Sardina pilchardus) is a small pelagic fish with a relatively high migratory capability that is expected to show low levels of genetic differentiation among populations. Previous genetic studies based on meristic and mitochondrial control region haplotype frequency data supported the existence of two sardine subspecies (S. p. pilchardus and S. p. sardina).Marine pelagic fishes exhibit rather complex patterns of genetic differentiation, which are the result of both historical processes and present day gene flow. Comparative multi-locus analyses based on both nuclear and mitochondrial genetic markers are probably the most efficient and informative approach to discerning the relative role of historical events and life-history traits in shaping genetic heterogeneity. The European sardine (RST pairwise comparisons) supported a single evolutionary unit for sardines. These analyses only detected weak but significant genetic differentiation, which followed an isolation-by-distance pattern according to Mantel test.We investigated genetic structure of sardine among nine locations in the Atlantic Ocean and Mediterranean Sea using allelic size variation of eight specific microsatellite loci. Bayesian clustering and assignment tests, maximum likelihood estimates of migration rates, as well as classical genetic-variance-based methods (hierarchical AMOVA test and We suggest that the discordant genetic structuring patterns inferred based on mitochondrial and microsatellite data might indicate that the two different classes of molecular markers may be reflecting different and complementary aspects of the evolutionary history of sardine. Mitochondrial data might be reflecting past isolation of sardine populations into two distinct groupings during Pleistocene whereas microsatellite data reveal the existence of present day gene flow among populations, and a pattern of isolation by distance. Understanding the rather complex population structure and dynamics of marine pelagic fishes requires discerning the relative influence of life-history traits and historical processes in shaping present-day population patterns e.g. -7). Mari. Mari7])Comparative analyses of both nuclear and mitochondrial genetic markers offer the best and most powerful approach to characterizing population genetic structure and diagnosing the evolutionary processes responsible for genetic differentiation in marine pelagic fishes e.g. ,17,21). ,21. 17,2Sardina pilchardus, Walbaum 1792) is a small pelagic fish that inhabits the coasts of the eastern North Atlantic Ocean , as well as the Mediterranean Sea, the Sea of Marmara, and the Black Sea [The European sardine  and S. p. sardina (Mediterranean Sea and Northwest African coast). Although no private mitochondrial control region sequence haplotype could be found for each proposed subspecies, they were suggested to be genetically distinct based on significant pairwise haplotype frequency differences [Morphological studies based on gill raker counts and head length ,30 foundferences . Moreoveferences . Accordiferences  between The sardine is heavily fished all over its distribution with global catches of 1.600,000 tons per year . In parIn this study, we analyzed allele size variation of eight polymorphic microsatellite loci in Atlantic and Mediterranean sardines. We used coalescent-based approaches for the estimation of the actual number of populations, and employed hierarchical AMOVA and isolation by distance tests to study population genetic differentiation. Our main objective was to explore whether microsatellites provide concordant genetic differentiation patterns with respect to mitochondrial control region sequence data . ComparaNA value ± standard deviation was 60 ± 18.60), and mean observed and expected heterozygosities of HO = 0.738 ± 0.13 and HE = 0.748 ± 0.14, respectively (Table SAR2F and 0.450 at locus SAR1.12 (mean FIS = 0.202 ± 0.18), and only two loci (SAR2.18 and SARA2F) were in Hardy-Weinberg (HW) equilibrium over all population samples , by mean allelic richness after rarefaction (NS = 27.3 ± 0.95), and by mean observed (HO = 0.747 ± 0.04) and expected (HE = 0.948 ± 0.00) heterozygosities  showed significant mean FIS values, indicating significant departures from HW equilibrium due to homozygote excess  genetic diversity measures were non-significant. The ANOVA test showed no differences for the mean number of alleles (NA) , the mean allelic richness after rarefaction (NS) , mean observed (HO)  and expected (HE)  heterozygosities, and the mean inbreeding coefficient (FIS)  between subspecies .Bayesian clustering analyses  detected= 4 Fig. . A BayesP < 0.8). The Bayesian assignment test correctly assigned 20.1% of the individuals to their own source location .The probability of assignment of individuals to the four or five possible populations as inferred using Bayesian clustering analyses  was geneRST = FST) was rejected (P < 0.000) based on the multilocus data set  over all loci suggested population structuring in sardines. Of 36 pairwise comparisons, only nine comparisons involving Nador, Barcelona and Kavala locations revealed significant values after correction for multiple tests (Table S. s. pilchardus) and the rest of the sampling sites (representing S. s. sardina) were non-significant.The null hypothesis of no contribution of the Stepwise Mutation model  to geneet Table , suggest samples . Three oP < 0.00) (Table S. p. pilchardus (Pasajes sampling site) versus S. p. sardina samples was not significant (P = 0.44). A possible a priori hypothesis of geographic structuring (organized as Atlantic Ocean versus Mediterranean Sea samples) was also not supported by the AMOVA (P = 0.07)  was significant  . Similarly, we found no significant correlation when using the Bayesian assignment DLR distances   Table . A two gnt P = 0.4. A possP two tails value of 0.031 for both tests), under the SMM model. No trace of genetic bottleneck was detected in Safi. Additionally, the test was performed using the Two Phase model  and theel . In theNe) of 12,818 ± 325 sardine individuals . Also the test rendered no significant variation of the number of emigrants among population samples . However a significant variation of immigrants among population samples  was detected. A one-way ANOVA was applied to test the null hypothesis of equal rate of immigrants between population samples. The analyses rendered a significant difference in the immigration rates among population samples , being Barcelona-Quarteira the only pairwise comparison that was significantly different (t > 1.998).The estimates of the population size parameter (Θ) ranged from 0.38 to 0.81 0.51 ± 0.13) (Table 1 ± 0.13 neration ). MigratThe study of population genetic variation of marine pelagic fish species has proven to be particularly challenging because of the biological peculiarities of these fishes including large effective population sizes and high dispersal capacities, as well as because of the apparent lack of physical barriers to gene flow in the marine realm ,46-48. MThe eight species-specific microsatellite loci used in this study showed high levels of polymorphism  and no sRST detected weak but significant genetic structuring among sardine population samples. Pairwise estimates of RST varied between 0.001 and 0.083, and were of the same level of magnitude to those reported for other marine fishes [RST values could be attributed to high levels of size homoplasy, as expected when using polymorphic microsatellites with high mutation rates in species with large effective population sizes [Overall e fishes -56. Theson sizes ,57,58. Hon sizes ,58.a priori hypotheses of genetic structuring rendered significant results in the AMOVA. Maximum likelihood estimates of migration rates showed that gene flow among population samples is high and even. All these results together support that sardine population samples are acting as a single significant evolutionary unit. The Mantel test detected positive and significant correlation between genetic differentiation (only when using RST) and geographical distance suggesting that a model of isolation by distance could explain the subtle genetic structuring of sardines within the evolutionary unit. Isolation by distance seems to be a rather common pattern in small-medium pelagic marine fishes . ,51. a prRST pairwise comparisons involved Mediterranean Sea versus Central Atlantic Ocean population samples. Theses results could reflect the existence of a phylogeographic discontinuity between the Atlantic Ocean and Mediterranean Sea, around the Gibraltar Strait and the Almeria-Oran front, as it has been postulated previously for different marine pelagic fish species . Ho,7. HoRSTes (e.g. . The infaponicus  or Thunn thynnus ) and forS. p. pilchardus and S. p. sardina) as previously reported based on meristic studies [RST pairwise comparisons, AMOVA test, and estimations of migration rates) based on microsatellite data. However, these results need to be taken with caution since one of the subspecies (S. p. pilchardus) was only represented by a single location (Pasajes). A more thorough sampling of sardine at North Atlantic locations would be mandatory to further test the validity of the two subspecies using microsatellite allele frequency data.The existence of two different subspecies  and three localities  in the Mediterranean Sea (N = 140)  of S. pilchardus were PCR amplified following optimized reaction conditions [Genomic DNA of newly analyzed specimens was extracted from fresh muscle following standard phenol-chloroform procedures as previously reported . Specifinditions . ForwardNA), and number of alleles standardized to those of the population sample with the smallest size (NS) [FIS) and linkage disequilibrium for each locus and sardine sampling site were assessed using GENEPOP version3.3 [P values from multiple comparisons were corrected using a Bonferroni correction [S. pilchardus subspecies were tested using a one-way ANOVA test.Microsatellite genetic diversity was quantified per locus and per sampling site as the observed and expected heterozygosities , number ize (NS) , using bize (NS)  and FSTAize (NS)    with the highest posterior probability and to estimate admixture proportions. Simulations were conducted using an admixture model and correlated allele frequencies between populations (MCMC consisted of 5 × 105 burn-in iterations followed by 2 × 106 sampled iterations). Additionally, the inference of the best value of K was also based on the modal value of ΔK [Ks was from one to nine, and five trial runs of STRUCTURE were carried out for each putative K.Several alternative methods were used to determine sardine population genetic structure. The program STRUCTURE 2.0  uses a mue of ΔK . The ran6 MCMC cycles, and we let α (the prior mean of the number of populations) be a random