Aedes albopictus (the Asian tiger mosquito) and its vector competence for numerous arboviruses represent a growing threat to public health security. Molecular markers are crucially needed for tracking the rapid spread of this mosquito and to obtain a deeper knowledge of population structure. This is a fundamental requirement for the development of strict monitoring protocols and for the improvement of sustainable control measures.The dramatic worldwide expansion of Wild population samples from putative source areas and from newly colonised regions were analysed for variability at the ribosomal DNA internal transcribed spacer 2 (ITS2). Moreover, a new set of 23 microsatellite markers (SSR) was developed. Sixteen of these SSRs were tested in an ancestral (Thailand) and two adventive Italian populations.FST estimates suggested the absence of great differentiation. The ancestral nature of the Thai population was corroborated by its higher level of variability.Seventy-six ITS2 sequences representing 52 unique haplotypes were identified, and AMOVA indicated that most of their variation occurred within individuals (74.36%), while only about 8% was detected among populations. Spatial analyses of molecular variance revealed that haplotype genetic similarity was not related to the geographic proximity of populations and the haplotype phylogeny clearly indicated that highly related sequences were distributed across populations from different geographical regions. The SSR markers displayed a high level of polymorphism both in the ancestral and in adventive populations, and The two types of genetic markers here implemented revealed the distribution of genetic diversity within and between populations and provide clues on the dispersion dynamics of this species. It appears that the diffusion of this mosquito does not conform to a progressive expansion from the native Asian source area, but to a relatively recent and chaotic propagule distribution mediated by human activities. Under this scenario, multiple introductions and admixture events probably play an important role in maintaining the genetic diversity and in avoiding bottleneck effects. The polymorphic SSR markers here implemented will provide an important tool for reconstructing the routes of invasion followed by this mosquito. Aedes (Stegomyia) albopictus is a vector of numerous arboviruses including Dengue, Chikungunya, and a variety of epizootic viruses [http://www.issg.org/database/species/search.asp?st=100ss).The Asian tiger mosquito,  viruses -3. It is viruses , and it Ae. albopictus vary according to its geographic origin. Given that these traits may be genetically determined and may influence pathogen transmission, it is of paramount importance to define the geographic origin of invading populations.Originally a native of the tropical forests of Southeast Asia, it has spread during the last 30–40 years to both tropical and temperate regions worldwide . Global Ae. albopictus populations are infected by the symbiont Wolbachia [Furthermore, as the threat represented by this mosquito is growing due to the lack of sustainable control measures and of the progressive spread of insecticide-resistance ,16, effiolbachia , whose polbachia .In the present study we used the ITS2 region to address the presence of intraspecific differentiation (genetic barriers) among groups of populations from different ancestral and recently colonized geographic areas. Moreover, we developed additional novel microsatellite markers for this mosquito species. We provide evidence of their usefulness for the analysis of the genetic composition of populations.Ae. albopictus were collected across the species range (Table Nine wild population samples of ge Table . Two samThe Rimini laboratory strain (F35) was used for the construction of the microsatellite-enriched libraries. This strain was established from mosquitoes collected in Rimini (Emilia Romagna) at the Centro Agricoltura Ambiente ‘G. Nicoli’ . Three of the nine wild samples previously described, Ban Rai, Cesena and Brescia, were also screened to assess the variability of the newly developed microsatellites markers.2, Reaction buffer , 200 µM dNTP, 10 pmol of each primer and 1 unit Taq DNA polymerase (Invitrogen). Amplifications were performed using an Eppendorf MasterCycler with the following cycle conditions: an initial denaturing step at 94°C for 2 min; twenty-five cycles of denaturation at 94°C for 20 s, annealing at 54°C for 15 s, extension at 72°C for 25 