Exobasidium maculosum, is an emerging disease that has rapidly increased in prevalence throughout the southeastern USA, severely reducing fruit quality in some plantings. The objectives of this study were to determine the genetic diversity of E. maculosum in the southeastern USA to elucidate the basis of disease emergence and to investigate if populations of E. maculosum are structured by geography, host species, or tissue type. We sequenced three conserved loci from 82 isolates collected from leaves and fruit of rabbiteye blueberry (Vaccinium virgatum), highbush blueberry (V. corymbosum), and southern highbush blueberry (V. corymbosum hybrids) from commercial fields in Georgia and North Carolina, USA, and 6 isolates from lowbush blueberry (V. angustifolium) from Maine, USA, and Nova Scotia, Canada. Populations of E. maculosum from the southeastern USA and from lowbush blueberry in Maine and Nova Scotia are distinct, but do not represent unique species. No difference in genetic structure was detected between different host tissues or among different host species within the southeastern USA; however, differentiation was detected between populations in Georgia and North Carolina. Overall, E. maculosum showed extreme genetic diversity within the conserved loci with 286 segregating sites among the 1,775 sequenced nucleotides and each isolate representing a unique multilocus haplotype. However, 94% of the nucleotide substitutions were silent, so despite the high number of mutations, selective constraints have limited changes to the amino acid sequences of the housekeeping genes. Overall, these results suggest that the emergence of Exobasidium leaf and fruit spot is not due to a recent introduction or host shift, or the recent evolution of aggressive genotypes of E. maculosum, but more likely as a result of an increasing host population or an environmental change.Emerging diseases caused by fungi are increasing at an alarming rate. Exobasidium leaf and fruit spot of blueberry, caused by the fungus  Many emerging plant diseases are caused by pathogens that have found new hosts or have been introduced to new areas, often as a result of human-mediated movement . Other pVaccinium virgatum), highbush blueberry (V. corymbosum) and southern highbush blueberry (V. corymbosum hybrids), but cultivars of rabbiteye blueberry seem to be the most susceptible. The fruit stage of the disease can result in losses of 60–70% in some plantings [Exobasidium maculosum [V. angustifolium) in Nova Scotia, Canada [Exobasidium on lowbush blueberry in New Brunswick, suggesting the presence of the pathogen in northeast North America for at least 100 years [E. maculosum populations from the southeastern USA are genetically distinct from populations from lowbush blueberry in northeastern North America based on the large subunit of the rDNA (LSU) [Vaccinium hosts have not been successful due to difficulty in reproducing disease symptoms [Exobasidium leaf and fruit spot of blueberry is an emerging disease that has rapidly increased in prevalence since 2011 throughout the southeastern USA in the commercial blueberry production regions of Georgia, North Carolina, and Mississippi ,15. The lantings . The cauaculosum . Althougaculosum , it was , Canada , and Bur, Canada  describe00 years . HoweverNA (LSU) ; but, reNA (LSU) , was notsymptoms , so it iExobasidium includes a diverse group of biotrophic plant pathogens that infect members of the Ericales, including Symplocus, Rhododendron, Camellia, and Vaccinium, including wild and commercial blueberry and cranberry species within Vaccinium section Cyanococcus. Exobasidium species cause a variety of plant deformities including localized leaf and fruit spots, systemic shoot infections characterized by reddened leaves, or tumors  on leaves, stems, flowers, shoots, and buds [Exobasidium species germinate to produce hyphae in culture [E. maculosum produces annual, localized infections that are characterized by chlorotic, circular, and sunken lesions with white, felt-like spots on the abaxial side. Basidiospores are produced from the felt-like hymenial layer. In culture, basidiospores of E. maculosum bud to form abundant yeast-like conidia [E. maculosum and the disease cycle of Exobasidium leaf and fruit spot of blueberry are not well understood.The genus and buds ,18,20–22and buds . Asexual culture . E. macu conidia . CurrentE. maculosum in the southeastern USA represents a different population or a distinct species from the isolates causing disease on lowbush blueberry in northeastern North America. The objectives of this study were to use a multilocus sequencing approach to estimate genetic diversity in E. maculosum to characterize demographic processes (founder effect vs. population expansion) underlying disease emergence, and to determine if populations are structured based on geography, blueberry host species, or host tissue (leaf or fruit) type.Our goals are to understand why Exobasidium leaf and fruit spot is emerging in the southeastern USA, to identify factors that may be driving population divergence, and to determine if E. maculosum were collected from cultivars of rabbiteye blueberry (V. virgatum), highbush blueberry (V. corymbosum) and southern highbush blueberry (V. corymbosum hybrids) from commercial fields in Georgia and North Carolina, USA. Isolates of Exobasidium causing leaf spot on lowbush blueberry (V. angustifolium) and isolates of E. rostrupii causing leaf spot on cranberry (V. macrocarpon) were obtained from infected leaves sent from Maine and New Jersey, USA, respectively. Leaf spots were excised with a sterile scalpel and tissue was affixed to the lid of a Petri dish with petroleum jelly, allowing basidiospores to fall or eject onto potato dextrose agar (PDA). After 8–12 hrs incubation in the dark at 23°C, single basidiospores were collected using a sterile dissecting needle and transferred to a new dish of PDA. Fruit spots were excised and surface sterilized in 10% sodium hypochlorite, plated on PDA, and incubated in the dark at 23°C. Single colony forming units likely derived from a single conidium (yeast cell) of Exobasidium isolates were obtained using a quadrant streaking protocol [Exobasidium causing leaf spot on lowbush blueberry in Nova Scotia, Canada were obtained from the collection of Nancy Nickerson . For this study there were 82 isolates of E. maculosum from blueberry from the southeastern USA (Georgia and North Carolina), 6 isolates of Exobasidium from lowbush blueberry from northeastern North America (Maine and Nova Scotia), and an isolate of E. rostrupii from cranberry for use as an outgroup for phylogenetic analyses , and calmodulin (CAL). For ITS, the primers used were ITS1 (5'- TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') [EF-1α, the primers used were EF1-728F (5'-CATCGAGAAGTTCGAGAAGG-3') and EF1-986R (5'-TACTTGAAGGAACCCTTACC-3') [CAL, the primers used were CAL-228F (5'- GAGTTCAAG GAGGCCTTCTCC C -3') and CAL-737R (5'-CATCTTTCTGGCCATCATGG-3') [EF-1α at 60°C) for 30 s, extension at 72°C for 5 min, followed by a final extension at 72°C for 5 min. PCR products were purified with QIAquick spin columns (Qiagen Sciences). All DNA fragments were sequenced at the Georgia Genomics Facility, University of Georgia, Athens, GA using the Applied Biosystems Automated 3730 DNA Analyzer with Big Dye Terminator chemistry and Ampli-Taq-FS DNA Polymerase. All gene regions were sequenced in both directions. Sequences were aligned and manually edited in Geneious v6 . There was a large amount of sequence variation among isolates so we tested the consistency of our sequencing results by randomly choosing six isolates for resequencing and comparison with the initial results. Sequences of ITS, EF-1α, and CAL were deposited in GenBank under accession numbers KR262338-KR262425, KR262258-KR262337, and KR262175-KR262257, respectively.Three nuclear loci were PCR amplified and sequenced from each isolate. Regions sequenced included the internal transcribed spacer region (ITS) of the ribosomal DNA, elongation factor 1-alpha (ATGC-3') ; for EF-TACC-3') ; and forATGG-3') . The PCRE. maculosum and E. rostrupii sequences, the conserved 5.8S region of the ITS rDNA and coding regions of CAL and EF-1α were identified by aligning a representative sequence of each region by BLAST in the NCBI database (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Searches were made using the blastn (for ITS) and blastx (for CAL and EF-1α) algorithms that search a nucleotide database and a protein database using a translated nucleotide query, respectively. The two best fungal species hits were aligned to each gene region and used to identify the conserved or coding regions for each locus. For EF-1α and CAL, translation of the coding regions was performed in Geneious v6. For the entire E. maculosum population, the number of segregating sites in each locus, the number of substitutions in the 5.8S rDNA, the number of synonymous substitutions and nonsynonymous substitutions in coding regions of EF-1α and CAL, and dN/dS ratios for EF-1α and CAL were determined in DnaSP v5 [For DnaSP v5 .E. rostrupii was used as the outgroup. DT-ModSel [E. rostrupii. BI was performed with parameter settings suggested by the best-fit nucleotide substitution models. The Markov chain Monte Carlo (MCMC) search was run with four chains for 3,000,000 generations generating 30,001 trees; the first 6,000 trees were discarded as "burn-in" of the chains.Phylogenies were inferred for each locus using maximum likelihood (ML) in PAUP*4b v10  and BayeT-ModSel  was usedExobasidium from Maine and Nova Scotia (northeastern North America or NE), E. maculosum from Georgia and North Carolina (southeastern USA or SE), E. maculosum from Georgia (GA), E. maculosum from North Carolina (NC), and E. maculosum and Exobasidium from lowbush blueberry (NE+SE). The number of unique haplotypes, haplotype diversity (hD), pairwise nucleotide diversity (π) [θw) [D [F [Haplotype networks were constructed for each locus using parsimony in the program TCS v1.21 with a 92% similarity cutoff . Isolatesity (π) , Wattersπ) [θw) , and the[θw) [D  and Fu aw) [D [F  in DnaSPw) [D [F . The minw) [D [F  that occKST* [Snn [n = 4; SE, n = 72; NC, n = 12; GA, n = 60), original host within the southeastern USA , or tissue type . The statistics were determined using the combined loci dataset. KST* is estimated by determining the proportion of genetic diversity within populations compared to the diversity in the total population. Hudson’s nearest-neighbor statistic (Snn) is a measure of how frequently the most similar sequence or "nearest neighbor" is from the same assigned population. Snn approaches one when populations are highly differentiated and near one-half when populations are panmictic [Two measures of genetic differentiation, KST*  and SnnT* [Snn  were useanmictic . SignifiE. maculosum from the southeastern USA, 6 isolates from V. angustifolium in northeastern North America, and an isolate of E. rostrupii from V. macrocarpon (cranberry) collected from New Jersey, USA that was used as an outgroup. Haplotype diversity (hD) was very high across all loci for all populations. In the combined dataset, each of the 76 isolates had unique multilocus haplotypes with a haplotype diversity (hD) of 1.00 (EF-1α with nucleotide diversity (π) of 0.024 and 182 segregating sites among the 949 sequenced nucleotides  occurring within the entire population. Lower levels of recombination were observed for ITS and CAL with 7 and 8 unique