variable. The first 1 × 105 cycles were discarded as burn-in.The program STRUCTURAMA  infers pWe finally applied a Bayesian assignment test as implemented in the program GENECLASS 2.0 , which pFST, IAM [RST, SMM [RST values (ρRST) for testing the null hypothesis of no contribution of SSM to genetic differentiation (ρRST = FST), and the alternative hypothesis that genetic differentiation is caused mainly by SMM-like mutation  [P < 0.000), and thus, further analyses of genetic differentiation between samples were mostly based on RST pairwise comparisons, as estimated by the program RST-CALC [The relative contributions of mutation and genetic drift to genetic differentiation of sardine populations could be determined by comparing the variance in allelic identity  and allRST, SMM ). The prRST, SMM  generate > FST,) . The tesRST-CALC .P-values were Bonferroni adjusted [RST as estimated in RST-CALC [DLR) as computed in SPASSIGN [To determine the amount of genetic variability partitioned within and among populations, an analysis of molecular variance (AMOVA)  was perfadjusted . The Manadjusted . The logRST-CALC  or genetSPASSIGN .To detect possible genetic bottlenecks (i.e. significant heterozygote excess) in any of the analyzed population samples, we assumed the SMM, IAM, and TPM, and applied the Wilcoxon sign-rank test as implemented in the software BOTTLENECK .Neμ, were Ne is the effective population size and μ is the mutation rate per site) and the migration rate, M  among sardine population samples based on the maximum likelihood method [FST estimates and a UPGMA tree were used as starting parameters for the estimation of Θ and M. The MCMC run consisted of ten short and two long chains with 5,000 and 50,000 recorded genealogies respectively, after discarding the first 100,000 genealogies (burn-in). One of every 20 and 200 reconstructed genealogies was sampled for the short and long chains, respectively. To test the null hypothesis that the number of emigrants/ immigrants between the Atlantic Ocean and Mediterranean Sea has equal rates, a nested mixed-model ANOVA was performed using two variables (basin and location of origin), with emigrant and immigrant rates as repeated measurements.The program MIGRATE v 2.1.0  was usedd method . A subseThe author(s) declares that there are no competing interests.EGG carried out the DNA genotyping, performed the statistical analyses and drafted the manuscript. RZ conceived the study, supervised the genetic studies, and contributed to the writing of the manuscript. Both authors read and approved the final manuscript.Sardina pilchardus population samples. The data provided summarizes the statistical analyses , expected (HE) and observed (HO) heterozygosities and FIS = Wright's statistics) for each locus and sampling site.Summary statistics for eight microsatellite loci of Click here for fileSardina pilchardus. The table shows the allele frequency and allele size values for each locus and sampling site.Allele frequency and allele size for eight microsatellite loci and each sample of Click here for file


Chamaedorea ernesti-augusti) of Central America, whose wild-collected leaves are becoming progressively more important to the global ornamental industry. We use microsatellite markers to describe the population genetics of this species in Belize and test the effects of climate change and deforestation on its recent and historical effective population size.Developing a greater understanding of population genetic structure in lowland tropical plant species is highly relevant to our knowledge of increasingly fragmented forests and to the conservation of threatened species. Specific studies are particularly needed for taxa whose population dynamics are further impacted by human harvesting practices. One such case is the fishtail or xaté palm (ca. 10,000 years ago, but fail to detect changes attributable to Mayan or contemporary deforestation.We found high levels of inbreeding coupled with moderate or high allelic diversity within populations. Overall high gene flow was observed, with a north and south gradient and ongoing differentiation at smaller spatial scales. Immigration rates among populations were more difficult to discern, with minimal evidence for isolation by distance. We infer a tenfold reduction in effective population size C. ernesti-augusti are genetically heterogeneous demes at a local spatial scale, but are widely connected at a regional level in Belize. We suggest that the inferred patterns in population genetic structure are the result of the colonization of this species into Belize following expansion of humid forests in combination with demographic and mating patterns. Within populations, we hypothesize that low aggregated population density over large areas, short distance pollen dispersal via thrips, low adult survival, and low fruiting combined with early flowering may contribute towards local inbreeding via genetic drift. Relatively high levels of regional connectivity are likely the result of animal-mediated long-distance seed dispersal. The greatest present threat to the species is the potential onset of inbreeding depression as the result of increased human harvesting activities. Future genetic studies in understory palms should focus on both fine-scale and landscape-level genetic structure.Populations of  In plants, tools from molecular ecology and population genetics have been used to characterize genetic patterns that result from geographic and biological barriers to pollen and seed dispersal, and to investigate their significance in the evolutionary history of a species -5. DespiWithin palms, understory species are the least represented. Only a handful of studies have focused on the basic population genetics of this group -11. UndeChamaedorea is one of the largest and most species-rich of the Neotropical Arecaceae, with ca. 96 species widely distributed in the Americas . In . In 63]]linators -66 but slinators , thrips n Belize . Thrips n Belize -73 and cn Belize ,75. In B flowers . The larruit set . While rruit set . SpecifiChamaedorea and other palm genera have local aggregations of specific cohorts or sexes [C. ernesti-augusti species is random with respect to one another, staminate and pistillate individuals are significantly aggregated at the community level [C. ernesti-augusti may also be a consequence of reduced fruit set observed in some localities within Belize or the result of female plants having restricted fertility patterns [Chamaedorea species, with a similar genetic pattern to C. ernesti-augusti, have females that only reproduce for a few years within their life cycle (C. elatior) or have populations with a subset of females dominating successful reproductive events (C. tepejilote) [Complex demographic patterns within populations, such as sex-biased distributions and differences in flowering times within populations, can also result in high levels of inbreeding ,76. Chamor sexes ,77-80. Aty level . If therty level ,81,82. Tpatterns . Two othChamaedorea palms .Demographic bottlenecks decrease local survival in other elegans; ) and couty level . This spty level . Most inty level ,84. Low C. ernesti-augusti populations, genetic structure through most of the Holocene would have created a southern and a southern division, with the far southeast, which has a distinct geological and geographical history, forming either an outlier or part of the southern region. Climatic change during the late Pleistocene and early Holocene have influenced the population genetics of other taxa [Ne) that coincides with major climatic and landscape changes in Belize and neighbouring regions in the early Holocene [Chamaedorea palms in the region [C. ernesti-augusti migrated into Belize when tropical forests expanded at 10,000 BP [If recurrent landscape changes were indeed a predominant barrier to gene flow of her taxa . We knowher taxa , but lesHolocene ,46. Prioe region . The gen0,000 BP . Its curChamaedorea species that moved into Belize. Although most of the theoretical and empirical evidence of long-distance seed dispersal events comes from trees [et al [C. ernest-augusti have a stratified dispersal model, where local movements occur by one set of mechanisms  and accidental or sporadic long-distance transport  occur by another.Long-distance seed dispersal has likely played an important role in om trees ,87-90, tom trees . As Cains [et al  suggesteC. ernesti-augusti populations found in the understory of closed mature tropical broad-leaf forests within Belize, as well as other congeners, except C. seifrizii, which has a different distribution pattern. The impact of Mayan deforestation would result in a more recent bottleneck and yield a second subdivision within the southern region. Populations along the border with Guatemala have also recently decreased as a result of fragmentation and poorly regulated harvesting in recent years.Massive deforestation by the Ancient Maya over a period of 1,500 years in the same region probably reduced all western C. ernesti-augusti has low heterozygosity, and has experienced at least one major reduction in its effective population size, there was no evidence of a recent mode-shift in allele frequencies across all loci in Belize patterns expected after significant changes in Ne [Population genetics theory in tropical species predicts that restricted pollen movement or human-induced barriers among populations can create an isolation-by-distance pattern ,93. It aes in Ne . We did C. ernesti-augusti to survive and re-expand locally. In combination with occasional long-distance seed dispersal, overlapping generations in C. ernesti-augusti could also provide sufficient time and opportunity to buffer more recent biases in allele distribution at the regional level ..C. ernesChamaedorea species  also suene flow , this ma species .C. ernesti-augusti can be roughly divided into five genetic clusters with a north to south gradient. Subdivisions largely agree with expectations of landscape changes during the Holocene (i.e. no subsequent division of El Pilar-Chiquibul and Sibun-Manatee after Mayan deforestation). The hierarchical analysis in STRUCTURE . Columbia is connected to southern areas in the Bladen Reserve, following a limestone continuum that is disrupted towards the rest of the southern populations, but there is no evidence from either BayesAss or GeneClass of recent migration between these populations. As with Chiquibul, Pueblo Viejo and its forest extension into Guatemala have been affected by slash-and-burn agriculture during the past 30 years. Currently only pockets of forest remain, perhaps pressuring dispersers to migrate towards larger forest remnants. Interestingly, Temash has the highest numbers of alleles, a relatively high number of private alleles, and a high self-recruitment rate. It is one of the most