Gonyostomum semen (Raphidophyceae). The study covered a 2-year period including all phases of the bloom. Amplified fragment length polymorphism (AFLP) was used to determine the genetic structure and diversity of the population. Our results showed a significant differentiation between samples collected during the two blooms from consecutive years. Also, an increase of gene diversity and a loss of differentiation among sampling dates were observed over time within a single bloom. The latter observations may reflect the continuous germination of cysts from the sediment. The life cycle characteristics of G. semen, particularly reproduction and recruitment, most likely explain a high proportion of the observed variation. This study highlights the importance of the life cycle for the intraspecific genetic diversity of microbial species, which alternates between sexual and asexual reproduction.The field of genetic diversity in protists, particularly phytoplankton, is under expansion. However, little is known regarding variation in genetic diversity within populations over time. The aim of our study was to investigate intrapopulation genetic diversity and genetic differentiation in the freshwater bloom-forming microalga  The field of genetic diversity in microorganisms has been under expansion since the development of recent molecular tools. Yet, little is known about the mechanisms involved in the generation and maintenance of genetic diversity in microbes in general, and about the dynamics of populations in particular. Due to their short generation time and huge population size, microbes are interesting and suitable organisms when studying the mechanisms involved in genetic differentiation and population dynamics. In addition, microorganisms show an extreme diversity, and live in a large variety of environments. In aquatic systems, autotrophic protists can make up an important part of the biomass and are major contributors to primary production. Some species can form blooms, which can be considered as large ephemeral populations because they occur within a few weeks or months .Bloom-forming phytoplanktonic species have been intensely studied in their ecology, the environmental conditions enhancing the bloom formation, and capacity to overgrow other species. Nevertheless, few studies have focused on investigating their intraspecific genetic diversity. Indeed, due to their largely asexual proliferation and low morphological variation, phytoplankton blooms have often been assumed to be either monoclonal or of extremely low genetic diversity. However, several recent studies have shown surprisingly high intraspecific genetic diversity in both marine and limnic species  Diesing – a nuisance freshwater raphidophyte, responsible for dense blooms that can dominate the phytoplankton community by up to 95% . We used amplified fragment length polymorphism (AFLP)  when the water column was mixed.In 2009, vegetative cells of in April . GonyostA total of 182 strains were successfully isolated, cultured without cyanobacterial or protist contamination  and genotyped . The surG. semen clonal isolates for each sampling date ranged between 0.066 and 0.102. In 2009, the gene diversity increased during the bloom . The October value of gene diversity was similar to those observed at the end of the bloom in 2008  were observed between samples collected in 2008 and samples from all the sampling dates of 2009 with values ranging from 0.103 to 0.180 (P < 0.01) from all other sampling dates, and FST values ranged from 0.061 to 0.130. The only exception was the second date of sampling, which was not significantly different from the first sampling date. The last five dates of sampling during the bloom did not show significant genetic differentiation between each other.nd 0.180 . Highly to 0.180 . IndividThe population structure analyses performed using the software STRUCTURE suggested the presence of a single population. Using the In this study, for the first time, we describe changes in genetic diversity during frequent intervals over time in a single lake phytoplankton bloom, as well as between two consecutive blooms. Despite a predominance of asexual reproduction, we observed an increase in genetic diversity coupled with a loss of genetic differentiation between samples over time. In addition, we found significant genetic differentiation between the populations sampled for two consecutive years in the same lake.Ditylum brightwellii, showing the extreme diversity occurring within a single bloom. In the same study the D. brightwellii population had similar genetic diversity values as observed for the G. semen population in the current study. If we assume a similar genotypic diversity for G. semen as the one observed for D. brightwellii, the probability of isolating two identical clones with our sampling design (20 cells per sampling date) was very low, which is in accordance with our results.The genotypic diversity observed for each sampling date concurs with previous studies demonstrating that phytoplankton blooms are not monoclonal events  was observed between the isolates from 2008 and 2009, of similar magnitude as the differentiation measured between populations of D. brightwellii sampled from different sites 20 km apart by G. semen within Lake Bökesjö occurred at a range similar to the differentiation occurring between populations in different lakes or sampling locations in phytoplankton species and also in some macroorganisms.Highly significant differentiation  clustered in separate populations mainly corresponding to the year they were isolated.Temporal differentiation of phytoplanktonic populations has been observed in previous studies, mostly in marine diatoms. In contrast to the present study, these studies have focused on marine species and had long time intervals between sampling. For instance, G. semen produces resting cysts after sexual reproduction, which overwinter in the sediment in order to survive winter conditions that are unfavourable for vegetative growth . Nor can it be explained by genetic recombination, as there was no evidence of sexual reproduction (i.e. presence of fusing cells) until September. Instead, an alternative explanation is that the increase of genetic diversity is caused by a continuous germination of new genotypes throughout the spring and early summer. In a field study, G. semen from the sediment (resting stages) to the water column can occur during the entire growth season. In that study, G. semen cells were observed in recruitment traps until September. On the other hand, germination experiments , an endogenous clock and temperature  with high significance. FST analyses heterozygosity between pre-defined populations, STRUCTURE does not use prior information concerning the samples. Hence, STRUCTURE analysis is often considered more conservative, and detects a higher level of population structure.In our study, the Bayesian population analysis by STRUCTURE identified one population. Thus the STRUCTURE analysis did not discriminate different populations despite high This study addresses fine-scale temporal differences both within and between consecutive blooms. With the strains successfully isolated and cultivated, we found significant genetic differentiation between two consecutive populations of a microalga within a single lake. Furthermore, we observed variation of genetic diversity during the bloom along with differentiation between sampling dates, possibly due to the germination of resting cysts, leading to an increase of genetic diversity over time. We propose that for phytoplankton, intrapopulation genetic diversity may be closely related to their life cycle. Studies on different phytoplankton groups are necessary to understand how life cycle can have large implications on genetic diversity patterns over time. Also, caution should be taken when interpreting data from comparative studies on diverse populations as the bloom state of the populations must be considered before drawing any conclusions.Gonyostomum semen cells were collected in Lake Bökesjö  at 55°34′N, 13°26′E. Lake Bökesjö is a small humic lake  situated in a beech forest in the nature reserve Häckeberga .G. semen bloom), and between April and October 2009. In 2008, samples were taken from the shore on four occasions within 3 weeks, for cell isolation only. In 2009, to cover all the phases of the bloom, samples were collected on nine occasions with 3-week intervals. Sampling started in April, corresponding to the initiation of cyst germination, and ended in October when the resting cysts were formed and the bloom ended. At the deepest point of the lake, a HQ30d temperature/oxygen meter (Hach) was used to measure the temperature and oxygen concentration of the water column in order to determine the depth of the stratification. Samples for chlorophyll a analysis and phytoplankton abundance were collected in the epilimnion using a plexiglass tube sampler . For G. semen isolation, in October 2008, the cells were collected from the shore in the surface water using a 20 µm net. In 2009, during low abundance of G. semen (< 1000 G. semen cells ml−1), a fresh sample of phytoplankton was collected using a 20 µm net in the epilimnion. For the sampling during high abundance of G. semen (> 1000 G. semen cells ml−1), a water sample of the epilimnion column was collected using the plexiglass tube sampler, as G. semen clogged the net when present in high abundance.To compare the population genetic structure of two consecutive years, sampling was performed in October 2008  using an inverted microscope (Nikon Eclipse TS100). Epilimnetic water samples were collected and preserved with Lugol's solution. For the early and late phase of the bloom (low abundance of G. semen), 20 ml of sample was sedimented in a 20 ml counting chamber overnight. At high abundance, G. semen cells were counted in 1 ml of sample using a Sedgewick-Rafter counting chamber, the whole chamber was counted.The epilimnetic water was filtered through GF/C glass fibre filter for chlorophyll G. semen vegetative cells were done in the laboratory using an inverted microscope (Nikon Eclipse TS100) and a glass micropipette prepared with 100 µl capillary tubes . The cells were transferred and washed three times in a mix of50% of sterile filtered Bökesjö water and 50% of artificial MWC medium (−1). Subsequently, the cells were transferred to individual wells of a 96-well Nunc plate  containing 200 µl of medium–lake water mix.Single cell isolations were performed to obtain a final number of approximately 20 clonal cultures per sampling date . The isoC medium  modified−2 s−1, with 12 h:12 h light : dark cycle. When the cell concentration had reached approximately 2000 cells ml−1, 20 ml of culture was harvested by centrifugation at 500 g for 15 min. The pellets were transferred to a 1.5 ml tube and centrifuged at 500 g for 10 min and the medium was discharged. The pellets were frozen at −80°C until DNA extraction.The strains were cultivated in increasing volumes of mix 50% MWC-50% sterile filtered Bökesjö-water at 20°C with a light 20 µmol mg for 15 min at 4°C. The upper layer was transferred to a 1.5 ml tube, one volume of 5 M NaCl and two volumes of ice-cold isopropanol were added. The DNA was precipitated by centrifugation at 20 000 g for 15 min at 4°C. The pellet was washed twice with ice-cold ethanol (70%) and centrifugation at 20 000 g for 5 min at 4°C. The ethanol was discharged and the samples were dried. The DNA was resuspended with 50 µl of TE buffer. The DNA concentration of the samples was estimated by measuring absorbance of a subsample diluted 10 times at 260 nm using a . For each sample, the quality of the DNA was determined using the 260/280 ratio. Only samples of high DNA quality, i.e. with a 260/280 ratio of 2.0, were used for downstream analyses. The DNA samples were stored at −80°C until genotyping.DNA extraction was performed using a CTAB-base method with the following steps. The pellets of cells were resuspended with 0.7 ml of CTAB lysis buffer [100 mM Tris-HCl (pH 8), 1.4 M NaCl, 20 mM EDTA, 2% (w/v) cetyltrimethylammonium bromide (CTAB), 0.4% (v/v) β-mercaptoethanol, 1% (w/v) polyvinylpyrollidone (PVP) . The samples were diluted 10 times, and dried at room temperature before sending them for capillary electrophoresis analysis. All the samples were then analysed with a MapMarker 1000 bp size standard with an ABI37730XL capillary electrophoresis machine at the Uppsala Genome Center, Sweden. To check for the effect of the presence of bacteria on the AFLP genotyping, a few samples containing only the supernatant (containing bacteria) of G. semen culture were extracted and genotyped. A few AFLP peaks were observed in the samples containing only the supernatant. These fragments were not observed in the genotype of the G. semen strain from which the supernatant was removed. These results indicate that the bacteria content did not affect the genotyping of G. semen strains.Amplified fragment length polymorphism (AFLP) analyses were performed on the samples according to FST values to estimate genetic differentiation between sampling dates, the P-values were determined using 1000 permutations, and the FST were considered significant for P < 0.05. The number of genetic populations were determined using the software STRUCTURE 2.3.3  and AFLPscore version 1.4  and the


