Pseudoplatystoma are very important species due to both their high commercial value and their ecological role as voracious predators. They undertake lengthy migratory movements during their life-cycle, this including reproductive migration which occurs from October to December in the rainy season. In the present study, seven microsatellite loci were analyzed to access genetic variability in two samples of P. reticulatum from the Upper Paraguay Basin. The loci were highly polymorphic (mean = 7.28). According to all analysis, the two samples of P. reticulatum revealed pronounced genetic differentiation. Fst value was 0.2290, Rst value 0.1067 and AMOVA 22.90% (Fst ) and 10.67% (Rst ), all being highly significant (p < 0.001). The division of the fishes into two groups was confirmed by microsatellite multi-locus Bayesian assignment testing. The results obtained present evidence of genetic structuring in a P. reticulatum population. Catfishes of the genus  Pseudoplatystoma belong to the family Pimelodidae, the 93 species of which occurring in the major river basins of South America . Studies using microsatellites have revealed pronounced genetic differences even among populations isolated by short geographic distances (Pseudoplatystoma reticulatum (“cachara”) collected in the Upper Paraguay River Basin, by using seven microsatellite loci originally developed for P. corruscans to access the genetic diversity of these samples. These data can be used as subsidies for setting up management policies for the development and conservation of these species.Microsatellite markers have been extensively used in studies on the genetics of fish populations. These markers have a co-dominant inheritance pattern, a high degree of polymorphism, and allow for easy analysis through experiments involving the polymerase chain reaction (PCR) (P. reticulatum were caught by gill nets at two points on the Paraguay River Basin. 31 specimens were collected from the Paraguay River (PRP) (16° 04' 00" S 57° 41' 00" W) and 21 from the Jauru River (PRJ) (15° 51' 00" S 58° 27' 00" W). Fin clips were the source of nuclear DNA. These were collected from freshly caught fish and immediately preserved in 95% ethanol. After collection, the fishes were sold. For the extraction of genomic DNA, about 0.1 mg of tissue was incubated in 200 μL of 5% Chelex (Sigma®) at 65 °C overnight.A total of 52 adult specimens of P. reticulatum samples were screened for variation at each of the seven microsatellite loci. Five of these loci have already been described by 2, 0.2 U of Taq-Pht DNA polymerase, 1X PCR buffer  and water. We used the following PCR profile for the loci Pcor01, Pcor02, Pcor05, Pcor08, Pcor21, Pcor23 and Pcor28: initial denaturation at 95 °C for 5 min, 30 cycles of 10 s at 95 °C, 15 s at an annealing temperature of 55 °C, 15 s at 72 °C and a final extension at 72 °C for 10 min. For the locus Pcor10, the PCR profile consisted of an initial denaturation at 95 °C for 5 min, 30 cycles of 30 s at 95 °C, 30 s at an annealing temperature of 48 °C, 30 s at 72 °C and a final extension at 72 °C for 10 min. Amplified products were resolved on 6% polyacrylamide gels stained with silver nitrate. Microsatellite alleles were identified by their size in base-pairs. Allele lengths were estimated by comparison with a 10 bp ladder (10 pb DNA Ladder - Invitrogen), using Kodak Digital Science 1D software.The HE, HO), inbreeding coefficient (FIS) and gene flow (Nm = 0.25(1-FST)/FST) were obtained with POPGEN 1.32 software , and their significance was tested with 1,000 permutations. Corrections of the significance level for multiple tests were performed following the Bonferroni procedure  was estimated by using the ‘no admixture' ancestral model with correlated alleles, thereby allowing for maximal population resolution, with K ranging from 1 to 6. Three independent runs of 500,000 Markov chain Monte Carlo (MCMC) generations and 100,000 generations of ‘burn-in' were used for each value of K. The true number of populations is expected to be the value of K that maximizes the estimated model log-likelihood, log (P(X|K))  to 11 (Pcor08), and allelic richness from 1.722 (Pcor28) to 9.503 (Pcor08) (Pcor28) to 0.870 (Pcor01). A total of 51 alleles were detected, of which 26 were private (Pcor28 - PRJ) to 0.8615 (Pcor01 - PRJ) and from 0.0556 (Pcor28 - PRJ) to 0.5556 (Pcor08 - PRJ and PRP), respectively  . Nei gen private . The pri private . Expecteectively . Signifiectively . The occanalyzed .P. reticulatum, estimated through the FST index for all the loci, was 0.2290 and was statistically highly significant , thereby showing the existence of strong genetic differentiation among the analyzed samples. The RST index estimated for all the loci was 0.1067, which was also statistically highly significant .Genetic differentiation between the two populations of Individual multi-locus genotypes were used to assign individuals to their respective population of origin. On considering the correct assignment of all individuals, 98.3% were correctly assigned to the PRP population and 99.1% to the PRJ population.FST = 77.10% and RST = 89.33%. The values for variability between populations were FST = 22.90% and RST = 10.67%. These were highly significant (p < 0.0001), thus revealing the strong structure of P. reticulatum populations.Hierarchical AMOVA revealed that most total genetic variance was to be found within populations K = 2 populations maximizing the estimated log likelihood in the model , consistent with the mean number observed in other fish species . HoweverA significant deviation of HWE (p < 0.05) was observed in seven loci within two samples analyzed . HWE depFIS was calculated for all loci. The results acquired displayed significant departure from HWE  and moderate (0.1067) values, respectively (p < 0.025), thus suggesting there is a strong genetic structure. The differences of values between indices were probably due to differences in the mutation models on which they were based. While FST is based on IAM, RST is based on SMM. The RST index may be the best for microsatellite analysis, and it is expected that RST values under a strict SMM pattern would be higher than those of FST 9GC(TC)9-; Pcor05 -(TC)8CC(TC)15- and Pcor10 -(GTCG)15(GT)9(CC-).There was significant populational structuring between the two samples of e of FST . HoweverMM model . This prP. reticulatum on using both indices (FST and RST), thereby depicting the occurrence of strong genetic structuring. The values of molecular variation among populations were 22.90% (FST) and 10.67% (RST), both with highly significant p values (p < 0.0001). Through structure analysis without admixture, it was shown that both populations were genetically distinct, with K = 2 populations maximizing the estimated log-likelihood in the model , 98.3% and 99.1% of the individuals were correctly assigned to the location from which they were sampled . These results are consistent with the values found by P. corruscans populations . FST ~ 0.1).The genetic flow of all loci in the two populations was estimated as ulations . Thus, oP. reticulatum analyzed. In five of these, frequency was higher than 10% (P. reticulatum analyzed. Furthermore, all the tests employed confirmed the strong structure in these populations. Thus, our work renders preliminary evidence of the genetic structure in P. reticulatum. As the results presented are consistent with those obtained for P. corruscans (P. reticulatum also presents homing behavior.Twenty-six private alleles were found in the two populations of than 10% . The exiP. reticulatum, the present data constitute a particularly important element when contemplating their management and conservation. It also places in evidence, the importance of preserving each population to further a positive outcome in the genetic conservation of these species.The ability to identify and define biological populations is crucial for taking informed decisions concerning conservation and management . Conside


