Cittarium pica, an abundant rocky shore trochid gastropod that is commercially harvested across its natural range, from the Bahamas to Venezuela.Regional genetic connectivity models are critical for successful conservation and management of marine species. Even though rocky shore invertebrates have been used as model systems to understand genetic structure in some marine environments, our understanding of connectivity in Caribbean communities is based overwhelmingly on studies of tropical fishes and corals. In this study, we investigate population connectivity and diversity of We tested for genetic structure using DNA sequence variation at the mitochondrial COI and 16S loci, AMOVA and distance-based methods. We found substantial differentiation among Caribbean sites. Yet, genetic differentiation was associated only with larger geographic scales within the Caribbean, and the pattern of differentiation only partially matched previous assessments of Caribbean connectivity, including those based on larval dispersal from hydrodynamic models. For instance, the Bahamas, considered an independent region by previous hydrodynamic studies, showed strong association with Eastern Caribbean sites in our study. Further, Bonaire  seems to be isolated from other Eastern sites.C. pica has some commonalities in pattern with more commonly sampled taxa, but presents features, such as the differentiation of Bonaire, that appear unique. Further, the level of differentiation, together with regional patterns of diversity, has important implications for the application of conservation and management strategies in this commercially harvested species.The significant genetic structure and observed in  Marine reserves are a primary mechanism to deal with threats facing populations and species in marine biodiversity hotspots such as the Caribbean Elacatinus evelynae, which support isolation of the Eastern and Western Caribbean regions across the Mona passage (between Puerto Rico and Hispaniola), as well as the differentiation of populations in the Bahamas from those to the south across the Exuma Passage Acropora palmata, Eastern and Western Caribbean populations appear to be genetically differentiated In the Caribbean marine province, four connectivity regions have been proposed based on complex hydrodynamic current models: Eastern Caribbean; Western Caribbean; Bahamas and Panama–Colombia Echinometra lucunter and EucidarisCittarium pica), a trochid gastropod that is a key primary consumer, distributed throughout the West Indies and on Central American shores where it inhabits crevices and small holes of exposed areas However, efforts to define connectivity through population genetic structure have been focused overwhelmingly on coral and fish populations C. pica has been included in the red list of endangered species of several Caribbean countries and territories, including Colombia, the U.S. Virgin Islands, Bermuda and Dominica C. pica is harvested for local consumption and sold as handcraft for tourists a ban for this species has not yet been established The population structure of this species is of special interest due to a short pelagic larval duration  which greatly limits the potential for dispersal, and because of economic significance, as it is commercially harvested C. pica through the analysis of mitochondrial DNA sequence variation. First, what degree of genetic structure occurs in C. pica and at what spatial scales? Do differentiated populations coincide with expectations from hydrodynamic dispersal models? Are phylogeographic breaks detected in C. pica consistent with previous data from other taxa and ecosystems, which would suggest a common underlying basis for differentiation? The existence of common breaks for Caribbean taxa supports the role of vicariance in the origins and evolution of Caribbean marine communities. Although the correlation between pelagic larval duration (PLD) and genetic structure is not always tight C. pica, often exhibit strong genetic structure. In marine animals, the extent of realized dispersal and gene flow are strongly influenced by mesoscale oceanography, the duration of planktonic larval development and larval behavior C. pica. Lastly, does a spatial trend in diversity levels occur in C. pica? For fishes and corals, because western Caribbean locations have more extensive coral reef ecosystems that may support larger population sizes, these regions are expected to have elevated genetic diversity Here we attempt to answer several questions related to the phylogeography and population genetics of C. pica. We sampled small crevices in well-exposed rocky intertidal areas where C. pica is commonly found within each region defined by region I. Panama-Colombia ; region II. Southwestern Caribbean ; region III. Eastern Caribbean ; region IV. Bahamas  . Individuals were collected during open fishing season for the species and hence permits were not required for the majority of sample locations. In Bonaire National Marine Park (STINAPA), samples were collected by local biologists with an authorization letter (intern permit) from the director of the park.The Caribbean region spans from an upwelling zone near Cape Canaveral, Florida in the north to the fresh water outflows of the Amazon and Orinoco rivers in the south, and from Central America in the west to the Atlantic Ocean in the east 4.291°N] . A totalDNA was isolated from sampled individuals by using either a QIAGEN DNeasy kit , or a cetyl trimethylammonium bromide (CTAB) isolation protocol http://fluxus-engineering.com/). This method combines features of Kruskall's distance-based algorithm for finding minimum spanning trees with maximum-parsimony A median joining algorithm was used to build a minimum spanning network to examine the evolutionary relationships among haplotypes, as implemented in Network 4.5  and the neutrality tests Tajima's D C. pica. A total of 37 distinct haplotypes were present among the 106 COI sequences, and 38 distinct haplotypes were found in the 16S sequence data. Overall values of nucleotide and haplotype diversity were substantial for both COI  and 16S .After exclusion of sequences with 20% of their total length or more constituting missing or low quality data with PHRED score <20 Haplotype relationships were visualized by median joining networks . Althougst values. The overall Fst is 0.168 for COI, corresponding to an effective number of migrants per generation (Nfm) of 2.48 under an island model of migration st was 0.358 for 16S, corresponding to an Nfm of 0.900. Exactly 50% of pairwise comparisons among sites for COI are significant (p<0.05), while 82% are significant for 16S, likely due in part to generally larger sample sizes for 16S  shows a small and insignificant proportion of the variance at both loci partitioned among these regions  and k = 2  for 16S  together with the Central Panama site (BV), while the second group contained the three southwestern sites  together with Bonaire (BN). In contrast to COI, several of the sites grouped together in this analysis are in fact significantly differentiated based on FST. As in both analyses several group number models were similar in the amount of variance explained, we also present the group configurations for these alternative outcomes , and h values ranged between 0.784 and 0.972 (havg  = 0.909). The highest values of π for COI were identified in Eastern sites (WPR and USVI) and the Bahamas (BSS), whereas the lowest values were observed in BN and BV with Southwestern sites  intermediate , while h ranged between 0.581 and 0.982 (havg  = 0.836). However, in contrast to COI the highest values of π for 16S were found in Bonaire (BN) and in Southwestern sites  with the lowest values in BV and BSS  and Southwestern  regions. Connectivity within these regions was demonstrated by low and insignificant values of FST  are strongly differentiated from the nearby Panama site (BV), in all analyses, consistent with the influence of coastal oceanography. The Panama-Colombia oceanographic gyre may be contributing to the isolation of Central Panama (BV) from other Central American sites. Previous studies suggested that regional current patterns from the northwestern coast of Panama to Belize might lead to mtDNA differentiation in coral reef fishes C. pica, another important factor promoting differentiation between these regions is the absence of suitable rocky intertidal habitat in areas to the west of the Panama Canal on the Caribbean shore of Panama, which limits recruitment between central and western Panama.On larger scales, however, connectivity is generally low, as significant genetic differentiation is apparent across regions and between sites at both loci sampled. The patterns of differentiation in some cases do appear consistent with boundaries of connectivity regions in the Caribbean defined by hydrodynamic models. The majority of FST values that indicate differentiation within connectivity regions, and a lack of differentiation between some sites in different regions, and is supported by SAMOVA analysis which indicates alternative groupings of sites which maximize the among groups variance component. Two particular exceptions to the pattern expected from the hydrodynamic connectivity model are noted.However, the overall structure observed is not best explained by the four broad regions of isolation defined by hydrodynamic models of dispersal within the Caribbean Acropora palmata) and in the goby Elacatinus evelynaeFirst, while the Bahamas is genetically distinct from most sites, as predicted by the model, it is not significantly differentiated at the COI locus from sites in the Eastern region, and is grouped with these sites in the SAMOVA analysis for 16S. The Antilles current  may mediST values are large and significant in nearly all pairwise comparisons involving this site, and SAMOVA supports Bonaire as a separate group for COI. Several private haplotypes were found for COI in Bonaire, supporting the isolation of this region in C. pica. Yet, values of Fst are reduced in comparisons with sites in the Southwestern region  and in several comparisons are insignificant across both loci. For 16S, the structure that maximizes among-groups genetic variance places Bonaire with the Southwestern region sites. These results could be due to a more recent separation of these sites, or higher connectivity between them, possibly mediated by the Caribbean current.Secondly, while Bonaire (BN) falls within the eastern region defined by hydrodynamic models, the separation of Bonaire from the most of the other regions is supported by genetic data. This result contrasts with previous studies in corals in which populations from Bonaire or nearby Curacao group with other eastern populations in the context of an east-west genetic break in the Caribbean Diadema and the red rock urchin, Echinometra lucunterC. pica, is not always easily predictable by features of life-history such as PLD A number of studies support the notion that the Caribbean region is a coherent marine biogeographic province with reduced or no phylogeographic barriers to promote genetic differentiation C. pica populations exhibited moderate to high values of genetic diversity in the majority of analyzed sites. These values are similar to others reported for Caribbean, Atlantic and Pacific gastropods for mitochondrial genes C. pica is harvested for local consumption and sold as exotic handcraft to tourists, and a fishery ban for this species has not been established yet. Uniformly positive values of neutrality tests in this region may indicate a bottleneck in these populations, although it is unclear whether this result could be related to anthropogenic factors.Genetic diversity is an important indicator of the level of fitness and sustainability in marine populations, and C. pica the high degree of population structure, the existence of common connectivity areas among neighboring countries (<300 km), the partial concordance of these areas with previous areas established for coral and fishes and spatial trends in genetic diversity are important factors to consider in future conservation and management plans. Conservation partners can be established between areas of strong connectivity and reduced differentiation. In addition, the presence of areas of reduced diversity (Southwestern sites) can be used as scientific evidence for the future implementation of a fishery ban for C. pica and for replenishment protocols in these areas Access to genetic information and its integration into present management and conservation policies is only a reality for a small number of species. For Table S1Summary of genetic parameters by population. Summary of genetic parameters include: geographic coordinates, GenBank accession numbers, number of COI and 16S sequences per location (N), number of observed haplotypes (H), haplotype diversity (h), nucleotide diversity (π), and neutrality tests: Tajima's DT and Fu's Fs. Note: Significant p-values (p<0.05 and p<0.01) are indicated with one or two asterisks based on permutation test results.(0.01 MB DOCX)Click here for additional data file.