isolated localities geographically, and also the wettest part of Belize, but acidic soils form a continuum on the east coast. The genetic connections between Temash southern sites are not yet clear, but it is possible that Mayan populations left Temash undisturbed [C. ernesti-augusti history within Belize.In the south, isturbed ,47, thusisturbed . A genetC. ernesti-augusti, it is most critical to maintain population sizes and allelic diversity within populations. One important step toward the conservation of C. ernesti-augusti is the enforcement of the existing harvesting rate regulations from the Belize Forest Department, which specify one leaf cut per individual per year [Chamaedorea species become locally extinct due to overharvesting [C. ernesti-augusti in some parts of Guatemala [Given the genetic pattern of high local inbreeding, and the potential importance of connectivity and migration through long distance dispersal in per year . Sustainper year . Decreasrvesting ,106, whiuatemala , documenChamaedorea ernesti-augusti is characterized by a combination of high levels of regional gene flow, moderate to high allelic richness, ongoing local genetic differentiation, and low levels of heterozygosity. These patterns are in part explained by the tenfold reduction in its effective population size during climate change in Belize at ca. 10,000 BP, and by reproductive and demographic patterns within populations. In C. ernesti-augusti, high gene flow patterns with ongoing differentiation and historical changes in effective population size probably reflect the complex composition of old and new forests in Belize. We suggest that species like C. ernesti-augusti have probably survived recurrent habitat changes by a combination of long-distance dispersal that maintains genetic connectivity, and reproductive and demographic patterns that maintain allele richness at a local spatial scale. We hypothesize that thrips may actually be a critical driver of genetic patterns observed within populations in this genus, while seed dispersal by birds or mammals probably plays an important role across regions.C. ernesti-augusti is inbred, inbreeding depression is probably offset over time by the overall genetic connectivity across regions, which may have allowed this species to survive deforestation patterns during the Mayan occupation of Belize. Populations in Belize can be roughly divided into five genetic groups clustered into south and south, which correspond to geographic barriers, including differences in soil composition. Although the detrimental effects of a future reduction in gene flow should not be discounted, changes in demography due to overharvesting or localized ecological degradation are more likely to be critical for preventing genetic erosion in C. ernesti-augusti. Characterization of the direct relationship between demographic and genetic patterns, estimates of seed and pollen-mediated gene flow, and the population genetics of Central American and Mexican populations, will offer additional insights into the effects of regional and localized environmental changes through time, and shed light into specific mechanisms, such as long-distance seed dispersal. We are currently investigating genetic patterns in congeners in Belize and C. ernesti-augusti in Mexico, to determine whether the patterns observed in the present study are consistent across the species and in other species of Chamaedorea, or are unique to Belizean populations of C. ernesti-augusti.Although C. ernesti-augusti across Belize were sampled for a total of 138 adults , and observed heterozygosity (Ho) corrected for sample size [Genetix v4.05 [Micro-checker v2.2.1 [QIAGEN DNEasy Mini kits  were used to isolate DNA from leaf tissue. We characterized nine nuclear microsatellites to measure genetic variation following Cibrián-Jaramillo et al . Datasete Toolkit. Linkagee Toolkit and devie Toolkit, therefo[Arlequin. Heterozple size . All genix v4.05 . The prer v2.2.1 , which cr v2.2.1 .C. ernesti-augusti suffered a bottleneck or a founder effect after climate change in the Holocene and/or after Mayan deforestation, populations should show a bias in the distribution of allele frequencies. Alleles at low frequency (<0.1) would be less abundant and rare alleles would be lost, often resulting in an excess of heterozygotes [AT), the number of alleles with a frequency greater than 5% (AT5%), less than 50% (AT<50%), and the number of private alleles (AP) [et al [If the effective population size of ozygotes ,116. Allles (AP) . The dis) [et al .C. ernesti-augusti in Belize together) of size N1 that changed in size exponentially or linearly in the past (ta generations ago) up to the current population of size N0. The posterior distribution of the demographic and genealogical history is given by a prior distribution of demographic and mutational parameters. These are tf = ta/N0, r = N0/N1 and q = 2N0m, where m is the locus mutation rate, tf is the time at which the population started changing in size, scaled by N0, the population size at present (time of sampling), and N1 is the population size in the past. Given the lack of information on the evolution of microsatellites in palms, we varied the microsatellite mutation rate  in order to examine the congruence of posterior estimates of N1 to N0, and t. We also varied the generation time with five and 10 years using a mutation rate of 10-3. We used the exponential change model with the remaining parameters as defaults for all runs. An exponential model is more suitable for modelling population changes over a shorter time scale (e.g. as a result of a sudden expansion). Each run was conducted for 200 million generations with a 104-step thinning interval.We also examined traces of a major population change and, most importantly, the time in generations since the population size changed, using the coalescent-based Markov chain Monte Carlo method  implemenN1. Those were established as 4.0 3 0 0.5; 3.5 3 0 0.5; 3.0 3 0 0.5; 3.0 3 0 0.5; and 3.0 3 0 0.5, for αN1, σN1, βN1, and τN1, respectively. In order to examine the congruence among independent runs with the same initial assumptions, we chose the scenario with m = 10-3 and a generation time of 10 years, and conducted five independent runs with the remaining parameters as described. We chose 10 years to take into account the fact that not all individuals reproduce yearly, not all seeds survive and seeds are sourced from a few plants [.We used a 50% burn-in before combining the posterior parameter estimates. Priors were the same across all runs except for the hyperprior means and variances for log w plants . PosteriST[f-statistics [Arlequin.We estimated an overall measure of differentiation across all localities using θST. To testatistics  with PilK) given the sampled genotypes. All individuals were clustered first without providing information on their geographic origin. Ten independent runs were performed, with values of K ranging from 1 to 10, a burn-in of 200,000 generations and a subsequent 300,000 Markov chain Monte Carlo (MCMC) steps. Allele frequencies were assumed to correlate . Because we assumed that each individual draws a fraction of its genome from each of the K clusters, we set a uniform prior on admixture and α  as recommended by [FST <0.05) [a priori information on geographic origin, and a third hierarchical analysis run was used to test the most likely number of clusters within each expected north and south region, until a single cluster was found (K = 1). θST was calculated within localities that had subpopulations. In all sets of runs, the most probable number of genetic clusters was determined from the approximation of the posterior probability for different values of K from independent runs for each K. The lowest log probability of the data Pr (X|K), in combination with the smallest variance for each value of K, was used as a measure of the most likely number of clusters, as recommended by [K) was also used as an estimator of the most likely number of clusters [STRUCTURE  was usedended by . AdmixtuT <0.05) . A seconended by . The secclusters .m, using a coalescent approach. This approach assumes that the estimated clusters are a result of gene flow in very recent generations [C. ernesti-augusti could decrease the accuracy of each estimate, but not necessarily change the relative proportions of m among localities.We tested for recent subdivision using BayesAss+ v1.3 . This meerations . Defaulterations . The preet al [Small and contrasting sample sizes may confound estimates in this method, so we compared BayesAss+ with individual assignments using the frequency criterion of Paetkau et al , implemeet al . The freet al .[Limited dispersal can result in a pattern of genetic differentiation that increases with geographic distance. Isolation by distance (IBD) was tested with a Mantel test for matrix correspondence ,126. Gen.ACJ participated in the design of the project, performed the microsatellite genotyping, analyzed the data, and wrote most of the manuscript. CDB participated in the project design, aided in laboratory work, and contributed to the writing and revision of the manuscript. NCG, SB, and RB initiated the study and participated in its design, the collection of samples, the interpretation of the results, and the revision of the manuscript. SR and MMT participated in the collection of samples, DNA extractions, and preliminary experimental protocols. DB guided and developed preliminary laboratory protocols and contributed to the writing and revision of the manuscript. WJH and RD provided financial support, laboratory facilities, and assisted with interpretation of the results.AMOVA in south and south regions. AMOVA [s. AMOVA  among reClick here for fileK = 5Graphical output of Structure for . The most likely number of clusters estimated without a priori information on population origin. Results for five genetic clusters (K = 5) from a run of K = 1 to K = 10. Bars represent the proportion of assignment of each individual to a genetic cluster (membership coefficient Q).Click here for fileGenetic clusters estimated within south and south regions. Box plots of the likelihood lnP(X|K) across numbers of clusters without a priori information on population origin for A) North and B) South of Belize. The line inside the box represents the median of the likelihood distribution, the box lower and upper edges represent the 25th and 75th percentiles, respectively, the bars represent the 5th and 95th percentiles, and open circles are far outside values.Click here for file


