Acropora species that display different patterns of distribution and abundance in the Indo–Pacific Ocean. Our results show that the relationship between rarity and genetic diversity is not a positive linear association because, contrary to expectations, some rare species are genetically diverse and some populations of common species are genetically depleted. Our data suggest that inbreeding is the most likely mechanism of genetic depletion in both rare and common corals, and that hybridization is the most likely explanation for higher than expected levels of genetic diversity in rare species. A significant hypothesis generated from our study with direct conservation implications is that as a group, Acropora corals have lower genetic diversity at neutral microsatellite loci than may be expected from their taxonomic diversity, and this may suggest a heightened susceptibility to environmental change. This hypothesis requires validation based on genetic diversity estimates derived from a large portion of the genome.Among various potential consequences of rarity is genetic erosion. Neutral genetic theory predicts that rare species will have lower genetic diversity than common species. To examine the association between genetic diversity and rarity, variation at eight DNA microsatellite markers was documented for 14  Ne – the number of breeding individuals). Thus, in populations with large Ne, high levels of genetic variation are maintained, and this maximizes adaptive potential  (Pavona gigantea) restricted to the far Eastern Pacific Ocean  . These pn = 39) , but othn = 39) . Until nn = 39) ; and oneAcropora  is the model group for this study because an extensive literature exists on the global ranges of Acropora species and we have abundance data that enable us to estimate means global census sizes. Acropora are extremely susceptible to coral bleaching, changes in water quality, disease, and predation  encompassing 25 populations from 11 geographic locations to obtain insights into their genetic diversity. We test the null hypothesis that rare species have lower genetic diversity than closely related common congeners, and we generate new a hypotheses pertaining to the susceptibility of Acropora corals to environmental change.This project is the first to perform a comparative analysis of genetic diversity over a large number of coral taxa. We examine the level of genetic diversity in 14 species of Samples of 14 species . Estimates of global census size were calculated according to Richards et al. (A. pichoni*).To determine which species have a restricted global distribution, the maximum global range of the 14 species included in this study was quantified using the WorldWide  Wallace . Longitus et al. . For easAcropora species occur, and the difficulty in identifying rare corals to the species level. Thus, for some of the rare species examined in this study, local populations are so small that it is not feasible to obtain larger population samples. Hence, the small sample sizes and somewhat limited number of species examined prevent a rigorous test of the association between genetic diversity and rarity; however, this study provides a foundation from which the level of genetic diversity in rare corals can be further explored.The population sample sizes of the 14 species included in this study range from 6 to 34 individuals . It is iAll molecular samples examined in this project have matching skeletal voucher specimens that were identified by the author and verified by Dr. Carden Wallace. Small branches (2–5 cm) were collected from individual colonies and stored in absolute ethanol. To minimize sampling across multiple recruitment cohorts and asexually derived clone mates, colony sizes and spacing were standardized . DNA was extracted from approximately 20 mg of coral branch according to Underwood et al. . PrecipiAcropora  was documented using markers previously developed for 2O. PCR profile consisted of the initial denaturation step of 15 min followed by 35 cycles of 94° for 30 sec, 50° for 90 sec, and 72° for 60 sec. The mix was incubated at 60°C for 30 min. Three microliters of the PCR product was electrophoresed in a 2% TAE-agarose gel in 1× TAE buffer to assess the yield. Successful products were then cleaned using the Sephadex resin in the Whatman Unifilter 800 system. One microliter of the purified PCR product was transferred to a skirted 96-well plate and sent for genotyping at the JCU Advanced Analytical Centre. Fragment analysis was conducted on the Amersham MegaBase. To minimize genotyping errors, all automated scorings of alleles were checked manually, and rerunning the clean PCR product cleared uncertainties.Microsatellites were pooled into three multiplex reactions . Each PChttp://www.macrogen.com) for sequencing using SP6 and M13F vector primers.In cases where over two alleles were detected in genotyping, the quality of genotyping results was cross-checked using standard cloning and sequencing techniques. Unlabelled microsatellite PCR products were cloned using the ligation kit, pGEM T easy   and incubated for 1–4 h at room temperature or overnight at 4°C. The bacterial cells were transformed with a ligated vector using 60 μL of NM522 competent cells. Cultures were spun in a benchtop centrifuge for 5 min at 4000 rpm. The supernatant was removed and DNA was isolated using the plasmid isolation protocol in the RBC Hyfield Plasmid Mini Kit. The concentration of DNA was determined using a spectrometer and a minimum of 1 μg of purified DNA was dried and sent to Macrogen Inc.  were established using a chi-square test  was assessed in Microchecker v 2.2.3 . The relationships between range size/census size and allelic richness/expected heterozygosity were initially examined with Pearson's Correlation Coefficient in R, and regression analysis was used to compare the goodness of fit (r2). Assumptions of linearity, normality, and homogeneity of variances were assessed through examination of residuals and variables. The significance of linear (ŷ = a + bx) and polynomial relationships (ŷ = a + bx + cx^2) was also examined in multiple regression.Statistical differences in genetic diversity and level of inbreeding among rare and common species were determined using the Kruskal–Wallace test implemented in SPSS 17 with a single outlier excluded  did not (57% of the loci were polymorphic).A total of 531 individuals in 14 species of yped see . Thirty-yped see ; howeverA. austera population where a single MLG was repeated four times , A. jacquelineae* from Kimbe Bay (16%); and A. kirstyae* and A. spathulata* from Orpheus Island . The remaining populations of five species were fully sexually produced .The population with the highest degree of clonality was the Majuro ur times  and the A. microphthalma, A. valida, A. austera, A. kirstyae*, A. kimbeensis*, and A. pichoni*. Cloning and sequencing verified that ≥2 alleles were present for locus Amil2_022 in A. valida and A. kimbeensis*, but not for any of the other loci or species. This suggests that this locus has undergone duplication in these species, rather than these species being polyploid. The existence of multiallelic profiles is not accommodated in computer programs that treat codominant markers, so this prevented the inclusion of locus Amil2_022 data in the analysis of the population within which they occur; however, it was included for the other species that showed ≤2 alleles per individual at this locus.Genotyping showed more than two peaks (three to five) in 15% of the individuals sampled (73 of the 531) , suggestP < 0.05. Two common species  have the greatest proportion of loci with significant heterozygote deficits . Significant heterozygote deficits were also detected in rare species  and null alleles were encountered 45 times , while the Majuro A. austera population remained in deficit after null alleles were corrected. No null alleles were detected in A. rongelapensis* nor A. jacquelineae*. Significant heterozygote excess was detected in A. papillare* at 3 loci and in A. rongelapensis* at 6/7 loci with 100% observed heterozygosity recorded at 3 loci.Heterozygote deficits due to null alleles were corrected in 73% of cases. In one case, heterozygote deficit was due to large allele dropout, whereby shorter alleles are preferentially amplified, resulting in the less efficient amplification of large alleles; however, these data were corrected. For the remaining 27% of cases, significant deficits remained after correction for null alleles, suggesting that there are additional reasons for the deficits or there were not enough data to correct the null alleles. Correction reduced P < 0.05). Among common species, there was no strong link between loci with statistically significant genotypic linkage disequilibrium and populations in HWE disequilibrium. For example, two locus pairs  were linked in all A. microphthalma populations; however, only one third of the A. microphthalma populations showed significant deviations from HWE (A. tortuosa* and A. kirstyae*). The presence of linkage disequilibrium in association with heterozygote deficits may indicate inbreeding or it may be a sign that members of different populations have been sampled .Significant genotypic linkage disequilibrium was found in 18 of the performed tests  could be detected in the level of expected heterozygosity  nor allelic richness  between species that are numerically rare versus common. We tested whether there was a significant difference in the level of expected heterozygosity and allelic richness in species that are geographically restricted versus widespread, and again no significant difference (P < 0.05) was apparent . We further examined the strength of linear and nonlinear relationships between genetic diversity and rarity metrics and the strength of the relationship  or between geographically restricted and widespread species .After correction for null alleles, pulation . There wAcropora spp with varying degrees of rarity. Contrary to expectations of neutral genetic theory, the rare Acropora species studied here do not have significantly lower genetic diversity (or higher levels of inbreeding) than common species. Thus, the association between the degree of rarity and genetic diversity in Indo–Pacific Acropora is not a simple positive linear relationship. For example, A. walindii* is restricted to Kimbe Bay in Papua New Guinea  at three loci despite having an exceptionally small estimated global census size of only  . The proposition that some rare species have hybrid ancestries is further supported by a phylogenetic analysis of nuclear and mitochondrial DNA that shows that some rare species are monophylotic for mitochondrial DNA and polyphyletic for nuclear DNA  may be more vulnerable and have a higher probability of local extinction following disturbance. Local extinction (the disappearance of a species from part of it range) and ecological extinction  are precursors to global extinction. Data here suggest that some populations of common Acropora species may be more vulnerable to local extinction events than previously thought. For example, the A. valida population at Heron Island had low genetic diversity, significant heterozygote deficits, and some alleles are fixed. Heterozygote deficits are commonly caused by clonality, null alleles, inadvertently sampling disparate populations ; however, we cannot exclude the possibility that Wahlund effects may have contributed to the observed deficits.Populations with low genetic diversity  alleles per locus. This level of genetic diversity is just over half of the “conservative mean” presented for non-Acropora corals and more closely resembles those reported from isolated high latitude populations .An unexpected outcome of this study was our finding that despite being a taxonomically diverse group in the Indo–Pacific, our overall mean estimate of genetic diversity is quite low in comparison with the mean non-r et al.   are listed by the IUCN as “Vulnerable,” meaning that they have an elevated risk of extinction risk this century (see Carpenter et al. The conservation implications of this study are found in both theory and practice. Theoretically, we show that there is a large amount of variability in the genetic diversity of