Schistosoma japonicum remain poorly understood, as over forty species of mammals are suspected of serving as reservoir hosts. However, knowledge of the population genetic structure and of the full-sibship structuring of parasites at two larval stages will be useful in defining and tracking the transmission pattern between intermediate and definitive hosts. S. japonicum larvae were therefore collected in three marshland and three hilly villages in Anhui Province of China across three time points: April and September-October 2006, and April 2007, and then genotyped with six microsatellite markers. Results from the population genetic and sibling relationship analyses of the parasites across two larval stages demonstrated that, within the marshland, parasites from cattle showed higher genetic diversity than from other species; whereas within the hilly region, parasites from dogs and humans displayed higher genetic diversity than those from rodents. Both the extent of gene flow and the estimated proportion of full-sib relationships of parasites between two larval stages indicated that the cercariae identified within intermediate hosts in the marshlands mostly came from cattle, whereas in the hilly areas, they were varied between villages, coming primarily from rodents, dogs or humans. Such results suggest a different transmission process within the hilly region from within the marshlands. Moreover, this is the first time that the sibling relationship analysis was applied to the transmission dynamics for S. japonicum.The transmission dynamics of  Schistosoma japonicum involves two obligatory host stages, with asexual reproduction within a molluscan host and sexual reproduction within a mammalian host. Having over 40 species of mammals suspected of being potential reservoirs complicates the transmission patterns. Understanding the complex transmission patterns is further hampered by the ethical and logistical difficulty in sampling adult worms from mammalian hosts. However, the two free-swimming larval stages, cercariae  and miracidia , are available, and elucidating the genetic composition of parasites at theses two stages could provide information of infection processes. Here we sampled cercariae during April 2006, miracidia during September-October 2006, and cercariae during April 2007 in three marshland and three hilly villages in Anhui Province of China, and, using microsatellite markers, analyzed the population genetic structure and, for the first time, the familial relationships of parasites at different stages. We found contrasting population structures of parasites, and host species-associated diversities and transmission patterns of parasites between and within two regions. Moreover, we demonstrate that the successful application of sibship analyses to infection process provides an alternative approach to the dissection of transmission dynamics. Adult worms  typically reside in the mesenteric venules of the vertebrate host, thus leading to an ethical and logistical difficulty in sampling S. japonicum adult worms, particularly from humans and valuable domestic mammals. It is feasible, however, to sample the parasite progeny miracidia, which are hatched from eggs that are passed in a host's faeces. As a single S. japonicum miracidium , after infecting a snail and subsequently undergoing several rounds of asexual reproduction, may give rise to thousands of clonal cercariae, different hosts may be predicted to be infected with the same genotypes of cercariae through water contact. Therefore, under such conditions, one pair of adult worms found in one mammalian host may be genetically identical to a pair found in other hosts. Clonal genotypes of schistosome adult worms, for example S. mansoni, have been reported to be present within a single host A storage system for schistosome larval DNA at room temperature with a Whatman FTA® card - a chemically-treated paper that extracts the DNA from a specimen - has been successfully developed S. japonicum infection in mammals and snails and/or the gene flow of S. japonicum miracidia between definitive host species, have suggested a high possibility of different reservoirs (cattle in marshland areas versus rodents in hilly areas) between two regions in Anhui of China. This raises the question of how the parasites were transferred between the suspected reservoir host species and intermediate host snails. As understanding such transmission dynamics could inform the design and focus of control measures, in this study we aimed to elucidate the extent of parasite gene flow between two larval stages  based on gene frequencies and the transfer of parasites from definitive to intermediate hosts using, for the first time for this species, sibship analyses. Therefore, we collected S. japonicum larvae samples during prevalence surveys implemented in April 2006 , September–October 2006  and April 2007  in three marshland and three hilly villages. We then, by using the previously identified polymorphic microsatellite loci Our previous research S. japonicum infection in intermediate host snails and definitive host mammals in the sampled villages here have been reported elsewhere The details of F-statistics FIS (f) measures the within-host population magnitude of departures from Hardy-Weinberg equilibrium expectations while FST measures the genetic differentiation of parasites between hosts. For each species, the estimator f (within-host) and the estimator FST (among hosts within species) were calculated over all loci according to F-statistics obtained above, 95% confidence intervals were obtained by bootstrapping over loci with the number of replicates set to 20000. Standard indices of genetic diversity of the parasite  population  were calculated. The observed heterozygosity (Ho) and the expected heterozygosity (He) and their standard deviations were computed with Arlequin The population structure of larval parasites for each species of definitive host, or for snails, within each village was defined as containing 2 levels: (1) a parasite infra-population (within-host population), the population of larvae found within an individual host; and (2) a parasite  population (within-species population), all larvae found from the individuals of the same species. Wright's FSTs, a measure of short-term genetic distance, between pairs of two larval populations, miracidia (from each host species) vs cercariae (from snails), were calculated with Arlequin Population pairwise a priori pedigree information, have been successfully employed to the partitioning of individuals into families S. japonicum larvae, to the estimation of full-sib relationships between the sampled larval multi-locus genotypes. All cercariae and miracidia sampled from the same village were pooled and then, by running Colony independently, partitioned simultaneously into full-sib family groups of variable size (group-likelihood approach). As no information on typing errors or mutation rate of the larval samples was available, a small error rate of 1% at each of six loci, as suggested by Microsatellite markers, due to their high polymorphism, are particularly well suited to studies of relatedness and kinship assessment The mean number of genetically unique adult worm pairs per host and its standard deviation were calculated for each species, and the significant difference among host species was tested with the non-parametric Kruskal-Wallis (for over two independent samples) or the Mann-Whitney U test (for two samples) using SPSS 11.0 software Ethical clearance for the project was obtained from the Scientific Committee at the Anhui Institute of Parasitic Diseases, which is responsible for Schistosomiasis control within the Anhui province where targeted villages are located. As the anti-schistosomiasis work, including health education and testing and treatment of humans, has been conducted in the sampled areas each year, and no detailed personal information is revealed in any subsequent data analyses, oral informed consent, approved by the Scientific Committee and witnessed by local village leaders, was obtained from all adults and from parents or guardians of minors who were involved in the project, and from owners whose domestic animals were sampled, and the names of all participants were then registered by the research team. The approach of sampling stools from domestic animals and from rodents was approved by the Scientific Committee and the work was conducted in accordance with the Anhui Institute of Parasitic Diseases' guidelines for animal husbandry. In brief, domestic animals were released shortly after obtaining stool samples, and those infected with the parasite were given a treatment at 25mg/kg bodyweight if possible. Control advice was also given to the owners of infected dogs or cats. Captured rodents were humanely euthanized with ether in black bags if positive in stool examination; otherwise, they were released at their original point of capture the following day.S. japonicum cercariae in the marshland, a significant within-host population heterozygosity deficiency (f), namely a departure from the assumption of Hardy-Weinberg equilibrium, was found in two cases in 2006 with one in Guanghui and the other in Heping. With the exception of cercariae from Longshang and Yuantou in 2007, there was a substantial genetic differentiation among snails observed across the marshland (FST = 0.269–0.471) and the hilly region (FST = 0.103–0.359).As seen in FST≤0.021) and of significant within-host population heterozygote deficiency (f). However, within the hilly region, a marked within-host heterozygote deficiency was only found in parasites from dogs in Longquan and from humans in Yuantou. For all host species, with the exception of dogs in Longquan, the FST values ranged from 0.094 to 0.274, indicating low gene flow of parasites among hosts. It was also noted that most of the 95% confidence intervals of the FST values were much wider in the hilly than in the marshy villages.As displayed in As displayed in From S. japonicum infection within snails (the number of cercariae genotypes per infected snail) was higher in the marshland than in the hilly region in both years  and 25 (61.0%) were inferred in dogs and rodents, respectively. In Longshang, only 24 pairs were estimated, with 14 pairs harboured in either of humans and rodents. In Yuantou, 19 pairs were estimated, out of which the numbers of pairs in humans, dogs, and rodents were 14, 12 and 6, respectively see .FST (or highest gene flow) were observed between the miracidial populations from cattle and the cercarial populations from snails, ranging from 0.002 to 0.017 across both years. In the hilly region, as illustrated in The transmission of parasites from definitive to intermediate hosts was inferred based on the familial memberships of miracidia from mammals and of cercariae from molluscs. As displayed in In the hilly village Longquan, there was evidence to suggest that parasites may be transmitted primarily from dogs and/or rodents to intermediate host snails, as most of the cercariae were successfully inferred from the two species. In Longshang, a substantial proportion of cercariae, up to 58.6% in 2006 and 84.9% in 2007, were estimated to come from rodents. However, in Yuantou a considerable number of cercariae (48.4–51.8%) were not traced back to their original reservoirs, with the remainder attributed to dogs, humans and/or rodents see .The results from the population genetic and sibling relationship analyses of parasites at two larval stages demonstrated contrasting population genetic structures of parasites between the two regions, and