We investigated variation in the degree of melanism in the common frog  by comparing the degree of melanism (i) among twelve populations spanning over 1500 km long latitudinal gradient across the Scandinavian Peninsula and (ii) between two populations from latitudinal extremes subjected to larval temperature treatments in a common garden experiment.The thermal benefits of melanism in ectothermic animals are widely recognized, but relatively little is known about population differentiation in the degree of melanism along thermal gradients, and the relative contributions of genetic STP) and neutral marker loci (STF) revealed that the ST P >STF, indicating that the differences cannot be explained by random genetic drift alone. However, the latitudinal trend observed in the wild was not present in the common garden data, suggesting that the cline in nature is not attributable to direct genetic differences.We found that the degree of melanism increased steeply in the wild as a function of latitude. Comparison of the degree of population differentiation in melanism (As straightforward local adaptation can be ruled out, the observed trend is likely to result from environment-driven phenotypic plasticity or ontogenetic plasticity coupled with population differences in age structure. In general, our results provide an example how phenotypic plasticity or even plain ontogeny can drive latitudinal clines and result in patterns perfectly matching the genetic differences expected under adaptive hypotheses. Melanins and carotenoids are two classes of pigment compounds that are responsible for much of the variation in animal coloration . Melaninagouti) [Intraspecific variation in melanism, when present, can express itself as distinct color morphs including completely dark, or melanistic, individuals ,18,19, oagouti) ,29. Thesagouti) ,30-33).Since melanism is genetically based in numerous taxa ,23,24,26Rana temporaria). Amphibians are a particularly well-suited group of organisms to study the genetic basis of pigmentation due to the large color variation they exhibit within and between species [Here, we studied the existence and possible genetic basis of a latitudinal cline in the degree of melanization in the common frog  it species , (iii) e species ,27, and  species . Further species -41.Rana temporaria favouring elevated heating rates towards higher latitudes. Based on this hypothesis we predicted that the level of melanism would increase towards north and that the pattern would at least partially be genetically based and independent of rearing environment.The aims of our study were twofold. First, we investigated the presence of a latitudinal trend in the degree of melanization in wild-caught adult common frogs. Second, we used a common garden experiment to test whether the observed cline in melanism was genetically based, or reflected either plastic response to environmental heterogeneity or ontogenetic plasticity with geographical differences in age structure. To these ends, we compared the level of dorsal melanism in twelve populations along over 1500 km latitudinal gradient across the Scandinavian Peninsula, and conducted a common garden study applying larval thermal treatments using two latitudinally extreme populations. We hypothesized that a latitudinally ordered thermal adaptation might have occurred in The common frog is the most widely distributed anuran amphibian in Europe: it can be encountered from Spain to the Ural Mountains in Russia, from sea level to altitudes above 2000 m . In the Data on wild adults was collected during the breeding seasons of 1998-1999 as part of other studies ,40,41,43A common garden experiment was conducted for two populations along the latitudinal gradient across the Scandinavian Peninsula Figure , Table 1ad libitum. To assure homogenous water quality, the animals were reared throughout the aquatic stage in reconstituted soft water (RSW) [Drosophila flies and small crickets. In late November, the juveniles were moved to another climate chamber where the conditions were gradually changed to resemble wintering conditions . They were allowed to overwinter until late February, when the conditions were gradually changed to correspond to the summer conditions again. The juveniles were then maintained under similar conditions as above. Nine months after metamorphosis, all juveniles were photographed for the estimation of the degree of melanism . Snout-vent length of the juveniles was measured from the digital photographs.The eggs and hatchlings from each full-sib family were raised in two 3 L vials (ca. 200 eggs in each) until Gosner stage 25 . Temperaer (RSW)  renewed Mortality limited the available data in the southernmost population mostly to full-sib families. Since also the data on the northernmost population came from full-sib families, we restricted our analysis below to family effects, and did not attempt to estimate sire and dam effects.Estimation of the relative area covered by the permanent melanistic spots was done similarly with the wild-caught and common garden samples. The individuals' (thawed carcasses in the wild-caught and live specimens in the common garden sample) dorsal sides were gently pressed against a transparent plexiglass panel to obtain a nearly flat surface for photographing. Digital photographs were taken under similar circumstances (different sets for wild-caught and common garden samples). A ruler or millimeter paper was placed in every photograph for scaling. Color calibration was not employed, but melanistic and non-melanistic areas were clearly distinguishable in the images, as shown by the relatively high repeatability estimate for the degree of melanism (see Results).2. 24 images were measured twice in order to estimate the repeatability for length and the degree of melanism.The digital photographs were processed with Image Pro Plus 4.5 software . We first defined the area  approach. Sex, latitude, snout-vent length (used as a rough proxy for age), the interaction of sex and latitude, and the interaction of sex and snout-vent length were included as fixed effects, and population as a random effect. The model was fitted to the arcsine transformed data and predictions of the effect of latitude and snout-vent length were back-transformed for purposes of visualization. 95% highest posterior density intervals (HPDI) obtained with the functions mcmcsamp and HPDinterval of the lme4 package were used as a confidence measure. We also calculated the degree of phenotypic divergence (STP) [Latitudinal trends in the degree of melanism in adults caught from the wild were tested by fitting a linear mixed model using the ce (PST)  between h2 the heritability. ST Pwas estimated for two scenarios, with heritability values 2 h= 1 and 2 h= 0.5. The variance components were estimated using the lmer function. We fitted a linear mixed model with sex and snout-vent length as fixed effects and population as a random effect. 95% confidence intervals for the ST Pestimates were obtained by non-parametric bootstrap, resampling data 5000 times.where ST F- describing the degree of neutral genetic divergence [ST Pseparately both for the six populations and for all twelve populations.An estimate of vergence ,52 - calvergence . We calclmer function of the lme4 package. Population, snout-vent length, larval temperature treatment, and the interaction of population and larval temperature treatment were included as fixed effects and family as a random effect. The model was fitted to the arcsine transformed data. 95% highest posterior density intervals were again used as a confidence measure. The inclusion of family effect to the model corrected for the non-independence of full-sib measurements, and allowed us to estimate the upper limit for heritability 2 h[We investigated the population (i.e. among population additive genetic) effects in the degree of melanism in metamorphosed juveniles reared in the common garden setting. We again fitted a linear mixed model in R using the ility h2  for the where 23,24 = 427.26; 95% CI: 0.98-1.00) for snout-vent length and 0.81  for the degree of melanism. The snout-vent length was 16.9-27.1 mm in the common garden juveniles and 53.3-90.6 mm in the adults from the wild.The repeatability for the juveniles reared in common garden was 1.00  assuming 2 h= 1, and 0.53 (95% CI: 0.43-0.63) assuming 2 h= 0.5. ST Pfor the six populations for which the degree of neutral marker divergence, STF, was available, was 0.42 (95% CI: 0.32-0.52) for 2 h= 1, and 0.59 (95% CI: 0.48-0.69) for 2 h= 0.5. ST Fpublished in [We found an increasing latitudinal trend in the degree of melanism in the adults caught from the wild, with suggestive sex differences so that melanism in females increased more steeply with latitude than in males Figure , Table 2ished in  for thesSnout-vent length did not have a significant effect on the degree of melanism in juveniles reared in common garden Table . There wThe thermoregulation hypothesis, stating that dark individuals have a fitness advantage in low temperatures as compared to light individuals due to their ability to heat up faster , is perhST Qand STF, respectively, is often used to infer the relative roles of natural selection and random drift in among-population divergence [ST Qexceeds ST Fthere is evidence of divergent selection, when the reverse is true there is support for stabilizing selection, and when ST Qand ST Fare not significantly different, random genetic drift as the only cause of phenotypic divergence cannot be excluded [ST Qestimates have not been available, ST Pvalues calculated from purely phenotypic data have been used as surrogates [STP, was significantly higher than the ST Festimate published in [ST Pvalue estimated assuming 2 h= 0.5 was used. Hence, looking naïvely at the phenotypic trend and divergence, without a further genetic analysis or study of population demography, the results suggest that the populations might have diverged under natural selection consistent with thermal adaptation hypothesis of melanism. However, when looking at the genetic population effects, not only do we find that there is no direct genetic basis for the latitudinal trend, but also that the observed population effects are to the opposite direction of the cline. Our results are thus in line with the criticism of the use of ST Pin studies of wild populations [Comparison of quantitative and neutral marker differentiation, as measured by vergence ,52. The excluded . When QSrrogates ,57. In oished in  for our ulations .The problem of disentangling genetic and environmental effects in studies of geographic or temporal phenotypic variation is a general one ,59,60. TEnvironmental plasticity itself can be either adaptive or non-adaptive . AlthougOur study comes with a number of caveats. The sample size in the common garden data was small in terms of populations, families and offspring. This was reflected e.g. in the fact that the confidence interval for the upper limit for heritability was very wide and prohibited any firm conclusions on heritability. However, the small sample size is taken into account in the confidence intervals and highest posterior density intervals, and hence the results are robust e.g. as to finding that in the common garden there was no population difference similar to that observed in the wild. The low number of offspring in the common garden analysis resulted partly from low survival in the southern population, but although mortality was thus high, there is no reason to suspect that it would have influenced the results. It is also true that the age structure of the wild and common garden data did not overlap, a fact reflected in the difference between the ranges of snout-vent lengths (see Results). Finally, while environmental conditions can be controlled in the laboratory, this also often unavoidably distorts them from natural conditions. For example, while we had two larval temperature treatments, neither of them arguably fully corresponded to the normal temperature conditions common frogs experience in the wild - while temperatures in the experiment were constant, in the wild they fluctuate daily and seasonally and vary spatially. In addition, the temperature treatments were limited to larval stage, ending after metamorphosis. Although these issues should be kept in mind when interpreting our results, the conclusions we have drawn should be justified in the light of the available evidence and information.Latitudinal and altitudinal clines in the degree of melanism provide an opportunity to study both the evolution of coloration and evolutionary processes in general. A genetic basis is necessary for any adaptive explanation for such a cline to be applicable. While we observed a positive correlation between latitude and the degree of melanistic pigmentation in the common frog, there was no straightforward genetic foundation for it. This emphasizes the potential role of environment in the degree of melanism, while leaving population demographic explanations and the possible genetic basis of environmental plasticity to be addressed in future studies.AL, KIJ and JM conducted the field study, and FS and AL the common garden experiment. GH measured the degree of melanism in the wild-caught frogs. JSA conducted the analyses and had primary responsibility of preparing the manuscript, with contributions and comments from all of the other authors.