CYP11A1 gene in the testes has been characterised. CYP11A1 is located in a region where a QTL influencing boar fat androstenone levels has been detected in a Large White pig population. Clarifying the role of CYP11A1 in boar taint is important because it catalyses the initial step of androstenone synthesis and also of steroid synthesis.Previously, in boars with extreme androstenone levels, differential expression of the CYP11A1 at approximately 1300 kb upstream from SNP H3GA0021967, defining the centre of the region containing the QTL for androstenone variation. In this study, we partially sequenced the CYP11A1 gene and identified several new single nucleotide polymorphisms (SNP) within it. Characterisation of one animal, heterozygous for CYP11A1 testicular expression but homozygous for a haplotype of a large region containing CYP11A1, revealed that variation of CYP11A1 expression is probably regulated by a mutation located downstream from the SNP H3GA0021967. We analysed CYP11A1 expression in LW families according to haplotypes of the QTL region's centre. Effects of haplotypes on CYP11A1 expression and on androstenone accumulation were not concordant.A genome-wide association study located CYP11A1 is not solely responsible for the QTL influencing boar fat androstenone levels. As a conclusion, we propose to refute the hypothesis that a single mutation located near the centre of the QTL region could control androstenone accumulation in fat by regulating the CYP11A1 expression.This study shows that testicular expression of  Boar taint refers to an unpleasant odour and flavour of meat which occurs in a high proportion of uncastrated male pigs and is primarily due to the accumulation of androstenone and skatole in fat tissue ,2. AndroCurrently, only a few studies have tried to identify QTL for androstenone accumulation -9. It isSus scrofa 7 (SSC7) using GWAS (Genome Wide Association Studies) near the position 66 Mb on the "Sscrofa9" version (April 2009) of the pig genome sequence. Examination of the gene content in this QTL region, suggested CYP11A1 as an obvious candidate gene. Moe et al. [CYP11A1 in the testes of boars with either extremely high and or low levels of androstenone in fat. Moreover, a previous study reported one polymorphism in exon 1 of CYP11A1 significantly associated with androstenone levels in Yorkshire boars [CYP11A1 in boar taint. If testicular expression of CYP11A1 is found to influence the QTL for androstenone variation, it would be difficult to select against this QTL without encountering reproduction problems.Le Mignon et al.  identifie et al.  had alrere boars . This gere boars . TherefoOn the INRA experimental farm, 98 LW sows were inseminated with 56 LW boars, chosen as unrelated as possible. Each boar inseminated one or two sows. A total of 580 male piglets were raised in pens till they reached 110 kg of live body weight and then slaughtered in a commercial slaughterhouse. A total of 480 animals were measured for backfat androstenone levels.3) were collected from the inner part of the testicular tissue, containing Leydig cells.Six litters were produced by inseminating LW sows with semen from two commercial LW boars. Twenty-two animals were produced and then slaughtered at 24-25 weeks of age. Testicular samples were collected immediately after slaughter, frozen in liquid nitrogen and stored at -80°C. To obtain testicular samples, testes were decapsulated to remove connective tissues, fasciae and the main blood vessels. Samples  kits according to the manufacturers' instructions, and treated with DNase to remove contaminating DNA. RNA concentration was determined using the NanoDrop ND-1000 spectrophotometer . First strand cDNA synthesis was conducted using SuperScript™-II Rnase H- Reverse Transcriptase . According to the manufacturer's instructions, 0.5 μg of total RNA from each sample was used as a template with dN9 random primers , in a total volume of 100 μL.Samples were disrupted, homogenised and ground to a fine powder by rapid agitation for 1 min in a liquid-nitrogen-cooled grinder with stainless steel beads before RNA extraction. Total RNA was isolated from testis using CYP11A1expression was determined by real-time PCR on cDNA from testes. Experiments were performed on the ABI 7900HT (Sequence Detection System 7900HT) in a 384-well plate. All measurements were performed in duplicate on the same plate and no reference sample was used. Primers were designed in exon 3 (TGTTTCGCTTCGCCTTTGA) and exon 4 (CCCAGGCGCTCTCCAAAT) of CYP11A1 cDNA. Transcript's concentrations were corrected with respect to the housekeeping gene, TOB2B (GGGATGTCTGAAGAAGTACGAAAC//CATTCCTACAAGCCATTCCTTACG). Data was analysed with ABI software to obtain Ct values (threshold cycle). Four points of dilutions of a mix of cDNA were used for each gene and for each tissue, to determine PCR efficiency (E). As efficiency levels were similar for all measured genes (including the reference gene), results are expressed as E(Ct_ref - Ct_gene) × 1000 in arbitrary units.The level of CYP11A1 transcripts, possible alternative transcripts were compiled from databases and we checked that the alternative transcript (AK235955 or DB787788) with a 3' shortcut exon 3 was absent in the testicular cDNA.Before quantifying CYP11A1 gene related sequences is underway and sub-clone sequences are being captured by a blast procedure (http://www.ncbi.nlm.nih.gov/genome/seq/BlastGen/BlastGen.cgi?taxid=9823) from the "traces-other" database.Sequencing of BAC CH242-402H17 containing To find polymorphisms, PCR products were produced with genomic DNA from several animals. To sequence the PCR products, an aliquot (1 to 12 μL) was purified by a single treatment (45 min at 37°C followed by 30 min at 80°C) using 0.5 U of Shrimp alkaline phosphatase (Promega) and 0.8 U of exonuclease I (New England Biolabs). Sequencing was done with a 3730 ABI capillary DNA sequencer using a Big Dye terminator V3.1 cycle sequencing kit.Differences between two groups of animals were assessed with a heteroscedastic Student's t-test as proposed by MS-Excel (Microsoft Corporation)."Sscrofa9" provisional genome sequence), identified by GWAS analysis [CYP11A1-GRAMD2 region from HSA15 . The information was completed and compared to the provisional annotation of the "Sscrofa9"version available on the Ensembl web site (http://www.ensembl.org/). We found that the assembly between 66.0 and 66.4 Mb did not coincide completely although the SNP order was correct  and the sequencing of BAC CH242-402H17 is underway. We performed several assemblies of sub-clone sequences starting with exon sequences. To capture the 5' flanking sequence, we used human sequences and in particular a regulatory region (HSA15:74665162-74667983). We were able to propose an initial assembly (HQ687747) with four genomic fragments schematized on Figure CYP11A1 gene is composed of nine exons in most mammals. Pig intron 1 (3205 bp) is significantly shorter than the human counterpart (19261 bp) but longer than the mouse one (975 bp). To characterise polymorphisms, the corresponding human 19 kb long intron 1 was sequenced in several animals chosen from the 480 LW animals. One SNP (R according to IUPAC codification) and one microsatellite were identified in the distant 5' flanking sequence. SNP (Y) patented by Greger [CYP11A1 region.e et al.  had alrey Greger , was fouCYP11A1 expression in animals from this LW population but no samples were available. Fortunately, expression of CYP11A1 in the testis of animals from other LW families could be estimated by real-time PCR. The results are shown in Table CYP11A1 expression ranged between 200 and 600 A.U. in the seven animals analysed, while in family B, two animals with a very high level of CYP11A1 expression (1425) and two animals with a low level (250) were found. Moreover four microsatellites were genotyped around CYP11A1 [see additional file CYP11A1expression level. Nevertheless, it is very likely that this latter sow (65472) is homozygous for the entire haplotype over this region [see additional file It would have been interesting to analyse variations of the testicular CYP11A1) and for two microsatellites (located 5' of CYP11A1 and SW1418). With the exception of M12, M13 and M14, all the markers were homozygous (data not shown). This female appeared to be homozygous for a large region (M1-M11) including SNP H3GA0021967. We examined the haplotypes of 480 LW animals for 14 SNP (M1 to M14) from the CYP11A1 region and found that 14 animals shared the same two SSC7 chromosomal regions present in sow 65472. Nevertheless, genotyping for three markers inside the CYP11A1 gene did not detect any animal carrying the 65472 sow's haplotype. Thus, we believe that the number of genotyped SNP is sufficient to assume that sow 65472 is homozygous for a large region (M1-M11) including SNP H3GA0021967.Sow 65472 was genotyped for the 21 SNP  could control androstenone accumulation in fat by regulating the CYP11A1 expression.Since sow 65472 is considered as heterozygous for We examined the haplotypes between M11-M14 in 480 LW animals Table . The GGACYP11A1 expression