the definitive host-associated genetic diversities and transmission patterns of parasites between and within two regions. The observations from both the gene flow and the estimated proportion of full-sib relationships of parasites between two larval stages indicated that the cercariae identified within snails in the marshland may mostly come from cattle, whereas in the hilly areas, they were varied between villages, coming from rodents, dogs or humans.f) and among-host (FST) genetic variance of miracidial populations each from species of cattle, water buffalo, goats or dogs, showed a similar pattern, consistent with high gene flow among hosts and high within-host inbreeding. As water contact activities for mammals may vary from species to species, or even from individual to individual, the observed high gene flow among hosts and the high inbreeding within hosts would suggest that, within each village all host individuals may each sample a various number of cercariae originating from the same parasite population, namely the common infection ‘foci’. As previously reported 2 among villages. An infection focus was found both in Guanghui and Xingzhuang, to which humans and domestic animals have free accessibility. In Heping, there were two infection foci, separated by a Yangtze River branch. It would be suspected that these foci each may comprise a large ‘well-mixed’ parasite population, in terms of allelic or genotypic composition. Within the hilly region, in contrast, most miracidial populations from rodents (with lower mobility), dogs or humans (with higher mobility) showed a pattern of low gene flow among hosts and of no or low within-host inbreeding. This may mainly due to the existence of several separated ‘less-mixed’ foci within each of the three villages. As previously reported An adult within-host population, i.e. the group of the parasites present within one host at a particular time S. japonicum between both The highest genetic diversity (genotype-based) was observed in miracidial populations from cattle than from any other mammals in each marshland village; whereas within hilly villages it was observed in parasites from dogs or humans, instead of rodents. Such difference among species within each village could be, to some extent, explained by several factors. First, definitive hosts from the more dispersing species or the species with a larger home range, for example, humans, dogs, and bovines compared to rodents, could harvest a higher number of genetically distinct larval parasites. As predicted, the results of this study did show that hosts with high movement, such as cattle in the marshland and dogs or humans in the hilly region, appeared to harbour the highest numbers of genetically unique adult worm pairs. Second, species with longer life-spans are likely to obtain more genetically diverse infections then species with shorter life-spans. In the marshland the highest estimated number of worm pairs were observed in cattle with an average of more than 10 years of lifespan, whereas in the hilly region the lowest in rodents with an average of less than 2 years of lifespan. Although humans may have been exposed over an even longer time period than the cattle, a lower index in humans at the time point of the survey could have resulted from the annual testing and treatment required of people in such endemic areas vs parasites from rodents in the hilly region). Multiple infection of schistosomes in snails has been well documented for S. mansoni and S. haematobium in various ecological and epidemiological transmission settings S. japonicum infections among snails was found in two hilly villages. The difference in the proportion of multiple infections and the extent of over-dispersion of infections among snails between the two regions could, on one hand, result from heterogeneities in the exposure, mainly associated with definitive host excreting movements. In the marshland, for example, bovines with a longer life span seem to be ideal ‘genetic mixing bowls’ for the parasites and then may cause the large ‘well-mixed’ infection foci via daily herding and discharging huge amounts of faeces in snail infested areas; whereas in the hilly region, it would be suspected that such separated ‘less mixed’ foci could be attributed to small territory-bound and short-lived rodents. On the other hand, multiple infection within snails could also be an artifact caused by the somatic mutation for S. japonicum at the asexual stage The higher genetic diversity  of cercarial populations at the level of villages in the marshland than in the hilly region was consistent with those observed for miracidial populations  in Yuantou. Annual selective chemotherapy, health education and improvement in sanitation may have been greatly reduced the role of humans in the transmission S. japonicum in the Philippines S. japonicum within such hilly areas could be reasonably inferred as follows: ‘clonal’ rodents maintain the parasite life cycle within their own territory, which may include one or more infection foci; with the existence of such several territories in each village, dogs get infected in one or more and, due to higher mobility, spread the parasites among territories; dogs could also get infected outside the village and then spread them; new infection territories would develop when immigrant parasites infect and establish within local rodents.The contrasting genetic structures of miracidial populations between two regions strongly indicated different main reservoirs between them. Within the marshland region, a higher genetic diversity of schistosomes in cattle than in other species also indicated a possibility of cattle as a main reservoir in such areas. This is further supported by both the observed Pairwise There are, however, two potential limitations inherent within this study. One is with regard to sample size Nevertheless, this is the first time that the potential number of genetically unique adult worm pairs within mammal hosts has been estimated using their offspring miracidia, although the concept of estimating such a parameter has been raised by Criscione and colleagues


Evidence of multiple genetic criteria of mate choice is accumulating in numerous taxa. In many species, females have been shown to pair with genetically dissimilar mates or with extra-pair partners that are more genetically compatible than their social mates, thereby increasing their offsprings' heterozygosity which often correlates with offspring fitness. While most studies have focused on genetically promiscuous species, few studies have addressed genetically monogamous species, in which mate choice tends to be mutual.Rissa tridactyla. We found that pairs were more genetically dissimilar than expected by chance. We also identified fitness costs of breeding with genetically similar partners: (i) genetic similarity of pairs was negatively correlated with the number of chicks hatched, and (ii) offspring heterozygosity was positively correlated with growth rate and survival.Here, we used microsatellite markers to assess individual global heterozygosity and genetic similarity of pairs in a socially and genetically monogamous seabird, the black-legged kittiwake These findings provide evidence that breeders in a genetically monogamous species may avoid the fitness costs of reproducing with a genetically similar mate. In such species that lack the opportunity to obtain extra-pair fertilizations, mate choice may therefore be under high selective pressure. Numerous traits influence mate choice that may produce non-random mating patterns in many species. Although most studies have focused on morphological and behavioral traits -4), therParus caeruleus), females acquired extra-pair fertilizations that enhanced the heterozygosity and fitness of their offspring [Lamprotornis superbus), the benefits of extra-pair fertilizations may differ according to the genetic similarity of the extra-pair partner [In species with biparental care, both sexes should be choosy in selecting a mate ,18. Blomffspring . In supe partner . HoweverRissa tridactyla) is a long-lived, monogamous seabird with no extra-pair fertilizations [The black-legged kittiwake  heterozygous mates , (2) geOur second aim was to examine the fitness consequences of breeding with genetically similar or dissimilar individuals. We searched for possible relationships between genetic characteristics of pairs and fitness components such as clutch size and hatching success ,30. BecaMicrosatellites are generally assumed to be neutral genetic markers and have been widely used to estimate relatedness, individual heterozygosity and population level of inbreeding -34. If hWe monitored 348 genotyped adults in 2003 and 2004: 241 were seen alive in 2003 and 289 in 2004. Adults formed 74 pairs in 2003 and 72 in 2004; the remaining adults for each year corresponded to unpaired adults or adults paired with non-genotyped mates. All these adults were included in the bootstrap analyses because they were alive in the considered year and thus potentially available for pairing.Phmxy of the parents was closely related to all indices of offspring heterozygosity , confirming that Phmxy might be a reliable estimate of the probability of a given pair of producing homozygous offspring. Furthermore, Phmxy of pairs used in our study exhibited a wide, six-fold range of variation .For chicks with genotyped parents, we found that H, SH and IR, but results did not differ with all loci. The "preference of heterozygous mates" hypothesis (hypothesis 1) predicted assortative mating by heterozygosity. However, male and female H were not correlated for any year . Results were the same for SH  and IR . Heterozygosity of paired and unpaired individuals did not differ .To correct for linkages between loci, OHW loci and K31 were excluded from calculations of The "genetic similarity avoidance" hypothesis (hypothesis 2) predicted that observed pairs comprise less genetically similar individuals than expected by chance. To test this, for each year we ran 10,000 bootstraps using the observed individuals. For each run, we used either a calculation across all loci, or excluding OHW loci  predicts that mates share more alleles than expected by chance. This means that observed pairs should comprise more genetically similar individuals than expected by chance. We would then expect to have a p-value for nd Table .272 = 0.01, p = 0.91, n = 74). However, for pairs that laid eggs, the number of hatched chicks was correlated with Phmxy , with the mean number of chicks hatched being lower in more genetically similar pairs .We used only 2003 data to determine the number of eggs laid and the number and proportion of eggs hatched, because the reproductive success of some of the pairs in 2004 and subsequent years may have been affected by other experiments after pair formation. Genetic similarity indices were computed without OHW loci and K31. The number of eggs laid was not correlated with pair genetic similarity . The hatching rank*chick heterozygosity interactions were non-significant for all heterozygosity indices (p > 0.6) and were thus discarded from the models. Chick survival was positively correlated with chick heterozygosity for all indices , but became non-significant when removing OHW loci and K31 from the computation of chick heterozygosity .Chicks were not genotyped for K67 (because of low variability). In 2005, we assessed the survival and growth in body weight, tarsus and wing length of 82 chicks until age 25 days. These life history parameters were highly correlated to hatching rank . Hatching success was negatively correlated