Erysiphe necator, was introduced into Europe more than 160 years ago and is now distributed everywhere that grapes are grown. To understand the invasion history of this pathogen we investigated the evolutionary relationships between introduced populations of Europe, Australia and the western United States (US) and populations in the eastern US, where E. necator is thought to be native. Additionally, we tested the hypothesis that populations of E. necator in the eastern US are structured based on geography and Vitis host species.The grape powdery mildew fungus, E. necator collected from the eastern US, Europe, Australia, and the western US. Phylogeographic analyses show that the two genetic groups in Europe represent two separate introductions and that the genetic groups may be derived from eastern US ancestors. Populations from the western US and Europe share haplotypes, suggesting that the western US population was introduced from Europe. Populations in Australia are derived from European populations. Haplotype richness and nucleotide diversity were significantly greater in the eastern US populations than in the introduced populations. Populations within the eastern US are geographically differentiated; however, no structure was detected with respect to host habitat . Populations from muscadine grapes, V. rotundifolia, are genetically distinct from populations from other Vitis host species, yet no differentiation was detected among populations from other Vitis species.We sequenced three nuclear gene regions covering 1803 nucleotides from 146 isolates of E. necator follows a pattern consistent with plant-mediated dispersal, however, more exhaustive sampling is required to make more precise conclusions as to origin. E. necator shows no genetic structure across Vitis host species, except with respect to V. rotundifolia.Multilocus sequencing analysis of the grape powdery mildew fungus is consistent with the hypothesis that populations in Europe, Australia and the western US are derived from two separate introductions and their ancestors were likely from native populations in the eastern US. The invasion history of  Cryphonectria parasitica, the fungus that causes chestnut blight. Its introduction from Asia [Castanea dentata) and markedly altered the species composition of forests throughout eastern North America. Source pathogen populations are expected to be more diverse than introduced populations because introduced populations have smaller effective population sizes due to losses in genetic diversity from population bottlenecks and genetic drift associated with small founder population sizes [Introduced pathogens have led to devastating epidemics in naïve host populations that lack evolved defences, as demonstrated by the plant pathogen rom Asia  practicaon sizes ,3. Howevon sizes ,5. Whereon sizes ,7. Additon sizes -10. HoweErysiphe necator (formerly Uncinula necator), an obligate parasite of Vitis species that was introduced into Europe and, eventually, all other wine-producing regions of the world. Historical records support the hypothesis that the source of the introduction is eastern North America [Vitis that have relatively high levels of resistance to many diseases and pests of grapevines, including powdery mildew [E. necator most likely dispersed long distances by the movement of grapevines, which were frequently traded between continents in the mid-1800's and later. E. necator remains dormant as mycelium in dormant buds, or as sexual spores in cleistothecia in the bark of vines [The focus of this research is the invasion history and population structure of the grape powdery mildew fungus,  America . Powdery America . Easterny mildew ,14. Aftey mildew  and Austy mildew . E. necaof vines ,18.E. necator to date have been limited to introduced populations in Europe and Australia where two distinct, yet sympatric, genetic groups have been consistently found [CYP51), the internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA) [TUB2) [ITS sequence [Population genetic studies on ly found -25. The A (rDNA) , and bet) [TUB2) . In Indisequence ,26. Smalsequence ,23,25,26sequence . Group Asequence ,23. Grousequence ,22,29, bE. necator in introduced populations, we tested the hypothesis that A and B were derived from separate introductions, as opposed to diverging after their introduction. To address this question, it was essential to study the population structure in eastern North America, the putative source population. Because no information was available on the population genetics of E. necator in North America, our major second objective was to describe the diversity and population structure in the eastern Unites States (US). We tested the hypothesis that if the eastern US population was a potential source of introductions, haplotypes found in introduced populations of Europe, Australia, and the western US would also be found in the eastern US. Moreover, we predicted that populations in the eastern US would have greater haplotype and nucleotide diversity than introduced populations. Finally, we tested the hypotheses that the population in the eastern US is structured by geography, Vitis host species, or host habitat (wild or cultivated Vitis).We had two major objectives for this study. First, to understand the evolutionary processes that led to the existence of groups A and B of E. necator from diverse wild and cultivated Vitis species collected from the eastern US  and from cultivated V. vinifera from the western US, Europe, and Australia , beta-tubulin (TUB2), and translation elongation factor 1-α (EF1-α). We were unable to amplify IGS from isolates sampled from P. quinquefolia. However, E. necator var. ampelopsidis from P. quinquefolia is markedly divergent with 94.9%, 93.0%, and 91.7% similarity to the consensus sequence of isolates from Vitis spp. for ITS, TUB2, and EF1-α, respectively. For comparison, the lowest sequence similarity within E. necator from Vitis spp. was 99.8%, 99.5%, and 99.4%. Among isolates of E. necator from Vitis spp. there were 37 segregating sites and 45 multilocus haplotypes  from Pabulin TUB, and traD [ITS/IGS and EF1-α do not deviate from neutral evolution , Watterson's theta (θw), and pairwise nucleotide diversity (π). Haplotype richness is significantly greater in the eastern US than in the introduced populations for each locus and the multilocus haplotypes (Table w) in the eastern US population for TUB2, EF1-α and for all three loci combined. There is greater pairwise nucleotide diversity (π) for EF1-α in the eastern US population. However, π is greater in the introduced population for ITS/IGS.We estimated several population genetic parameters in the eastern US and introduced populations, including haplotype richness  as isolates in group B from Europe (Table IGS sequence that is found only in group B (data not shown). We did not sequence additional loci for these isolates from the western US because all were like group B for IGS.To determine evolutionary relationships among isolates, we constructed networks for the three gene regions and multilocus haplotypes Figures  and 2. AV. rotundifolia belonged to two multilocus haplotypes  and not shared by isolates from any other host species.The majority of haplotypes for the individual loci, and especially for the multilocus network, are represented by individuals from the eastern US populations, demonstrating that populations in the eastern US are more diverse than in Europe, Australia and the western US. The sample size from the eastern US population is larger; however, this difference is accounted for in the comparisons of diversity estimates by rarefaction analyses. Although isolates from the central US are represented by diverse haplotypes, they are mostly derived haplotypes at or near the tips of the multilocus network, which suggests that this region is peripheral to the centre of diversity. All isolates obtained from 33) was common in the southeastern US in isolates from diverse wild and cultivated host species, including V. vinifera, vinifera hybrids, V. aestivalis, and V. riparia  forming a discrete lineage in group B; none of these multilocus haplotypes was found in eastern North America. However, group B does not differ from the eastern US population at all loci , we conducted tests of geographic subdivision on isolates collected only from V. vinifera and vinifera hybrid hosts, which are found abundantly among the three regions, to avoid confounding host and geography. Geographic subdivision was still evident when the analyses only included isolates from V. vinifera and vinifera hybrid hosts .  