Cavitation resistance to water stress-induced embolism determines plant survival during drought. This adaptive trait has been described as highly variable in a wide range of tree species, but little is known about the extent of genetic and phenotypic variability within species. This information is essential to our understanding of the evolutionary forces that have shaped this trait, and for evaluation of its inclusion in breeding programs.P50), growth and carbon isotope composition in six Pinus pinaster populations in a provenance and progeny trial. We estimated the heritability of cavitation resistance and compared the distribution of neutral markers (FST) and quantitative genetic differentiation (QST), for retrospective identification of the evolutionary forces acting on these traits.We assessed cavitation resistance . QST was significantly lower than FST, indicating uniform selection for P50, rather than genetic drift. Putative mechanisms underlying ST<FSTQ are discussed.In contrast to growth and carbon isotope composition, no population differentiation was found for cavitation resistance. Heritability was higher than for the other traits, with a low additive genetic variance (h The climatic niches of forest tree species are moving faster than the maximum rate of migration, measured by palynology or gene flow analysis, as a direct consequence of the current increase in temperatures due to global warming From a physiological point of view cavitation resistance is an important trait to estimate drought tolerance. Indeed, dysfunctions of the vascular system of the tree, such as xylem embolism due to cavitation events, is likely to be a key factor governing the mortality of these long-lived organisms 2 = 0.9) between these two traits. Secondly, global surveys of cavitation resistance in woody species showed that xeric species are more resistant to embolism than hydric species A direct causal link between survival (fitness) and cavitation resistance during extreme drought has been highlighted, based on two lines of evidence suggesting that cavitation resistance is an important adaptive trait. Firstly, assessments of the correlation between cavitation resistance and lethal water potential At the intraspecific level, cavitation resistance can be analyzed by provenance or progeny trials. The few studies carried out to date (reviewed in Pinus pinaster Ait.), a species with a fragmented distribution in the western part of the Mediterranean region. The scattered distribution of this species may have prevented or limited gene flow between different groups of populations, promoting high levels of genetic divergence between ecotypes due to genetic drift Pinus pinaster populations have been subjected to diversifying selection for cavitation resistance. More specifically, this experiment aimed to address the following questions: what is the level intraspecific variation and heritability for cavitation resistance? Can we separate the relative roles of drift and selection in population differentiation for this trait?The aim of this study was to provide the first estimates of heritability, additive variance and population differentiation for cavitation resistance-related traits. We carried out a case study of maritime pine  with single-tree plots. Each block contained at least one tree from each half-sib family. There were 600 seedlings per block, giving 9,000 seedlings for the entire experiment.We carried out a provenance-progeny trial, in which young trees (six-year-old plants) were planted in December 2003 at the INRA forestry station in the Aquitaine Region . The mean annual temperature at this site is 13.2°C and mean annual rainfall is 836 mm (1984–2006). The soil is a sandy podzol with a water table rising to about 0.5 m below the surface in winter and descending to a depth of 2 m in late summer. Seedlings were grown in the nursery from open-pollinated seeds collected from 24 natural populations (or ecotypes) in France, Spain, Morocco and Tunisia, to cover the fragmented distribution of Pinus pinaster species. In each population, eight families  were randomly selected for further analysis.The assessment of cavitation resistance is a intensively time-consuming process Pinus pinasteri.e., much longer than the longest tracheid). Bark was removed from all segments before measurements.We collected branches, according to the sampling procedure described below, during winter 2009, before 10 am, to avoid native embolism. Needle water potential was lower than -1 MPa, far from the minimum needle water potential in summer (−2 MPa) of Px) was first set to a reference pressure (−0.5 MPa) and maximal conductance (kmax) was determined by measuring the flux of a reference ionic solution (10 mmol dm−3 KCl and dm−3 mmol dm−3 CaCl2 in deionized water) through the sample. The centrifugation speed was then set to a higher value for 3 min to expose the sample at a more negative pressure. Conductance (ik) were measured 4 times for each step, and the average was used to compute percent of loss of xylem of conductance (PLC in %) following PLC = 100 (1−ik/kmax). The procedure was repeated for at least eight pressure step with a −0.5 MPa step increment until PLC reached at least 90%. The percent loss of xylem conductance as a function of xylem pressure (MPa) represents the sample's vulnerability curve (Cavitation resistance was measured on 240 genotypes (6 populations * 8 families * 5 offsprings), with the Cavitron technique ty curve . Rotor vik) (see 50P) and the slope of the curve at 50P (50S).kmax is the highest conductance measured for each sample , ik is the mean conductance at a given pressure concerned, X is the percentage loss of conductance of interest (in %), XP (in MPa) is the pressure inducing X% loss of conductance and XS (in MPa.% −1) is the slope of the tangent at the XP abscissa point on the curve. Analysis has been performed for pressures and slopes corresponding to X = 12, 50 and 88 % loss of conductivity .Based on a sensitivity analysis (data not shown) and graphical checking , we retaδ13C in %o) was obtained as previously described h) was calculated as the difference between these two measurements (in 2004 and 2005).Carbon isotope ratio  of fixed block effects, pop is the vector (number of populations) of random population effects, f is the vector (number of mother trees) of the random genetic effects of mother tree within the population, ε is the vector  of residuals, X is called the design matrix, 1Z and 2Z are the incidence matrices linking the observations to the effects. A variance was fitted for each random effect: Genetic analysis was conducted with the following mixed model:Variance or covariance components were estimated by the restricted maximum likelihood (REML) method, assuming a normal distribution of the random effects. The significance of variance components were tested using log-likelihood ratio tests. We included population as a random effect to draw inference at species levels X = 100√(Variance)/MeanX where X is the trait considered, and CV is the coefficient of variation. Each variance component is expressed with a CV . The variance of each component was extracted from the asymptotic covariance matrix. The significance of mean population difference was estimated using the same model (Eq. 2) with a proc GLM with a Student-Neuman-Keuls post hoc test.Variance components were standardized by the trait mean P50 and δ13C were converted from negatives to positives. Genetic correlations between traits were evaluated by calculating Pearson's coefficient on the family Best Linear Unbiased Predictor estimation  caused by demographic and other processes not related to selection . Eight polymorphic nuSSRs were selected from those previously developed by FST  rather than RST  because genetic drift affects allele frequency but not mutation rate. STF values for each locus were estimated with Genepop The estimate of phenotypic differentiation between populations, STQ and STF, to disentangle the effects of genetic drift from those of selection, we develop a new test to avoid previously reported limitations STQ >STF and STQ <STF. We explicitly derive the FST and QST distribution using in both case a parametric bootstrap as follows.FST* is a parametric bootstrap replicate of FST. First, nuSSR loci were randomly resampled with replacement, to estimate the sampling variance of STF. Each of this STF replicate (FST* as the “drift distribution”.For the comparison of QST (Eq 4), by simulating the distribution of each variance component (i factor. It is obtained by multiplying idfe).dfei is the effective degree of freedom for the variance component of i factor. dfi is the observed degree of freedom. i factor. i-1 factor . n is the total size of the sample. j is the number of level of factor i. We calculate a QST* for each replicate from QST* as the “phenotypic distribution”, although QST is a standardized measurement of additive genetic variance between populations.We estimated the sampling variance of FST* and ST*Q distributions, using nonparametric and free distribution two-sample test for equality of the 2.5 and the 97.5 quantile  around −3.25±0.006 MPa (see BP) was significant for δ13C and Δh but not for P50 . Cavitation resistance-related traits had much lower coefficients of variation than Δh . It should be noted that CVs for δ13C are not comparable with those of other traits, because they are estimated relative to a standard For each population, vulnerability curves showed similar sigmoid shape with the air-entry  had the highest Δh values , whereasP50  and micro-environmental sensitivity  (2ns) for P50 was higher (0.44±0.18) than those estimated for δ13C (0.21±0.10) and Δh (0.35±0.06), showing that cavitation resistance was genetically controlled, although the standard error was high, probably due to the small number of progenies per mother tree analyzed. The CVs of P50 and Δh presented contrasting patterns, with a lower coefficient of additive variation for P50 (CVA = 4.4%) than for Δh (CVA = 16.2%), suggesting limited evolvability of P50.Heritabilities and normalized measurements of trait dispersion (i.e. CVs) were estimated to evaluate the within-population additive variance, evolvability  . Narrow-QST and FST comparisons have three possible outcomes QST > FST, the degree of differentiation for quantitative traits exceeds that attainable by genetic drift alone (ii) if QST and FST are not significantly different, the observed degree of differentiation for quantitative traits could have been reached by genetic drift alone, and (iii) if QST<FST the observed degree of differentiation is lower than expected from genetic drift alone. Consistent with previous reports FST* and QST* presented skewed distributions (h and P50 had a QST* distribution different from the FST* distribution (P = 0.003 and P <0.0001 respectively). For, δ13C, the difference between QST* and FST* values was centered on 0 (see P = 0.88). Conversely, differences between the QST* and FST* distributions for P50 were centered on -0.18, suggesting that the studied populations