to genetic similarity of pairs in 2003 for two loci (Phm index for K32 and RBG20). Chick heterozygosity and survival until 25 days (in 2005) were positively correlated only for RBG29 . For chick growth, we found that the parameters associated with the interactions Age*Heterozygosity and Age*Heterozygosity*Rank were significant in explaining chick growth in body weight and wing length when chick heterozygosity was estimated through three of the nine loci . Similarly, the parameters associated with the interactions Age*Heterozygosity and Age*Heterozygosity*Rank were significant in explaining chick growth in tarsus length when chick heterozygosity was calculated with two loci (H index for K31 and K32).For mating patterns in 2003, we found that pairs were formed of genetically dissimilar individuals according to three of the 10 loci  and saddlebacks (Philesturnus carunculatus), in which pairings were random in relation to genetic similarity of mates [Our main objective was to examine predicted relationships between genetic variables and mating patterns in a genetically monogamous species. Selective pressures on mate choice may be stronger in such species, which do not mitigate the costs of homozygous offspring with extra-pair fertilizations. To our knowledge, there is only one other study of this kind of genetically monogamous species, the New Zealand robin , sand l agilis, ), mice [ agilis,  and huma agilis, . In contesticus, , and Seyllensis, ), or witpoleuca, , and grea minor, ), while inaceus, ).As expected, we also found deleterious effects of genetic similarity and homozygosity of offspring. This set of results  is statistically independent of results about mating patterns, but are biologically linked and consistent. Genetically similar pairs hatched fewer offspring than more dissimilar pairs Figure , which iThe use of multi-locus estimates of heterozygosity and genetic similarity has been criticized by authors arguing that global heterozygosity-fitness correlations may be driven by certain loci that are physically linked to fitness-affecting loci ,36,44. HLittle is known about mate choice in kittiwakes, and the pattern we describe here may result from different mechanisms. It could be due to a passive process with, for example, genetically similar pairs being more likely to fail in their breeding attempt, which in turn would make them more prone to divorcing than successful pairs. In our population, this seems unlikely since divorce after reproductive failure is not systematic . FurtherPachiptila desolata) have been shown to recognize their mates through odors [Active choice of genetically dissimilar mates has been demonstrated in mammals . This island supports a large declining population of black-legged kittiwakes . We stuNest sites were observed twice daily from mid-May to late-August to assess individual attendance and reproductive success. We arrived at the field site too late to assess the arrival dates of adults. During each visit, we recorded the color-band combination of attending adults, the stage of nest building, the presence of eggs and chicks and the behavior of adults in the form of incubation, nest building and copulation, variables that we used to confirm pair identity. Each chick was marked at hatching and banded at 25 days old. Adults received five bands . We could thus precisely determine laying and hatching dates of focal pairs, as well as the hatching rank of chicks . Copulation behavior was also monitored in order to determine the sex of every paired adult; for unpaired adults, the sex was identified by morphological values such as bill length, tarsus length, bill width and head+bill length . We sampWhole genomic DNA was extracted from each blood sample using a "salting out" protocol described in ), modifiLarus novaehollandiae scopulinus [Samples were genotyped at 10 microsatellite loci. Loci K6, K16, K31, K32, K67 and K71 were first described in the black-legged kittiwake , see Tabopulinus , see Tabopulinus , RBG18 aopulinus ) were alThe forward primer in each primer pair was synthesized with a modified 19- to 20-bp universal tail  added to the 5' end of the oligonucleotide . We usedAmplification products were separated on a 48-well 25-cm 6% polyacrylamide gel on a LI-COR 4200 LR automated sequencer, using Base ImagIR™ . Allele sizes for specific samples at each locus were determined relative to the M13 phage single nucleotide ladder. These samples were later used as internal size standards to score new genotypes, using Gene ImagIR™ 4.05 software . For quality control purposes, we reprocessed a minimum of 10% of the samples for all markers, and only kept markers giving reliable scores. Two markers  were calculated in GENEPOP Version 3.1 [Mean number of alleles (A) and observed and expected heterozygosities  and RBG29 (p = 0.005). Because of its lower heterozygosity, K67 was not genotyped in all samples. Thus, for each analysis, we present the results obtained using all loci, or excluding K16, K67 and RBG29, which are designated as the "OHW" loci, for "out of Hardy-Weinberg" equilibrium. Only K31 and K32 were genetically linked (p < 0.05 after correcting for multiple tests). Since our genetic similarity analyses are based on bootstrapping on the multi-locus genotypes  and not on alleles, both loci were kept. We also conducted every allelic-based analysis with all ten loci and without "OHW" loci and K31 (7 remaining loci).Phm). For each locus (l), this probability is equal to:The genetic similarity between two given pair members (x and y) was estimated by the probability for this pair of producing homozygous offspring , and 0 otherwise. An index based on such probabilities was first proposed by Mathieu et al. [following Lynch & Ritland's ) notatiou et al. , but hasu et al.  showed tPhm across all loci, a weighted average of the Phmxy(l) is calculated using the formula proposed by Mathieu et al. [To obtain a value for u et al. :pl is the probability of an individual being homozygous by chance at locus l. Therefore Phmxy will be close to 1 if the pair has a high probability of producing a homozygous offspring , and close to 0 otherwise.where H , the standardized heterozygosity SH and the internal relatedness IR, all described in [©1983, 1989 Inprise Corporation; Additional file We used three different indices to estimate individual global heterozygosity: the direct heterozygosity ribed in ). All th©1983, 1989 Inprise Corporation) that was previously tested for no significant differences with Queller & Goodnight estimations obtained from the IDENTIX software [Phmxy and, given the configuration of our datasets, it makes our Monte-Carlo analyses much easier than previously published programs.Genetic indices were performed using a program computed . These bootstraps gave a set of values that we used as the distribution under the assumption of random mating . The observed values of the genetic indices were then tested for significant departure from the expected distribution obtained by the bootstraps. When bootstrapping, we used only adults seen in the considered year, and in each bootstrap we randomly made the same number of pairs that had been genotyped in the population using all live adults, as they were all considered as potentially available for pairing. Allelic frequencies were calculated for this sub-sample of individuals. For Hypothesis 2, that mates are genetically less similar than expected by chance, statistical significance was assessed by the proportion of the bootstraps having a mean genetic index lower than the observed mean. We statistically tested Hypothesis 3 that mates share more alleles than expected by chance, by assessing the proportion of the bootstrap values with a mean genetic similarity index higher than the observed mean . By bootstrapping on observed individuals and not on randomly created individuals generated via allelic frequencies, we might also expect to correct for biases due to linkage disequilibria or genotyping errors. Individuals with incomplete genotypes had thus the same probability of being included in observed pairs as in bootstrapped pairs. Thus, biases will be similar for observed and bootstrapped pairs.® package . Correlations between the two indices of genetic similarity, or between male and female heterozygosity, were analyzed through standard linear models. To analyze the links between reproductive success (number of eggs laid and the number and proportion of eggs hatched) and genetic variables, we used a generalized linear model  since the dependent variables never showed more than three levels. For each of these three levels, the genetic variables and the residuals from the model were normally distributed. Similarly, we analyzed the correlations between chick survival and heterozygosity using generalized linear models, since both chick survival  and hatching rank (A or B) are binary effects. For each rank and each survival value, the residuals from the model procedure were normally distributed.General statistical analyses were made using SAS2), heterozygosity (H) and hatching rank (R) as explaining variables. We added A2 because growth of morphological traits is non-linear. For each variable and each heterozygosity index, we ran several models and kept only the one with the lowest AIC value. Given that the three growth variables are correlated, and given that the different indices of heterozygosity are also correlated, the structure of the model with the lowest AIC was always the same, in the form of Growth variable = A + H + R + A*H + A*R + H*R + A*H*R + A2 + Chick identity as a random parameter. We will only give the results obtained with the latter model, since the difference in AIC between this model and other models was always higher than 2. The significance of the interaction A*H was then tested in each rank separately.For the analyses of growth variables , we have repeated measures of individuals, having measured each chick every 5 days. We therefore conducted mixed linear models with chick identity as a random parameter in order to account for non-independence. We made mixed linear models with tarsus length, wing length or body weight as dependent variables, chick identity as a random parameter, and chick age (A), age*age (AHM carried out the genetic analyses, monitored the birds on Middleton Island and drafted the manuscript with RHW. ED and RHW conceived the study and participated in its design and coordination. JFW and FH helped in monitoring the birds and in the conception of the study in the field. SLT and AMR participated in the microsatellite genotyping and scoring. SAH is responsible of the long-term monitoring on Middleton Island and gave access to the study area. All authors read and approved the final manuscript.Table 2with effect size of heterozygosity. Extended version of the Table Click here for fileCode of the program Nausicaa. This file contains the complete code of the program used for calculation of genetic indices and Monte-Carlo analyses.Click here for fileGNU-GPL Licence. GNU-GPL Licence for the program Nausicaa contained in additional file Click here for file


