Petroica australis rakiura), for which reduced microsatellite diversity was previously observed. We genotyped 17–24 birds from a reintroduced island population (including the 12 founders) for nine genes, seven of which were polymorphic. We observed a total of 24 single-nucleotide polymorphisms overall, 15 of which were non-synonymous, representing up to five amino-acid variants at a locus. One locus (TLR1LB) showed evidence of past directional selection. TLR7. The levels of TLR diversity that we observe are sufficient to justify their further use in addressing conservation genetic questions, even in bottlenecked populations.Toll-like receptors (TLRs) are an ancient family of genes encoding transmembrane proteins that bind pathogen-specific molecules and initiate both innate and adaptive aspects of the immune response. Our goal was to determine whether these genes show sufficient genetic diversity in a bottlenecked population to be a useful addition or alternative to the more commonly employed major histocompatibility complex (MHC) genotyping in a conservation genetics context. We amplified all known avian TLR genes in a severely bottlenecked population of New Zealand's Stewart Island robin ( The evolutionary consequences of the amount and distribution of genetic diversity among individuals are a major focus of population and conservation genetics. Widely-used markers such as microsatellites are useful for estimating migration and delineating management units, but are not necessarily relevant to processes affecting evolutionarily significant functional diversity Escherichia coli) involves TLR4Staphylococcus aureus) has been shown in mammals to be linked to both TLR1TLR2TLR1LA and TLR1LB, and TLR2A and TLR2BTLR2 in mammals TLR2 and TLR1 or TLR6) have both been reported TLR15) specific to birds and thought to be involved in response to other Gram-negative bacteria including Salmonella entericaToll-like receptor (TLR) genes are an ancient part of the genome of many animals. They mediate innate immune responses via recognition of pathogen-associated molecular patterns (PAMPs) such as dsRNA of some viruses, or lipopolysaccharide of Gram-negative bacteria Growing evidence from studies of model organisms has indicated that variation in binding regions of TLR loci can translate to functional variation in pathogen recognition Due to their key role in host defence, observed variation in TLR genes likely represents an important component of functional genetic diversity of wild populations Petroica australis rakiuraP. a. rakiura declined to <300 individuals in approximately 1% of its former habitat since the introduction of mammalian predators to Stewart Island over the last four centuries P. a. rakiura by specifically examining diversity in the 12 Ulva Island founders Recent investigations of TLR diversity have focussed on model and abundant species p = 1/2N.offspring, following DNA samples had previously been collected from ten of the Ulva Island genetic founders . To deduce the missing founders' genotypes, we included up to seven offspring from each, as well as the other parents of those offspring. For loci where the sampled parent or offspring were homozygous, we could infer the genotypes of the unsampled parents based on a smaller number of offspring genotypes see . For othP. a. rakiura, we were able to use primers from their main set  – finchTLR21F5′ TTGACAACAACCTGCTCACTG 3′ and finchTLR21R5′ TACGCAGCTCGTTCTTGG 3′ (831-bp product). Our TLR21 primers improve identity with passerines, as ambiguities in the multi-species avian alignment Alcaide and Edwards main set  to amplitive set . TLR4 diTLR2 using previously published primers TLR2A and TLR2B resulting in numerous polymorphic sites. In order to design locus-specific primers to allow us to amplify the two genes separately, a coamplified TLR2A and TLR2B PCR product obtained using primers avTLR2F & RTLR2A and TLR2B) by sequencing seven clones from one individual and comparing the results to published sequences on Genbank using a BLAST search PauTLR2AF5′ CCAAAGTATTACCCGAGCAATAGTG 3′, and PauTLR2BF5′ CCTAAGATCGCCAGCATTTTAG 3′) were designed in a region of high dissimilarity between the two genes (16% and 18% respective similarity to the other gene). These forward primers were each paired with avTLR2RSequence data for TLR7 also suggested coamplification of duplicate loci by the presence of eight nucleotide sites (0.8% of 947 bp) that appeared heterozygous for all genotyped individuals (with uniform chromatogram peak heights). These SNPs also did not appear to follow Mendelian inheritance, as this dataset included 15 parent/offspring trios in which all individuals were heterozygous , further indicating coamplification of duplicate loci. Three additional sites at TLR7 showed variable heterozygosity  among sampled birds suggesting that one or both genomic copies of this gene may be variable. Due to the close similarity between duplicate copies of TLR7 in P. a. rakiura, locus-specific primers could not be designed. However, as a preliminary investigation into the structure and diversity of this gene we cloned PCR products from two unrelated individuals amplified using avTLR7-2 F & R2 concentration of 2.0 mM (included in the enzyme buffer), initial hot-start at 95°C for 10 min and extension time of 2 min at 68°C. The Velocity and Phusion reagent setups followed the manufacturer's protocols, and thermocycling conditions did not vary from our standard protocol below, with the exception of a 98°C denaturation temperature. A total of 24–33 clones was sequenced for each individual. Sequencing 24 clones for an individual gives a 99.6% probability of observing all haplotypes at least once, if there are two heterozygous gene-copies (i.e. maximum of four unique haplotypes), based on multinomial probability Sequence data for 2, 1× Taq buffer and 500 nM each primer (Sigma-Aldrich). Thermocyling conditions on an Eppendorf Mastercycler pro S consisted of 94°C for 3 min, followed by 35 cycles of 94°C for 40 s, locus-specific annealing temperature  for 40 s, and extension at 72°C for 80 s, with a final extension step of 72°C for 10 min. Amplified products were purified by excision of the target band from a 1% agarose 1× TAE gel stained with SYBR Safe DNA gel stain (Invitrogen) and cleaned up using the DNeasy Ultra-Sep gel purification kit (Omega Bio-Tek). Purified PCR products were sequenced in both directions on an ABI 3730xl Genetic Analyser . Correct amplification of the target genes was confirmed by BLAST search.Unless described otherwise, all amplifications were carried out in a total volume of 15 µl and contained 0.6 U BioTaq DNA polymerase (Bioline Ltd), 200 µM each dNTP, 1.5 mM MgClSequences were edited and aligned using Sequencher v5.0 (Gene Codes Corporation), using IUPAC ambiguity codes where double-peaks were observed. All SNPs for all individuals were confirmed by careful examination of chromatograms; any putative SNPs or haplotypes observed in only one individual were re-amplified and sequenced. None of the individuals included in the analyses (and reported in the results) had any missing data for any SNPs. Sequence data from this manuscript is available on Genbank (accession numbers provided in N = 12 birds). Observed heterozygosity (HO) and expected heterozygosity (HE) as well as nucleotide diversity (π), were calculated using Arlequin 3.5 Allelic phasing of SNPs was undertaken using a combination of PHASE v2.1.1 C. mexicanus published by Alcaide and Edwards N = 51 for TLR1LA, 13 for TLR1LB, 4 for TLR21, and 8 for TLR2A, TLR2B, TLR4 and TLR15TLR5, as Alcaide and Edwards C. mexicanus for this locus. Sequences were aligned using ClustalW codon alignment The McDonald-Kreitman statistic P. a. rakiura (TLR7 could not be genotyped due to high similarity between duplicate copies (see below). We observed 24 SNPs across all loci, of which 15 were non-synonymous . A non-conservative TLR4, TLR7, TLR15 and TLR21, The majority of SNPs we observed were diallelic, the exception being one triallelic site in TLR2A contained at least one non-synonymous base substitution. For five of the genotyped loci , all observed SNP haplotypes translated as different amino acid variants. For TLR2B and TLR21, multiple observed SNP haplotypes resulted in two and three amino-acid variants respectively. Although it is possible that synonymous substitutions have functional consequences All polymorphic TLRs except HO = 0.417; HE = 0.477, p-values for all comparisons >0.05, N = 12, After deducing the genotypes of unsampled founders from those of their offspring, we observed between two and five amino-acid variants per polymorphic locus among the Ulva Island founders , we observed that the proportion of non-synonymous fixed differences between species for TLR1LB (51%) was approximately double the proportion of non-synonymous polymorphism within species (24%), a statistically significant result . The direction of the relationship at this locus indicates possible evidence of divergent positive selection at some time during the evolutionary history between the two species. Although there was no evidence of positive selection at the other loci we tested , all loci except TLR3 and TLR21 showed the same pattern of excess fixed substitutions . Therefore, the error rate resulting from PCR, cloning and sequencing steps is low, as was expected given our use of high-fidelity polymerases ; from individual “RU152” we observed two sequences of type I (N = 15 and 2 clones) and four sequences of type II  . None of the observed TLR7 clone haplotypes contained stop codons.Cloning results suggested a genomic duplication of ases see . Overall clones) . This la species . Althougications , which iVariation in genes responsible for front-line immunity is likely an important target of natural selection, and can be used to obtain crucial insight into maintenance or loss of functional genetic diversity, and therefore adaptive potential, of threatened populations. Our study is the first to examine genetic diversity of Toll-like receptor genes in a bottlenecked population. Despite previous microsatellite evidence indicating reduced genetic variation in this population, we observed putatively functional variation in seven out of nine genotyped loci in this native New Zealand passerine. This variation in TLR loci highlights their promise as additional potential indicators of functional genetic diversity for the population and conservation genetics of bottlenecked species.C. mexicanus and lesser kestrel Falco naumanni  compared to levels reported for similarly-sized samples of ion e.g. . It is iTLR4, TLR5, TLR7 and TLR15, P. a. rakiura. While neutral diversity is more easily lost during the population bottleneck, certain amino-acid variants that may have functional consequences, either at present or in the past, may have been retained.Despite observing a number of functional variants for most TLRs, the overall number of SNPs detected in this population was low . TLR locP. a. rakiura sequence diversity to that of C. mexicanus indicated clear evidence of directional selection for TLR1LB since these two species diverged . In threatened populations, genetic diversity predictions for immune-related genes can be unclear, as an increase in the frequency of alleles with non-synonymous substitutions is predicted under heterozygote advantage, frequency dependent selection, or repeated rounds of directional selection (as would occur in a Red Queen-type scenario). Depending on the context, these processes may result in reduced genetic diversity after population bottlenecking The overall pattern and context of our data suggests that variation at these loci has functional significance and, despite the limited sample size here, our analysis suggested that selection has been important in the evolution of at least one gene. For example, comparing per se will facilitate future studies into the effects of genetic drift and selection on these alleles. Innate immune responses are expected to play an important role in the evolution of resistance to novel diseases in wild populations, an expectation that was upheld in a recent study of the consequences of a Mycoplasma outbreak in wild house finches Although our study does not allow us to predict specifically how the observed levels of diversity will relate to future evolutionary potential of the population, the presence of diversity TLR1 and TLR2 are known to be duplicated in birds TLR7 is probably duplicated in passerines TLR2A and TLR2B have conserved and divergent regions TLR7 duplicates were too similar in P. a. rakiura to design locus-specific primers, so unfortunately this gene could not be easily genotyped. Overall, while uncertainty in the number of co-amplifying genes and pseudogenes can be a major challenge of working with MHC in some taxa Most TLR loci were present in only single genomic copies, in contrast to avian studies of MHC which can find coamplification of many loci as well as genomic copy-number variation among individuals within a population  TLR7 duplication has not been observed in any other vertebrate taxon to date TLR7 in other passerines and closely-related avian orders to determine when duplication occurred relative to the passerine radiation.Our results were consistent with at least two genomic copies of P. a. rakiura, and that individual genotypes could be easily assigned for nine genes. Non-synonymous variation was present within the relatively small population of founder birds. The high proportion of non-synonymous variation for some genes suggests that these loci have been, and may still be, subject to natural selection, and therefore provide additional loci for addressing questions of functional genetic diversity in this and other threatened species. By presenting gene sequences, primers and diversity estimates for a bottlenecked population, we further enable study of TLRs in other conservation contexts, especially where the complexity of MHC may be prohibitive for economic and/or technical reasons.In summary, we found that the full family of TLRs could be successfully amplified in our bottlenecked population of Figure S1Neighbour-joining tree of TLR7 sequences available for nine avian genera.(DOCX)Click here for additional data file.Table S1McDonald-Kreitman tests contrasting substitution and mutation rates for a comparison of P. a. rakiura TLR sequences with those of C. mexicanus.(DOCX)Click here for additional data file.Table S2Alignment of clone haplotypes obtained for two individuals amplified at TLR7 showing character states at variable sites; all other base positions were monomorphic.(DOCX)Click here for additional data file.


