZ and GSPFA.We provide baseline information on a number of new introductions of B. tryoni and other similar pests  to provide evidence for developing phytosanitary measures for domestic and international markets.Future DNA sequencing studies will now be relatively easier to implement using newly available next‐generation high‐throughput amplicon sequencing technologies  has been submitted to GenBank: accession numbers KY550463 ‐ KY550654.


Salvelinus namaycush) inhabiting a large, pristine, postglacial lake , with up to five discernible genetic clusters having distinctions in body shape, size, colouration and head shape. However, genetic differentiation was low (FST = 0.017) and genetic clustering was largely incongruent between several population- and individual-based clustering approaches. Genotype- and phenotype-environment associations with spatial habitat, depth and fish community structure (competitors and prey) were either inconsistent or weak. Striking morphological variation was often more continuous within than among defined genetic clusters. Low genetic differentiation was a consequence of relatively high contemporary gene flow despite large effective population sizes, not migration-drift disequilibrium. Our results suggest a highly plastic propensity for occupying multiple habitat niches in lake trout and a low cost of morphological plasticity, which may constrain the speed and extent of adaptive divergence. We discuss how factors relating to niche conservatism in this species may also influence how plasticity affects adaptive divergence, even where ample ecological opportunity apparently exists.The study of population differentiation in the context of ecological speciation is commonly assessed using populations with obvious discreteness. Fewer studies have examined diversifying populations with occasional adaptive variation and minor reproductive isolation, so factors impeding or facilitating the progress of early stage differentiation are less understood. We detected non-random genetic structuring in lake trout ( Ecological speciation occurs when divergent natural selection in different environments results in reproductive isolation and ultimately new species formation . Its speSalvelinus alpinus) and threespine stickleback (Gasterosteus spp.), morphs are at the intermediary stages of ecological speciation. These morphs exhibit varying degrees of genetic differentiation, moderate to relatively high gene flow, adaptive variation to limnetic and benthic feeding niches, and potentially reversible reproductive isolation )al lakes ,14. The al lakes , but appal lakes ,18 and ual lakes ,20. Conval lakes . Finallyal lakes . Overall2), deep (180 m), postglacial lake in northern Canada [Salvelinus fontinalis) and walleye (Sander vitreus) [Mistassini Lake  ,25. For vitreus)  suggestevitreus) ,28. WhilWe firstly tested and provided evidence that Mistassini Lake harboured multiple genetically- and morphologically-distinguishable clusters of lake trout associated with spatial habitat, depth and fish community structure, through the use of morphological analyses, catch locations, bycatch data and microsatellite DNA surveys. Yet genetic differentiation was low and genetic clustering was largely incongruent among several population- and individual-based clustering approaches. Moreover, genotype- and phenotype-environment associations were either inconsistent or weak. We thus also evaluated genetic differentiation in relation to effective population sizes and the extent (and possible asymmetry) of contemporary gene flow, and considered additional ecological factors within Mistassini and other large postglacial lakes that might inhibit genetic differentiation in lake trout and other postglacial fishes. Our work contributes to a better understanding of how intraspecific phenotypic plasticity might affect the trajectory and progress towards ecological speciation.Between June 12 and July 29, 2013, 636 lake trout were sampled throughout Mistassini Lake using monofilament nylon gillnets and angling, in order to obtain genetic (n = 636) and morphological data (n = 281). Gillnet design was based on previous lake trout research in large lakes , expected (HE) and observed (HO) heterozygosity for each locus were computed using FSTAT (v.2.9.3.2.) [We tested for the presence of null alleles, large allelic size dropout, or scoring errors associated with allelic stuttering issues in our dataset using MICROCHECKER (v.2.2.3) . Descrip.9.3.2.) . To ensu.9.3.2.) . We then.9.3.2.)  (i) unde.9.3.2.) . If the ST and multiple glacial lineages. We ran STRUCTURE with no a priori information under models of K = 1–20, a burn-in period of 500,000 followed by 2,000,000 iterations, and replicated 20 times each per K. We estimated the most likely K value based on the ad hoc ΔK statistic of Evanno et al. [q) for each individual using the program CLUMPP (v.1.1.2) [ST) [ST for both body and head morphological clusters (see below) to determine if genetic differentiation was higher among morphological vs. genetic clustering.We employed both non-spatial and spatially informed (see below) approaches to characterize genetic clustering and structure. Non-spatial analyses were performed using three different methodologies see ; howevero et al.  as impleo et al.  and the v.1.1.2) . Becausev.1.1.2)  we estimv.1.1.2)  as a sup.2) [ST)  between ST rather than FST, which in young postglacial lakes can reflect population divergence predating lake age [ST > FST in Mistassini Lake  typically has lower standard errors than RST [ST for most typical sample sizes [Genetic differentiation may be influenced by stepwise mutations that are accounted for in measuring Rlake age . We asseini Lake . Nonethethan RST , (ii) isthan RST , and (iile sizes .I) [To complement non-spatial clustering analyses and provide insight into individual-level genetic relationships, we tested for associations between geography  and the distribution of individual genetic