Dating of population divergence is critical in understanding speciation and in evaluating the evolutionary significance of genetic lineages, upon which identification of conservation and management units should be based. In this study we used a multilocus approach and the Isolation-Migration model based on coalescence theory to estimate the time of divergence of the Spanish and Eastern imperial eagle sister species. This model enables estimation of population sizes at split, and inference of gene flow after divergence.Our results indicate that divergence may have occurred during the Holocene or the late Pleistocene, much more recently than previously suspected. They also suggest a large population reduction at split, with an estimated effective population size several times smaller for the western population than for the eastern population. Asymmetrical gene flow after divergence, from the Eastern imperial eagle to the Spanish imperial eagle, was detected for the nuclear genome but not the mitochondrial genome. Male-mediated gene flow after divergence may explain this result, and the previously reported lower mitochondrial diversity but similar nuclear diversity in Spanish imperial eagles compared to the Eastern species.Spanish and Eastern imperial eagles split from a common ancestor much more recently than previously thought, and asymmetrical gene flow occurred after divergence. Revision of the phylogenetic proximity of both species is warranted, with implications for conservation. The dating of speciation events and investigation of the evolutionary processes involved are major themes in evolutionary biology with important implications for conservation biology. For endangered species, knowledge of these historical processes is critical for correct interpretation of current patterns of genetic variation, and guides assignment of conservation priorities and elaboration of subsequent conservation strategies ,2.The classical approach to dating speciation events has been based on the molecular clock, which has proven to be an appropriate tool for addressing a wide range of evolutionary hypotheses . HoweverNew likelihood and Bayesian methods based on coalescence theory have recently been developed for estimating species divergence times, potentially overcoming some of the above handicaps e.g. ,11,12]. ,12. 11,1Aquila adalberti Brehm) has fluctuated between threatened and vulnerable in the last 20 years, and it is currently classified as "Vulnerable" in the IUCN red list of threatened species  was -3.12, suggesting the adequacy of a model that includes migration. The proportion of A. adalberti replaced by A. heliaca migrants, scaled by mutation rate, was estimated in m1 = 0.99   Figure . If the 2 × m1/μ  and estiA. heliaca (Eastern) plumages in both adult and juvenile A. adalberti individuals, observed in the field at low frequencies , might indicate retained ancestral polymorphism consistent with a recent separation of imperial eagles.Analysis of two independent data sets indicated that the Eastern and Spanish imperial eagles split from a common ancestor much more recently than has been suggested ,20, and If imperial eagles remained isolated during repeated glacial maxima in two different glacial refugia , our datA. clanga (spotted eagle) and A. pomarina (lesser spotted eagle) . The absence of female-mediated gene flow could also be a consequence of the lower fitness of heterogametic female hybrids , as haspomarina  and docupomarina ,44. Malepomarina ,45), as A. heliaca plumages in both adults and juveniles occurring in the A. adalberti population; Spanish Ministry of Environment, unpublished data) and the low level of morphological, ecological or behavioural divergence, these taxa might be conservatively classified as allospecies based upon the criteria of Helbig et al. [The recent split and occurrence of subsequent gene flow have implications for the taxonomic and conservation status of both imperial eagles. Although the asymmetric gene flow pattern might still indicate some level of reproductive incompatibility, full reproductive isolation is contradicted by our observations of gene flow. Based on the scarcity of diagnostic characters . A random subset of 15 individuals of each species was analysed for eight microsatellite loci  [A. adalberti historic sample was included to avoid the possible influence of the recently arisen genetic structure in the analysis [A. heliaca in the period analysed, as the species has always been globally abundant and widely distributed. A complete description of the samples is available upon request.Thirty-four museum specimens of ears ago , 33 contividuals , and 50 A) splits at time t into two descendant populations (sizes N1 and N2) which may subsequently establish gene flow between them at rates m1 and m2 . The model assumes selective neutrality and no further population subdivision within each population. It also assumes that population sizes do not change over time. Nielsen and Wakeley [1 ≠ m2), θ is the population parameter 2Neμ, and X is the data. It was necessary to apply some assumptions to these parameters, described by the prior distributions, to estimate the posterior distribution. Firstly, we assumed that all parameter values were equally likely (uniform distribution), so that posterior probabilities are proportional to the likelihood function. Secondly, to ensure that the posterior distribution was a real probability distribution, we constrained the parameter space, i.e. we set maximum values for the priors of the parameters. By doing so, the results can be interpreted in a Bayesian framework as well as in a likelihood framework. The model was modified [The basic IM model ,50-52, a Wakeley  implemenmodified . More res sizes N and N2 w2 for the microsatellite dataset, and both m1 and m2 for the mitochondrial dataset). We finally performed three independent runs of 5 × 108 steps with a burn-in of 2 × 106 steps for the set of eight microsatellite markers, and three independent runs of 2 × 107 steps with a burn-in of 1 × 106 steps for the control region sequence dataset, using narrow priors after results of the preliminary analyses.A critical issue with the use of MCMC approach is whether the parameter space has been fully explored and the stationary distribution has been adequately sampled. As recommended by Hey and Nielsen  we firstAnalyses were performed in a Pentium 4, 3.2 GHz, 1 GB processor integrated in a supercomputer with 80 DELL 750 nodes interconnected by Gigabit Ethernet to a data server 1750 with two processors, Xeon 3 GHz and 438 GB SCI disk, in RAID 5 configuration at the Centro de Supercomputacion de Galicia (CESGA), Spain. Each of the three final runs for each marker took around 138 hours for the microsatellite dataset and around 27 for the mtDNA dataset in a single processor.The author(s) declares that there are no competing interests.BMC carried out the genotyping of individuals, analysed the microsatellite raw data and performed the analysis with the IM software. BMC and JAG conceived and elaborated the design of the study. JAG coordinated the study, supervised the drafting of the manuscript and read and approved the final manuscript. All authors have read and approved the final manuscript.


Desmognathus monticola, was discovered on the Ozark Plateau, ~700 km west of its broad continuous distribution in the Appalachian Mountains of eastern North America. Using Nested Clade Analysis (NCA) we test whether the Ozark isolate results from population fragmentation  or long distance dispersal (a human-mediated introduction).In most regions of the world human influences on the distribution of flora and fauna predate complete biotic surveys. In some cases this challenges our ability to discriminate native from introduced species. This distinction is particularly critical for isolated populations, because relicts of native species may need to be conserved, whereas introduced species may require immediate eradication. Recently an isolated population of seal salamanders, D. monticola is restricted to less than 2.5% of the distribution in the extreme southern Appalachians, where genetic diversity is high for other co-distributed species. By intensively sampling this genetically diverse region we located haplotypes identical to the Ozark isolate. Nested Clade Analysis supports the hypothesis that the Ozark population was introduced, but it was necessary to include haplotypes that are less than or equal to 0.733% divergent from the Ozark population in order to arrive at this conclusion. These critical haplotypes only occur in < 1.2% of the native distribution and NCA excluding them suggest that the Ozark population is a natural relict.Despite its broad distribution in the Appalachian Mountains, the primary haplotype diversity of D. monticola from the Ozarks is not native to the region and may need to be extirpated rather than conserved, particularly because of its potential negative impacts on endemic Ozark stream salamander communities. Diagnosing a species as introduced may require locating nearly identical haplotypes in the known native distribution, which may be a major undertaking. Our study demonstrates the importance of considering comparative phylogeographic information for locating critical haplotypes when distinguishing native from introduced species.Our analyses suggest that the isolated population of  Species introduced by human activities are one of the leading threats to biodiversity -4. A criDesmognathus, endemic to eastern North America, includes 19 recognized species; their primary diversity is centered in the southern portion of the Appalachian Mountains [The Plethodontidae is the most species rich family of salamanders ,12, and ountains ,12 , a large stream-dwelling species that is widespread in the Appalachian Mountains, more than 700 km to the east  in the Ozark Plateau or an introduced species that could threaten local endemic species if it spreads (see Discussion).The Ozark Plateau of east-central North America is a karst uplift separated from the Appalachian Mountains by the low elevation flood plain of the Mississippi River. The Ozark Plateau harbors many endemic species, including a unique salamander fauna ,24. Ther Plateau ,26, but  Plateau ,28 sincen Ozarks . This pot Figure . This isD. monticola to be one of the most common salamander species found in fishing bait shops [There is a wealth of biogeographic evidence that suggests a relatively recent faunal connection between the Ozarks and the Appalachians, and many conspecifics or sister species of fish -32, crayit shops , so thiscox1), we test whether this isolate is the result of a recent human-mediated introduction or represents a relict from historical faunal connections between the Appalachian Mountains and the Ozark Plateau. If the Ozark population originated through fragmentation and contraction of a previously more widespread distribution, then we would expect it to exhibit significant genetic divergence from Appalachian populations. Alternatively, if the Ozark population results from a recent introduction through human activities, we would expect little or no genetic divergence from Appalachian populations, despite the large geographic separation between these highland regions. These predictions are ideally suited for testing with Nested Clade Analysis, which uses the geographic distributions of ancestral (interior) haplotypes relative to younger (tip) ones to draw inferences about processes that have shaped spatial patterns of genetic variation.Using NCA, based on a portion of the mitochondrial gene cytochrome oxidase-1  in northern Georgia, and a correct diagnosis of the Ozark population is highly dependent on including critical haplotypes from this region in the NCA. We discuss our results in the context of developing sampling strategies based on comparative phylogeography of co-occurring species when assessing the history of populations that are suspected of being introduced.Adequately sampling genetic diversity is critical for testing phylogeographic hypotheses ,38. Althted taxa -43. We fcox1 fragment among our seven Ozark samples. The 100 Appalachian samples