recombination events occurring within the combined population, respectively.A total of 1775 nucleotides were sequenced for 82 isolates of  of 1.00 . The mosleotides . HoweverD and Fu and Li’s F were estimated for ITS, EF-1α, and CAL (p ≤ 0.05), indicating positive selection at or near this locus. Most estimates for the other loci in each population were negative. The slightly negative, non-significant values observed at all other loci are indicative of purifying selection. Selection was further investigated by determining if substitutions within coding regions of EF-1α and CAL were synonymous or non-synonymous and if the 5.8S region between ITS1 and ITS2 was conserved. BLASTn analysis of the ITS nucleotide sequence from E. maculosum against the GenBank database resulted in two best hits of E. inconspicuum  and E. arescens . BLASTn analysis of EF-1α nucleotide sequence from E. maculosum against the GenBank database resulted in two best hits of E. rhododendri  and E. gracile . Only two non-parsimonious segregating sites were observed within the 5.8S region among all isolates. The putative coding regions and translated amino acid sequences of E. rhododendri and E. gracile were used to determine the putative coding regions and amino acid sequence of EF-1α for E. maculosum. Out of the total 182 segregating sites within the coding region, 176 were synonymous, and only 6 resulted in an amino acid change  and Rhizoctonia solani AG-3  were used to identify the putative coding regions and amino acid sequence for CAL in E. maculosum. Out of 40 segregating sites in the coding region, only 8 resulted in amino acid changes. The dN/dS ratios for EF-1α and CAL were 0.03 and 0.20, respectively, which indicates that although nucleotide diversity is high, purifying selection is acting on these genes to conserve the amino acid sequence of the proteins.Tajima’s  and CAL  to deterd change . FurtherExobasidium population from lowbush blueberry in northeastern North America represents a distinct species, ML and BI phylogenies were generated for each of the three loci. The best-fit model of DNA evolution for ITS and EF-1α was equal-frequency Tamura-Nei with invariable sites and a gamma distribution (TrNef+I+G) [CAL was the generalized time reversible with a gamma distribution (GTR+G) [P = 0.001), thus loci were not combined for phylogenetic analyses. For ITS, isolates from lowbush blueberry in northeastern North America (NE) clustered in a well-supported clade with 0.99 posterior probability (pp) and 100% bootstrap support (bs); however, two isolates from Georgia (GA) also clustered with this group   clustered into a single clade (To determine if the Nef+I+G) , and for (GTR+G) . Based ois group . Additio100% bs) . For CALphyletic . Only thα or CAL . Furtherle clade .E. maculosum. Each network contained an extensive number of haplotypes with very few repeated haplotypes (EF-1α networks (CAL network (P < 0.001) between the NE and SE populations based on estimates of population structure KST* and Snn on the combined dataset . Isolates collected from fruit and leaf spots were not genetically distinct based on gene trees, haplotype networks or estimates of population structure  indicated recombination between the NE and SE populations within each locus  and within the combined dataset (Rmin = 58). There was also extensive recombination within each population  for each locus and the combined dataset  and a high level of recombination within and among loci . Within ITS, we identified 77 segregating sites for E. maculosum, whereas there are very few, if any, segregating sites for the ITS region for most fungal species [Batrachochytrium dendrobatidis, collected from South Korea and other parts of Asia where at least 50 ITS haplotypes have been identified [E. maculosum within the housekeeping genes EF-1α and CAL. Although the sequences were diverse, we found purifying selection was acting within the coding regions. Historically, synonymous substitutions that do not change the amino acid sequence of proteins were thought to have no functional consequences, and thus were neutral and of little biological significance [E. maculosum populations [E. maculosum genome for the presence of DNA repair and RIP machinery and to identify if this high level of diversity occurs within segments of the genome or whether it is widespread. The extreme genetic diversity we observe in E. maculosum indicates that the species has a high evolutionary potential providing it with an adaptive advantage [The extremely high genetic diversity observed for  species ,44. In f species . Though entified . Similarificance . Recent ificance . Synonymificance . Selectiulations ,52. Withdvantage .E. maculosum. The number of recombination events that occurred both within and among loci for the entire population suggests that E. maculosum populations are outcrossing. Most Exobasidium species produce both sexually-derived basidiospores, through a thick-walled basidium that is formed on the tissues of their hosts, and asexually-derived, yeast-like conidia [Exobasidium some species have a homothallic, or self-compatible, mating system, rather than a heterothallic, or self-incompatible, mating system. Single-conidial isolates of E. vaccinii and E. japonicum inoculated onto V. vitis-idaea and Azalea indices, respectively, formed the sexually derived basidia and basidiospores [Exobasidium are homothallic. We know that E. maculosum undergoes sexual reproduction because it produces basidia and basidiospores [E. maculosum is homothallic or heterothallic, as fungi with both homothallic and heterothallic mating systems have the ability to outcross [In addition to a high population mutation rate, recombination contributes to the high genetic diversity in  conidia . Sundstr conidia  showed tiospores ,56. Howeiospores ,16, but outcross ,58.Exobasidium species have been studied primarily at the genus level using morphological and molecular systematics [Exobasidium species at the population level, which has allowed for a better examination of the phylogeography and evolutionary history of E. maculosum. One aim of this research was to understand why Exobasidium leaf and fruit spot is emerging on commercial blueberry in the southeastern USA. Often, emerging diseases arise when pathogen populations become more aggressive, are found on novel hosts, or occur in new geographic locations [E. maculosum, haplotype diversity reached the highest value possible (hD = 1) with each isolate represented by a unique multilocus haplotype, demonstrating extremely high genetic diversity [E. maculosum have been established in the southeastern USA for much longer than the recent emergence. Additionally, the geographic population structure detected within the southeastern USA suggests that E. maculosum has had a historical presence in this region. Causes for the recent emergence of this disease are not clear, but could be the result of increasing populations of blueberry hosts within North Carolina and Georgia. Acreage of commercial blueberry plantings within the southeastern USA has increased rapidly within the last 15 years. In 1999, the area of harvested blueberry in Georgia was 4,400 ha and increased to 5,463 ha in 2013, with a similar trend occurring in North Carolina . Another explanation for disease emergence is a change in the environment, such as increasing winter temperatures or greater precipitation. Since E. maculosum can exist in a yeast form, populations could have persisted for years on the leaf phylloplane or as endophytes of Vaccinium hosts without causing symptoms, but became pathogenic when environmental or host plant conditions became conducive. This switch from non-pathogen to pathogen has been hypothesized for fungi that are able to exist as both endophytes and pathogens, such as those collected from grasses [tematics ,54,59,60ocations . When diocations . In popuiversity . These r grasses ,65.D and Fu and Li’s F to determine if populations had undergone population bottlenecks or expansions. In populations where bottlenecks have occurred, there is a reduction in low-frequency polymorphisms that generates positive Tajima’s D values [D and Fu and Li’s F will be significantly negative across multiple loci, which is caused by an excess of recent mutations that produce a star phylogeny [D and Fu and Li’s F estimates showed no evidence for population bottlenecks or expansions in E. maculosum as most estimates did not significantly deviate from neutrality. It has been shown, however, that inferring demographic history in the presence of mutation rate heterogeneity is difficult [E. maculosum has remained constant further supporting our conclusion that disease emergence is the result of an environmental change or a change in the host population.To better understand the demographic history of this pathogen, we used the neutrality estimates Tajima’s D values ,67, wherhylogeny ,69. Tajiifficult . OverallV. angustifolium and in the southeastern USA on commercially produced blueberry hosts including V. virgatum, V. corymbosum, and V. corymbosum hybrids [Exobasidium collected from V. angustifolium in northeastern North America and E. maculosum from commercial species in the southeastern USA were genetically distinct [E. maculosum, but not distinct species. Well-supported reciprocal monophyly of the NE population was observed for EF-1α but not for CAL or ITS, possibly due to incomplete lineage sorting or hybridization. The haplotype network for CAL showed reticulation between the NE and SE population suggesting historic or contemporary recombination. Ongoing gene flow between populations may be preventing speciation between the populations, or the populations are undergoing speciation or have recently speciated, but not enough time has passed for reciprocal monophyly to occur for the majority of gene trees.Exobasidium leaf and fruit spot disease is found in the eastern Canadian provinces of Nova Scotia, New Brunswick, Quebec, and Ontario  and Main hybrids . Exobasidistinct , but it distinct  is incredistinct –74. UsinE. maculosum were genetically structured by original blueberry host species, host tissue (leaf or fruit), or geography. We found that the main driver of population structure in E. maculosum is geography. Within the southeastern USA, genetic differentiation was not detected based on original blueberry host species or host tissue type suggesting that these factors are not driving population structure. This is also supported by the gene trees and haplotype networks. However, significant differentiation was detected between E. maculosum populations from northeastern North America and the southeastern USA. The genetic structure between these populations was suggested by previous results [E. maculosum. All isolates from northeastern North America were collected from lowbush blueberry (V. angustifolium), which is genetically distinct from the blueberry species sampled in the southeastern USA [KST* was significant between E. maculosum isolates from Georgia and North Carolina suggesting that geography is a main driver for population divergence within the southeastern USA and that there is limited dispersal of E. maculosum between these regions.To better understand the emergence and spread of this disease we determined if populations of  results . Differetern USA ,76. InteS1 FigChanges of amino acids at a single position are highlighted in red, and sites highlighted in shades of grey indicated amino acid variation.(PDF)Click here for additional data file.