in LW families according to haplotypes specifically of the QTL region. Haplotypes of the region between markers M11 and M14 in 22 animals characterised for CYP11A1 expression are shown in Table CYP11A1 expression level had haplotype TGAG, the fourth haplotype characterised in the 480LW population and for which it was not possible to evaluate its effect on androstenone accumulation. Nevertheless these two animals have a paternal haplotype GGAG or TAGG which could have a contrary effect on androstenone level. Moreover, we found six animals GGAG/TAGA expected as +/- and seven animals TAGG/TAGA expected as +/+ for androstenone accumulation with no significant difference in CYP11A1 expression  could control androstenone accumulation in fat by regulating the CYP11A1 expression.This study suggests that the variation of The authors declare that they have no competing interests.AR and KF performed real-time PCR, sequencing, and data processing. AR made the main contributions to the data analysis, data interpretation and drafting of the manuscript. GLM contributed very significantly to data interpretation. CL and JR supervised the experimental design and contributed to data interpretation and manuscript evaluation. Genotyping data acquisition was supervised by CL. All authors read and approved the final manuscript.Presentation of genotypes of animals from the two families evaluated for the CYP11A1 expression. The data provide genotypes of eight markers allowing the characterisation of SSC7 haplotypes in a large region around CYP11A1 of animals from the two main LW families evaluated for CYP11A1 expression (families A and B)Click here for file


Sphyrna lewini, is a large endangered predator with a circumglobal distribution, observed in the open ocean but linked ontogenetically to coastal embayments for parturition and juvenile development. A previous survey of maternal (mtDNA) markers demonstrated strong genetic partitioning overall  and significant population separations across oceans and between discontinuous continental coastlines.The scalloped hammerhead shark, FST = 0.035) over an order of magnitude lower than the corresponding measures for maternal mtDNA lineages (ΦST = 0.749). Nuclear allelic richness and heterozygosity are high throughout the Indo-Pacific, while genetic structure is low. In contrast, allelic diversity is low while population structure is higher for populations at the ends of the range in the West Atlantic and East Pacific.We surveyed the same global range with increased sample coverage (N = 403) and 13 microsatellite loci to assess the male contribution to dispersal and population structure. Biparentally inherited microsatellites reveal low or absent genetic structure across ocean basins and global genetic differentiation (S. lewini, and indicate that females are philopatric or adhere to coastal habitats while males facilitate gene flow across oceanic expanses. This study includes the largest sampling effort and the most molecular loci ever used to survey the complete range of a large oceanic predator, and findings emphasize the importance of incorporating mixed-marker analysis into stock assessments of threatened and endangered shark species.These data are consistent with the proposed Indo-Pacific center of origin for  Physeter macrocephalus) in the North Atlantic As with other sexually reproducing species, reproductive behavior in sharks has implications for population structure. Females make the greater investment in reproduction with large ova, long gestation times, and time spent transiting to coastal nursery grounds. Males contribute less energy to reproduction and are expected to exhibit promiscuity Population structure of coastal-oceanic sharks is something of an enigma, conforming neither to the expectations of sedentary coastal fishes with pelagic larvae, nor the oceanic migrants such as billfish and tunas. Unlike most teleost (bony) fishes, sharks are viviparous, producing small numbers of highly developed young that are capable of swimming and navigation soon after parturition. As a result, many shark species are dependent on shallow coastal habitat for birthing and offspring development Carcharadon carchariasIsurus oxyrhinchusCarcharhinus plumbeusSphyrna lewini, is a large (up to 420 cm) viviparous (live-bearing) shark with a circumglobal distribution in tropical and warm-temperate waters. S. lewini gives birth to 13–30 pups following an 8–10 month gestation S. lewini can occasionally be found in the open ocean, and documented oceanic movements exceed 1500 km S. lewini populations have declined worldwide as a result of overfishing and bycatch Sampling large marine predators is challenging, and few studies have documented phylogeography on a global scale, most of these focusing exclusively on marine mammals but see . Only thS. lewini revealed significant genetic structure between oceans and between discontinuous coastlines within oceans et al.An earlier analysis of maternal (mtDNA) markers in e) of the haploid, uniparental mitochondrial DNA (mtDNA) compared to diploid, biparental nuclear DNA (nDNA) e mean that even in the absence of sex-biased migration, mtDNA structure may be greater than nDNA structure due to the differential rate at which the markers attain drift-migration equilibrium.An ideal system for untangling male and female components of dispersal would include both maternally- and paternally-inherited markers. Lacking markers for the Y sex chromosomes  have ruled out high connectivity across ocean basins for female lineages. Nonetheless the circumglobal range and single-species status of et al.S. lewini with subsequent dispersal westward into the Atlantic and eastward into the Central and Eastern Pacific. We address this hypothesis with increased sampling and the application of microsatellite data, which in conjunction with mtDNA data has substantial power to resolve population history.Population structure and phylogeography: Duncan Scalloped hammerhead specimens  were acquired in Hawaii by fishing under permit #2008-99 issued by the State of Hawaii's Division of Aquatic Resources to the Hawaii Institute of Marine Biology, and elsewhere were bought from commercial fishermen or purchased from fish markets between 1999 and 2008. Specimens were collected from multiple locations in each of three ocean basins, including (i) Pacific: Tropical East Pacific, Pacific Panama, Hawaii, the Philippines, Taiwan, and Eastern Australia; (ii) Indian: Western Australia, Seychelles, and South Africa; and (iii) Atlantic: Western Africa, Gulf of Mexico, and East Coast USA (South Carolina). Collection sites were grouped together if they were within close geographic proximity and statistical analyses indicated no significant differences between sites; two collections of tissue specimens  were made from proximate locations and grouped to create a single Gulf of Mexico collection. Similarly, two sites in Baja California consisting of 32 and 24 specimens were grouped together with five specimens from Pacific Panama to create one Tropical East Pacific (TEP) sample. When possible, specimens were collected from juvenile sharks (fork length <60 cm) within a proposed nursery area to avoid the confounding effect of sampling adults in feeding areas where distinct breeding populations may overlap 3) were stored in 20% dimethylsulfoxide (DMSO) saturated salt (NaCl) buffer Taq DNA polymerase , 1× Taq buffer, 0.25–0.0625 µM of each primer, 200 µM each dNTP, and 2.0 mM MgCl2. PCR amplification on a MyCycler  consisted of an initial denaturation at 95°C for 4 min, followed by 30 cycles of 1 min at 95°C, 30 s at optimal annealing temperature (eneMapper Software 4.0 (Applied Biosystems).Tissue samples  according to the algorithm implemented in stat 2.9.3.2 jmp 7  to statistically estimate degree of decrease in observed heterozygosity and allelic richness per site with increasing distance from the putative center of origin for S. lewini, the Indo-West Pacific, by using straight-line distance from Indonesia in kilometers as our dependent variable.We tested for deviation from Hardy–Weinberg Equilibrium (HWE) using GFST generated in ArlequinFST values generated from highly variable multiallelic data can be problematic because the maximum FST is constrained by the mean within-subpopulation heterozygosity FST = 1), making comparisons between microsatellites and other genetic markers difficult to interpret. To address the downward bias in FST estimates with highly polymorphic loci, we applied the FST standardization approach ecodeData 0.1. Pairwise FST estimates differ in magnitude but provide the same qualitative patterns and significance values as standardized F′ST estimates  are represented in a data set, we plotted the change in the log probability of successive K values (ΔK) mp 7. ΔK cannot be used for K = 1, but when more than one population is likely, ΔK shows a mode at the actual K more consistently than L(K) and is particularly effective for resolving large microsatellite data sets where individuals may be admixed K = 1−11 with 10 repetitions each.Global patterns of population subdivision were also examined using Structure. We then assessed the magnitude of genetic differentiation between the K subpopulations in Arlequin with a hierarchical Analysis of Molecular Variance , which partitions genetic variation within sites (FST), among sites within groups (FSC), and among groups (FCT).Patterns of pairwise differentiation and individual gene pool assignment were used to group collection sites according to the number of populations indicated by SF′ST). F′ST is a standard measure of population differentiation representing the proportion of genetic diversity resulting from allele frequency shifts between sampling sites, with no consideration for genetic distance between individual alleles. For this reason, direct comparisons of F′ST with mitochondrial ΦST, which takes into account both haplotype frequency and genetic distance between haplotypes, can be problematic. We therefore used Arlequin to generate a mitochondrial analogue for pairwise F′ST (herein identified as mtFST) that describes S. lewini population structure solely in terms of haplotype frequencies , with broad priors for all, and reduced them in repeated runs to better sample the posterior distribution. Once several replicates converged on the same answer, we recorded the maximum likelihood estimate (MLE) for each parameter and 95% highest posterior density interval or HPD. We converted migration parameters 1m and 2m into the number of migrants per generation (Nm) using the equation Nm = (θ m)/4.We estimated migration rates  consistent with departure from equilibrium  and