from 47 populations covering the entire latitudinal distribution of D. monticola represented 18 unique cox1 haplotypes  with NCA ,45  haplotypes to determine whether the Ozark population is native or introduced, we sequentially ran additional NCAs that excluded Appalachian samples that were 0.000% (haplotype F), and ≤ 0.733%  divergent from the Ozark population. Excluding only identical Appalachian haplotypes (Haplotype F = 0.56% of the distribution), still leads to the inference that the Ozark population was introduced. However, excluding Appalachian haplotypes F, C, and I, leads to a significantly small Dc for clade 2-2 . Since the Ozark population is separated from the remaining haplotypes by a larger than average number of mutational steps, and is allopatric from clades 2–3 and 2–4, this leads to the inference of allopatric fragmentation at the level of clade 3–2. This demonstrates that in this case it is necessary to locate and include in the NCA, haplotypes that are ≤ 0.733% divergent from the Ozark population, in order to properly diagnose the Ozark population as introduced. The distributions of these critical haplotypes  are localized to a small region in northern Georgia that comprises < 1.2% of the geographic distribution of To date most genetic studies of introduced species have focused on evolutionary genetics ,49, geneD. monticola are consistent with patterns exhibited by many co-distributed species. The large majority of the distribution of D. monticola represents a single lineage  that likely represents a recent range expansion from the southernmost highlands of the Appalachian Mountains all the way to the most northern extent of the distribution , this could effectively guide further sampling. For example, if the known native distribution of a terrestrial species extends across a region that is dissected by many rivers that are barriers to gene flow for other co-distributed terrestrial species, then initial analyses could compare the population in question to individuals from the land between each river . This could help to narrow down which part of the distribution is genetically most similar to populations in question, and could help to focus subsequent sampling.D. monticola recently discovered in the Ozark Plateau is introduced. We found that this population shares identical cox1 haplotypes with populations of D. monticola from four counties in the northeastern corner of the state of Georgia in the southern Appalachians (~1000 km away). Nested Clade Analysis of the cox1 fragment shows that this highly disjunct population results from a long distance dispersal (human-mediated introduction). Furthermore, we sequenced an additional 1.6 kb of a more variable region of mitochondrial DNA, upstream from our cox1 fragment, for select individuals. This region includes the gene NADH dehydrogenase subunit 2, tRNATrp, tRNAAla, tRNAAsn, tRNACys, tRNATyr, the origin for light-strand replication, and the beginning of cox1. We found only a single nucleotide substitution between the Ozark population and our samples from northeast Georgia .Based on our analyses it appears that the population of Desmognathus, Gyrinophilus, and Pseudotriton are vernacularly referred to as "spring lizards", and are commonly used as fishing bait for catching large species of centrarchid fishes such as largemouth bass . "Spring lizards" are collected from the wild and sold in bait shops by the thousands [D. monticola to be the most common salamander sold [Ambystoma mavortium) are also extensively collected and sold as fishing bait in the central and western United States. Populations of A. mavortium have been introduced to localities in California (~2000 km from their native distribution) where they hybridize with the rare California tiger salamander, Ambystoma californiense [Aneides vagrans, which was accidentally introduced to western Canada from central California through shipments of bark from oak trees in the early 1900s [How did a southern Appalachian salamander get transported to the western Ozarks? In the southeastern United States, stream-dwelling plethodontid salamanders of the genera housands . In factder sold . Unfortuorniense . Anotherly 1900s . These sly 1900s .Desmognathus monticola have a negative ecological impact in the Ozark Plateau? The Ozark Plateau is home to four stream/spring-dwelling species of plethodontid salamanders of the genus Eurycea:E. lucifuga, E. longicauda melanopleura, E. spelaea, and E. tynerensis, and the latter two (E. spelaea and E. tynerensis) are actually species groups that represent a major endemic Ozark radiation [Desmognathus and Eurycea are broadly sympatric in the Appalachian and Ouachita Mountains, the Ozark Plateau is geologically distinctly different from these regions [Eurycea as well as D. monticola are quite limited. Desmognathus monticola are large territorial salamanders and are very aggressive towards both conspecific and heterospecific stream-dwelling salamanders [Desmognathus occur sympatrically [Desmognathus monticola is larger and more robust than any of the Ozark Eurycea, and large adults would likely be the competitively-superior salamanders in a spring or stream. Although predation by large stream-dwelling Desmognathus on other salamanders in the wild is rare [D. monticola could have a negative impact on Ozark Eurycea simply by excluding them from moist habitats at the periphery of springs and streams, a habitat feature that can be quite limited in arid areas of the Ozark Plateau.Do introduced adiation . Althoug regions  and cont regions -70. The amanders . It has trically , ecologitrically -78. Desm is rare , this sp is rare . TherefoDesmognathus monticola are currently known from only two locations. The salamanders included in this study are from a small spring ca. 1 meter wide that issues from the underside of a dirt bank and flows approximately 8.5 meters before reaching a much larger stream (Spavinaw Creek). Since the original discovery in 2003 we have visited this site four times and have found up to 25 individuals, ranging in size from very small newly metamorphosed juveniles, to very large adults (75+ mm SVL), in a single survey. Among the largest individuals, we found one gravid female, 55 mm SVL, with enlarged (ca. 2.5–3.0 mm in diameter) ovarian follicles (ca. 35 ova). Therefore, it appears that D. monticola are breeding and reproducing at this location. Recently, a second Ozark D. monticola location was discovered by members of the Arkansas Herpetological Society. Three adult D. monticola were found in a small spring entering Spavinaw Creek  approximately 2.5 km upstream of the locality included in our analyses . We presently know little about the abundance of Desmognathus monticola in the Ozarks and additional research on the ecological interactions between this introduced species and co-occurring species of Eurycea are necessary to determine the level of impact that it has on local salamander fauna.Ozark Desmognathus monticola, recently discovered on the Ozark Plateau, is not native to the region. This population may need to be extirpated because of its potential negative impacts on endemic Ozark stream-dwelling salamanders. This conclusion could only be realized once nearly identical haplotypes from the known native distribution were included in our analyses. Locating the most genetically similar individuals (the putative source population) in the known native distribution of a species to effectively test the origin of an isolated population can be a labor-intensive and costly undertaking. We propose that considering comparative phylogeographic patterns can greatly facilitate this process and expedite diagnoses of native and introduced species.Our analyses of mitochondrial DNA indicate that the highly disjunct population of seal salamanders, Desmognathus, vouchers were only taken for the first six individuals and were deposited at Arkansas State University Museum of Vertebrates . Tail tips were taken in the field from subsequent specimens because at the time we did not know the species was introduced. These were compared to a large database of D. monticola cox1 sequences from GenBank [Salamanders from the Ozark population were collected from 30-September-2003 to 4-April-2005. Due to the limited distribution of the Ozark  GenBank , specimecox1, 515 bp not including primers) was amplified using the primers COX1F (5'-GGTATTGAGGTTTCGGTCTG-3') and COX1R (5'-CTTAGTCTCTTAATTCGAGC-3') [96 cleanup kit (Montáge™) or EXOSAPIT (USB Corp.). Big Dye (ABI) was used for cycle sequencing reactions sequenced on an ABI 3730 capillary sequencer. Sequencher ™ 3.1 (Gene Codes Corp.) was used to align and edit sequences. The alignment was unambiguous and the translation was checked in MacClade [D. monticola, was based on the Global Amphibian Assessment [DNA was extracted from fresh frozen tissues and ethanol preserved tissues using Qiagen DNeasy extraction kits. A portion of the mitochondrial gene cytochrome oxidase 1 (GAGC-3')  with staMacClade . SequencMacClade  was usedsessment . We omitsessment ,82. The D. monticola was investigated with Nested Clade Analysis [The origin of the Ozark population of Analysis ,45 in orAnalysis , and theAnalysis ,88. The Analysis  was usedcox1, cytochrome oxidase 1; Dc, clade distance; Dn, nested clade distance; I-T, interior to tip; JFBM, John Ford Bell Museum; MVZ, Museum of Vertebrate Zoology; NCA, nested clade analysis; PF, past fragmentation; RE, range expansion; RGF, restricted gene flow.ASUMZ, Arkansas State University Museum of Zoology; bp, basepairs; D. monticola and participated in subsequent field trips. JAW collected invaluable material from across the state of Georgia and edited the manuscript. SET made the initial identification of AB's discovery and coordinated additional fieldwork and contributed to the preparation of the manuscript. All authors read and approved the final manuscript.RMB coordinated the study, collected and analyzed sequence data, and primarily prepared the manuscript. KHK contributed many Appalachian samples, performed the NCA, and contributed to the preparation of the manuscript. DRV assisted in collecting sequence data and contributed to the focus and preparation of the manuscript. AB discovered the Ozark population of D. monticola samples used in this study.Locality information, museum and Genbank accession numbers for the Click here for file