We have developed a spatially explicit model of a biological population to study the emergence of spatial and temporal patterns of genetic diversity in the absence of predetermined subpopulation boundaries. We propose a 2-D cellular automata model showing that an initially homogeneous population might spontaneously subdivide into reproductively incompatible species through sheer isolation-by-distance when the viability of offspring decreases as the genomes of parental gametes become increasingly different. This simple implementation of the Dobzhansky-Muller model provides the basis for assessing the process and completion of speciation, which is deemed to occur when there is complete postzygotic isolation between two subpopulations. The model shows an inherent tendency toward spatial self-organization, as has been the case with other spatially explicit models of evolution. A well-mixed version of the model exhibits a relatively stable and unimodal distribution of genetic differences as has been shown with previous models. A much more interesting pattern of temporal waves, however, emerges when the dispersal of individuals is limited to short distances. Each wave represents a subset of comparisons between members of emergent subpopulations diverging from one another, and a subset of these divergences proceeds to the point of speciation. The long-term persistence of diverging subpopulations is the essence of speciation in biological populations, so the rhythmic diversity waves that we have observed suggest an inherent disposition for a population experiencing isolation-by-distance to generate new species.A commonly held view in evolutionary biology is that  A commonly held view in evolutionary biology is that new species form in response to environmental factors, such as habitat differences or barriers to individual movements that sever a population. We have developed a computer model, called EvoSpace, that illustrates how new species can emerge when a species range becomes very large compared with the dispersal distances of its individuals. This situation has been called isolation-by-distance because remote parts of the range can take different evolutionary paths even though there is no particular place where we would expect different populations to separate. When the extent of genetic difference between individuals is coupled with decreasing offspring viability , EvoSpace predicts that sharp spatial boundaries can emerge in arbitrary locations, separating subpopulations that occasionally persist long enough to become reproductively incompatible species. The model shows an inherent tendency toward spatial self-organization, in contrast with the traditional view of environmentally forced origins of new species. We think that isolation-by-distance is a common aspect of the evolutionary process and that spatial self-organization of gene pools may often facilitate the evolution of new species. Allopatric speciation, in which a species range becomes severed and leads to population fragments that are not linked by gene flow, has been viewed as the most common means of speciation The most common framework for understanding the process of biological speciation is geographical. For example, instances of speciation are typically allocated among three categories based on the extent of geographical separation between the daughter species. parapatric speciation, one species becomes two where the daughter species occupy contiguous ranges. This has most often been modeled as a consequence of habitat variation and divergent local adaptation by subpopulations e.g., Sympatric speciation, in which the ranges of the daughter species overlap, has similarly been modeled as a consequence of microhabitat variability and niche-partitioning e.g., The two other geographical categories of speciation involve divergence between subpopulations in the face of gene flow, and it has been less clear what the compelling “fingerprints” of these processes might look like when observed after the fact. In the second category, A common theme among all three of these categories is that speciation is induced by divisive, external factors and that the inherent tendency of biological populations is to remain unified in the absence of these factors. In other words, the conventional wisdom is that it is the environment that tears species apart, and that in the absence of local dispersal barriers or environmental heterogeneity, biological populations tend to sustain functional and genetic cohesion. One well-known, but rare, situation where this view breaks down is in the case of ring species Isolation-by-distance can also lead to symmetry-breaking in the distribution of genetic variation across the species range, leading to the emergence of discretely different and spatially segregated subpopulations Most previous spatial models of speciation have assumed predetermined subdivisions , habitat variation-inducing localized selection differences, or both N×N grid, where cells were either unoccupied or occupied by one individual. An individual contained genetic information in the form of a set of chromosomes and could migrate and mate with other individuals within a certain distance. A chromosome consisted of a string of characters from the set {A, C, G, T} representing the nucleotide bases. The number and length of chromosomes were the same for all individuals. During mating, an offspring was constructed by randomly selecting and combining two haploid genomes from the two diploid parents, possibly introducing random mutations in the process. Thus reproduction in our model was sexual, because genomes from two parents were combined to produce the offspring and the two genomes within a parent exhibited recombination through the independent assortment of chromosomes during gamete formation; but individuals were also hermaphrodites, as any adult could potentially mate with any other adult.We have implemented a generalized cellular automaton model of evolutionary processes, called EvoSpace. The simulated population was distributed across an dispersal distance. Then, they could choose a mate within similar bounds. Finally, after mating, each offspring was placed in a random cell in the vicinity of one of the parents while the parents died, so that only one generation lived on the grid at each time step. The mean reproductive rate in the population was regulated each generation to buffer swings in population size resulting from a variety of factors, such as stochastic mortality (see below). This was achieved by randomly removing individuals or generating additional offspring, reflecting a constant carrying capacity of the environment. Simulations began with a population of genetically identical individuals, amounting to 80% of the grid cells (20% of locations remained empty space), and the system was allowed to evolve for hundreds to millions of generations.At every generation (time step in the model), migration, mating, and mutations created genetically distinct offspring, but resulted in only minor changes in the spatial structure of population genetic variation. First, individuals could randomly move on the grid world within bounds determined by a survival probability was a decreasing function of the genetic difference between merging gametic genomes. Expressing the genetic difference between two chromosomes as a fraction between 0  and 1 , offspring resulting from gametes with a genetic difference greater than a threshold θ had zero survival probability . The negative relationship between gamete genomic difference and offspring viability was a simple representation of Dobzhansky-Muller reproductive incompatibility. Orr For the experiments described in this paper, the habitat across the grid environment was homogeneous, so location did not influence the fitness of individuals. However, we introduced one important dependency: the offspring's The outbreeding depression function was central to the exploration of speciation in this model, as reproductive isolation between sexual species lies at the core of the concept of speciation. It was, in fact, the fundamental criterion embodied in the definition most commonly assumed in the context of evolutionary biology: the Biological Species Concept (BSC) δ = 1.5, and 20 cells when δ = 5 , because of the 25% probability that two bases were identical at any particular position of the DNA sequence. In the case of a population of genetically identical genomes—the starting condition for our simulations—the plot showed a single sharp peak at 0. As mutation led to genetic divergence, peaks traveled to the right in the mismatch distribution.To assess the pattern of genetic diversity in our model system, we measured the genetic difference between two randomly selected, haploid gametes, as though these gametes were about to merge in fertilization and produce more or less viable offspring. We then analyzed this information with δ = 5), genetic differences within the population did not grow far beyond the θ0 = 0.05 threshold, where outbreeding depression began to impact offspring viability. Under these conditions, the population mixed across the grid rapidly enough to remain a single, genetically coherent population. This was represented as a distinct and stable peak in the histogram at a genetic difference level of 5%. No pair of genomes was found with a genetic difference greater than 10% when δ = 5. All aspects of the model's behavior described here were repeatable for different runs of the model under the same conditions.Observing time-sequence movies of these mismatch distributions under different model conditions illustrated the spatiotemporal patterns of gain and loss of genetic diversity, especially as it revealed the origin and existence of distinct subpopulations . The series of snapshots in δ = 1.5, but in the absence of outbreeding depression , representing all within-subpopulation comparisons. In effect, each emergent subpopulation functioned like a single panmictic population. The traveling waves peeled off this peak as existing subpopulations divided. As these peaks moved to the right of θ0, the subpopulations being compared experienced an increasingly stringent demographic disadvantage, because when individuals from diverging subpopulations mated with each other, their offspring were decreasingly viable. Divergence continued as the demographic cost of outbreeding increased. Nevertheless, sometimes a peak became established to the right of θ. This represented a set of comparisons between gametes from reproductively isolated subpopulations, since their hybrid offspring could not be viable anymore. In summary, we interpret the traveling waves to reflect discrete genetic subpopulations, new species when they persisted past θ = 0.6, emerging through the spatial microevolutionary dynamics within a population.The patterns that we detected in intraspecific dynamics were greatly enhanced by combining the outbreeding depression function with the shorter dispersal distance . Along wθ  was quite sensitive to the interrelationships of the spatial scale of the simulation , the mutation