Thymelicus that represent a gradient of diverging ecological traits. We analyzed landscape effects on their gene flow by deriving inter-population connectivity estimates based on different species distribution models (SDMs), which were calculated from multiple landscape parameters. We then used SDM output maps to calculate circuit-theoretic connectivity estimates and statistically compared these estimates to actual genetic differentiation in each species. We based our inferences on two different analytical methods and two metrics of genetic differentiation. Results indicate that land use patterns influence population connectivity in the least mobile specialist T. acteon. In contrast, populations of the highly mobile generalist T. lineola were panmictic, lacking any landscape related effect on genetic differentiation. In the species with ecological traits in between those of the congeners, T. sylvestris, climate has a strong impact on inter-population connectivity. However, the relative importance of different landscape factors for connectivity varies when using different metrics of genetic differentiation in this species. Our results show that closely related species representing a gradient of ecological traits also show genetic structures and landscape genetic relationships that gradually change from a geographical macro- to micro-scale. Thus, the type and magnitude of landscape effects on gene flow can differ strongly even among closely related species inhabiting the same landscape, and depend on their relative degree of specialization. In addition, the use of different genetic differentiation metrics makes it possible to detect recent changes in the relative importance of landscape factors affecting gene flow, which likely change as a result of contemporary habitat alterations.To understand how landscape characteristics affect gene flow in species with diverging ecological traits, it is important to analyze taxonomically related sympatric species in the same landscape using identical methods. Here, we present such a comparative landscape genetic study involving three closely related Hesperid butterflies of the genus  In the theory of island biogeography, McArthur & Wilson Understanding the effects of the landscape matrix on realized dispersal and functional population connectivity is also a major focus of landscape genetics Many studies assess landscape effects on gene flow in only a single species. However, to understand how landscape effects on gene flow differ between species, and to take effective conservation actions, it is important to analyze multiple species in the same landscape using identical methods Generalist species can be found in a broader variety of ecosystems, showing higher abundances and broader spatial distributions. In contrast, specialist species demanding certain habitat conditions are often geographically restricted to specific habitats and usually occur in lower local abundances Thymelicus (Hubner 1890). The three species include the generalist T. lineola, which occurs in high abundances and shows strong dispersal propensity; the specialist T. acteon which is sedentary and occurs restricted to specific habitats; and T. sylvestris, which lies in between these two extremes in terms of habitat specificity and dispersal abilities. Using these three species, we (i) investigate the impact of ecological traits on species-specific functional landscape connectivity and (ii) determine the overall relevance of landscape characteristics for connectivity in each species, as well as the individual importance of topography, climatic conditions and land-use parameters. We hypothesized that species-specific landscape effects on gene flow should follow the cline of specialization in the three Hesperid butterflies, with strongest landscape effects on genetic differentiation in the most specialized T. acteon and weakest landscape effects in the generalist T. lineola.In this study, we present a comparative landscape genetic analysis involving three closely-related butterfly species, to assess the impact of landscape parameters  on the genetic structure of sympatric species with different ecological traits. We re-analyzed a molecular dataset taken from a previous study The research was conducted under permission, to collect butterflies and to work in several protected areas, by the local authorities of Saarbrücken , Koblenz , Luxembourg, and Metz . Imagoes of the respective species were stored in liquid nitrogen until genetic analysis.Thymelicus species, acting as valuable retreats and stepping stones Our study area is located in the south-west of Germany and includes adjacent parts of France and Luxembourg . The samT. sylvestris, T. lineola and T. acteon are closely related to each other with T. lineola and T. acteon being most distant related and where T. sylvestris clusters to a monophylum with T. acteon  sampled at 12 locations which were distributed across the same study area. Sample sizes ranged from 17 to 44 individuals per species and location. Thymelicus sylvestris and T. lineola were sampled at identical locations, while T. acteon was not found at four of the sampled locations and the data set was supplemented by one additional location  as well as their spatial delineation was evaluated for each species separately using the genetic clustering method implemented in the software Genelandi) genetic differences can occur without any obvious landscape pattern , which in turn would lead to (ii) erroneous conclusions on isolation-by-distance IBD/isolation-by-resistance IBR analyses on spatially independent structured data. Geneland assigns geo-referenced individuals to genetics clusters (K) that maximize Hardy-Weinberg-and Linkage-Equilibrium. K was treated as unknown to allow Geneland to vary K within a given range between 1 and the maximum number of populations depending on the species (i.e. 7 in T. acteon and 11 in both T. sylvestris and T. lineola). Markov Chains were run for 3,000,000 generations and sampled every 1000th generation, after an initial burn-in of 300 samples after thinning (10%). Markov Chains with these settings were run 10 times independently and the iteration with the highest log posterior probability was chosen for inferring the most likely K and individual assignments.Prior to inferring landscape effects on genetic differentiation, the number of genetic groups  incorporating topographic, bioclimatic and/or land use features. We then used resulting SDMs as resistance surfaces to derive inter-population connectivity estimates based on electrical circuit theory, and statistically compared these connectivity estimates to actual genetic differentiation. SDMs are increasingly applied for resistance surface parameterization in landscape genetic studies Thymelicus species in the study area, presence data were taken from personal observations of JCH, D. Louy and T. Schmitt (Germany) covering the years 2003–2012. Further presence data were added from high resolution records downloaded from the GBIF database (www.gbif.org). The final datasets comprised 67 records for T. sylvestris, 62 for T. lineola and 28 records for T. acteon. Given their specific habitat demands and the sampling effort that was performed across the study area for either species , we selected a subset of variables . Finally, CORINE land use related data was obtained from the European Environmental Agency (www.eea.europa.eu). We either used CORINE 2006 data to assess current habitat availability as well as CORINE 1990 data for assessing recent land use changes. All environmental layers were re-sampled to uniform grid resolution of 90 m.For construction of the SDMs, we used freely available GIS based environmental layers. Bioclimatic data based on monthly averaged temperature and precipitation data with 30 arc seconds spatial resolution was obtained from the Worldclim Database  to predict spatial patterns of environmental suitability. The SDM output represents the likelihood of species potential occurrence across a geographic area of interest  to 1 (fully suitable). For model evaluation, the area under the receiver operating characteristic curve (AUC) was used We defined a set of hypotheses based on the available environmental data and generated five variable sets for comparing landscape effects on species-specific gene flow  and associated unit resistors  that connecting two nodes and calculates resistance distances between focal locations based on a nodal analysis algorithm as described in The resulting SDMs were used as conductance surfaces  using linear regression models as well as multiple regressions on distance matrices (MRDM) ecodist package for R was used Resulting resistance values among locations were statistically compared to estimates of genetic differentiation , while T. acteon showed highest genetic diversities . Thymelicus sylvestris showed an intermediate level of genetic diversity, as compared to its congeners . The genetic differentiation was low in T. lineola , while we detected highest genetic differentiation for T. acteon . Again, Thymelicus sylvestris showed an intermediate level of genetic differentiation, with a rather low among-population variance  (No significant deviation from Hardy-Weinberg equilibrium was detected for any population in the respective species. Genetic diversity was comparatively low in <0.0001) .eneland runs stabilized long before the end of the Markov Chains, indicating that convergence was reached  was most important, followed by a set of temperature related variables . Using estD, the IBD scenario produced the best model  however with a weak relationship . Furthermore, MRDM showed no landscape related signals for either estimate of genetic differentiation in T. lineola, suggesting that gene flow in this species is not affected by any spatial or landscape features at this scale. The most specialized species, T. acteon showed no significant IBD, but significant IBR for two scenarios (land use & land use change) with both stF and estD under multi-model inference. These signals become also prominent using MRDM for inference, even though models were slightly insignificant at p = 0.05 . The combined results from AIC and MRDM suggest that land use and land use change both affect genetic differentiation among T. acteon populations. Genetic differentiation in Thymelicus sylvestris corresponded most strongly to the connectivity estimates derived from the SDM incorporating all variables  using Fst and the information-theoretic approach. The climate related scenario was also within the most reliable models under AICc . However, MRDM suggested that land use and land use change were also important for explaining genetic differentiation in this species. The opposite becomes obvious using Dest as differentiation metric. Here, the information theoretic approach reveals climate, land use and land use change as highly informative, with climate being most important . Surprisingly, the scenario covering the entire variable set contributed nearly no information . In addition, MRDM highlighted only climate as significantly related to genetic differentiation. In summary, the combined results of different differentiation metrics and inference methods suggest that the climatic conditions across the study site deliver the most important and stable relationship for adjusting gene flow in the intermediate species, with additional effects of land use. Classical IBD received less support against IBR models  might have become more influential in the most recent times. Land use as well as changes in land use patterns (i.e. assessed over a 16yr period) influences the connectivity of T. acteon populations. In contrast, T. lineola populations were panmictic, lacking any landscape related effects on genetic differentiation at this spatial scale.The obtained results show strong genetic differentiation and high genetic diversities in the specialist species Our data illustrate that closely related species representing a gradient of ecological traits  also show a gradient of changing genetic structures and even more interesting of changing landscape genetic associations . This hiT. acteon is concordant with its patchy occurrence predicted in our SDMs , land-use-change might be even more important, when addressing land-use-change over an even larger time period than the 16 years used here. Unfortunately, there is no information available to assess past land-use-changes covering this large geographical extent further into the past. Keeping time-lags between fragmentation and genetic responses accompanying these fragmentations in mind  are highest in T. acteon, the generalist T. lineola represents opposing genetic features: the species shows a broad ecological amplitude and a much higher mobility eneland results.In contrast to the specialist T. sylvestris, has an abundance like T. lineola but shows a sedentary dispersal behavior comparable to that of T. acteonstF vs. estD/multimodel inference vs. MRDM), landscape resistance based on the climate scenario was most important, delivering a consistent strong signal across the different inference methods used . In T. sylvestris the climate related SDM revealed high resistances along river valleys as well as on the higher