s, followed by a final extension at 72°C for 5 min.DNA was extracted according to Baruffi et al.  from indThe PCR products were separated on 1.5% agarose gels, stained with ethidium bromide and photographed under ultraviolet light. Amplification products were ligated into the TOPO 2.1 vector and transformed into competent One Shot cells using the TOPO TA cloning kit (Invitrogen). DNA inserts were sequenced on both strands.Ae. albopictus sequences in GenBank. The ITS2 sequences were aligned using MUSCLE 3.8 [a priori definition of the number of groups (K) of populations that exist and generates F-statistics  using an AMOVA approach. Different numbers of groups (K) were tested, and a simulated annealing procedure permitted the identification of the composition of each of the K groups that maximizes the FCT index . The program was run for two to eight groups (K = 2 to K = 8) each time with the simulated annealing process repeated 100 times, starting each time with a different partition of the population samples into the K groups. A phylogenetic analysis was also performed including the unique ITS2 sequences or haplotypes identified in the nine population samples by Arlequin 3.5.1.2 [Ae. flavopictus ITS2 outgroup sequence (GenBank accession number: AF353559.1) were aligned using MUSCLE 3.8 [http://tree.bio.ed.ac.uk/software/figtree/).The boundaries of the ITS2 region were determined by comparison with the available SCLE 3.8 . NucleotSCLE 3.8 . GeograpSCLE 3.8 . This ap 3.5.1.2 . The hapSCLE 3.8 . The KimSCLE 3.8 . The relhttp://www.genetic-id-services.com) for library construction. The library was enriched for four different di- and tri-nucleotide microsatellite motifs, namely (CA)n, (GA)n, (AAC)n, and (ATG)n. Insert DNA from individual clones was amplified by PCR following GIS guidelines. The PCR products were purified using QIAquick columns (Qiagen) and sequenced using the DYEnamic ET Terminator Cycle Sequencing Kit (Amersham Bioscience) with the universal M13 primers (M13f 5′-aggaaacagctatgaccatg-3′ and M13r 5′-acgacgttgtaaaacgacgg-3′). Clones with inserts less than 200 bp were not considered for sequencing. Sequencing products were resolved on the Applied Biosystems model 377 DNA Sequencer (Applied Biosystems). The resulting sequences were screened for the presence of microsatellite motifs using WebSat [Total genomic DNA was extracted individually from 64 mosquitoes  from the Rimini strain using the method reported by Baruffi et al. . The DNAg WebSat . All theg WebSat .2, 270 µM dNTPs (Invitrogen), 1 U Taq polymerase (Invitrogen), and 10 μM of each primer, one of which was 5′ labelled with a fluorescent dye, in a final volume of 15 μl. PCR cycling conditions were 94°C for 3 min, 30 cycles of 94°C for 30 s, 57-60°C for 30 s, and 72°C for 30 s, followed by a final extension step of 72°C for 10 minutes. Aliquots of PCR products were separated by electrophoresis on 2% agarose gels stained with ethidium bromide, and visualized under UV light. Each PCR product was then diluted 1:10 in ddH2O water and 1 μl of this dilution was added to 9 μl of a mixture of deionized formamide and GeneScan-500 ROX size standard (Applied Biosystems). After denaturation for 4 min at 94°C the fragments were resolved on an ABI PRISM 310 Genetic Analyzer (Applied Biosystems) with the GENESCAN software package (Applied Biosystems). To avoid genotyping errors, the program TANDEM [Microsatellite sequences that shared one or both the 5′/3′ flanking regions in common were discarded to exclude potential multi-locus microsatellites families. The sequences that were found to be unique within the library were subjected to BLASTn analysis against the nr database to identify eventual hits with previously described sequences. Primer pairs were designed for the flanking regions of the identified SSR sequences using Primer 3 . Prelimim TANDEM  was useda), number of effective alleles (ne), number of private alleles (np) and AMOVA were computed using GenALEx 6.5 [O and HE, respectively), gene diversity (HS) and FST values were computed using MICROSATELLITE ANALYSER (MSA) V.4.05 [n) and the deviations from Hardy-Weinberg expectations were computed using GENEPOP version 4.2 [The parameters of intra- and inter-population genetic diversity, such as observed allele size, number of actual alleles (nALEx 6.5 . Observe) V.4.05 . Linkagesion 4.2 ,55. For sion 4.2  for eachsion 4.2 .ITS2 sequences were obtained from an average of three individuals from each of the nine population samples