Chalcides mionecton from Morocco and used Bayesian analyses of nuclear and mitochondrial DNA (mtDNA) loci to examine: (i) their concordance with morphological patterns, (ii) support for species delimitation, (iii) timing of speciation, and (iv) levels of gene flow between species. Four digit individuals were found at sites between Cap Rhir (in the south) and the northern extreme of the range, whereas five-digit individuals were found in two disjunct areas: (i) south of Cap Rhir and (ii) the north of the range where they were often syntopic with four-digit individuals. The pattern of variation in generalized body dimensions was largely concordant with that in digit number, suggesting two general morphotypes. Bayesian analyses of population structure showed that individuals from sites south of Cap Rhir formed one genetic cluster, but that northern four- and five-digit individuals clustered together. Statistical support for delimitation of these genetic clusters into two species was provided by a recent Bayesian method. Phylogenetic–coalescent dating with external time calibrations indicates that speciation was relatively recent, with a 95% posterior interval of 0.46–2.66 mya. This postdates equivalent phylogenetic dating estimates of sequence divergence by approximately 1 Ma. Statistical analyses of a small number of independent loci provide important insights into the history of the speciation process in C. mionecton and support delimitation of populations into two species with distributions that are spatially discordant with patterns of morphological variation.Delimitation of species is an important and controversial area within evolutionary biology. Many species boundaries have been defined using morphological data. New genetic approaches now offer more objective evaluation and assessment of the reliability of morphological variation as an indicator that speciation has occurred. We examined geographic variation in morphology of the continuously distributed skink  Traditionally, most species have been described using morphology, but morphological analyses cannot easily demonstrate isolation and so genetic analyses are generally required . StatistAdditional approaches can provide other important insights into speciation. Algorithms implemented within programs such as IMa2  the evidence for genetic species within C. mionecton, (ii) whether these reflect morphological differences, and (iii) the timing of divergence and degree of gene flow between these putative species.Chalcides mionecton were captured from 16 sites extending from the north to the south of its range during field trips carried out between June 2006 and June 2008 . We used individuals that had been collected for previous research projects  and Juan Grande in Gran Canaria, and C. coeruleopunctatus from Calera and Vallehermoso in La Gomera and from Valverde and Frontera in El Hierro.Canary Island skinks belong to the same Western ionecton . Divergets e.g., ; see Brots e.g.,  for moreExternal morphology of the 192 museum specimens was recorded. The numbers of digits was recorded from both the manus and the pes. The following 12 body dimensions were measured to the nearest 0.1 mm using digital calipers: snout–vent length (SVL), head length (HL), head depth (HD), head width (HW), jaw length (JL), snout–thorax (ST), snout–hind limb length (SHL), fore–limb length (FLL), rear–limb length (RLL), fourth toe on fore–limb length (FTFL), fourth toe on hind limb length (FTHL), and body width at thorax (BWT). The following three scalation characters were also recorded: the number of ventral scales rows between the rostral scale and the cloaca, the number of scales counted around the mid-trunk, and the number of infra-digital lamellae on the fourth toe of the hind limb.  Bilateral measurements were recorded from the right-hand side.10 body dimensions against log10 SVL were used to adjust each body dimension to its value at the overall mean log10 SVL. Discriminant function analyses (DFA) were computed on these size-adjusted values, with individuals grouped by sampling locality. This method provides functions that maximize among-group relative to within-group variation, and therefore allows investigation of whether body dimensions differ between regions. DFAs were also applied to the three scalation characters.Sexes were analyzed separately. The pooled within-group coefficients from the least-squares regressions of logcytb). Two nuclear gene fragments were also amplified: (i) 371 bp of the c-mos gene and (ii) 713 bp of the RAG-1 gene. Polymerase chain reaction (PCR) products were sequenced by GATC Biotech  and Source BioScience . Wherever possible, several individuals were sequenced from each site for the NADH  and c-mos (n = 84) fragments in order to gain insights into within-site genetic diversity. One or two individuals were sequenced per site for cytb (n = 21) and RAG-1 (n = 20).DNA was extracted from tail-tips using a standard phenol–chloroform procedure. Primers were designed or used from previous studies (Appendix S2). The following mtDNA fragments were amplified: (i) 737—795 bp (depending on primers used) of NADH dehydrogenase subunits 1 and 2 and three intervening tRNAs (this fragment will be referred to as “NADH”) and (ii) 655 bp from the cytochrome b gene . Multilocus genetic structure within mos and RAG-1 sequences . We obtained the multilocus maximum likelihood values of the per locus Watterson's estimator of population mutation rate  was used to specify upper bounds on uniform prior distributions. Specifically, population sizes were specified from the uniform distribution, U, splitting time, t (in generations) was specified from U, and migration rates per mutation (m) were specified from U. The rationale for using these prior upper bounds is outlined in the IMa2 documentation (http://lifesci.rutgers.edu/∼heylab/ProgramsandData/Programs/IMa2/Using_IMa2_8_24_2011.pdf). The mutation rate for the NADH sequence (per locus per year) was calibrated using a 1.1 Ma divergence between Chalcides from the Canary islands of La Gomera and El Hierro  approach implemented in the program IMa2  was usedrate see . This isμ and is the probability of migration in a specific direction, per gene copy per generation). A likelihood ratio test (LLR) (Probabilities of differences between population sizes and migration rates were obtained by pairwise comparisons of posteriors. Population migration rates were obtained as 2NM (where M = mst (LLR)  was used0) was assigned from the gamma prior G, while the other divergence time parameters were assigned from a Dirichlet prior  to G. Not surprisingly, this led to slightly different posterior estimates of these parameters, although the posterior probability for the two species model was unaffected. Analyses were run several times to check for consistency .The program BPP  was usedcytb sequences from C. mionecton from all sites (n = 21), together with the nine Canary Island Chalcides described above, were included in the analysis. Nuclear loci showed negligible diversity across the Canary Island taxa and so we used only the mitochondrial locus for the phylogenetic analyses  was used for robust estimation of divergence times between genetic clusters identified by BAPS . The metC. mionecton, (ii) Southern C. mionecton, (iii) C. sexlineatus (Gran Canaria), (iv) C. viridanus (Tenerife), (v) C. coeruleopunctatus (La Gomera), (vi) C. coeruleopunctatus (El Hierro). Time priors were placed on the species tree, with 1 time unit = 10 Ma. The general prior on divergence times was specified using a Birth–Death model, with the birth–death rate specified from the uniform distribution U and death rate relative to birth rate specified from U. The times of the most recent common ancestors (tMRCAs) of different species groups were constrained using geological information. The oldest emerged parts of the Canary island of El Hierro have been dated at 1.12 Ma with the island likely to have been colonized soon after its subaerial emergence  node. Two other nodes were also constrained albeit more loosely. The age of the root was specified from the gamma distribution G, and the ancestor of all Canary Islands sequences was specified from G, following previously published estimates (Species were defined as follows: (i) Northern genetic cluster of mergence . The gamstimates .Chalcides, and on each of the two forms of C. mionecton  distribution, and the standard deviation from the exponential distribution Exp(0.33), for all partitions. The HKY+G model was specified with kappa parameters assigned from G and alpha parameters from U, for all partitions. Monophyly constraints were imposed on sequences from the same island, on La Gomera and El Hierro together, on all Canary Island cton see .7 generations with a sampling interval of 1000, and the first 3000 samples were discarded as burn-in. Convergence diagnostics as well as posteriors on the gene and species trees were obtained using Tracer (v1.4.1) (MCMC chains were run for 3 × 10(v1.4.1)  and Tree(v1.4.1) .To date, it has been common to use Bayesian phylogenetic methods to date sequence divergence. In these analyses, time calibrations are applied to sequences and sequence divergence times are estimated. We also used this approach  for comparison with the *BEAST phylogenetic–coalescent analysis described above. The priors within the analysis were the same as for the *BEAST analysis, but applied to tMRCAs of the sequences within the relevant species.All individuals obtained from sites south of Cap Rhir (sites 11–16) were found to have five digits on the manus and the pes, except for one individual at site 16 which possessed four digits on the pes only . IndividFor female body dimensions, the first discriminant function (DF1) represented 35.4% of the total variation among sites, while the second function (DF2) represented 28.1%. Values were similar for males: DF1 = 36.2%, DF2 = 24.6%. Patterns of pooled within-group correlations between body dimensions and standardized DFs were quite similar between sexes . Howeverr = 0.76, P = 0.003) and female DF1  were significantly associated with digit number, but correlations were not significant for male DF1  or female DF2 . DFAs on males and female scalation characters revealed no differences between northern and southern forms (results are not shown for conciseness).The northern populations with one or more five-digit individuals were ostensibly intermediate between the remaining four-digit northern populations and the five-digit southern group . There wC. mionecton from different regions .For mtDNA, substantial numbers of mutational steps separate  regions A. The cybed here B and C. k = 5 genetic clusters by BAPS provided the highest log likelihood (lnL = −1507.5). Higher values of k produced decreasing log likelihoods . However, all analyses assigned all southern individuals to a single cluster, that is, an increase/decrease in the value of k simply increased/decreased