Siganus spinus in the western Pacific. This species is a culturally important food fish in the Mariana Archipelago and subject to high fishing pressure. Our primary hypothesis was to test whether the individuals resident in the southern Mariana Island chain were genetically distinct and hence should be managed as discrete stocks. In addition to spatial sampling of adults, newly-settled individuals were sampled on Guam over four recruitment events to assess the temporal stability of the observed spatial patterns, and evidence of self-recruitment. We found significant genetic structure in S. spinus across the western Pacific, with Bayesian analyses revealing three genetically distinct clusters: the southern Mariana Islands, east Micronesia, and the west Pacific; with the southern Mariana Islands being more strongly differentiated from the rest of the region. Analyses of temporal samples from Guam indicated the southern Mariana cluster was stable over time, with no genetic differentiation between adults versus recruits, or between samples collected across four separate recruitment events spanning 11 months. Subsequent assignment tests indicated seven recruits had self-recruited from within the Southern Mariana Islands population. Our results confirm the relative isolation of the southern Mariana Islands population and highlight how local processes can act to isolate populations that, by virtue of their broad-scale distribution, have been subject to traditionally high gene flows. Our results add to a growing consensus that self-recruitment is a highly significant influence on the population dynamics of tropical reef fish.We used microsatellite markers to assess the population genetic structure of the scribbled rabbitfish  For most marine organisms, a pelagic larval stage provides the primary mechanism for dispersal among spatially fragmented habitat patches . The degBecause the pelagic larval duration of many fish larvae can be more than one month , coral rTraditionally, the most widely used approach for estimating connectivity has been population genetics  in the western Pacific, with a particular focus on the islands of the southern Mariana archipelago  to local (southern Mariana islands) scales. Prevailing large-scale current patterns indicate an east–west flow between islands in this region and analysis of gene flow pathways will identify any genetic discontinuities and possible barriers to dispersal. The lack of any obvious physical barriers would implicate oceanographic conditions in such a case. Sampling from multiple locations around Guam provides insights into whether significant structure is evident at the island scale. Such information is important in the context of formulating effective conservation management plans for this species; (2) in conjunction with the local-scale spatial sampling we also sampled genetic variability within and between multiple cohorts at several sites on Guam, to assess the temporal stability of gene flow, degree of self-recruitment, and evidence of sweepstakes reproductive success. Many connectivity studies on coral reef fish that have included temporal sampling have focused on small, long-lived site-attached fish  the spatial genetic population structure of see e.g.,, whereasS. spinus individuals were collected from nine islands across the western Pacific (S. spinus recruits (<50 mm FL) were collected from four discrete recruitment events: November 2007, July 2008, September 2008, and October 2008 from nine locations , tissue samples from 971  Pacific ; Table 1ocations ; Table 2ocations  allowed All fish were collected from shallow reef-flats using hand spears, with the exception of the Philippine samples, which were obtained from the Dumaguete fish market. Individuals were kept in ice slurry before being processed. Tissue samples, taken as fin clips, were preserved in 95% ethanol (EtOH) and stored at room temperature while awaiting further analysis.S. spinus congener S. fuscescens  were carried out individually in a 10 µl reaction volume containing: 1 µl 10× Taq polymerase buffer, 0.2 µl Taq DNA polymerase, 0.2 µl 10mM dNTPs, 0.5 µl 25mM MgCl2, 5.1 µl ddH2O, 1 µl of genomic DNA (∼10–40 ng µl–1), and 1 µl each of forward and reverse primers. For loci Sfus-8, Sfus-21, Sfus-56, and Sfus-113, primer concentrations were 5 µM; for locus Sfus-98, primer concentration was 2.5 µM. Thermocycling conditions for the PCRs consisted of an initial step of 80°C for 1 min and a denaturing step of 94°C for 2 min. Ten cycles of 30 sec at 94°C, 30 sec at 56°C, and 30 sec at 72°C were followed by 20 cycles of 30 sec at 94°C, 30 sec at 54°C, and 30 sec at 72°C, with a final extension step of 4 min at 72°C. Capillary electrophoresis was performed on pooled PCR products using an AB 3730 DNA Analyzer , and scored with GeneMapper v. 3.7 . All PCR reactions and genotyping were performed by the Australian Genome Research Facility, Brisbane, Australia.Genomic DNA was extracted using DNeasy Blood and Tissue kits . Samples were genotyped at five microsatellite loci originally designed for the scescens . The locfstat v. 2.9.3  for each locus pair across all populations. One hundred and twenty-eight samples were regenotyped to determine the study-specific error rate and resolve suspected null homozygotes.The mean number of alleles per locus, allelic richness, and number of observed alleles were calculated using v. 2.9.3 . Expectev. 2.9.3 . Departup v. 3.4 ; signifi-checker  was usedFST genetic differentiation measures and Bayesian assignment methods. Global and population pairwise FST values between all sampled sites were calculated using FreeNA   permutations of alleles across clusters, also using arlequin. Results were visualized by performing a principle coordinate analysis (PCoA) on a genetic distance matrix constructed using pairwise FST values. Patterns of isolation-by-distance were investigated by plotting genetic distance against geographic distance and tested with a mantel test using 999 permutations implemented in GenAlEx. Cavalli-Sforza and Edwards’ (1967) chord distance (DC) was used as it is insensitive to departures from HWE. Geographic distances were calculated as the shortest distance by sea between the midpoints of sampling locations.Population structure was assessed using standard g FreeNA , with an (AMOVA)  implemen v. 3.11 . Signifistructure v. 2.2.3 (K) based on the likelihood of the individual belonging to each subgroup, as determined by the individual's allele frequencies at each locus. K was set from 1 to 18 and each model run was repeated independently 10 times using a run length of 100,000 MCMC repetitions and a burn-in period of 100,000. The model was run with admixture, correlated allele frequencies, and with prior location options  was employed to assess spatial genetic discontinuities between sampling locations. geneland uses a Bayesian clustering model similar to structure, but also includes geographic sampling location data as a weak prior to produce spatially explicit maps of genetically homogenous clusters. Again, 10 independent runs were used for each K, with K ranging from 1 to 18, using 1,000,000 MCMC repetitions with a burn-in period of 200,000 and a thinning value of 1000.To investigate the presence of subtle genetic structure and spatial genetic discontinuities, we used Bayesian assignment methods to determine the most likely number of homogenous genetic clusters of all sampled individuals. First, v. 2.2.3  was used options . The mos. 0.56.4 . Second,v. 3.2.1 , 2008 pageneclass2 . PCoA was used to visualize the results and nonparametric multivariate analysis of variance (MANOVA)  within cohorts and decreased relatedness between cohorts if sweepstakes reproduction is evident, as a result of the high variance of an individuals’ chance of successfully reproducing. Similarly, one would also expect to see decreased heterozygosity in recruit populations compared to adult populations   was usedmer v. 6 . Tests wulations .STF values. Samples were grouped into the three clusters determined by the region-wide analyses, including Guam recruits as a fourth cluster, and pairwise comparisons were made between all clusters using nonparametric MANOVA in primer.Finally, all recruit samples were added to the region-wide dataset and PCoA was performed on pairwise Sfus-8) to 2 (Sfus-56 and Sfus-9). Both Sfus-56 and Sfus-9 were monomorphic within some sample groups. Allelic richness ranged from 2.61 to 4.56 and was generally lower in the Guam recruit samples than the region-wide samples. A total of three private alleles were present in the dataset  and analysis of pairwise FST estimates showed no differences between either dataset. Bayesian analyses using datasets including and omitting Sfus-8 also resulted in congruent outcomes, indicating the presence of null alleles had no tangible effect on the results.Significant departures from HWE were detected at locus e groups  and A2. h FreeNA , with anFST= 0.023, P < 0.001) with 41% of pairwise FST comparisons significant , with genetic distance increasing with greater geographical separation. Moreover, at the within-island scale none of the 16 pairwise FST estimates was significantly different from zero, indicating little genetic differentiation within islands. When samples were grouped at the island level, AMOVA showed a significant proportion of genetic variance  was partitioned among islands , with the southern Mariana samples grouping out along PCoA Axis 1.There was considerable genetic differentiation across the study region  were also excluded from both the East Micronesia and West Pacific populations, and thus, could be assigned as originating from the southern Mariana Islands population . The EasRXY values for adults and recruits also showed no significant differences between life-history stages , or sample sites . There was also no evidence of sweepstakes reproduction. Permutation tests in fstat comparing mean heterozygosity and FST between adult and recruit samples revealed no differences between adult and recruit groups . When considering only the recruit samples, there was no difference between the eight