collected in Thailand, Réunion and Italy (Table P < 0.05) of the total variance. When the three main geographical population groups  are accounted for  of the haplotypes are present in only one population. Only three haplotypes are shared by two populations . Haplotype 23 is shared by three Italian populations , and haplotype 13 is present in four populations .Ae. albopictus library, 92 contained tandem DNA repeats with at least 5 repeat units. BLASTn analyses showed that one or both flanking regions were shared in 42 sequences and they were thus excluded from the subsequent characterization. Within the remaining 50 sequenced clones, five harboured more than one tandem DNA repeat, resulting in the identification of 55 microsatellite motifs. Among these, trinucleotide motifs were the most abundant (28), followed by dinucleotide (22), mononucleotide (3), heptanucleotide (1) and hendecanucleotide motifs (1). Among the trinucleotides motifs, CAA was highly abundant (17) followed by TGA (10). Among the dinucleotide motifs, TG was predominant (14) followed by CT (8). Thirty-seven of these microsatellite motifs contained perfect repeat arrays. Twelve microsatellites contained imperfect repeats and the other six microsatellites contained compound repeats.Out of a total of 109 sequenced clones obtained from the Forty-five microsatellite sequences were considered for further validation, as the others lacked sufficient flanking regions to design primers. After the PCR screening on 20 individuals from the Rimini laboratory strain, 23 SSR sequences were validated as loci Table . The nucp) were detected in all populations, with the highest average value in the Ban Rai sample (2.25). Analysis of molecular variance (AMOVA) across the three populations indicates that the greatest portion of variance, 76.62%, is found within individuals, while 16.80% is among individuals and only the remaining 6.57% is among populations. The FST values between each pair of populations are statistically significant and indicate that the degree of differentiation between the two Northern Italian samples, Cesena and Brescia (FST = 0.061), is not very different from those between these two Italian samples and the Ban Rai sample (FST = 0.072 in both comparisons).On the basis of a preliminary screening performed on the Rimini strain, 16  of the 23 validated SSR loci Table . The aveAe. albopictus are crucially needed to track the rapid spread of this mosquito that is causing increasing concern due to its status as a vector of several arboviruses of public health importance [ST estimates indicate that the differentiation between the two Italian samples is slightly smaller (FST = 0.061) than that between the Italian and Thai samples (FST = 0.072), suggesting the absence of great differentiation between ancestral and derived populations. The ancestral nature of the Thai population is also corroborated by its high level of variability.In the absence of a fully annotated genome, molecular markers for portance ,9. The tportance . The FSTTaken together these genetic data raise questions on the way this mosquito is spreading and becoming established worldwide. Clearly, our data indicate that the diffusion of this mosquito does not conform to a progressive expansion away from the native Asian source area, but supports a relatively recent and chaotic propagule distribution mediated by human activities. Under this scenario, multiple introductions and admixture events probably play an important role in maintaining the genetic diversity and in avoiding bottleneck effects.Ae. albopictus populations collected in different eco-geographic areas. Most of the genetic variation was detected at the individual level and this contributed to the high genetic variation observed within populations. This genetic feature, also revealed using other markers [In this study we have exploited nuclear molecular markers that have allowed us to highlight the presence of high intraspecific variability and a low/moderate level of differentiation of  markers ,34,36,59The highly polymorphic SSR markers here implemented, together with the set previously described ,59,60, r