the number of northern clusters  (k = 2. This produced a lower likelihood (lnL = −1914.1) but the two genetic clusters represented northern individuals from sites 1–10, and southern individuals from sites 11–16  . This ises 11–16 .P > 0.05). Population migration rates from the north to the south were also very low (P < 0.001). Marginal posteriors on current population size parameters (θ) were represented by single peaks. For the southern form, θS was small: posterior mean = 0.401, 95% HPD (0.125–0.731), compared with θN (northern form): posterior mean = 1.007, 95% HPD (0.390–1.730). The difference between θS and θN was significant (Pr [θ N> θ S] = 0.997). Various exploratory analyses with different priors indicated that the data were relatively uninformative about the ancestral population size parameter, θA, or the population splitting time.To facilitate interpretation, we will report on migration rates in the natural (forward) direction of time rather than coalescent time. Population migration rates from the south to the north are negligible A, with a, 0.158])A, but he0 were 0.0064 .The posterior probability of the north–south species delimitation was 1.00. As for IMa2, the posterior on Θ for the northern form indicated a larger population size , compared with the southern form . Note that BPP and IMa2 are parameterized differently and so parameters are not directly comparable between programs. The posterior mean and 95% HPD for τC. mionecton  species node (P = 0.98). The corresponding posterior mean speciation time was 1.43 Ma , which was approximately 1 Ma older than the posterior north–south speciation time within C. mionecton (see above). However, this does not represent a north–south split between sequences. The ancestral node that is common to the two mtDNA lineages, comprising sequences from northern (7–10) and southern sites (11–16), is quite well supported (P = 0.94). It has a posterior mean and posterior interval of 1.58 and  Ma, respectively, which is quite similar to that obtained under the phylogenetic–coalescent analysis.The *BEAST coalescent analysis provided strong posterior support for the –2.66 Ma)B. ComparSpecies delimitation can require a combination of genetic, morphological, and biogeographical information in order to be convincing. Here, coalescent analyses of genetic data alone demonstrate negligible gene flow between northern and southern groups of contiguously distributed populations, indicating that they form separate metapopulation lineages  is dated at 1.6 Ma which is not too different from the phylogenetic–coalescent date for divergence of northern and southern forms. While this highlights important differences between the dating approaches, detailed future comparisons between methods would be more informative in cases where it is appropriate to use a single topology for each analysis, rather than integrating over the range of possible topologies.Until recently, many phylogeography studies have used phylogenetic dating of sequence divergence times . We showic study  by an evC. mionecton, we find that digit number varies in a quite complex manner with five digits being more common throughout the far north of the range than previously thought  One hypothesis is that the four-digit morphology arose due to mutations that appeared in the northern form after N–S speciation. Gene flow between northern populations allowed these mutations to spread north, while restricted gene flow between species impeded southern movement. This scenario suggests that these morphological differences originated recently. However, it does not explain why the distribution of the four-digit morphology is interrupted by a northern area in which only five-digit individuals were found. It has been proposed that digit loss is evolutionary labile, with complete loss of digits over approximately 3.5 Ma in some Australian skink lineages  gene.It is interesting to speculate why some individuals from the most northerly populations should show morphological similarity with the most southern populations, despite delimitation of northern and southern individuals into discrete genetic clusters. The most parsimonious explanation is that the ancestral state in this species is five digits. Other morphological states found in the extreme north and south, such as a wider thorax, would also be considered ancestral but for clarity we focus our argument on digit number. The sister group to e digits  which woe digits  interestlineages . Rapid es remain . This seC. mionecton. However, it does provide information on current distributions and divergence times. There was no evidence of unsuitable habitat between the southernmost four digit sample from Cap Rhir and the adjacent five-digit sample from Taghazoute (separated by a distance of around 25 km), so it is probable that the two forms come into contact without significant hybridization. C. mionecton populations, would appear plausible.This paper does not aim to reconstruct the historical biogeography of C. mionecton therefore appear to represent good species under the unified species concept, but these species are not diagnosed by digit number or other external morphological characteristics.To summarize, we are able to examine key features of the speciation process, namely timing of divergence and gene flow between lineages, using only a small number of loci, two of which have low substitution rates. Bayesian methods provide strong statistical support for divergence of two lineages in the Pleistocene after which very few genes were exchanged. Previously described subspecies of


D. andersoni and D. variabilis in sympatric populations from northwestern North America. Recently, D. variabilis has expanded its range westward into the natural range of D. andersoni. Using a sample of 235 D. andersoni and 62 D. variabilis, we identified 31 individuals as putative hybrids: four F2 individuals and 27 backcrosses to D. andersoni (as defined by NewHybrids). We found no evidence of hybrids backcrossing into D. variabilis. Furthermore, all hybrids presented 16S mtDNA signatures characteristic of D. andersoni, which indicates the directionality of the hybrid crosses: female D. andersoni × male D. variabilis. We also discovered 13 species-specific AFLP fragments for D. andersoni. These loci were found to have a decreased occurrence in the putative hybrids and were absent altogether in D. variabilis samples. AFLP profiles were also used to determine the levels of genetic population structure and gene flow among nine populations of D. andersoni and three of D. variabilis. Genetic structure exists in both species  as well as significant estimates of isolation by distance .Hybridization in ticks has been described in a handful of species and mostly as a result of laboratory experiments. We used 148 AFLP loci to describe putative hybridization events between  An estimate of 10% of animal species and 25% of plant species are found to be capable of hybridization and/or introgression, with some taxa more prone than others Mallet . HybridiHowever, successful hybridization between species has been described in numerous wild and domesticated plants and animals  species have shown hybridization to be possible in some of these species . We use genetic markers to explore whether equal genetic mixing occurs among parent species, or if one-way introgression into one of the parent species has occurred. Furthermore, we also characterize the population genetic structure of D. andersoni along its natural range in the northwest intermountain region of North America, and report genetic structure in disjunct western populations of D. variabilis.In this study, we use molecular markers to report the occurrence of hybridization between Dermacentor andersoni (Stiles), the Rocky Mountain wood tick, and Dermacentor variabilis Say, the American dog tick, are hard ticks of the family Ixodidae. Both are three-host ticks; each life stage feeds on a different host and molting between stages occurs off of the hosts. Mating occurs on the host and engorged females drop to lay eggs , and lagomorphs . In contrast, adult D. variabilis are found primarily on wild and domestic canids , as well as on felids, mustelids (badgers and weasels), bears, raccoons, skunks, rabbits, voles, and opossums. They rarely use larger animals like deer, horses, and cattle. The immature stages (larvae and nymph) of these two ticks show almost no overlap in host preferences, with D. andersoni using a wider host range that includes many species of chipmunks and ground squirrels and, less frequently, marmots, lagomorphs, voles, Peromyscus, and wood rats. Meanwhile, immature D. variabilis are found overwhelmingly on voles and rarely on Peromyscus mice  with all life stages capable of overwintering  according to the manufacturer's protocol with the modifications described by Scoles . GeneticAFLP fragments were scored against the MapMarker® X-Rhodamine Labeled 50–1000 bp size standard electrophoresed on an ABI 3730 Genetic Analyzer and analyzed with GeneMapper® Software v.4.0 . To reduce the probability of errors due to homoplasy between loci and/or automated scoring mistakes, a conservative approach to scoring was defined. First, loci were selected when at least one individual possessed a band of ≥1500 relative fluorescent units (RFUs). This step reduced the number of usable fragments by about 75%. Furthermore, it assured that only loci presenting a strong signal were considered for analysis. Second, only loci separated by at least ± 1 bp were scored. This was done to avoid a software bias that consistently scores the taller of two bands within 1 bp of each other. Third, given that PCR favors amplification of small fragments only loci between 100 and 500 bp were scored. This lowered the probability of homoplasy between loci, which is a problem with small size markers (<100 bp)  a subset of duplicates was started at the preselective amplification stage (step 2) and taken through the rest of the procedure; and (2) a second subset was duplicated only for selective amplifications (step 3). Scores were compared for duplicate samples and the error rate was calculated as the number of differences divided by the number of comparisons . If two loci showed LD in at least 50% of the populations tested, then they were considered in LD for the entire dataset.All pairs of loci were tested for linkage disequilibrium (LD) in Arimer v.5.2.9  was performed using PD. andersoni and D. variabilis, we used the program Structure v.2.3.3  was set to two, corresponding to each parental species. An admixture model was selected using a burn-in of 25,000 permutations followed by 100,000 repetitions. All runs demonstrated convergence before the end of the burn-in, suggesting good performance of the Markov chain Monte Carlo method. Based on the population analyses of AFLPs, ticks from pure populations were expected to have extremely high assignment probabilities . In contrast, hybrid individuals were expected to display much lower Q-values. To be conservative, hybrids were classified as those individuals with assignment probabilities between 50% and 90%. We then tested a wider range of K values (1–10) to determine whether putative hybrids clustered separately from either parent species. This wider analysis was also useful for the study of population structure (below), which we confirmed with the ΔK method . We did the same for D. variabilis . Given the large number of loci (148), a burn-in period of 75,000 repetitions was defined, and 100,000 iterations were run thereafter.The NAnderson , the sofD. andersoni, but none specific to D. variabilis. We examined the distribution of these loci among the putative hybrids and pure D. andersoni and D. variabilis individuals. We compared the occurrence means using an ANOVA and an a posteriori Tukey test. We expected a lower occurrence of these alleles in the putative hybrids than in pure D. andersoni if genetic material is introgressing from D. variabilis individuals. All 13 alleles were absent in all D. variabilis samples.In the AFLP analyses, we identified thirteen loci specific to ntrogress, an R-based script  in each species, we