were less differentiated than would be expected in the presence of drift alone . No relationship between P50 and Δh was found at either phenotypic or genetic level. A significant negative phenotypic correlation between Δh and δ13C was detected . This correlation was barely significant at the genetic level .We found a significant positive phenotypic correlation between absolute value of QST- FST comparison). We can therefore reject the hypothesis of diversifying selection. We suggest instead that uniform selection has shaped the phenotypic variability of this trait. Uniform selection could be seen as a stabilizing selection acting within each population with the same selection optimum in each population despite the steep climatic gradient We reliably estimated for the first time the genetic variability of cavitation resistance, a functional trait that allows plants to survive under severe drought. We also provided evidence of natural selection acting on this trait. These results were based on the greatest number of genotypes ever measured to date in an experimental design (BP = 1%). The few studies published to date (reviewed in P = 6.6%), but consistent with the range reported for wood properties of maritime pine, such as mean ring density, lignin content and fiber morphology , given that substantial variability has been described between species. For instance, Delzon et al (2010) showed that cavitation resistance ranged from −3 to −12 MPa in a sample of 40 coniferous species. This variability was interpreted as the effect of natural selection rather than phylogenetic legacy Despite the steepest climatic gradient  and strong phylogeographic structure between the six studied populations In contrast, the population differentiation observed for growth and water-use efficiency (WUE) was significant and consistent with previous results for this species The weak but significant positive correlation found between absolute value of cavitation resistance and water-use efficiency (carbon isotope composition) suggested that drought-tolerant genotypes had lower water-use efficiency. In dry environments, genotypes that allocate more carbon to the construction of cavitation-resistant wood in order to avoid runaway embolism might be able to maintain higher stomatal conductance and hydraulic conductance at low leaf water potential, resulting in a decrease in water-use efficiency. Our results are consistent with previous findings QST<FST) may result from an underestimation of QST variance FST estimate was more than five times greater that the QST estimate. We thus believe that this difference is biologically meaningful and not due to a statistical artifact Juniperus genus), that cavitation resistance gave strong phylogenetic conservatism, suggestive of uniform selection for the maintenance of ancestral traits.The phenotypic distribution of cavitation resistance was significantly lower than the expected distribution under the drift hypothesis. This may be interpreted as a consequence of uniform selection . This inference (QST being greater than FSTQuercus suber (PST/FST comparison). However, the distribution (QST-FST) of this trait was shifted to the right .The causal mechanisms underlying uniform selection or leading to evolutionary stasis are not well understood Lethal or sublethal mutations may limit the variability of the genes they affect, thereby controlling trait variation. In conifers, cavitation resistance is known to be determined by xylem anatomy, including, in particular, the characteristics of intertracheid pits If selection acts on a trait that is negatively correlated with another trait (or traits) also under selection, than the decrease of rate of evolution for the first trait is proportional to the strength of the correlation Arabidopsis thaliana, there is no evidence of canalized trait in plants nowadays. For cavitation resistance, two arguments lead us to consider canalization as the most likely mechanism: (i) low additive genetic variance between populations (VBP) and (ii) the similarity of cavitation resistance values among all the Pinus species This hypothesis suggest that cavitation resistance is canalized to buffer the variation of this key hydraulic trait against all kinds of disturbance, being of genetic  and/or environmental nature 2ns = 0.4) and its additive coefficient of variation (CVA = 4.4%). These values suggest that this trait may respond to truncation selection frequently practiced in breeding. However, for a given selection intensity, genetic gain for cavitation resistance would be limited by the low additive variance, although long-term artificial selection experiments have shown that quantitative traits have a non negligible mutational variance Variance component analysis for cavitation resistance resulted in the first estimates of the heritability of this trait  for height increment is consistent with previous reports and accounts for the genetic gain achieved for this trait over successive generations in breeding programs 13C, a previous study 2ns = 0.17 vs. 0.21 here), but with estimation based on a diallel cross of limited size, with a narrow genetic background restricted to 12 elite trees from south-western France.The much higher CVin situ phenotypic variability of cavitation resistance and the extent of phenotypic plasticity using provenance trials installed under different edapho-climatic environments. Further studies are also being pursued to dissect the genetic architecture of cavitation resistance to determine the number, map location and effects of Quantitative Trait Loci controlling part of the variation of this trait.Phenotypic variation is a fundamental prerequisite for evolution because natural selection acts on phenotype. Adaptation and evolution via natural selection requires the presence of genetic variation among individuals in a population upon which natural selection can act. Intra-population genetic variability can thus be seen as the fuel for future adaptation. However, an environmentally induced shift in phenotype is also a major component of the variation we see in nature. Recent studies Figure S1Genetic (right panel) and phenotypic (left panel) correlation between traits. For ease of interpretation, we have converted all the negative values to positive values . For the genetic correlation, all Pearson correlations (r) were computed over the best linear unbiased prediction (BLUP) . For phenotypic correlation, all Pearson correlations were computed over the BLUP family plus BLUP population and the grand-mean, to ensure that the order of degree of freedom remained the same and the block effects are removed. P50, pressure at 50 % loss of conductivity in MPa, Δh the annual increment between 2004 and 2005, in mm, δ13C is the isotope discrimination for carbon 13 in ‰.(TIF)Click here for additional data file.Table S1Review of intraspecific studies for cavitation resistance estimated using P50 or related parameters (as indicated in the table). Npop: number of populations used, Nind: number of individuals per population used to assess cavitation resistance. The table is divided in two parts, the first part corresponds to provenance or progeny trials, and the second to “in situ” studies.(DOC)Click here for additional data file.Table S2Result of the principal component (PC) analysis (PCA) for climatic data of Pinus pinaster populations . The eigenvalues of PC1 = 7.65, PC2 = 3.059, PC3 = 0.97, PC4 = 0.86. W is mean wet ground days (days). I is mean Martonne's index (Pi/(Ta+10)). iP is the mean precipitation (mm.days−1). C is percent of cloud cover (%). S is the mean of wind speed (m.s−1). V is the water vapor pressure in air (hPa). VPD is the water vapor pressure deficit of air (hPa). Tmin is the minimum temperature (°C). ΔDT is the mean diurnal temperature range (°C). Tm is mean temperature (°C). GR is mean global radiation (W.m2). Tmax is the maximum temperature (°C). H is mean soil water deficit . ETP is mean Truc's potential evapotranspiration (mm).(DOC)Click here for additional data file.


F-statistics to explain contemporary drivers of population genetic differentiation despite high gene flow. We investigate range-wide population genetic structure of the California spiny (or red rock) lobster (Panulirus interruptus) and find slight, but significant global population differentiation in mtDNA  and seven nuclear microsatellites , despite the species’ 240- to 330-day pelagic larval duration. Significant population structure does not correlate with distance between sampling locations, and pairwise FST between adjacent sites often exceeds that among geographically distant locations. This result would typically be interpreted as unexplainable, chaotic genetic patchiness. However, kinship levels differ significantly among sites , and ten of 17 sample sites have significantly greater numbers of kin than expected by chance (P < 0.05). Moreover, a higher proportion of kin within sites strongly correlates with greater genetic differentiation among sites . Sites with elevated mean kinship were geographically proximate to regions of high upwelling intensity . These results indicate that P. interruptus does not maintain a single homogenous population, despite extreme dispersal potential. Instead, these lobsters appear to either have substantial localized recruitment or maintain planktonic larval cohesiveness whereby siblings more likely settle together than disperse across sites. More broadly, our results contribute to a growing number of studies showing that low FST and high family structure across populations can coexist, illuminating the foundations of cryptic genetic patterns and the nature of marine dispersal.We combine kinship estimates with traditional  F-statistics  with more direct measures such as kinship or parentage analyses  and genetic structure  measures of population connectivity, it is possible that direct analyses may identify recruitment patterns that cannot be detected using traditional F-statistics  among populations of bicolor damselfish (Stegastes partitus) in Exuma Sound, Bahamas, but parentage analysis identified high levels of self-recruitment at two of the eleven sampled locations. The direct identification of parent–offspring pairs resulted in very different management advice for this species than interpretation based on the FST data alone.Direct evidence of coancestry between individuals provides a particularly valuable complement to . et al.  found liPanulirus interruptus , spends at least the first 8 months of its life in the plankton, during which time it can presumably disperse across its entire geographic range. Species without barriers to dispersal are expected to exhibit no detectable neutral genetic population structure. Here, we use the California spiny lobster as a model to test the intuitive assumption of genetic homogeneity in species with extended PLD. We demonstrate the utility of individual-based estimates of genetic exchange in the interpretation of connectivity based on F-statistics.To date, most marine kinship studies have understandably