The Maldives are an 850 km-long string of atolls located centrally in the northern Indian Ocean basin. Because of this geographic situation, the present-day Maldivian population has potential for uncovering genetic signatures of historic migration events in the region. We therefore studied autosomal DNA-, mitochondrial DNA-, and Y-chromosomal DNA markers in a representative sample of 141 unrelated Maldivians, with 119 from six major settlements. We found a total of 63 different mtDNA haplotypes that could be allocated to 29 mtDNA haplogroups, mostly within the M, R, and U clades. We found 66 different Y-STR haplotypes in 10 Y-chromosome haplogroups, predominantly H1, J2, L, R1a1a, and R2. Parental admixture analysis for mtDNA- and Y-haplogroup data indicates a strong genetic link between the Maldive Islands and mainland South Asia, and excludes significant gene flow from Southeast Asia. Paternal admixture from West Asia is detected, but cannot be distinguished from admixture from South Asia. Maternal admixture from West Asia is excluded. Within the Maldives, we find a subtle genetic substructure in all marker systems that is not directly related to geographic distance or linguistic dialect. We found reduced Y-STR diversity and reduced male-mediated gene flow between atolls, suggesting independent male founder effects for each atoll. Detected reduced female-mediated gene flow between atolls confirms a Maldives-specific history of matrilocality. In conclusion, our new genetic data agree with the commonly reported Maldivian ancestry in South Asia, but furthermore suggest multiple, independent immigration events and asymmetrical migration of females and males across the archipelago. Am J Phys Anthropol 151:58–67, 2013. © 2013 Wiley Periodicals, Inc. The Indian Ocean has been an important corridor for human migrations, and it has been travelled since the classical era Hourani, . NortherCypraea sp.) that were used as a currency throughout the classical old world and even reached West Africa  using a multiplex single-base primer extension  assay as described in Quintans et al. . In addiThirty-two SNPs at informative positions in the Y-chromosome phylogenetic tree  was used according to the manufacturer's description to type 17 autosomal microsatellite markers and the Amelogenin locus that was used to confirm the reported gender of each participant.http://PhyloTree.org , and corrected for multiple testing using the Holm-Bonferroni procedure . Then, we binned the gene flow values into four classes: (1) significantly low; high Fst, (2) nonsignificant below 20; Fst > 0.025, (3) between 20 and 30; 0.025 > Fst > 0.004, and (4) 30 or higher; Fst < 0.004. No first or second degree familial relatedness was found after pairwise checking for allele mismatch in 17 autosomal STRs. The correlation of genetic distance with geographic distance was assessed using a Mantel test in Alleles In Space  and 16086C-16209C-16256T (n = 5), respectively. Most of the mtDNA haplotypes observed in the Maldives fell into haplogroups M6, M30, M36, M39, M41, M66, R5, U1, and U2 . All these haplogroups have previously been detected in the Central reference population, that is, South Asia. Four individuals that clearly belonged to haplogroup M2 (defined by 16223T-16274A-16319A), also carried the haplogroup M2b defining mutations 16320T and 195C but lacked other M2b defining variants such as 152C, 182T, 14766C, 16169.1C, and 16189C, therefore most likely reflecting an intermediate split within the M2b haplogroup; we have thus tentatively assigned these individuals to haplogroup “pre-M2b.” Interestingly, all haplogroup M39b individuals in our sample carried a six bp deletion within HVS2 (affecting pos. 105–110), a mutation which is rare but recurrent, as it has previously only been reported on the background of Native American haplogroup D1 , C(M130), F*, G(M201), H(M69), J(M304), K*, L(M20), Q(M242), and R(M207) . The mosA quantitative analysis of admixture proportions Wang,  from mtDVamong populations; P < 10−4) in the AMOVA analyses. This substructure is not related to a dialect difference between northern and southern islands . Pairwise population comparisons for autosomal markers reveal low pairwise Fst values that indicate little differentiation between islands .The six island populations have gene diversity values  that ind islands . Only thVamong populations = 8.3% (P < 10−4) . Moreover, there was no significant correlation of mtDNA HVS1 variation with geographic distance within the archipelago as a whole .Diversity estimates for the island samples and for the combined sample  are some < 10−4) . The pai < 10−4) . The Ali < 10−4)  did not P < 10−3) of the genetic variance in Y-STRs is explained by among-population variance . In addition, no significant correlation with geographic distance across all islands within the archipelago was found .Y-STR diversity is reduced in all island populations as well as in the Maldives as a whole . This isvariance : male- mvariance . First, variance . An AMOVThe wide range of haplogroups for both mtDNA and Y present in our Maldivian sample suggest a diverse origin. However, our results from both qualitative and quantitative analyses of the haplogroup distribution of uniparentally inherited markers indicate that the varieties of genomes sampled in the Maldives do not differ significantly from those found in the Central reference populations that contain the countries India, Pakistan, and Sri Lanka. In contrast, the most abundant haplogroups in the East reference population  are almost completely missing in our Maldivian samples.Female-mediated gene flow from the Western region was also limited. Frequent Western mtDNA haplogroups JT, L, R01, and HV are almost completely absent in our Maldivian sample. Based on the quantitative admixture results, a minor contribution from West Asia to the Maldivian Y-chromosome pool is likely. However, this is caused predominantly by sharing of Y-haplogroup J2(M172), a haplogroup that is more frequent in West Asia compared to South Asia. This haplogroup is present at relatively low frequencies in South Asia, predominantly in upper-caste populations  for individuals with a similar haplotype observed in Madagascar using Mitotool (Fan and Yao, A previous simulation study of seafaring across the Indian Ocean suggested that there could be an important role for the Maldives in the settling of Madagascar by people from Southeast Asia (Fitzpatrick and Callaghan, The ancestry of mtDNA- and Y haplogroups in present-day South Asia is a highly complex mix of tribal, caste, religious, and language groups, and the structure along those cultural identities is likely to be subtle (Majumder, The Dhivehi language is the southernmost Indo-Aryan language, and sharing of specific haplogroups with Indo-Aryan Sinhalese populations mostly from northern India and from Sri Lanka could point to a common origin of these populations. The Indo-Aryan-speaking higher castes in northern India in particular show moderately high levels of admixture with West Eurasian populations that include Indo-Aryan speakers (Bamshad et al., We observed low frequencies of some haplogroups in the Maldives that are typically found outside South Asia. This may indicate that occasional migration from outside South Asia to the Maldives did occur. We found one Maldivian with mtDNA haplogroup M71, which has been described to occur at a low frequency in Southeast Asia (Peng et al., Diversity estimates in autosomal and mtDNA markers reveal normal to mildly reduced diversity levels that are typical of other island populations (Jorde et al., We report a strong genetic link between the Maldives Islands and the Indian sub-continent. We exclude the sharing of haplogroups between Southeast Asia and the Maldives, which questions the previously suggested central role of a Maldive Islands' stopping point for migration from South East Asia to Madagascar. The Maldives were more likely used for supplies than for settling a population, which is no surprise considering the limited landmass of coral atolls. We also dismiss female-mediated gene flow from West Asia. Male-mediated gene flow from West Asia to the Maldives could have occurred, although it cannot be separated from such gene flow through South Asia. The wide range of Y-chromosomal and mtDNA haplogroups mirrors the haplogroup diversity in South Asia. We find a subtle substructure within the Maldives that is not directly related to geographic distance or dialect. Reduced diversity of Y-chromosomal markers on each atoll combined with reduced male-mediated gene flow between atolls suggests independent founder effects for each atoll. Reduced female-mediated gene flow between atolls confirms a Maldives-specific history of matrilocality.