re-ran pairwise FST. A comparison of pairwise FST values calculated from the subset of 9 loci and from the full data set was non-significant . As such, we present results from the full 13 loci here. There was no evidence of linkage disequilibrium among pairs of loci after correction for multiple comparisons.Microsatellite fragments were analyzed for 403 specimens from eleven sampling locations. Milibrium . To testoH = 0.537−0.785, and allelic richness ranged from rA = 6.91−8.54 with the exception of the Gulf of Mexico . The East Pacific and the Gulf of Mexico sites had the lowest heterozygosity and allelic richness (rA = 4.89−6.91). Allelic richness showed significant decrease with increasing distance from Indonesia , while the decrease of observed heterozygosity was lower and not statistically significant .Observed heterozygosity among geographic sampling sites ranged from F′ST) and uncorrected (FST) microsatellite based estimates of pairwise population differentiation revealed a general pattern of high contemporary gene flow across ocean basins and along continental margins (average F′ST≈0), with some noteworthy exceptions , however no comparisons differentiated Hawaii from the three sampling sites in the West Pacific . No population structure was detected across the Indian Ocean . Likewise Indian Ocean samples are undifferentiated from Taiwan, Philippines, and Eastern Australia in the West Pacific , indicating contemporary gene flow bridging the Sunda Shelf barrier. South Africa was weakly differentiated from most other Indo-Pacific populations except for the Seychelles, with which it shares a continuous continental shelf . While the degree of differentiation indicates some isolation of the South African site, this site shows allele frequencies similar to Hawaii across a distance of 17,600 km . Between the Indian and Atlantic Oceans, South Africa is differentiated from West Africa . Low but significant structure also differentiates West Africa and South Carolina , indicating limited contemporary gene flow across the Atlantic. The Gulf of Mexico was highly differentiated from every other site sampled , including the proximate South Carolina site in the West Atlantic . The highest level of allelic differentiation separates the Gulf of Mexico from the TEP . The broad mixing of microsatellite alleles across the Indian and Pacific oceans is clearly illustrated in the radial dendrogram of pairwise FST genetic distances . Geographical structuring is as follows: Cluster C1 is by far the most widespread, represented in every sampling site from the TEP and Hawaii throughout the Indo-Pacific and Atlantic to South Carolina. Taiwan, Philippines, and South Africa assign primarily to this cluster, though this could be due to low sample size. One small, relatively isolated cluster (C3) is predominantly observed in the Indian Ocean, with high representation in the Seychelles. In South Carolina, about half of the individuals were assigned to C1, the widespread cluster found in the Indo-Pacific, and about half to the predominantly Atlantic C2 cluster shared between West Africa and the Gulf of Mexico. The low level of admixture in South Carolina indicates that samples may have been drawn from a common feeding area where distinct breeding populations may overlap. The Gulf collections were the most homogeneous of any sampling site, with nearly 100% of individuals assigned exclusively to C2. The TEP sample also exhibited high population homogeneity, with the majority of individuals assigned to a single genetic cluster (C4).Structure and tested these for within- and between-group variation. Individuals from the East Pacific and the Gulf of Mexico had the greatest probability of self-assignment and were accordingly analyzed as independent sub-populations. As the remaining nine sites showed extensive admixture, groupings were not immediately obvious and collection sites were therefore clustered by major biogeographic province, these being the Indo-Pacific  and Atlantic FSC = 0.002, P = 0.098). However, 4.88% of the variance was partitioned among groups , indicating that our groupings reflect actual population structure. Global genetic structure was low but significant . Though our data indicate gene flow both across the Sunda Shelf and around the tip of South Africa, the presence of a unique multi-locus lineage primarily found within the Indian Ocean , we grouped sites into four subpopulations as indicated by SF′ST) and matrilineal mtDNA (mtFST and ΦST) are contrasted in F′ST and mtFST in S. lewini . ΦST and mtFST were highly similar except in a limited number of pairwise comparisons where the magnitude of ΦST was elevated relative to mtFST . This effect was noted particularly in pairwise comparisons that include the Seychelles, West Africa, and West Australia. Because divergent haplotypes coupled with genetic isolation could create a signal of haplotypic distance that increases the magnitude of ΦST relative to mtFST, these data indicate some genetic isolation of Indian Ocean sampling sites relative to the rest of the globe, results similar to those indicated by Structure.Genetic distances derived from biparentally-inherited microsatellite DNA (m/4) between 0.03 and 7.22. Although 95% posterior probability densities (PPDs) of these estimates were large with several infinite upper boundaries, all PPDs had strong peaks with probabilities falling to zero as m approached zero. We report both Nm1 (estimated migrants per generation from population 2 into population 1) and Nm2 (estimated migrants per generation from population 1 into population 2) for each pair of sites examined, and compare these mixed-marker values to the migration rates for mitochondrial data alone (M) from Duncan et al. 1/Nm2 were consistently equivalent to or lower than M, while across ocean basins Nm1/Nm2 values were higher than M, sometimes by an order of magnitude.IMa revealed maximum likelihood estimates of the number of migrants per generation  and the Gulf of Mexico are relatively young  The distribution of maternal lineages indicates strong restrictions to dispersal between discontinuous coastlines, whereas biparentally-inherited markers reveal much higher connectivity and in some cases nonsignificant population structure across ocean basins. Male-mediated dispersal and gene flow has likely facilitated the contemporary connectivity observed among global Sphyrna lewini, as indicated by contrasting genetic distances derived from matrilineal mtDNA , and between Hawaii and all West Pacific sites  include evidence gleaned from biparental inheritance, yet even with 50% input from matrilines, the differences between marker types clearly signify male-mediated dispersal.Although divergent S. lewini females. One caveat to our conclusions is that genetic isolation is not always synonymous with restricted physical movement in sharks. Lemon sharks (Negaprion brevirostris) show philopatry to specific sites C. carcharias; S. lewini, but telemetry studies would be necessary to link the sex-biased gene flow observed here with reduced transoceanic movement by females.This male-biased dispersal pattern is consistent with the premise that shark reproductive strategies more closely resemble those of sea turtles and marine mammals than other fishes (Musick 1999). It is likely that female reliance on coastal habitat for reproduction can explain the relatively limited vagility of et al.Sphyrna lewini in the same region , and decreases progressively with distance from that region . Genetic structure shows the opposite pattern, with lower population structure within the Indo-Pacific and increasing differentiation towards the ends of the global range in the East Pacific and Gulf of Mexico. These sites each show the lowest heterozygosity, admixture, and allelic richness, as well as the highest pairwise differentiation .The statistical parsimony analysis in Duncan e region . These distances  shows thtructure shows almost zero admixture in the Gulf population, while the adjacent South Carolina (West Atlantic) site shows a near 1∶1 mix of Gulf (green) and Indo-Pacific (red) types  population. Sd) types . IMa indd) types . This paS. lewini and not the cryptic Sphyrnid species found in the same region et al.S. lewini tagged in the Gulf of Mexico (sex unknown) has been recaptured to date, not far from the catch site Reasons for the isolation of the Gulf of Mexico from the proximate West Atlantic in the current study are hard to pinpoint, though it is clear that these samples belong to S. lewini crosses multiple well-documented marine biogeographic barriers and includes seamounts, coralline archipelagos, pelagic blue-water environments, and coastal brackish-water estuaries. In S. lewini, maximum population differentiation between East Pacific and West Atlantic populations is likely due to the difficulty of maintaining circumglobal gene flow, in particular the bridging of large stretches of open water separating the Tropical East Pacific from the Central Pacific, the cold upwelling water around South Africa, and the oceanic gap between East and West Atlantic The circumglobal tropical distribution of et al.et al. 2007), possibly because their high adult vagility allows for connectivity across the Sunda Shelf, even during lowered sea levels. While the scalloped hammerhead shows a population level separation across this barrier with mtDNA, that lack of microsatellite DNA structure over the same area demonstrates male mediated dispersal between the Pacific and Indian Oceans  is a large, mobile predator with a coastal-pelagic life history and global range. Both mtDNA and microsatellite data indicate that modern populations of this tropical species originated in the Indo-West Pacific, then subsequently dispersed into the Atlantic and East Pacific. Although the ability to traverse pelagic habitat indicates high rates of dispersal, female S. lewini display site fidelity to single coastlines, archipelagos, or individual nursery areas and show no evidence of ongoing trans-oceanic movement. Male S. lewini, by contrast, disperse long distances across the open ocean with clear consequences in reproduction and the transmission of gametes, though the frequency of these migrations is unknown. Populations at the ends of the range in the TEP and the Gulf of Mexico have differentiated over time, and are prevented from contact by the Isthmus of Panama. Currently scalloped hammerheads are exposed to massive fishery mortality on a global scale, and their coastal-pelagic life history makes them vulnerable to inshore and estuarine fishing mortality as well as offshore commercial operations. In 2008, the IUCN raised the global status of S. lewini from “threatened” to “endangered” S. lewini is considered to be a single genetic stock The scalloped hammerhead shark (S. lewini highlights the need for analyses of genetic markers with multiple lines of inheritance. Single-marker assays using either female or biparentally-inherited loci alone conducted on species with complex reproductive behavior and life history may give a misleading picture of management units S. lewini, as well as strong genetic partitioning in the West Atlantic and in other regions of the world, leads us to recommend drastic reevaluation of management units and conservation strategies for this species.In regards to management strategies, the complex population structure observed in