performed an Analysis of Molecular Variance (AMOVA) as defined by Excoffier et al.  between populations, we used the RELATE function in Primer v.5.2.9 , 9999 permutations were performed.The number of polymorphic loci, expected heterozygosity, and overall mean expected heterozygosity were estimated for each population using the package Gr et al.  using ArD. andersoni and 64 individuals from three populations of D. variabilis (D. andersoni and four in D. variabilis) showed weak evidence of linkage disequilibrium. However, none of the 11 loci exhibited significant LD in more than half of the test populations. We ran all subsequent analyses in this study with and without the 11 loci and found minimal changes in the final results. Thus, LD does not appear to be a problem and we present results generated with all 148 loci.A total of 148 AFLP loci were scored unambiguously for 235 individuals from nine populations of riabilis . All of R = 0.966, P = 0.001). Population polymorphism levels varied between 50% and 95.95% for D. andersoni and between 65.54 and 78.38% for D. variabilis (HE = 0.284 (SE = 0.050) for D. andersoni and HE = 0.232 (SE = 0.009) for D. variabilis  demonstrated that these species are clearly separated with AFLP markers  and recovered very similar admixture patterns for the same 18 ticks. The NewHybrids analysis identified these same 18 individuals plus additional 13 potential hybrids that were not identified by Structure. These 13 individuals were collected from the same six populations mentioned above except for Corwin Springs, MT. The NewHybrids analysis estimated zero F1 individuals, four F2 individuals, 27 backcrosses to D. andersoni, and zero backcrosses to D. variabilis. In no instances did Structure or NewHybrids predict genetic admixture back into the D. variabilis populations.Results of the assignment test in Sndersoni . These pD. andersoni were found among D. andersoni, D. variabilis, and the 31 putative hybrid samples . In all 13 cases, these alleles are specific to D. andersoni, not found in D. variabilis, and found sporadically in the putative hybrids. A posteriori comparison of all pairs of means showed differences among the three groups of samples (α = 0.05). Consistent with the above results, all putative hybrids had 16s mtDNA sequences characteristic of D. andersoni (data not shown).Significant differences in mean count for thirteen species-specific loci to ntrogress analysis, 23 loci (15.5%) showed positive selection, whereas 12 loci (8.1%) demonstrated negative selection as defined by Luttikhuizen et al.  or interfere (negative selection) with genomic introgression between species. In our In et al. . This maD. andersoni . This result suggests moderate genetic connectivity among populations of this species . In light of the low IBD value, we tested the removal of potential hybrids from the AMOVA to determine if the presence of hybrids was determining the observed population structure. This procedure produced almost no change in ΦST .Genetic differentiation among populations was significant for  species . A similD. variabilis . However, this estimate is based on just three populations at the extreme edge of the distribution of this species.Significant population structure was also found for D. andersoni and D. variabilis. Hybrids between these two species have been described previously in laboratory experiments . These samples clearly show levels of admixture in their AFLP profiles, which are most likely the result of hybridization between the two tick species. Four individuals (two from Lolo Pass and two from Corwin Springs) were identified as F2 generation hybrids. The remaining 27 individuals of the possible hybrids presented different degrees of backcrossing to D. andersoni. We discovered 13 loci specific to D. andersoni. As expected, these markers display a lower count in the putative hybrids than in pure D. andersoni individuals. This suggests that some of these alleles are being lost due to hybridization between these two species. However, a third species of Dermacentor tick, D. albipictus, is also found in the sampled regions  broaden their host preferences in a hybrid zone, feeding on both mammals and birds  . The potential for hybrids to survive and quest across a longer transmission season could have important implications for pathogen transmission. Hybridization could also be a mechanism for moving nontransmissible symbionts between these two species and may explain why the non-transmissible Rickettsia peacockii, a symbiont of D. andersoni, has been found in both species (Scoles unpubl. data). This may also have implications for vector competence as it has been suggested that the microbiome of a tick can affect its vectoral capacity. These and other potential effects on their role as vectors provide a logical focal point for investigating the range expansion, hydridization, and subsequent introgression of D. variabilis genes into populations of D. andersoni.In light of these examples, it seems possible that hybridization between D. andersoni is a three-host tick that must quest for a new host at each life stage. This species exhibits little host specificity and parasitizes a broad range of terrestrial mammals , the level of host specificity, and the ecology of each species involved   and the significant but low value of isolation by distance (ρ = 0.066) are comparable to those found in I. pacificus  demonstrated the potential for high levels of population differentiation within this species  were genetically different (D. variabilis populations (D. variabilis was first reported in the early 1970s, when it was first described in Washington and Idaho (Stout et al. D. variabilis transplants may originate from a wide variety of source material in central and eastern North America. Unintentional translocations might simply reflect the genetic variation found in widely separated source populations. Second, female ticks can lay more than 6000 eggs per reproductive season, and only a few individuals are needed to establish a new population. Under these conditions, strong founder effects are to be expected and could explain the low levels of gene flow observed in our results. Further examination of these recent populations is needed to understand whether D. variabilis is ecologically established in this region and not simply repopulated each year by humans.The level of genetic differentiation in ifferent , indicatulations . This is


Carcharhinus melanopterus) in French Polynesia. Five demes were sampled from five islands with different inter-island distances (50–1500 km). Whether dispersal occurs between islands frequently enough to prevent moderate genetic structure is unknown. We used 11 microsatellites loci from 165 individuals and a strong genetic structure was found among demes with both F-statistics and Bayesian approaches. This differentiation is correlated with the geographic distance between islands. It is likely that the genetic structure seen is the result of all or some combination of the following: low gene flow, time since divergence, small effective population sizes, and the standard issues with the extent to which mutation models actually fit reality. We suggest low levels of gene flow as at least a partial explanation of the level of genetic structure seen among the sampled blacktip demes. This explanation is consistent with the ecological traits of blacktip reef sharks, and that the suitable habitat for blacktips in French Polynesia is highly fragmented. Evidence for spatial genetic structure of the blacktip demes we studied highlights that similar species may have populations with as yet undetected or underestimated structure. Shark biology and the market for their fins make them highly vulnerable and many species are in rapid decline. Our results add weight to the case that total bans on shark fishing are a better conservation approach for sharks than marine protected area networks.The population dynamics of shark species are generally poorly described because highly mobile marine life is challenging to investigate. Here we investigate the genetic population structure of the blacktip reef shark ( Sharks have been considered keystone species driving the evolution of marine ecosystems Effective conservation planning for any organism can partly rely on defining the spatial extent of populations, the levels of exchange (connectivity) among them and their distribution in space Several studies that have found significant genetic heterogeneity among populations of shark species have included samples between oceans e.g.  leading 2 of ocean French Polynesia is a prime example of a fragmented marine environment for a shark species known to inhabit coastlines; there are 130 islands in French Polynesia spread across 2,500,000 kmWe tested for the presence of genetic structure among 5 shark demes from the reef and coastal habitat surrounding 5 different islands with varying inter-island distances (50 to 1500 km). We use the word ‘deme’ here to define a group of individuals that have been sampled around the same island and because these may not be populations per se.Locations of sampling sites in French Polynesia are detailed in Table S1 and were taken and adapted from DNA samples were extracted following the PURAGENE™ DNA Purification Kit procedure for 5–10 mg fresh tissue. PCR was conducted using 4 multiplexes for 11 microsatellite loci, and following QIAGEN® Multiplexes PCR Kit procedure. The loci characteristics and thermal cycling conditions used are described in A first set of analyses was computed to test the quality of the 11 markers. Microcheckerv2.2.3 FST and RST) We used one genotypic clustering approach and two allele-based statistics approaches . We used the program Bottleneck We used ARES FST values and the log of geographic distances, as suggested in Because of the low number of demes, the correlation between geographic distance and genetic differences was examined by jackknifing over populations, using Isolation By Distance RST/pRST test described in Considering the stepwise mutation model, we used the program SPAGeDI Table S3, which has a histogram describing differences in allele frequency between demes. Differences in allele frequencies are used here to estimate genetic differences between demes. These raw frequencies, for each deme and each locus, are very different in some of our cases. As an example, the allele 220 in the locus LS20 is present 5.7% of the time in the Rangiroa deme but 42.4% of the time in the Fakahina deme.Heterozygosity ranged from 0.077 (Cli2) to 0.865 (LS53) with a mean of 0.492 Table 1.The DAPC results for the 21 principal components found show a clear genetic structure for all demes.Overall, individuals are associated with their original deme. The membership probability is particularly strong for Fakahina as a majority of individuals are 100% associated with the Fakahina deme. A large number of unsampled demes are likely reinforcing the structure of isolated demes. However, individuals among the 4 other demes also show strong membership probability to their original deme, but with more individuals having mixed signals than it was the case with Fakahina. There is very little genetic signal from Fakahina in other demes. In contrast, Maria has mixed individuals with signal mostly from Moorea and Rangiroa. Some outliers are observed in all clusters, suggesting potential recent migrations. These results are depicted in FST results show high values for the Fakahina deme  and for the Maria deme . FST results are highly significant for all deme pairs (p = 0.01). Fakahina RST values are of 0.301 versus Moorea, 0.437 versus Tetiaroa, 0.111 versus Rangiroa and 0.081 versus Maria . Rangiroa RST values versus Moorea and Tetiaroa are small (0.0041 and 0.126) but significant (p = 0.017 and 0.008). The Moorea versus Tetiaroa RST value is 0.054, and is non-significant (p = 0.063).Pairwise 2 = 0.739); the slope after jackknifing over the 5 demes (0.3654) has a large standard error  indicated that 8% of the variation was caused by variation among geographic clusters, 38% by the variation within individuals and 54% by the variation