There is substantial ethnic, cultural and linguistic diversity among the people living in east Africa, Sudan and the Nile Valley. The region around the Nile Valley has a long history of succession of different groups, coupled with demographic and migration events, potentially leading to genetic structure among humans in the region.17. The genotype data from the Sudanese populations was combined with previously published genotype data from Egypt, Somalia and the Karamoja population from Uganda. The Somali population was found to be genetically distinct from the other northeast African populations. Individuals from northern Sudan clustered together with those from Egypt, and individuals from southern Sudan clustered with those from the Karamoja population. The similarity of the Nubian and Egyptian populations suggest that migration, potentially bidirectional, occurred along the Nile river Valley, which is consistent with the historical evidence for long-term interactions between Egypt and Nubia.We report the genotypes of the 15 Identifiler microsatellite markers for 498 individuals from 18 Sudanese populations representing different ethnic and linguistic groups. The combined power of exclusion (PE) was 0.9999981, and the combined match probability was 1 in 7.4 × 10We show that despite the levels of population structure in Sudan, standard forensic summary statistics are robust tools for personal identification and parentage analysis in Sudan. Although some patterns of population structure can be revealed with 15 microsatellites, a much larger set of genetic markers is needed to detect fine-scale population structure in east Africa and the Nile Valley. The marker panel has also been used in population structure and admixture studies of humans [Since the introduction of a standardized set of forensic microsatellite markers , over 10f humans -21, and f humans . Admixtuf humans . Howeverf humans ,23,24.In this study, we report the genotypes of 15 autosomal STR markers (the markers in the AmpFlSTR Identifiler PCR Amplification Kit) for different populations in Sudan, and compute commonly used forensic summary statistics. We infer population structure for the Sudanese population and for an expanded set of populations (compiled from previous studies) from Uganda , Egypt  and SomaThe geographic locations of the sampled populations are indicated on a map of Sudan Figure , and sam-18. Five of the 15 loci .For the combination of the 18 Sudanese populations, allele frequencies for the 15 STR loci are shown in Table act test ) had devet al. [Ιn), which describes the information content of a particular genetic marker for ancestry inference (it ranges from zero (no information) to the natural logarithm of the number of populations (maximum information)). We computed Ιn values for the 15 Identifiler STRs  from the present study. The mean, across markers, (and standard deviation) Ιn was 0.167 (0.070) for the Sudanese populations, and 0.154 (0.066) for the Sudanese populations and the populations from Egypt, Somalia and Uganda (the Karamoja). To determine the relative informativeness of the 15 Identifier microsatellites, we computed In for 377 microsatellites (of which 274 were tetranucleotide microsatellites) for two groups of African populations from Rosenberg et al. [et al. [In (across markers) was 0.234 (0.098) for the HGDP sub-Saharan group and 0.106 (0.054) for the HGDP Niger-Congo group, which was a significant difference . The In statistic depends on both marker information about ancestry and the level of differentiation between the investigated populations. Because the same markers were used for both the HGDP sub-Saharan group and the HGDP Niger-Congo group, the difference in In between the two groups was due to the greater level of differentiation between populations in the HGDP sub-Saharan group. The In values for the Sudanese populations and the larger group of northeast African populations  were found to lie between the values for the HGDP Niger-Congo group and the HGDP sub-Saharan group. This result is not surprising, considering that the sampled populations from Sudan belonged to different linguistic groups, although the groups are not as differentiated as the Pygmy, the San and the Niger-Congo-speaking populations in the HGDP sub-Saharan group. We concluded that the CODIS STRs contain information on population structure in the same range as many other microsatellites. However, it is possible to select more informative STR loci for population structure inference; for example, the top 15 most informative loci of the 377 microsatellites [In values of 0.489 (0.062) and 0.249 (0.041) respectively. This observation can be explained by the fact that the Identifiler STRs have been selected to have high levels of variation, because of the potentially high mutation rates of these STRs, in order to be powerful tools for identifying individuals, but the high level of variation also makes it challenging to separate alleles that are identical by state from those identical by descent [Rosenberg et al.  developeg et al. ; six sub [et al. , the meatellites  .The greatest number of distinct alleles , consideFor pairs of Sudanese populations, the greatest mean number of alleles that are private to pairs of populations was found for the Zagawa-Nuba pair 0.131 ± 0.064), followed by the Zagawa-Nilotic pair (0.080 ± 0.051) and the Zagawa-Copt pair  [P < 0.001, Mann-Whitney U-test), these from southern Sudan and Uganda into the 'red' cluster , and those from Somalia into a (partly) separate 'yellow' cluster . The mixed ancestry of basically all individuals is probably a consequence of the limited number of markers (and of the set of markers not being particularly informative about ancestry) rather than an indication of recent admixture, a behavior of admixture analyses that has been reported previously  . Althougg et al. ). Using e Figure .The number of unique alleles Figure  was greaThe patterns of population structure we found in northeast Africa, in particular the similarity of Nubian (a northern Sudanese group that speak Nilo-Saharan languages) and the Egyptian population. is consistent with the historical evidence for long-term interactions between Egypt and Nubia, probably resulting in genetic flow between the two regions. However, the Nubian group and the Karamoja group from Uganda share a relatively large number of private alleles Figure , potentiEven though most of the genetic variation was seen within populations and between individuals, some genetic variation was found between groups Table . For theEven though the 15 Identifiler microsatellites are not the most informative markers for inference of ancestry, these markers contain sufficient information to differentiate (to some degree) between distinct geographic and linguistic groups within Sudan and within a larger collection of northeast African populations. We conclude that geography, language and culture have played an important role in shaping patterns and structure of genetic variation in Sudan, and that these patterns may have been shaped by long-term occupation by Nilo-Saharan groups and more recent migration from North Africa and Eurasia to the Nile Valley, but a larger number of markers would be needed for fine-scale population structure inference. This study confirms that the set of 15 Identifiler microsatellites is a suitable tool for personal identification and parentage analysis in Sudan, despite the levels of population structure.The study was described to each participant before sampling, and informed consent was obtained from each participant.In total, 498 unrelated subjects 366 men, 132 women) were recruited from different geographic regions of Sudan. For each individual, we collected blood and (self-reported) information about the gender and ethnic background of the participant and their parents and grandparents. For the calculation of forensic summary statistics, we excluded seven people because of a potential first-degree relationship, which was inferred using Relpair v2.0.1 , leaving32 women lSTR® Identifiler™ PCR Amplification Kit; Applied Biosystems) following the manufacturer's recommendations [XL Genetic Analyzer; Applied Biosystems). For each run on the analyzer, an allelic ladder, positive and negative controls, and an internal lane standard  were included. Allele calling was performed . We followed the International Society for Forensic Genetics (ISFG) recommendations on the analysis of DNA polymorphisms. The recommended nomenclature was used, and we followed the guidelines on quality control [Genomic DNA was extracted from DNA storage cards , following the manufacturer's instructions. Fifteen STR loci  were co-amplified for each of the 498 subjects (AmpFhttp://www.promega.com/geneticidtools/powerstats/). Testing for deviations from Hardy-Weinberg equilibrium was performed using Arlequin, version 3.11; (http://cmpg.unibe.ch/software/arlequin3/) [In, [In statistic is based on information theory, and evaluates the efficiency of a marker for assigning individuals to one of K populations. Following Rosenberg et al. [Standard summary statistics for forensic applications, including allele frequencies, PE, power of discrimination, and polymorphism information content, were computed  . To asse3/) [In,  for eachg et al.  for a paN is the number of alleles for the locus, K is the number of populations, ijpis the (parametric) frequency of allele j in population i, jpis the mean frequency of allele j across populations and 'log' denotes the natural logarithm. The minimal In value is 0 , and the maximal value is log K . In was computed for the combined dataset of northeast African populations from Sudan, Egypt, Somalia and Uganda. For comparison of the level of informativeness for the 15 Identifiler microsatellites, we computed In values for 377 microsatellites genotyped in the HGDP [In values were computed for two groups of populations from the HGDP: six African populations , referred to as the 'HGDP sub-Saharan group', and a subgroup of three populations from the HGDP who all speak Niger-Congo languages , referred to as the 'HGDP Niger-Congo group'.where the HGDP . AverageK), from K = 2 to K = 10. The 10 replicates for each choice of K was summarized . The distance matrix was visualized using PCA.Based on the genotype data from the 15 microsatellites, we inferred population structure for northeast African populations using the clustering software Structure ,40. We uumpp.html), with truct.html). Popularuct.html between http://rosenberglab.bioinformatics.med.umich.edu/adze.html[We computed the mean number of distinct alleles, the mean number of private alleles and the mean number of private alleles for pairs of populations  for theadze.html, we treaadze.html was usedadze.html) and theThe authors declare that they have no competing interests.HB and MJ conceived of the study; HB collected samples with help from HH; HB conducted the genotyping; HB, CS and MJ analyzed the data; and HB, CS and MJ wrote the paper with input from HH. All authors read and approved the final manuscript.