focused on parent–offspring identification in reef fishes with fairly short larval durations. Here, we show that kinship analyses can also be useful at the opposite end of the potential dispersal continuum: the California spiny, or red rock lobster, Panulirus interruptus, exhibits high site fidelity during its adult phase  to Bahia Magdalena, Mexico. Throughout its geographic distribution, P. interruptus plays an important ecological role in structuring both kelp forest and intertidal communities , Baja California Sur, Mexico, in the south  using species-specific primers PintCOI-F (5′-GCTTGAGCTGGAATGGTAGG-3′) and PintCOI-R (5′-CACTTCCTTCTTTGATCCC-3′), which were designed from GenBank sequence #AF339460 , with an initial denaturation step of 95 °C for 4 min, 35 cycles of denaturation (95 °C for 30 s), annealing (56 °C for 30 s) and extension (72 °C for 30 s), followed by a final extension step of 72 °C for 10 min. PCR products were treated with 0.75 units of Exonuclease I and 0.5 units of Fast Alkaline Phosphatase  per 7.5 μL of PCR product and incubated at 37 °C for 60 min, followed by deactivation at 85 °C for 10 min. Purified DNA fragments were sequenced in the forward direction with fluorescently labelled dideoxy terminators either on an ABI 3730XL capillary sequencer  by the Advanced Studies of Genomics, Proteomics and Bioinformatics (ASGPB) Center at the University of Hawai'i at Mānoa or an ABI 3130XL Genetic Analyzer (Applied Biosystems) at the Hawai'i Institute of Marine Biology EPSCoR Sequencing Facility. Unique sequences and sequences with ambiguous nucleotide calls were also sequenced in reverse to confirm sequence identity. Sequences were edited, aligned and trimmed to a uniform size using sequencher 4.8b . The alignment did not contain any indels or frameshift mutations.We amplified a 494-bp fragment of cytochrome h) for each sampling site as described in Nei . We investigated population structure using an analysis of molecular variance (amova) as implemented in arlequin. We used an analogue of Wright's FST (ΦST), which incorporates a model of sequence evolution, for both our complete data set and pairwise population comparisons  in P. interruptus, we also calculated Dest_Chao as an absolute measure of differentiation between sites. The magnitude of FST is inversely proportional to heterozygosity ] and the natural log of Euclidean distance  and haplotype diversity , 2 μm total dNTPs, 0.1 U GoTaq polymerase (Promega Corp.), 6 μm each of forward and reverse primer and 20 ng of genomic DNA. PCR was carried out using a Bio-Rad DNA Engine Dyad Thermal Cycler with the following conditions: an initial denaturation step of 95 °C for 3 min, 35 cycles of denaturation (94 °C for 40 s), annealing at primer-specific annealing temperatures   and expected (He) heterozygosities at each location, as well as allelic richness (A), rarefied to 25 individuals, using the Excel Microsatellite Toolkit 3.1.1 , linkage equilibrium, scoring errors] followed Selkoe & Toonen  as detaiet al. , but exp1.1 Park  and fstat Goudet , respectrham's θ . Consisthao Jost  in additFST and Dest_Chao patterns, we calculated kinship coefficients  was conducted with 10 000 unrestricted permutations of the raw data and type III sums of squared differences. Significance is determined by evaluating a pseudo-F value  in ML-Relate, following the scale for the index of relatedness  to be able to compare our kinship results with a relatedness index. We also tested the relationship of the mean pairwise FST, mean pairwise Dest_Chao and local FST for each site with the proportion of closely related lobsters at that site .To investigate site-specific patterns in kinship, we counted the number of closely related individuals within each site where the kinship coefficient was greater than or equal to the equivalent of that expected for quarter-siblings (0.047). Following Selkoe et al.'s  and Bustet al.'s  analysiset al.'s  coanceste et al. , and we e et al.  metric aDest_Chao for each sampling location. We interpolated these values beyond the specific sites we sampled using an inverse weighted distance (IWD) algorithm in the Spatial Analyst extension in arcgis 10. To test the hypothesis that upwelling is a potential driver of both increased kinship and, in turn, site-specific genetic structure for P. interruptus in this region, we overlaid on this map known areas of consistent upwelling in Baja California, Mexico . We tes  was high, ranging from 0.88 to 0.95 (8.3–20 effective haplotypes), with a mean of 0.92 (12.5 effective haplotypes). In contrast, nucleotide diversity (π) was relatively low, ranging from 0.005 to 0.018, with a mean of 0.010. The number of individuals sequenced (N), haplotype diversity (h), effective number of haplotypes (heff) and nucleotide diversity (π) for each site are listed in We sequenced COI for 931 individuals across 17 sites, which yielded 238 haplotypes. Haplotype diversity  was low, but significant (P = 0.001). Statistically significant pairwise ΦST values were 0.01–0.04, and in general, Dest_Chao values were an order of magnitude higher than ΦST, both for global Dest_Chao (0.025) and for each of the pairwise comparisons, ranging up to 0.300  was between 0.86 and 0.90, while observed heterozygosity (Ho) exhibited a slightly wider range from 0.76 to 0.87  and a global genetic differentiation test . Global partitioning was also detected in analyses of individual loci, with a significant global FST  at each individual locus, except A110.Significant partitioning of the samples among locations was detected in the microsatellite loci with both a global fixation test , Punta Banda (PBDA), Punta Baja and Bahia Tortugas (BTG)]. These sites also stood out by having the highest proportion of significant differences when compared to the other sites. Three of these four sites also had the highest local FST values . There was no pattern of IBD in the data set, whether we examined all sampling locations together, or for any of the specific subregions, as described for the mtDNA results  ranged from 0.034 to 0.057 (k) at each site . Kinship coefficients were significantly greater for within-site than for among-site comparisons . In total, 10 of 17 sites had significantly greater numbers of closely related pairs of individuals than expected by chance (P < 0.05) in at least one of four kinship categories: five sites had an excess of individuals in the ‘nearly identical’ category (0.57 > k > 0.375), five sites had an excess in the ‘full-sib’ group (0.375 > k > 0.1875), five sites had an excess in the ‘half-sib’ category (0.1875 > k > 0.09375), and four sites had an excess in the ‘quarter-sib’ bin (0.09375 > k > 0.047)  and Dest_Chao  and Dest_Chao  with the proportion of kin stayed approximately the same, while the relationship of local FST with the proportion of kin strengthened . The relationships of mean pairwise FST, Dest_Chao and local FST with the proportion of related individuals  from ML-Relate were all significant and stronger than the relationships between these summary statistics and proportion of kin  overlap the regions of strong upwelling intensity  between mean kinship at each site and the closest distance between each site and the edge of an upwelling zone [(mean kinship + 1) = −0.005 ln (distance to upwelling + 100 km) + 1.028, r) versus distance from upwelling as well , demographic or colonization history, migration or some combination of these factors, especially for populations that may not have reached migration–drift equilibrium. Direct interpretation of summary statistics  in the context of gene flow can be problematic . However, this pattern would also result from extreme differential reproductive success among individuals, so that a recruiting cohort is entirely made up of offspring from only a few individuals (sweepstakes recruitment, Hedgecock et al. et al. High levels of within-site kinship could also be driven by a temporally stable pattern of self-recruitment, either through larval retention or through larval dispersal with subsequent recruitment back to the natal site. The prospect that larvae stay in the plankton for 240–330 days and return to settle near their site of release seems unlikely at first. However, the site-specific kinship patterns in our data match theoretical predictions for a species that has evolved a long PLD to avoid predation during the larval phase rather than to facilitate broad dispersal of larvae (Strathmann et al. et al. et al. et al. et al. et al. The four sites with the highest proportion of kin in Baja California, Mexico, are located in areas of persistent upwelling (Zaytsev et al. et al. et al. et al. P. interruptus specifically, Pringle (Panulirus argus, resulted in a 60% or greater decrease in the average distance a larva is predicted to settle from its release site compared with simulations of larvae that remain on the surface (Butler et al. P. interruptus has much higher relative survival rates in the presence of kelp than in surrounding areas where kelp is absent (Mai & Hovel et al. Spiny lobsters are known to have relatively large larvae capable of dynamic movement. Both the phyllosoma and puerulus stages show evidence of active movement in the pelagos, with phyllosoma exhibiting diel vertical migrations as well as horizontal movements (Kittaka  Pringle  found boF-statistics leaves us without a clear indication of what is driving the pattern of genetic differentiation. The addition of kinship analyses reveals how alleles are shared between individuals, rather than just among populations, and provides an independent test of the hypothesis that population genetic structure as measured by F-statistics is a result of population connectivity. In this case, the majority of locations contained an excess of closely related individuals. This supports the inference that either self-recruitment or some form of coordinated larval delivery is driving population-level genetic differences in a species that would be expected to be broadly dispersive throughout its range, given its extremely long PLD. In combination with regional oceanographic data and larval dispersal behaviour, kinship analyses provide evidence for a mechanism of differentiation in an otherwise murky population genetic data set. The ability to directly test hypotheses about what drives population genetic substructure in high gene-flow species by independent means, such as kinship or coalescent analyses, provides greater confidence in the underlying causes of population substructure than summary statistics alone. As the ease of developing greater numbers of genetic markers increases, individual-based analyses such as relative kinship indices can provide a valuable complement for understanding patterns in traditional population genetics data sets.Here, we present a novel approach to understand contemporary drivers of population differentiation in systems with high gene flow. In isolation, the population-level data present a commonly documented scenario among marine species: no evidence for any particular regional separation or isolation-by-distance patterns, but low and significant pairwise differences among populations. While the agreement between nuclear and mitochondrial markers confirms that the results are not due to statistical artifact, the nature of