present .Vitis species are found throughout eastern North America [E. necator from cultivated grapes and from four of the most common wild species: V. riparia, V. aestivalis, V. labrusca, and V. rotundifolia  were derived mostly from backcrossing to V. labrusca and are grown in colder climates of eastern North America. In contrast to the diversity of hosts in eastern North America, most other major wine-producing regions are dominated by V. vinifera, which is native to Eurasia [E. necator [Cultivated varieties grown throughout eastern North America are also diverse. Interspecific hybrids derived from crosses between the European wine grape, ed vines . Cultiva Eurasia  and high necator .E. necator from the eastern US  and western US . In fact, the isolates we sequenced from Australia were identified as having distinct genotypes [V. vinifera is the only host plant E. necator was sampled from in Europe and Australia because this is the dominant species present in these regions. Therefore, we did not sample from wild species outside of the eastern US.We sampled  regions -24. Amonenotypes , represeParthenocissus quinquefolia, also in the Vitaceae, were collected in Ithaca, NY, USA for comparison with powdery mildew from Vitis species. Powdery mildew from P. quinquefolia is considered variety ampelopsidis of E. necator [Parthenocissus species exhibit host specialization, although some can infect V. vinifera but with greatly reduced growth compared to isolates from V. vinifera [Isolates of powdery mildew from  necator . Isolatevinifera .et al. [V. vinifera 'Cabernet Sauvignon' grown in a greenhouse. Leaves were surface-sterilized in 0.6% sodium hypochlorite for 1.5 min, rinsed twice with sterile distilled water and air dried in a sterile laminar flow hood. Leaves were kept in Petri dishes containing 20 ml of 2% water agar. Colonies of E. necator were initially isolated by touching a mildew colony from an infected leaf to a surface-sterilized leaf. Asexual spores (conidia) from the resulting colonies were transferred 6-12 days later with a sterile pipette tip to another surface-sterilized leaf at least once to rid the colonies of contaminants prior to DNA extraction. Isolates were maintained by transferring to new leaves approximately once per month.Mildew isolates were maintained as described by Evans et al.  on young2 piece of office tape  to the colony multiple times until the tape was covered in fungal tissue. The tape was placed in a 1.5 mL microcentrifuge tube with 100 μL of 5% chelex [For DNA extraction, conidia and hyphae were collected from colonies 2-3 weeks after inoculation by touching a 1-cm% chelex ,58, vortITS/IGS, TUB2, and EF1-α. ITS [TUB2 [E. necator, whereas IGS and EF1-α were identified in E. necator in this study. For ITS, we developed primers ITSEnF: 5′-AAGGATCATTACAGAGCGAGAGG-3′ and ITSEnR: 5′-GGATGACCGGACAAAGGTG-3′. For TUB2, we designed primers Bt2c: 5′-CAGACTGGCCAATGCGTA-3′ and Bt2d: 5′-AGTTCAGCACCCTCGGTGTA-3′ based on the published sequence (GenBank accession no. AY074934) [IGS region in E. necator with the conserved ascomycete primers IGS-12a and NS1R [EF1-α was identified in E. necator by designing degenerate primers to conserved regions of EF1-α in Sclerotinia sclerotiorum (GenBank accession no. DQ471086) and Botrytis cinerea (GenBank accession no. DQ471045). We then developed primers EF1-5: 5′-ATAGCGACGATGAGCTGCTT-3′ and EF1-6: 5′-TCGAAAAGGTTTGTTGCAGA-3′ for improved PCR amplification. The PCR reactions for ITS, IGS, and TUB2 were carried out in a total volume of 25 μL. Reaction components included 2.5 μL of 10 × PCR buffer , 2.5 μL dNTPs, 1.25 μL of 10 μM forward and reverse primers, 0.75 U ExTaq , and 1 μL DNA template. Cycling conditions included an initial denaturation at 95°C for 2 min followed by 35 cycles with a denaturation step at 94.5°C for 1 min, annealing at 56°C for 1 min, extension at 72°C for 1 min, followed by a final extension at 72°C for 5 min. PCR products were purified with QIAquick spin columns (QIAGEN). The PCR reaction for EF1-α was carried out in a total volume of 50 μL with all components added at 2 × the volumes used in the reactions for the other loci. Thermal cycling was carried out as described for the other loci. The EF1-α PCR products were purified by electrophoresis in a 1% agarose gel, excision of the band and purification with the QIAEX II Gel Extraction Kit (QIAGEN). All DNA fragments were sequenced at the Cornell University Life Sciences Core Laboratories Centre using the Applied Biosystems Automated 3730 DNA Analyzer with Big Dye Terminator chemistry and AmpliTaq-FS DNA Polymerase. All gene regions were sequenced in both directions in at least one isolate. Sequences of EF1-α, ITS, TUB2 and IGS for haplotype 1  . We idenand NS1R , then deE. necator var. ampelopsidis as an outgroup. Outgroup haplotypes could not be incorporated into the network with TCS due to high divergence.Sequences were aligned and manually edited in SeqMan . Haplotype networks were constructed for each locus and for combined multilocus sequences by statistical parsimony with the program TCS 1.21 ,61. HaplhR), the total number of haplotypes; Watterson's theta (θw), which is a measure of nucleotide polymorphism equivalent to 2Neμ (in a haploid population) and an estimate of the effective population size [hR, θw, and π. This was repeated 1000 times and the median estimates for each parameter were recorded. We conducted a one-tailed test to determine if eastern US populations were more diverse than introduced populations. P-values were estimated as the proportion of the null distribution that was less than the observed diversity estimate for the introduced population.To compare diversity between source and introduced populations we estimated several population genetic parameters. They include: haplotype richness  measures how often the most similar sequence or sequences ('nearest neighbour') is from the same designated population [Snn estimates the proportion of nearest neighbours that are from the same population versus from a different population. With two populations, for example, a value close to 1 suggests that the two populations are highly differentiated, because almost every sequence would be most similar to other sequences from the same population, whereas a value of 0.5 would be expected if populations are not genetically structured because the closest sequences would be most similar to those from either population with equal probability. We also estimated differentiation with HST, a powerful measure of population subdivision that estimates FST among haplotypes [Snn and HST were calculated by using DnaSP v5. P-values were estimated by permutation tests with 1000 replications.Differentiation among geographic regions, host habitats, and host species in eastern North America was estimated on combined multilocus sequences. For these analyses labrusca hybrids were grouped with pulation . This stplotypes . Both SnMTB participated in the design of the study, conducted the research, analyzed the data, interpreted the results, and wrote the manuscript. MGM conceived the study, participated in the design of the study, performed some of the analyses, assisted with interpreting the results, and critically revised drafts of the manuscript. Both authors read and approved the final manuscript.Table S1. Origin, collection date, and multilocus haplotypes of Erysiphe necator isolates.Click here for file


Viral population evolution dynamics of influenza A is crucial for surveillance and control. In this paper we analyzed viral genetic features during the recent pandemic caused by the new influenza human virus A H1N1, using a conventional population genetics approach based on 4689 hemagglutinin (HA) and neuraminidase (NA) sequences available in GenBank submitted between March and December of 2009. This analysis showed several relevant aspects: a) a scarce initial genetic variability within the viral isolates from some countries that increased along 2009 when influenza was dispersed around the world; b) a worldwide virus polarized behavior identified when comparing paired countries, low differentiation and high gene flow were found in some pairs and high differentiation and moderate or scarce gene flow in others, independently of their geographical closeness, c) lack of positive selection in HA and NA due to increase of the population size of virus variants, d) HA and NA variants spread in a few months all over the world being identified in the same countries in different months along 2009, and e) containment of viral variants in Mexico at the beginning of the outbreak, probably due to the control measures applied by the government. In April 2009 the Mexican Secretariat of Health reported an outbreak of respiratory disease. A new human influenza virus A H1N1 with molecular features of North American and Eurasian swine, avian, and human influenza viruses was identified -6. In DeST), coancestry coefficient (FST) and migration (Nm). These indexes refer to: π, average proportion of nucleotide differences between all possible pairs of sequences in the sample; θ, proportion of nucleotide sites that are expected to be polymorphic in any suitable sample from this region of the genome. Both indexes are used to assess polymorphisms at the DNA level and monitor diversity within or between ecological populations, and examine the genetic variation in related species or their evolutionary relationships [ST and GST are two equivalent genetic statistics used to measure differentiation between or among populations; FST is used when there are only two alleles at a locus, and GST with multiple alleles; common used values for genetic differentiation are: 0 to 0.5 small; 0.05 to 0.15 moderate; 0.15 to 0.25, great, and values above 0.25 indicate huge genetic differentiation, while negative values are due to small sample size [Analysis of genetic diversity within and between populations were calculated using DnaSP v4 -10 and iionships . FST andple size  and thusple size ,12. The ple size .The previously described genetic diversity analyses were performed with A H1N1 Influenza Database  with seqUp to 3462 sequences (1779 of HA and 1683 of NA) with 2208 VV (1216 of HA and 992 of NA) from 31 countries were used, interestingly 80% were recorded between April and July  were high when this country was paired with Mexico, France, Greece or New Zealand (seen as full or empty dots or triangles), while the values of genetic flow (Nm) were higher when USA was compared to Chile, Germany, Russia, China, Philippines or Australia (seen as shadowed areas or star peaks). Following the same explanation for the other four countries, it can be seen that some showed high or low degree of differentiation for FST and GST but opposed for Nm. Thus, the highest flow is seen in USA followed by Japan, China and Spain, and the lowest was found in Mexico. Interestingly, in the image obtained when samples from April-December were used, a different pattern can be seen: USA shows a moderate flow with all countries used