variation across the lake. The advantage of these spatial analyses is that they do not rely on HWE or linkage equilibrium and thus are useful for detecting gradients and complex spatial-genetic structures . We wereI) ,48. AlthI) , which iI) ; and (iiI)  and assuNe) for each defined genetic cluster using the linkage disequilibrium method implemented in LDNe (v. 1.31) [Ne estimates up to 25–30% [m, within the last few generations) between cluster pairs using all individuals with BayesAss (v.3.0) [7 Markov chain Monte Carlo (MCMC) iterations with a burn-in period of 106 and sampled at a frequency of 100. Prior to each run, mixing parameters  were optimized until acceptance rates were between 20–35%, as recommended in the manual. To assess whether MCMC had converged successfully for each run and overall, we used the program TRACER (v.1.6.0) [ST to reach halfway to a new equilibrium:We estimated the contemporary effective population size (v. 1.31) . Lake tro 25–30% . We esti (v.3.0) . Five sev.1.6.0) . Whetherv.1.6.0)  equationm = mean gene flow into each cluster.To characterize body morphological variation, we took a full-bodied standardized photograph of each trout immediately after capture using a digital camera (Nikon D3100) with a UV filter mounted on a tripod. Many tissue samples were donated from local fishers, so we were only able to take photos of a total of 281 of 636 trout. We placed each fish on its right side on a flat piece of plywood with the dorsal, caudal and anal fins in open positions; if fish were bent or distorted, photographs were discarded. Eighteen landmarks  . TpsRelwBody size (mass) and the relationship between mass and total length (TL) was compared among genetic clusters using linear models. In the latter model, the natural logarithm (ln) of mass was regressed onto the ln of TL, genetic cluster and their interaction. A subsequent pairwise comparison of the inferred slopes (mass/length curves) was conducted using ‘lsmeans’ package (v.2.17) . Mass wa2 test in the ‘vcd’ package (v.1.4–1) [Colour frequency distributions were compared using a χv.1.4–1) . The colq values for each individual. We used a global permutation test of the RDA result to test for a relationship between genetic cluster and ecological variables, as well as a permutation test on canonical axes with 1,000 steps [2 values were calculated as a measure of ‘goodness-of-fit’. The RDA was complemented with a similar multinomial regression analysis which, in contrast, incorporated all individuals weighted based on their highest q value. Because the RDA and multinomial regression analyses yielded largely congruent results, only results for the former are reported in the paper  and/or by water depth, using a redundancy analysis (RDA) within the ‘vegan’ package (v.2.3–1) , which i00 steps ; R2 valuBased on recorded gillnet bycatch data, we tested whether genetic clusters were associated with fish community structure using a contingency test. Bycatch abundance and diversity captured within the same net/panel as each trout provided a reasonable proxy for prey availability and the extent of interspecific competitor species. The relationship between genetic clustering and fish community structure was visualized using a RDA.ST values and either the absolute difference in (i) mean scores for the first seven RWs for body morphology and first three RWs for head morphology, (ii) median depth, (iii) prey abundance, or in (iv) colour frequencies.We further tested for associations between the extent of genetic differentiation among defined clusters and either morphological differentiation, depth, fish community structure (abundance), or colour dissimilarity, by using Mantel tests implemented in the ‘vegan’ R package. Mantel tests always compared two pairwise distance matrices: cluster pairwise FP-values to adjust for any potential type I errors following the false discovery rate procedure [Unless otherwise stated, we corrected multiple comparison rocedure ; signifiO across loci of 0.813 ; the remaining 17 deviations from HWE (P = 0.000–0.044) were spread across all five clusters and across nine loci. We also detected significant LD in only seven of 171 locus-by-cluster tests , and found little evidence for LD when accounting for clusters  was confirmed for the 16 samples independently run and scored three times. The 19 loci averaged 29.5 alleles per locus (range of 13–72) with an average HST of 0.017 and significantly greater than zero. Pairwise estimates of FST between clusters ranged from 0.012 to 0.036  were lower and ranged from 0 to 0.002  but no significant local structure . Screen- and bar-plots of the eigenvalues  to 4,199  to 177 generations (clusters 1–3), suggesting migration-drift equilibrium has been reached.Point estimates of The seven body RWs explained 78% and the first three head RWs explained 70% of the total variation . Major shape differences are visualized in MCLUST defined three separate body shape clusters (B1-3) with a ΔBIC = 42, and five head shape clusters (H1-5) with a ΔBIC = 6 . Cluster 4 individuals were larger than all others ; cluster 3 was larger than clusters 1, 2, and 5 . The slope for the mass/length relationship of cluster 4 was significantly steeper than cluster 1, 2 and 3 . Clusters 2, 3, and 5 were predominately (between 73–80%) of black colouration  but these relationships were best described as weak . Each morphological cluster contained individuals in varying frequencies from all genetic clusters (Genetic clusters were associated with body and head clusters (body: clusters .2adj = 0.12). A global permutation test revealed relationships between ecological variables and genetic clustering (P = 0.001). A subsequent canonical axes permutation test revealed that only the first three RDA axes explained this relationship . RDA1 (50% of the variation) was driven primarily by depth and was especially important for distinguishing cluster 1 as occupying deeper water than all other clusters ; clusters 3 and 4 showed the converse pattern (both P < 0.001), whereas cluster 2 was captured in equal numbers among basins ; only individuals in cluster B1 were captured at a greater mean depth compared to B2 individuals . Cluster H5 individuals were captured at a greater mean depth than all other morphological clusters  and cluster H1 was captured more deeply than cluster H3 (P = 0.029).As depth influenced genetic clustering, we also examined whether it affected morphological clustering. Depth was only a weak predictor of body shape cluster , though the association was weak (Cramer’s V = 0.168). Cluster 1 was rarely captured with other fish species, clusters 2 and 3 were associated with burbot (Lota lota) and Lake whitefish, cluster 5 with walleye, and cluster 4 with all bycatch species . Specifically, pairwise FST was positively associated with both dark colours in which clusters with the greatest genetic differentiation also exhibited the greatest absolute difference in the frequencies of individuals that were black and brown. Additionally, some evident positive trends between increasing FST and increasing morphological differences were detected  influenced primarily by depth and less so by lake geography ; (ii) associated with some variation in body size, colouration, and fish community structure, but was (iii) inconsistently associated with body/head shape differences linked to trophic ecology and locomotor mode. Rather, genetic clusters displayed high morphological variation, and all contained individuals belonging to the here-identified three body shape clusters or five head shape clusters.One obvious phenotype-environment association detected was camouflage body colouration and depth. In contrast to other, clear postglacial lakes inhabited by lake trout morphs, Mistassini is tea-coloured with a relatively shallow secchi depth . CorresFunctionality of different head shapes also corresponded somewhat with depth. Lake trout with dorsally located eyes, blunt snouts and short heads (H5) were routinely captured in deep water (median = 142 m), whereas individuals with more streamlined heads (H1 –H4) were captured in shallower water median = 9–27 m); the former head shape is specialized for benthic foraging or more vertical oriented piscivory, the latter is associated with piscivory or insectivorous preferences in shallower waters –27 m; th. ConversThese general body shape-depth/habitat associations have similarities and differences with other lake trout morphs. The shallow-water, streamlined, long and narrow caudal peduncle ‘lean’ morph in other lakes  is similDespite some evidence for phenotypic-environment associations and drift-migration equilibrium conditions, we found only weak support for positive relationships between genetic and ecological or morphological differentiation and hence a putatively adaptive basis for occupying distinct habitat niches. Although some genetic clusters varied slightly in the frequencies of different morphologies, they all contained individuals exhibiting all three body types and all five head shape types. Furthermore, colouration and depth associations were not absolute: all five clusters contained individuals with each identified main colour and were captured in areas of differing fish-community structure. When compared to collated data from other large, postglacial lakes harbouring lake trout morphs, such relatively weak or little association between morphological and genetic differentiation appears common .Ne that a large lake size confers towards sustaining multiple evolving populations of a large-bodied predator fish, and (ii) the different habitat niches available in larger lakes that lake trout can exploit, including shallow and deep water habitat, extensive shoreline distance, multiple basins and multiple prey fish species . At Compared to the tributary spawning nature of brook trout and walleye, which generates considerable spatial isolation (tens to hundreds of kilometres) and a more complex lifecycle, Mistassini lake trout spawn along lake shorelines, and local fishing experts have described the spawning sites of various morphs in close geographic proximity (K. Marin 2013 pers. comm.). Although natal fidelity is well documented among postglacial lake-dwelling salmonids , the eviNe and m revealed significant asymmetries in m among genetic clusters, and hence the possibility that gene flow constrains adaptive divergence [m was significantly higher from the large genetic cluster occupying deeper waters (#1) to large and small clusters occupying mid-depths and shallow waters . Adaptive divergence for occupying some habitat niches in shallower waters may thus be constrained; alternatively, selection against migrants may be stronger against individuals from shallower than deeper genetic clusters.Consideration of vergence  in certaWhereas defined genetic clusters exhibited summer depth preferences, individuals from different clusters were captured throughout the water column. The striking morphological variation was more continuous within than among genetic clusters, suggesting a highly plastic propensity for occupying multiple habitat niches. Habitat use among individual lake trout has indeed been found to be highly variable but consistent between years –90.ST and a lack of migration-drift equilibrium, but rather, related to a high contemporary gene flow and large Ne. And, in contrast to other cases in north temperate fishes [Phenotypic plasticity is thought to both facilitate and constrain adaptive divergence and reproductive isolation during the process of ecological speciation ,91. Sympe fishes ,93, incle fishes , remarkae fishes .If low genetic differentiation in sympatric lake trout is constrained by plasticity, then a key question is why plasticity might more readily constrain later steps of ecological speciation in this species relative to congeneric Arctic charr. One possibility is that the evolution of lake trout was initially born out of specialization for terminal piscivory in cold-water lakes , whereasA = 29.51 and 17.26, respectively). Indeed, we found no evidence that Mistassini lake trout have experienced bottlenecks  and an independent study that screened Mistassini lake trout of unknown location (n = 46) using the same 19 microsatellite loci , have fotlenecks  and the ST) exceeded global FST Click here for additional data file.S2 File(PDF)Click here for additional data file.S3 File(PDF)Click here for additional data file.S4 File(PDF)Click here for additional data file.S5 File(PDF)Click here for additional data file.S6 File(PDF)Click here for additional data file.S7 FileNe) and gene flow Mistassini lake trout.Effective population size ((PDF)Click here for additional data file.S8 File(PDF)Click here for additional data file.S9 File(PDF)Click here for additional data file.