Neisseria are utilized to demonstrate the potential of an approach where these population genetic tools are combined with an phylogenetic analysis. The statistical tools introduced here are freely available in BAPS 5.2 software, which can be downloaded from http://web.abo.fi/fak/mnf/mate/jc/software/baps.html.The evolution of bacterial populations has recently become considerably better understood due to large-scale sequencing of population samples. It has become clear that DNA sequences from a multitude of genes, as well as a broad sample coverage of a target population, are needed to obtain a relatively unbiased view of its genetic structure and the patterns of ancestry connected to the strains. However, the traditional statistical methods for evolutionary inference, such as phylogenetic analysis, are associated with several difficulties under such an extensive sampling scenario, in particular when a considerable amount of recombination is anticipated to have taken place. To meet the needs of large-scale analyses of population structure for bacteria, we introduce here several statistical tools for the detection and representation of recombination between populations. Also, we introduce a model-based description of the shape of a population in sequence space, in terms of its molecular variability and affinity towards other populations. Extensive real data from the genus  The study of bacterial population biology is complicated by the fact that, although bacteria are largely asexual, they can also exchange genetic materials through homologous recombination. Unlike eukaryotes, recombination in bacteria is not an obligatory process. Furthermore, the recombination mechanisms are subject to many biological and ecological factors that can vary even within different populations of the same species. Although increasing evidence for homologous recombination has been found in many bacterial species, determining the frequency of recombination and understanding the influence that it exerts upon the evolution of bacterial populations remains a challenging work. In this article, we provide a dynamic picture of recombination within and between closely related bacteria species. Through an integration of several Bayesian statistical models, our method highlights the importance of a quantitative estimation of recombination. Our analyses of a challenging multi-locus sequence typing (MLST) database demonstrate that combined analyses using both traditional phylogenetic methods, explorative MLST tools and Bayesian population genetic models can together yield interesting biological insights that cannot easily be reached by any of the approaches alone. Whether or not certain populations  are more likely than others to undergo recombination, either as donors or recipients of DNA, is not well understood, though there are several biological reasons why this might be the case. Such preferential recombination, which we may intuitively describe as Discovery of such gene flow currents is scientifically interesting in its own right, as a means for characterizing populations and reflecting upon accumulated taxonomic understanding of their heterogeneity. However, there are other potential uses for detailed knowledge concerning the genetic structure of a bacterial population, e.g. when it can be connected to patterns of virulence and antibiotic resistance.Statistical analysis of molecular variation and reproductive isolation in natural populations is in many cases far more challenging for bacteria than for eukaryotic organisms, due to difficulties in acquiring broad-coverage samples and the putatively complex admixture events With the above-mentioned difficulties, it is hardly surprising that a Bayesian statistical approach based on explicit admixture models has recently gained popularity in studies of bacterial populations genetic shape of an identified population. However, it is important to notice that a population identified by BAPS may have a different interpretation in different evolutionary contexts. The BAPS models target for identifying molecular evidence that links a particular group of strains together in terms of sufficiently similar nucleotide frequencies. Thus, such a population may for instance arise in the analysis due to common ancestry within a clonal complex. In contrast, a population can also be identified from the traces left by recombination events which have imposed considerable gene flow between separate lineages of strains. Also, under certain circumstances a more heterogeneous population may arise analogously to long branch attraction in phylogenetics, in particular, when very limited numbers of strains from the corresponding lineages are present among the analyzed samples. All these three cases are illustrated in our analyses. As BAPS is capable of capturing a variety of distinct biological signals hidden in molecular data, interpretation of the identified populations must be done with care, using preferably both complementary phylogenetic methods and auxiliary knowledge about the strains under investigation.Despite the success of the standard MCMC approach in a variety of studies of bacterial populations see e.g. , it is cNeisseria meningiditis and Neisseria lactamica species. We also present analyses of simulated data to demonstrate the potential of our Bayesian approach to handle large databases and complex genetic population structures. Our analyses illustrate that biological insights to complex data are best gained by combining several complementary methods of analysis.To illustrate the levels of complexity at which our methods can operate, we consider a population sample of 5086 strains that have been identified as a priori unknown. In our statistical approach presented later we consider in detail the inference of these from molecular data. Here we aim to estimate the strength of gene flow via a stochastic characterization of the rates of admixture between the Assume that the target population consists of Let Assuming that a particular strain within population To arrive at a statistical characterization of the rates of admixture among the populations under the above framework, let a priori. Modern Bayesian statistical framework utilizing state-of-the-art MCMC computation can in principle be thought to provide a suitable setting for fitting such models to MLST sequence data. However, the computational complexity associated with the models suggests that formal posterior inferences would remain beyond the bounds of computational tractability even for only moderately sized population samples. This is crucial, as to study such problems we require large samples with a broad coverage of the genetic variation in the underlying population. Therefore, we consider here an approximate inference strategy to estimate It follows from the definition of Assume we have a sampled set of Let In a series of earlier works in a priori uncertainty about the underlying genetic structure in terms of a probability distribution over the space Let Here we use a Bayesian estimate of the genetic structure provided by the posterior modeThe marginal likelihood for the observed sequence data given any structure However, to explicitly specify the terms Given a plausible representation of the underlying genetic population structure based on (4), our aim is to obtain a model-based characterization of the rates of admixture between the populations, such that an estimate may be derived for the gene flow weight matrix The most recent version of the BAPS software (5.2) contains an implementation of the admixture estimation algorithm introduced by Let We now combine the statistical tools from The latest version of BAPS (5.2) contains an implementation of the estimation procedure leading to (7), such that high-resolution images of the directed graph The above presented statistical models and tools provide means for assessing the number of genetically isolated populations and the extent of recombination among them. However, this leaves open questions related to the underlying genetic population structure. In particular, the model summary estimates do not provide any information on the area occupied by the population in sequence space, which we term its genetic shape. By a genetic shape we refer both to the molecular heterogeneity present in a population, as well as the genetic affinities of its members towards other identified populations. We will illustrate that an investigation of the genetic shapes in this sense can yield useful characterizations of the population, pinpoint interesting subgroups of strains, and eventually provide clues to relate the genetic structure to some auxiliary information.Let The difference (8) can be interpreted as the amount of information we lose in the prediction of the molecular characteristics in To numerically characterize the genetic shape of a population using the values of (8) for The simulated MLST data sets were generated by assuming a tentative gene flow graph The assumptions for the data generation are based on a simplified, yet reasonable evolutionary model of bacterial populations. We assumed that each population has a common local ancestor, and further back in time these local ancestors originated from a common ancestor of the whole population, termed as a global ancestor. This assumption enables a tree representation of the evolutionary relationships among the populations . It is istage-1 mutation, and a mutation that occurred afterwards as a stage-2 mutation. We assumed the infinite-site model of mutation, which implies that at most one mutation per site can occur in the DNA sequences When ignoring recombination, the strains in a population will differ from each other only through the accumulation of point mutations. The mutations may have accumulated in two consecutive stages. In what follows, we referred to a mutation that occurred prior to the local ancestors as a To simplify the problem, we considered recombinations that always lead to changes of DNA, so that they can be detected as admixture events. This corresponds to assuming that the DNA introduced by admixture needs to be distinctive compared to the homologous sites that have been observed within the population. Given the tree representation of the population evolution, such recombinations are restricted to occur at stage-1 mutation sites only.Following these assumptions described above, the expected numbers of stage-1 and stage-2 mutation sites can be obtained byWe specified a putative gene flow graph Sampling a data set according to the putative population structure consists of three steps. First, a global ancestor of 500 segregating nucleotides was randomly simulated and for each of the six population a local ancestor was generated by randomly altering each nucleotide of the global ancestor with the probability 0.8. The sample strains for each population were generated by randomly mutating the local ancestor with the probability 0.2. The strains that have been recombined were randomly selected according to the parameter Using the procedure described above, a population data set can be simulated for each of the selections of abc Z, adk, aro E, fum C, gdh, pdh C and pgm genes are obtained and unambiguously characterize the strain. The strain sequences are generally reported to the publicly accessible MLST strain databases , which are currently hosting a fast growing number of bacterial genera and also a few eukaryotic organisms.To illustrate the presented methods with a real data set, we applied BAPS 5.2 to MLST bacterial data. MLST(multi-locus sequence typing) is an approach to the unambiguous characterization of bacterial strains. The internal sequence of seven housekeeping genes, which include the Neisseria species for validation as homologous recombination is known to be frequent in both N. meningitidis and N. lactamica species N. meningitidis and 263 strains of N. lactamica. The data was accessed for analysis from the Neisseria MLST database at 17/3/2006 We chose the Neisseria populations, using the procedure as follows:For such MLST type sequence data, we utilized the genetic linkage model Decide the number of clusters Select randomly For each chosen cluster, sample with replacement a random number of strains. The number of sampled strains follows the uniform distribution in the range of .Clustering the data generated in step 3 using BAPS 5.2.By repeating the scenario multiple times (we use 5 repeats) for each N. meningitidis which show significant gene flow towards the N. lactamica species, and also compared their similarity in terms of admixture tendency.Conditional on the identified population structure, comparative rates of admixture between the populations can be further estimated and summarized in a gene flow graph. We plotted the genetic shapes of several populations in To investigate whether the signals of admixture varied