Rhinichthys osculus) from northern California, USA that has rapidly increased in abundance but remained restricted to a 25-km stretch of river since its introduction in the mid-1980s. Field and laboratory analyses indicate that invasion success of speckled dace is constrained by the combined effects of multiple predators. The role of bottleneck effects associated with the introduction has not been studied. We assayed variation in seven microsatellite loci and one mitochondrial DNA gene in the introduced population and nine putative source populations to identify the source population and evaluate bottleneck effects. The Trinity River system was supported as the source owing to its genetic similarity and geographic proximity to the introduced population. Consistent with a bottleneck, the introduced population exhibited reduced allelic and haplotype richness in comparison to source populations. Estimates of the genetically effective number of individuals founding the introduced population using nuclear coalescent analyses and a mitochondrial simulation procedure were highly concordant in suggesting that the initial colonizing group was comprised of about 10 individuals. A bottleneck effect in an exotic species exhibiting limited invasion success has rarely been documented and thus introduction of speckled dace represents an important model system for future investigation. Establishing a relationship between genetic diversity and factors limiting invasion success in this system  will help determine the extent to which genetic diversity loss has constrained invasion success in speckled dace.Molecular evaluations of successful invaders are common, however studies of introduced species that have had limited invasion success, or have died out completely, are rare. We studied an introduced population of speckled dace ( Exotic species are considered second to habitat loss and fragmentation as a threat to global biodiversity . Exotic Genetic diversity is considered essential for establishment and spread of exotic species . For praEstimating the number of individuals founding introduced populations using genetic data provides a powerful approach for evaluating bottleneck effects and genetic diversity loss associated with exotic species introductions e.g., . Given gRhinichthys osculus), a small  cyprinid fish, which has had limited invasion success since their introduction to the Van Duzen River  in the mid-1980s  and pikeminnow (Ptychocheilus grandis) at their downstream limit in the Van Duzen River. Competition for microhabitat and predation by benthic sculpins, combined with predation by the water-column-occupying pikeminnow, appear to prevent spread of speckled dace in this system  for the introduction. Establishing a source population provides a benchmark to evaluate genetic changes in introduced populations . AccuracSpeckled dace were collected from the introduced population (Van Duzen River) and nine putative source populations . CollectA total of 71 (introduced) and 491 (source) speckled dace were genotyped with seven microsatellite loci see . MicrosaTests for conformance to Hardy–Weinberg proportions for each locus in each population were conducted using the Markov chain Monte Carlo approximation of Fisher's exact test implemented in ARLEQUIN 3.1 . Loci weFST) between all population pairs and permutation tests of their significance were implemented in FSTAT V2.9.3.2 (Q), the proportion of each individual's genome assigned to each group. Estimates of the number of genetic clusters present in the data were generated by calculating the log probability of the data (ln Pr[X|K]) and ΔK  indicated that run lengths were sufficient for convergence. Graphical depictions of STRUCTURE results were generated using DISTRUCT (Estimates of nuclear genetic differentiation (V2.9.3.2 . PHYLIP V2.9.3.2 . Branch V2.9.3.2  using thV2.9.3.2  for the V2.9.3.2 . DAPC peV2.9.3.2  was used) and ΔK  assumingt groups . All STRDISTRUCT .The effective number of individuals founding the introduced speckled dace population was estimated using maximum likelihood methods implemented in COALIT and NFCONE . RequireP), allelic richness (A), observed heterozygosity (HO), and Hardy–Weinberg expected heterozygosity (HE). HP-RARE 1.0 (Ap), and standardized allelic richness (AR), equalized to a sample size of 26 genes using rarefaction. We compared allelic richness, standardized allelic richness, and expected heterozygosity using a two-way analysis of variance (ANOVA) with population and locus considered as random factors. We specifically evaluated the contrast between the introduced population and the nine populations within the species’ native range and a pairwise contrast between the introduced and the most likely source population.ARLEQUIN 3.1  was usedRARE 1.0  was usedb gene. Amplifications were conducted using primers L14724 and H15915 under the following conditions (h), and nucleotide diversity (π) were estimated using ARLEQUIN 3.1.We sequenced a total of 186 individuals from the introduced population and nine putative source populations for a 759-bp fragment of the mitochondrial cytochrome nditions : 35 cyclnditions . HaplotyEstimates of the genetically effective number of founders for the introduced population were generated using computer simulations to model the effects of the founding event on haplotype richness. Random samples of founder individuals were drawn (with replacement) from a list of haplotypes and their counts in the source population. A probability distribution was constructed describing the proportion of draws, out of 100,000 trials, which contain two haplotypes, the number detected in the introduced population, assuming the introduced population was founded by 2, …, 20 individuals. The analysis was first run assuming the source was FG (eight haplotypes), the only location containing both haplotypes found in the introduced population. A second analysis was conducted assuming the source consisted of a pooled sample of the three most likely ancestral populations , to account for haplotype sampling error. Our analysis follows that of All nuclear loci were highly polymorphic, ranging from 6 to 48 alleles, with an average of 22 alleles per locus across all populations. On average, <2% of the microsatellite genotypes were missing from the final dataset, and missing data were not characteristic of loci or populations. Of the 70 tests for conformance to Hardy–Weinberg proportions (10 populations at seven loci), five were significant following Bonferroni correction for multiple tests . MICROCHECKER suggested deviations were primarily due to null alleles; however, no single locus or population consistently departed from expectations, eliminating locus- and population-specific factors as causes for the deviations. A total of two of 210 pairwise tests for linkage disequilibrium were significant following Bonferroni correction for multiple tests .FST) indicated that the introduced speckled dace population was most similar to native populations from the Trinity River (TR and CY), 0.0659 and 0.0541, and South Fork Trinity River (HA and FG), 0.1410 and 0.0815, and divergent from all other native populations, 0.2281–0.4807 (K indicated K= 2 suggesting our data consisted of two clusters (L[K]) arrived at a plateau at K= 6, suggesting another lower level of structure featuring six clusters , depending on parameter inputs. Intrinsic rate of increase had a modest influence on the founder number estimate. When about 10 . The larld equal .The proportion of polymorphic loci ranged from 0.57 to 1 in the native populations and was 0.86 in the introduced population . StandarF= 9.03, df 1,54, P= 0.004 for allelic richness; F= 10.57, df 1,54, P= 0.002 for standardized allelic richness), but expected heterozygosity in the introduced population did not differ detectably from the native populations  (P= 0.025), a marginal difference for standardized allelic richness (P= 0.085), and no difference for heterozygosity (P= 0.748).Both allelic richness and standardized allelic richness were lower in the introduced population compared to the nine native populations (= 0.210) . A pairwA total of 186 mitochondrial DNA sequences were aligned, and 88 variable nucleotide positions defined 61 haplotypes see . The intThe presence of only two haplotypes in the introduced population contrasted with a range of 4–12 haplotypes present in the native populations . The lacOur results show the Trinity River system  was the most likely source for the introduced speckled dace population. This result was indicated by parallel trends in estimates of pairwise genetic differentiation, neighbor-joining trees, multivariate plots, and Bayesian clustering analyses. All methods show high levels of microsatellite and mitochondrial similarity between the Trinity River system samples and the introduced population and marked divergence of this founder–source combination from all other populations we examined from the native range. The geographic proximity of the Trinity River system and the introduced population further supports the results of the genetic analyses .Several lines of evidence support the hypothesis that the introduced population has experienced a severe bottleneck. First, the introduced speckled dace population exhibited divergence from all other populations, including all of the likely source populations, in the analysis of microsatellite variation. Divergence is an indicator of a bottleneck, which causes an increase in frequency of rare alleles among individuals surviving the bottleneck . Diverger≥ 1.0 seems reasonable, which suggests a genetically effective founding population of 10. Furthermore, overall genetic drift in a growing, colonized population is influenced most strongly during the early generations when the population is small and there is little density dependence. During such episodes, it is not unrealistic to expect that the variance in reproductive success amongst a small group of founders could be reduced relative to that in later generations. Accordingly, though we have estimated the effective number of founders, this quantity should be close to the census number of (successful) founders.Over broad ranges of carrying capacity and intrinsic rates of increase, the microsatellite data suggest an effective founder number between 7 and 17. Variation in our estimate could be primarily attributed to the assumed intrinsic rate of increase following introduction . We hypoComputer simulations using mitochondrial DNA haplotypes suggested that the genetically effective number of founders was two. Although smaller than the nuclear-based estimates, mitochondrial DNA is expected to provide an underestimate in comparison to nuclear markers for several reasons. First, due to maternal inheritance, mitochondrial DNA has a smaller effective population size than nuclear