among individuals. When an AMOVA was performed for either 4 or 5 groups, the ‘among populations’ value was 7%. The Isolation by Distance shows that genetic and geographic differences are correlated  = 0.984) and for Tetiaroa-Fakahina  = 0.977) /extrapolate  the number of samples used for the analysis to 40. The estimated values of allelic richness ranged from 68 for Rangiroa to 44 for Fakahina and Maria, 57 for Moorea and 53 for Tetiaroa. Despite these differences in allelic richness, no evidence of a bottleneck was found, using the IAM, SMM and TPM . The microsatellite allele frequency distribution was L-shaped for each locality, indicating that these were all at mutation-drift equilibrium for the loci used. The FST and RST estimators can provide valuable information about the genetic structure of populations with some provisos FST and RST results include the presence of evolutionary forces like genetic drift In combination, the FST values are high, suggesting strong genetic differences among demes. This is especially true for the Fakahina and Maria demes, which are further from Moorea, Tetiaroa and Rangiroa than any of these are from each other. The Isolation by Distance analysis shows that genetic differentiation  mostly increases with the distance separating demes. With only 5 sampled demes, it is hard to make general conclusions based on the Isolation by Distance results. Pairwise FST values are slightly higher though for Fakahina than for Maria, even though Maria is furthest the other islands sampled. This could be due to differences between these islands in the degree of isolation. The island closest to Fakahina is more than 200 km away while 4 islands in the Tuamotu chain are within 100 km of Maria. The proximity of islands in the Tuamotu chain to Maria is likely promoting some gene flow. The effective population size estimates of Fakahina and Maria are exactly the same based on allelic richness and so probably do not affect the comparison of their FST values.Even given the above, in the case presented here pairwise FST values are known to underestimate genetic differentiation in the case of strong genetic structure RST, potentially indicating that the gene flow is not sufficient to counter the effect of a higher level of genetic divergence. The only significant RST/pRST results are associated with Fakahina. Variance is high for the results among loci but this result is consistent with the particular geographic isolation of Fakahina. Migration rates are unlikely to be sufficient at Fakahina to keep allele sizes from diverging from the other demes. When these new microsatellite alleles appear in a deme, it means that gene flow has been especially low for many generations.The Fakahina deme appears more differentiated when using the FST results this suggests structuration by genetic drift with some level of gene flow. Surprisingly, RST values associated with Maria (except for Maria-Fakahina) are small and non-significant. The small RST values associated with Maria deme probably come from loci that do not fit the SMM behind the RST calculation. We suggest the RST values between demes are misleading in comparison to the results of FST value comparisons. Our case for this is that the FST values, the results of the Bayesian approach and the allele distribution (see Table S3) all suggest that Maria deme is strongly differentiated from any other deme.The patterns are not consistent though – there are never more than 4 loci significant for the same pair of demes. As with the Carcharhinus limbatus in Tests for sex-biased dispersal seem to hint at a pattern of male-based dispersal (non-significant result) but the sample sizes are likely too low to detect a pattern. Most of the outliers found in the clustering analysis are indeed males, supporting the suggestion that male-based dispersal is a life-history trait of these populations. This is aligned with the findings of other similar studies, like that of Understanding the complex process driving genetic structure can be tricky, and results must be treated with caution  and juveniles/sub-adults (as future adults) need to be protected to ensure shark populations are self-sustaining. Range sizes for sharks are difficult to assess but are highly likely to be larger than the proposed areas management agencies and conservationists plan to have in a marine protected area (MPA) network. Further, shark populations are naturally small, at least in fragmented environments. Protecting large areas will not result in the population and biomass increases seen in teleost fish protected in MPAs. For these reasons it is important to consider shark populations in conservation planning but MPAs are not likely to be the best solution for protecting sharks. The management ‘best practice’ approach to protecting most shark populations is a total ban of shark fishing everywhere this is possible. The Bahamas, French Polynesia, and Palau are examples of locations where such a ban has already been implemented. These nation-wide regulations with other recently implemented shark sanctuaries represent steps in the right direction. Other management actions like MPAs remain critical and can have positive effects on shark populations but are insufficient to avoid the severe declines caused by shark fishing.Table S1Locus description with sequence, repeat type and hybridization temperatures. All ‘Cli’ are taken from (TIF)Click here for additional data file.Table S2Extrapoled allelic richness and means. Tetiaroa was excluded because of a low number of samples (12).(TIF)Click here for additional data file.Table S3Allele frequencies for each deme ordered by size (left) and presented as histograms.(TIF)Click here for additional data file.


Argiope bruennichi expanded its range recently and we asked whether rapid settlement of new sites exposes individuals to a risk of inbreeding. We sampled four geographically separated subpopulations, genotyped individuals, arranged matings and monitored hatching success. Hatching success was lowest in egg-sacs derived from sibling pairs and highest in egg-sacs derived from among-population crosses, while within-population crosses were intermediate. This indicates that inbreeding might affect hatching success in the wild. Unlike expected, differential hatching success of within- and among-population crosses did not correlate with genetic distance of mating pairs. In contrast, we found high genetic diversity based on 16 microsatellite markers and a fragment of the mitochondrial COI gene in all populations. Our results suggest that even a very recent settlement secures the presence of genetically different mating partners. This leads to costs of inbreeding since the population is not inbred.Few studies investigated whether rapid range expansion is associated with an individual's short-term fitness costs due to an increased risk of inbred mating at the front of expansion. In mating systems with low male mating rates both sexes share potential inbreeding costs and general mechanisms to avoid or reduce these costs are expected. The spider  Inbreeding, defined as the mating between two related individuals, increases the occurrence of homozygous deleterious alleles. The loss of heterozygosity leads to a decrease in the fitness of offspring, known as inbreeding depression In mating systems with classical sex roles . However, some species are tolerant to short-term inbreeding as for example Stegodyphus lineatusOedothorax apicatusAnelosimus cf. jucundusInbreeding is particularly likely if a small number of individuals split off from the original population and establish a new population representing only a fraction of the gene pool of the source population Argiope bruennichi (Araneae) that unites a mono- and bigynous mating system and has recently extended its range from southern Europe and Asia to Northern Europe A. bruennichi can be considered a successful disperser. In combination with the observation that the species has started its range expansion from a large source population, it is likely that newly established populations even by small numbers of individuals encompass some genetic variation. A. bruennichi disperses aerially by ballooning and bridging to move within habitats. This passive mode of dispersal, particularly ballooning, entails a large component of chance as individuals can only influence direction by selecting certain wind conditions to fly A. bruennichi spiderlings hatch simultaneously from large clutches after winter and likely disperse in groups from the same brood when conditions are favourable. This will lead to a situation in which many siblings from a single female are likely present in a meadow that also contains other families. Spiderlings may disperse short or long distances. This scenario creates both, inbreeding risk as siblings encounter one another and costs of inbreeding (note that costs of inbreeding require a population that is not inbred). Such a scenario match data derived from mating experiments and field observations that demonstrated selection to avoid the costs of inbreeding Here, we use the spider A. bruennichi egg-sacs and juveniles from four similar sized populations located near the northern edges of the species range. We assessed genetic diversity by analysing 16 microsatellite loci and a part of the mitochondrial COI gene. Furthermore, we assembled mating pairs that stemmed from the same egg-sac, from different egg-sacs of the same population or from two different populations and correlated the genetic distance of the mating partners with mating behaviour and hatching success. We predicted differences in mating behaviour with increasing relatedness of the mating partners and expected genetic distance to be positively correlated with hatching success.We collected While the sampled populations are all located within the recently colonised range of the species, they likely differ in their short-term settlement history in that they may have been populated early in the invasion process or in recent years.Argiope bruennichith century with the exception of an isolated group around Berlin 2 .A. bruennichi possess paired mating organs. Females have two copulatory openings that are connected by two ducts to independent sperm storage organs (spermathecae) As typical of entelegyne spiders, Argiope species as well Females show a highly aggressive mating behaviour. All females attack males during copulation and 80% of males are cannibalised by the female after mating We collected egg-sacs and juveniles from four geographically separated populations in the northern part of Germany , Quarrendorf , Buxtehude , and Hamburg-Moorfleet ) between the end of April and the beginning of June 2010. There were no specific permissions required for these locations and the sampling did not involve endangered or protected species.The collected egg-sacs were produced in 2009 and had overwintered. Several hundred spiderlings hatch out of the same egg-sac * 36 * 6 cm) after they moulted to maturity. Mating trials were conducted in the frames, where females built their typical orb-webs. All spiders were sprayed with water five days a week. Males were fed with approx. 15 Drosophila spec., subadult and adult females with three Calliphora spec. on two days a week. After individuals' final moult, both females and males were weighed on an electronic balance (Mettler Toledo AB54-S) to the nearest 0.001 mg. All males and females used in the mating experiments were frozen at −80°C and preserved for genetic analyses (see below). Males were preserved after a single copulation and females were kept in the laboratory to produce egg-sacs until they died a natural death.387 individuals were raised from eggs in the laboratory until they reached adulthood. Each spider was individually labelled so that it was known from which population and from which egg-sac it derived. Males were kept in individual 250 ml plastic cups, whereas subadult females were housed in 330 ml plastic cups and were transferred in individual Perspex frames , between non-siblings from the same population (N = 45) and between non-siblings from different populations (N = 62). Egg-sacs were collected from two populations (Buxtehude and HH-Moorfleet) so that we derived 15 maternal lines . Females and males from these matrilines were randomly assigned to one of the three mating trials. Spiders that were collected as juveniles were only used in the treatment where we arranged matings between different populations. 