sampling sites . There was also no evidence of differentiation between cohorts that settled at different recruitment events using either AMOVA , indicating temporal stability of gene flow over the 11-month sampling period.Results indicate genetic homogeny across Guam and Saipan both spatially and temporally. Comparisons using AMOVA did not reveal any genetic differentiation between adults and recruits . PCoA aner AMOVA  or pairwt= 1.84, P= 0.133), but were significantly differentiated from the two other region-wide clusters . The similarity between Guam recruit samples and the Southern Marianas Island cluster in the region-wide analysis is suggestive of limited gene flow between the Southern Marianas Islands and the rest of the region.Consistent with no evidence of spatial or temporal genetic structure within Guam, when the recruit samples from Guam were added to the region-wide dataset, the PCoA ordination indicated clearly that the Guam recruit samples were most closely related to the Southern Marianas Island cluster . MoreoveS. spinus populations across the West Pacific, with the Southern Mariana Islands strongly separated from the rest of the region. The stability of the Southern Mariana Islands population was confirmed by temporal sampling of newly settled fish from four separate recruitment events over a period of 11 months. Recruitment cohorts were genetically indistinguishable from each other and from the resident adult population on Guam and Saipan. These results indicate limited connectivity with the rest of the West Pacific and highlight the likely significance of self-recruitment in maintaining the unique genetic signature of reef fish from the Southern Mariana Islands.We found significant large-scale spatial genetic structure in FST and Bayesian analyses, indicating significant isolation from the rest of the region. The East Micronesian and West Pacific populations exhibited less, but nevertheless still significant, differentiation from each other. Genetic structure between populations was detected when using Bayesian analyses with either prior sampling group information (structure) or spatially explicit geographical coordinates (geneland). These models have been criticized for coaxing data into forming cluster groups by weighting results according to sample location. However, in the case of structure, simulation and empirical evidence has shown that the inclusion of sampling information does not lead to detection of genetic structure when genetic structure is not present , East Micronesia , and the West Pacific . The Southern Mariana Islands were strongly separated from the rest of the region in all  present . AdditioAcanthurus triostegus , which could indicate a consistent barrier to dispersal between East and West Micronesia. However, interannual variation was observed within Pohnpei, which may suggest populations of this species are not temporally stable.Our study is the first to document significant genetic structure between the southern Mariana Islands and its nearest neighbors in Micronesia, and this has potentially profound considerations for future management of reef fish populations in the region. Previous genetics-based studies within the Pacific Ocean basin have generally focused on connectivity from a phylogeographic perspective. These studies used more highly conserved allozyme and mtDNA markers, which reflect historical patterns of connectivity that may not be representative of contemporary patterns of gene flow. Contrasting patterns of genetic structure were nevertheless observed across a range of fish species, although most of the cited studies reported an absence of genetic structure . One stuiostegus . PopulatS. spinus among islands in Okinawan waters separated by 430 km. In contrast, S. guttatus sampled concurrently did exhibit genetic structuring. The authors attribute this disparity to the early life-history characteristics of S. spinus with their larvae having greater dispersive potential than S. guttatus. A similar result was obtained when S. argenteus, a species with early life-history characteristics similar to S. spinus  share more alleles with other recruits or with adults from the same site. This suggests the southern Mariana Islands should be managed as a single demographically connected population. Temporal stability amid low levels of gene flow suggests persistent self-recruitment may be responsible for maintaining the distinct genetic structure found across the southern Mariana Islands . Such a dynamic demographic structure may dramatically reduce the chance of finding parent–offspring pairs. Nonetheless, given that, under certain conditions, very few successful migrants per generation can lead to genetic homogeny among populations, our results provide strong evidence for treating the southern Mariana Islands as an isolated, predominately self-recruiting population . However, the preserves are opened seasonally to allow fishing for newly settled S. spinus recruits. The effects of directly fishing recruitment pulses . While clearly enough to maintain genetic homogeneity, the actual extent of larval exchange between islands remains unknown. Such information is critical for providing effective conservation of exploited marine resources.


Here we characterise a panel of new microsatellite markers for the common marine flagellate Oxyrrhis marina. Nine microsatellite loci were used to assess genotypic diversity at two spatial scales by genotyping 200 isolates of O. marina from 6 broad geographic regions around Great Britain and Ireland; in one region, a single 2 km shore line was sampled intensively to assess fine-scale genetic diversity. Microsatellite loci resolved between 1–6 and 7–23 distinct alleles per region in the least and most variable loci respectively, with corresponding variation in expected heterozygosities (eH) of 0.00–0.30 and 0.81–0.93. Across the dataset, genotypic diversity was high with 183 genotypes detected from 200 isolates. Bayesian analysis of population structure supported two model populations. One population was distributed across all sampled regions; the other was confined to the intensively sampled shore, and thus two distinct populations co-occurred at this site. Whilst model-based analysis inferred a single UK-wide population, pairwise regional STF values indicated weak to moderate population sub-division (0.01–0.12), but no clear correlation between spatial and genetic distance was evident. Data presented in this study highlight extensive genetic diversity for O. marina; however, it remains a substantial challenge to uncover the mechanisms that drive genetic diversity in free-living microorganisms.Free-living marine protists are often assumed to be broadly distributed and genetically homogeneous on large spatial scales. However, an increasing application of highly polymorphic genetic markers ( Free-living marine protists commonly have broad distributions A further reason to limit generalisations about the distributions and genetic diversity of free-living protists is the diverse array of life history strategies displayed by such organisms Oxyrrhis marina (Alveolata: Dinophyceae) is an ideal model to study population structure/dynamics and evolutionary processes in free-living marine protists as it is common in coastal habitats O. marina is genetically differentiated on broad spatial scales O. marina as a useful model for the study of biogeographic processes within free-living marine protists c oxidase I, COX1, and 5.8S internal transcribed spacer rDNA, 5.8S-ITS) indicated contrasting patterns of genetic diversity among broad geographic regions; in particular most samples isolated from coastal waters of the Northwest Atlantic were identical at these loci Oxyrrhis are poorly documented To date, most population-genetic studies of marine protists have focussed on a relatively small number of ecologically or economically important species such as harmful algae, coccolithophores and diatoms e.g. . HoweverO. marina and use these to (1) compare the level of genetic diversity at microsatellite loci with previous studies based on sequence variation of common phylogenetic markers and (2) assess the extent of genetic structure at two spatial scales, first on a fine-scale (∼1–2 km), within a single shore and second at a regional scale , among a range of coastal areas of Great Britain and Ireland. We uncover high levels of genetic diversity and, for the first time in this taxon, detect fine-scale spatial genetic structure. These results have clear implications for researchers using O. marina as an experimental model Here, we develop the first panel of polymorphic microsatellite markers for Oxyrrhis marina samples were isolated from 15–100 ml of seawater obtained from coastal, predominately intertidal, habitats by the authors or collaborators (see Acknowledgements). Samples were located at six general regions around Great Britain and Ireland: (1) southern England (n = 21), (2) southern Wales (n = 20), (3) northern Wales (n = 81), (4) western Ireland (n = 23), (5) western Scotland (n = 41), and (6) eastern Scotland (n = 14), with the emphasis at the site in northern Wales  were established. Isolates were on-grown for 1–2 weeks in 50 ml volumes (with low concentrations of D. primolecta); subsequently samples were pelleted by centrifugation and then stored at −20°C. DNA for genotyping was extracted using a high-salt method D. primolecta) and treated with penicillin/streptomycin (100 µgml−1) and gentamycin (50 µgml−1) to minimise bacterial growth. Cultures were maintained in the dark for ∼2 weeks (to prevent growth of D. primolecta) or until O. marina grazed the prey to extinction. Absence of D. primolecta was confirmed by PCR using universal eukaryote 18/28S rDNA primers O. marina cell concentrations were ∼5×104 ml−1; cultures were then harvested by centrifugation, and DNA was extracted using a phenol:chloroform method Clonal isolates were obtained following methods outlined in Sau3AI (Boehringer-Mannheim) were used to digest 8–10 ìg of genomic DNA (pooled from the two isolates detailed above). Digested DNA was subsequently ligated