Pterois volitans) invasion on NW Atlantic ecosystems, little genetic information about the invasion process is available. We applied Genotyping by Sequencing techniques to identify 1,220 single nucleotide polymorphic sites (SNPs) from 162 lionfish samples collected between 2013 and 2015 from two areas chronologically identified as the first and last invaded areas in US waters: the east coast of Florida and the Gulf of Mexico. We used population genomic analyses, including phylogenetic reconstruction, Bayesian clustering, genetic distances, Discriminant Analyses of Principal Components, and coalescence simulations for detection of outlier SNPs, to understand genetic trends relevant to the lionfish’s long-term persistence. We found no significant differences in genetic structure or diversity between the two areas . In fact, our genomic analyses showed genetic homogeneity, with enough gene flow between the east coast of Florida and Gulf of Mexico to erase previous signals of genetic divergence detected between these areas, secondary spreading, and bottlenecks in the Gulf of Mexico. These findings suggest rapid genetic changes over space and time during the invasion, resulting in one panmictic population with no signs of divergence between areas due to local adaptation.Despite the devastating impact of the lionfish ( Pterois spp.) invasion of the northwestern Atlantic (NW Atlantic) is remarkable for its speed and magnitude1 and considered among the world’s more critical conservation issues during the last decade2. The lionfish invasion is the first recorded invasion of a marine fish species in United States Atlantic waters3. Lionfish are now the most abundant fish predators in many reefs within the Wider Caribbean 6, including those at depths of 30–350 m, with densities that, in some cases, far exceed those in their native ranges7. The success of these invasive predators is likely related to a combination of niche availability in the introduced area, ability to colonize a wide variety of habitats and broad thermohaline tolerance, high fecundity, wide-ranging dispersal, venomous defences, and absence of natural predators in the newly colonized environments8. The rapid increase in lionfish abundance in the Wider Caribbean has been linked to direct and indirect impacts on invaded ecosystems7, such as significant declines in native fish biomass due to lionfish’s predatory success on native species9 and their purported ability to displace large reef fishes, including groupers9. Lionfish are high-efficiency predators that feed primarily on post-settlement reef fishes, which they disorient by blowing water jets on them before capture10. Prey in the invaded range have not developed defence strategies against this predatory mechanism and so succumb easily10. In addition, female lionfish are capable of spawning year-round, every 2–9 days (season dependent), with an average annual output of more than 2 million eggs and several tens of generations. This tremendous output of buoyant gelatinous egg masses that drift with marine currents, provides opportunities for widespread dispersal over short time-periods11.The Indo-Pacific lionfish  and P. miles , were introduced in the NW Atlantic23, P. volitans is the most ubiquitous species, occurring throughout the US east coast, Caribbean Sea, and the Gulf of Mexico24. Although some molecular data did not detect signs of mitochondrial introgression and/or hybridization between the two potential species16, the most recent morphological and molecular information revealed that P. volitans is a recent hybrid species between the Indian lineage of P. miles and a Pacific lineage encompassing P. lunulata and P. russelii25.During the last years, genetic approaches using mitochondrial DNA have been applied to investigate the lionfish invasion. Barcode analyses suggested that although two lionfish species, P. volitans, conducted across the Wider Caribbean, supported the chronological records of the invasion and confirmed the colonization routes followed by the species27. While these studies discarded the hypothesis of multiple introductions from native sources during the invasion process, they revealed successive bottlenecks over the course of the invasion. Across the Wider Caribbean, only nine d-loop haplotypes were identified among the total 1,248 samples sequenced27, a number that contrasts with the 37 different haplotypes sequenced in the native range at the Indo-Pacific in only 70 specimens20. D-loop analyses showed that the first bottleneck occurred from the native range to the NW Atlantic, although the NW Atlantic area (east coast of the US), identified as the entrance point of lionfish, displayed the highest nucleotide and haplotype diversity and number of haplotypes within the Wider Caribbean, with the nine different haplotypes16. The second bottleneck occurred within the invasive range when of the nine haplotypes found at the NW Atlantic only four secondarily spread to the Caribbean Sea. Finally, three of the four haplotypes found in the Caribbean Sea invaded the Gulf of Mexico27. Hence, the lionfish invasion initially contrasts