Clupea harengus) in the Baltic Sea are no exception; historically, very low levels of population differentiation (FST ≍ 0.002) have been found, despite a high degree of interpopulation environmental heterogeneity in salinity and temperature. Recent exome sequencing and SNP studies have however shown that many loci are under selection in this system. Here, we combined population genetic analyses of a large number of transcriptome-derived microsatellite markers with oceanographic modelling to investigate genetic differentiation and connectivity in Atlantic herring at a relatively fine scale within the Baltic Sea. We found evidence for weak but robust and significant genetic structuring (FST = 0.008) explainable by oceanographic connectivity. Genetic differentiation was also associated with site differences in temperature and salinity, with the result driven by the locus Her14 which appears to be under directional selection (FST = 0.08). The results show that Baltic herring are genetically structured within the Baltic Sea, and highlight the role of oceanography and environmental factors in explaining this structuring. The results also have implications for the management of herring fisheries, the most economically important fishery in the Baltic Sea, suggesting that the current fisheries management units may be in need of revision.Marine fish often show little genetic structuring in neutral marker genes, and Atlantic herring ( Intraspecific biodiversity forms the basis for evolution and is important for the long-term viability of populations and thereby also for sustainable harvesting of species of commercial importance . Hence, In the marine environment, gene flow by drifting larvae and migrating adults may be extensive, and accordingly, local adaptation in marine fish has traditionally been viewed as rare, with weak population structuring . HoweverConsidering the ongoing depletion of most fish populations , spatialhttp://www.itameriportaali.fi/en/itamerinyt/en_GB/itamerinyt/).The Baltic Sea in the eastern Atlantic Ocean offers a test bed where several environmental factors show steep geographic gradients, e.g. in salinity, temperature and community composition . There iPlatichthys flesus) had a number of genes that were expressed at significantly different levels in North Sea fish compared to Baltic Sea fish in a reciprocal transplantation experiment mimicking natural local salinities. Likewise, Atlantic cod (Gadus morhua) seem to be adapted to local salinity  is found throughout the Baltic Sea, allowing for investigation into interactions between the environment and the genome. Most extant herring populations in the Baltic are spring spawning. They produce pelagic larvae that are potentially free to drift with the sea currents  that are consistent with strong positive selection. Divergence between Baltic and North Sea herring populations is pronounced, and samples from the Kattegat cluster separately from Baltic Sea and Atlantic samples, but more closely to Baltic Sea samples . Total DNA was extracted from a small segment of fin clip for each individual using a silica-based method , 4 μL 2xPhusion® Flash High-Fidelity PCR Master Mix (Finnzymes), 2.2 μL water, and 1 μL of DNA (~2 ng of DNA). The PCR program was: 98°C for 1 min, followed by 34 cycles of 98°C for 1 s, 58°C for 12 s, 72°C for 20 s, and a final extension at 72°C for 1 min. The PCR products were diluted 1:80, and run on a MegaBace 1000 capillary sequencer. Genotyping was performed using Fragment Profiler 1.2 software , and automated allele binning was checked manually.In total, 68 microsatellite loci were genotyped, consisting of 51 transcriptome-derived loci , confidence intervals of 95%, a false discovery rate (FDR) of 0.05 (implemented within Lositan), with both the Infinite Alleles and the Stepwise mutation models. BayeScan was run with burn in = 50 000, thinning = 50, sample size = 1000, number of iterations = 300 000, number of pilot runs = 20, length of pilot runs = 5000, and an FDR of 0.05 (implemented within BayeScan). Outlier loci were considered to be those that were identified by both methods as being significant outliers. Based on the results of the outlier testing, three datasets were created consisting of: (i) all 60 loci, (ii) 59 loci, excluding the most severe outlier (Her14), and (iii) the most severe outlier, Her14, alone.Two selection test methods were used: Fdist  implemenhttp://blast.ncbi.nlm.nih.gov/Blast.cgi). We used blastn searches against the nucleotide collection (nr/nt), reference RNA sequence (refseqrna), and the three-spined stickleback (Gasterosteus aculaetus) genome, and blastx searches against non-redundant protein sequences (nr) and reference proteins (refseq_protein).BLAST searches were performed for loci Her14 and CPA107 using the NCBI website  and expected heterozygosity (HE) were calculated for the three different datasets and for each sampling site using FSTAT version 2.9.3.2  using FIS values were tested for each locus in each site using GenePop on the Web  plots were generated using the cmdscale function in R . The results files were then used in Structure Harvester  were performed with and without the detected cluster for K = 2–10 for the following datasets: (i) all loci detected as outliers under putative positive selection by either Lositan or BayeScan, (ii) all loci detected as outliers under putative balancing selection, (iii) all loci detected outliers under either positive or putative balancing selection, (iv) all loci except those detected as outliers under putative positive selection, (v) all loci detected as outliers under putative balancing selection, and (vi) all loci except those detected as outliers.t tests  to test for differences between the mean locus specific FST values, expected heterozygosity, and allelic richness of each marker type.Markers derived from the transcriptome, and thus gene-associated and potentially affected by directional or purifying selection, may show a different level of differentiation compared to loci derived from genomic libraries, and putatively selectively neutral. In order to test this we performed Welch Two Sample Oceanographic connectivity offers a mechanistically derived measure of the potential exchange between populations which may explain patterns of genetic differentiation . We modej and ending in grid cell i, and then summarised in a 5168 × 5168 connectivity matrix. We then multiplied the connectivity matrix by itself 30 times to estimate connectivities among spawning locations for 30 dispersal events, which may be viewed as a multiple-generation connectivity and correspond better to gene flow than single dispersal events  for locations closest to the sampling sites. Two different averages were used: mean values for the month of April , and mean values for the 2 weeks spanning the estimated spawning time . In addition, an estimate of fisheries pressure based on The mean temperature and salinity at 5–10 m depth were estimated using Helcom data , or due to bottleneck effects such as those induced by high fishing pressure . We testFST) for the three different datasets and environmental distances. Increased environmental distance may result in increased genetic distance as individuals or populations adapt to different environmental factors in different regions .In order to test for associations between specific alleles and environmental variables, and to further substantiate both the outlier and environmental correlation tests, MatSAM v.2  was usedFST values were comparable and whether the location DE-RUGEN is consistently distinct, or whether the observed genetic structure may be caused by temporal fluctuations. These comparisons were made using subsets of the nine loci used in the present study that were also used in previous studies : CPA104, CPA107, CPA112, Her14, Her37, Her41, Her63, while locus CPA114 fell in the direction of putative balancing selection (Low FST relative to HE). Of these, two loci (CPA107 and Her14) were identified as significant outliers after FDR correction (Table S3). BayeScan identified the same two loci as putatively under positive selection, and 20 other loci as potentially being under putative balancing selection (Table S4). It should be noted that while the statistical detection of positive selection is relatively easy and robust with low levels of neutral FST as found here, the detection and interpretation of negative or balancing selection (i.e. lower FST than expected) is difficult and much less robust.The loci detected as outliers by Lositan were the same under the Infinite Alleles and Stepwise mutation models. Those that fell outside the 95% confidence intervals were mostly in the direction of positive selection  = 1.000) was shown to be considerably more of an outlier than CPA107  = 0.963), hence our decision to run specific Her14 analyses. The blastn nr/nt search for locus Her14 against the nucleotide collection only showed hits for the microsatellite repeat region of the sequence, with short flanking regions . The blastn search against the three-spined stickleback genome, reference RNA sequences, and the blastx searches also showed no good hits. Locus CPA107 showed no good blastn nr/nt, genome, refseqrna, or blastx hits.Locus Her14 . Estimates of FIS and P-values for departure from HWE (heterozygote deficiency) showed that no single locus had consistent departure from HWE in all populations (Table S6). Most populations (9 out of 15) showed a homozygote excess, with no apparent geographic pattern .Allelic richness and expected heterozygosity averaged across the 60 loci ranged between 4.804–5.751, and 0.467–0.551, respectively (Table S5). The one-way FST was 0.008 (P < 0.001), and ranged from zero to 0.075 for individual loci, with locus Her14 having the highest (FST = 0.075) and locus Her37 the next highest estimate . Pairwise FST values (Table S8) using all 60 loci showed significant genetic structure between the majority of the sites , and