Incidental entanglement in fishing gear is arguably the most serious threat to many populations of small cetaceans, judging by the alarming number of captured animals. However, other aspects of this threat, such as the potential capture of mother-offspring pairs or reproductive pairs, could be equally or even more significant but have rarely been evaluated. Using a combination of demographic and genetic data we provide evidence that i) Franciscana dolphin pairs that are potentially reproductive and mother-offspring pairs form temporal bonds, and ii) are entangled simultaneously. Our results highlight potential demographic and genetic impacts of by-catch to cetacean populations: the joint entanglement of mother-offspring or reproductive pairs, compared to random individuals, might exacerbate the demographic consequences of by-catch, and the loss of groups of relatives means that significant components of genetic diversity could be lost together. Given the social nature of many odontocetes (toothed cetaceans), we suggest that these potential impacts could be rather general to the group and therefore by-catch could be more detrimental than previously considered. By-catch is among the most serious threats to non-target marine fauna globally Pontoporia blainvillei), endemic to the Western South Atlantic Ocean and possibly the most impacted cetacean in the region We seek to evaluate potential impacts of by-catch to specific family groupings in cetaceans, focusing on the rare Franciscana dolphin , reproductive group, or are unrelated individuals. This knowledge will inform us about potential demographic and genetic impacts to Franciscana dolphins, and possibly other social cetaceans.Tissue samples from 245 individuals were obtained mostly from incidentally entangled Franciscana dolphins in fishing gear in Argentina during 2000 through 2009. At least 14 dolphins were by-caught simultaneously, in pairs in the same net, in Bahia Samborombon South (BSS) and Cabo San Antonio (CSA) . In addiTo investigate the genealogical relationships between all simultaneously entangled and captured dolphins, we extracted genomic DNA, confirmed visual sexing with molecular techniques, sequenced a 560 bp mitochondrial DNA (mtDNA) fragment in the control region, and genotyped all samples using 12 microsatellite markers optimized for this species . All genp = 0.05) for departure from HWE and for LD were corrected for multiple comparisons with the sequential Bonferroni correction STF (haplotype frequencies only) and STΦ statistics (using the Kimura 2-parameter correction), computed using Arlequin v3.1Φ- or F-statistics was tested using the null distribution generated from 10,000 non-parametric random permutations of the data. This estimation was done between BSS-CSA and between CSA-BASS, given the coastal habits of these dolphins and that the three sampling sites are separated along the same coastline. In addition, we assessed the degree of partitioning in our total sample without a priori definition of putative populations using a Bayesian clustering algorithm on the microsatellite data with STRUCTURE v2.3.1 6 burn-in steps, 107 total steps) for each value of K (1≤K≤6), for a total of 60 runs  the adult dolphin was a female and both dolphins had the same mtDNA haplotype. The fourth pair was composed of an adult male and a juvenile male with different mtDNA haplotypes. The other three by-caught pairs were adult female-male pairs ; one of them with individuals sharing their mtDNA haplotype. All of the captured and released pairs consisted of an adult female and an adult male with different mtDNA haplotypes, whereas the trio (group 11) consisted of an adult female and male with different haplotypes and a calf that shared her haplotype with the adult female .STF  = 0.054, p<0.001; STΦ  = 0.068, p = 0.031), and less significant structure between CSA and BASS . The microsatellite data showed no evidence of HW disequilibrium or LD, the STRUCTURE runs showed convergence and were concordant with a hypothesis of three genetic partitions, evidenced by the plateau in the log likelihood values at K = 3, also coincident with the maximum ΔK for K = 3  and display relatedness values rest ∼0.5, whereas all other pairs are not significantly related (pr est>0.05) and all display relatedness values rest∼0, with the exception of individuals 1 and 2 in pair 11 (rest∼0.2)  . Likelih of 0.05 .Orcinus orca) and pilot whales  Our combined evidence suggests that small family groups of Franciscana dolphins involving mature females swim together and are by-caught simultaneously along the Argentinean coast. Specifically, our genetic data suggest that mother-offspring associations (pair 11) and unrelated adult pairs that are potentially reproductive  were captured-released together, suggesting that they form temporary (or long term) bonds, and are therefore at risk of simultaneous entanglement. Mother-offspring pairing is well known for cetaceans, and female-male pairing among adult dolphins is typically attributed to reproduction, in agreement with our suggestions that these adult pairs could be reproductive associations X2p>0.05; Fisher>0.05p for both BSS and CSA and the entire region). What our data suggest is that the consequences of multiple entanglements could be quite serious when pairs that are potentially reproductive and mother-offspring pairs are lost together, since they contribute more significantly to the population growth rate and persistence than random individuals We have not observed a gender bias in the chance of by-catch for adult individuals or pairs of individuals in any of our study areas or the region as a whole  Phocoena phocoena), through Bayesian modeling approaches From the ‘first principles’ of demography, it is well established that the juvenile and adult survival elasticities  are typically high for long-lived species such as marine mammals From a genetic perspective, harvest will inevitably change the make up of impacted populations (i.e. their genetic diversity and effective population size) and their relationship with other populations (i.e. population subdivision parameters) potentially add to the known depletion impact of by-catch. Whether the loss of mother-offspring and reproductive pairs actually does result in an “extra” impact to the depletion effect of by-catch could be evaluated through demographic and genetic modeling, which is a next step on our research efforts.We realize that our finding of mother-offspring pairs and adult pairs forming temporal bonds and being by-caught simultaneously does not directly translate into an evaluation of the impact of loosing these social groupings on the population persistence. Rather, our data highlight relevant genetic and demographic aspects associated with by-catch, which may have been previously overlooked, and that could There appears to be some geographic segregation to the types of associations found in our study, although this is not conclusive due to the small sample size. Most mother-offspring pairs were documented in BSS and no records were obtained in CSA. Coupled with recent evidence of strong population structure between BSS and other sites in Argentina Our data provides evidence that mother-offspring and reproductive pairs of Franciscanas are impacted by by-catch, and highlight potential synergies from genetic and demographic impacts on this and possibly other social cetaceans. On the one hand, by-catch removes large numbers of individuals from their populations, which is in itself a serious demographic and genetic impact Although the potential impacts of by-catch are surely manifold and difficult to quantify, we believe that our approach combining field, demographic and genetic evidence can provide a more comprehensive picture of this threat than estimating the number of by-caught animals alone. Moreover, including population structure evaluations as part of a threat assessment strategy seems particularly relevant for highly mobile species, given the potential for population connectivity across large marine areas. One of the caveats when using genetic data to make kinship inferences is that the data need to show enough variability to allow statistical testing under some frameworks. In our example, the magnitude of the type II error rates we observe in the likelihood ratio tests with our empirical data is likely a consequence of a relatively low number of loci and/or alleles, or a relatively small sample size of our empirical data. To test this, we have used KINGROUP to run simulations with higher numbers of alleles, loci and bigger population sizes, and in fact observed a marked reduction in the type II error rates (data not shown).While we base our assumptions of demographic and genetic consequences of mother-offspring and reproductive pair by-catch on what are general principles in demography, modeling exercises using empirical data would certainly contribute to this issue, and are among the next steps in our research efforts.Table S1Microsatellite amplification conditions.(DOC)Click here for additional data file.Table S2Runs for the Bayesian analysis of population structure. Burnin steps maximize the chances of reaching a high probability region in the probability space before the actual estimation.(DOC)Click here for additional data file.Table S3Runs for the Bayesian analysis of population structure. Burnin steps maximize the chances of reaching a high probability region in the probability space before the actual estimation.(DOC)Click here for additional data file.Table S4Runs for the Bayesian analysis of population structure. Burnin steps maximize the chances of reaching a high probability region in the probability space before the actual estimation.(DOC)Click here for additional data file.Text S1Supporting Text.(DOC)Click here for additional data file.