rate, and other factors. For example, if the mutation rate was too high, overall genetic diversity increased rapidly until it was too hard to find viable mating pairs within the mating neighborhood and the whole population went extinct. If the mutation rate was too low, the degree of genetic difference generated between subpopulations did not reach the threshold of speciation before at least one of the subpopulations went extinct. We are working now to examine systematically the likelihood that biological populations would evolve within the region of phase space associated with these interesting dynamics.We have further found that the development of new, stable peaks to the right of genetic cluster plots  gene pool mixing and outbreeding depression under isolation-by-distance. The emergence of sharp boundaries between genetic subpopulations is consistent with previous computational models incorporating isolation-by-distance isolation by distance. there is complete continuity of distribution, but interbreeding is restricted to small distances by the occurrence of only short range means of dispersal. Remote populations may become differentiated merely from The presumed pattern of isolation-by-distance is a smooth, monotonically increasing relationship between geographic distance and genetic distance The model of evolutionary genetics presented here is a very simple and generic one. It does not depend on idiosyncratic forms of selection or particular population structures. Instead, it is based on fundamental and common building blocks of biological populations  that are stochastically affected by mutation, mortality, reproductive success, and dispersal. The interesting behavior of the model emerges dynamically due to the constraints of isolation-by-distance and outbreeding depression. Therefore, we conjecture that the tendency for spatial self-organization and parapatric speciation may occur universally in biological populations. This is not a claim that our model is the exclusively correct model of speciation; rather, we are suggesting that the dynamic of diversification illustrated by our model may exist even under conditions that suppress the realization of emergent population substructure, such as great dispersal distances in relatively small species ranges. We expect that the inherent tendency for spatial diversification amplifies the effects of environmental heterogeneities, and we plan to explore this interaction with further developments of EvoSpace. Thus we do not deny that habitat variation and dispersal barriers can play important roles in instances of speciation, but we think that the inherent dynamic identified here may generally drive diversification/speciation in a way that is molded to these external constraints.In conclusion, our model reveals an aspect of intraspecific evolutionary dynamics that emerges when dispersal distances are sufficiently short relative to a species range. Localized subpopulations regularly form and diverge from one another while maintaining their identities as clusters of genetically similar individuals. Subpopulations that grow so large as to embody too much genetic diversity tend to subdivide through spatial segregation, just as the original population does in our model. If the degree of outbreeding depression grows as gametes' genomes become increasingly different, the pattern of genetic and spatial population subdivision becomes better defined, and some subpopulations can diverge to the point where they complete a process of parapatric speciation. The behavior of our model suggests that spatially extended populations regularly generate new subpopulations, each of which takes a path of genetic divergence with the potential of becoming a new species. Most of these embryonic subspecies become extinct before emerging as reproductively independent species, but the internal dynamic of this model constantly potentiates the production of new species.N×N grid that wraps around north-south and east-west, creating a torus. Typical grid sizes are 100×100 to 500×500 cells . Grid coordinates are Cartesian pairs r = , and distances between cells are simple Euclidean distances from the cell centers. Thus each cell has exactly four neighbors at distance 1, four more neighbors at distance √2, and so on. A simulation parameter δ correlates with the distance within which individuals can move, mate, or appear in a single generation. As the value of this parameter increases, so too does the mobility of the individuals within the grid world. Movements of individual genomes, carried by either diploid individuals or haploid gametes, occur in three stages: migration, mating, and offspring placement. (a) To begin with, each individual migrates to another site randomly determined by the combination of a Gaussian and a uniform probability distribution. Denoting by r the starting location of an individual, its new location r′ after migration is calculated in two steps. First, a distance d is drawn from a Gaussian distribution of mean and standard deviation δ: δG(d) = κ exp[−(d−δ)2/δ2], where κ is a normalization coefficient. Second, the location r′ is drawn from a uniform distribution within a disc D of radius d centered around r: migP(r′|r) = dD(r′−r), where dD(u) = 1/πd2 if ||u||≤d and 0 otherwise.  (b) Then, each individual is considered in turn to act as the “father” and sends a gamete to a potential “mother” r″ at a location chosen uniformly randomly in a circle of radius δ centered around the father's location r′: matP(r″|r′) = δD(r″−r′). (c) Finally, a newly formed offspring (see next section) settles into a grid cell r′′′ drawn uniformly randomly in a circle of radius 2δ around its mother's position offP(r′′′|r″) = 2δD(r′′′−r″).The simulated world is an n and length l (number of base pairs) of chromosomes are the same for all individuals and are both set at the start of a simulation; n = 2 and l = 200 for all results presented here. Reproduction in our current model is sexual, because genomes from two parents are combined to produce the offspring, but individuals are also hermaphrodites able to function as either the male or female in a sexual encounter. An offspring is constructed from two parents as follows. Each parent produces a haploid gamete by randomly selecting one of the chromosomes from each diploid pair . The number oid pair . These hgenetic difference H between the two merging gametic chromosomes in a diploid pair is expressed as the fraction (between 0 and 1) of all the base pairs containing different bases: H = B/nl. Thus H = 0 means that all bases in the child's gametes #1 and #2 are identical at every position, and H = 1 means they all differ at every position. The offspring's survival probability S can be set to be dependent on this genetic difference, according to the curve in θ0 and θ, such that 0≤θ0≤θ≤1, we set S = 1 for H≤θ0, S = 0 for H≥θ, and S = (θ–H)/(θ–θ0) for θ0<H<θ. Thus offspring composed of gametes with a genetic difference H greater than θ are nonviable and immediately removed from the grid at birth. In general, two random genome sequences are expected to have a genetic difference H = 0.75, since each nucleotide position would have a 25% chance of being occupied by the same base The The simulation begins at generation 0 with a population of genetically identical individuals. The initial population randomly fills a predetermined fraction of the maximum grid occupancy. For the experiments described in this paper, the grid occupancy fraction is 80%. For example, a grid of 400×400 = 160,000 cells starts with 128,000 individuals.migrates to a random cell r, according to the probability δG described above. (b) Then, individuals reproduce. The algorithm iteratively considers each member of the population once to be the “father” in a mating. For each father, a list of potential mates is created from those individuals in all cells within a radius δ of the father, and one of those individuals is randomly selected to be the “mother.” Mating then proceeds as described above. The number of potential offspring resulting from each mating is given by a Poisson distribution with mean 1. (c) Finally, each offspring is placed in a random cell within a radius 2δ of the mother. If no cell within this range has room for the offspring according to the maximum per-cell occupancy m, then some or all offspring of this mating could be lost. No parents survive into the next generation, so overcrowding can be the result only of offspring from matings that have already taken place during earlier processing of the current generation. Since each individual is the father in exactly one mating, and the mother in an average of one mating, and each mating has an average of one offspring, the population size should stay about the same from generation to generation. Certain factors, however, could result in the population size shifting from the target size, which is addressed in the next phase.The creation of the next generation for the simulation is a three-phase process. (a) First, each individual adjustment, the algorithm makes an effort to keep the population close to the initial target population size. This may require additional “make-up” births from randomly selected parent pairs, if offspring viability reduced the population size due to high genetic difference of parents, or if localized overcrowding resulted in the loss of some offspring. More rarely, if the population ends up above the target level, the algorithm randomly selects individuals for culling.In a supplementary step, Given any two diploid individuals on the grid, we draw one haploid genome from each individual by randomly selecting one chromosome in each diploid pair they contain . The genetic distance (as plotted in θ0 (0.05 for the results presented here) representing the within subpopulation comparisons. However, if clusters B and C also happened to be 0.4 distant, two peaks would be superimposed and would obscure the number of subpopulations. A peak typically represents a set of comparisons between two subpopulations defining the degree of divergence between the subpopulations. The peaks move to the right in the mismatch distribution as long as both subpopulations persist and evolve along different trajectories, and the peaks stop moving when divergence hits the maximum value of 75% expected under the Jukes-Cantor mutation model A mismatch distribution reveals the shape of genetic diversity in a sample through pairwise comparisons of genetic differences, which are displayed in a frequency histogram x-axis and genetic distance on the y-axis Click here for additional data file.Video S2Short dispersal distance without outbreeding depression(7.23 MB MPG)Click here for additional data file.Video S3Longer dispersal distance without outbreeding depression(6.15 MB MPG)Click here for additional data file.