to 50 pmol of phosphorylated linkers 6 oligonucleotides (MWG Biotech). After a series of differential stringency washes in 2× SSC and 1× SSC, the enriched DNA was made double stranded and amplified in a 25 ìl PCR 4SO4, 0.01% (v/v) Tween 20, 0.2 mm each dNTP, 1.5 mm MgCl2, 25 pmol primer S61 and 1.25 units of Taq polymerase (ABgene). The thermal profile of the PCR was: 95°C 5 min, 25–30× , 72°C 10 min. The DNA was purified using a QIAquick PCR purification kit (Qiagen), ligated into pGEM®-T vector (Promega) and transformed into JM109 E. coli competent cells (Promega). Recombinant clones were identified using black/white screening on S-gal (Sigma) agar/ampicillin plates. Plasmids containing an insert with a microsatellite were identified by two or more amplified products after PCR primed with 50 pmol SP1 and 25 pmol of (nonbiotinylated) microsatellite oligonucleotide (either (CA)12 or (TCAC)6 see xl (Applied Biosystems). Primers flanking microsatellite regions were designed using Primer3 software To construct the partial genomic library, 40 units of aT°C 45 s, 72°C 45 s], 25× , 72°C 10 min; aT is the locus-specific annealing temperature (4)2SO4, 0.01% (v/v) Tween 20, 0.2 mM each dNTP, 2.0 mM MgCl2, 5–50 ng template DNA, 3 pmol each primer, and 0.25 uTaq polymerase (ABgene). PCR fragments were fluorescently labelled with either 6-FAM, NED, PET or VIC fluorescent dyes (Applied Biosystems). PCR products were pooled with a 500 bp (LIZ) size standard (Applied Biosystems), separated by capillary electrophoresis on an ABI3130xl and sized using genemapper software (Applied Biosystems). Error rates were assessed by genotyping  a subsample of 48 individuals twice. The occurrence of null alleles and systematic scoring errors was assessed using Microchecker Microsatellite alleles were amplified by PCR in a 10 ìl reaction volume on a Dyad DNA Engine (MJ Research Inc.). PCR conditions were 95°C for 1 min, 5× [95°C 30 s, perature . Each PCaN), expected heterozygosity (eH), Wright's f  assigned to a population (or populations if they are admixed) on the basis of their multilocus genotypes, assuming unlinked loci and HWE conditions within populations. Typically, the number of distinct populations (K) is estimated from the model of K that maximises the probability of the data ), but for complex population structures a modal value of ln P(D) may not be apparent. In such instances the value of K at the beginning of a ‘plateau’ of estimates of ln P(D) may be selected as this is the smallest value of K that captures the major structure of the data setad hoc measure: ΔK (the second order rate of change of ln P(D) with respect to K; ΔK corresponds to the most pronounced partition of the data set. Five independent runs of structure were carried out for the total data set for K = 1 to K = 10 using the admixture model and correlated allele frequencies Spatial genetic structure was assessed using the model-based clustering approach implemented by .3.1 see . Brieflyfstat was used to calculate the level of genetic differentiation - STFSTF estimates from zero assessed through 2,000 permutations of genotypes between populations. Isolation by distance (IBD) genetic structure was investigated by spatial autocorrelation ijF; spagedi version 1.2 structure (defined in the ijF were generated by jackknifing over loci.i.e. JM109 cells with plasmids) bearing putative microsatellite repeats, of which 171 were sequenced. PCR primers could be designed around 42 microsatellite loci; of these 25 suffered from severe stutter banding, 5 were monomorphic, and 12 were polymorphic. Nine polymorphic microsatellite loci  of 0.00–0.30 and 0.81–0.93 , with significant  heterozygote deficits identified in 24 instances (i.e. locus per region), notably in the samples from North Wales and Eastern Scotland and all regional sample groups at the locus Om06  and most  variable loci, respectively, with corresponding variation in expected heterozygosities  linkage disequilibrium: Om02–Om06 (South England); and Om03–Om09, Om02–Om03, and Om02–Om09 (West Scotland). The exception to this was the sample from North Wales, as every locus-pair combination (except for the eight comparisons involving Om08) was in significant linkage disequilibrium . This effect largely reflects the intra-site population subdivision since, when the North Wales sample was partitioned into the two distinct clusters identified by structure analysis  increased, but did not uncover a distinct modal value of ln P(D); in addition, it was difficult to identify an objective “plateau” to the variation in lnP(D) against K and thus no obvious partition of the data was evident using the value of lnP(D) itself. By contrast, ΔK provided clear support for a bipartition of the data set  between the two hypothetical clusters (identified by structure) within the North Wales site. There was little evidence for an IBD structure  throughout the general distribution of our samples, but rather an apparent clustering of similar genotypes within shores. Thus, isolates tended to be genetically similar (average ijF variable but positve) at a small spatial scale (∼1–2 km), but thereafter average pairwise kinship among isolates (ijF) remains close to zero irrespective of the distance class  as high as 0.97 were uncovered in 5 species from the genus AlexandriumeH vary between 0.08–0.82, 0.08–0.88, 0.21–0.68 and 0.55–0.93 in other potentially harmful dinoflagellates  O. marina, locus Om07 displayed a high frequency of non-amplifying samples, contributing to potentially high error rate without repeat genotyping, and another locus (Om06) showed a strong tendency toward heterozygote deficiency. Presumably, high frequencies of null alleles are a result of mutations in flanking sequence, but whether they indicate an elevated rate of mutation within nominal species or reflect the occurrence of cryptic species is unclear. High levels of cryptic genetic diversity certainly occur within O. marina, with present analysis of globally-distributed isolates culminating in two genetically divergent lineages (i and ii) that each consist of two distinct clades O. marina lineage i, which so far has been found to dominate samples from northern European coastal waters ii (data not shown), which has been proposed to be a separate species  and tend to overlook patterns of variation within localities. This sampling strategy probably reflects a commonly, though not universally  markers are used. A comparison of genotype data for O. marina from the study presented here reinforces the above points. That we identified more than 180 unique genotypes within our sample of 200 O. marina isolates from the coastal waters of Great Britain and Ireland is an obvious contrast to the single haplotype indentified across the same region based on sequence variation at COX1 and 5.8S-ITS The recent application of highly polymorphic genetic markers (such as microsatellites), in combination with intensive sampling strategies, have provided greater resolution to determine fine-scale spatial structure in populations of marine protists. In some cases studies have supported a lack of population differentiation; for example, microsatellite-based studies on several diatom species have failed to uncover significant genetic divergence among samples within regional seas Fine-scale divergence, below) individual model-based clustering did not uncover substantial spatial genetic structure across much of the coastal waters of Great Britain and Ireland (cf. STF were greater than 0.05), indicating that O. marina is not panmictic at this scale. Such levels of genetic differentiation are relatively low compared to other dinoflagellates  genotypic diversity at a local scale is high and (2) a conspicuous population division occurs at this location. Contrary to our expectation that one or few clonal genotypes may dominate, particularly in the 81 isolates collected from the 2 km stretch of shore in North Wales, we identified a large number of unique genotypes, as indeed have the few studies of other free-living protists that have genotyped many samples from the same location O. marinaO. marina, O. marina, which is a commonly employed ecological-model organism More generally, these observations raise important questions about the possible mechanisms that sustain the apparently high levels of diversity in populations of free-living protists. For example, large population sizes potentially harbour high levels of neutral genetic diversity simply as a function of effective population size and largely irrespective of the underlying selective regime i.e. ability to model linkage disequilibrium and Hardy-Weinberg equilibrium conditions) at this location as compared with elsewhere (see C. polykrikoidesOxyrrhis, since both mitochondrial and nuclear sequence variation between these isolates is absent Oxyrrhis marina has been documented to form ephemeral adherent cysts associated with the tidal cycle The partition of the sample from North Wales into two model populations, one of which was common with other UK regions, and the other of which was unique to this location, was unexpected. The occurrence of a unique, highly divergent population at this single site is certainly curious. It is possible that this pattern reflects the greater sampling effort at the North Wales site and the concomitant statistical power to resolve population structure (Oxyrrhis marina is not panmictic at spatial scales between 2 and ∼1000 km within Great Britain and Ireland. These data highlight extensive levels of genetic diversity, but raise questions about the mechanisms that maintain such variation. Studies, such as the one reported here, employing population genetic approaches at a range of spatial scales, are increasingly providing the molecular tools to estimate demographic parameters and explore the adaptive mechanisms behind spatial and seasonal variation in a broad range of free-living protists.To conclude, these new microsatellite loci have uncovered spatial structure at a greater resolution than afforded by sequence markers commonly-employed for phylogeographic analysis of free-living marine protists.