for comparison; while Mexico is the country with the highest differentiation. The in-between countries are Japan, China, Spain and Singapore; the latter country appears in figure ST, GST and Nm. Negative values for FST and GST indicate no differentiation; in some cases NA showed lower FST values that those of HA with a similar trend. Tajima's D provided negative values: -2.619 and -2.380 in the initial four months and -1.802 and -2.358 in the overall analysis, for HA and NA, respectively, indicating arousal of new polymorphisms as a consequence of population size expansion along 2009 [Genetic population indexes were compared in the countries with most sequences reported . Figure Figure Figures Our study shows that a high viral diversity during the 2009 pandemic took place, as compared, for example, to a study of HA performed in 1999-2000 with samples from French infected patients with A/H3N2, which showed an average of π = 0.0034 . TherefoST, GST and Nm of this new A H1N1 it was observed that the virus had different behaviors along 2009 when comparing paired countries; which was, in general, independent of their geographical proximity. The extremes were found in USA and Mexico; the former showed a high distribution of virus variants to and from several countries in the initial four months of the pandemic, becoming a worldwide dispersion towards the end of the year, while in Mexico minimal influx of variants was seen in the initial four months. This was probably due to the governmental actions taken in April to contain the influenza outbreak in the whole Mexican Republic [When integrating data of FRepublic  or to thST: coancestry coefficient statistics; GST: genetic differentiation index; HA: hemagglutinin; NA: neuraminidase; NN: migration index; VV: viral variants; VV57NA: viral variant 57 of neuraminidase; WHO: World Health Organization; π: nucleotide diversity; θ: haplotype polymorphism.FThe authors declare that they have no competing interests.FMH, DEJG, AMF and GVC collected data and carried out the bioinformatics analysis. GV, SKK and AF participated in biological interpretations of results and in the discussion. PM and MRV formulated the idea. All authors contributed in writing the manuscript.A H1N1 gene sequences used for the genetic diversity analysis. List of GenBank sequences of A H1N1, number of accession and country of origin.Click here for filePopulation genetic indexes among paired sequences of A H1N1 obtained from different countries. List of values (indexes) obtained for population genetic analysis among paired sequences from different countries after DnaSP v4 analysis.Click here for file


Large pelagic fishes are generally thought to have little population genetic structuring based on their cosmopolitan distribution, large population sizes and high dispersal capacities. However, gene flow can be influenced by ecological (e.g. homing behaviour) and physical  factors. In this regard, Atlantic bigeye tuna shows an interesting genetic structuring pattern with two highly divergent mitochondrial clades (Clades I and II), which are assumed to have been originated during the last Pleistocene glacial maxima. We assess genetic structure patterns of Atlantic bigeye tuna at the nuclear level, and compare them with mitochondrial evidence.RST pairwise comparisons, isolation by distance (Mantel) tests, Bayesian clustering analyses, and coalescence-based migration rate inferences supported unrestricted gene flow within the Atlantic Ocean at the nuclear level, and therefore interbreeding between individuals belonging to both mitochondrial clades. Moreover, departures from HWE in several loci were inferred for the samples of Guinea, and attributed to a Wahlund effect supporting the role of this region as a spawning and nursery area. Our microsatellite data supported a single worldwide panmictic unit for bigeye tunas. Despite the strong Agulhas Current, immigration rates seem to be higher from the Atlantic Ocean into the Indo-Pacific Ocean, but the actual number of individuals moving per generation is relatively low compared to the large population sizes inhabiting each ocean basin.We examined allele size variation of nine microsatellite loci in 380 individuals from the Gulf of Guinea, Canary, Azores, Canada, Indian Ocean, and Pacific Ocean. To investigate temporal stability of genetic structure, three Atlantic Ocean sites were re-sampled a second year. Hierarchical AMOVA tests, Lack of congruence between mt and nuclear evidences, which is also found in other species, most likely reflects past events of isolation and secondary contact. Given the inferred relatively low number of immigrants per generation around the Cape of Good Hope, the proportions of the mitochondrial clades in the different oceans may keep stable, and it seems plausible that the presence of individuals belonging to the mt Clade I in the Atlantic Ocean may be due to extensive migrations that predated the last glaciation. FST) )FST) -9, and tFST) ,10,11.Given this complexity, accurate determination of marine pelagic fish genetic structuring would ideally require both temporal (different years) and spatial (covering large areas) sampling, analysis of multiple loci  that improves the estimates considerably ,13, as wThunnus obesus  is a marine pelagic fish species characterized by large populations and a worldwide distribution  restricted to tropical and subtropical waters (except the Mediterranean Sea) [Bigeye tuna, ean Sea) ,23. Althean Sea) ,25. Catcean Sea)  indicateus Lowe, 839 is a Several studies ,26-28, rThe contemporary presence of individuals of both mt clades in sympatry across the Atlantic Ocean but not in the Indo-Pacific Ocean poses several interesting questions that require further investigation using nuclear data. If individuals of mt Clade I and Clade II show high genetic divergence at the nuclear level, the two lineages could represent distinct cryptic species. Alternatively, individuals from both mt clades could interbreed in the Atlantic Ocean, a process that would render homogeneity at the nuclear level. On the other hand, the presence of individuals of mt Clade I in both the Atlantic Ocean and Indo-Pacific Ocean allows testing whether gene flow around the Cape of Good Hope is still occurring at present within this clade, as well as the directionality of the process.Besides the above-mentioned historical factors, contemporary ecological factors such as e.g. phylopatric behaviour of adults could play an important role in shaping the current genetic structure of bigeye tuna. In order to discern the relative role of historical and ecological factors, the comparison of molecular markers of mt and nuclear origin is particularly informative. For instance, discordance between mt and nuclear markers in describing population structure have been used to asses sex-biased dispersal or philopatry -32. In tF-statistics, Mantel tests to determine for isolation by distance, Bayesian estimation of the number of populations, and coalescence-based estimation of the magnitude of gene flow. The main goal of these population genetic analyses was to determine genetic structure of bigeye tuna within the Atlantic Ocean based on nuclear data, and to compare it with mitochondrial evidence. We also tested the null hypothesis of genetic homogeneity (i.e. panmixia) worldwide, and estimated the magnitude and direction of gene flow between the Atlantic Ocean, and the Indo-Pacific region.In this study, we gathered the most comprehensive genetic data set on Atlantic bigeye tuna to date. We analyze allele size variation of nine polymorphic microsatellite loci in four Atlantic locations that were surveyed in a previous study , and anaFIS value. According to the geographical origin of the samples, most (83%) of FIS significant values were found to belong to Guinea and Guinea 2 sampling sites  [FST and RST pairwise comparisons between first and second year replicate comparisons at Guinea, Canary Islands, and Canada were each not significant  in order to test temporal stability of genetic variability. After Bonferroni correction, 12 out of 54 exact tests remained significant, which indicates their departure from Hardy-Weinberg equilibrium (HWE) (Table  and 2003on (AIC) . Two modon (AIC) . The besNS) and observed heterozygosity was similar among sampling sites for the same microsatellite locus . The total NS varied from 7.16 (for TTH217) to 18.77 (TTH4) , and included also one Indian Ocean, and one Pacific Ocean locations as outgroups. Genetic analyses were based only on those individuals that were correctly genotyped for at least seven loci (only nine out of 380 samples did not met this requirement and were excluded). The amount of genetic variability, in terms of average number of alleles (us Table . HoweverFST and RST estimates (P = 0.3). Multilocus pairwise estimates of FST showed nine significant comparisons after Bonferroni correction  or RST  estimates  were chosen to allow the existence of two possible populations with very similar allele frequencies [K = 1 .The number of populations  was estimated using Bayesian inferences. Although the prior parameters for the quencies , the hig= 1 Fig. . If the Ne) of 15175, 5825, and 4175 bigeye tuna individuals for the Atlantic Ocean, the Indian Ocean, and the Pacific Ocean, respectively  to census population size  of origin using both ML and BI methods . The ML ye tuna; ) are comye tuna; ,44, and ye tuna; . In ordele size; ), which Migration rates seem to be symmetric between the Indian Ocean and the Pacific Ocean Fig. . HoweverNe) and time since divergence (t) showed that these loci provided enough statistical power [Thunnus albacares [FIS locus-population comparisons involved Guinea samples of different years versus other Atlantic populations, being deviations attributed to homozygote