this result remained the same on removal of locus Her14. For locus Her14 alone, 40/105 site pairs showed a significant FST, and the great majority of these were between the sites SE-STROMSTAD, DK-FREDRIKSHAVN, DE-RUGEN, LV-LIEPAJA, and all other sites (38/40 of the significant results).Overall The multidimensional scaling (MDS) plot for all 60 loci  show thaThe Bayesian clustering analyses using Structure showed that when using the dataset of all 60 loci with no admixture, the sites DE-RUGEN, SE-STROMSTAD, DK-FREDRIKSHAVN, and LV-LIEPAJA stand out as a distinct cluster . DE-RUGEWhen only the 38 putatively neutral loci are run , both the DE-RUGEN, SE-STROMSTAD, DK-FREDRIKSHAVN, LV-LIEPAJA cluster, and the EE-NARVANLAHTI, FI-VAASA cluster are still identified . Again, DE-RUGEN remains more divergent than SE-STROMSTAD and DK-FREDRIKSHAVN from the Baltic Sea populations.FST values for the transcriptome versus the genome-derived microsatellite markers , however expected heterozygosity and allelic richness were both significantly lower in the transcriptome-derived loci .Comparisons of the different marker types indicated that there was no difference between the mean locus-specific The map illustrating the multiple-generation connectivities among the sites based on oceanographic modelling indicates three main regions of connectivity, one in the North around the Bothnian Bay, one in the East around the Gulf of Finland, and one in the West around the Kattegat/Skagerrak B.r = 0.317, P = 0.040), and 59 loci , but not with Her14 , 59 loci , and Her14 .Significant isolation by distance (IBD) was found using both the dataset with all 60 loci . The correlations with oceanographic connectivity remained significant when the DE-RUGEN site was removed , and only Her14 including DE-RUGEN remained significantly correlated to oceanographic connectivity when geographic distance was accounted for .Oceanographic connectivity seemed better than IBD at explaining the genetic differentiation: with reduced genetic differentiation where there was high oceanographic connectivity  were very similar . The two temperature estimates are less similar to eachother; mean April temperatures (ca. 0–6°C) tended to decrease towards the North, whereas mean spawning-time temperatures (ca. 2–10°C) were more variable between sites.F1,6 = 14.518, P = 0.009), and this correlation became a little stronger if the site DE-RUGEN is removed . There were no other statistically significant (P < 0.05) correlations (Table S9).The general linear models showed that locus Her14 heterozygosity was correlated with mean April temperature (FST) and environmental distances . However, when DE-RUGEN was removed, it became clear that both this site, and locus Her14 were again having a substantial effect on the results.In analyses using all sites, no statistically significant correlations were detected between genetic distance  and Her14  and locus Her14 datasets .P = 0.05, only three of the loci  showed such associations at P = 0.0001 (after a Bonferroni correction applied within the MatSAM software). It is noteworthy that these analyses show similar results for locus CPA112 associations with salinity as were found by P = 0.0000001), the frequency of allele 138 in locus Her14 was associated with latitude, distance from the entrance to the Baltic, April temperature, and April and Spawning time salinity , 0.006 , and indicate significant genetic structuring in herring within the Baltic Sea. As well as significant pairwise differentiation between the majority of the sites sampled, we also identified several distinct genetic clusters. We found oceanographic connectivity to be a better predictor than geographical distance at explaining the genetic differentiation. A single transcriptome-derived microsatellite (Her14) showed particularly high FST values of ≍ 0.008 , was a substantially stronger outlier with an FST of over double that of Cpa112 (Her14 FST = 0.075). Significant pairwise FST values between the majority of the study sites using all datasets  indicated that the genetic differentiation is not driven by a single outlier site. Indeed, clear patterns of isolation by distance confirmed that genetic differentiation is occurring throughout the Baltic region.Recent studies have demonstrated clear differentiation between herring in the Atlantic and the Baltic Sea, however these studies have provided limited information on structuring within the Baltic Sea . Genetic ≍ 0.008 , though FST. While Rügen was the most differentiated site, the other three sites had a large number of individuals that showed similar genotypes to the Rügen individuals. It is perhaps surprising that other nearby sites on the Swedish coast  do not show such similarity to Rügen. Our results suggest that oceanographic connectivity explains the genetic differentiation better than geographic isolation by distance does, and it can be seen from the biophysical model of ocean connectivity  clustering with nearby sites in the Kattegat, Skagerrak and on the Latvian coast . This cluster was apparent using Bayesian clustering, and also from multidimensional scaling plots of ectivity B that thectivity  observedltic Sea . Adult hltic Sea . The micFST estimates between comparable sites from sampling in 2002/2003  that do not cluster in the same group. In addition, there is little evidence for oceanographic connectivity occurring exclusively between these sites. It is possible that the similarity could be due to chance, or perhaps due to similar but unknown selective pressures in the two regions (e.g. pollution or disease). FST but significantly lower allelic richness and expected heterozygosity compared to microsatellites derived from genomic libraries. The lower diversity may be explained by purifying selection acting upon the transcribed genes, resulting in relatively more conserved genetic regions  in all three datasets (when DE-RUGEN was excluded). The FST-mean April temperature correlations remained after correcting for geographical distance (60 loci and Her14 datasets only) or oceanographic connectivity (60 loci dataset only), and after correcting for April salinity (60 locus and Her14 datasets only), indicating that the result is likely to be biologically meaningful. No correlations were detected between spawning-time temperature and FST or genetic diversity, and this is likely because the temperature at the spawning time is less reflective of differences between the sites: mean April temperatures reflect differences between the sites at a fixed time, while the spawning dates are not fixed between sites. Population differentiation (FST) correlated with salinity (spawning-time and mean April estimates), but only once DE-RUGEN was excluded. For both salinity measures, this result remained consistent after correcting for oceanographic connectivity (60 loci and Her14 datasets), geographical distance (though only for Her14 locus), and temperature. Association tests between allele frequency and environmental variation showed that one particular Her14 allele (138) increased in frequency with increasing temperature and salinity, and decreased in frequency with increasing distance from the entrance to the Baltic and decreasing latitude. While the latter two variables are difficult to disentangle as they co-vary, the associations between genetic differentiation at locus Her14 with temperature and salinity were not caused by isolation by distance or oceanic connectivity, implying that the temperature and salinity correlations can not be explained by a demographic effect.Genetic diversity was quite uniform throughout the Baltic and in most tests average FST and April temperature was only significant with DE-RUGEN removed. In contrast, in the Altogether, our results indicate that both temperature and salinity differences between sites may contribute to the patterns of genetic differentiation, although the correlation between The International Council for the Exploration of the Sea (ICES) currently divide herring in the Baltic region into five management units A: BothniFST and isolation by distance), as well as distinct genetic clusters of herring within the Baltic Sea. It is clear from our results, supported by the findings of previous studies  presently comprises several distinct populations. Herring spawning in the southern Baltic along the German-Polish-Lithuanian-Latvian coast are connected oceanographically due to the anti-clockwise circulation pattern , and posRügen herring undertake feeding migrations to the Skagerrak and return to Rügen in spring for spawning . GeneticAlthough we have shown genetic discontinuity, there are gaps in our geographical sampling regime, particularly the Gulf of Riga and the Polish coast, and we have not included Autumn spawning herring stocks in this study. More comprehensive sampling is required to hone our understanding of genetic differentiation in this region for a targeted study on Baltic management units. In particular this may help to resolve the issues surrounding the differentiation of Northern Baltic herring. However, we have shown that the Baltic herring do not represent a panmictic population, and in light of these findings, the management units in this region warrant reconsideration. Furthermore, our approach using a large number of gene-based molecular markers combined with oceanographic modelling and environmental data may prove useful for elucidating potential population units and subsequent management implications for other marine species. A recent study by