Rhinogobius sp. YB (YB), which evolved from amphidromous R. brunneus (BR). The fish fauna was surveyed at 30 sites in 11 rivers on Iriomote Island, Japan, the geography of which was characterized by terraces/tablelands with many waterfalls. We found that all YB individuals were distributed only above waterfalls (height 6.8–58.7 m), whereas BR, and other fishes, were mostly distributed below waterfalls. Mitochondrial DNA analysis showed that every YB local population above the waterfall was independently evolved from BR. In contrast, cluster analysis of nine morphological characters, such as fin color and body pattern, showed that the morphology of YB individuals held a similarity beyond the genetic divergence, suggesting parallel evolution has occurred relating to their morphology. Genetic distance between each YB local population and BR was significantly correlated with waterfall height (r2 = 0.94), suggesting that the waterfalls have been heightened due to the constant geological erosion and that their height represents the isolation period of YB local populations from BR . Each local population of BR was once landlocked in upstream by waterfall formation, consequently evolving to YB in each site. Although the morphology of YB had a high degree of similarity among local populations, finer scale analysis showed that the morphology of YB was significantly correlated with the genetic distance from BR. Consequently, there could be simultaneous multiple phases of allopatric/parallel evolution of the goby due to variations in waterfall height on this small island.Waterfalls may affect fish distribution and genetic structure within drainage networks even to the extent of leading evolutionary events. Here, parallel evolution was studied by focusing on waterfall and the landlocked freshwater goby  Geographic isolation is one of the causes of speciation. Biological populations of identical species may become isolated by geographic change, such as mountain building, or social change, such as emigration, leading to changes in selectivity, mutation, and/or genetic drift; this eventually leads to the evolution of distinctly different characteristics (Dobzhansky Gasterosteus), where a stream-resident ecotype  has repeatedly evolved from an anadromous ecotype    , a freshwater goby, is distributed in the mountain streams of the Ryukyu Archipelago, Japan  analyses of six R. sp. YB populations from four islands in the Ryukyu Archipelago partially supported Nishida's hypothesis, although the results and conclusion were still unclear  were established along 11 rivers on the northern part of the island . The stuRhinogobius sp. YB was restricted only above waterfalls. Thus, a niche analysis was conducted with a relationship between several environmental factors including presence/absence of waterfalls and the distribution of the fishes.In this study, we aimed to test assumptions that the waterfalls prevent the upstream migration of amphidromous/brackish fishes and the distribution of The assessment of the river environment was conducted in 5–15 June 2011. The weather was generally fine and the river condition was stable during the period. Ten factors were used to evaluate the environment at the site . PresencWaterfall heights were determined using the laser range-finder. Waterfalls less than 3 m in height were disregarded because there were so many waterfalls less than 3 m and amphidromous fishes were always well distributed above them.Multivariate analysis of variance (MANOVA) using the nine environmental parameters was used to determine whether integrated environmental conditions differed between the sites above and below/without waterfalls.Pseudobagrus ichikawai  (Watanabe and Ito Oncorhynchus masou ishikawae (pelagic fish)  was used for the analysis with the presence/absence of the species/taxa as the dependent variable (1 or 0) and the ten environmental factors  was also used to analyze species richness using the “glmmML” function in R . The best model was selected using the same method described above.R. sp. YB and/or R. brunneus were distributed   and Rhinogobius sp. DL (depressed large-dark type) (UR4 and ML2) were obtained as references for the phylogenetic analysis. In total, 182 samples  were obtained.Each individual was anesthetized using clove oil diluted in water (50 ppm), and part of the left pectoral fin was cut and preserved in 100% ethanol for mtDNA analysis (described below). At several sites, fin samples of R. sp. YB and R. brunneus was apparent, a quantitative morphological evaluation was conducted to test the extent the two fishes were really separated in morphology, to what extent the R. sp. YB individuals displayed similarity to each other among the sites, and whether the phenotypes corresponded to genotypes. Nine morphological characters (R. sp. YB and R. brunneus while under anesthesia. The nine characters were (a) darkened body, (b) black dots along the lateral line, (c) dense orange spots on the cheek, (d) thin extension of the first dorsal fin, (e) yellowness at the edge of the first dorsal fin, (f) yellowness at the edge of the second dorsal fin, (g) stripe pattern on the second dorsal fin, (h) yellowness at the upper backward edge of the tail fin, and (i) dark band on the base of the tail fin. A score of “0” or “1”was recorded depending on whether the character was absent or present, respectively, except in cases where a clear judgment was difficult and then a score of “0.5” was recorded.While the morphological/phenotypic difference of aracters  were evaa–i) was compared between R. sp. YB and R. brunneus for each sex  by the Mann–Whitney U-test. The level of significance was adjusted by the Bonferroni method (P = 0.05/18 = 0.0028).Score of each morphological character ; a–i wasCluster analysis was conducted with the nine morphological characters for each sex. The Euclidean distances between all possible pairs of the individuals were then calculated using Ward's method with the “hclust” function in R.ND5 and full cyt-b regions. Polymerase chain reaction (PCR) was conducted using the primer sets L12321-Leu . All sequences were deposited in DDBJ/GenBank (accession numbers: AB674598–AB674735). Data about the local populations were also deposited in GEDIMAP . We targeted partial A statistical parsimony network of haplotypes was obtained using the program TCS ver. 1.21  among the separate R. sp. YB local populations (study sites were discriminated), the R. brunneus population , the R. sp. CB population , and the R. sp. DL population  were determined using MEGA5.Genetic distances  of the simple liner regression model obtained above and the evolutionary rate of Rhinogobius species (R)  was calculated as E = R/C and was applied to each waterfall of known height .We also tested whether morphological difference was correlated with genetic distance between each R. sp. YB and R. brunneus coexisted (see Results), mature individuals were caught to compare the size between the two. We used a hand net to randomly catch 17 and 13 males and 20 and 12 females of R. sp. YB and R. brunneus, respectively. We measured the total length of each individual and compared the mean for each sex of R. sp. YB and R. brunneus by the Student's t-test. The level of significance was adjusted by the Bonferroni method (P = 0.05/2 = 0.025).At site YCR2 where P > 0.05).R. sp. YB, the species observed were all amphidromous or brackish water fishes. Rhinogobius sp. YB was seen only at sites above the waterfalls, whereas most other fishes were usually found at the sites below or without waterfalls. Several amphidromous species were recorded above waterfalls , but species richness in such sites was low as compared with sites below or without waterfalls. YCR2 was the only site where R. sp. YB and Rhinogobius brunneus coexisted.Over 15 species were observed during the snorkeling survey . SeveralR. sp. YB was positively correlated with the presence of waterfalls below the sites but not with other factors . Other factors were also selected in the best models, although none of these factors had a statistically significant effect.The GLM analysis and model selection by AIC showed that the presence of  factors . In contd in females, all morphological characters showed a significant difference between R. sp. YB and R. brunneus in both sexes  and cyt-b  sequences from R. sp. YB and R. brunneus were similar , so phylogenetic analyses were conducted using haplotypes that were a combination of sequences from the two regions .The variable rates of R. sp. YB, R. brunneus, R. sp. CB, and Rhinogobius sp. DL, respectively  and low within populations (9.7%). In contrast, the genetic variation in R. brunneus was low among populations  and high within populations (93.6%).The AMOVA also confirmed these findings. The genetic variation in R. sp. YB was 0.0002, showing that R. sp. YB individuals from YCR3, and/or their descendants, had drifted downstream to YCR2 , R. brunneus and R. sp. CB (0.0229), and R. sp. DL and R. sp. CB (0.0281).Genetic distances among the groups are shown in see also . In contR. sp. YB local populations and the R. brunneus population varied from 0.0026 to 0.0057 (P < 0.0001). Data from YCR2  were omitted from this analysis because the R. sp. YB population of YCR2 had probably drifted from the YCR3 population (see above), making it unsuitable for the analysis. Using this information, the erosion rate forming the waterfalls was calculated to be 0.67 mm/year. Thus, the formation period of the waterfalls was estimated at ca. 11,000–88,000 years .In both sexes, morphological Euclidian distance between two fishes was significantly positively correlated with genetic distance   was significantly smaller than that of R. brunneus  .The total length of adult male and female Rhinogobius sp. YB below waterfalls is notable because downstream migration via waterfalls should be possible and physical conditions of the habitat do not appear to affect distribution . Selection pressure from interspecific competition is low above the waterfalls because of the relative absence of other species (Rhinogobius sp. TO (Tokai area type), is distributed among ponds in central Japan and is reported to be threatened with extinction by alien species, such as the largemouth bass Micropterus salmoides  and R. brunneus are different species, and the results of mtDNA analysis clearly separated two different groups with a genetic distance of 0.0048 . The transporter hypothesis based on standing genetic variation and parallel evolution predicts that speciation is potentially more rapid than that expected from new mutations  also had high morphological distance from R. brunneus. The two fishes were genetically separated at YCR2, but the haplotypes of R. sp. YB at UR4 (YB15) and NS2 (YB33), sites that were separated by relatively low waterfalls, were also shared by R. brunneus (R. sp. YB individuals drifted or moved downstream to UR4 and NS2 and mated with R. brunneus individuals because the genetic features and morphology of two fishes are still relatively similar when compared with further evolved local populations, such as those at PN2 and YCR3. We cannot confirm whether R. sp. YB and R. brunneus are different species, but our results indicate that speciation/evolution of multiple phase is simultaneously occurring at different locations across the island because of its heterogeneous geography, that is, variations in waterfall height.Cluster analysis showed that respective brunneus . It is lR. sp. YB is the yellow color of the fins; we cannot provide a hypothesis as to why the fins became yellow at present. In guppies, however, males are colorful at low predation sites, whereas they are less colorful at high predation sites (Endler and Houde R. sp. YB. In either case, we suspect that the morphological differentiation of the two fishes might be attributed to presence/absence of interspecific interactions, also leading to the morphological similarity of R. sp. YB beyond the genetic divergence of this fish.One of the representative morphological characters of R. sp. YB is correlated with the process of waterfall formation on the island: each local population of R. brunneus was once isolated and landlocked upstream by waterfall formations, consequently evolving to R. sp. YB in each site.Cliffs are often formed from heterogeneous geologic strata when a harder rock type overlies a weaker rock that is then weathered (Larson et al. R. sp. YB on the island and possibly have affected the survival of aquatic organisms other than fish by limiting predator populations, as reported in freshwater shrimps (Covich et al. Waterfalls locally decrease the diversity of fish fauna/genes above the waterfalls (e.g., Martin-Smith and Laird R. sp. YB local populations above the waterfalls are the same species. We expect to follow up this study with one comparing the detailed ecology, behavior, and morphology among the R. sp. YB local populations, as well as conducting breeding experiments in the laboratory. If the local populations are the same species, then genetic differences do not necessarily define a species, at least in terms of mtDNA. If the populations were different species, it follows that indigenous species are being generated or have been generated above separate waterfalls on this small island.A question that remains to be answered is whether the separate