elevations of the low mountain ranges  in the landscape matrix where methodological shortcomings can be excluded . However, the obtained IBR models explain only up to 24% of the variance in our dataset. That in turn indicates that the remaining variance of our data can only be explained by additional factors such as ecological traits and habitat requirements. These can be even more relevant for butterfly species than habitat size and habitat isolation, e.g. as shown for the Heath butterfly Coenonympha tulliaFinally, the species standing in-between these two extremes, n ranges . These pThymelicus sylvestris, the prominent signal under stF arising from the SDM using all landscape variables becomes completely eliminated when using estD as a differentiation metric. The fact that different metrics can lead to different conclusions is also evident in the ongoing debate about the utility of different genetic differentiation measures stF -like metrics are more sensitive to recent demographic changes (which depends e.g. on effective population size) than metrics which are independent of effective population size, such as estDstF more sensible to effects of gene flow or drift in comparison to estD. Thus, from a landscape genetic perspective, using different types of differentiation metrics allows to test for the contribution of landscape effects in contemporary versus past times. If landscape composition change over time (and consequently the amount of gene flow mediated by the landscape), stF would respond much quicker to those changes while estD remains rather stable over time. In the case of Thymelicus sylvestris, this means that estD may highlight the landscape effect (here climate) of highest importance for gene flow in this species in former times, whereas stF highlights more recent landscape effects on genetic structure that involves also other landscape elements beside climate such as land use and topographical elements.Interestingly, in the case of T. sylvestris is indeed common but not very mobile, anthropogenic land transformations of the past decades might now lead to a stronger fragmentation of populations which ultimately lead to changes in the contributions of landscape factors shaping gene flow as shown elsewhere sensuT. acteon and T. lineola showing both consistent results across the different metrics underpinning their stable state in terms of their abundance (insentinent and widespread vs. sensible and endangered) and specialization .In contrast to the climate-only scenario, connectivity estimates involving all variables did not give highest importance to climatic factors. In particular, land use and slope contribute almost 54% of the total importance of this scenario, whereas the best performing variables from the climate scenario, bio11 (mean temp of coldest quarter) and bio7  that contribute together 54.9%, contributing under the full model just 12.7% of the total importance. This might highlight the change of landscape factors important to gene flow in this species. As T. sylvestris) also interact with the landscape but at coarser scales in comparison to specialist species (here T. acteon). However, in light of global change such species might be on the highest risk due to negative genetic effects such as inbreeding depression, because changes in the habitat matrix can push former meta-population into isolated remnants T. sylvestris comparing the genetic structure under either stF or estD. Further studies focusing on the degree of habitat specialization in addition to dispersal capabilities are needed, ideally conducted with closely related taxa in other areas. Such comparative studies will greatly expand our current understanding of landscape genetic relationships and ultimately lead to more effective conservation and management of biodiversity.Taxonomically close relatives serve as ideal model systems to study interspecific characteristics in ecological traits without confounding effects derived from different evolutionary histories. Yet, studies investigating the role of landscape on gene flow of closely related taxa inhabiting the same environment are still scarce. Our results reveal that even between sibling species, gene flow is affected by the landscape in very different ways. Thus, it is challenging to predict landscape genetic relationships in one species from a study involving another species, even if the two species are taxonomically closely related. Nevertheless, some generalizations are possible for specialist versus generalist species. In our study, the genetic structure of the generalist species with high dispersal propensities remained unaffected by the current landscape matrix, whereas specialist species were highly sensitive to fine scale habitat features. Changes of these features might therefore affect specialists more readily than generalist species with the negative consequences for their genetic setup. Species with an intermediate degree of specialization  Convergence of the MCMC after thinning (see methods for details). Values prior to burn-in (indicated as red dashed line) were not considered as chain does not reached convergence. B) Frequency of the estimated number of populations along the chain after burn-in.(DOC)Click here for additional data file.Figure S2Thymelicus sylvestris is shown at the upper half, T. acteon at the lower half). Note that just the most prominent isolation by resistance pattern is shown (i.e. climate in T. sylvestris and land use change in T. acteon).Scatterplots showing the differences of isolation by distance patterns with isolation by resistance patterns in the two species that show a spatial genetic structure ((DOC)Click here for additional data file.Table S1Geographic coordinates of the sampling locations. ID numbers correspond to those stated in (DOC)Click here for additional data file.Table S2Description of the landscape data used for resistance surface building depending on the scenario assumed. Note that the scenarios ‘land use’ and ‘land use change’ used the same data source. SDM refers to species distribution model.(DOC)Click here for additional data file.Material S1Thymelicus butterflies.Evolutionary history of the three (DOC)Click here for additional data file.


Salvelinus malma) spawn in nearly all accessible streams throughout the watershed, including those draining directly to an estuary, Chignik Lagoon, into larger rivers, and into lakes. Collections of Dolly Varden fry from 13 streams throughout the system revealed low levels of population structure among streams emptying into freshwater. However, much stronger genetic differentiation was detected between streams emptying into freshwater and streams flowing directly into estuarine environments. This fine-scale reproductive isolation without any physical barriers to migration is likely driven by differences in selection pressures across freshwater and estuarine environments. Estuary tributaries had fewer larger, older juveniles, suggesting an alternative life history of smolting and migration to the marine environment at a much smaller size than occurs in the other populations. Therefore, genetic data were consistent with a scenario where isolation by adaptation occurs between populations of Dolly Varden in the study system, and ecological data suggest that this isolation may partially be a result of a novel Dolly Varden life history of seawater tolerance at a smaller size than previously recognized.Numerous studies of population genetics in salmonids and other anadromous fishes have revealed that population structure is generally organized into geographic hierarchies (isolation by distance), but significant structure can exist in proximate populations due to varying selective pressures (isolation by adaptation). In Chignik Lakes, Alaska, anadromous Dolly Varden char ( Genetic population substructuring has been detected in Northern Hemisphere marine and anadromous fishes, often resulting from postglacial colonization . Patterns of population structure for streams draining into freshwater were relatively subtle and generally mirrored those of sockeye salmon in this system  in streams throughout the Chignik watershed, and one from an adjacent watershed Table , Fig. 2 z-test to compare the proportion of fish in each habitat that were larger than 80 mm FL and 115 mm FL, which are minimum size estimates of age 1+ and 2+ fish, respectively, from previous otolith analysis of Chignik Lakes Dolly Varden . Genotypes were scored independently by two researchers. Genotypes were compared, and fish with discrepancies were reamplified and rescored until discrepancies were resolved. Two quality control measures were employed. First, PCR amplifications were repeated for 8% of the samples, size-separated, and rescored to check and correct for laboratory errors. In addition, DNA was extracted a second time from approximately every 25th sample collected and genotyped to quantify laboratory error rates. Individuals with >2 missing genotypes were removed from further analyses.Genomic DNA was extracted using a DNeasy 96 Tissue Kit , and genetic variation was assessed at 11 microsatellite loci developed from ma Table . PolymerWe used the maximum likelihood method implemented in the program COLONY 2.0.3.5 Wang  to identhttp://www.montana.edu/kalinowski/) was then used to test for the presence of null alleles. Exact tests for deviations from Hardy–Weinberg and linkage equilibrium were conducted for each locus in the program GENEPOP 4  given by the program and the ΔK method , geographic distance, and salinity. Geographic distance was the shortest waterway distance between the stream mouths of each pair of collections and was estimated by hand using ARCGIS 10 . Mean values for salinity in ‰ for each Estuary stream were obtained from lagoon surface water near stream inlets every 10 days throughout the sampling period.Simple and partial Mantel tests with 1000 randomizations were used to test for relationships between genetic differentiation (pairwise FST) and geographic distances then implements the Monmonier's maximum difference algorithm  = 162595.5, Z = 4.78, P < 0.001. In addition, significantly more large individuals (>80 mm FL) were found in Freshwater streams (16.76%), compared to Estuary streams . Similarly, no individuals >115 mm fork length were found in Estuary streams, indicating few larger, older individuals in Estuary stream habitat compared to Freshwater streams where they comprised 8.6% of the fish encountered.Genetic samples were obtained from 1017 fry representing 13 collections in the Chignik system  after removing siblings and pooling collections from multiple years Table . AnalysiFST across the entire data set was 0.039 and pairwise FST values ranged from −0.001 for the Boulevard Creek – Alec Tributary 1 comparison, to 0.125 for the Cloud Creek – Indian Creek comparison , we used the number of sibling groups per 100 fish as a proxy for population size. This analysis revealed that several of the Freshwater streams and Estuary tributaries had similar sibling encounter rates, but the Freshwater populations did not show high levels of neutral genetic differentiation from nearby streams. Small Estuary and Freshwater streams are therefore both likely to have few spawning adults. However, even small numbers of spawners straying from Freshwater to Estuary streams would quickly erode the barrier to gene flow we observed between the two regions. It is therefore unlikely that the differentiation observed between Freshwater and Estuary populations is primarily caused by genetic drift or founder effects. Instead, these data are consistent with reproductive isolation facilitated by selection for alternate juvenile life histories in the Freshwater and Estuary habitats.FSTs between Estuary and Freshwater tributaries in Chignik were much greater than other estimates of FST for Dolly Varden from rivers that may be separated by several watersheds, and 100's of km of marine waters, such as rivers of Alaska's North Slope evaluated with similar techniques  during summer sampling indicates that there may not be suitable overwinter habitat in these streams, and rearing time in freshwater may be reduced. Poor environmental conditions  in winter in these streams may compel young Dolly Varden to leave Estuary streams in search of more tolerable habitat, possibly ascending Chignik River, a migration which would require travel through waters of salinity ranging 6–30 ‰. The ability for small Dolly Varden to survive in seawater is unstudied, but in closely related Arctic char Salvelinus alpinus, osmoregulatory capacity of juveniles varies by population . Under these circumstances, other studies have often shown little to no significant variation among collections. Most studies identifying highly divergent populations in anadromous fishes have been conducted on much larger spatial scales , or fine spatial scales where movement is confined by physical barriers (Whiteley et al. The Chignik watershed is an apparent hotspot for within species diversity, possibly as a zone of contact among divergent lineages. Extensive life history, morphological, or genetic diversity has been demonstrated in several species Narver . AlthougThis research highlights the importance of identifying both genetic and life-history diversity at appropriate spatial scales for management. Regional management of anadromous fishes is often driven by a single large stock, or an assumed metapopulation of stocks (Schtickzelle and Quinn