There are many different studies that contribute to the global picture of the ethnic heterogeneity in Brazilian populations. These studies use different types of genetic markers and are focused on the comparison of populations at different levels. In some of them, each geographical region is treated as a single homogeneous population, whereas other studies create different subdivisions: political , demographic , or ethnic . In this study, we performed an enhanced reassessment of the genetic ancestry of ~ 1,300 Brazilians characterised for 46 autosomal Ancestry Informative Markers (AIMs). In addition, 798 individuals from twelve Brazilian populations representing the five geographical macro-regions of Brazil were newly genotyped, including a Native American community and a rural Amazonian community. Following an increasing North to South gradient, European ancestry was the most prevalent in all urban populations . The populations in the North consisted of a significant proportion of Native American ancestry that was about two times higher than the African contribution. Conversely, in the Northeast, Center-West and Southeast, African ancestry was the second most prevalent. At an intrapopulation level, all urban populations were highly admixed, and most of the variation in ancestry proportions was observed between individuals within each population rather than among population. Nevertheless, individuals with a high proportion of Native American ancestry are only found in the samples from Terena and Santa Isabel. Our results allowed us to further refine the genetic landscape of Brazilians while establishing the basis for the effective application of an autosomal AIM panel in forensic casework and clinical association studies within the highly admixed Brazilian populations. Despite the nature of genetic markers  or their location in the genome , polymorphisms known as Ancestry Informative Markers (AIMs), present very high intercontinental allelic differentiation across populations e.g., ,2,3).,3.2,3]).The investigation of genetic ancestry profiles of human populations is a valuable tool to understand the dynamics of migrations and colonisation events, as well as to determine admixture patterns inside populations.http://pritch.bsd.uchicago.edu/software/STRAT.html), in addition to pre-selecting criteria to match controls with available cases , part, part5])In the field of forensic genetics, having a set of markers that may provide estimates of ancestral membership proportions or help identify the source population of the donor of a certain piece of evidence can help direct the criminal investigation ,8.Brazil is well known for the heterogeneous distribution of three main ancestral contributions from Native Americans, Europeans and Africans. These people met and mated among themselves in distinct ways, giving rise to a highly multiethnic admixed population. The European and African colonisation of the Brazilian territory, previously occupied only by Native Americans, started on the coast and gradually reached the interior. The progression of colonisation was highly diverse in different regions, as far as European, African and Native American parentages were concerned . This coThe first attempts to evaluate the ethnic diversity of Brazilians were based on blood groups and protein markers and provided a broad overview of the ethnic heterogeneity associated with the different geographic regions of the country e.g., ,14). Sin. Sin14])Apart from the variation in the number and/or type of markers that have been used to evaluate admixture processes in Brazil, consideration of the various sampling strategies that have been used until now is also important. In many studies, it is not possible to have a comprehensive view of the population gene pool because only some population subgroups are investigated, namely, groups classified based on (a) self- reported ancestry, (b) social stratus, (c) skin colour (d) or other phenotypic classifications.Considering the demography of Brazil in addition to the disparity of markers and sampling criteria that have been used in different publications, the ancestry of the Brazilians is far from fully known. Hence, additional analyses using larger random samples to cover new populations can be helpful in determining novel aspects of the genetic structure within Brazil’s five macro-regions. To accomplish this objective, we performed thorough analyses involving existing data ,21, the http://www.wma.net/en/30publications/10policies/b3/). The access to the Terena community living area was approved by FUNAI second authorisation n.º 016/CGEP/99.All samples involved in the study were anonymised DNA extracts previously obtained from healthy unrelated individuals. The samples were collected under written informed consent to participate in this study. This study was approved by the Ethic Committees at the State University of Rio de Janeiro (CAAE:0067.0.228.000-09). The current study complies with the ethical principles of the 2000 Helsinki Declaration of the 206 World Medical Association . The samples from Pernambuco were collected at the Laboratory of Human Identification, University of Pernambuco, from cases of paternity investigation. The samples from Espírito Santo and Manaus were obtained from students and professors at the federal universities of Espírito Santo and Amazonas, respectively. The samples from Mato Grosso do Sul were obtained from students at the State school João Ponce de Arruda. The samples from Alagoas and Santa Isabel were selected from previous research projects belonging to healthy unrelated individuals used as controls in association studies, which involved the State hospitals as well as the Federal University of Alagoas and the Research Institute FIOCRUZ . The Terena samples were those previously included in Manta et al. )The consistency of the results obtained among populations and within groups of populations, in addition to the expectations based on the available genetic and demographic data, precludes any significant distortion of the results obtained in the remaining populations we have studied. These samples are from paternity investigation cases preformed by private or judicial request, covering, therefore, people from both the lower and the higher social status that are living in the concerned states.Overall, the present results highlight the considerable amount of ethnic admixture that occurred throughout the country and that the current Brazilian population is significantly differentiated from the ancestral Native American, European and African populations that have been incorporated into its genepool during the last five centuries. The arrival of the Europeans stands out as the major event that shaped the genetic landscape of Brazil, which is especially evident in urban populations. Although demographically less representative of Brazil and spread throughout the country, there are still many rural populations and native communities that have maintained a strong Native American background. The African legacy is also a hallmark of all Brazilian populations, and in the present study represents more than 25% of the genetic makeup of most urban populations with the exception of the Northern and Southern regions (from 11 to 18%).Apart from the variation found among populations throughout the country, our study also underscores the tremendous diversity that can be found among individuals from the same population in terms of the three different ethnic contributions .In accordance with previous studies, our results emphasise the heterogenity of the Brazilian population from within and among populations, which justifies the need for additional studies involving more markers in new populations to allow an accurate assessment of the genetic ancestry for each subpopulation.Table S1List of genotypes found in 12 different populations included in the present work as well as data from Rio de Janeiro, Brazil, previously published by Manta et al., 2013.(XLS)Click here for additional data file.Table S2Allele frequencies for 46 AIM-Indels in twelve Brazilian populations.(XLS)Click here for additional data file.Table S3Gene diversities for 46 AIM-Indels in twelve Brazilians populations.(XLS)Click here for additional data file.Table S4ST) between African, European, Native American and Brazilian populations  and corresponding non-differentiation P values .Genetic distances (F(XLS)Click here for additional data file.Table S5Demographic significance of sampled populations based in 2010 IBGE data for Brazilian Regions and States.(PDF)Click here for additional data file.


Coralliophila abbreviata are corallivorous gastropods that live and feed on most of the reef-building corals in the tropical western Atlantic and Caribbean. Populations of C. abbreviata associated with the threatened acroporid corals, Acropora palmata and A. cervicornis, display different behavioral, morphological, demographic, and life-history characteristics than those that inhabit other coral host taxa, indicating that host-specific selective forces may be acting on C. abbreviata. Here, we used newly developed polymorphic microsatellite loci and mitochondrial cytochrome b sequence data to assess the population genetic structure, connectivity, and demographic history of C. abbreviata populations from three coral host taxa  and six geographic locations across the Caribbean. Analysis of molecular variance provided some evidence of weak and possibly geographically variable host-associated differentiation but no evidence of differentiation among sampling locations or major oceanographic regions, suggesting high gene flow across the Caribbean. Phylogenetic network and Bayesian clustering analyses supported a hypothesis of a single panmictic population as individuals failed to cluster by host or sampling location. Demographic analyses consistently supported a scenario of population expansion during the Pleistocene, a time of major carbonate reef development in the region. Although further study is needed to fully elucidate the interactive effects of host-associated selection and high gene flow in this system, our results have implications for local and regional community interactions and impact of predation on declining coral populations.Host-associated adaptation is emerging as a potential driver of population differentiation and speciation for marine organisms with major implications for ecosystem structure and function.  