excess. According to the ANOVA test only the genetic factor (the locus) and neither the time nor the combination of both factors was responsible for the deviation from HWE. On the other hand, the only case of departure from HWE due to null alleles was associated to the Guinea location. All these results together highlight the singularity of the Guinea location, and could be explained in terms of admixture of genetically distinct cohorts  within this site, owing to the fact that the Gulf of Guinea is known as the main spawning and nursery area for bigeye tuna in the Atlantic Ocean [Thunnus thynnus) [Negaprion brevirostris) [Despite cross-specific PCR amplification, all analyzed microsatellites showed relatively high levels of polymorphism. Simulations using various combinations of effective population size  , and lemrostris) .High values of genetic variability  characterized all studied locations. These values are similar to those previously reported for other scombroid fishes, such as the bluefin tuna ,54, yellFST and RST pairwise comparisons rendered low and non-significant values except a FST pairwise comparison between Canada2 and Canary). Additionally, the Mantel test failed to detect significant correlation between geographical and genetic distances, which discarded isolation by distance among populations within the Atlantic Ocean. Overall, these results support unrestricted gene flow among bigeye tuna populations within the Atlantic Ocean. Similarly, no genetic structuring was reported for bigeye tuna within the Pacific Ocean [Within the Atlantic Ocean, no significant genetic differentiation between temporal replicates or geographic locations was observed , but, butRST   . AlthougNm) have a large variance associated   showed large 95% confidence intervals that include FST values up to about 0.2. The presumed differentiation was most likely an artifact of the large number of alleles present in the sample. Although FST statistics [FST estimations assume that there is no error in the sample frequencies, but with many alleles per locus the precision of sample frequencies is questionable. In such cases, the use of both RST, which is independent of the mutation rate [FST.Most population genetic analyses performed in this study suggest lack of population structure, and relatively high levels of gene flow among bigeye tuna populations at a global scale in agreement with other population genetic analyses. However, nine out of 36 on Table . In orde) Figure . Estimatatistics  are wideatistics  with laratistics ,73,74. Fion rate ,76, and RST statistics, Bayesian cluster analyses, and coalescence-based migration rate inferences based on nuclear markers supported lack of trans-oceanic genetic structure, which contrasts with previously detected significant mt divergence of bigeye tuna between Atlantic Ocean and Indo-Pacific region due to the existence of two clades, one restricted to the Atlantic Ocean. Our results support interbreeding between individuals belonging to different clades in the Atlantic Ocean. Given the current distribution of bigeye tuna individuals belonging to the two clades, and the inferred asymmetrical nuclear migration rates between the Atlantic Ocean and the Indo-Pacific region, it is likely that little number of individuals  may currently move between the Atlantic Ocean and the Indo-Pacific region, maintaining the proportions of the mt clades in both oceans. Therefore, the current presence of individuals belonging to mt Clade I would have originated in an earlier interglacial period.Population genetic analyses based on microsatellite data of bigeye tuna populations showed unrestricted gene flow within the Atlantic Ocean, and supported the existence of a spawning and nursery region in the Gulf of Guinea. At a global scale, Thunnus obesus) were obtained from different locations in the Atlantic Ocean , the Indian Ocean (N = 49), and the Pacific Ocean (N = 50)  were re-sampled a second year to investigate temporal stability of genetic structure in these regions , in a total volume of 10 μl. Forward primers were labeled with fluorescent dye (Invitrogen), and amplified products were run for size detection on an ABI 3700 automated sequencer. Data collection and sizing of alleles were carried out using GeneScan v3.1.2 and Genotyper v3.1 software (Applied Biosystems). Approximately 10% of the samples were re-run to ensure repeatability in scoring.Genomic DNA was extracted from muscle tissue as previously described . After o thynnus , plus silbacares , and alrlbacares ,55 were NA), and number of alleles standardized to those of the population with the smallest sample size (NS) [FIS statistic estimations that detect deviations from HWE, and the linkage disequilibrium test were performed for each locus and sampling site using the program GENEPOP version3.3 [P values from multiple comparisons were Bonferroni corrected [Ne and t , leading to FST of 0.001 and 0.0025, which reflects the magnitude of FST and Ne values estimated from our empirical data. We also estimated the α error (type I) by performing a simulation of no divergence among samples . Results indicated that the probability for detecting population structure were high and statistically significant, corresponding to 76% and 72% for the Ne/t combinations of 9,000/18 and 10,000/20 and 100% for both the Ne/t combinations of 9,000/45 and 10,000/45, respectively. When FST was set to zero, the proportion of false significances (α) was 1%, which is lower to the intended value of 5%.For each locus and sampling site (including second-year samples), observed and expected heterozygosities , number ize (NS)  were calize (NS)  and FSTAize (NS) . Similarrsion3.3 . Signifirsion3.3 . P valueorrected . MICRO-Corrected  was usedorrected  was usedFST ; [RST (stepwise mutation model (SMM); [FST and RST estimates were calculated. In all instances with multiple tests, P values were adjusted using the Bonferroni correction [RST = FST), a total of 1000 allele size permutations were computed using SPAGEDI 1.1 [RST values (ρST). Mantel test was used to test correlation between geographical and genetic distances as implemented in GENEPOP version3.3 [FST/(1 - FST) or RST/(1 - RST) using the program ISOLDE in GENEPOP.A possible geographical pattern in the distribution of genetic variability was tested for each pair of sampling sites (36 pairwise comparisons including second-year samples) using l (IAM);  and RST l (SMM);  pairwisel (SMM);  and RST-l (SMM); , respectrrection . In ordeGEDI 1.1  to provirsion3.3 . The logHierarchical genetic structuring of microsatellite data (only first year sampling) was analyzed by assessing the relative contribution among groups, within groups and within population components for partitions of total molecular variance (AMOVA)  using ARF-statistics [K) with the highest posterior probability given the data (only first year sampling) was estimated using STRUCTURE [F-model) because it is considered the superior model for detecting structure among closely related populations [5 burn-in iterations followed by 1,5 × 106 iterations. We explored K in the range of one and nine and we performed 20 runs for each K value. In BAPS [6 MCMC cycles, discarding the first 1 × 105 cycles as burn-in.In order to complement and contrast the results obtained with the classical standard atistics  we also TRUCTURE , BAPS v3TRUCTURE  and STRUTRUCTURE . In STRUulations . MCMC co In BAPS , the Bay In BAPS  infers p In BAPS ,40. We rNeμ, where Ne is the effective population size and μ is the mutation rate per site) and the migration rate, M  among bigeye tuna populations. These analyses used the Brownian mutation model [FST estimates and a UPGMA tree were used as starting parameters for the estimation of Θ and M. For each locus in the data set, the ML was run for ten short and three long chains with 10,000 and 100,000-recorded genealogies, respectively, after discarding the first 10,000 genealogies (burn-in) for each chain. One of every 20 reconstructed genealogies was sampled for both the short and long chains. The Bayesian run consisted of one long chain with 50 millions-recorded parameter and genealogy changes after discarding the first 10,000 genealogies as burn-in for each locus. For all the analyses we used an adaptive heating scheme with 4 concurrent chains; the analyses were run on a cluster computer using up to 80 compute nodes. We conducted the analyses over two sets of data, the first one with microsatellite data structured into two groups equivalent to mt Clades I and II; and the second set of data structured according to geographical regions .The program MIGRATE v 2.4  was usedon model  and mutaon model  and the on model ,18, modeIn order to test whether past populations bottlenecks could be responsible of the observed difference between census and effective population sizes, we used the M_P_val program to calculate M, a statistic that estimates population bottleneck history using the ratio of the number of alleles to the range in allele size .FST estimation, we explored the range of estimated FST values for a range of migration rates. We simulated 20 times 100 datasets over the range of the number of immigrants per generation, Nem between 4 and 40 and report the 95% confidence intervals. The migration rate range covers the estimated migration rates of the real data using the coalescent inference method.In order to better understand the limitations of EGG carried out the DNA genotyping, performed the statistical analyses and drafted the manuscript. PB undertook software implementation, statistical and simulation analyses as well as interpretation and discussion of the results. RZ conceived the study, supervised the genetic studies and analyses, and contributed to the writing of the manuscript. All authors read and approved the final manuscript.