with the perception that avoiding genetic bottlenecks by the influx of genetic diversity through repeated introductions from the native range increases the invasion success and posterior spread within the invasive area29, as observed in a number marine invasions fuelled by multiple introductions from different native sources31. The study by Johnson and co-authors (2016), which pooled together all d-loop sequences obtained from the Wider Caribbean between 2007 and 2013, also detected significant differences in genetic structure among the three mentioned areas: NW Atlantic, Caribbean Sea, and the Gulf of Mexico  and the northern Gulf of Mexico, respectively. These two marine areas displayed highly significant differences in genetic diversity, structure and absence of connectivity across the Florida Strait for the d-loop marker in a previous study21.Despite the important insights revealed by the d-loop, using only one mitochondrial marker may prevent detection of fine-scale differentiation and connectivity within lionfish’s invasive range29 and to design management strategies for its control.We here apply Genotyping by Sequencing (GBS) techniques to identify over a thousand nuclear and independent single nucleotide polymorphic sites (SNPs) from samples collected over a short time. Our general aim is to understand the invasion progression by determining fine-scale population genomics between the NW Atlantic (east coast of Florida) and the northern Gulf of Mexico, and potential connectivity between them, knowing that the spatial genetic structure, temporal genetic trend, and levels of connectivity among areas is relevant to predict the potential long-term persistence of an invader32. Filtering for individuals with at least 75% of the called loci and loci that were present in at least 84% of individuals yielded a total of 1,654 SNPs and 162 individuals from the NW Atlantic and Gulf of Mexico  and 373 SNPs with significantly greater observed than expected heterozygosity   library of 229 lionfish samples, a total of 404,254 sequence tags were retained in TASSELp-value ≤ 0.01 was applied). No marker was identified to be under balancing selection from Arlequin .The main genetic descriptors obtained for all 1,220 lionfish SNPs are listed in Table WE Table . Our anaThe Maximum Likelihood (ML) tree reconstructed from 1,220 SNPs of 162 specimens from 13 locations did not show geographical clustering of specimens related to the sampling site and/or geographical area where they were collected  according to the ad hoc statistic ΔK  for the whole data setST statistics gave us more details of the pairwise differences between populations within the NW Atlantic and the Gulf of Mexico. The FST statistics were in general very low, and only two pairwise comparisons were significant  after FDR correction of the p-values when all 1,220 SNPs  for all 1,220 SNPs, 1,207 neutral SNPs, and 13 outlier SNPs also did not detect genetic differentiation associated with the NW Atlantic and Gulf of Mexico (“Among groups”), regardless of whether the two populations from the Florida Keys (Dry Tortugas- DT and Pulley Ridge- PR) were pooled or removed from the analyses, and most genetic variation was retained within individuals Table . NeverthPs Table  or only The discriminant analyses of principal components (DAPC) also showed a general pattern of low genetic differentiation, as that observed from previous analyses. According to the Bayesian Information Criterion (BIC) that compares different DAPC clustering solutions, two clusters were the optimal number to describe our data. The DAPC plot, including all sampling sites and all 1,220 SNPs, showed no clear separation of populations or clusters between the NW Atlantic and Gulf of Mexico Fig. . Only FGP. volitans specimens across the NW Atlantic and the northern Gulf of Mexico represents the first study using nuclear loci to explore the genetic structure of this invasive predator and is among the few studies applying genome-wide scanning, based on Next Generation Sequencing technologies, to investigate population structure of a marine invader . Although 18 nuclear microsatellite loci were isolated for P. volitans and P. miles a few years ago37, to our knowledge, those markers have not yet been used for population analyses.Our genomic data of 1,220 SNPs from 162 ST distances; the 13 outlier SNPs, FG and Ap showed significant differences with five additional sites, but that divergence was not mirrored by other analyses (e.g. Structure and DAPC). Only FG seemed to be genetically divergent for most analyses and databases. However, this location only includes four individuals, a sample size that cannot be considered representative of the genetic diversity and structure of this location, as demonstrated by the low number of accumulated alleles within these four individuals , such as those noticed here for lionfish, are becoming more commonly reported as the number of nuclear multilocus datasets increases (see examples in refs39) and can be explained by several non-exclusive causes40. Demographic asymmetry due to sex-biased dispersal can cause mito-nuclear discordance in motile animals, including marine fish species41. Although recent findings suggest that lionfish adults move more than initially thought42, they are not migratory, and buoyant eggs are the dispersal stage11, so different migratory behaviour between males and females cannot explain the pattern we found, and other hypotheses should be considered, including temporal genetic shifts and selection.Our findings, therefore, contrast with the discontinuity between the NW Atlantic and the Gulf of Mexico and the lower genetic diversity of the Gulf of Mexico observed in previous studies based on d-loop data45. Despite the importance of temporal genetic trends for the invasion dynamics, this point is overlooked in most studies of marine invaders, and it is assumed that genetic diversity remains stable over time34. The few studies