DNA. Second, unlike the nuclear DNA simulations, our mitochondrial DNA simulations did not account for loss of haplotypes via drift in the generations immediately following introduction. Lastly, mitochondrial data provide an estimate of the number of female founders, which could be considerably less than the total number of founders. Considering these factors, the nuclear and mitochondrial estimates are highly concordant.Our findings are most consistent with a single introduction for multiple reasons. First, our estimate of an actual founding number of 10 individuals is consistent with a single introduction. Second, significant losses of genetic diversity in the introduced population suggest a single introduction, as multiple introductions would likely serve to restore genetic diversity. Lastly, we found no evidence of admixture from multiple founding sources in the Bayesian cluster analysis. Thus, the introduced population of speckled dace in the Van Duzen River apparently contrasts with some successfully introduced populations that have had genetic diversity restored by multiple founding sources and large founder numbers . The absThe mitochondrial DNA analysis points to the South Fork Trinity River site FG as the most likely source, due to one shared haplotype occurring at high frequencies in FG and the introduced population but not found elsewhere . In contThe key limitation on the invasion success of speckled dace in the Van Duzen River appears to be their inability to evade multiple predators . The sevAnother possibility is that predator avoidance behaviors may be limited in the introduced population because the source population experiences relatively modest predation risk and, therefore, limited predator avoidance behaviors. The source river lacks pikeminnow and contains few sculpins in much of the river network. Geographic variation in antipredator behavior, according to the presence or absence of major predators in their native range, has been documented for other fish (e.g., three-spined stickleback: Finally, the ecological setting in the Van Duzen River may be such that the spread of speckled dace would be resisted regardless of the relative predator avoidance abilities of the introduced population. An experiment by The relative importance of genetic diversity loss associated with bottlenecking and source population effects could be resolved by conducting laboratory predator avoidance trials. The original multiple predator trials of


Acrocephalus arundinaceus) and the clamorous reed warbler (A. stentoreus) overlap in southern Kazakhstan and a previous study has documented hybridization in a sympatric population. In the present study, we first present a large set of novel microsatellite loci isolated and characterised in great reed warblers. Secondly, we evaluate whether hybridization in the sympatric breeding population has been followed by backcrossing and introgression.Hybridization is observed frequently in birds, but often it is not known whether the hybrids are fertile and if backcrossing occurs. The breeding ranges of the great reed warbler (arundinaceus–stentoreus hybrid birds that showed mixed ancestry (admixture proportions 0.495–0.619). Analyses of simulated hybrids and backcrossed individuals showed that the sampled birds do not correspond to first–fourth-generation backcrosses, and that fifth or higher generation backcrosses to a high extent resemble ‘pure’ birds at this set of markers.We isolated 181 unique microsatellite loci in great reed warblers. Of 41 loci evaluated, 40 amplified and 30 were polymorphic. Bayesian clustering analyses based on genotype data from 23 autosomal loci recognised two well-defined genetic clusters corresponding to the two species. Individuals clustered to a very high extent to either of these clusters (admixture proportions ≥0.984) with the exception of four previously suggested Acrocephalus warblers. The time to reach reproductive isolation is believed to be very long in birds, approximately 5 Myrs, and with an estimated divergence time of 2 Myrs between these warblers, some backcrossing and introgression could have been expected. However, there was no evidence for backcrossing and introgression suggesting that hybrids are either infertile or their progeny inviable. Very low levels of introgression cannot be excluded, which still may be an important factor as a source of new genetic variation.We conclude that these novel microsatellite loci provide a useful molecular resource for  Hybridization and introgression can lead to the creation of novel genotypes and phenotypes and are therefore important processes in the evolution of animals and plants 1) hybrids, then there is a possibility that these will backcross with at least one of the parental genotypes, with introgression as a consequence. If the resulting backcrossed individuals subsequently mate with the most similar parental genotype, novel genes and gene complexes can be particularly rapidly introduced into the new genetic background Studies of hybrid zones have indicated that natural hybridization is most likely to take place in intermediate habitats, which are often found at the ecological limits of the species' distributional ranges, and where both taxa are found in close proximity to each other Corvus corone ssp.) DenroicaGeospiza spp.) In birds, several well-characterised hybrid zones are known, e.g. between carrion and hooded crow  and the clamorous reed warbler (A. stentoreus) are closely related passerines in the family Sylvioidea A. a. zarudnyi) are at their south-eastern range limit, whereas clamorous reed warblers (subspecies A. s. brunnescens) are at their northern range limit Ase50), carried genetic material from both parental species The great reed warbler  and clamorous reed warblers (A. s. brunnescens) at Stone Lake  and Kremenevskyi pond , located 39 km apart in southern Central Kazakhstan, where they co-occur with a total population size of approximately 500 and 40 territorial males, respectively We studied great reed warblers  was taken from the brachial vein of 56 birds . Blood samples were stored in SET buffer . Genomic DNA was extracted using a standard phenol/chloroform protocol et al.MboI fragments were enriched separately for the following di- and tetra-nucleotide microsatellite motifs: (GT)n, (CT)n, (GTAA)n, (CTAA)n, (TTTC)n and (GATA)n and their complements, which had been denatured and bound to magnetic beads following Glenn & Schable A microsatellite-enriched library was constructed from a single female ‘pure’ great reed warbler sampled at Lake Kvismaren, Sweden (see e.g. Hasselquist 1998 Taeniopygia guttata) genome assembly  for 90 s, 72°C for 60 sec, followed by 60°C for 30 min and an ambient hold temperature (locus specific aT is given in eneMapper 4.0 (Applied Biosystems).A total of 181 unique great reed warbler microsatellite sequences were isolated , number of alleles and Wright's inbreeding coefficient (FIS).Each locus was tested for deviation from Hardy-Weinberg equilibrium using the software Ftructure , corresponding to the two species. However, we also tested K from 1 to 5, and as expected these analyses confirmed that K = 2 is the most likely K in our data  amplified and 30 (73%) were polymorphic in four unrelated great reed warbler individuals from Sweden .IS was low to moderate for all loci , and the Micro-Checker analyses suggested the presence of null alleles at this locus at a frequency 12% , for K = 1–5 was as follows: −3494.4, −3064.5, −3509.7, −3175.8, and −3256.8. Thus, K = 2 was the most likely number of genetic clusters in the group of individuals genotyped.The SThe individual admixture proportions indicate that there are no backcrossed individuals present in the sample and there is no evidence for introgression of genetic material between the species . The 28 The analyses of the artificial hybrid and backcross genotypes  suggest In the present study, we have presented a large set of novel microsatellite loci isolated from and characterised in the great reed warbler. In total, 181 loci were isolated and, among 41 tested for amplification, 73% were polymorphic in a small set of great reed warbler individuals. Moreover, we used a set of 23 autosomal microsatellites to evaluate whether ongoing hybridization in a sympatric breeding population in Kazakhstan has been followed by backcrossing and led to significant introgression of genetic material between the species. The clamorous reed warbler has expanded its range northwards in Kazakhstan during the last three decades and sympatric breeding populations have been recorded since 1981 tructure to recognise two genetic clusters corresponding to the two species, and all individuals either clustered to a very high extent to either of the species cluster (i.e. ‘pure’ species) or showed a mixed ancestry (i.e. hybrids). There was neither any evidence for backcrossed individuals nor introgressed genetic material in the population, suggesting that the hybrids are either infertile or their progeny inviable. We cannot of course exclude very low levels of backcrossing and old introgression events in the study populations  and clamorous reed warblers (AJ004788) is approximately 4% which may correspond to a divergence time of approximately 2 Myrs Hippolais icterina and H. polyglotta), and between collared and pied flycatchers  introgression occurs at low frequencies The We conclude that our novel microsatellite markers provide a useful molecular genetic resource for this group of birds, and that there is no evidence for backcrossing and introgression in the study population, which in turn suggests that hybrids between these two warbler species are either infertile or their progeny inviable. We cannot however exclude very low levels of introgression, which could potentially be an important factor as a source of new genetic variation in the species.Table S1Summary of BLAST analyses of 181 great reed warbler  microsatellite sequences and four other avian microsatellite loci used in the present study against the zebra finch genome assembly . BLAST hit statistics and chromosomal locations in the zebra finch genome are included.(PDF)Click here for additional data file.Table S2Characteristics of 181 great reed warbler  microsatellite loci isolated and characterised in this study. Primer sequences, expected and observed product size, melting (Tm) and annealing (Ta) temperatures, 5′-fluoro-label, and amplification success in four test individuals are shown.(PDF)Click here for additional data file.Table S3IS, and two-tailed Number of alleles, range of fragment length (bp), expected and observed heterozygosity, Wright's FP-value for deviations of Hardy-Weinberg equilibrium  in great and clamorous reed warblers. Also given are null allele frequencies in the great reed warbler.(PDF)Click here for additional data file.Figure S1Chromosomal locations of great reed warbler  microsatellite loci and four other avian microsatellite loci included in the present study  on the zTaeniopygia guttata) genome.(PDF)Click here for additional data file.