F. paralugubris.Animal societies are diverse, ranging from small family-based groups to extraordinarily large social networks in which many unrelated individuals interact. At the extreme of this continuum, some ant species form unicolonial populations in which workers and queens can move among multiple interconnected nests without eliciting aggression. Although unicoloniality has been mostly studied in invasive ants, it also occurs in some native non-invasive species. Unicoloniality is commonly associated with very high queen number, which may result in levels of relatedness among nestmates being so low as to raise the question of the maintenance of altruism by kin selection in such systems. However, the actual relatedness among cooperating individuals critically depends on effective dispersal and the ensuing pattern of genetic structuring. In order to better understand the evolution of unicoloniality in native non-invasive ants, we investigated the fine-scale population genetic structure and gene flow in three unicolonial populations of the wood ant F. paralugubris. Because of this spatial genetic heterogeneity, members of the same clusters were moderately but significantly related. The comparison of nuclear (microsatellite) and mitochondrial differentiation indicated that effective gene flow was male-biased in all populations.The analysis of geo-referenced microsatellite genotypes and mitochondrial haplotypes revealed the presence of cryptic clusters of genetically-differentiated nests in the three populations of The three unicolonial populations exhibited male-biased and mostly local gene flow. The high number of queens per nest, exchanges among neighbouring nests and restricted long-distance gene flow resulted in large clusters of genetically similar nests. The positive relatedness among clustermates suggests that kin selection may still contribute to the maintenance of altruism in unicolonial populations if competition occurs among clusters. Many social animals live in small family groups of closely related individuals, and the role of kinship in promoting the evolution of reproductive altruism and eusociality by kin selection has long been recognized -4. HowevThe evolution and maintenance of unicolonial populations constitutes one of the enduring major challenges for kin selection, and more generally for modern, gene-centred evolutionary theory -9. This Linepithema humile . Moryrhachis ,22,29,32Hamilton  first prThese models show that the impact of population genetic structure on the evolution of altruism critically depends on specificities of the life-cycle, and in particular on the patterns of dispersal and behavioural interactions of queens, males and workers. It is therefore important to obtain detailed empirical data on sex-specific dispersal and on cryptic genetic discontinuities that may correspond to cooperative units in unicolonial species. Such data will be useful to develop new models and design behavioural studies that will help to assess the role of kin selection in structured population.Formica paralugubris, a wood ant native to the European alpine area. Each nest contains hundreds of reproductive queens .The dispersal behaviour of males has been little studied so far. They may mate within their natal nest or fly away to mate on open meadows, or possibly in other nests . In addiF. paralugubris forms unicolonial populations, as demonstrated by the very high number of queens per nest, movement of queens and workers among neighbouring nests, and lack of aggression between workers from distant nests within populations , the region of NADH dehydrogenase from subunit 4 to subunit 6  [GenBank: EU600793], and the region from ND6 to ND1, which includes the cytochrome b (2427 bp and 2430 bp) [GenBank: EU600789–EU600792]. The primers used to amplify the DNA are listed in Table Apis mellifera, Formica fusca and F. truncorum, except for the primer COI-RLR-F which came from Roehrdanz & Degrugillier . Indivirnestmates (population) and rclustermates (population) estimate the probability by which an allele is more likely to be shared among nest- and clustermates than expected from the allele frequency in the population to which the nest or cluster belongs. The relatedness rnestmates (cluster) is the probability by which an allele is more likely to be shared among nestmates than expected from the allele frequency in the cluster.The relatedness among worker nestmates was estimated with the computer program Relatedness 5.0.8 . Nests wF-analysis of variance, as implemented in the computer program Hierfstat  - [(1 - Fst mit)/Fst mit)] by applying Wright's island model [Nf mf and Nm mm should not be considered as actual number of migrants [The use of both nuclear and mitochondrial markers allowed us to roughly estimate the relative contribution of each sex to gene flow. We estimated the gene flow among nests within populations from the genetic differentiation among all nests within each population at the biparentally-inherited nuclear microsatellites (by 1-Hs) ,47, we cä et al. , with thFst mit) , where Nnd model  modifiednd model . Becausemigrants , but as BH, LK and MC jointly conceived, designed and coordinated the study. BH carried out the sampling and laboratory analyses, analyzed the data and wrote a first draft of the manuscript. LK and MC supervised the analyses and MC finalized the manuscript. All authors read and approved the final manuscript.