Although coral reefs are among the most biologically diverse ecosystems on the planet, the magnitude of this diversity and the mechanisms that drive and maintain it are still poorly understood Leptoconchus that are associated with mushroom corals  in the Indo-West Pacific. Many of these species are found in the same geographical area and can only be distinguished based on host association and molecular data. On the other hand, Oliverio & Mariottini Coralliophila meyendorfii that displayed host-specific size structure. Further studies are needed to elucidate the life-history characteristics and environmental conditions that facilitate or oppose host-specific differentiation and speciation for these and other coral associated organisms.Similar to phytophagous insects, corallivores are ubiquitous members of coral reef communities that provide a link from foundational scleractinian coral species and their symbionts to higher trophic levels Coralliophila abbreviata are found on reefs throughout the Caribbean and tropical western Atlantic. These snails live and feed on the tissue of at least 16 species of scleractinian coral from five different families representing diverse growth forms and life-histories C. abbreviata. Supporting this assertion, populations of C. abbreviata display host-specific behavioral, morphological, demographic, and life-history characteristics across the Caribbean. Snail populations found on the branching acroporid corals, Acropora palmata and A. cervicornis, are larger C. abbreviata. However, high dispersal and gene flow via planktotrophic veliger larvae with a putative pelagic larval duration (PLD) of more than 30 days , may preclude host-specific adaptation and differentiation. We can thus use this system to investigate the interactive effects of potentially diversifying selection and homogenizing gene flow in the marine environment.C. abbreviata using de novo microsatellite markers as well as mitochondrial DNA sequence data. Our overall objective was to characterize the neutral genetic variation of C. abbreviata populations from different coral host taxa and geographical locations to assess a.) potential host-associated genetic differentiation, b.) the scale and patterns of gene flow across the Caribbean, and c.) the possible role of historical demographic fluctuations in shaping the observed patterns of genetic variation and population structure. An understanding of these processes is necessary to elucidate contemporary community interactions and to predict the potential impact of C. abbreviata on the persistence and stability of threatened host corals in the future. Further, this study contributes to the general understanding of the ecological and evolutionary processes that create and maintain biodiversity on coral reefs.Here, we assess the Caribbean wide population genetic structure of Coralliophila abbreviata were collected using SCUBA over a five year period (2001–2006) from three coral host taxa, 18 reef sites  and Montastraea spp. (MON). In Panama, however, C. abbreviata were not encountered on surveyed A. palmata and Montastraea spp. coral colonies and were thus collected from Mycetophylia spp. (MYC) corals, on which they were prevalent. All necessary permits were obtained for the described field studies, including: recreational saltwater fishing license issued to LJ by the Florida Fish and Wildlife Conservation Commission (Florida: Little Grecian and Sand Island); Special use permit 41529-2006-03 ; ANAM permit PETCA 58323 ; general collection permit issued to CARMABI Foundation by the Dutch Antillean Government ; permit from the Department of Fisheries, granted to IBB ; permission granted to IBB from the Bahamian Department of Fisheries (Bahamas: Green Turtle Cay). No other permits were required for collections.Individual C. abbreviata using either a standard CTAB extraction protocol or a Qiagen DNeasy Tissue Kit, following the manufacturer's instructions. Published primers (UCYTB151F and UCYTB270R) and PCR conditions were used to amplify a portion of the mitochondrial cytochrome b gene  and nucleotide diversity (π) were calculated for all sampled populations using the program ARLEQUIN v. 3.5 cyt b haplotypes were assessed by constructing a phylogenetic network using the median joining algorithm implemented in NETWORK v. 4.6 Genetic diversity estimates for FIS values and test for deviations from Hardy-Weinberg equilibrium (HWE) and linkage equilibrium for each locus. All microsatellite loci were checked for the presence of null alleles and errors due to stuttering and large allele dropout using the program MICROCHECKER For each microsatellite locus, the number of observed alleles, allele frequencies, and observed and expected heterozygosity were determined using the program GENEPOP v.4 F-statistics, and exact tests of population differentiation using both mtDNA sequences and microsatellite data in ARLEQUIN v. 3.5. Due to small sample sizes from several reef sites , pairwise ef sites , individef sites . For micMycetophylia spp. host corals in Panama (PAN MYC) was excluded from initial AMOVA tests of host-associated differentiation to avoid introducing error due to limited sampling. However, for exploratory purposes, we repeated the above tests including the PAN MYC sample.To test the hypothesis of host-associated differentiation, individuals from all localities were first pooled by coral host (ACR and MON) for analysis. Then, to tease apart potential effects of host and locality on measures of differentiation, populations were defined by host and locality  and including the Lesser Antilles, and the western Caribbean including the Florida peninsula , Mantel tests were performed for both mtDNA and microsatellite data sets in ARLEQUIN. The geographical distance matrix was constructed using the shortest distances between locations via major ocean surface currents (as reported by Ne) was set to 3,000 while the number of generations of drift (t) was varied to determine the lowest level of differentiation [FST] that could be detected in simulated populations with at least 90% accuracy. With a Ne greater than 2,000, there should be little to no effect of loss of alleles on the power estimates for small FST values To assess the power of the microsatellite data set to detect low levels of population differentiation, simulations were conducted in POWSIM v.4.1 ad hoc, the number of discrete populations (K) represented in a sample. It assigns individuals to populations and can identify migrants when prior population information is used. Here, the admixture, location prior  was averaged across replicates for each K value to estimate the most likely number of populations using Structure Harvester v 0.6.6 Finally, the Bayesian Markov Chain Monte Carlo (MCMC) clustering method implemented in the program STRUCTURE v. 2.3 cyt b sequences was compared to the distributions expected under models of pure demographic expansion 0, and θ1, and τ) were estimated by a generalized non-linear least-square approach with confidence intervals obtained through parametric bootstrapping  is in mutational units; τ = 2ut, where t measures time in generations and u is the sequence mutation rate. The sums of squared deviations (SSD) of bootstrapped replicates were used to calculate the significance of the fit between the observed and expected mismatch distributions Historical demographic trends were investigated with several distinct methods. First, the mismatch distribution, based on the number of observed nucleotide differences between pairs of mitochondrial DFScyt b sequences to test for deviations from selective neutrality and to refine inferences of demographic history, using ARLEQUIN. These statistics are expected to be zero for populations of constant size and in mutation drift equilibrium. Significant deviations from neutrality may be caused by selection or historic demographic fluctuations such as population bottleneck and expansion FS has been shown to be a particularly sensitive statistic for detecting sudden demographic expansion 5 neutral coalescent simulations.Second, Tajima's −8 substitutions per site per year. We chose the faster rate estimate as the conservative estimate for questions regarding the influence of historical events on contemporary genetic structure and gene flow. Operators were auto optimized. We ran three independent runs of 200 million MCMC steps sampled every 1000 steps after a 10% burn-in. The log and tree files for the three independent runs were combined using LOGCOMBINER v.1.6.1, discarding the burn-in and re-sampling every 1000 steps. Convergence and effective sample sizes (ESS) were evaluated in TRACER v1.5. After confirming that parameters showed good convergence and all ESS values were greater than 200, the BSP was constructed using the combined files in TRACER v1.5.Next, we used the coalescent-based approach implemented in the program BEAST v1.6.1 to construct a Bayesian skyline plot BSP; . Bayesiacyt b gene was sequenced and analyzed for 130 Coralliophila abbreviata individuals. The sequence alignment contained 55 polymorphic sites, resulting in 57 unique haplotypes. Haplotype diversity (h) was moderate to high across localities, ranging from 0.613 to 0.902  and nucleotide diversity (π) was low across populations, ranging from 0.003 to 0.005  of all observed haplotypes, was shared among all populations and was the most common haplotype found in each population. Eighty-four percent of the divergent haplotypes were singletons and separated from the ancestral haplotype by only 1–3 mutational steps, resulting in a star-like haplotype network  linkage disequilibrium between pairs of loci was detected and no loci deviated significantly from HWE after correction for multiple comparisons.Characteristics of each of the five polymorphic microsatellite loci, including the number of observed alleles, observed and expected heterozygosity, FST = 0.002; P<0.001) and small and marginally significant for the mtDNA . However, subtle but significant genetic structure among hosts was still indicated based on microsatellite data . Subsequent pairwise comparisons indicated that the differentiation was driven by differences between ACR and MONT populations .When the PAN MYC sample was included in the analysis and individuals were pooled by coral host taxon, no genetic structure was detected with the mtDNA  were significant (α = 0.05) before Bonferonni correction but none remained significant after correction and there were no significant pairwise comparisons derived from microsatellite data . No signFST = 0.0013 with 92% accuracy, 2.) host and locality (ten populations) when FST = 0.0020 with 92% accuracy, 3.) locality (six populations) when FST = 0.0017 with 95% accuracy, and 4.) oceanographic region (two populations) when FST = 0.0012 with 91% accuracy. The finest resolution of structure tested in this study was between pairs of populations defined by host and locality, with an average sample size per population of 31. At this level, the microsatellite data set could detect an FST of 0.0074 with 91% accuracy in simulated populations.Overall, POWSIM simulations demonstrated that the microsatellite data contained sufficient power to detect low levels of population structure. Population structure was detected for simulated populations defined by 1.) host (two populations) when cyt b haplotypes did not cluster by coral host taxon or geographic region . Finally, for the STRUCTURE analysis, all individuals had approximately the same probability