Plasmodium vivax is a leading cause of malaria around the world, only a handful of vivax antigens are being studied for vaccine development. Here, we investigated genetic signatures of selection and geospatial genetic diversity of two leading vivax vaccine antigens – Plasmodium vivax merozoite surface protein 1 (pvmsp-1) and Plasmodium vivax circumsporozoite protein (pvcsp). Using scalable next-generation sequencing, we deep-sequenced amplicons of the 42 kDa region of pvmsp-1 (n = 44) and the complete gene of pvcsp (n = 47) from Cambodian isolates. These sequences were then compared with global parasite populations obtained from GenBank. Using a combination of statistical and phylogenetic methods to assess for selection and population structure, we found strong evidence of balancing selection in the 42 kDa region of pvmsp-1, which varied significantly over the length of the gene, consistent with immune-mediated selection. In pvcsp, the highly variable central repeat region also showed patterns consistent with immune selection, which were lacking outside the repeat. The patterns of selection seen in both genes differed from their P. falciparum orthologs. In addition, we found that, similar to merozoite antigens from P. falciparum malaria, genetic diversity of pvmsp-1 sequences showed no geographic clustering, while the non-merozoite antigen, pvcsp, showed strong geographic clustering. These findings suggest that while immune selection may act on both vivax vaccine candidate antigens, the geographic distribution of genetic variability differs greatly between these two genes. The selective forces driving this diversification could lead to antigen escape and vaccine failure. Better understanding the geographic distribution of genetic variability in vaccine candidate antigens will be key to designing and implementing efficacious vaccines.Although  Plasmodium vivax causes tens of millions of malaria cases each year. Although some vaccines against P. vivax are being developed, little is known about the geospatial genetic diversity and selective constraints of the parasite surface antigens that these vaccines target. In order to create vaccines that are both efficacious and useful in diverse regions of the world, the strain diversity of these potential vaccine targets must be well understood. Specifically, we must understand whether and how the human immune system develops immunity against these antigens as well as understanding whether these antigens are similar in geographically diverse parasite populations. Here, using next-generation sequencing and population-genetic analyses, we found evidence of likely immune selection in specific regions of two leading vivax vaccine candidate antigens, PvMSP-1 and PvCSP. At the pvmsp-1 locus, we also found more genetic variability within populations than between populations, with some DNA sequences from geographically diverse populations being highly similar. In contrast, pvcsp sequences from geographically diverse populations are very distinct from one another, with specific sequence patterns occurring in certain geographic regions. Our findings provide new insights into the geographic genetic diversity of these two antigens and can help inform the development of effective P. vivax vaccines. Plasmodium vivax causes 80 to 300 million infections per year and over 2.5 billion people remain at risk of infection despite malaria elimination efforts P. vivax is growing due to reports of increasingly severe disease P. vivax malaria P. vivax and P. falciparum lends hope for effective vaccines against these parasites; however, because the majority of global malaria research funding targets P. falciparumP. vivax antigens are currently being considered for vaccine development P. vivax merozoite surface protein 1 (pvmsp-1) and circumsporozoite protein (pvcsp).P. vivax circumsporozoite protein has an immunogenic central repeat, consisting of two major types of nonapeptide repeats  flanked by highly conserved 5′ and 3′ regions  and we used the PacBio and Illumina platforms to sequence the complete pvcsp gene (n = 47) from Cambodian isolates pvmsp-1 and pvcsp, we extracted worldwide pvmsp-1 and pvcsp sequence data available in GenBank (n = 238 for pvmsp-1 and n = 412 for pvcsp) , and studied our sequence data alongside the sequences from GenBank msp-1. Finally, we compare the performance of Illumina and PacBio sequencing to traditional Sanger sequencing, and discuss the potential and challenges of next-generation sequencing for population genetic studies of malaria parasite antigens.To better understand the selective forces on, and geospatial genetic diversity associated with Clinical samples from a previous study were used for this study pvmsp-1 42 kDa region  was amplified using primers F: 5′-CAG GAC TAC GCC GAG GAC TA-3′ and R: 5′-GGA GGA AAA GCA ACA TGA GC-3′ and an Eppendorf Mastercycler  in 50 µL reactions containing 5 µL 10× Qiagen Hotstar Master Mix , 0.25 µL Qiagen Hotstar Taq, 300 nM forward primer, 300 nM reverse primer, 1 µL 10 mM dNTPs, and 5 µL 5–10 mM template. Cycling conditions were: 95°C×15 m; 35 cycles of 95°C×45 s, 55°C×45 s, 72°C×3 m; and 72°C×10 m. The pvcsp gene (PVX_119355) was performed by nested PCR. The outer step used primers F: 5′-GGC AAA CTC ACA AAC ATC CA-3′ and R: 5′-TGC GTA AGC GCA TAA TGT GT-3′. Reactions were as above except for 600 nM forward primer, 600 nM reverse primer, 1 µL 10 mM dNTPs, 5 µL 5–10 mM template, 6 µL of 25 mM MgCl2, and 28.75 µL H2O. Cycling conditions were: 95°C×15 m; 25 cycles of 95°C×45 s, 45°C×45 s, 72°C×3 m; and 72°C×10 m. The inner step used 600 nM of each of the primers F: 5′-AAA CAG CCA AAG GCC TAC AA-3′ and R: 5′-GAC GCC GAA AAT ATT GGA TG-3′ using 5–10 µL of the initial amplification. The cycling conditions were: 95°C×15 m; 25 cycles of 95°C×45 s, 54°C×45 s, 72°C×3 m; and 72°C×10 m.The pvmsp-1 and pvcsp amplicons were fragmented by acoustic shearing  using the following settings: 10% duty cycle, 5.0 intensity, 200 cycles per burst, and frequency sweeping mode. Forty-eight barcoded libraries were prepared using the NEXTflex multiplex library kit , each containing the pooled pvmsp-1 and pvcsp amplicons from one patient. Libraries were sequenced on the Illumina HiSeq2000, using the paired-end 100 base pair chemistry .pvmsp-1 short reads de novo and to determine SNP frequency within each assembly. For purposes of comparison and confirmation, we re-sequenced 9 pvmsp-1 amplicons with differing MAFs: 3 samples with all MAFs>90%; 3 samples with all MAFs between 60% and 90%; 3 samples with MAF<60% for at least one SNP. Sanger-sequence haplotypes were compared to predicted Illumina haplotypes. Based on these comparisons, only predicted pvmsp-1 haplotypes with MAF>60% at all polymorphic sites were used in our analysis.We used Lasergene SeqMan NGen v.3.1.1  to assemble pvcsp amplicons were sequenced using PacBio Circular Consensus Sequencing . One PacBio SMRT cell produced a total of 12103 reads with a minimum of 3× circular consensus coverage, which were used for this study. These were further filtered, removing truncated reads or reads with errors in the barcode. This left 8430 reads (3979 forward and 4451 reverse). Clustering attempted to minimize false positive haplotypes due to erroneous base calls and PCR slippage within the tandem repeat region. For each sample, haplotypes were created by clustering reads, allowing reads differing only by indels of 1 and 2 bases and low quality mismatches to collapse. Low quality was defined as either a mismatching base Q<30 or any Q<25 within an 11 basepair region centered on the mismatch, as has been applied previously to rigorous SNP discovery from shotgun data In addition to Illumina sequencing, pvcsp, and JX461286-JX461333 for pvmsp-1.The long-read haplotypes determined through consensus clustering were used as templates for short-read alignment using Bowtie2 v 2.1.0 http://cran.us.r-project.org/).Rarefaction curves of haplotypes were calculated using EstimateS v9.0. Individual-based curves using sampling without replacement were estimated pvmsp-1 and the whole-gene of pvcsp. Sequences from a recent publication GenBank was queried for population sets published prior to August 1, 2013, which included sequence data for the 42 kDa region of pvmsp-1 and the pvcsp constant regions using a 50 bp sliding window with a 25 bp step size. We also performed 1000 coalescent simulations with recombination to determine a 95% confidence interval and centile for each Tajima's D estimate pvmsp-1 42 kDa region amplicons reported here and by others, 15 Plasmodium knowlesi pkmsp-1 isolates from Thailand pvmsp-1 relative to pkmsp-1, and were not considered. We could not obtain MK estimates for pvcsp sequences due to numerous insertions and deletions relative to pkcsp.Population datasets with >25 sequences that were collected over a span of ≤4 years were included for analysis of selection. We used DnaSP v5.1 to perform tests of selection pvcsp repeats, we performed pairwise comparisons of untranslated repeat units within individual pvcsp sequences For analysis of FST). Pairwise fixation values between pvmsp-1 populations were calculated in DnaSP. Site-specific fixation values for pairwise comparisons among Cambodia, NW Thailand, S Thailand, India, and Turkey were generated using the analysis of molecular variance (AMOVA) function within Arlequin v3.11 Interpopulation heterogeneity was first assessed using Wright's fixation index  Neighbor-joining trees for NN) NN, 1000 jackknife samplings were constructed for both pvmsp-1 and pvcsp VK210 and VK247 populations using Fast UniFrac pvmsp-1 haplotypes, we constructed a median-joining network using NETWORK v4.6  pvcsp due to the variable length of CR haplotypes.To cluster geographic groups, we calculated Hudson's nearest-neighbor statistic (Spvmsp-1 42 kDa-fragments (pvmsp-1 42 kDa haplotypes (26 unique haplotypes) with a major allele frequency of >60% at all polymorphic sites (pvmsp-1 haplotypes in this region of Cambodia (Table S1) containing either the whole-gene or 42 kDa-region sequence information.We Illumina sequenced ic sites Table 1.P. vivax population and a Thai P. knowlesi population p = 0.00427) in the intervening region but not in the HARBs (data not shown) or the entire 42 kDa region (p = 0.681), suggesting that the intervening region is under long-term selective pressure   , demonstrpvmsp-1 fragment that are considered vaccine candidates were each assessed for diversity in the Cambodian population The three regions of the pvmsp-1 42 kDa region contains potential vaccine candidates pvmsp-1 42 kDa diversity, we calculated Wright's Fixation index (FST) FST values between naturally evolving parasite populations  approached zero, showing a high degree of genetic similarity, while comparisons with populations that have undergone a recent bottleneck (S Thailand and Turkey) showed a high degree of genetic distance due to their limited number of haplotypes. Similarly, FST values calculated for each variable site demonstrate a high degree of homogeneity in pairwise comparisons between the Cambodia, NW Thailand, and India populations across all sites, and substantial heterogeneity between S Thailand and Turkey across all sites . This is evidence that balancing selection maintains a similar range of alleles in the pvmsp-1 42 kDa region of multiple geographically diverse naturally evolving P. vivax populations.Although the n index FST TableNN), which assesses how frequently a variant's nearest neighbor is from the same population pvmsp-1 42 kDa sequences from naturally evolving populations in Cambodia, India, and NW Thailand showed no evidence of strong geographic clustering  region, resulting in inappropriately short CRs. In contrast, PacBio long reads allowed the gene to be sequenced in its entirety and, after clustering, predicted 47 pvcsp