Sciurus vulgaris) on the Channel Island of Jersey using mitochondrial DNA (mtDNA) control region sequence and nuclear microsatellite genotypes. Our findings reveal two different genetic origins and a genetic architecture reflective of the introductions 120 years ago. Genetic structure is marked within the maternally inherited mtDNA, indicating slow dispersal of female squirrels. However, nuclear markers detected only weak genetic structure, indicating substantially greater male dispersal. Data from both mitochondrial and nuclear markers support historic records that squirrels from England were introduced to the west of the island and those from mainland Europe to the east. Although some level of dispersal and introgression across the island between the two introductions is evident, there has not yet been sufficient gene flow to erase this historic genetic “footprint.” We also investigated if inbreeding has contributed to high observed levels of disease, but found no association. Genetic footprints of introductions can persist for considerable periods of time and beyond traditional timeframes of wildlife management.Wildlife populations have been introduced to new areas by people for centuries, but this human-mediated movement can disrupt natural patterns of genetic structure by altering patterns of gene flow. Insular populations are particularly prone to these influences due to limited opportunities for natural dispersal onto islands. Consequently, understanding how genetic patterns develop in island populations is important, particularly given that islands are frequently havens for protected wildlife. We examined the evolutionary origins and extent of genetic structure within the introduced island population of red squirrels ( Wildlife populations have been relocated and translocated by people for centuries, both accidentally and intentionally Atkinson . AccidenFor recently introduced populations, their genetic make-up and spatial distribution of genetic diversity depends on a number of factors, including the number of founding individuals and their evolutionary origin, relative breeding success between them during the initial phases of establishment, rate of increase in population size and subsequent expansion, and the extent of gene flow within the introduced population Freeland . UnderstOligosoma suteri) in the 15 years immediately following their reintroduction to Korapuki Island, New Zealand. However, many human-mediated wildlife introductions have taken place across much longer timescales, with many founded well over a century ago. Whether genetic footprints of such events persist across such time periods could have far reaching implications for the long-term management of wildlife. Islands provide ideal settings in which to study how introduced populations are founded and become established and consequently how patterns of genetic structure develop. Their isolated nature restricts the extent of natural colonization  on Jersey, to look for a historic genetic footprint of the introduction. We use mitochondrial DNA (mtDNA) sequence data and nuclear microsatellite DNA markers to trace the evolutionary origins of the founding individuals and examine current genetic structure in light of the known ecological history of the population over the past few hundred years. This introduced red squirrel population is an ideal system to interpret patterns of genetic structure because the history of the introduction is well documented and the island's ecological and land-use history over the past 200 years has remained relatively unchanged. Furthermore, the island has remained free of invasive North American gray squirrels (Sciurus carolensis) and so the negative effects of competition and diseases that they can bring have not had an impact on this red squirrel population.Here, we apply these ideas to a recently introduced population of red squirrels  red squirrel disease monitoring program, dead squirrels were collected on Jersey by members of the public and taken to the JSPCA. For this study, 169 Jersey squirrels that presented dead or had died as a result of illness or injury from July 2008 to January 2011 were examined. For all individuals, a full post-mortem examination was carried out and tissue samples were taken for DNA, along with location details. Cause of death was attributed to one of three groups: ill health, predation/trauma, or road traffic accident. Sick (classified as “diseased”) individuals were those with evidence of clinical illness and “amyloid individuals” were those that were found to have amyloid deposits detected at histopathology within examined sampled tissues. Thirty-four samples from the Isle of Wight were collected on an ad hoc basis by wildlife and forestry personnel between 1995 and 2007. One hundred and ten samples from France were collected from individuals that were victims of road traffic accidents throughout northern and western France. Specimens were stored frozen at −20°C and tissue samples for DNA analysis were collected and stored in ethanol at −20°C.3) was used, 500 μL of digestion buffer was used with 30 μg of Proteinase K, the pellet was washed twice in 70% ethanol and air dried until all ethanol was removed, and the DNA pellet was dissolved in DNA grade H2O rather than EDTA (Ethylenediaminetetraacetic acid) TE (Tris ETDA) buffer.DNA was extracted as described in Nicholls et al.  with theTaq reaction buffer (16 mmol/L (NH4)2SO4, 67 mmol/L Tris-HCl), 1.5 mmol/L MgCl2, 0.1 mmol/L each dNTP, 0.2 mmol/L each primer, 0.002 U Taq  and 2 μL template DNA under the following conditions 94°C for 4 min, then 30 cycles of 94°C for 30 sec, 49°C for 30 sec, 72°C for 1 min with a final extension of 72°C for 10 min. Polymerase chain reaction (PCR) products were purified and sequenced in both directions by Macrogen Genomics Europe in The Netherlands. mtDNA control region sequences for 110 individuals from France were provided for this study by A. Dozières, of which five were resequenced to corroborate results.A 500-bp fragment of mtDNA control region was amplified from 72 Jersey individuals and 10 Isle of Wight individuals using the primers Sq070F 2SO4, 67 mmol/L Tris-HCl), 1.5 mmol/L MgCl2, 0.1 mmol/L each dNTP, 0.2 mmol/L each primer, 0.002 U Taq (Bioline), and 0.4 μL template DNA under the following conditions 94°C for 4 min, then 30 cycles of 94°C for 30 sec, 56°C or 58°C for 30 sec, 72°C for 1 min with a final extension of 72°C for 10 min. All markers were multiplexed prior to analysis . Consensus sequences were created in Bioedit v7.0.9.0 Hall . AdditioGenotypes were scored using Genemapper software v3.7 . Observed and expected heterozygosities were calculated per locus in GenAlEx6 , regression estimators (r = 0.843), internal relatedness (r = 0.898), standardized heterozygosity (r = −0.821), and heterozygosity–homozygosity by loci (r = 0.848)  Wang . This me Ritland . To obta Ritland . TrioML  Ritland . A one-wU (M-W-U) tests were used to compare between diseased and nondiseased groups of individuals on Jersey. Kruskal–Wallis rank sum tests and Mann–Whitney U tests were carried out within the R statistical computing software  incorporating the logarithmic proportion of an individual's genome, its longitude and an interaction between the two factors were carried out. As an additional assessment of the data to test for an association between genotype and amyloidosis, locus-by-locus tests were carried out using a chi-squared test for each allele at each marker to establish if any of the markers genotyped are associated with amyloidosis. A Bonferroni correction was applied to account for multiple tests.Only two mitochondrial control region haplotypes were detected on Jersey with eight variable sites between them (Genbank accession numbers JX853758–JX853759), indicating that they have separate evolutionary origins. The two Jersey haplotypes are located in two distinct clades, one is predominantly British (JE162), the other predominantly European (JE120) . The phyThe prevalence of the two haplotypes found on Jersey reveals that there is a geographic pattern to where the mitochondrial haplotypes occur on the island . The BriA total of 169 individuals were successfully genotyped at most microsatellite loci. All microsatellite loci used were polymorphic and none had a null allele frequency greater than 0.25 . There wThe number of clusters that STRUCTURE and GENELAND infer varies from 1 to 9 depending on the input parameters. Applying an Evanno et al.'s  correctiThe uncorrelated allele frequency model and incorporation of spatial data appear to be the most realistic model for our data, based on our assumption that allele frequencies are unlikely to be correlated and the number and locality of clusters on the island. Therefore, to assess the introgression of the two introductions that we confirmed as having occurred using mtDNA haplotypes, the results from the uncorrelated allele frequency model are presented here. However, all of the model combinations under which both STRUCTURE and GENELAND were run gave broadly similar results. P = 0.001, with adjusted 5% level P = 0.00104), indicating that there is nonrandom mating occurring in these populations. The French population was within HWE, but only marginally so (P = 0.0021), suggesting some level of nonrandom mating.When only 20 unrelated individuals were tested across all three populations, no single marker was consistently out of HWE, but when all individuals were included in an analysis, the Jersey and Isle of Wight populations were both significantly out of HWE , which indicates a consistency among the microsatellite loci for heterozygous individuals at one locus to be more likely to be heterozygous at other loci – a characteristic of the data set, which supports its use as a proxy for assessing levels of inbreeding among individuals.We detected a significant heterozygosity–heterozygosity correlation , with both island populations having significantly higher mean levels of inbreeding than the mainland population in France (Tukey's Test Isle of Wight-France P = 0.015 and Jersey-France P = 0.008), but the level of inbreeding between the two island populations was not significantly different (P = 0.685). In contrast, levels of unbiased expected heterozygosity between the three populations were not significantly different .We observed a significant difference in the levels of individual inbreeding coefficients between populations  or unbiased expected population heterozygosity .Tests among the individuals on Jersey for an association between inbreeding and cause of death, presence of disease, or amyloidosis revealed that there is no association between them when using either TrioML estimates  showed a significant association with the prevalence of amyloidosis. However, after application of a Bonferroni correction, which decreased the significance level of the