Danaus plexippus) are best known for undertaking a spectacular long-distance migration in eastern North America; however, they also form non-migratory populations that breed year-round in milder climates such as Hawaii and other tropical locations. Prior work showed an inverse relationship between monarch migratory propensity and the prevalence of the protozoan parasite, Ophryocystis elektroscirrha. Here, we sampled monarchs from replicate sites within each of four Hawaiian Islands to ask whether these populations show consistently high prevalence of the protozoan parasite as seen for monarchs from several other non-migratory populations. Counter to our predictions, we observed striking spatial heterogeneity in parasite prevalence, with infection rates per site ranging from 4–85%. We next used microsatellite markers to ask whether the observed variation in infection might be explained by limited host movement and spatial sub-structuring among sites. Our results showed that monarchs across the Hawaiian Islands form one admixed population, supporting high gene flow among sites. Moreover, measures of individual-level genetic diversity did not predict host infection status, as might be expected if more inbred hosts harbored higher parasite loads. These results suggest that other factors such as landscape-level environmental variation or colonization-extinction processes might instead cause the extreme heterogeneity in monarch butterfly infection observed here.Host movement and spatial structure can strongly influence the ecology and evolution of infectious diseases, with limited host movement potentially leading to high spatial heterogeneity in infection. Monarch butterflies ( Much work during the past two decades has focused on understanding the spatial ecology of host-pathogen interactions. Some studies have shown that genetic variation in traits affecting host resistance and pathogen virulence can generate spatial variation in infection patterns Host dispersal patterns can have important consequences for spatial processes and the ecology and evolution of host-pathogen interactions Ophryocystis elektroscirrha, hereafter called OE) infecting monarch butterflies (Danaus plexippus) on the island chain of Hawaii. Monarchs inhabit islands and continents worldwide and occupy a subset of the range of their larval milkweed host plants Asclepias physocarpa, Calotropis gigantea, and C. procera. Hawaiian monarchs are smaller than North American migratory monarchs Here, we examined spatial heterogeneity in the occurrence of an obligate protozoan parasite , we further used neutral genetic (microsatellite) markers to examine evidence for host population structure. In particular, we asked whether genetic evidence indicates that host movement within and among islands might be limited, such that between-site variation in prevalence could be attributed to locally structured host sub-populations that are isolated from other patches. Finally, we asked whether measures of host neutral diversity (as indicators of genome-wide heterozygosity) might correlate negatively with parasite infection probability at the individual or patch level, as suggested by prior work in Soay sheep, sea lions and several other species, whereby animals with greater genome-wide diversity can better resist parasite infections than more inbred hosts In this study, we sampled monarchs and recorded Calotropis or Asclepias spp.) occur  across four islands in Hawaii: Hawaii (Big Island), Oahu, Maui, and Kauai ; Table 1.) occur . Sites wThe field collections for this project did not involve endangered or protected species. We collected at three different private sites  after receiving permission from S. and A. Montgomery, S. Marques, and E. Kilpatrick. The remaining collection sites consisted of roadsides, parks, or unprotected areas. No permits were necessary to collect these monarchs in Hawaii . All butterflies were transported to the University of Georgia, Athens, GA, under permission from the United States Department of Agriculture (USDA PPQ-526 Permit #11-04112 and Permit #06-01690 to S. Altizer).Adult monarchs were captured using an aerial net between 0900 and 1600 hr. Following capture, monarchs were stored individually in glassine envelopes and held at 14°C for up to 6 hr prior to sampling. We recorded sex and forewing length to the nearest 0.01 mm. Wing condition, which qualitatively reflects age or distance traveled, was recorded in two ways. First, we recorded wing damage on a 0–4 scale, based on the number of wings with evidence of tears or other physical damage as might be caused by predators or contact with hard surfaces. Second, we recorded wing wear on an ordinal scale of 1–5, based on the level of scale loss (from newly emerged to nearly transparent wings) following Cockrell et al. OE spores transferred to a 2.5 cm-diameter transparent sticker pressed against adult abdomens  to examine the main effects of year, island, and site  on variation in monarch infection status  as a binomial variable. We also included the island*year interaction effect, and individual-level predictors of sex, forewing length, wing damage and wing wear in the full model. Prior to analysis we excluded data from sites for which fewer than 5 samples were available. In a separate analysis, we investigated whether site-level variation in patch size, land use type , and host plant species explained variation in average prevalence measures (with details provided in Supporting Information).To investigate host genetic differentiation, we used the software Arlequin 3.5.1.2 Samples for each site were resampled with replacement using Poptools We used the software STRUCTURE version 2.3.2.1 FST and RST statistics. These statistics are commonly used to calculate genetic differentiation, with levels of 0 indicating panmixia, and values higher than 0 indicating genetic differentiation. RST was developed as a more appropriate statistic for microsatellite markers, based on its use of a stepwise mutation model FST statistics FST and RST values were significantly different from 0. To further examine population genetic structure, we analyzed the correlation between site collection time and measures of genetic differentiation using Mantel tests implemented in the vegan library version 2.0-0 We also examined population genetic structure using OE across all sites and years . In particular, the outer islands of Kauai and the Big Island showed the highest average infection levels . By comparison, average prevalence was much lower on Oahu . Although we also observed a significant main effect of year , with infection prevalence higher for 2010 than for 2007 or 2009 .On average, 35.5% of monarchs were heavily infected with N = 885; . We deteN = 885; –2, with  or 2009 , differe or 2009 , as supp2 = 40.38, d.f. = 4, P<0.001). Of the 16 sites monitored through 2010, 9 were assessed for two or more consecutive years for monarch presence and parasite prevalence .At a finer scale, we detected strong within-island heterogeneity in the proportion of infected monarchs , and theevalence . Althougevalence , a separOE infection. At the individual level, males  had higher infection prevalence than females  and this effect was highly significant . Forewing length was negatively associated with infection status, such that infected monarchs had smaller wings than healthy butterflies . Wing wear  also predicted variation in infection probability , such that infected monarchs were more likely to show greater wing scale loss. Wing damage (as an index of tatter), however, was not associated with monarch infection status .Our multivariate logistic regression analysis further controlled for individual-level variables that might explain variation in A separate analyses of variance based on average prevalence by site and year (N = 28) showed that no site-level measurements  were significant predictors of variation in parasitism (results presented in Supporting Information). Although collection times (within the 0900–1600 hr range) varied among sites, there was no correlation between collection time and average infection prevalence .RST demonstrated that differences among sites, rather than among islands, are responsible for much of the observed variation in allele frequencies  and one of the Oahu sites (East Side). However, RST values for these comparisons were not significant. For thoroughness, we also looked at differentiation among islands and found similar results, with slight genetic differentiation detected between Oahu and the Big Island, as well as slight differentiation detected using FST between the Big Island and the other islands  or RST . This lack of a correlation indicates that differences in collection times are not responsible for the small amount of genetic variation found.An AMOVA analysis using quencies . Therefog Island . The sit islands . Thus, a islands . The lac islands . We perfF6,98 = 0.65, P = 0.69; F6,98 = 0.44, P = 0.85; F6,98 = 0.66, P = 0.68; Mean heterozygosity levels among sites ranged from a low of 0.333 in Palia to a high of 0.474 in North Shore (both of these sites are within Oahu) and did not significantly differ among sites within islands .No site-level measures of genetic diversity  were found to correlate significantly with site-level averages of parasite prevalence . At the Parasite prevalence was highly variable among and within the Hawaiian Islands. These results are unexpected, because the non-migratory status of monarchs of the Hawaiian Islands would lead us to predict that parasite prevalence should be relatively high across all sites. Instead, patterns identified here suggest that factors other than migratory behavior can play a major role in driving heterogeneity in parasite prevalence in this system.RST and FST statistics, we found no evidence of population structure using the program STRUCTURE. Moreover, parasite prevalence was not explained by variation in genetic diversity, heterozygosity, and allelic richness among sites. Thus, it appears that population genetic variation cannot explain the observed heterogeneity in parasite prevalence in this system. Instead, spatial environmental heterogeneity or ecological metapopulation processes might play stronger roles in determining infection heterogeneity in this host-parasite interaction.In this paper, we examined whether population sub-structuring might be responsible for the among-site variation in parasite prevalence. In particular, limited host movement might allow for local inbreeding and the loss of genetic diversity in sites with small populations, which is known to increase pathogen susceptibility in other systems Spatial variations in patch size, isolation and quality have been shown to alter parasite transmission and spatial spread in other host-pathogen systems. For example, empirical work demonstrated that landscape-level heterogeneity in habitat quality, host species diversity, and major geographic features such as water bodies affected the spatial spread and prevalence of pathogens ranging from rabies virus in raccoons to Lyme disease in white-footed mice Metapopulation ecology could offer a different perspective for understanding how spatial processes cause infection heterogeneity in the monarch-pathogen interaction In conclusion, we observed drastically varying prevalence of a protozoan parasite in monarchs inhabiting the Hawaiian Islands, despite high levels of butterfly gene flow and a lack of host population structure. The impact of site-level characteristics and landscape heterogeneity, in addition to colonization-extinction processes, are promising directions that could provide insight into the dynamics of this host-parasite interaction.Figure S1Structure plot showing that K (number of distinct populations)  = 1. Monarchs on the Hawaiian Islands for one admixed genetic population .(TIFF)Click here for additional data file.Table S1Field collection site variables. Latitude and longitude, site type, site area and perimeter , and milkweed species  were recorded. Only sites with 5 or more monarchs sampled are shown below, as sites with fewer monarchs were excluded from analyses.(DOCX)Click here for additional data file.Table S2Monarchs used for genetic analysis by sampling site and sampling year.(DOCX)Click here for additional data file.Table S3Microsatellite loci used in this study. Locus name, multiplex reaction, fluorescent label, primer sequences, repeat motif and primer annealing temperature (TA). Number of alleles and allele size range were determined by Lyons et al (2012).(DOCX)Click here for additional data file.Table S4Observed (oH) and expected (eH) heterozygosity at the seven Hawaiian sites at each locus as calculated by Arlequin 3.5.1.2.(DOCX)Click here for additional data file.Table S5Pairwise RST and FST values between four islands, as calculated in Arlequin version 3.5.1.2.(DOCX)Click here for additional data file.File S1Analysis for site-level characteristics and infection prevalence.(DOCX)Click here for additional data file.