32 females from population HH-Moorfleet and Buxtehude were paired with males from the same family ; 45 females from population HH-Moorfleet and Buxtehude were paired with males from the same population that hatched out of a different egg-sac and 62 were mated to males that originated from different populations . Each mating pair was allowed a single copulation. Mating trials began by introducing the adult male into the frame threads of the female's web. Trials were terminated after the first copulation. If no mating occurred until one hour had passed, a new male was introduced to the female. A female was presented with a maximum of three males. It never happened that a female was not mated after introducing the third male. During every mating trial, we noted the times of male's first contact with the web and the female, the beginning and duration of courtship and copulation, the insemination duct the male copulated into and the occurrence of sexual cannibalism or male escape from a female attack.Mated females were transferred from the frame into 500 ml plastic cups where they built their egg-sacs. We obtained egg-sacs from 95 females, each of which produced 3.37±0.18 egg-sacs on average. All egg-sacs were weighed on the day of their construction and were visually inspected. Some egg-sacs were damaged or not completed. We selected all intact egg-sacs and left them to hatch. After the young had hatched from the eggs, egg-sacs were preserved and all eggs and spiderlings were counted under the microscope. Hatching success of all intact egg-sacs was determined by the following calculation: total number of spiderlings//100).A. bruennichi. We were able to determine genetic distances in seven sibling pairs, 11 within-population pairs and in 31 among-population pairs.We used microsatellite typing to estimate genetic distance  between individuals within and among the four populations of For this, we extracted DNA with the 5 PRIME ArchivePure DNA Kit according to the manufacturer's protocol .A. bruennichiST value and pair-wise FST values among populations were calculated using Microsatellite Analyser (MSA) 4.05 We genotyped our specimens for a set of 16 previously developed microsatellite loci for Due to the presence of null alleles  for the microsatellite screened, detected with the software Microchecker 2.2.3 Most data were analysed with the statistical program JMP 7.0.2. Non-normally distributed data  were analysed with the non-parametric Kruskal-Wallis test. Significant differences between groups were specified with the Dunn test 2 = 13.12, p = 0.0014; After a period of incubation, egg-sacs were opened and unhatched eggs and spiderlings were counted to determine hatching success. Hatching success was highly variable in all three treatments. As expected, the average hatching rate was lowest for sibling matings , followed by within-population matings  and was highest when the pair originated from different populations . The observed heterozygosity of the four populations ranged from 0.31 to 0.38 and was much lower than the expected heterozygosity  and differentiation between all population pairs were significant  and the frequency of cannibalism  were independent of the genetic distance between the mating partners. Furthermore, the genetic distance between a male and a female did not affect the time required until copulation occurred .We pooled all mating pairs regardless of their origin and tested whether the number of shared alleles between female and male of a mating pair correlated with components of their mating behaviour. The duration of copulation . However, we did not detect significant differences between unrelated pairs derived from the same (0.61±0.03) or from different populations  and male of each pair  revealed no significant association with hatching success either .Using all mating pairs, the genetic distance between female and male of each pair did not significantly predict hatching success of their egg-sacs . The interaction between the mutation and genetic composition of mating pairs suggests more complex genetic interactions and might be one possible explanation of the higher reproductive success of among-population pairs compared to the within-population pairs.A. bruennichi suggests that they are potent ballooners although it is unclear whether all hatchlings of an egg-sac balloon or whether a proportion stays. Published accounts are inconsistent in this respect It remains an open question how relevant incompatible matings are in natural populations that may show a much larger intermixture of genotypes through long distance dispersal. The rapid range expansion of A. bruennichi an admixture of different lineages occurs resulting in the introgression of Asian alleles in populations of Northern Europe As the calculation of heterozygosity based on the microsatellite data set revealed a conspicuous difference between observed and expected heterozygosity of the four populations that did not relate to the genetic distance data, the presence of null alleles was tested for each locus and was confirmed in some loci. Null alleles occur through a failure of amplification during PCR leading to an over-estimation of homozygotes. Therefore we chose the mitochondrial COI gene as an additional marker to better trace the genetic diversity of the four populations. However, a DnaSP analysis of the COI gene data confirmed the similar genetic diversity within the four populations. A comparison with other studies showed that null alleles seem to be widespread in spiders A. bruennichi spiders and that there should be strong selection for inbreeding avoidance. The genetic data suggest that active partner choice would be beneficial even in small and recently founded populations as the genetic diversity is high and consequently the probability of finding a compatible partner is generally high. However, reduced hatching success in pairings of spiders derived from egg-sacs of the same population was not mirrored in the genetic distance data. Incompatibilities other than those caused by inbreeding may be responsible for the reduced hatching success.In conclusion, our results show that sibling matings lead to severe inbreeding depression in A. bruennichi, the probability for individuals of encountering siblings cannot yet be estimated accurately. Studies are under way to close this gap by identifying the genetic population structure of natural populations close to and during the mating season on a small spatial scale. Furthermore, future experiments are of interest to test whether A. bruennichi has evolved pre-copulatory avoidance mechanisms to prevent or at least reduce costs of inbreeding depression in the field. During field studies, we commonly observed that males reject virgin females in the field without any obvious reasons Due to the experimental exclusion of ballooning and missing data on small scale population sub-structuring in


Average coancestry, or the degree of genetic similarity of individuals, increased from 0.81% to 1.44% during the period 1993 to 2005, at the same time that Ne decreased from 263 to 93. The observed trend for fe was irregular throughout the experiment in a way that fe was 68, 87, 77, 92, and 80 in 1993, 1996, 1999, 2002, and 2005, respectively. Simultaneously, fg , the most informative effective number, decreased from 61 to 35. The index of genetic diversity (GD) which was obtained from estimates of fg , decreased about 2% throughout the period studied. In addition, a noticeable reduction was observed in the estimates of fne from 595 in 1993 to 61 in 2005. The higher than 1 ratio of fe to fg indicated the presence of bottlenecks and genetic drift in the development of this population of Zandi sheep. From 1993 to 1999, fne was much higher than fe , thereby indicating that with respect to loss of genetic diversity, the unequal contribution of founders was more important than the random genetic drift in non-founder generations. Subsequently, random genetic drift in non-founder generations was the major reason for fe > fne . The minimization of average coancestry in new reproductive individuals was recommended as a means of preserving the population against a further loss in genetic diversity. The aim of this study was to monitor changes in genetic size of a small-closed population of Iranian Zandi sheep, by using pedigree information from animals born between 1991 and 2005. The genetic size was assessed by using measures based on the probability of identity-by-descend of genes (coancestry,  The long term survival of a population depends on the maintenance of sufficient genetic variation for individual fitness and population adaptability . In the In managed populations, selection is the main factor responsible for the loss in genetic diversity. Whereas genetic drift is expected to occur in populations undergoing selection, Ne; Ne is a measure of ‘identity-by-descent' (IBD) of genes, and determines the level of inbreeding, as well as the degree of genetic variation lost from populations due to random genetic drift. It has been frequently shown that Ne is often much less than the census size of the population  were purchased from various sheep farms in the region of the breeding station. In general, the flock was reared by following conventional industrial procedures. The mating season commences in August. Ewes on heat undergo artificial insemination (AI), with the restriction that mating between very closely related animals is avoided. The maximum number of ewes allocated to each AI ram is no more than 25-head per breeding year. Animals that do not conceive by AI are allocated to natural servicing. In this case, ewes are assigned to ram-breeding groups with an average mating ratio of 10-15 ewes per ram. Lambing commences in December. Coat-color in new-born is black, but gradually changes with age, in such a way that black, light-brown and gray adult animals are to be found.Pedigree information of the tagged individuals has been recorded since 1991, thereby providing an opportunity to study the genetic structure of the population. The analysed pedigree consisted of 6035 animals . Analysis involved populations born every three years from 1991 to 2005. Coancestry  is defined as the probability that any two alleles, sampled at random , are identical copies of an ancestral allele.et al. (2008) and in the form proposed by Gutiérrez et al. (2009) based on the individual increase in inbreeding (ΔFi). ΔFi coefficients are computed as Fi is the individual coefficient of inbreeding and t is the equivalent complete generations  in the reference population relative to the base population is approximated as: GD = 1 - 1/2fg .f) in the population studied during the experiment is shown in Ne, fe, fg, fne, and GD are shown in Ne was non-significant (p > 0.05), although there was a decrease from 263 to 93 during the analysed period. Likewise, fe showed a non-significant trend (p > 0.05) during the experiment and was 68, 87, 77, 92 and 80 in 1993, 1996, 1999, 2002 and 2005, respectively. In contrast there was a significant decrease (p < 0.01) in both fg and fne. For fg, this being from 61 in 1993 to 35 in 2005, with a reduction of approximately 2% in the corresponding diversity index (GD), and for fne from 595 in 1993 to 61 in 2005. The ratio of fe to fg increased significantly (p < 0.01) from 1.11 to 2.28 during the period. Individual and cumulative genetic contributions of the first 41 most influential founders in the population studied appear in The evolution in coancestry . Coancestry highlights the degree of genetic similarity of individuals in a breeding population. High coancestry means low diversity, with less ability in selecting better animals, as the difference between individuals is narrower. Although, coancestry is an informative parameter, and has been shown to be useful for