Acropora cervicornis and A. palmata, have been devastated by white-band disease (WBD), resulting in the listing of both species as threatened under the U.S. Endangered Species Act. A key to conserving these threatened corals is understanding how their populations are genetically interconnected throughout the greater Caribbean. Genetic research has demonstrated that gene flow is regionally restricted across the Caribbean in both species. Yet, despite being an important site of coral reef research, little genetic data has been available for the Florida Acropora, especially for the staghorn coral, A. cervicornis. In this study, we present new mitochondrial DNA sequence data from 52 A. cervicornis individuals from 22 sites spread across the upper and lower Florida Keys, which suggest that Florida's A. cervicornis populations are highly genetically interconnected (FST = −0.081). Comparison between Florida and existing mtDNA data from six regional Caribbean populations indicates that Florida possesses high levels of standing genetic diversity (h = 0.824) relative to the rest of the greater Caribbean (h = 0.701±0.043). We find that the contemporary level of gene flow across the greater Caribbean, including Florida, is restricted (A. cervicornis in Florida, the relatively high genetic diversity and connectivity within Florida suggest that this population may have sufficient genetic variation to be viable and resilient to environmental perturbation and disease. Limited genetic exchange across regional populations of the greater Caribbean, including Florida, indicates that conservation efforts for A. cervicornis should focus on maintaining and managing populations locally rather than relying on larval inputs from elsewhere.Over the past three decades, populations of the dominant shallow water Caribbean corals,  Acropora cervicornis) and the elkhorn coral . Since WBD was first observed in the late 1970s, record losses of up to 95% of live Acropora cover have been observed throughout the Caribbean Coral reefs have declined rapidly over the past three decades, due in large part to the loss of dominant reef-building corals Acropora is knowledge about the extent to which populations of each species are interconnected via larval dispersal. Genetic exchange over large spatial scales (i.e. hundreds of kilometers) might allow distant healthy populations to rescue damaged reefs; whereas, restricted gene flow would indicate that populations rely on local recruitment and require local management. Information about the genetic make-up of Caribbean Acropora populations is also important since both species rely heavily on asexual fragmentation to propagate locally Acropora populations have too few individuals to spawn consistently and successfully. The genetic make-up of Acropora populations may also affect their resiliency. For example, recent research indicates that 6% of A. cervicornis individuals are resistant to WBD One key to designing appropriate management strategies and conserving the remaining Caribbean Acropora species indicates that gene flow is geographically restricted among populations separated by 500 km or more A. palmata can be subdivided into distinct Western and Eastern Caribbean subpopulations A. cervicornis detected fine-scale genetic differences among populations separated by as little as 2 km Acropora argues for regionally-based management A. cervicornis suggests that the scale of dispersal and thus management at some locations may need to be much smaller  in this species Recent genetic research on both Caribbean Acropora is lacking is the Florida Keys reef tract, which is the largest continuous barrier reef in the U.S. and a focal point for U.S. coral research in the Caribbean. The Florida Keys reef tract sits downstream of most Caribbean reefs, which makes it a possible sink for immigrant larvae from upstream source populations A. palmata support the genetic relationship between Florida and Western Caribbean, clustering Florida in the Western Caribbean subpopulation with Panama and Mexico, as well as with the Bahamas A. cervicornis also suggest a genetic connection between the Western Caribbean and Florida, but too few samples (n = 5) were available to estimate the extent of population genetic structure between Florida and the greater Caribbean.One area where population genetic information from the Caribbean A. cervicornis population. Florida is at the northernmost limit of this species in the Caribbean A. cervicornis have a sparse and patchy distribution and have been heavily impacted by WBD A. cervicornis throughout the Keys, which is possible given the observation of genetic isolation over distances as short as 2 km in other A. cervicornis populations To date, the five genets from Florida used for the study by Vollmer and Palumbi (2007) represented the entirety of our knowledge about the genetic state of this Acropora cervicornis individuals from 22 sites across the Florida Keys to evaluate the population genetic structure within the Florida Keys and compare these data to published sequence data from across the greater Caribbean. Genetic comparisons between Florida and the rest of the Caribbean allow us to estimate genetic connectivity within Florida and between Florida and the greater Caribbean and to evaluate the genetic diversity within Florida relative to other Caribbean populations. Based on oceanographic models and genetic data from A. palmata, we hypothesize that the Florida Keys reef tract is a sink for A. cervicornis larvae and genetic diversity from upstream sources, predominantly the Western Caribbean. We also evaluate the possibility of local recruitment and genetic structure within the Florida Keys reef tract, given previous observations of genetic structure in A. cervicornis over distances as small as 2 km Here we use mitochondrial DNA sequence data from 52 A. cervicornis, whereas 13 haplotypes represent introgressed haplotypes from A. palmata   were useP>0.5). The high frequency of introgressed haplotypes in Florida was matched only by Panama, with an introgression frequency of 60%. Other Caribbean populations have introgressed haplotype frequencies between 19%–47%. Belize was an exception with no introgressed haplotypes (n = 20). Introgression frequencies of haplotypes varied significantly across the seven Caribbean samplings .2) do not suggest significant deviations from neutral expectations including population bottlenecks or expansions, except in the Bahamas and Turks and Caicos Islands where R2 was significant.Nucleotide and haplotype diversity values for the native mtDNA haplotypes were similar among populations across the greater Caribbean . BahamasThe haplotype network shows the relatively high levels of mtDNA diversity of native (n) and introgressed (i) haplotypes and their distributions across the four Caribbean regions . For theWith respect to the phylogeographic distribution of the seven native haplotypes observed in Florida, four haplotypes had broad geographic distributions , whereas the remaining three haplotypes had geographically restricted distributions. Of the geographically restricted haplotypes found in Florida, which are likely to reflect recent gene flow, two were shared between Florida and the Western Caribbean (n5 and n8), providing evidence of a link between these two regions. The other haplotype was exclusive to Florida (n18).A. cervicornis across the Caribbean. Haplotype i2 was the most common introgressed haplotype and was found in all four regions; it is also the most common and ancestral haplotype in A. palmataA. palmata. Regarding the four introgressed haplotypes found in the Florida sample, two haplotypes had broad geographic distributions (i2 and i11), and two were restricted to one (i13) or two (i10) regions. Haplotype i10 is a restricted haplotype shared between the Eastern Caribbean and Florida; however, this could reflect gene flow in either A. cervicornis or A. palmata. Because of the high variance in introgression frequency across populations, the distribution of introgressed haplotypes may not accurately reflect gene flow among populations of A. cervicornis; rather, it may represent differential gene flow between A. palmata and A. cervicornis occurring in different regions.Thirteen introgressed mtDNA haplotypes were detected in ST = −0.024, P = 0.66) or the native only dataset  was observed, and both upper and lower Keys had similar levels of introgressed haplotypes. As a result, the upper Keys and lower Keys samples were combined into a single Florida population for the Caribbean-wide population genetic analyses.Within Florida, due to the small sample sizes within sites, AMOVA and analysis of population structure at the level of reefs were not possible. To evaluate gene flow among the Florida Keys, collection sites were classified as upper Keys or lower Keys according to geography . No evidP = 0.02), but not among populations within regions (P = 0.11). In contrast, AMOVA for the complete dataset indicates structure among populations within regions (P<0.001) but not among regions (P = 0.66). This demonstrates that the inclusion of introgressed haplotypes in the analysis increases the variation among populations, but obscures the regional structure revealed in the native haplotypes.Hierarchical AMOVA was used to compare the levels of population genetic differentiation among regions . Curacao was the most distinct population with all pairwise