of originating from each cluster, regardless of the K value , at which point a large population expansion began . Estimates of contemporary gene flow based on the mitochondrial sequence data should, therefore, be interpreted cautiously as genetic homogeneity may reflect historic rather than modern day demographic processes. Despite this caveat, it is probable that contemporary gene flow among C. abbreviata populations throughout the Caribbean is high enough to prevent significant genetic subdivision. We base this assertion on the observed lack of genetic structure across large spatial scales in both the mtDNA and microsatellite datasets (i.e. lack of IBD) and the life-history of the species . Furthermore, coalescent-based estimates of ancestral population size indicate that demographic expansion occurred approximately 250,000 years ago , providing ample time under most scenarios for allele frequencies to have diverged if gene flow had been restricted post-expansion In addition to continued gene flow, large effective population sizes can maintain genetic homogeneity among populations over long periods of time after an expansion and subsequent demographic isolation. In such large populations, genetic drift has a smaller influence and it takes longer to reach drift-mutation equilibrium C. abbreviata based on AMOVA analyses of microsatellite and mitochondrial cyt b sequence data . Also in the Florida Keys, individually tagged snails have been observed to move among and feed on multiple coral host taxa in a manipulative field experiment The biological, ecological, and evolutionary importance of the observed low levels of host-associated differentiation is difficult to gauge based on current information Adaptive genetic polymorphisms may be maintained in a panmictic population through balancing selection when alternative genotypes exhibit greater fitness in specific habitats C. abbreviata snails were originally collected from both A. palmata and Montastraea spp. coral colonies. Regardless of the original coral host, recovered snails feeding on A. plamata grew faster than those feeding on Montastraea spp. corals. Thus, plasticity in snail growth under different environmental conditions  appears to contribute to the observed host-associated differences in population size structure. However, the relative importance of host-associated adaptation via genetic polymorphisms and phenotypic plasticity in the evolutionary ecology of C. abbreviata remains to be determined. These processes have important implications in regards to the propensity of C. abbreviata populations to diverge as coral community structure and other environmental conditions change across space and time. Further research is thus needed to fully assess the potential influence of selection acting on genotypes when occupying diverse coral hosts.Baums et al. C. abbreviata in the Caribbean. The mtDNA-based analyses consistently supported a scenario of Pleistocene demographic expansion preceded by a reduction in population size for C. abbreviata. The shallow cyt b gene genealogy with a single dominant haplotype and many new mutations (singleton haplotypes), resulting in moderate/high haplotype diversity (h = 0.773) and low nucleotide diversity (π = 0.4%) suggested a single colonization/founder event or a selective sweep followed by a rapid demographic expansion. Based on the mismatch distribution, the expansion began during the Pleistocene, approximately 219,000–365,000 years ago. This time frame was in agreement with the mean tMRCA and onset of expansion  determined through Bayesian skyline analysis . We hypothesize that C. abbreviata colonized the greater Caribbean region during the mid-late Pleistocene and subsequently expanded with the expansion of reef habitat and potential prey. Indeed, Johnson et al Sea level fluctuations during Pleistocene glacial cycles isolated basins and altered current patterns leading to demographic contractions and expansions in tropical marine taxa across the Indo-Pacific Coralliophila abbreviata sampled from different host coral species and geographic localities. Although C. abbreviata have not diverged into host races or sibling species, we found evidence of weak host-associated differentiation. Coralliophila abbreviata populations exhibited stronger differentiation by coral host taxon than by geographic locality. The biological, ecological, and evolutionary importance of the observed extremely low levels of host-associated differentiation remains to be seen. Overall, the results of this study indicate that C. abbreviata constitutes a large, interconnected meta-population throughout the greater Caribbean that expanded during the Pleistocene, likely due to the large-scale expansion of reef habitat during that time. However, direct field-based demographic surveys and experiments as well as additional molecular studies are needed to determine the magnitude and ecological importance of dispersal and connectivity among coral hosts. Future molecular studies should be based on extensive sampling of sympatric host-associated populations across multiple spatial scales using additional neutral and adaptive markers to fully elucidate the interactive effects of host and locality on the population genetic structure, ecology, and evolution of C. abbreviata.We used five highly polymorphic microsatellite loci and 366 bp of the mitochondrial cytochrome b gene to assess the population genetic structure, connectivity, and demographic history of Acropora spp. corals have declined drastically throughout the Caribbean over the last three decades due to a variety of natural and anthropogenic stressors, resulting in their listing as ‘Threatened’ species under the U.S. Endangered Species Act in 2006. Predation by C. abbreviata may represent a profound threat to the persistence and recovery of remnant populations of the Caribbean acroporids C. abbreviata is thus important for coral conservation efforts. Whereas Baums et al. A. palmata, the results of this study indicate that gene flow is high across the Caribbean for C. abbreviata. Thus, snail populations are decoupled demographically from local and regional population fluctuations of the threatened acroporid corals as snails may be supplied from distant locations and maintained on alternative coral prey. Targeted snail removal, therefore, may be necessary to ensure the persistence and/or recovery of particularly vulnerable Acropora colonies such as small fragments or remnant colonies, nursery transplanted colonies and new recruits. However, removal efforts may be offset by input from other local and regional sources and any potential control strategies need to be designed accordingly.The Supporting Information S1Detailed methodology for microsatellite development, amplification, and testing.(PDF)Click here for additional data file.Table S1Sample sizes of Coralliophila abbreviata by region, locality and reef for mitochondrial cyt b sequences (mtDNA) and microsatellite markers (Msats) with sampling coordinates.(PDF)Click here for additional data file.Table S2AMOVA results for tests of geographic differentiation among sampled populations of Coralliophila abbreviata.(PDF)Click here for additional data file.Figure S1STRUCTURE plots representing the probability of membership to each hypothetic population for 311 individual Coralliophila abbreviata based on five polymorphic microsatellite loci. Shown are plots for K = 2 (a), K = 4 (b), and K = 10 (c), where individuals are grouped by sampling locality .(PDF)Click here for additional data file.Figure S2STRUCTURE results: mean (±SD) of estimated Ln probability of the data for each K value.(PDF)Click here for additional data file.Figure S3Mismatch distribution. The observed number of pairwise nucleotide differences (open circles) for mitochondrial cyt b sequences plotted with the expected number of pairwise nucleotide differences under a model of sudden demographic expansion (solid line) and the 95% confidence intervals for the model estimation (dashed lines).(PDF)Click here for additional data file.


Dermochelys coriacea is the most widely distributed sea turtle species in the world. It exhibits complex life traits: female homing and migration, migrations of juveniles and males that remain poorly known, and a strong climatic influence on resources, breeding success and sex-ratio. It is consequently challenging to understand population dynamics. Leatherbacks are critically endangered, yet the group from the Northwest Atlantic is currently considered to be under lower risk than other populations while hosting some of the largest rookeries. Here, we investigated the genetic diversity and the demographic history of contrasted rookeries from this group, namely two large nesting populations in French Guiana, and a smaller one in the French West Indies. We used 10 microsatellite loci, of which four are newly isolated, and mitochondrial DNA sequences of the control region and cytochrome b. Both mitochondrial and nuclear markers revealed that the Northwest Atlantic stock of leatherbacks derives from a single ancestral origin, but show current genetic structuration at the scale of nesting sites, with the maintenance of migrants amongst rookeries. Low nuclear genetic diversities are related to founder effects that followed consequent bottlenecks during the late Pleistocene/Holocene. Most probably in response to climatic oscillations, with a possible influence of early human hunting, female effective population sizes collapsed from 2 million to 200. Evidence of founder effects and high numbers of migrants make it possible to reconsider the population dynamics of the species, formerly considered as a metapopulation model: we propose a more relaxed island model, which we expect to be a key element in the currently observed recovering of populations. Although these Northwest Atlantic rookeries should be considered as a single evolutionary unit, we stress that local conservation efforts remain necessary since each nesting site hosts part of the genetic diversity and species history.The leatherback turtle  Natural populations are dynamic systems facing variations in time and space that are directly or indirectly related to environmental changes. Consequently, population genetics deals with non-equilibrium states, meaning that alongside long-term adaptive processes, other complex mechanisms have to be incorporated such as the balance of gene flows among populations, changes in the sizes of populations, population dispersals to gain new or depleted habitats, and movements between breeding and feeding areas. Among population dynamics models, the metapopulation concept has been extensively considered and refers to an assemblage of ephemeral interacting subpopulations (i.e. including emigration and immigration events) that persist over time in a dynamic balance of local declines and increases Metapopulation theory also addresses demography and structure of subpopulations, and thus their extinction