investigating temporal trends of genetic structure in introduced marine species showed variable outcomes51. Whereas some invasive ascidians suffering massive seasonal die-off events maintained stable levels of genetic diversity and homogeneous structure over time due to the re-establishment of populations from the survivors or recolonization from nearby sites51, other introduced species exhibited changes in genetic architecture over short time periods. For instance, in the introduced colonial ascidian, Perophora japonica, a genetically isolated population from Plymouth (South England) displayed a linear reduction in mitochondrial genetic diversity and large haplotype frequency changes over a 9 year-monitoring period, due to either genetic drift and/or selection46. Rapid allele frequencies changes over time, high heterozygous deficiency, and inbreeding were also detected in isolated populations of the colonial ascidian Botryllus along the coast of Israel49, but genetic isolation was not always associated with genetic diversity loss in this species. Invasive Botryllus populations along the Californian coast, isolated from other genetic sources and highly influenced by genetic drift and selection, maintained stable levels of genetic diversity thanks to high mutation rates generating a complex pattern of allele gains and losses47. Nevertheless, marine invasive populations are, in many cases, characterised by high levels of genetic diversity due to multiple introductions from genetically distinct sources28. An outstanding example of multiple cryptic introductions and genetic admixture within the invaded range, which might be related to the high invasion success, is that of the European green crab, currently one of the most important aquatic invaders established across all temperate shores around the world36.A plausible cause of discordance between lionfish studies using different markers is temporal changes in the genetic structure over the invasion process. Population genetics theory anticipates fast genetic changes in introduced populations characterized by bottlenecks, founder effects, strong genetic drift, and new selective pressures in the introduced environments20, which would result in a small initial effective population size. Moreover, changes in genetic structure are expected to occur faster in mitochondrial than nuclear DNA because mitochondrial DNA, a haploid, maternally inherited molecule, has an effective population size of one-quarter that of nuclear DNA and therefore is more sensitive to diversity changes associated with genetic drift. For this reason, the comparison of the NW Atlantic d-loop data (collected between 2007 and 2009) and the Gulf of Mexico (collected between 2011 and 2013)27 should be taken with caution since it assumes that NW Atlantic populations remained static over a six-year period with no changes in haplotype frequencies due simply to genetic drift. This assumption of temporal stability might obscure the most recent pattern of genetic diversity in lionfish. In this sense, the analyses presented here, based on 1,220 nuclear SNPs from samples collected during a brief period of 20 months seems to be a more reliable way to determine lionfish’s current genetic structure.In lionfish, d-loop data revealed strong bottlenecks and scientists discarded the idea of multiple introductions into the NW Atlantic from the native range53. Different environmental pressures between the NW Atlantic and the Gulf of Mexico could also have favoured some lionfish mitochondrial haplotypes over others, thus shaping the spatial distribution of haplotypes during the invasion process. However, the sampling scheme used in our study and the lack of new mitochondrial sequences do not allow mitochondrial selection and/or adaptation hypotheses to be tested. None of the 1,220 tags containing the SNPs were identified as mitochondrial fragments, and the different analyses performed did not reveal evidence of local adaptation and/or nuclear selection across the lionfish’s invasive range. In some marine species with long-dispersal potential, outlier SNPs unravelled significantly finer genetic structure than neutral markers, suggesting the existence of local adaptation57. For example, in the European hake, Atlantic and Mediterranean populations showed sharper divergence in analyses using outlier SNPs than neutral SNPs54, a pattern of higher resolution that was also found in other fish species at small geographical scales of a few hundred kilometres when analysing outlier SNPs56. In some marine invaders, local adaptation also seemed to play an important role in shaping populations’ genetic structure, showing either latitudinal clines in outlier allele frequencies36 or significant correlation with environmental variables such as salinity and water temperature57. Nevertheless, in lionfish, differential selection between the NW Atlantic and the Gulf of Mexico, and mito-nuclear interactions remains as an open question because although we did not find strong evidence of local adaptation, 1,220 SNPs still represent a small proportion of the species’ genome, and selection on non-explored genomic areas could be possible.Besides neutral temporal trends, differential selection can also promote discrepancy patterns between mitochondrial and nuclear DNA. Mitochondrial selection under divergent environmental conditions plays an important role for the distribution of mitochondrial variants in other marine fish speciesThe SNP data here presented yield valuable information of the genetic trend in the lionfish invasion, which could potentially be affected by genetic changes over time and across space, although other hypotheses such as different selective pressures between mitochondrial and nuclear DNA cannot be completely discarded. Additionally, we perceive some limitations in our study that should be taken in consideration for further investigations. For instance, we noticed that population analyses based on SNPs should