Studies from a wide diversity of taxa have shown a negative relationship between genetic compatibility and the divergence time of hybridizing genomes. Theory predicts the main breakdown of fitness to happen after the F1 hybrid generation, when heterosis subsides and recessive allelic (Dobzhansky-Muller) incompatibilities are increasingly unmasked. We measured the fitness of F2 hybrids of African haplochromine cichlid fish bred from species pairs spanning several thousand to several million years divergence time. F2 hybrids consistently showed the lowest viability compared to F1 hybrids and non-hybrid crosses , in agreement with hybrid breakdown. Especially the short- and long-term survival (2 weeks to 6 months) of F2 hybrids was significantly reduced. Overall, F2 hybrids showed a fitness reduction of 21% compared to F1 hybrids, and a reduction of 43% compared to the grandparental, non-hybrid crosses. We further observed a decrease of F2 hybrid viability with the genetic distance between grandparental lineages, suggesting an important role for negative epistatic interactions in cichlid fish postzygotic isolation. The estimated time window for successful production of F2 hybrids resulting from our data is consistent with the estimated divergence time between the multiple ancestral lineages that presumably hybridized in three major adaptive radiations of African cichlids. The loss of genetic compatibility is one of the causes of reproductive isolation in classical models of speciation . HybridiGenetic incompatibilities are expected to accumulate the longer two taxa diverge independently from each other. This process, which typically extends over hundreds of thousands to millions of years, can be simulated by producing hybrid offspring from species pairs that have evolved in isolation from one another for varying amounts of time. The positive relationship between incompatibility and divergence time has been confirmed experimentally in many different taxa –13 and wTigriopus californicus is well-studied in this respect. Hybrids of divergent populations show increased fitness with increasing genetic/geographic distance in F1 hybrids, whereas in F2 hybrids the opposite is observed. Fitness decreased significantly with increasing genetic/geographic distance consistent with the D-M model [Arabidopsis populations [The main breakdown of hybrid fitness should only be observed after the F1 generation, when recessive D-M factors are increasingly expressed , 20. How-M model . Then ag-M model . In othe-M model , or in hulations . These mulations –29).Although cichlid fish are one of the best-studied model systems for speciation, data from F2 hybrids on the incompatibility-divergence time relationship are, as yet, missing. Here, we explore the fitness of second generation hybrids of African haplochromine cichlids bred from species pairs with increasing genetic differentiation, spanning several thousand to 2.7/3.8/7.4 million years (MY) divergence time , both sexually reciprocal F1 hybrid crosses were available  to produce F2 offspring. For all other crosses, sexually reciprocal matings could not be obtained (probably due to courtship-related behaviours). Fish were maintained at the Eawag Center for Ecology, Evolution and Biogeochemistry Kastanienbaum, Switzerland. For breeding, fish were kept in 100x40x40 cm. All aquaria were part of a large water recirculation system, in which water chemistry, temperature, light conditions, and feeding regime were kept constant over the entire duration of the experiment, including the rearing of grandparental, F1 and F2 crosses. Dry food was provided daily and twice a week fish were fed with a blend of shrimps, peas and Spirulina powder. The light regime in the aquaria was 12L:12D and water temperature was kept at 24–26 °C. Stones and plastic pipes were provided for males to become territorial and to motivate courtship. Offspring from non-hybrid  crosses were generated and raised contemporaneously with F1 hybrids, but because F2 hybrids were generated from F1 hybrids, F2 data were taken at a later time point. However, any variation in fitness between these groups should be due to differences in their pedigree because rearing conditions were kept identical.Crosses involved seven different cichlid fish species in different pairwise combinations, bred from laboratory populations. F2 hybrids were produced from a total of 14 F1 hybrid families representing seven different cross types . The F1 All animal work conducted here adhered to the relevant national and international guidelines. The Veterinary Office of the Canton of Lucerne (Switzerland) granted permission to conduct this research (permit number LU04/07). The amount and time of handling of the fish was kept to a minimum to ameliorate suffering and distress. No animal was sacrificed for this study.Four different components of post-mating isolation were measured. All were previously measured in the F1 hybrid generation of the same cross types, i.e. involving the same two parental species : fertiliAverage inviability of homospecific, F1 hybrid and F2 hybrid crosses was calculated by first averaging across replicated F2 clutches bred from the same F1 family, then averaging across replicated F2 families of the same cross type, and then across the different cross types within homospecifics, F1 hybrids and F2 hybrids (for sample sizes see http://ncbi.nlm.nih.gov/genbank) and were extracted from Stelkens et al. [Genetic distances between species are uncorrected p-distances based on D-loop sequences from NCBI GenBank . Details on all crosses, genetic distances and corresponding divergence times, as well as the number of available F1 families to produce F2, and the number of F1 and F2 hybrid families for the estimation of post-mating isolation components, can be found in 2,20 = 5.34, p = 0.015), 14-day survival , 180-day survival  and cumulative fitness . Only fertilization rate was not significantly different between groups . F2 hybrids consistently showed the highest inviability in all components of fitness, except for fertilization rate  and cumulative inviability  increased significantly with genetic distance. For hatching  and 180-day inviability  the relationship was strong but non-significant. Fertilization failure was not predicted by genetic distance .Inviability of F2 hybrids increased with genetic distance between the grandparental populations . 14-day Many studies have investigated the accumulation of genetic incompatibilities in first generation hybrids , 33, 34.F2 hybrids consistently showed the lowest viability across all components of fitness (except for fertilization rate). Using a cumulative fitness measure that encompasses all life stages from fertilization to survival to 6 months, F2 hybrids showed a fitness loss of ca. 21% compared to the average fitness of the F1, and a fitness loss of even 43% compared to the grandparental crosses. Especially the short- and long-term survival (2 weeks to 6 months) of post-hatchling F2 hybrids was reduced compared to the survival of F1 and parental crosses. F1 hybrids on the other hand did not suffer from a significant decrease in fitness compared to their parents (though this may not be surprising since we could only use F1 crosses viable enough to produce F2 offspring). Interestingly neither F1 nor F2 hybrids suffered from fertilization failure. In fact, fertilization success in the F2 was significantly higher than fertilization success in F1, indicating that the cell-physiological formation of zygotes does not represent a barrier to gene flow between the species studied here.Drosophila [Solanum [Our results are consistent with theory on hybrid breakdown in the F2. Incompatible alleles at different loci, inherited from the parental species, may not be harmful in the heterozygous condition, i.e. in the F1, but can be deleterious when homozygous, a condition, which should be more frequent in higher generation hybrids , 19, 20.osophila  and Sola[Solanum .et al. [An important limitation of our study with respect to estimating reproductive isolation is that we do not have data on F1 (or F2) hybrid sterility, which could considerably contribute to hybrid breakdown. We acknowledge a further caveat in our study: species divergence times were estimated with a single mitochondrial region (D-loop). Given our knowledge of the ease with which mitochondrial genomes can introgress, it is possible that our estimates of genetic distance underestimate the original splitting between the sympatric species pairs, which would make the increase of incompatibility with divergence less steep. Another source for inaccuracy is the phylogentic dependence of the species pairs used (five out of seven crosses include the same species). Correcting for phylogenetic relatedness did not change the conclusions on the increase of incompatibility with divergence time in F1 hybrids of the same crosses , but weet al. [heartstrings mutation described for zebrafish which has been shown to be caused by a single recessive mutation [Heartstrings has also been observed in sunfish hybrids and has been suggested as a candidate for a Dobzhansky–Muller incompatibility locus in vertebrates [Our study was not designed to identify the genetic basis of reproductive isolation, but a specific deformation indicates a possible cause for hybrid failure. The deformation was already observed by Stelkens et al.  in F1 hymutation . Heartsttebrates .There is growing evidence that interspecific hybridization is an important force in evolution, potentially being a source of genetic variation and evolutionary novelty , 39. MolS1 Tableet al. [Genbank accession numbers of D-loop sequences used to calculate genetic distances. If no sequences were available for certain species, sequences of closely related species (in brackets) belonging to the same clades characterized by incomplete mitochondrial DNA sorting were used. Data extracted from Stelkens et al. .(PDF)Click here for additional data file.