considerably over the seven genes, we performed a bootstrap analysis where a single gene at a time was excluded when inferring the rates of admixture. The analysis was performed conditional on the clusters identified using the original complete data set. The relative importance of each gene could then be tentatively summarized by calculating for each cluster the average changes in incoming and outgoing gene flow while treating the estimates from the complete data as a baseline.Neisseria data was performed using MEGA v.4.0.2 Neisseria data was performed using the default options in the online version 3 available at http://eburst.mlst.netPhylogenetic analysis of the i.e. e.g. as We reported the partition accuracy with respect to different choices of see e.g.  is summasee e.g. . In the We may look further into the gene flow graph prediction only if the genetic structure (i.e. the true partition) is correctly identified. We used Hamming distance as a measure of gene flow structure accuracy and the result is shown in We used a simulated data set for illustration of genetic shapes represented as the density estimation in (8). The data set was generated with N. lactamica species and the remaining 29 populations are labeled as N. meningitidis species. For accessing the robustness of the identified population structure, the partition determined using the whole data set was compared with the partition using bootstrap data generated according to the simulation scenarios. In total 32 BAPS populations are identified, where three populations  belong to the Neisseria data set are summarized in N. meningitidis populations (11 and 19) are constantly influencing the genetic makeup of one population of N. lactamica (population 29). The admixture arrows are uniformly directed from N. meningitidis towards N. lactamica, implying that N. meningitidis might donate genetic materials into N. lactamica, while the gene flow in a reversed direction is not supported by the analysis.The results of admixture analysis for the gdh seems to affect the admixture consistency most, as in this case the changes of outgoing (incoming) gene flow reach the maximum value in 20 (13) of the 32 clusters. In contrast none of the clusters is experiencing the largest gene flow changes when either the gene adk or aroE is excluded, suggesting that recombination signals on these two genes are more marginal.The admixture estimates for the 32 clusters obtained under the bootstrap analysis over the genes are summarized in N. lactamica populations , relative to N. meningitidis populations 11 and 19 separately. The densities for population 29 have a tendency towards zero, suggesting a close genetic affinity with populations 11 and 19. In contrast, the densities of populations 8 and 32 are much further away from zero, implying a distinctive difference in their genetic makeup compared to N. meningitidis populations 11 and 19. This result is consistent with the admixture pattern presented in We plotted in Neisseria database resulted in 253 groups and 1165 singleton strains. The biggest group consists of 795 strains and there are additionally four groups containing more than 200 strains. N. lactamica strains , only one forms a group in the eBURST analysis  which according to the inferred gene flow network is the most prominent recipient of genetic material from a multitude of sources, is seen  to incluN. lactamica fell into three populations: 8, 29 and 32. N. lactamica population. The role of meningococcal strains in populations 11 and 19 in this is evident, in that the recombinant N. lactamica strains  apparently originate close to these populations in the main meningococcal radiation.In the BAPS analysis, strains identified as N. lactamica strains and the main meningococcal radiation. Hence these STs may be considered as examples of the so-called fuzzy fringes which have been proposed for recombinogenic species Population 32 is intermediate on the tree between the majority of Neisseria database was completed within roughly 95 CPU hours on a standard PC with a 2.8 GHz Pentium 4 processor. As a comparison, our initial experiments with the STRUCTURE software In the present work we have introduced statistical tools implemented in the BAPS software that enable analyses of bacterial population structures on a previously unprecented scale, as the computational complexity of the earlier standard Bayesian methods prevents their application to large databases associated with complex patterns of admixture. This is particularly important when at least a moderate number of recombination events have plausibly taken place in a population, as a statistically valid characterization of the population structure then requires fairly extensive sampling of strains. It was also noted that a standard MCMC-based approach is not expected to yield a viable strategy for such analyses in practice, due to both the time constraints as well as the statistical accuracy of the resulting estimates. The BAPS analysis (inference about the populations and the levels of admixture) of the The presented methods can be effectively utilized in a variety of contexts, where the genetic population structure is relevant, e.g. for the investigation of epidemiological questions and experimentally derived features of bacterial strains. For instance, outlying groups with specific characteristics with respect to virulence or antibiotic resistance may be detected from large population samples.The concept of a genetic shape, which was introduced here to represent the molecular variability of an identified population and its affinity towards other populations as a whole, is an intriguing characteristic associated with considerable potential for further theoretical research. Namely, the average change of log predictive likelihoods between populations can also be interpreted as the change in ‘free energy’ associated with a gene flow event. The larger this quantity, the more likely the ‘reaction’ of gene flow could occur spontaneously. However, it is not trivial to determine the minimal energy level that triggers such events. From the analysis of simulated data (results not shown) we are expecting that such an energy threshold depends on the identified population sizes. In particular, the analogy with physics-based characterizations of molecular interaction systems could yield mathematical ways to predict horizontal transfer events.Although this integrated approach advocated here provides a feasible means of handling data from thousands of strains and a multitude of genes, several issues remain. Firstly, if a very large number of genes are considered, it is likely that not all of them will be present or functional in overall in a heterogeneous population at a genus level. Under such circumstances it would be necessary to develop further the Bayesian model for a population structure and admixture to take into account that not all molecular information is shared by the sampled strains. Secondly, the scalability of the stochastic learning algorithms should be improved to ensure that models could still be fitted to data without access to supercomputing facilities. Given the present rate of improvement in sequencing facilities, it is likely that the need for such large-scale analyses will be a reality within a relatively short time-span. In order to meet these needs in the future, we are currently investigating several theoretical approaches to develop further the statistical population genetic tools available in BAPS.N. lactamica and N. meningitidis that have arisen on multiple occasions and have occurred for clearly separate lineages of the two species. As the former is a non-pathogen and N. meningitidis represents a pathogen of considerable importance in human health, exchanges of genetic material between them might have consequences for our understanding of their evolution. Moreover, the diversity and the extent of recombination indicated among the N. meningitidis populations highlight that it is necessary to consider these pathogens as a heterogeneous population, and that multiple pathways of evolution may arise among them as a response to treatment strategies, including antibiotics and vaccines, as also recently discussed in The findings from the combined phylogenetic and population genetic analyses suggest possible events of convergence between


Calidris alpina schinzii, an endangered shorebird.Inbreeding and loss of genetic diversity are expected to increase the extinction risk of small populations, but detailed tests in natural populations are scarce. We combine long-term population and fitness data with those from two types of molecular markers to examine the role of genetic effects in a declining metapopulation of southern dunlins The decline is associated with increased pairings between related individuals, including close inbreeding . Furthermore, reduced genetic diversity seems to affect individual fitness at several life stages. Higher genetic similarity between mates correlates negatively with the pair's hatching success. Moreover, offspring produced by related parents are more homozygous and suffer from increased mortality during embryonic development and possibly also after hatching.Our results demonstrate strong genetic effects in a rapidly declining population, emphasizing the importance of genetic factors for the persistence of small populations. Fragmentation of natural habitats is associated with population declines of many species. The resulting small and isolated populations are threatened by extinction for several reasons (reviewed in ). Such pet al )Parus major confirms that inbreeding can affect the entire life cycle [Using field observations of parentage and molecular data, we demonstrate an increased frequency of pairings between related individuals during the 12 year study. Such matings, including incestuous inbreeding, resulted in more homozygous offspring with reduced survival during early development (before hatching) and possibly also later in life (recruitment). A recent study of a natural population of great tits fe cycle . Althougfe cycle  show thafe cycle . Our finfe cycle ,13.Melospiza melodia (a passerine bird with relatively short generation time), pedigrees revealed that 61% of the overall inbreeding was caused by matings among distant relatives [We found that at least 4% of all pairings represented matings between first-order relatives. However, the frequency of close inbreeding does not necessarily provide a good approximation of the population level of inbreeding . Becauseelatives . Regardlelatives . If dunlInbreeding depression may be most difficult to detect when its effects are greatest. For example, if deleterious genes are expressed very early in development, only less inbred offspring may be left to sample . We circet al [Although microsatellites are generally considered selectively neutral (non-coding) markers, this and other studies show that heterozygosity at microsatellite loci may correlate with measures of fitness (reviewed in ). Such het al  proposedet al ,52), butet al ,51-53.We have shown that a declining population of a long-lived, endangered vertebrate suffers from substantial negative genetic effects. Our results highlight that ignoring genetics may underestimate the extinction risk of natural populations and thus lead to inappropriate conservation measures .DB conceived and managed the study including data collection, statistical analyses and writing of the manuscript. AP carried out the molecular genetic analyses. AP, ML and LF helped with data collection and writing of the manuscript. All authors read and approved the final manuscript.