P = 0.0255) in whether an individual has amyloidosis, along with the interaction between the proportion of the genome that is English and longitude (P = 0.0260). However, comparison of regression lines between the two groups when the proportion of the genome that is English is plotted against longitude reveals that there is no difference between the groups (P = 0.7518).The GLM indicates that the proportion of an individual's genome that is English is a significant factor , it seems likely that strong genetic structure existed within the newly established population, a situation enhanced by the two founding events occurring at geographically separate introduction sites. However, how has this genetic structure changed over the last 120 years? Following establishment and a period of population growth, the extent of structure could have either remained the same (or increased) if no interbreeding occurred between the two differently sourced introductions, or diminished with interbreeding, potentially becoming erased entirely.Remarkably, despite the length of time since the initial introductions for movement of individuals to have occurred, there remains a distinct east–west structure reflected in both mtDNA and nuclear markers that corresponds to historic accounts  and 3. HWhile the mtDNA loci revealed substantial genetic structure, the extent of structure revealed by nuclear loci was low. Our analyses using both STRUCTURE and GENELAND included specifying allele frequencies between clusters as being either uncorrelated or correlated. We ruled out the latter, having little reason to suspect allele frequencies between the two different source populations to be correlated. Furthermore, correlated models have been shown to be unreliable at resolving the true number of clusters even when the data are simulated under that model (Guillot et al. Alongside this result of panmixia, however, we were also interested in assessing to what extent the two introductions have introgressed. Consequently, we reran STRUCTURE under the admixture model, allele frequencies uncorrelated, with prior information about population for those individuals that we obtained mtDNA genotype data for included and allowed for a high level of admixture by setting MIGRPRIOR to 0.4. That genetic structure has remained intact for so long is perhaps due to the availability and connectivity of suitable habitat on Jersey. Land use on Jersey has remained relatively stable since the mid 1800s, with the majority of land used for farming and the remainder for human habitation. Despite a lack of extensive woodland on the island, those wooded areas that do exist are of high habitat quality for red squirrels, with a mixture of seed trees providing a reliable year-round source of food. However, there was a dramatic reduction in tree cover during the Second World War, before trees were rapidly replanted and regenerated in the post-war years, so that today the island contains many mature trees Le Sueur . In addiIsland populations are predicted to be significantly more inbred than mainland populations Frankham , and inbAlthough the population and individual measures of inbreeding from this study do not align regarding the difference in the levels of inbreeding between the three populations, both measures consistently find no association between the level of inbreeding and prevalence of amyloidosis or other diseases, therefore we must conclude that there is no association between inbreeding and amyloidosis or disease. Within any population, even those that are not inbred, there will be individuals that are more inbred than others and it has been shown that it is often these inbred individuals that are more susceptible to disease (Wayne et al. t-test comparing regression lines of the proportion of the genome that is English against longitude found no difference between those individuals with amyloidosis and those without. The GLM also found no significant association between amyloidosis and locality, although it was significantly associated with the proportion of an individual's genome that is English. The finding that more individuals with amyloidosis have a higher proportion of English genetic origin could further support the hypothesis that there is a susceptibility allele within the population of English origin. However, we also note our finding that amyloidosis is significantly associated with the interaction between an individual's geographic location and the proportion that an individual's genome is English.Despite not finding an association between individual genotype and amyloidosis within Jersey's red squirrels, our genetic analyses reveal an intriguing spatial pattern to the prevalence on the island. Of the 34 individuals found to have amyloidosis, most were found longitudinally down the center of the island – a distribution which broadly coincides with the area at the center of the island where our results suggest the two differently sourced introduced populations meet – a putative hybrid zone . OutbreeTwo introductions of red squirrels occurred on the island of Jersey, one from England and one from mainland Europe, likely France, comprising two evolutionarily different source populations, which most likely contributed to high initial levels of genetic structure. Our study has shown that at nuclear loci, this structure has subsequently diminished over the course of the past 120 years, largely as a consequence of male-mediated gene flow, but that it still remains detectable and reflects a genetic footprint consistent with the historic introduction events. Our results do not provide evidence that inbreeding has contributed to the unusually high prevalence of amyloidosis on the island, although this island population is significantly more inbred than the population on mainland France. The spatial distribution of those Jersey individuals with amyloidosis appears to coincide with a putative zone of mixing between east and west introduced populations, although we find no statistical support for any genetic association. Nevertheless, our findings advocate an important message to wildlife managers planning to introduce red squirrels or other small mammal species to island refuges – patterns of genetic structure caused by multiple introduction events can persist for considerable periods of time, well beyond the traditional timeframe of most population management plans.


Phoxinus phoxinus). To this end, we isolated and genotyped exon 2 of two MHCIIB gene duplicates (DAB1 and DAB3) and 1′665 amplified fragment length polymorphism (AFLP) markers in nine populations, and characterized local bacterial communities by 16S rDNA barcoding using 454 amplicon sequencing. Both MHCIIB loci exhibited signs of historical balancing selection. Whereas genetic differentiation exceeded that of neutral markers at both loci, the populations' genetic diversities were positively correlated with local pathogen diversities only at DAB3. Overall, our results suggest pathogen-mediated local adaptation in European minnows at both MHCIIB loci. While at DAB1 selection appears to favor different alleles among populations, this is only partially the case in DAB3, which appears to be locally adapted to pathogen communities in terms of genetic diversity. These results provide new insights into the importance of host–pathogen interactions in driving local adaptation in the European minnow, and highlight that the importance of adaptive processes driving MHCIIB gene evolution may differ among duplicates within species, presumably as a consequence of alternative selective regimes or different genomic context.Host–pathogen interactions are a major evolutionary force promoting local adaptation. Genes of the major histocompatibility complex (MHC) represent unique candidates to investigate evolutionary processes driving local adaptation to parasite communities. The present study aimed at identifying the relative roles of neutral and adaptive processes driving the evolution of MHC class IIB (MHCIIB) genes in natural populations of European minnows (Phoxinus phoxinus). We highlight that the relative importance of neutral versus adaptive processes in shaping immune competence may differ between duplicates as a consequence of alternative selective regimes or different genomic contexts.Using next-generation sequencing, the present manuscript identifies the relative roles of neutral and adaptive processes driving the evolution of MHC class IIB (MHCIIB) genes in natural populations of a cyprinid fish: the European minnow ( In natural environments, heterogeneity in selection due to habitat fragmentation, climate fluctuations, and pathogen regime variation play a major role in shaping populations' genetic diversity, and eventually can lead to local adaptation (Kawecki and Ebert Genes of the major histocompatibility complex (MHC) are among the best candidates to study the genetics of the adaptive immune response . This species frequently suffers from infectious diseases, such as the salmon anemia virus, or furunculosis caused by the bacteria Aeromonas salmonicida. The polymorphism observed at MHC class I and IIB is maintained through pathogen-mediated selection  or Atlantic salmon   is a cyprinid occurring in most European freshwater systems. Local adaptation in growth and survival of P. phoxinus to a specific pathogen, the trematode Diplostomum phoxini, has been highlighted in the early 1990′s . Bacterial DNA was extracted from 500 μL of digested lysate using phenol chloroform (pH 7.5), sodium acetate (15 μmol/L), isopropanol, and ethanol precipitation procedure. DNA was resuspended in sterile water and concentration was adjusted to 10 ng μL−1. DNA from fish was extracted using the Gentra Puregene Qiagen kit according to the manufacturer instructions. DNA concentration was adjusted to 50 ng μL−1.One hundred and seventy-six European minnows were collected in nine populations from the Western Swiss Alps  during sP. phoxinus, a subset of four individuals from six populations  were used. We first amplified the 3′-end of exon 2, intron 2 and the 5′-end of exon 3 using the primers FishC12S and Fish12R from Ottova et al. . PCR for the two duplicates of MHCIIB exon 2 characterization were performed in a 25 μL reaction volume containing 1.5 mmol/L MgCl2, 1× PCR buffer (Qiagen), 0.4 μ mol/L of each primer, 100 μ mol/L of each dNTP, 1 unit of Taq Polymerase (Qiagen), and 50 ng of genomic DNA. Thermal cycling was carried out on a Biometra T Professional Standard Thermocycler , with an initial denaturating step of 95°C for 3 min, followed by 39 cycles of 30 sec of denaturing at 95°C, 1 min annealing at 52°C and 1 min 30 sec of extension at 72°C, and a final extension of 5 min at 72°C. PCR products were cloned into the pGEM T-easy vector . Between four to eight positive clones for each duplicate of MHCIIB exon 2 were purified with the Wizard SV PCR clean-up system (Promega) and sequenced using the Big Dye 3.1 Terminator cycle sequencing kit  on a ABI 3100 PRISM genetic analyzer (Applied Biosystems) using SP6 and T7 standard primers and universal procedures. Sequences from clones were read using BioEdit 7.09 . Start and end of each exon 2 duplicates were deduced by comparison with known cDNA and gDNA sequences of D. rerio .In order to amplify the two duplicates of MHCIIB exon 2 and bacterial 16S rDNA in a partial 454 GS FLX Titanium run , we pooled 18–20 individuals per population and randomly assigned each population five base pair tagged PCR primers . These primers were then used in locus-specific PCRs to amplify DAB1 and DAB3. The same tagging strategy was used to amplify bacterial 16S rDNA from the nine populations. PCRs for both MHCIIB exon 2 and 16S rDNA were conducted on Biometra T Professional Thermocyclers (Biometra) in a final volume of 25 μL, using 1 × Q-solution (Qiagen), 1× buffer (Qiagen), 1.5 mnol/L MgCl2 (Qiagen), 0.2 mmol/L dNTPs , 0.2 μmol/L of each primer, 2 units of Taq (Qiagen), and 20 ng of genomic DNA. Cycling conditions included an initial denaturation step at 95°C for 3 min followed by 35 cycles at 95°C for 30 sec, annealing at 52°C (DAB1 and