Petunia species are closely related, present high genetic similarity, and field observations suggest natural hybridization between them. Here we described helpful tools for evolutionary studies addressing genetic population, interspecific hybridization, and plant speciation. Based on previously described microsatellites of Petunia hybrida we were able to identify private alleles characterizing species and their putative hybrids. These profiles could also be useful to study gene flow, population structure, genetic conservation and landscape.Despite extensive morphological diversity and different floral syndromes, two wild  Petunia genus grows in South American grasslands and comprises both recently diverged wild species and commercial species. In this work, we analysed two closely related species: Petunia exserta, which has a narrow endemic range and grows exclusively in rocky shelters, and Petunia axillaris, which is widely distributed and comprises three allopatric subspecies. Petunia axillaris ssp. axillaris and P. exserta occur in sympatry, and putative hybrids between them have been identified. Here, we analysed 14 expressed sequence tag-simple sequence repeats (EST-SSRs) in 126 wild individuals and 13 putative morphological hybrids with the goals of identifying differentially encoded alleles to characterize their natural genetic diversity, establishing a genetic profile for each taxon and to verify the presence of hybridization signal. Overall, 143 alleles were identified and all taxa contained private alleles. Four major groups were identified in clustering analyses, which indicated that there are genetic distinctions among the groups. The markers evaluated here will be useful in evolutionary studies involving these species and may help categorize individuals by species, thus enabling the identification of hybrids between both their putative taxa. The individuals with intermediate morphology presented private alleles of their both putative parental species, although they showed a level of genetic mixing that was comparable with some of the individuals with typical P. exserta morphology. The EST-SSR markers scattered throughout the Petunia genome are very efficient tools for characterizing the genetic diversity in wild taxa of this genus and aid in identifying interspecific hybrids based on the presence of private alleles. These properties indicate that these markers will be helpful tools in evolutionary studies.Identifying the genetic basis of speciation is critical for understanding the evolutionary history of closely related wild species. Recently diverged species facilitate the study of speciation because many genetic and morphological characteristics are still shared by the organisms under study. The  Genetic diversity and population structures are relevant for understanding the evolutionary history, breeding systems, geographical distributions and ecological requirements of species. In evolutionary studies, molecular markers that differentiate close species and their putative hybrids greatly increase our understanding of the genetic basis of speciation and the effects of introgression on species integrity.Petunia genus encompasses 14 taxonomically accepted wild species, which are distributed exclusively in southern South America  of sandstone towers where they are protected from direct rain and sunlight, P. axillaris ssp. axillaris individuals grow in open and sunny habitats   scattered throughout the genome. Initially, we selected 34 primers based on their chromosomal location and polymorphic index content to test the cross-transferability among taxa and putative hybrids. Only 14 were successfully amplified and were subsequently used to estimate genetic variability. Polymerase chain reactions (PCRs) were conducted in a ﬁnal volume of 10 μL containing ∼10 ng of genomic DNA as a template, 200 μM of each dNTP , 1.7 pmol of ﬂuorescently labelled M13(-21) primer, 3.5 pmol of reverse primer, 0.35 pmol of forward primer with a 5′-M13(-21) tail, 2.0 mM MgCl2 (Invitrogen), 0.5 U of Platinum Taq DNA polymerase (Invitrogen) and 1× Platinum Taq reaction buffer (Invitrogen). The PCR conditions were as follows: an initial denaturation at 96 °C for 3 min; 32 cycles of 96 °C for 15 s, 50–52 °C for 30 s and 72 °C for 1 min; and a ﬁnal extension cycle at 72 °C for 7 min. The forward primers were FAM-, NED-, or HEX-labelled. The amplified product was visualized on a 2 % ultra-resolution agarose gel stained with 2 μL 0.001 % of GelRed (Biotium Inc.). The DNA fragments were denatured and size-fractionated using capillary electrophoresis on a MegaBACE 1000 automated sequencer  with an ET-ROX 550 internal size ladder . The manufacturer's software was used to identify the alleles. The primer sequences, repeat motif, fragment size range as estimated by the number of base pairs, and respective annealing temperatures are shown in Table S1 [Supporting Information].The genomic DNA of each individual was used to amplify the previously characterized EST-SSR markers (A), gene diversity, allelic richness (RA) and number of private alleles (E) per locus. We estimated the frequencies of null alleles and the polymorphic information content using the CERVUS 3.0.3 software  shared by all loci and a locus-specific component (α) shared by all populations using a logistic regression approach. Selection is assumed to occur when the α component is necessary to explain the diversity observed at a locus.The tests for outlier loci were performed with the BAYESCAN 2.1 software  based onTo investigate the genetic similarity between taxa, we carried out a principal coordinates analysis (PCoA) using the GENALEX 6.4 software , 2012. WP. axillaris ssp. axillaris and P. exserta that presented intermediate morphological characters, such as corolla colour, reproductive organs size and relative position, as previously described   as performed in GENETIX version 4.05 (K) was evaluated from 1 to 10, with 10 independent runs per K value, to determine the maximum value of the posterior likelihood [ln P(D)] and the best value of K. Each run was performed using 2.5 × 105 burn-in periods and 106 Markov chain Monte Carlo repetitions after burn-in, and the convergence was assessed. The optimal value of K was calculated using the maximum value of ΔK , followed by P. exserta and P. axillaris ssp. parodii  and P. axillaris ssp. subandina . The majority of the private alleles found in P. exserta or P. axillaris ssp. axillaris was present in their putative hybrids. In total, 12 private alleles were shared between P. axillaris ssp. axillaris  or P. exserta  and their hybrids Supporting Information—Table S2][see .The 14 EST-SSR loci revealed 143 alleles in 126 sampled individuals (Table P = 1), PM88 (P = 0.829), PM74 (P = 0.983) and PM192 (P = 1) were under selection with negative α values, which suggests the occurrence of a balancing or purifying selection. However, this result should be considered carefully because we did not sample a representative population.The frequency of null alleles varied from 20 to 79 % across all taxa. Null alleles were present at high frequency (>5 %) in all taxa. The tests for outlier loci among all 126 individuals revealed that PM177 ; whereas individuals of P. axillaris ssp. parodii and P. axillaris ssp. subandina preferentially composed the second group (Group B). Group A was divided into two subgroups, one corresponding to P. exserta individuals and only two individuals of P. axillaris ssp. axillaris and another composed of only individuals of the P. axillaris ssp. axillaris. Some P. axillaris ssp. axillaris and P. exserta individuals clustered with Group B . These loci were obtained from an EST database with sequences that were isolated preferentially from the roots and floral organs of Petunia hybrida . Here, we found that all taxa contained private alleles, which indicated that EST-SSR loci are a useful tool for evolutionary analyses of these species. The morphological and ecological differentiation between P. axillaris and P. exserta is clear, and although they share plastid haplotypes, there is no doubt about the placement of these lineages at the species level . These results provide evidence that the putative hybrids evaluated may not be F1 hybrids and could have backcrossed with the parental species or crossed among themselves. Studies analysing the morphology of individuals, in addition to canonical morphological data coupled with analysis of these markers, can be useful for understanding the evolution of these closely related wild species. In the STRUCTURE analysis  and it is possible that the hybrids have habitat or ecological restrictions. Another more controversial possibility is that speciation with gene flow occurred, and some of the morphological traits were not uniform as demonstrated to other species  gene, and the differences between P. axillaris subspecies are associated with different genotypes at this locus , while PM192 and PM74 are located in transcription factors Petunia species are an excellent model for this area of study. Further experiments, especially analyses of morphology, gene flow and crossbreeding systems, are necessary to answer the remaining evolutionary questions about the Petunia species and speciation processes within this genus. Understanding the processes involved in the diversification and speciation of wild Petunia species is not only important for ecological and environmental issues but may also be useful for breeding commercial hybrid species; the EST-SSRs tested here constitute useful tools for these purposes.The effects of morphological differences on the feeding strategy of the pollinators and adaptive genetic differentiation to maintain the processes of speciation involving close related species with the ability to hybridize in nature raise interesting questions, and Petunia genome are very efficient tools for characterizing the genetic diversity in wild taxa of this genus and are able to classify interspecific hybrids based on the presence of private alleles. These factures indicate that the markers presented here are helpful tools for evolutionary studies.The EST-SSR markers scattered throughout the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and the Programa de Pós-Graduação em Genética e Biologia Molecular da Universidade Federal do Rio Grande do Sul (PPGBM-UFRGS).This work was supported by the C.T. and L.B.F. planned, designed and led the project; C.T., J.B. and A.L.A.S. conducted the experiments; C.T., A.L.A.S. and S.L.B. ran the analyses; C.T., A.L.A.S. and L.B.F. wrote most of the text; L.B.F. and S.L.B. provided reagents and equipment to develop the experiments. All authors contributed in the preparation of the study and have commented on and approved the final manuscript.None declared.The following additional information is available in the online version of this article —Table S1. Characteristics of 14 EST-SSR markers observed in wild Petunia individuals. Forward and reverse primer sequences, repeat motifs, annealing temperatures (Ta), EST GenBank accession numbers, allele numbers (A) for each marker and putative function described in literature are presented for each marker.Table S2. Frequency of private alleles found in each taxa and in putative hybrids of P. axillaris ssp. axillaris and P. exserta.