Paramuricea clavata. Thirty four percent of the larvae were sired by male colonies surrounding the brooding female colonies, revealing that the bulk of the mating was accomplished by males from outside the studied area. Male success increased with male height and decreased with increasing male to female distance. The parentage analyses, with a strong level of self-recruitment (25%), unveiled the occurrence of a complex family structure at a small spatial scale, consistent with the limited larval dispersal of this species. However, no evidence of small scale SGS was revealed despite this family structure. Furthermore, temporal genetic structure was not observed, which appears to be related to the rather large effective population size. The low level of inbreeding found suggests a pattern of random mating in this species, which disagrees with expectations that limited larval dispersal should lead to biparental inbreeding. Surface brooding and investment in sexual reproduction in P. clavata contribute to multiple paternity (on average 6.4 fathers were assigned per brood), which enhance genetic diversity of the brood. Several factors may have contributed to the lack of biparental inbreeding in our study such as (i) the lack of sperm limitation at a small scale, (ii) multiple paternity, and (iii) the large effective population size. Thus, our results indicate that limited larval dispersal and complex family structure do not necessarily lead to biparental inbreeding and SGS. In the framework of conservation purposes, our results suggested that colony size, proximity among colonies and the population size should be taken into consideration for restoration projects.Dispersal and mating features strongly influence the evolutionary dynamics and the spatial genetic structure (SGS) of marine populations. For the first time in a marine invertebrate, we examined individual reproductive success, by conducting larval paternity assignments after a natural spawning event, combined with a small-scale SGS analysis within a population of the gorgonian  Dispersal plays a key role in the spatial partitioning of genetic diversity within and among marine populations and strongly influences the evolutionary dynamics of the populations through natural genetic processes . Mating features, such as individual reproductive success, are also key determinants which contribute to the spatial genetic structure (SGS) of the species.Reproductive success relies on the rates of fertilization, larval survival, and recruitment, which vary depending on the reproductive strategies, environmental factors, and stochastic events. For marine invertebrate species, releasing one or both set of gametes, fertilization success depends on many factors such as population density, proximity between gamete sources, gamete dispersal, egg size, sperm concentration and longevity, and current regime  is a key species of the highly diverse Mediterranean coralligenous assemblages  or stochastic . The genetic analysis of larvae would also allow estimating the genetic heterogeneity of larval pool, just after fertilization, and its potential impact on genetic structure. Finally, the analysis of SGS among colonies at a local scale may reveal the dispersal abilities of this species and indicate whether sexual reproduction within populations is random or if some inbreeding occurs. The sessile and gonochoric characters of P. clavata are well suited for such study as the sexes can be determined on spatially located individuals. Through surface brooding, maternity can be established and eggs can be easily collected in the field.For the first time, we evaluate individual reproductive success of a marine invertebrate during a natural spawning event along with the SGS of a population. This study was based on paternity assignment of larvae collected on female colonies of P. clavata dwells on a rocky cliff. Within an area of 2 m² (1 × 2 m), the coordinates of all the 87 colonies were determined to generate a map of the study area  A where audy area B and theudy area ]. The frudy area . The sexudy area . Additioudy area , where tudy area , by usinudy area . We randudy area , except Total genomic DNA was extracted from 104 colonies and 385 larvae  followinPID) , which may cause multiple sampling of the same colony, we computed the unbiased probability of identity (P = 0.552). For all the following analyses involving the broods only the mature colonies (from C1 to C3) were considered as the putative parental population, whereas all colonies (from C0 to C3) were considered for the analyses among colonies. The details for each colony  are given in We defined stage classes among the colonies based on the maximum colony height and their reproductive status. We classified the sampled colonies into 20 cm size intervals , spanning the entire measured size range (from 3 to 92 cm), in order to test for kinship relationships among and within colony stage classes and for temporal structure analysis among colony stage classes. As we were able to determine the sex from colonies of 15 cm, the 29 colonies measuring between 3 and 14 cm were assigned to the first stage class C0 and were assumed as sexually immature. The second stage class C1 was composed of 37 colonies measuring between 15 and 30 cm in height; the third stage class C2 grouped 26 colonies measuring between 31 and 50 cm. Finally, the last stage class C3 gathered all the colonies measuring more than 51 cm, . Among the 71 sexually mature colonies , 33 colonies were identified as female and 38 as male. The sample displayed a sex ratio not significantly different from 1:1 ] and private allelic richness [Ap(g)] were computed with a rarefaction procedure using HP-RARE  implemented in GENETIX. GENEPOP v.4.0  to establish the relationship between male reproductive success, computed as the number of larvae a male sired, and the main factors that can affect it such as colony height (hereafter height) and the male to female distance (hereafter distance). This relationship was investigated on the basis of each female colony in relation to all male colonies.6 iterations  were computed for each K value (K varying from 1 to 15) under the admixture model, the assumption of correlated allele frequencies among clusters and using the recessive allele option to cope with null alleles ] as a function of K across the 20 runs to select the most likely K value  and spatial population structure among colonies using the Bayesian approach implemented in STRUCTURE v.2.3  method in FREENA (Chapuis and Estoup We computed Weir and Cockerham's  θ estimaI) (Moran ij) described in Loiselle et al.  Moran  and the e et al. . DistancSignificance levels were adjusted using a false discovery rate (FDR) correction for multiple tests . For all the colonies, based on six loci, observed and expected heterozygosities were both equal to 0.74. Linkage disequilibrium was found for one pair of loci in sample A10 brood (Parcla 09–Parcla 17) and three pairs of loci in sample B4 brood  after FDR correction; otherwise no global linkage disequilibrium was observed. The multilocus FIS value was equal to 0 (not significant at a 5% FDR) for all the colonies, 0.04 (significant at a 5% FDR) for the larval pool, and 0.04 (significant at a 5% FDR) for the larval pool combined to parental population  whereas variance of RXY was significantly higher than expected (P = 0.042).All loci were polymorphic over the 100 colonies analyzed, with the number of alleles ranging between 7, for Par_d, and 22, for Parcla 10, Parcla 12, and Parcla 14, with a mean value of 16 alleles per locus. Among the broods, the observed and unbiased expected heterozygosities, based on five loci, ranged from 0.50 for J4-1 brood to 0.77 for A3-1 and A3-2 broods and from 0.48 for J4-1 brood to 0.74 for C5 brood, respectively . ObservePaternity was assigned for 132 larvae out of 385 (34.3%). On an average 35.2 ± 16.9% of larvae per brood were assigned a father among the 38 sampled male colonies . MultiplMale reproductive success, ranging from 0 to 19 , was affOver the 100 colonies, 98% of colonies  were involved in at least one parentage relationship  and 18% and 98% of colonies were involved in a full sibship and a half sibship, respectively. The details of parentage assignments per stage class are given in The Bayesian clustering method did not reveal neither spatial nor temporal genetic discontinuity among all the colonies as only one cluster was detected with STRUCTURE .FST value was 0.14 when comparing the 12 broods, while it was 0.12 when comparing the 12 broods and putative parental population. The corresponding differentiation test was highly significant. Pairwise FST values between pairs of broods ranged from 0.006 for A3-1 versus A3-2 and J4-1 versus J4-2 to 0.212 for C5 versus K9 whereas pairwise values between pairs of broods and the putative parental population ranged from 0.037 with A3-2 to 0.107 with C5 . All pair-wise FST between broods were significant (after FDR correction) except A3-1 versus A3-2 and J4-1 versus J4-2 which corresponded to two different broods collected from the same female colony . Pairwise FST values between the four stage classes varied between −0.011 (C1 vs. C3) and 0.001 (C0 vs. C1 and C0 vs. C3) and were not significantly different from pairwise FST corrected for null alleles . All pairwise values between stage classes were not significant after FDR correction, suggesting a lack of temporal structure among colonies within the studied site.Over all loci  with C5 . No signI and φij values were not significant and the correlograms were confined within the 95% confidence intervals of no association between geographical distances and pairwise genetic comparisons between individuals .The spatial autocorrelation analyses failed to detect any spatial structure over all the distance categories as MP. clavata planulae were sired by males surrounding the brooding female colonies within 5 m2. This result suggests that a part of mating takes place among colonies within close proximity but can extend beyond this area as all the putative fathers were not identified. Indeed, considering our exhaustive sampling scheme of the studied area and the high detection power allowed by microsatellites, the nonidentified fathers should be present outside of this area. The presence of alleles in the larval pool that did not match any of the examined male genotypes (data not shown) is a parallel source of evidence supporting this conclusion. At a larger spatial scale (400 m²), similar results were encountered on the surface brooding octocoral Pseudopterogorgia elisabethae in the Caribbean , (b) the clear pattern of increase on gonadal production with colony height exhibited by P. clavata, a pattern that is much less evident in P. elisabethae, and (c) the higher production of gonads in P. clavata in contrast to that in P. elisabethae  , (b) the high fertilization rates observed in P. clavata  the low number of larvae analyzed per brood compared to the mean number of eggs produced by a mean size female colony . Moreover, 25 parent–offspring dyads were detected which implies a strong level of self-recruitment (25%) at small scale. The pattern of multiple mating led to the local recruitment of about two offspring per colony. Therefore, the present study revealed the occurrence of a complex level of family structure. This result is in agreement with: (a) the negative phototaxis behavior of the larva and its short swimming period  larvae may randomly settle within the site where free available space is found within the area and thus “larva” shadows  and suggests that sweepstakes effects did not occur in the studied population  the fact that sperm limitation appears to be of limited relevance at the studied scale, (ii) the multiple paternity, and (iii) the large effective population size. The results on inbreeding disagree with early expectations that limited larval dispersal should lead to biparental inbreeding in brooding species Carlon . HoweverP. clavata to further investigate this hypothesis.Given the moderate level of inbreeding and the lack of spatial and temporal genetic structures at the studied site, we hypothesize that the heterozygote deficiencies found within other locations at larger scales (Mokhtar-Jamaï et al. P. clavata populations is mainly driven by local processes. These results confirm the previous recommendations for conservation plans at local scales (Mokhtar-Jamaï et al. The results of the present study provide useful and complementary information for the conservation of the red gorgonian. The achievement of mating among colonies close to each other, implying that mating partly takes place locally, and the occurrence of self-recruitment suggest that the functioning of Paramuricea clavata populations are affected by the combined effects of mass mortality events (Coma et al. P. clavata populations and if it may have an adaptive value, it should be taken into consideration for restoration project. However, the hypothetical “role” of half-sib family structure within populations should be first investigated.