probability Demographic events and migrations also result in contrasted signatures of genetic diversity. A decrease in the effective population size results in an excess of gene diversity at neutral loci, because the rare alleles that were lost contributed little to the heterozygosity of the ancestral population Dermochelys coriacea, Vandelli, 1761) is a pelagic marine species widely distributed in tropical and subtropical waters and is currently classified as ”critically endangered„ with a constantly declining global population trend A good understanding of the history, magnitude and drivers of past changes is necessary if we hope to adequately assess the current status of threatened species and populations and make future projections of their likelihood of extinction or recovery In this study, we aimed to investigate the recent demographic history and the current fine-scale structure of the NW Atlantic Ocean RMU using the most recent markers, including some recently published (i) The small-scale structure of these rookeries and the strength of migrations among the rookeries in order to achieve a precise evaluation of nest-site fidelity and geographic level of gene flow within the NW Atlantic RMU,(ii) The historical baselines of effective population sizes, in order to understand the possible extent of recent demographic changes and their significance for current and future population status.Skin biopsies , or blood samples (with a heparinised syringe in the venous sinus in the hind flipper) were collected from nesting leatherbacks during oviposition between 1990 and 2010. Three sets of samples were considered, and corresponded to the three following rookeries: (i) Awala-Yalimapo (AY), Western French Guiana, at the border with Suriname (n = 52); (ii) Cayenne (CAY), East French Guiana, 300 km east of AY (n = 95); and (iii): Martinique (n = 56) and Guadeloupe (n = 12) in the French West Indies (FWI), 200 km apart and 2,000 km northwest of French Guiana . Total D8 and (GT)8 biotinylated microsatellite probes and the other for tetranucleotide sequences using (GATA)4 and (GACA)4 biotinylated microsatellite probes. The selected fragments were amplified by PCR then cloned into the pGEM-T vector (Promega©). Plasmids were introduced into XL-1 blue cells and transformed cells were cultivated on agar plates (incubation temperature = 37°C) containing 100 µg/ml of X-galactosidase and ampiciline. About 500 clones containing inserts were sequenced with the ET Dye terminator Cycle Sequencing Kit (Amersham Biosciences) following the manufacturer's recommendations for sequencing in an automated MegaBACE 1000 DNA analysis system. Repeated microsatellite motifs were found using the Gramene Project SSR tool We built two microsatellite enriched genomic libraries 2 (BioLine©), and 0.5 µM (for P186 and Nigra 200) or 1.0 µM of each primer. Other loci  were analyzed using MegaBACE 1000, in which primers were synthesized with a M13 tail and fluorescent complementary sequences were added in the PCR reactions Besides the four new markers developed in this study (see above), we also analyzed P186, Dc99 2 (BioLine©), and 0.4 µM of each primer. The amplification program for both markers consisted of 10 min at 94°C, followed by 35 cycles of 30 s at 94°C, 30 s at 51°C for CR and at 55°C for Cyt-b, 1 min at 72°C, and a final extension step of 15 min at 72°C. PCR products were sent for sequencing to Beckman Coulter Genomics . The consensus and the sequences were aligned with Clustal W implemented in MEGA 5.05 The mtDNA control region (CR) was entirely amplified for 68 samples  using the primers LCM 15382 and H950 GIMLET software We checked for occurrence of linkage disequilibrium among the 10 microsatellite loci with GENEPOP 1.2 A Bayesian clustering approach implemented in STRUCTURE 2.3.1 adegenet in R 2.13.0 find.clusters. In all analyses, 40 principal components (PCs) were retained, corresponding to the number of principal components that explained 90% of the cumulative variance.We also used a multivariate method to make assumptions regarding data structure. Unlike STRUCTURE, multivariate models do not assume that populations are in Hardy-Weinberg equilibrium. Accordingly, a Discriminant Analysis of Principal Components DAPC,  was perf−4 to 9.5×10−3 were previously set in several marine turtle species We used MSVAR1.3 http://beast.bio.ed.ac.uk). A preliminary analysis was performed using the Coalescent prior, and constant population size was also run in BEAST in order to estimate the population size. We used these results to set the population size prior in the EBSP analysis, using a uniform prior and the 95% CI estimated with the constant population size. In order to compare models and check if the EBSP results were different from a constant size model we calculated a Bayes Factor  Ne) over time. We applied a strict molecular clock and a piecewise-constant Bayesian skyline tree prior. A mutation rate of 2% per site per Million Years Ago (MYA) was considered Both CR and Cyt-b gene sequences were analyzed for haplotype and nucleotide diversities with DNAsp 4.20.2 Since leatherbacks from AY had been sampled over a long period, two preliminary approaches were implemented in order to control a putative bias resulting from genetic drift during this period: (i) differentiation among rookeries was calculated using RST and FST indexes between 2 periods: samples collected in 1990–2000 vs. those collected in 2001–2010, and no structuration was evidenced; (ii) STRUCTURE was used to investigate the number of ancestral stocks within this sample. It revealed that a single stock (K = 1) was the most probable solution. Consequently, all the samples from AY were considered as a single rookery.p>0.05) after Bonferroni correction. Regarding the 4 new microsatellite markers developed specifically for this study , Dc008 presented the highest allelic richness , while Dc005 presented the lowest allelic richness (AR = 4.35). The number of alleles per locus ranged from 4 (Nigra32) to 29 (LB141) (All the 10 microsatellite loci were polymorphic and linkage disequilibria were not significant ( (LB141) . Gene di (LB141) . Conside (LB141) . Dc008 pThe analysis of stocks using STRUCTURE indicated that the most probable number of populations  was 1, therefore failing to recover any ancestral structure. The DAPC results were similar, and although the lowest value of BIC indicated a K = 6 (with a possible range from K = 3 to 7), individuals from all three sample sites were assigned in all six groups. Therefore DAPC also suggested a single ancestral stock.When analyzing all ten loci with model genotypes that were either original or adjusted with PIM model Observed and expected heterozygosities, and Inbreeding coefficients, are shown in We found a high rate of gene flow among rookeries, with 13 to 33 migrants per generation . In analyses which excluded the loci with null alleles, the number of migrants between AY and CAY was seen to increase to 80, whereas it remained in the same range (12) between AY and FWI. Whatever the set of data used, emigrants from CAY and FWI to AY were twice as numerous as immigrants from AY to other rookeries.10 BF>30). The sizes of populations estimated with the constant coalescent model were concordant with MSVAR analysis, indicating three small effective populations of approximately 100 females. Additionally, by the parameter onePhaseProb revealed that a low occurrence of microsatellites mutating as a single step (ranging from 0.14 to 0.16), indicating that in the most cases the loci analyzed do not follow the single step mutation model, but rather change length in a>1 repeat unit.Sensitive Bayesian methods implemented in MSVAR showed dramatic declines in effective population sizes, with ancestral effective population sizes ranging from 120,000  to 1,600,000 (CAY) shrinking to current effective population sizes ranging from 70 (FWI) to 120 (CAY) . This coRegarding the CR (711 bp), a total of five haplotypes were evidenced, all of which were present in AY, with Dc_A5 and Dc_C3 being newly reported and exclusive of this rookery. All three rookeries shared the other three haplotypes, revealing the presence of Dc_C2 for the first time , Table 5Leatherback turtles exhibit complex life traits, including female homing and migration, migration patterns of juveniles that remain little known to date, and climate that has been shown to strongly influence resources, breeding success and sex-ratio. Based on a comprehensive integrated approach combining microsatellite and mitochondrial DNA, our study provides new insights into the population dynamics of leatherbacks in the Northwest Atlantic, considered as one of the world's largest populations This work puts forward the complexity of analytic choices with concurrent approaches. We used three methods based on Bayesian inference, namely MSVAR, BEAST and MIGRATE, to explore recent demographic history and changes in the evolution of effective population size in distant rookeries with contrasted numbers of nesting females. MSVAR has been shown to be a relevant tool to detect expansions and declines in different species One other key point in our study was a high estimated occurrence of null alleles in our dataset. Null alleles result in lower heterozygosity and consequently impact the structuration signal among populations, overestimating the distances among clades The concurrent use of different methods to analyze sequences of the control region and autosomal microsatellite variability in this study has revealed that the Northwest Atlantic stock of leatherback turtles derives from a single ancestral origin, but shows a current genetic structure at a small geographic scale that is related to the distribution of nesting rookeries.The sequencing of the entire Cyt-b revealed evidence of only two haplotypes, probably the same as those previously described and based on shorter sequences (876 bp) Among our five CR haplotypes, only two have been previously described Lepidochelys olivacea) Chelonia mydas and the hawksbill Eretmochelys imbricata turtles in the wider Caribbean region Microsatellite markers revealed a low genetic diversity compared to other marine turtle species Following the idea of recent megaufauna extinction and the controversial ”blitzkrieg„ hypothesis The recovery of populations suggested by both recent increases of effective population sizes and positive trends of nesting activities Metapopulation functioning implies that some groups are separated by habitat types that are not relevant for feeding and/or breeding activities Although the metapopulation theory implicitly refers to non-migrating species, this model has also been explored in migrating species, and namely in birds Assessments of genetic diversity based largely on neutral variation provide essential information about population history and demography Maintaining a high level of genetic diversity is assumed to be essential for the conservation of viable populations