Rana chensinensis) to test whether the Tsinling Mountains and the nearby Daba Mountains impose major barriers to gene flow.Amphibians in general are poor dispersers and highly philopatric, and landscape features often have important impacts on their population genetic structure and dispersal patterns. Numerous studies have suggested that genetic differentiation among amphibian populations are particularly pronounced for populations separated by mountain ridges. The Tsinling Mountain range of northern China is a major mountain chain that forms the boundary between the Oriental and Palearctic zoogeographic realms. We studied the population structure of the Chinese wood frog (FST values ranging from 0.0175 to 0.1625 with an average of 0.0878. Furthermore, hierarchical AMOVA analysis attributed most genetic variation to the within-population component, and the between-population variation can largely be explained by isolation-by-distance. None of the putative barriers detected from genetic data coincided with the location of the Tsinling Mountains.Using 13 polymorphic microsatellite DNA loci, 523 individuals from 12 breeding sites with geographical distances ranging from 2.6 to 422.8 kilometers were examined. Substantial genetic diversity was detected at all sites with an average of 25.5 alleles per locus and an expected heterozygosity ranging from 0.504 to 0.855, and two peripheral populations revealed significantly lower genetic diversity than the central populations. In addition, the genetic differentiation among the central populations was statistically significant, with pairwise R. chensinensis. High population connectivity and extensive juvenile dispersal may account for the significant, but moderate differentiation between populations. Chinese wood frogs are able to use streams as breeding sites at high elevations, which may significantly contribute to the diminishing barrier effect of mountain ridges. Additionally, a significant decrease in genetic diversity in the peripheral populations supports Mayr's central-peripheral population hypothesis.The Tsinling and Daba Mountains revealed no significant impact on the population genetic structure of  FST value of 0.224 in a toad species  among populations separated by less than 16 kilometers [Amphibians in general are poor dispersers and highly philopatric with strict habitat specificity and physiological requirements ,2. Pond lometers .Rana luteiventris) highlighted the impact of mountain ridges on amphibian population structure [Genetic differentiation among amphibian populations appears to be relatively high for populations separated by mountain ridges -11. Usintructure . They fotructure . Evidenctructure .The Tsinling Mountain range in northern China is one of the few major mountains on Earth that extend from east to west. With the combined altitudinal and latitudinal differences, a great reduction in gene flow across the range is expected. Its peak, Mt. Taibai, reaches 3767 meters above sea level (a.s.l.) and most parts of the ridge are higher than 1800 meters a.s.l. The range divides two major water drainages in East Asia, the Yellow River system to the north and the Yangtze River system to the south. The fauna at the northern and southern sides of the Tsinling Mountains is so different that the Tsinling is recognized as the boundary between the Oriental and Palearctic zoogeographic realms . ApproxiRana chensinensis) is a common species in central and northern China with a wide distribution , which are located at the periphery of the species' distribution  and the central sites were generally higher, ranging from 0.1206 to 0.3130  values against geographical distance (in kilometers) is presented in Figure PMantel < 0.001, R2 = 0.3116). The two peripheral populations (11 and 12) had reduced genetic diversity and thus high pairwise FST values with the central populations, and they also had large geographical distances from the central populations. Therefore, including them in the analysis might have artificially enhanced the correlation. To further test the impact of the Tsinling Mountains and eliminate the potential impact of the peripheral populations, a second IBD analysis that included only the nine Tsinling sites was conducted. It also showed a weak but significant positive correlation  [Rana temporaria across Sweden [HE of 0.504 to 0.855 in different populations.A high level of genetic diversity was detected at microsatellite DNA loci in Chinese wood frogs. Two commonly used indices of genetic diversity, allelic diversity and heterozygosity, are comparable to or higher than those found in other similar studies of anurans. For example, Funk et al. (2005) found 5 – 16 alleles per locus and expected heterozygosity of 0.23 – 0.66 in various populations of the Columbian spotted frog (ventris) , while Js Sweden . We founFST ranging from 0.0175 to 0.1625, and with an average of 0.0878  from different sides of mountain ridges within distance of only 10 kilometers [FST values up to 0.453 between populations within a major mountain valley and a distance of 60 kilometers in tiger salamanders (Ambystoma tigrinum) [FST values were observed when the two peripheral sites were also included (up to 0.3130), these values may have been inflated by the low genetic diversity of the peripheral sites.Low to moderate genetic differentiation between the ten central sites was observed, with pairwise igrinum) . While hNm of 0.03 to 0.34 between sites within distance of 15 kilometers for the natterjack toad  [FST values. Hedrick (1999) analytically demonstrated that, when the amount of within-population variation becomes high, the FST values necessarily become low [A moderate to high level of migration is the most likely explanation for the low to moderate differentiation between sites. Despite geographical distances of 50 – 100 kilometers and a major mountain range between them, the migration rates between site-pairs from different sides of the Tsinling Mountains ranged from 0.17 to 1.26 individuals per generation with an average of 0.79  . Additiocome low . Other ecome low  whereas come low , and theThe Tsinling and Daba Mountain ranges do not present significant barriers to gene flow in Chinese wood frogs. Both AMOVA and Monmonier's maximum difference algorithm have proven to be powerful tools for detecting the impact of landscape features on population genetic structure ,23. NeveNm values in this study support this explanation  is probably the primary cause of the diminished mountain effect, and extensive juvenile dispersal may be responsible for such gene flow. Most mature female Chinese wood frogs produce 400 to 900 eggs annually, with some producing up to 2000 eggs, which is higher than other closely related wood frog species ,24. Thusmporaria , althougmporaria . Funk etmporaria .Chinese wood frogs are capable of using slow moving mountain streams as breeding sites at high elevations and are not restricted to breeding in ponds. For example, at one of our collecting sites, site 12, the frogs bred in a stream at an elevation of 2480 meters a.s.l. This life history trait is perhaps crucial for the Chinese wood frog to maintain a low to moderate population differentiation with the presence of major mountain ridges. Mountain ridges have significant impact on pond breeder population structure, which is well established by numerous studies -11. NevePopulations at the periphery of a species' distribution tend to have lower genetic diversity than the central populations , and LesRana chensiensis). Some amphibian species, particularly those with large numbers of juveniles, may have high population connectivity and hence be able to maintain relatively large gene flow across natural or man-made geographical barriers. Mountain ridges likely only impose a weak barrier effect to stream breeders. Life history traits and properties related to population genetic structure should be carefully evaluated before using amphibian species to assess environment quality, such as habitat fragmentation [Our study presents an exception to the currently accepted hypothesis that mountain ridges impose major barriers to amphibian movement. The Tsinling Mountains surprisingly demonstrated no statistically detectable barrier to the gene flow among populations of the Chinese wood frog  were collected between the1st and 10th of April, 2008. The Chinese wood frog typically takes two  to three  years to reach sexual maturity [Samples from twelve breeding sites were collected from the Tsinling and Daba Mountain region. Eight  were collected between the 1maturity  and the Adult frogs were collected from a single pond at each site during their breeding season. Two toes from two limbs were clipped from each individual and the toe samples were preserved in 95% ethanol. Ten individuals from each site were euthanized and preserved as reference voucher specimens. All other frogs were immediately released at the point of capture after toe clipping. The toes from a total of 523 individuals were collected for this study and the detailed sample site and sample size information is provided in Table 2+, New England Biolabs), 200 μM of each dNTP, 0.25 μM of each primer and 0.5 U of Taq DNA Polymerase™ (New England Biolabs). The PCR cycling parameters were: 95°C for 5 minutes, then repeated 30 times at 95°C for 30 seconds, 52°C for 30 seconds and 72°C for 30 seconds, and a final extension at 72°C for 5 minutes. The PCR products were denatured and electrophoresed on 6% denaturing polyacrylamide gels and visualized on a FMBIO laser scanner (Hitachi). The alleles were scored relative to a TAMRA™ size standard marker  and against the frog sample that was used to design the primers [Total genomic DNA was isolated from the toe samples using the standard phenol/chloroform method. Polymerase chain reaction (PCR) amplifications were performed using thirteen  polymorphic microsatellite DNA markers developed by Zhan & Fu (2008) . One pri primers .A) and their frequency (F), the observed heterozygosity (HO) and the expected heterozygosity (HE) were calculated using the computer program POPGENE 32 [Ar) [) [Genetic diversity within sites was measured with five indices. The number of alleles  , which i 32 [Ar) . In addi 32 [Ar)  was empl [Ar) [) . Signifi [Ar) [) .FST (= θ) [FST values were calculated using ARLEQUIN. Pairwise significance tests for FST were performed by permutation and resampling of multilocus genotypes among pairs of samples. To ensure small standard deviations, 10000 permutations were performed to allow for the significance at the 1% nominal level after sequential Bonferroni corrections. Additionally, difference in allelic richness (Ar) and expected heterozygosity (HE) between sites was also tested using a non-parametric test (Mann-Whitney U test) in SPSS version 12 for Windows. The statistical significance for population pairs was evaluated at the level of 0.05 after sequential Bonferroni correction.The degree of population subdivision was determined from mutilocus estimates of ST (= θ)  for all Nm) between sites were calculated based on multilocus genotypes with Bayesian inference using the program MIGRATE (version 2.4.1) [θiMi, where θi equals xμ and Mi equals mi/μ. Among the parameters, x is the inheritance parameter; μ is the mutation rate per locus per generation; and mi is the immigration rate. For our analysis, x was set as 4, the value commonly used for nuclear gene data, and other parameters were estimated from the data by the program. A Brownian motion mutation model was used. We used 10 short chains  and 3 long chains  with 50,000 iterations discarded as an initial 'burn-in' for the Bayesian search strategy. The replicates index was set as '= YES: 5' and the randomtree index was set as '= YES'. A static heating scheme was employed with 'heating = ADAPTIVE: 1{1.0 1.5 3.0 6.0}'. The simulation was repeated 5 times to ensure consistency. For an initial run, we used θ (FST) to find the starting parameters. In the subsequent runs, we changed the random number seed and the Bayesian estimates of θ and M from the previous run as the new starting values. The results from the separated runs were nearly identical and the estimates from the last run were used.To illustrate the dispersal patterns between breeding sites, the migration rates (n 2.4.1) . The numFST/(1-ST F) [Isolation by distance (IBD) was examined by testing the correlation between Rousset's (1997) (1- FST)  and geogThe impact of mountains on population structure was tested with a locus by locus analysis of molecular variance (AMOVA)  using ARFST genetic matrix. Putative genetic boundaries were identified across the geographical landscapes [We also employed Monmonier's maximum difference algorithm  to highlndscapes . The datAZ performed most of the lab work, data analyses and manuscript preparation. CL collected most of the specimens. JF conceived the project. All authors contributed equally to this work in discussing research strategy and development. All authors read and approved the final manuscript.Rana chensinensis)Genetic diversity at 13 microsatellite loci for 12 sites of the Chinese wood frog  and migration rate between sitesEstimates of population genetic differentiation (pairwise . The file provided the original data of the pairwise FST and estimates of numbers of immigrants and numbers of emigrants between sites.Click here for file