Carcharhinus longimanus, is a highly migratory, large-bodied, pelagic shark listed by the IUCN  Red List as "vulnerable" throughout its range and “critically endangered” in the western north Atlantic. In 2014, the species was protected globally under Appendix II of CITES , limiting and regulating trade. This study used partial sequences of mitochondrial DNA (mtDNA) control region to determine the population genetic structure of oceanic whitetip sharks across the Atlantic and Indian Oceans. 724 base pairs were obtained from 215 individuals that identifed nine polymorphic sites and defined 12 distinct haplotypes. Total nucleotide diversity (π) was 0.0013 and haplotype diversity (h) was 0.5953. The Analysis of Molecular Variance (AMOVA) evidenced moderate levels of population structure (ɸST = 0.1039) with restricted gene flow between the western and eastern Atlantic Ocean, and a strong relationship between the latter region and the Indian Ocean. Even though the oceanic whitetip is a highly migratory animal the results presented here show that their genetic variability is slightly below average of other pelagic sharks. Additionally, this study recommends that at least two populations in the Atlantic Ocean should be considered distinct (eastern and western Atlantic) and conservation efforts should be focused in areas with the greatest genetic diversity by environmental managers.Information regarding population structure and genetic connectivity is an important contribution when establishing conservation strategies to manage threatened species. The oceanic whitetip shark,  International Union for Conservation of Nature (IUCN), which quantifies the conservation status of most taxa, currently lists over a thousand species of elasmobranchs [The effects of unsustainable fishing on populations of sharks and rays (elasmobranchs) has been well-documented globally , and stuobranchs , 5. The obranchs .Carcharhinus longimanus, is a pelagic shark with worldwide distribution, found mainly in tropical and subtropical open waters [C. falciformis and the blue shark Prionace glauca, C. longimanus was once considered a highly abundant oceanic shark [The oceanic whitetip shark, n waters , 8. Histic shark –11. Howeic shark  coveringic shark , was recC. longimanus as "vulnerable" throughout its range and “critically endangered” in the western north Atlantic [The IUCN Red List currently lists Atlantic , and in Atlantic . In the Atlantic , 17. RetAn aspect that is fundamental to sustainable use of marine resources and quantification of productivity of ocean ecosystems is the identification and maintenance of distinct stocks . The conC. longimanus from the Atlantic and Indian Oceans by using control region of mitochondrial DNA with an intention to provide a framework for future assessments and tracking studies.Population declines, such as that observed in the oceanic whitetip shark over the last 50 years, raises questions about the maintenance of genetic variability and the putative loss of evolutionary lineages. These issues are more relevant in species with population structure where the distribution of genetic characteristics is geographically limited. Therefore, this study aimed to describe the genetic variability and population structure of C. longimanus were collected by fishery observers on Portuguese, Brazilian and American commercial pelagic longline vessels in several regions of the Atlantic and Indian Oceans. Tissue samples, including small muscle or fin fragments (<1cm3), were collected opportunistically during the normal fishing operations of the vessels, and were subsequently frozen in 95% ethanol. Tissue samples from the north Atlantic were collected in The Bahamas as part of an ongoing study of oceanic whitetip movement and life history. All samples were collected under the Cape Eleuthera Institute research permit (MAF/FIS/17 and MAF/FIS/34) and in accordance with Stony Brook University and Cape Eleuthera Institute regulations developed within the guidelines of the Association for the Study of Animal Behaviour and the Animal Behavior Society [C. longimanus before the species effectively became part of CITES in September 2014, all samples used in this study were collected before that date.Samples of  Society . SamplinA total of 215 specimens were sampled for this study. Specifically, 206 were sampled from various regions of the Atlantic Ocean, including 28 from the Northwest Atlantic, 51 from the Western Equatorial Atlantic, 54 from the Northeast Tropical Atlantic, 50 from the Meso-Atlantic, 17 from the Southeast Atlantic and six from the Southwest Atlantic. In addition, nine samples were collected from the Indian Ocean .Genomic DNA was extracted using the NucleoSpin® Tissue XS Kit . Partial sequences of the control region of the mitochondrial DNA were attained according to Mendonça et al. . Individh), nucleotide diversity (π), and number of pairwise nucleotide differences among populations were calculated using ARLEQUIN 3.5.1.3 software [C. longimanus index ΦST was calculated using the Analysis of Molecular Variance (AMOVA) [ΦST estimates were tested with 1.000 non-parametric bootstrap pseudo-replicates using the ARLEQUIN 3.5.1.3 software, and the p-values were adjusted for simultaneous pairwise comparisons using the sequential Bonferroni correction [The relative nucleotide composition, number of polymorphic sites, haplotype diversity (software . To esti (AMOVA)  under th (AMOVA) . The ΦSTrrection . The AMOrrection . The misrrection .C. longimanus analyzed, we obtained a total matrix with 724 bp with 9 polymorphic sites, yielding 12 haplotypes  and in the Meso-Atlantic  and one  were the most common and were found in all sampled regions, representing 75.8% of the analyzed oceanic whitetip shark specimens. Numbers of polymorphic sites, haplotypes, nucleotide diversity and haplotypic diversity for different samples are shown in Tables In the overall analyses, we found low relative levels of haplotype diversity ɸST = 0.1039, P <0.001) was observed when populations were grouped in two geographic regions: Eastern  and Western  Atlantic Ocean. In this case, the indices related to genetic variability were h = 0.716 and π = 0.00165 in West Atlantic, and h = 0.471 and π = 0.00104 in the East Atlantic. Due to the low sampling rate in the the Indian Ocean, the AMOVA did not detect a statistically significant structure when left as a distinct group. However, in the FST pairwise analysis an absence of structure was observed between the Indian Ocean and the East Atlantic groups , which was found in all sampled regions .Cetorhinus maximus), crocodile shark (Pseudocarcharias kamoharai), the whale shark (Rhincodon typus), pelagic thresher shark (Alopias pelagicus) and the white shark (Carcharodon carcharias) [Ginglymostoma cirratum) [Carcharodon carcharias) [Negaprion acutidens) [The results show low levels of genetic diversity for the oceanic whitetip shark in the Atlantic and also in the Indian Ocean, although the latter has been evaluated with a small number of individuals and in a relatively small area. Population genetic studies with marine vertebrates, such as bony fishes, cetaceans and other elasmobranchs (including pelagic sharks) also tend to report low values of nucleotide and haplotypic diversity in the Atlantic Ocean –39. Althcharias) , 40–44. charias)  and sickΦST = 0.10 with sequences of mtDNA control region and FST = 0.1057 with microsatellite marker, between the northwest Atlantic and southern Africa.Considering two distinct groups for the oceanic whitetip shark in the Atlantic Ocean and based on the hypothetic population groupings and genetic structuring simulations, restrictions to gene flow with moderate genetic divergence were observed, with one population occurring in the West and another in the East Atlantic. Population structure between the eastern and western portions of an oceanic basin was observed by Cardeñosa et al  for the C. longimanus as rates of haplotype and nucleotide diversity are approximately 35% higher in western Atlantic. The haplotype network in star contraction generated from the haplotype H3 suggests an expansion event, possibly from the individuals of the eastern Atlantic, where the highest rates were observed in diversity with higher frequency on the H3.It would not be surprising to observe panmitic populations given the migration potential of this species. However, ocean currents and their interactions produce the oceanic barriers, organismal dispersal potential, behavior and organismal environmental tolerances which all contribute to patterns of diversification in the oceans , 46. SciCurrently, very little is known about the movements and habitat use of oceanic whitetips in the Atlantic. The most comprehensive movement study of this species used Pop-up satellite archival tags applied to 11 mature females and one mature male,.near Cat Island in the central Bahamas. In that study, the maximum individual displacement from the tagging site dispersed 290–1940 km after 30–245 days at liberty, with individuals moving to several different destinations. The tag affixed to the male shark never reported. All of the tagged oceanic whitetips remained within 500 km of their tagging location for the first 30 days of their tracks and none of them moved away from the northwestern Atlantic .The shortest route between the western and eastern Atlantic is between Brazil and Guinea-Bissau, requiring an oceanic crossing measuring ~2400 km. Although this distance does not seem to be a barrier to potential oceanic whitetip migration, such a trans-Atlantic route does not seem to be an aspect of the female behavior of the species. In those regions the oceanic whitetip shark populations in the western and east Atlantic are genetically differentiated from one another at mitochondrial loci, at least by female lineages. Philopatry is one of the factors that could also influence the reduction of gene flow across the Atlantic, by the migration of females to their original birth place . PhilopaFST, despite low statistical significance, highlight the dispersal capability of C. longmanus. Considering the movement of individuals between Indian and eastern Atlantic, the absence of significant genetic structure can indicate the existence of only one genetic stock of oceanic whitetip sharks around the African continent, both in the eastern Atlantic and western Indian Ocean. The warm Agulhas current passes the Cape of Good Hope and is incorporated into the cool Benguela current. The Agulhas current is one of the strongest currents in the world, and it may help the transport of oceanic whitetip sharks from the southwest Indian Ocean to the Southeast Atlantic. However, the question on whether the flow of these sharks can also occur in the reverse order remains unknown.The haplotype network denotes genetic differences among individuals without showing correlations between the different branches and their geographic distributions, with a characteristic pattern of an unstructured population. Although dispersion of fishes between the Indian and Atlantic Oceans are rarely observed, mtDNA data show that some species of tropical and sub-tropical fish found in the Indian Ocean may cross the barrier of the Benguela current . The watMore genetic studies and their importance in the evolutionary history of elasmobranchs with oceanic distribution, as well as for the management of commercially exploited shark species, are needed. Phylogeographic studies presenting extensive sampling and appropriate geographic scales including habitats with different ecological characteristics  have a greater chance of detecting evolutionarily significant units, which would facilitate the development and application of appropriate conservation measures .C. longimanus, ubiquitous in all tropical oceans, with its susceptibility to incidental longline capture, make it prone to population declines, prompting the IUCN to designate it “vulnerable”. Considering the important relationship between the sustainability of a natural population and their genetic diversity, there is an urgent need to uncover information about global levels of genetic diversity and geographical distribution as well as patterns of gene flow and the detection of significant evolutionary units.Though shark populations are often impacted by overfishing, pelagic species, including Here we present evidence that two populations of oceanic whitetip sharks exist in the Atlantic, specifically one in the western and another in the eastern regions of the basin. Despite their highly migratory nature, barriers to the gene flow of oceanic whitetips in the Atlantic result in two genetically distinct and demographically independent populations. This structure should be incorporated into assessments and monitoring of this species. The factors that restrict gene flow in the Atlantic may be absent between the east Atlantic and parts of the Indian Ocean; as there appears to be connectivity between those two regions. Isolated populations, especially if reduced by fishing or other sources of mortality, are vulnerable to inbreeding as reproducing adults have an elevated probability of mating with relatives . Nonrand