conservation purposes  depends on both their total number (m), as well as the disequilibrium among their expected contributions to the gene pool. If all the founders contribute equally, then fe = m, but when this is not so then fe < m. As shown in fe, are those with a high contribution to the current population. They may have a considerable impact on the trait being selected, if they possess beneficial alleles for the selected trait to pass on to their progeny. In populations with minimum inbreeding, the effective number of founders is expected to be one-half of effective population size. Other situations (fe ≥ Ne) indicate that the breeding structure has undergone certain changes since the founder generation . While fe takes into account the unequal founder contributions, in populations which have undergone a bottleneck, the effective number of founders is overestimated, since the effect of such possible bottlenecks is not taken into consideration, especially in animal breeding, where selection for specialized types of animals imposes strong bottlenecks on the populations. Moreover, there is another important handicap when calculating fe, brought about by the phenomenon, ‘converged genetic contribution of founders' (qk's) will be very similar, whereupon fe will approach a relatively constant value within a few generations, seeing that founder contributions will remain stable. Even in the presence of selection, fe will approach a constant value as long as new founders are not added to the population. fe will also become constant, without further change. In simulations of true random mating, this will happen to a large degree within five generations, although, in real life, populations tend to undergo a certain degree of positive assortative mating which slows this down . Thus, a management program based on the maximization of fe, while partially effective in the initial generations, will be completely ineffective thereafter.The effective number of founders (ounders' . When rafg), which, besides dealing with the total loss in diversity, is directly related to genetic diversity itself. As shown by fg is fg = (1/2c), where c is the mean coancestry of all the individuals in the current population, including the relationship of each individual to itself. The value shows that as the population becomes more related, as would happen with any closed population, fg decreases. A significant decrease in fg reveals a reduction in the genetic size of the population as regards founder genes. As expected, during the experiment, the decrease in estimates of fg was accompanied by a drop in the corresponding GD index which comprises all the causes of reduction in genetic diversity. It is noteworthy that while fg is the more accurate description of the amount of founder variation present in a population, it does not account for mutation or migration, and, if mutations do occur, a slightly higher amount of heterozygosity could be present.The most informative effective number is that of founder genomes (The ratio of the effective number of founders to the effective number of founder genomes, which was higher than 1 throughout the experiment, indicated the presence of bottlenecks and genetic drift in the development of this population of Zandi sheep.fne, it is possible to clarify which, as regards the loss of genetic diversity, is the more important, random genetic drift accumulated in non-founder generations or the unequal contribution of founders. Where fe > fne, the reduction in genetic diversity is more related to genetic drift accumulated in non-founder generations, whereas if fne > fe, the unequal contribution of founders is the major reason for the loss of genetic diversity. In our population and during the period 1993 to 1999, fne was much larger than fe, thereby indicating unequal contribution of founders to be the more important. Afterward, random genetic drift in non-founder generations was the major reason for the loss of genetic diversity as fe > fne.Last effective number, viz. effective number of non-founder genomes, accounts only for the effect of genetic drift in non-founder generations. By using In general, the results obtained here show that in small-closed populations, especially those undergoing managed selection, genetic diversity can be lost at a rapid rate, with two important consequences, first, the loss in heterozygosity, followed by inbreeding depression, and second the loss of allelic variants, thereby limiting long-term responses to selection. Although inbreeding could not be avoided or indefinitely limited, care must be taken in minimizing its rate of occurrence. Various methods have been proposed to minimize the loss of genetic diversity. The minimization of group coancestry has proved to be the most efficient method to conserve genetic diversity . Minimiz


Haliotis diversicolor supertexta, of the family Haliotidae, is one of the most important species of marine shellfish in eastern Asia. Over the past few decades, this species has drastically declined in Korea. Thus, hatchery-bred seeds have been released into natural coastal areas to compensate for the reduced fishery resources. However, information on the genetic background of the small abalone is scarce. In this study, 20 polymorphic microsatellite DNA markers were identified using next-generation sequencing techniques and used to compare allelic variation between wild and released abalone populations in Korea. Using high-throughput genomic sequencing, a total of 1516  reads containing simple sequence repeats were obtained from 86,011 raw reads. Among the 99 loci screened, 28 amplified successfully, and 20 were polymorphic. When comparing allelic variation between wild and released abalone populations, a total of 243 different alleles were observed, with 18.7 alleles per locus. High genetic diversity  was observed in both populations. A statistical analysis of the fixation index (FST) and analysis of molecular variance (AMOVA) indicated limited genetic differences between the two populations . Although no significant reductions in the genetic diversity were found in the released population compared with the wild population (p > 0.05), the genetic diversity parameters revealed that the seeds released for stock abundance had a different genetic composition. These differences are likely a result of hatchery selection and inbreeding. Additionally, all the primer pair sets were effectively amplified in another congeneric species, H. diversicolor diversicolor, indicating that these primers are useful for both abalone species. These microsatellite loci may be valuable for future aquaculture and population genetic studies aimed at developing conservation and management plans for these two abalone species.The small abalone,  Haliotis diversicolor supertexta, which belongs to the Haliotidae family, is a small (maximum shell length of 10–12 cm) abalone that varies in color and is a commercially important species distributed throughout the southern coasts of Korea, Japan, China and Taiwan [H. diversicolor supertexta would aid in the development of a more effective fishery management strategy for this species. However, no information is available on the genetic diversity of wild and released populations of H. diversicolor supertexta.Pacific abalones are gastropod mollusks that are highly valued, especially in eastern Asia. d Taiwan . To compd Taiwan . Howeverd Taiwan . Thus, gd Taiwan . In reced Taiwan . Knowledd Taiwan . Thus, aH. diversicolor [H. diversicolor supertexta must be developed and screened to identify a suite of loci that are powerful and efficient for conducting further population genetic analyses, including assignment tests, pedigree analyses and mapping studies.Monitoring genetic variation among marine resources necessitates the development of genetic markers. Among the available genetic markers, simple sequence repeats (SSRs) or microsatellites (MS) are an extremely valuable tool for population genetic studies as well as conservation and management of genetic resources because of their useful properties, which include high levels of polymorphism, codominant inheritance and good reproducibility –10. MS mrsicolor –13; howersicolor ,15. TherHistorically, MS markers were developed by screening small-insert genomic DNA libraries or repeat-enriched libraries . These tH. diversicolor supertexta using 454 GS-FLX pyrosequencing, and we examined the genetic variability at these loci in a wild and released populations of this species. Additionally, the applicability of these markers in another congener species, Haliotis diversicolor diversicolor, was evaluated via cross-species amplification experiments. The polymorphic MS markers described here will be useful in future genetic studies aimed at understanding the genetic status and facilitating the conservation of two Pacific abalones, H. diversicolor supertexta and H. diversicolor diversicolor, in Korea.In the present study, we developed 20 novel polymorphic MS primer sets for vs. 314.1 bp). This process eliminates repetitive sequences and creates longer reads. We obtained a longer average read length compared with previous studies, with average read lengths of 369 bp in the water strider Gerris incognitus [Agkistrodon contortrix [Pachyornis elephantopus [The raw 454 sequence data from a 1/8 plate run included 26.9 Mbp containing 86,011 reads or sequences with an average length of 314.1 bp . Millions of base pairs of genomic sequence would be useful for both MS-related and MS-unrelated research , and thecognitus , 215 bp hantopus . Longer hantopus . The lenhantopus . Therefohantopus .H. diversicolor supertexta samples. The primer sequences, repeat motifs, annealing temperatures, fluorescent labels and GenBank accession numbers for the 20 new MS loci are summarized in Of the 66,910 unique sequences, 1516 (2.26%) contained simple sequence repeats, and 1143 (75.4%) contained a minimum of five di-, tri- or tetra-nucleotide repeat motifs, which were suitable for use as polymorphic MS markers. Motifs containing five to six repeats were the most abundant (78.6%), followed by seven to nine repeat motifs (17.7%) and motifs with more than ten repeats (3.7%). Among these, 244 sequences with a minimum of seven di-, tri- or tetra-nucleotide repeat motifs were used to develop MS primers. To design the primers, sequences that were of adequate length (more than 300 bp) and unique sequences flanking the MS array (minimum of 100 bases) were selected. Thus, 99 MS loci  were selected for subsequent polymorphism screening. Of these 99 MS loci, 28  were amplified successfully (as viewed on agarose gel) in the initial evaluation of the MS primers. The remaining 51 primers did not generate the desired amplification products in any of the eight samples despite retesting under modified PCR conditions. Additionally, amplifications of 20 loci produced faint or inconsistent bands, which may have been due to nonspecific PCR amplification. Further screening revealed that 20 (20.2%) loci were polymorphic in the eight H. diversicolor supertexta. An additional 899 sequences that contained MS were also obtained. Comparing the number of loci detected across different taxa is difficult because genomes vary substantially in their MS frequency. In this study, only 2.26% of all contigs obtained using 454 sequencing contained a MS. Using an identical MS isolation method, MS were detected in more than 35.2% of contigs in the soybean aphid Aphis glycines [Gerris incognitus [Megalobrama pellegrini [H. diversicolor supertexta. The number of MS detected is likely to be inflated by the occurrence of multiple reads covering the same sequence [The development of polymorphic MS markers using 454 sequencing technology is an outstanding new method. An increasing number of studies are using this technique to identify MS because it is less expensive and less time-consuming than traditional methods . Using 4glycines , more thcognitus  and morellegrini . Therefosequence . The incH. diversicolor diversicolor. All the primer pairs effectively amplified their target sequences using the same PCR conditions because these species are closely related