DAB3 loci) or 58°C  for 1 min, 72°C for 1.5 min, and a final extension step at 72°C for 5 min. PCR product concentrations were estimated simultaneously on agarose gel and with a Nanodrop spectrometer . PCR products with similar concentrations were pooled and purified using the MinElute PCR Purification kit (Qiagen). Purified PCR products' concentrations were estimated a second time in order to produce a final pool with equimolar quantities of PCR products , 16S rDNA sequences were submitted to the Bayesian Ribosomal Database Project (RDP) Classifier . Altogether with primers and tag, read length exceeded the length that 454 run cold sequences, resulting in reads whose length was shorter than expected. All sequences were thus truncated to 220 bp in order to maximize the number of sequences for further analyses  markers using the same protocol as described in Collin and Fumagalli . AFLP weIn order to explore nonpathogenic and pathogenic bacterial composition between sampling locations, we conducted a permutational multivariate analysis of variance based on dissimilarities, that is, beta nonpathogenic and pathogenic bacteria diversity (ADONIS), using populations as a factor. Betadiversity was calculated using the nonpathogenic bacteria and pathogens' abundance matrices . We finally conducted a canonical correspondence analysis (CCA) in order to analyze the correlation between pathogen composition and various environmental factors characterizing each sampling location and which have been shown to influence bacteria distribution  and nonsynonymous (dN) substitutions was calculated according to Nei and Gojobori . We then used CODEML to implement four site models: two neutral models (M1a and M7) and two models allowing for positive selection (M2a and M8)  for DAB1 and 58 codons (174 bp) for DAB3, in which peptide-binding region (PBR) represented the most polymorphic codons as they bind pathogenic antigens /(dmax−dmin), where dx represents the pairwise Euclidean distance between two populations, dmax the maximum Euclidean distance between two populations, and dmin the minimum Euclidean distance between two populations identified in the matrices. We first tested for isolation by geographical distance (IBD) with Mantel tests both for neutral AFLP and MHCIIB (separately for DAB1 and DAB3). In order to estimate if MHCIIB differentiation exceeds neutral differentiation along geographic clines, we tested whether isolation by distance (IBD) patterns in MHCIIB persisted when accounting for neutral population differentiation using a partial Mantel test. We tested for correlation between differentiation at AFLP and at MHCIIB loci (DAB1 and DAB3 separately) using a Mantel test in order to highlight a potential effect of divergent selection which would increase levels of genetic differentiation at immune loci compared to neutral markers. We also aimed at obtaining the full neutral distribution of IBD slopes, in order to directly compare this distribution to IBD slope values observed at the MHC, hence providing a cross-test of IBD not only using neutral population-based data but individual neutral data. We estimated individual-based AFLP Fst matrices between pairs of populations using a subset of randomly chosen AFLP markers among the 1665 available. The regression slopes between Fst matrices and geographical distance matrices were obtained from Mantel tests. We then compared the distribution of neutral regression slopes  with values obtained from MHC loci. All Mantel and partial Mantel tests were implemented with 1000 permutations in each case using the “vegan” library in R  TV presence–absence in each population for DAB1 and DAB3 and (2) AFLP band presence–absence in each population. DAB1, DAB3, and AFLP distance matrices were then standardized to a 0/1 scale using the following formula:  and salmonids , with sequence similarities ranging from 79% to 92%. As reported also from other species . Average, minimum and maximum read lengths and quality scores frequencies are reported in t-tests: df = 8.832, t = −3.685, P = 0.005; df = 10.438, t = −4.219, P = 0.002). In concordance with these results, the Z-test of selection showed evidence for diversifying selection on PBR codons as the rate of nonsynonymous substitution (dS) over synonymous substitution (dN) was significantly higher than expected under neutral expectations . The maximum likelihood method implemented in CODEML revealed that models allowing for positive selection (M2a and M8) better explained DAB1 and DAB3 sequences evolution than neutral models (M1a and M8)  and 11 (DAB3) codons matching human PBR codons . Mean DActations . We test and M8) . Models R = 0.330, P = 0.037 and R = 0.403, P = 0.025). DAB3 showed a tendency of IBD . The nonsignificance of this correlation was driven by three outlier population pairs  for which low levels of genetic differentiation was not explained by the geographic distance separating them. The three outlier population pairs are highlighted by black circles in R = 0.515, P = 0.004). The pattern of IBD at DAB1 persisted when controlling for neutral genetic differentiation , and stayed nonsignificant for DAB3 . Again, when removing the three outlier pairs of populations and controlling for neutral differentiation, significant IBD was found also at DAB3 .Isolation by distance was observed at neutral loci and DAB1 . Again, removing the three outlier pairs of populations resulted in significantly correlated genetic differentiation at AFLP markers and DAB3 . The neutral distribution of regression slopes ranged from −0.263 to 0.839, which includes values observed at MHC loci .Patterns of differentiation at neutral and MHCIIB duplicates were significantly correlated for DAB1, but not for DAB3 , Staphylococcus sp., Flavobacterium sp., unclassified Clostridiales, unclassified Enterobacteriaceae, unclassified Pseudomonadaceae, and unclassified Oxalobacteraceae. We therefore partitioned the bacteria into three different data sets: “pathogenic bacteria,” “nonpathogenic bacteria”, and “all bacteria.” The species richness index calculated for these three data sets ranged from zero to three for pathogens, from two to 36 for nonpathogens, and from three to 39 for all bacteria. We examined the overall bacteria phylum composition for each sampling location , while in other locations Flavobacterium pathogens (Bacteroidetes) predominated  was positively associated to higher altitudinal sites and topography. Pathogen compositions at other sampling locations were characteristic of highly productive (eutrophic) sites at lower elevation.To investigate how pathogenic bacteria affect location . We founocations . Il locaominated . ADONIS ominated . As mentF = 12.015, P = 0.010). ANOVA results of linear regression models are summarized in We used linear models and ANOVAs to explore the relationship between amino acid polymorphism at MHCIIB and species richness of pathogenic bacteria, nonpathogenic bacteria, and all bacteria. The percentage of polymorphic amino acid sites for the two MHCIIB duplicates ranged from 80% to 100% for DAB1, 82% to 100% for DAB3 at PBR codons, and at non-PBR codons from 20% to 27% for DAB1 and 19% to 36% for DAB3. Only the percentage of polymorphic amino acid sites within PBR codons at DAB3 showed significant positive association with pathogen species richness , though they appeared to be driven by different aspects of pathogen-mediated selection. Patterns of genetic differentiation at DAB1 suggest that selection favored different alleles in each population, resulting presumably in better immune defense against local pathogens. This pattern was also found for DAB3, but only partially when removing the three pairwise comparisons with largest geographic distance. Contrary to its paralog, genetic diversity at DAB3 was correlated with local pathogen diversities. This suggests that although locally adapted alleles were favored by selection also at DAB3, selection for locally adapted genetic diversity overpowered the selection for locally adapted alleles. The interplay between these different selection processes may explain the breakdown of the overall pattern of IBD at DAB3.The present study aimed at examining whether the cyprinid fish Bacteroidetes, Actinobacteria, and Proteobacteria  a strong spatial structure between populations and (2) a role for diversifying selection, with selection on DAB1 favoring different, locally adapted alleles in every population. DAB1 genetic diversity seems thus to be the result of diversifying selection promoting different locally adapted alleles frequencies in different populations. Similar results were observed on MHCIIB in recent studies on great snipes .Contrarily to DAB1P. phoxinus have already been demonstrated in other cyprinid species  display a similar MHCIIB architecture with each two of the duplicates present in European minnows highlighted in the present study. In S. cephalus DAB3 also presents a greater diversity than DAB1. Although these data support the results found in the present work, several studies on other cyprinid fish reported the inverse pattern  understand how pathogen-mediated selection shapes diversity at two MHCIIB copies. Despite providing important insights into these questions, our study also shows the technical limitations of this approach.Our study used a combination of molecular evolution and environmental genomics to understand how pathogenic bacteria communities and their environmental characteristics influence MHCIIB genetic diversity in nine populations of European minnow. We also used genome-wide neutral AFLP markers to compare levels of genetic differentiation at both neutral and selected markers. This study attempted to (1) characterize MHCIIB in First, the bacteria diversity survey was based on water samples collected from a single water sampling event. Even though this may represent some limitation, this approach gave some important information about pathogen distribution and abundance that can be a starting point for further studies on European minnows or other species. Indeed, most studies aiming at detecting pathogen-mediated selection, often do not report diversity, spatial distribution, or abundance of pathogens Wegner . Even thWe demonstrated that both copies presented historical evidence for positive selection, but only one copy (DAB3) presented evidence for contemporary pathogen-mediated selection. DAB1 may also be subjected to this type of selection, but as fish and water samples have been sampled during the summer, we can hypothesize that this copy may be involved in the recognition of pathogens that were not sampled at that time of the year, or other pathogen taxa.Our next-generation sequencing of MHCIIB copies presents several limits as compared to more classical approaches. The major limitation of our approach was the population-level tagging rather than individual-based sequencing. Even though it provided a rapid and cheap way to sequence a large number of individuals, the frequency of the identified variants could not be reliably inferred. Several hypotheses related to pathogen-mediated selection could not be tested such as a potential heterozygote advantage or frequency dependent selection. Nevertheless, several recent publications successfully used a population-based 454 sequencing approach similar to the one presented in our study to assess population-level MHC variation (Spurgin et al. Duplication events and the fate of duplicates in the face of both neutral processes and selection are still sparsely understood. In this context, the present study demonstrates how host–pathogen interactions can be the driver of genetic diversity in immune-related genes, and how alternative selective regimes may influence the processes driving the evolution of gene duplicates within the same species. Further studies with higher technical resolution examining the duplication history of MHC genes in cyprinid fish in Europe and the origin of duplicates' diversity should provide new insights into the relative role of historical and contemporary selection, and neutral processes on host–pathogen related local adaptation.