Calonectris diomedea. Microsatellite fragments were analysed from samples collected across almost the entire breeding range of the species. To help disentangle the taxonomic status of the two subspecies described, the Atlantic form C. d. borealis and the Mediterranean form C. d. diomedea, we analysed genetic divergence between subspecies and quantified both historical and recent migration rates between the Mediterranean and Atlantic basins. We also searched for evidence of isolation by distance (IBD) and addressed spatial patterns of gene flow. We found a low genetic structure in the Mediterranean basin. Conversely, strong genetic differentiation appeared in the Atlantic basin. Even if the species was mostly philopatric (97%), results suggest recent dispersal between basins, especially from the Atlantic to the Mediterranean (aprox. 10% of migrants/generation across the last two generations). Long-term gene flow analyses also suggested an historical exchange between basins (about 70 breeders/generation). Spatial analysis of genetic variation indicates that distance is not the main factor in shaping genetic structure in this species. Given our results we recommend gathering more data before concluded whether these taxa should be treated as two species or subspecies.Dispersal is critically linked to the demographic and evolutionary trajectories of populations, but in most seabird species it may be difficult to estimate. Using molecular tools, we explored population structure and the spatial dispersal pattern of a highly pelagic but philopatric seabird, the Cory's shearwater  Dispersal is a central concept in population and evolutionary biology Larus michahellisHydrobates pelagicus, and The Mediterranean Sea became separated from the Atlantic Ocean during the Messinian salinity crisis, approximately 5.5My ago and the present day species inhabiting the Mediterranean are mostly the result of subsequent colonization, mainly from the Atlantic Ocean Calonectris diomedea throughout its breeding range. In particular we investigate differences between the two described taxa, the larger one breeding in the Atlantic (mean mass 790 g.) and the smaller one, breeding on the Mediterranean islands (mean mass 650) In this study we investigate patterns of genetic variation at nuclear loci in a large-range pelagic seabird, the Cory's shearwater The aim of this study is to explore population structure and the spatial dispersal pattern in the Cory's shearwater and to infer short and long term dispersal between the two ocean basins. For that purpose, we genetically analyse the largest and most comprehensive data set so far used in this species, i.e. 387 individuals sampled from 27 breeding colonies from most Mediterranean and some Macaronesian colonies.All animals were handled in strict accordance with good animal practice as defined by the current European legislation, and all animal work was approved by the respective national and regional committees for scientific capture , Ministerio de Medio Ambiente y Medio Rural (Spain), Govern Balear (Spain), Centre de Recherches par le Baguage des Populations d'Oiseaux, (France), Department of Environment (Greece) and Istituto Nazionale per la Fauna Selvatica ).C. edwarsii (not considered here) appears to be distinct and is widely treated as a separate species Calonectris diomedea diomedea shows some morphological, ecological, vocal and genetic differences from the north Atlantic subspecies C. d. borealisCory's shearwater is a pelagic seabird that breeds mainly on islands, throughout the Mediterranean Sea as well as the Atlantic Islands of Berlenga, Selvagens, Canaries, and Azores. New colonies have also been discovered recently along the coasts of Galicia We visited 27 Cory's Shearwater breeding colonies throughout almost all of its breeding range in the Mediterranean and Atlantic regions  and tookPuffinus mauretanicus2 (depending on the primer pair) and 1–2 µl of template DNA. The thermocycling conditions were as follows: 94°C for 2 min, followed by 34 cycles of 95°C for 30 seconds, 50°C–60°C for 30 seconds and 72°C for 30 seconds, with a final extension of 72°C for 5 min. Specific annealing temperatures and magnesium concentrations for each locus are shown in Table S1. We checked the amplification and purification results by loading 1–2 µl of product in a 1.5% agarose gel. Reactions were loaded together and the length of the DNA fragments were analysed directly from PCR product using an ABI 3100 automated sequencer  and the ABI software GeneMapper v. 3.7 and visually rechecked. Alleles were scored as PCR product size.Total DNA was isolated from blood samples by overnight incubation at 55°C in SET buffer with 30 µl SDS 10% and 2.5 units/ml of proteinase K followed by a standard phenol/chloroform protocol IS) and tested for deviations from the Hardy-Weinberg equilibrium. Using permutations (>1000) we tested for the occurrence of non-random associations of pairs of loci (i.e. linkage disequilibrium). A deficit in heterozygotes can be mimicked by null alleles; this was checked by assuming that some of the homozygotes were heterozygotes for the null allele and that individuals failing to amplify were homozygous for the null allele. Additionally with the software FreeNA ST using the ENA correction method and using the original data. The ENA correction method was found to efficiently correct for the positive bias induced by the presence of null alleles on FST estimation and provide accurate estimation of FST in presence of null alleles For 24 breeding colonies (sample sizes ≥9), we measured the mean number of alleles per locus, and the intrapopulation genetic diversity for each population was evaluated in terms of allelic richness as well as observed and unbiased expected heterozygosity ST pairwise distance matrix between sampling localities To asses genetic differentiation we used F-statistics K) and estimates the probability of the data (X) being associated to this specific number of populations ). Estimation of K was based on Evanno's method 6 Markov Chain Monte Carlo (MCMC) repetitions and a burn-in period of 100,000 iterations. For visualising and compare different Structure results for different K values we used the software the Software Distruct 1.1 Population structure was further analysed using the Bayesian assignment method implemented in Structure v.2.3 To reveal if there was a genetic structure within the data set we also conducted an analysis of molecular variance AMOVA,  with theeμ, where Ne denotes the effective population size and μ the mutation rate per locus per generation), and the mutation- scaled immigration rate (M) . We ran Migrate using a Brownian motion mutation model with constant mutation rates and starting parameters based on FST calculations An uniform prior distribution  was used to estimate theta, and a uniform prior distribution  was used for M. The priors were chosen based on the performance of multiple trial runs with different prior values. Runs visited a total of 2,000,000 parameter values including a 500,000 burn-in period, and sampled the parameter value every 20 iterations. To assist with convergence, we used the ‘static heating scheme’ option with four concurrent chains. We evaluated convergence by looking at the effective sample size, the autocorrelation, and the posterior distribution histograms over all loci. We report the median values of theta and M, and the 0.025 and 0.975 posterior distribution values as 95% confidence interval estimates To avoid overparametrization (i.e. to include more parameters in the model than can be estimated from the data), we pooled all Mediterranean samples and all the Atlantic samples to obtain an estimate of the magnitude and direction of dispersal rates between the Atlantic and the Mediterranean. We used two methods: first, we estimated the migration rate (m) using the Bayesian assignment algorithm implemented in BayesAss To avoid estimation problems caused by very different sample sizes between groups, for both analyses BayesAss and Migrate, we randomly reduced the larger Mediterranean data set to 100 individuals.Patterns of differentiation among populations were visualized by factorial correspondence analysis of multilocus scores (MCA) computed using Genetix v. 4.05  ST (θ) matrix of genetic distances among colonies to calculate a new matrix θ/(1−θ) of genetic distances. A matrix of geographical distances was also calculated as the natural logarithm of the shortest distance from colonies over the sea (shearwaters do not fly over land). To search for evidence of isolation by distance, we first compared the genetic and geographical distance matrices using Mantel tests with software R and the ape package We used the previously estimated Slatkin's FSpatial analyses were carried out at two different scales: a) in the global range, including both Atlantic and Mediterranean colonies and b) at the Mediterranean scale, only including those individuals from Mediterranean colonies.IS values significantly greater than zero in most populations  as when using the original data (0.086). FST pairwise genetic differentiation between Mediterranean colonies mostly showed small genetic differences between colonies  but unexpectedly very different from all other Mediterranean colonies (mean FST: 0.16). Another striking feature came from FST pairwise genetic differentiation between the four Atlantic colonies, which could be divided into two clearly different groups: there were no differences between individuals from Canaries and Berlenga (FST = 0.02) but these birds were statistically significant from those from Azores and Selvagem  these differences were lower than expected. Differences between mtDNA and microsatellites might be a simple consequence of different coalescent time for markers  ST = 0.18 and 0.17, respectively), being statistically significantly distinct from almost all Mediterranean colonies  were identified when applying the Bayesian clustering approach implemented in Structure , 3, alth were ideST = 0.15, P<0.0001). The hierarchical AMOVA showed the highest significant FCT value when the sample sites were divided into the four groups corresponding to the two Atlantic groups, Spargiotto and Barretini, and all the other Mediterranean colonies ; we detected some recent gene flow between basins, about 3% from then Mediterranean to the Atlantic  and about 10% from the Atlantic to the Mediterranean , suggesting that low but effective dispersal has occurred recently between subspecies. When analysing the long term gene flow using Migrate, theta values in the Mediterranean and Atlantic basins were 10.173 (0.667–18.346) and 4.503 (0.000–9.6731), respectively. We detected some historical dispersal between basins, being M =  9 (95% CI 0.000–21.333) and M = 12.333 (95% CI 0.000–26.667) the estimated scaled-migration rates from the Atlantic to the Mediterranean and from the Mediterranean to the Atlantic, respectively. However the results should be treated with caution because 95% confidence interval estimates include zero in both cases. We estimated that each generation ∼90 individuals migrated from the Atlantic to the Mediterranean and 55 individuals migrated from the Mediterranean to the Atlantic, with a mean of 70 individuals exchanged between basins per generation.When all Atlantic and Mediterranean colonies were analysed, the slope of the regression between distance matrices was estimated to 0.027, with a slightly significant association between matrices (P = 0.025) and suggesting a low isolation by distance pattern. We can see in borealisWe found that much greater genetic population structuring was present in the Atlantic than in the Mediterranean. The similarities between individuals from Berlenga and Canaries on one hand, and between Selvagem and Azores, on the other hand, were not evident with mtDNA analysis and are surprising if we bear in mind the geographical locations of these archipelagos. Nevertheless, previous comparisons of morphometric data from adult birds from Berlenga and Selvagem Grande did detect significant differences in all characters measured (including eggs) except for wing-length, even though both populations belong to the subspecies Patterns of genetic variation in Cory's shearwater revealed differences between the Atlantic and Mediterranean colonies which were lower than expected, especially compared to previous studies of Mt DNA. Capture-recapture data in this species suggests mainly local recruitment and, less frequently, low-to-medium-range dispersal Small genetic differences between subspecies should not simply be interpreted as evidence of high connectivity From a morphometric point of view both Atlantic and Mediterranean taxa are clearly differentiated Phylogenetic analyses of mitochondrial DNA suggested that both subspecies formed reciprocally monophyletic groups and they estimated the gene flow between basins to be less than 1 female per generation Only a slight IBD pattern appeared when Atlantic and Mediterranean colonies of Cory's shearwaters were analysed, suggesting that the geographical distance between breeding colonies may not be the primary determinant of population divergence. The IBD pattern completely disappeared when analyzing only Mediterranean colonies, and this may not be due to a difference on sample sizes between analyses Previously the Almería-Oran Oceanic Front (AOOF), rather than the Straits of Gibraltar, was proposed as the phylogenetic break between Cory's shearwater taxa Our results are consistent with the limited capture-mark-recapture data and suggest that in Cory's shearwater: i) distance is not the main factor in shaping population structure, ii) local recruitment is the most frequent dispersal event, and iii) there are rare but recurrent long-distance dispersal events. We conclude that dispersal between subspecies may not be negligible, thus, in contrast to recent work, we recommend gathering more data to determine whether these taxa should be considered two different species or subspecies.Table S1Amplification conditions for microsatellite primers. Conditions were designed for microsatellite amplification in Balearic shearwater and used in Cory's shearwater; for primer sequences see González et al. (2009).(DOC)Click here for additional data file.