based on taxonomy. Cross-species amplification is effective only if primer sequences are conserved between species, and generally, the number of amplified loci tends to decrease proportionally with an increasing divergence between species [Additionally, the cross-species amplification of 20 MS markers was performed in another subspecies,  species . We noteH. diversicolor supertexta (each n = 60) collected from the coastal waters of Donggwi  were screened for variation by the 20 newly uncovered polymorphic MS loci. The 20 primers yielded variable profiles, and reruns were performed for 26.7% of each of the sample sets to ensure that the allele scoring was reproducible. No differences were observed, indicating that genotyping errors did not affect allele scoring. Samples that failed to amplify after the rerun were not included, which reduced the likelihood that our results were affected by poor DNA quality.Two wild and hatchery-bred populations of FST relative to neutral expectations, indicating that these loci may be subject to selection. To minimize the detection of false positives, we either assumed that null alleles were present at these seven loci or that they were under directional selection because they were detected by both methods with high statistical support. Thus, these loci were eliminated from subsequent analyses.A MICRO-CHECKER analysis revealed that 13 loci  could have been influenced by one or more null alleles in the wild and released samples; our data demonstrated that seven loci  were affected by null alleles in both samples, an effect that could be problematic for population genetic analyses that assume Hardy-Weinberg equilibrium (HWE). The global outlier test using both populations with LOSITAN under both infinite allele and stepwise mutation models detected two loci (KHds8 and KHds9) that had an excessively high or low p > 0.05). The mean observed and expected heterozygosities in the wild samples were 0.809 and 0.814, respectively, and the corresponding parameters in the hatchery-bred released samples were 0.697 and 0.801, respectively. There were no significant differences in expected and observed heterozygosities between the wild and released populations .All 20 MS loci were highly polymorphic in both populations. A total of 243 different alleles were observed and the average number of alleles per locus was18.7. The number of alleles varied from eight at the KHds6 locus to 39 at KHds14 . Not allIn this study, a high level of genetic diversity  in the wild population was detected. A similarly high genetic diversity has been reported in many other marine bivalves ,32 incluFIS) varied among the markers from −0.040 (KHds16) to 0.515 (KHds31) in the released samples and from −0.131 (KHds7) to 0.108 (KHds19) in the wild samples. The average FIS, including all markers, was 0.123 in the released samples and 0.013 in the wild samples (Inbreeding coefficients ( samples .p < 0.004), indicating no distinct differences. Similar unique alleles were observed in the wild and released populations (45 vs. 44). Allele frequency distributions indicated that 48.6% (118/243) of alleles in the released sample were rare alleles (frequency <5%), whereas 50.6% (123/243) of alleles in the wild sample were rare alleles. Rare alleles were detected at most loci in both populations. An examination of pair-wise linkage disequilibrium revealed that all 20 MS loci were in linkage equilibrium (p > 0.004).The allele frequency distributions were varied between the wild and released populations. However, the results of a heterogeneity test of allele frequency distributions in wild and released samples showed no significant heterogeneity across all loci after sequential Bonferroni correction for multiple tests (p > 0.05) using either the sign test, standardized differences test or the Wilcoxon signed-rank test, suggesting that neither the wild nor released populations have experienced a recent bottleneck.To examine the departure from mutation-drift equilibrium based on heterozygosity excess (or deficiency) in both populations, a bottleneck analysis was conducted using Bottleneck software under a two-phase model of MS mutation. None of the populations displayed a significant heterozygosity excess (p < 0.004) were found at six loci  in the released samples, and all were deviations from HWE due to heterozygote deficiency.Significant deviations from HWE after Bonferroni correction .Single-locus p = 0.000), 6.74% (p = 0.000), and 0.2% (p = 0.960), respectively  of the 13 MS loci yielded results similar to those from the FSTAT analysis; variation within individuals, among individuals within populations, and among populations was 93.1% (ectively . The AMOFST) and analysis of molecular variance (AMOVA) indicated no significant genetic differentiation between the wild and released populations. However, the genetic difference was confirmed by the genetic diversity parameters for each population mentioned above. In the H. diversicolor supertexta populations of Donggwi , the seeds released for stock abundance had a different genetic composition, although no significant reductions in diversity were observed compared with the wild population  were sampled from the coastal waters of Donggwi  in May and June of 2010. Many hatchery-raised seeds  have been released in this water since 2007 as part of a stock enhancement program begun by the JPFI. Hatchery-raised abalone seeds were produced in part from the brood stock captured and reared for reproduction at the JPFI and purchased by the Jeju Special Self-Governing Province. No details regarding the founding and maintenance of the purchased hatchery-raised seeds are available; however, their original parents were maintained at a farm on the coast of Jeju, Korea. Samples were selected by experienced researchers based on the shell characteristics of wild animals; for released specimens, a clear green shell coloration 2 cm in length on the top of the shell  and/or metal tags on the shells (which were applied as part of the stocking practice) were also used for identification. We considered the impact of stocking on the wild population to be negligible in the case of abalone with shell lengths over 5 cm (4–5 years of age). For cross-amplification, eight individuals of H. diversicolor diversicolor were collected from the coast near Donggwi on Jeju Island, Korea, in March 2011. Species identification was verified by morphological distinctions [3) were placed in absolute ethanol and kept frozen at −20 °C until DNA extraction.One wild and one released population of inctions . Tissue H. diversicolor supertexta. A whole-genome shotgun library was generated from 2 μg of genomic DNA with the GS DNA Library Preparation Kit  according to the manufacturer’s protocol. The DNA library was titrated by sequencing using the Genome Sequencer FLX system . Based on the results of the titration sequencing run, an appropriate amount of the DNA library was used to set up the emulsion PCR. Subsequently, the clonally amplified DNA fragments bound to the capture beads were enriched and sequenced on 1/8 of a plate in a Genome Sequencer FLX Titanium instrument .For MS isolation, the TNES-urea buffer method  was usedTo characterize the MS loci via genotyping, total DNA from sections of the mantle musculature of each abalone sample was extracted using a MagExtractor-Genomic DNA Purification Kit  for an automated DNA extraction system, the MagExtractor MFX–2100 . The extracted genomic DNA was stored at −20 °C until further use.H. diversicolor supertexta were assembled into contigs using Newbler software, verion 2.3 . A PERL script was run to select sequences longer than 300 bp with a minimum of five repeats of di-, tri- or tetra-nucleotide repeat motifs. Of the reads that were identified, a subset that had a minimum of seven repeats was selected. For these reads, a PERL script was also used to design the primers, and the following criteria were used to identify loci that could be reliably amplified: (i) a GC content of 30%–90%; (ii) a product size of 90–250 bp; (iii) a primer length of 18–20 bp; and (iv) a melting temperature of 58–68 °C. Primer redundancy was tested using NCBI BLAST [The resulting raw sequences from BI BLAST .The newly designed PCR primer pairs were tested for consistency in PCR amplification and polymorphisms; these tests were performed on a sample set of eight abalones collected from Donggwi on Jeju Island, Korea. PCR amplifications were performed using an ABI 9700 Thermal Cycler System (Applied Biosystems) in 25 μL solution containing 12.5 μL of 2× Multiplex PCR Pre-Mix , 100 ng of template DNA and 10 pmol of each primer. The forward primer from each pair was 5′-end-labeled with 6-FAM and HEX dyes (Applied Biosystems). PCR reactions were run for 15 min at 95 °C followed by 30 cycles of 20 s at 95 °C, 40 s at 54 °C, and 2 min at 72 °C with a 3-min final extension at 72 °C. The annealing temperature (54 °C) was 4 °C–5 °C below the Tm estimated from the nucleotide compositions of the primer pairs served as the annealing temperature. The PCR amplification was considered to be successful based on the presence of a visible band after running 5 μL of the PCR product on a 5% denaturing agarose gel. The 1 kb Plus DNA Ladder molecular weight marker  was used as a standard to assess the product size. If no amplification was detected, that primer set was excluded from further analysis by multiplex PCR. Some loci could not be amplified from any of the samples, and some yielded only faint bands even after adjustment of the PCR conditions. We excluded these loci from further testing. For the remaining loci, genetic variation was examined in all samples collected. MS polymorphisms were identified using an ABI PRISM 3100 Automated DNA Sequencer (Applied Biosystems), and alleles were designated by PCR product size relative to a molecular size marker . Fluorescent DNA fragments were analyzed using GENESCAN, version 3.7  and GENOTYPER, version 3.7  software packages (PE Applied Biosystems). The populations were multiplexed for genotyping by pooling samples tagged with different dyes within a well. We assessed the reliability of the primers by repeating the amplification and genotyping 16 samples from each population (26.7%).H. diversicolor supertexta were assessed for cross-amplification in another congener species, H. diversicolor diversicolor, using eight individuals.Finally, all of the newly developed polymorphic MS loci in FST-outlier detection method. To access genetic diversity, the number of alleles per locus (NA), size of alleles in base pairs (S), frequency of the most common allele (F), and number of unique alleles (U) were determined for each local population at each locus using the GENEPOP program, version 4.0 [FIS) [FST; 1000 permutations) [Populations were screened for variation at the newly developed MS loci. MICRO-CHECKER 2.2.3  was usedsion 4.0 . GENEPOPsion 4.0  was used.0 [FIS)  per locu.0 [FIS) , compone.0 [FIS) . The sigtations)  and to atations) . Signifitations) . Becausetations)  was emplH. diversicolor supertexta. In this study, we report the development of 20 polymorphic MS markers for H. diversicolor supertexta using 454 pyrosequencing. This study is among the first few studies to validate the next-generation sequencing method of MS acquisition for a commercially important fishery species. No significant genetic difference was detected between the wild and released populations based on an analysis of 13 MS loci, with none of null alleles being detected in either population. Continued monitoring of the genetic variance between wild and released abalone stocks using DNA markers is essential for the successful implementation of an artificial enhancement project and the conservation of natural Korean H. diversicolor supertexta resources. Moreover, primers for all polymorphic loci can be used in the related species H. diversicolor diversicolor.In conclusion, despite the release of hatchery-bred seeds into natural coastal areas to compensate for the reduced fishery resources in Korea, to date, no detailed genetic information is available on the genetic diversity of wild and released stocks of