comparisons being significant and four of six comparisons remaining significant after sequential Bonferroni correction. For the native dataset, significant population structure was observed between Florida and all other Caribbean populations (FST = 0.117±0.011), except Panama (FST = 0.042); however, after sequential Bonferroni correction, none of the comparisons remained significant . For this dataset, Belize was the most distinct population with four of six pairwise comparisons being significant after Bonferroni correction, largely due to the absence of introgressed haplotypes in the Belize sample. In the complete dataset, significant population structure was observed between Florida and three of the four Caribbean populations, but after sequential Bonferroni correction only two comparisons are significant (Belize and Turks and Caicos).Pairwise Fnificant . For theAcropora cervicornis populations across the greater Caribbean  in our dataset. However, we detected relatively high levels of genetic structure between Florida and other populations in the Caribbean (average native pairwise FST = 0.105ST = 0.042), whereas Curacao was most different (native FST = 0.157). Native FST values for all other populations with Florida were between 0.097–0.126. Florida and the Western Caribbean share geographically-restricted haplotypes, suggesting that the Western Caribbean has historically been an upstream source of genetic diversity for Florida.Our data indicate that Florida's Acropora cervicornis across large swaths of the Caribbean support the previous findings of Vollmer and Palumbi's (2007) multi-locus Caribbean-wide survey. In addition to the genetic structure among Caribbean populations observed in the previous study (ΦCT = 0.249) and supported here (ΦST = 0.143), the data demonstrate significant structure among regional groupings . Despite this population structure, analysis of shared haplotypes indicates a historical and possibly on-going connection between A. cervicornis populations in the Western Caribbean and Florida, which Vollmer and Palumbi (2007) tentatively suggested with a small sample of Florida A. cervicornis . While Florida, the Greater Bahamas and the Eastern Caribbean also share some haplotypes, strong genetic connections are not evident between these regions. Although the Bahamas is much closer to the Florida Keys (200 km) than the other populations, the strong Gulf Stream current appears to act as a barrier to gene flow. Shared haplotypes between Florida and the greater Caribbean are likely to be the result of gene flow to Florida, rather than from Florida into the other Caribbean regions due to prevailing currents Evidence presented here for restricted gene flow in A. cervicornis larvae should be on the order of tens of kilometers at most. Sharing of haplotypes between Western Caribbean and Bahamas (1200 km apart) may also be accomplished via a similar route of connectivity along the reefs of Cuba Ocean currents support a link between the Western Caribbean and Florida A. cervicornis in Panama and Florida share similar characteristics that favor introgressive hybridization, although it is not clear what those characteristics might be. The rarity of A. cervicornis in Florida could increase the likelihood of inter-specific fertilization, but this cannot explain the high introgression frequency in Panama, which has dense thickets of A. cervicornis.Significant differences in the frequencies of introgressed haplotypes among populations provides another strong indication that gene flow among these populations is geographically restricted. With free exchange of larvae among populations, introgression frequencies would homogenize, but the data demonstrate that this is not the case. The extremely high introgression frequencies in Florida and Panama distinguish these populations from elsewhere in the Caribbean, including the Greater Bahamas. While the similarity in introgression frequencies between Florida and Panama may be due to ongoing gene flow, we consider this unlikely given the distance and genetic structure of both populations with Belize, an intermediate population. It is possible that Acropora coral (3–5 days) and reef corals in general AcroporaMontastrea corals A. palmata using microsatellites have shown strong genetic structure across the greater Caribbean with a genetic break between the Western Caribbean and the Eastern Caribbean occurring at the Mona Passage between Puerto Rico and the Dominican Republic A. palmata support a connection between Florida and Western Caribbean reefs A. cervicornisA. cervicornis, in A. palmata there appears to be a strong genetic connection between Florida and the Bahamas Montastrea species within the same genus revealed that one species, M. annularis, exhibits high population differentiation while the other, M. faveolata, appears panmictic between the Western Caribbean (Yucatan), Eastern Caribbean (Puerto Rico) and Florida Acropora and Montastrea, genetic data suggest that related coral species with similar life-histories and dispersal potentials can have contrasting population structures. Future research is needed to explain these differences.Given the relatively short dispersal potentials of the Caribbean Elacatinus spp) Stegastes partitus) Pseudopterogorgia elisabethae) Strombus gigas) Panulirus argus) Differing degrees of population genetic structure have also been detected in a variety of other Caribbean reef taxa and ascribed to a variety of causes. Within the Florida Keys, structure was observed in damselfish over a few meters and attributed to either a local genetic bottleneck A. cervicornis within the Florida Keys. This is surprising, given that our samples were collected over a distance of 200 kilometers and a range of environmental conditions. Previous research on A. cervicornis by Vollmer and Palumbi (2007) detected multiple instances in which A. cervicornis populations separated by 2–15 kilometers were genetically distinct. In Florida, however, no population genetic differences were detected between the upper Keys and lower Keys in either native or introgressed haplotypes. The absence of population genetic structure within Florida may indicate that gene flow is high across the Florida Keys reef tract, but alternate explanations are possible. In particular, barriers to gene flow between reefs may exist at a small geographic scale, but due to the limited sampling available from this diminished A. cervicornis population such fine-scale analyses could not be applied. Similar haplotype frequencies between upper and lower Keys may also result from recent mortality due to WBD that may be exhibiting positive selective pressure on resistant genotypes A. cervicornis indicates that no such structure exists . Thus, it may well be the case that Florida is characterized by having high gene flow within the region.Our data revealed no significant population structure among P>0.5). The absence of localized differences in introgression frequencies provides additional support for high gene flow across Florida Keys reef tract; however, the low abundance of A. cervicornis may have resulted in a higher proportion of hybrid recruits. Additional investigation of the geographic patterns of hybridization and introgression may shed light on this matter.It is not entirely clear why Vollmer and Palumbi (2007) detected such fine-scale differences among reefs elsewhere. Much of the fine-scale genetic differences in their dataset were driven by highly localized introgression signatures at one or more of the mtDNA or nuclear intron loci surveyed, including the putative mtDNA control surveyed here. Florida is distinctive for having the highest frequencies of introgressed mtDNA haplotypes detected to date across the greater Caribbean, but interestingly these high frequencies do not differ between the upper and lower Keys  is comparable to, and even higher than, the rest of Caribbean (h = 0.7012) do not indicate that there has been a significant loss of gene diversity (i.e. a genetic bottleneck) associated with the recent declines of A. cervicornis in Florida due to WBD. This may not be a surprise given that it should take multiple generations of random genetic drift for population size reductions to be reflected in genetic diversity estimates Diadema antillarum), which suffered an analogous decline of up to 97% throughout the Caribbean as a result of disease in the early 1980s, also retained high genetic diversity in an mtDNA marker surveyed for individuals collected between 1987 and 1999 The relatively high genetic diversity and the results of tests for population size fluctuations  is the effective number of females, which is equivalent to Ne (the effective population size) because A. cervicornis is hermaphroditic, and u is the mutation rate per generation]. To determine the neutral mutation rate of the putative mitochondrial control region (u), we used the current estimated divergence time between A. palmata and A. cervicornis of 3.6–2.6 mya, which corresponds to 350,000 to one million generations ago using a generation time of 3–8 years A. palmata and A. cervicornis sequences of 814 bp, we estimated a neutral mutation rate between 2.106×10−8 and 7.371×10−9 mutations per basepair per generation. Given the estimated theta (per site) for Florida (0.00171), this corresponds to an estimated range of effective population size of 40,600–116,000 individuals within the Florida