Chelonibia testudinaria is specific on marine turtles whereas C. patula is a host generalist, but rarely found on turtles. It has been questioned why C. patula, being abundant on a variety of live substrata, is almost absent from turtles. We evaluated the genetic ) and morphological differentiation of C. testudinaia and C. patula from different hosts, to determine the mode of adaptation exhibited by Chelonibia species on different hosts. The two taxa demonstrate clear differences in shell morphology and length of 4–6th cirri, but very similar in arthropodal characters. Moreover, we detected no genetic differentiation in mitochondrial DNA and AFLP analyses. Outlier detection infers insignificant selection across loci investigated. Based on combined morphological and molecular evidence, we proposed that C. testudinaria and C. patula are conspecific, and the two morphs with contrasting shell morphologies and cirral length found on different host are predominantly shaped by developmental plasticity in response to environmental setting on different hosts. Chelonibia testudinaria is, thus, a successful general epibiotic fouler and the phenotypic responses postulated can increase the fitness of the animals when they attach on hosts with contrasting life-styles.Turtle barnacles are common epibionts on marine organisms.  Chelonibia testudinaria  has long been known to inhibit predominantly on the carapace, flippers and skin of marine turtles Caretta caretta  Chelonibia patula  that exhibits distinct shell morphology  and ultimately speciation Chelonibia testudinaria and C. patula exhibit host specific adaptation through speciation, differential divergence in genome or phenotypic plasticity, investigation on the genetic divergence pattern between the two species is the prerequisite. Here, we attempted to determine the mode of adaptation in the two taxa using an integrated genetic  and morphological  analyses on Chenolibia testudinaria and C. patula populations from different hosts and geographic locations.To understand whether Mitochondrial markers, on one hand, have been proven to be useful tools to delineate barnacle species due to their fast evolving nature and are commonly applied in barnacle phylogenetic and population genetic studies C. patula is conical and with smooth surface . From SIMPER analysis, the most significant characters (>10% contributed differences) were the length of the cirri IV, V and VI and the opercular length. Chelonibia patula had a relatively larger orifice length and cirri IV, V and VI were almost twice as long as C. testudinaria  for all three mitochondrial markers, except the Nei’s genetic diversity based on 16S rRNA . The results of AMOVA demonstrated that the “between species” variances were, whether significant or not, small for all three markers. Most of the variance occurred at the “within population” level  and 308 (90.6%) respectively. The Nei’s genetic distance (expected heterozygosity) estimate based on AFLP ranged from 0.33 to 0.37 (P>0.05) and pairwise distance calculated based on allelic frequency  >0.05; nMDS ordination ST value significantly higher than that across all loci  = 0.31), when the FDR was set at 0.05 (While 120 AFLP bands on average (<200 bands) were scored for each primer combination in the present study, the biases on the outlier identification introduced by size homoplasy of AFLP was assumed to be small see . A total to 0.37 . There w at 0.05 . This loChelonibia patula and C. testudinaria exhibit distinct morphological differences in external shell characters and length of cirri. However, the arthropodal characters, which are important characters in species identification, are indistinguishable between the two species. Furthermore, the two species do not exhibit significant differentiation in mitochondrial gene sequences nor AFLP genotypes. We found little evidence to support the species separation of the two taxa and we therefore, propose that Chelonibia patula  is a junior synonym to C. testudinaria . C. testudinaria/C. patula should be regarded as a host generalist live broadly on sea turtles, decapods, gastropods and even sea snakes C. testudinaria constitutes of three major lineages in the world’s oceans, the eastern Pacific, western Pacific and Atlantic (including the Mediterranean) based on the divergence pattern in mitochondrial COI C. testudinaria and C. patula collected in SE Asia and Taiwan belong to the western Pacific group , induced by physical environments and predation pressure, revealed ∼5% of AFLP loci exhibiting significantly higher level of differentiation than expected under a neutral model Fundulus heteroclitus  detected a smaller number of SNP loci, 1.4–2.5%, that are possibly influenced by selection in the population inhabiting polluted areas In the present study, only one out of the >300 AFLP loci  is significantly deviated from neutral expectation. This value appears to be significantly smaller than those reported in other studies using similar approaches Chelonibia testudinaria on turtles examined in the present study have a much depressed shell, and the radii between the shell plates have oval-shaped depressions that can house dwarf males C. testudinaria living on marine turtle are patchily and sparely distributed. The presence of dwarf males in those oval-shaped depressions of external shells can increase the mating success. On the contrary, Chelonibia living on benthic crustaceans have a higher shell with larger orifice and the radii between the shell plates are smooth. In general, Chelonibia barnacles on turtles are relatively larger than those on benthic crustaceans. Such difference in size is likely due to moulting of the host crustaceans. After moulting the epibiotic barnacles would be removed together with the old exoskeleton. Adult Portunus pelagicus , for example, moults every 2–3 weeks Chelonibia populations on turtles and crustaceans may be related to age rather than phenotypic responses. In contrast, morphological characters such as the pits on the shell plates, are found on specimens from the turtles and crustaceans of similar size  can dive to depth of 10 m or more and can stay submerged for about 20 minutes before swimming back to the surface for a short breath −1 for Chelonia mydasChelonibia living on marine turtles can reduce drag under strong currents. The pits in the shell radii may facilitate the settlement of cyprids and development of dwarf males in a rapid flow environment , all the morphological parameters measured (except shell length) was divided by the shell length before subsequent analysis to reduce the error due to size differences between samples. Due to great size differences in body sizes between the barnacles on crustaceans and on turtles, absolute value on morphological parameters was not used for statistical analysis as the resulted variations between the two species may merely due to size but not on the morphological differences of the species. Variations in the morphological parameters were analyzed using multivariate analysis  was conducted to generate two dimensional plots of the scutum and tergum geometry between species from different geographical locations. Analysis of similarity (ANOSIM) was conducted to test the differences in opercular dimension between species and SIMPER (Similarity Percentage) analysis was used to detect the significant discriminating opercular diameters.A total of 79 individuals of ollected . Barnacld tergum  and the d tergum  were meaThe somatic body was dissected and the arthropodal characters  were examined under scanning electron microscopy , while a Bayesian inference (BI) tree was inferred by the program MrBayes v.3.12 jModelTest version 0.1.1 a priori parameters in BI analysis, in which two Markov-chain-Monte-Carlo (MCMC) searches with random starting points were conducted in 10,000,000 generations; trees were sampled every 10,000 generations. The burn-in value was set to retain the last three-quarter of the sampling trees, from which the posterior possibility was calculated to illustrate the statistical support for nodes. Five haphazardly chosen sequences from each of the five sampling populations of C. testudinaria in C. caretta (FJ385728-30) used as the outgroup. The sequences were trimmed to fit the shortest sequence length  analyzed in the alignment.To compare the COI sequences obtained from this study with those of rlequin version 3.1 http://www.graphpad.com/quickcalcs/ttest1.cfm?Format=SD) was used to test whether the diversities between the two species were significantly different. Locus-by-locus analysis of molecular variance (AMOVA), which was suggested to better accommodate any effect of missing data, was performed to demonstrate the partitioning of genetic variance among populations and between the two species, and to test if the partition is statistically significant through 10,000 permutations using Arlequin.To infer the intraspecific population structure, haplotype networks of the three mitochondrial markers were generated by TCS version1.13 C. patula and C. testudinaria and determine whether there are loci under selection.Other than mitochondrial genealogical markers, the genome-wide genotyping method amplified fragment length polymorphism (AFLP) was used to elucidate the relationship between The quality of the genomic DNA of each specimen was first checked using agarose gel electrophoresis to ensure there was a clear, intact band (nuclear genome) near the wells. The AFLP profile of specimens with good DNA quality (76 samples) was generated according to enographer version 2.1.4 (http://sourceforge.net/projects/genographer). Viewing the gel under the intensity of 5, we scored the bands falling within 100 to 500 bp. The scoring threshold was set to 0.5 and the band width one (bp). A matrix of presence/absence binary data was then obtained. As recommended by Banding profiles of the specimens were visualized and scored using Gsurv version 1.0 C. patula and C. testudinaria, and to test whether there is significant population differentiation between the species through 10,000 permutations, assuming Hardy-Weinberg genotypic proportion. The pairwise distances (i.e. 1- relatedness coefficients) among each genotyped specimen were graphically visualized through nMDS generated by PRIMER.AFLPST values significantly higher than overall mean values across the loci, were identified by BayeScan version 2.01 ST values that reflect the locus-wise difference of allele frequency, was used to infer the selection packageThe AFLP candidate loci under selection pressure, which are the outlier loci demonstrating FAppendix S1Chelonibia patula and C. testudinaria specimens and their respective GenBank accession nos. Refer to Detail information of (DOC)Click here for additional data file.