include: sizes over 10 individuals to retain the potential maximum genetic diversity within populations, representative populations from the native range to shed light on the current impact of bottlenecks in genetic diversity across the whole invasive range, and populations from the Caribbean Sea to clarify the most important connectivity routes within the invaded area.28, the lionfish invasion is an example of how reductions in genetic diversity do not necessarily compromise population establishment and spreading28 and points out the importance of primary (pre-border) and secondary (post-border) introductions, e.g., secondary introductions to the Caribbean Sea and later to the Gulf of Mexico27. The potential of lionfish to overcome these initial steps of the invasion with low genetic diversity at the mitochondrial DNA27, and to homogenize nuclear genetic structure across the invaded area (as shown in this study with SNPs), should be considered when developing theoretical models on the expected geographical spreading of this invasion58 and implementing appropriate strategies for its management and control.As demonstrated by previous publications based on mitochondrial DNA, which detected strong bottlenecks during the first introduction steps and invasion progressionP. miles invasion in the Mediterranean Sea. As P. volitans across the Wider Caribbean, P. miles has quickly colonized wide areas of the eastern Mediterranean59, which adds an additionally threaten in a small sea that is at the same time a hotspot of marine biodiversity and one of the world’s most impacted seas.Finally, the lionfish invasion to the Wider Caribbean can be used as a lesson to anticipate the genetic trend and potential impacts of P. volitans samples were collected over a 20 month period, between June 2013 and February 2015, from thirteen locations along Florida’s eastern coast (NW Atlantic and Florida Keys) and the northern Gulf of Mexico, at depths between 4 and 62 meters. Sampling sites and number of individuals genotyped are detailed in Table No endangered or protected species were involved in this study. Lionfish were sampled opportunistically by the authors from lionfish derbies or state and federal collections (as stated below); only dead lionfish were obtained. Lionfish were collected by a number of organizations in areas open to fishing with a spear or permitted by methods utilized. These fish were collected as a result of other activities such as tournaments, commercial harvest, and general fisheries surveys, and were sampled opportunistically for this study. No permits were required to collect lionfish beyond a state saltwater fishing license, which was in possession of divers at each collection. In the case of lionfish collected from offshore Florida, no fishing license is required. The University of Miami Institutional Animal Care and Use Committee (IACUC) did not require a protocol for this study since only dead specimens were donated to the University. State and Federal government organizations, although exempt from IACUC requirements, follow best practices to minimize pain and suffering of specimens. These are approved Institutional Animal Care and Use Committee protocols via the American Veterinary Medical Association Guidelines for the Euthanasia of Animals and the American Society of Ichthyologists and Herpetologists Guidelines for Use of Fish in Research.TM Broad Range dsDNA Quantitative Solution according to the manufacturer’s instructions. After quantification, 100 ng of DNA from each sample was dried down in 96-well plates in a SpeedVac concentrator. Samples were then rehydrated overnight with 5 µl of ultrapure milliQ water before further processing.Genomic DNA was extracted from tissue clips using silica columns. DNA quality was assessed via agarose gel electrophoresis, and DNA concentrations were quantified using Biotium AccuBlueApeKI. A total of 50 ng of genomic DNA per sample was digested at 75 °C for 2 hours. Unique barcoded adapters were used for library construction as described in60. A total of 229 DNA samples were pooled together and fragments approximately 300 bp in length were selected with magnetic beads. Primers complementary to the adapters were then used for library amplification60. Before sequencing, library quality was checked in an Agilent 2100 Bioanalyzer. The GBS library including the 229 individuals was sequenced in two lanes of an Illumina Hi Seq. 2500 using 75 bp single end reads at Elim Biopharmaceuticals, Inc. Hayward, CA.Genotyping by Sequencing libraries were constructed using the restriction enzyme 32 for species without a reference genome was used to call SNPs using Bowtie61. The software identifies SNPs found on single non-overlapping “tags” (64 bp sequences) initiated at the restriction sites. Only SNPs that had a minimum of five reads across all samples were retained to reduce the impact of sequencing errors. Loci with significant linkage disequilibrium  identified in TASSEL and those with significantly greater observed than expected heterozygosity  were removed from the database before performing further analyses. SNPs were then filtered to select individuals with at least 75% of the called loci and loci that were present in at least 84% of individuals. Maximum heterozygosity during filtering was set at 0.5 to avoid excess of heterozygotes due to sequencing errors. All sequences containing selected SNPs were blasted  against the mitochondrial DNA of Salmo salar and the Genbank database to identify mitochondrial fragments.The UNEAK GBS analysis pipeline in TASSELThe HapMap file including the whole dataset of SNPs here analysed, a coverage file and the genepop file including allele frequencies have been deposited in PANGAEA (10.1594/PANGAEA.886118).62 and Lositan63, were used to identify non-neutral SNPs, as candidate markers under selection, based on an FST-outlier detection method and coalescence simulations. Arlequin uses simulations based on observed heterozygosity (Ho) to create a null distribution of FST values and associated p-values for each locus. We performed a total of 20,000 simulations, with 100 demes, under a finite island model. This model was chosen due to the general lack of genetic structure (see Results). FDR correction of the p-values was applied to detect significant outliers; we also considered a more conservative approach with significance at p < 0.01 since strong corrections can increase type II error thereby assuming neutrality in SNPs that are not neutral55 . Lositan, on the other hand, creates a distribution based on the relation between FST values and expected heterozygosity (He). We performed a first run using all loci to estimate mean FST values with 20,000 simulations, 99% confidence interval, infinite alleles mutation model and false discovery rate of 0.1%. After the first run, loci in the confidence interval were removed, and “neutral” FST values were recalculated. A third run was finally performed using all loci, and the neutral FST values previously calculated were implemented to detect outliers. Finally, outliers recovered from both software programs, Arlequin and Lositan, were considered as candidate SNPs under selection.Two different software programs, ArlequinIS, and the Hardy Weinberg Equilibrium were calculated for all markers per population using Arlequin 3.5.1.262 and the “adegenet” package in R64.General descriptors of genetic diversity as mean number of alleles, observed heterozygosity (Ho), expected heterozygosity (He), fixation index F65 to explore potential clustering of individuals related to different geographical areas and/or sampling sites. The ML tree was then visualized and edited in Figtree 1.4.0 (http://tree.bio.ed.ac.uk/software/figtree/).A Maximum Likelihood (ML) tree, including all genotypes obtained, was reconstructed in RAxML with a GTR+ G model and 100 rapid bootstrap replicates66, was used to investigate the optimal number of major homogeneous genetic clusters (K) found within our datasets under the null hypothesis of genetic homogeneity. Because Bayesian analysis can be computationally very intense and long, an initial fast run was performed with a K from 1 to 13 with five independent replicates, 20,000 Markov chain Monte Carlo (MCMC) per replicate, and a 2,000 burn-in period to get a general idea about the maximum number of clusters expected. Then, a definitive run was performed with K from 1 to 8 with five independent replicates, 100,000 MCMC per replicate, and a 10,000 burn-in period. We used an “admixture model” and correlated gene frequencies as implemented in Structure. The five independent runs were averaged using the clumpak server67 (http://clumpak.tau.ac.il). The K value was determined by comparing the rate of change in the likelihood of K, using the ad hoc statistic ΔK in Structure Harvester 0.6.9468.A Bayesian clustering analysis, performed with the software Structure 2.3.470, we could not a priori assign these two sites (PR and DT) to one or the other area. Therefore, we performed three different AMOVA analyses: the first analysis included PR and DT within the NW Atlantic pool, the second included them within the Gulf of Mexico pool, and the third one excluded these two sites from the analysis. After testing differences between major marine areas, pairwise FST distances based on allele frequencies between all sampling sites were calculated with the same software. The significance of AMOVA and FST values was assessed after 50,000 non-parametric permutations of individuals among populations and/or populations between geographical areas and under the null hypothesis of genetic homogeneity. FDR correction of these p-values was applied for FST multiple testing71.Analyses of Molecular Variance (AMOVA), based on allele frequencies, were performed to specifically explore the potential genetic break between the two genetically different regions previously identified from mitochondrial DNA, the NW Atlantic and the Gulf of Mexico. The locations of PR and DT, at the Florida Strait, are rich mesophotic reefs and part of the Florida Keys reef complex but far inside the Gulf of Mexico. Since the genetic break between the NW Atlantic and the Gulf of Mexico shifts at different points of the Florida Strait depending on the speciesSignificant difference in genetic diversity (Ho and He) between the two marine regions, the NW Atlantic and Florida Keys , and the Gulf of Mexico  was evaluated with a t-test.72 were computed for the complete dataset. DAPC does not assume any underlying population genetic model and is not as affected by Hardy Weinberg disequilibrium as other methods based on genetic distances (e.g. FST and AMOVA) and Bayesian clustering analyses. We used collection sites as populations with the “adegenet” package in R64. DAPC extracts multivariate information from genetic datasets by first performing a principal component analysis (PCA) on predefined groups (collection sites in this case) and then using the PCA factors as variables for a discriminant analysis (DA), which seeks to maximize the inter-site component of variation. Thus. DAPC allows the visual identification of genetic clusters and can outperform more computer-intensive approaches, such as Structure, in detecting genetic structure72. Since the number of principal components (PCs) retained may have large impact on the DAPC output, the optimal number of PCs to be retained was first explored by the cross-validation method implemented by this package.Additionally, discriminant analyses of principal components (DAPC)ST and DAPC was comparatively applied to three different SNP datasets: for all isolated SNPs, for neutral SNPs, and for candidate SNPs under selection (outliers).To understand whether selection and/or local adaptation within the lionfish’s invasive range is an important driver of the genetic structure, the searching strategy explained before for the Bayesian clustering analysis, AMOVA, FSupplementary Information