Keys.The effective population size of et al. (2008) A. cervicornis in the Florida Keys. Clearly, more precise estimates of current census population sizes are needed. One reason for the discrepancy between census and effective population size estimates is that the effective population size reflects effective number of genets rather than ramets, and there are likely to be many ramets per genet in this asexually reproducing species. In addition, effective population size is often much smaller than the census size due to gender imbalance (not a factor in hermaphroditic species), variance in reproductive success A. cervicornis and the tendency of effective population size estimates to reflect the long-term average (harmonic mean) population size This estimate is far less than the 2007 abundance estimates of Miller A. cervicornis along these reefs, but over time the effect of genetic drift in a small population may result in a future genetic bottleneck. Even with relatively high genetic diversity, successful reproduction will occur at the level of individual reefs and requires that multiple genotypes are present. Future research should address the extent of localized genotypic diversity on Florida Keys reefs with additional sampling and long-term monitoring.The high standing genetic diversity in the Florida Keys is a hopeful sign for future resilience of A. cervicornis in the Florida Keys comprise a unique population within the Caribbean and should be treated as a distinct management unit for conservation. Monitoring of genetic diversity should continue for the Florida Keys region as the effect of a genetic bottleneck may be lagging behind the observed decrease in population size resulting from WBD. Furthermore, the genetic make-up may shift if WBD takes a greater toll on disease susceptible genotypes The significant levels of population structure detected between Florida and other regions in the Caribbean reveal that ongoing rates of recruitment to the Keys from reefs elsewhere are low. Restricted gene flow between Florida and other Caribbean populations indicates high dependence on local larval retention within the Florida Keys on the whole. Overall, our data suggest that the Collection of coral samples for this project was approved by the National Oceanic and Atmospheric Administration and was conducted under permit numbers FKNMS-2008-006 and FKNMS-2007-061.A. cervicornis specimens were collected July-September 2008 from 22 sites spanning the Florida Keys from southwest of Key West to Key Largo , which reflects the biological reality of the typically small patch sizes within Florida. Tissue samples were collected by sampling a small (1 cm) branch tip from staghorn coral colonies spaced at least 10 meters apart to minimize collection of clones produced by asexual fragmentation within each patch . Tissue samples were preserved in Chaos DNA extraction buffer and stored at room temperature. Extraction of DNA from the samples was conducted using a modified phenol-chloroform procedure A. cervicornis, consisting of mtDNA sequence data from 148 individuals from six geographic regions plus Florida , and thus signatures of population structure are dominated by introgression A. palmataA. cervicornis; however, preliminary analyses of these loci indicate that they are confounded by introgression and homoplasy . In order to avoid the confounding effects of introgressed alleles, we used mtDNA sequence data, which allowed for identification of introgressed and native haplotypes. Polymerase chain reaction (PCR) amplifications and DNA sequencing of the putative mtDNA control region were carried out according to Population genetic analyses were conducted using the putative mitochondrial control region Due to the small sample sizes within Florida (n = 1–6 samples per site), it was not possible to examine population structure between each site, rather the sites were more broadly classified into two regions, upper Keys (n = 22) and lower Keys (n = 30), for analyses of structure within Florida . SamplesAcropora cervicornis hybridizes and exchanges genes with its congener A. palmataA. palmata into A. cervicornisA. palmata) or native to A. cervicornisA. cervicornis obscures native population structure across the Caribbean, but adds to the genetic structure between local populations (i.e. reefs) due to strong differences in introgression frequencies among local staghorn coral populations. To account for the differences between native mtDNA haplotype variation (i.e. reflecting intra-species gene flow only) and variation in the complete dataset including introgressed genes (i.e. reflecting intra- and inter-specific gene flow), we split the mtDNA data into two datasets for analyses: one complete dataset including all haplotypes (i.e. native and introgressed haplotypes) and one dataset including only native (non-introgressed) haplotypes. Native and introgressed haplotypes in the sampling were identified after Vollmer and Palumbi  It has been shown that A. palmata lineage. Using the native haplotypes we tested for deviations from neutral expectations using standard tests: Tajima's D 2 statistic DNA sequence polymorphism for each population was characterized using DnaSP 4.0 ST (ST) values were calculated between each population. Significance was determined by 10,100 permutations and P-values were adjusted using sequential Bonferroni ST.Hierarchical Analysis of Molecular Variance (AMOVA)


Caffrogobius caffer was investigated across a known major oceanographic barrier, Cape Agulhas, which has previously been shown to strongly influence genetic structuring of South African rocky shore and intertidal marine organisms. Given the variable and dynamic oceanographical features of the region, we further sought to test how the pattern of gene flow between C. caffer populations is affected by the dominant Agulhas and Benguela current systems of the southern oceans.Oceanography and life-history characteristics are known to influence the genetic structure of marine species, however the relative role that these factors play in shaping phylogeographic patterns remains unresolved. The population genetic structure of the endemic, rocky shore dwelling C. caffer. Fifty-five haplotypes were recovered and in stark contrast to previous phylogeographic studies of South African marine species, C. caffer showed no significant population genetic structuring along 1300 km of coastline. The parsimony haplotype network, AMOVA and SAMOVA analyses revealed panmixia. Coalescent analyses reveal that gene flow in C. caffer is strongly asymmetrical and predominantly affected by the Agulhas Current. Notably, there was no gene flow between the east coast and all other populations, although all other analyses detect no significant population structure, suggesting a recent divergence. The mismatch distribution suggests that C. caffer underwent a population expansion at least 14 500 years ago.The variable 5' region of the mtDNA control region was amplified for 242 individuals from ten localities spanning the distributional range of C. caffer to maintain gene flow across its distributional range, including a long pelagic larval stage. We have shown that life-history characteristics can be an important contributing factor to the phylogeography of marine species and that the effects of oceanography do not necessarily suppress its influence on effective dispersal.We propose several possible life-history adaptations that could have enabled  With no obvious physical barriers to gene flow, marine species with large ranges are expected to show genetic homogeneity over long stretches of ocean – a panmixia paradigm . Many stThe South African coastline offers a unique setting to study the relative importance of oceanography on the genetic structuring of marine organisms. There are two contrasting current systems that meet as the continental shelf widens between Cape Agulhas and the Cape Peninsula Figure . These aUpogebia africana) and caridean shrimp  with long-lived, actively swimming larvae exhibit at least one genetic break .Sequences were edited and aligned by eye with BioEdit ver7.0.4.1 . Sequencequin3.1 . All hapST values were calculated to take the number of mutational changes between the haplotypes into consideration . Lar. LarC. ces [e.g. ,49, whices [e.g. . Since pes [e.g. , the widOur results show that successful dispersal across major oceanographic barriers such as Cape Agulhas is possible for some marine species and that availability of habitat is another key determinant in population structuring of marine organisms along the South African coast. We have shown that life-history characteristics could in fact be an important contributing factor to the population genetic structuring of marine species in southern Africa and illustrate that the influence on the effective dispersal of marine organisms is not necessarily suppressed by the effects of oceanography as has recently been advocated ,15,16.MN carried out the majority of the field and laboratory work and wrote the draft version of the manuscript. CAM participated in field work, helped with the interpretation of data and finalisation of the manuscript. RCKB collected samples, helped with statistical analyses and contributed to writing the manuscript. SvdH conceived the study, helped with sampling and data analysis and contributed to writing the final manuscript. All authors have read and approved the final manuscript.