haplotypes within the 43 samples. Reported error rates for PacBio sequencing have been high, especially for indels pvcsp haplotypes, individual haplotypes were used as a template for alignment of Illumina reads from the same clinical isolate. The addition of Illumina reads corrected only a single 1-bp deletion in a single haplotype. Therefore, after clustering, PacBio-predicted haplotypes have an error rate of 1/(∼1200 basepairs/sequence ×47 sequences), or approximately 0.002%.We sequenced the complete pvcsp sequences (n = 27), and 12 cohorts of CR sequences (n = 385) pvcsp CR haplotypes in this part of Cambodia, and that there are significantly fewer pvcsp CR variants in this region of Cambodia than pvmsp-1 42 kDa variants  or Tajima's D test  is important for parasite invasion of hepatocytes EWTPCSVTCGVGVRVRRR, similar to previous reports In contrast to s D test Table 1.pvcsp CR, we assessed the dN/dS ratio for Cambodian VK210 and VK247 p<0.001) and VK247  repeats. This is consistent with the finding that VK210 and VK247 isolates from around the world consistently demonstrate a depressed dN/dS ratio, suggesting that the VK210 and VK247 repeat regions are both under strong purifying selection To better understand the selective forces acting upon the P. falciparum csp is thought to evolve by slipped-strand mispairing pvcsp repeats, we studied the mismatch distribution of pairwise genetic distances between untranslated repeat units within each VK210 and VK247 repeat array type in Cambodia. Consistent with another study pvcsp repeats have a high proportion of identical or nearly identical repeats (data not shown). This finding is consistent with a continuous and rapid expansion and contraction of repeats by slipped-strand mispairing, which may be a mechanism to evade host immunity The CR of pvcsp CR, but did not define the correlates of differentiation between populations A recent study assessed global genetic diversity in the pvcsp CR, we modeled CR repeat expansion, contraction, and substitution using MS_Align, which calculates an event-based genetic distance between CR haplotypes –pvmsp-1, the VK210 and VK247 trees revealed striking geographic clustering by country and continent. We quantified clustering using Hudson's SNN, and observed strong genetic differentiation between most geographically diverse parasite populations, in contrast to pvmsp-1  Latin American repeat arrays contained either a 5′ (GDRADGQPA)4 or an internal (GDRADGQPA)3–4, while very few (11/278) of the Asian sequences contained one or both of these features. Similarly, for VK247 repeat arrays, all (34/34) Latin American sequences began with a single EDGAGDQPG repeat, while only one (1/44) Asian sequence began with this repeat. These sequence features may represent a reliable method to assign sequences to a geographic region.We were able to define the peptide sequence basis of the clustering observed among pvmsp-1 and pvcsp sequences from Cambodia, (2) identifies a signature of putative immune-mediated, frequency-dependent selection in the pvmsp-1 42 kDa region and the pvcsp CR, and (3) provides the most comprehensive evaluation to date of geospatial genetic diversity for these genes. We also demonstrate the feasibility of using a next-generation sequencing approach to study the genetic diversity of malaria antigens.This study (1) presents the first population set of P. vivax amplicon sequence data from clinical isolates. This work represents a first step into this largely unexplored territory. As a relatively new technology, next-generation sequencing methods must be validated before use in molecular epidemiological studies. We provide evidence that the dominant Illumina-predicted pvmsp-1 haplotypes are consistent with Sanger sequencing, and are fit for comparison with population sets generated by traditional sequencing methods. Methods for predicting multiple haplotypes from short-read sequencing are under development and will need further validation. We also demonstrate the ability of combined PacBio-Illumina haplotypes to predict pvcsp VK210 and VK247 haplotypes out of individual mixed infections. As next-generation sequencing methods are utilized more frequently for population genetic studies of infectious diseases, the methods introduced here will be further improved and will help to provide greater insight into Plasmodia population genetics.A distinguishing feature of this study is the use of next-generation sequencing methods to generate pvmsp-1 42 kDa intervening region is under strong immune pressure in multiple panmictic populations. Results from the MK test suggested that this region is under sustained selective pressure  balancing selection.We found compelling genetic evidence that the pressure Table 2;P. vivax proteins.Because PvMSP-1 is a merozoite surface antigen, it is highly accessible to antibodies and complement. The predicted structure of the 42 kDa region shows that the 33 kDa fragment covers the 19 kDa fragment pvmsp-1, Tajima's D and FST were inversely correlated. Populations with strong evidence of high Tajima's D in the pvmsp-1 intervening region showed a low genetic differentiation by FST. This suggests that in naturally evolving populations, diversification of this region is extensive and maintains a similar range of genetic diversity despite geographic distance. Populations that have undergone a recent bottleneck show a low Tajima's D with relatively few variants and strong genetic differentiation from more diverse populations. This suggests that if strain-specific immune responses are important in vaccine efficacy, vaccines may work more effectively if other interventions can be used to bottleneck the population, thus decreasing its genetic diversity For pvcsp CR P. falciparum NANP repeats of varying lengths P. cynomolgi csp CR The central repeat region (CR) is a primary immunodominant region of PvCSP. Though alignment-based methods to assess for selection  cannot be employed in a tandem repeat region, there is wide-ranging evidence that selective pressures shape the genetic composition of the pvcsp isolates from Brazil pvcsp isolates pvcsp VK210 and VK247 isolates have a strong bias toward synonymous substitutions. This signature of purifying selection is consistent with reports from pfcsppvcsp locus in both VK210 and VK247 variants. This phenomenon may be responsible for immune evasion Our analysis of the two CR array types, VK210 and VK247, also suggests that selection is occurring in this region. In pairwise comparisons of nucleotide and amino acid differences we observed a positive skew showing decreased differences among repeat units. This finding is consistent with Patil et al.'s study of P. falciparum malaria, substantial differences are seen in the effects of immune selection between these genes and their orthologs. Previous reports have shown that the functionally similar pfmsp-1 42 kDa fragment has relatively low nucleotide diversity and lacks evidence of balancing selection by Tajima's D pfcsp, on the other hand, shows a high level of nucleotide diversity P. vivax, and this highlights the need for P. vivax-specific studies to determine appropriate candidate vaccine antigens.Although these two vivax genes are orthologs of well-characterized vaccine candidate antigens from pvmsp-1 42 kDa region underscores the importance of selecting an appropriate parasite population for population-genetic studies. We did not observe signatures of balancing selection in pvmsp-1 populations from S Thailand or Turkey. This is likely due to bottlenecks secondary to robust malaria control measures employed in S Thailand Finally, our analysis of the pvcsp, but not pvmsp-1, showed strong clustering by geography  the depressed number of non-synonymous mutations suggests purifying selection, (2) the differences in CR genotypes between geographic locations suggests directional selection, and (3) the genetic composition of the repeats suggests rapid expansion and contraction, possibly due to immune selection. It is unclear what drives the first two signatures of selection. We hypothesize a model in which purifying selection within a population limits the amino acid composition of repeats due to functional constraints of the protein, while directional selection between populations is driven by environmental factors.It is interesting that the CR of pvcsp CR sequences is the mosquito vector. The circumsporozoite protein is expressed in the mosquito during oocyst development One environmental factor that may explain both the purifying and directional selection of parasite pvmsp-1 and pvcsp, the observation itself has significance for vaccine design. The malaria vaccine field is just beginning to unravel how antigenic diversity within a single parasite population can reduce vaccine efficacy pvmsp-1 may necessitate a highly multivalent pvmsp-1 vaccine, while the dramatic interpopulation variability seen in pvcsp suggests that a PvCSP-based vaccine that is effective in one part of the globe may not be effective in other regions. Thus, a thorough understanding of the geospatial genetic diversity of candidate vaccine antigens must inform antigen selection for vaccine design.Regardless of the cause of the differing patterns of geospatial genetic diversity we observed in pvcsp sequences: JX461243-JX461285 and KJ173797- KJ173802pvmsp-1 sequences: JX461286-JX461333Figure S1Geographic distribution of P. vivax populations contributing to this study. In total, we identified 13 populations with pvmsp-1 42 kDa fragment sequences and 13 populations with pvcsp central repeat or whole-gene sequences. These populations were collected from 14 countries, pictured above. For countries with n≥10 isolates, the total number of pvmsp-1 and pvcsp isolates is marked.(TIF)Click here for additional data file.Figure S2FST values at polymorphic sites within the pvmsp-1 42 kDa intervening region. Available parasite populations with n>25 individuals  share 42 variable sites within the 42 kDa intervening region of pvmsp-1. FST values for each variable site were calculated in a pairwise manner between all five populations. FST values approaching 0 indicate limited inter-population variability at that site, while values approaching 1 indicate substantial inter-population variability. Coordinates are reported for every third polymorphic site.(TIF)Click here for additional data file.Table S1pvmsp-1 and pvcsp sequences included in this study. The PlasmoDB gene identifier is PVX_099980 for pvmsp-1 and PVX_119355 for pvcsp. *Indicates the year the sequences were made available in GenBank. †Indicates study unpublished but sequences available in GenBank.(DOCX)Click here for additional data file.