Theory predicts that lower dispersal, and associated gene flow, leads to decreased genetic diversity in small isolated populations, which generates adverse consequences for fitness, and subsequently for demography. Here we report for the first time this effect in a well-connected natural butterfly metapopulation with high population densities at the edge of its distribution range.We demonstrate that: (1) lower genetic diversity was coupled to a sharp decrease in adult lifetime expectancy, a key component of individual fitness; (2) genetic diversity was positively correlated to the number of dispersing individuals  and adult population size; (3) parameters inferred from capture-recapture procedures  correlated much better with genetic diversity than estimates usually used as surrogates for population size  and dispersal .Our results suggest that dispersal is a very important factor maintaining genetic diversity. Even at a very local spatial scale in a metapopulation consisting of large high-density populations interconnected by considerable dispersal rates, genetic diversity can be decreased and directly affect the fitness of individuals. From a biodiversity conservation perspective, this study clearly shows the benefits of both in-depth demographic and genetic analyses. Accordingly, to ensure the long-term survival of populations, conservation actions should not be blindly based on patch area and structural isolation. This result may be especially pertinent for species at their range margins, particularly in this era of rapid environmental change. Habitat destruction and fragmentation result in decreased habitat patch size and increased habitat patch isolation. Consequences of this common and now well-studied phenomenon include, amongst others, modified community composition and structure, smaller population sizes and decreased population connectivity . Both poPolyommatus coridon (Poda)  situated in southern Belgium, at the northern edge of the species distribution range .P. coridon. Indeed, a longer life span will allow a greater number of matings (both sexes of P. coridon mate more than once). Furthermore, P. coridon females with a higher lifetime expectancy will lay more eggs since they lay eggs singly and continually until they die, but need to attain a certain maturity after hatching before being able to lay eggs, and lay fewer eggs in early life compared with later on )Ne) remains difficult, detecting inbreeding effects in population sizes of several hundreds to several thousands of individuals may suggest that Nes were only a tiny fraction of census population sizes (N) in this study area. The same pattern of decrease in genetic diversity in small and isolated local populations was recently detected within a metapopulation of lesser kestrel  (Fleischer) , a vagile and migratory raptor with high dispersal potential )A positive correlation between genetic diversity and mean lifetime expectancy was detected at a very local scale in a metapopulation consisting of large high-density populations interconnected by considerable dispersal rates. Even in such a system, genetic diversity can be decreased and directly affect the fitness of individuals. From a biodiversity conservation perspective, this study clearly demonstrates the benefits of both in-depth demographic and genetic analyses and highlights that in order to ensure the long-term survival of the populations conservation actions should not be solely based on patch area and structural isolation. This result may be especially pertinent for species at their range margins, particularly in this era of rapid environmental change.P. coridon populations was studied with the CMR methodology. The five study sites, delimited on the basis of favourable habitat and topographic features, were visited as often as possible (weather permitting). Every encountered imago was captured with a net, individually marked with a permanent pen and immediately released. For each (re)capture, the following data were recorded: tag number, sex, age (estimated through wing wear), date and hour, site and patch.During the entire 2003 flight period (end of June to beginning of September), the demography of five φt, capture rate pt, recruitment rate bt, daily population size Nt, and total population size N) were obtained for the five populations separately, by analyzing the large CMR data set  using the constrained linear models method, with AIC-based [Estimates of demographic parameters  is currLTEt), i.e. the number of days a newborn butterfly is expected to live, was computed from a virtual life table constructed from estimates of daily survival rate (φt) using the formula:Daily lifetime expectancy . For each butterfly population, the mean LTE at birth was computed by weighting daily values of LTEt by daily number of births . Its 95% confidence interval was computed from the distribution of 1000 random drawings of φt and Bt from their variance-covariance matrices.with Numbers of dispersing individuals (immigrants and emigrants) were calculated directly from the CMR data set. The number of immigrants was used to quantify the dispersal rate as it reflects the potential amount of gene flow between populations.8G; 816, (CA)8T; 848, (CA)8RG. Only primers with 100% reproducible bands were considered. Primers 809, 816, and 848 produced, respectively, 9, 11, and 14 bands that could be unambiguously scored, and were reproducible and polymorphic. Of the 34 polymorphic loci, four were dropped from analyses due to linkage disequilibrium. Results of analyses based on biased and unbiased allele frequencies [Samples (between 25 and 31 per population) were collected, during the 2003 field season, using a non-invasive sampling technique: a tiny fraction of the wing or one leg per butterfly was sampled and analysed using dominant DNA neutral markers, i.e. intersimple sequence repeat . Reactioquencies  were verHe, (2) average gene diversity over all loci Div, and (3) percentage of polymorphic loci PPL. Allele frequencies were calculated from null homozygote frequencies assuming panmixia and corrected for dominance [He was calculated from the null allele frequency and based on the Hardy-Weinberg equilibrium. PPL (99% criterion) and genetic structuring (θst) were calculated using the same program. We also used genotype data directly to obtain Div, the mean number of differences between all pairs of genotypes divided by the number of loci, and to derive Fst from the variance components (AMOVA) using ARLEQUIN [He and Div are independent estimates of genetic diversity since the former is based on allele frequencies and the latter on haplotype identity. He and PPL are probably correlated by nature because both depend on the presence or absence of polymorphism at the locus level. A Mantel test was used to assess the association between Nei's unbiased (1978) genetic matrix and the geographical distance matrix.Genetic diversity was quantified for each population by three measures: (1) expected heterozygosity ominance  using TFARLEQUIN . He and The following habitat patch descriptors were measured: area, connectivity, a summary variable describing microclimatic conditions, abundance of nectar sources, and host plant abundance.. Structural connectivity of a habitat patch was quantified according to its relation to all other patches in the study system:Patch area and inter-patch distances were calculated using aerial photographs (year 2000) with ArcGIS 9 software djk is the distance between patch j and patch k, and Ak the area of patch k [P. coridon study system [H. comosa abundance.where  patch k . Plant sy system . Microcly system ). This fHe, Div, and PPL) and the mean lifetime expectancy LTE, and the correlation of these four variables with (1) population size N and dispersal rate (number of immigrants and emigrants), (2) surrogates of population size , and (3) surrogate of dispersal rate . As there was no a priori reason to believe that these would be linear correlations, rank (Spearman), instead of linear (Pearson), correlation coefficients were used throughout this study. No correction procedure for multiple testing has been used because we did not base our conclusions on a sharp interpretation of the significance level of each correlation, but on the general trend emerging from redundant correlations, as already mentioned.We quantified the correlation between the three estimates of genetic diversity (He) and LTE, was sensitive to the error present in the estimates of these variables. This procedure had a double aim: (1) to take into account the unequal precision of each population-specific estimate of average He and LTE, and (2) to test the robustness of the genetic data. Concerning He, a subset of the 30 loci was randomly selected and the mean computed for each population; subsets of 30 loci to only one locus were considered. On the other hand, LTE was computed for each population from randomly generated values of demographic parameters φt and Bt using their respective variance-covariance matrices. The Spearman's correlation coefficient r between population He-LTE means was then computed. This procedure was repeated 1000 times to construct the distribution of r. The mean r was reported as covariation measure between the two parameters studied. Statistical significance of this correlation (showing that it significantly differed from zero), however, was based on the probability of obtaining the observed proportion s (in the 1000 simulations) of significant correlations by chance. The distribution of s under the null hypothesis of no correlation was also obtained by resampling, with observed values for the site populations randomly shuffled, breaking any existing correlation. This shuffle procedure was repeated another 1000 times for each of the 1000 simulations and the p-value for the test was computed as the proportion of random associations with a proportion s of significant correlations greater than or equal to the observed s.However, we designed a bootstrap procedure  to assesSV and MB designed the research; SV performed the research; SV and NS analysed the data; SV, NS, and MB wrote the paper. All authors contributed equally to this work.