Taeniopygia guttata castanotis and T. g. guttata are native to Australia and the Lesser Sunda Islands, respectively. The Australian subspecies has been domesticated and is now an important model system for research. Both the Lesser Sundan subspecies and domesticated Australian zebra finches have undergone population bottlenecks in their history, and previous analyses using neutral markers have reported reduced neutral genetic diversity in these populations. Here we characterize patterns of variation in the third exon of the highly variable major histocompatibility complex (MHC) class I α chain. As a benchmark for neutral divergence, we also report the first mitochondrial NADH dehydrogenase 2 (ND2) sequences in this important model system.Two subspecies of zebra finch, Despite natural and human-mediated population bottlenecks, we find that high MHC class I polymorphism persists across all populations. As expected, we find higher levels of nucleotide diversity in the MHC locus relative to neutral loci, and strong evidence of positive selection acting on important residues forming the peptide-binding region (PBR). Clear population differentiation of MHC allele frequencies is also evident, and this may be due to adaptation to new habitats and associated pathogens and/or genetic drift. Whereas the MHC Class I locus shows broad haplotype sharing across populations, ND2 is the first locus surveyed to date to show reciprocal monophyly of the two subspecies.Despite genetic bottlenecks and genetic drift, all surveyed zebra finch populations have maintained high MHC Class I diversity. The diversity at the MHC Class I locus in the Lesser Sundan subspecies contrasts sharply with the lack of diversity in previously examined neutral loci, and may thus be a result of selection acting to maintain polymorphism. Given uncertainty in historical population demography, however, it is difficult to rule out neutral processes in maintaining the observed diversity. The surveyed populations also differ in MHC Class I allele frequencies, and future studies are needed to assess whether these changes result in functional immune differences.The online version of this article (doi:10.1186/s12862-015-0546-3) contains supplementary material, which is available to authorized users. Island colonizing species have long been an important target for evolutionary studies. The transition into novel ecological environments sets the stage for ecological adaptation, and sometimes, speciation . On the The loss in genetic variation may have especially profound effects on the immune system where genetic diversity is thought to be particularly important for certain loci . ReducedA classic target of immune evolution studies has been the genes of the major histocompatibility complex (MHC) , 14. TheGallus gallus domesticus). The chicken was described to have a “minimal essential” MHC containing a single dominantly expressed MHC Class I locus  [Carpodacus erythrinus) MHC class I exon 3 [GenBank: FJ392790]   and scarJ392790]  with theBottleSim v2.6 [To test whether patterns of variation could be explained by genetic drift acting on our MHC class I sequences during colonization of the Lesser Sunda Islands, we ran bottleneck simulations in Sim v2.6 . We usedSim v2.6 . Post boPHASE software [DnaSP v5 [s) and nonsynonymous (πa) site nucleotide diversity (Table a is still high (πa = 0.033), indicating maintenance of nonsynonymous diversity  of the neutral loci sampled in Timor birds . Domesticated and wild Australian populations showed the lowest levels of divergence, but the two populations were nevertheless significantly differentiated . These patterns can also be observed in the TCS network . There are however, numerous 25) alleles found in wild population but not represented in our captive sample. Likewise, 31 alleles found in captive birds were not detected in wild-derived samples. This is further reflected by the large number of unique amino acid sequences found in each population as well as the significant genetic differentiation among captive and wild birds  and slight, but statistically significant, divergence between wild and domesticated populations . The relatively low levels of diversity and significant population divergence are reflected in the large number of shared alleles between Australian and Domestic samples and separation of Australian and Timor samples in our ND2 haplotype network  is relatively low: Australia = 0.003, Domestic = 0.006 and Timor = 0.007 (Table omegaMap [Anas platyrhynchos) and scarlet rosefinch (Carpodacus erythrinus) allowed us to determine which of our sites were putative residues of the PBR and whether these sites exhibited signatures of positive selection. Our sequences contained 17 putative PBR residues, seven of which exhibited strong signatures of positive selection in each population , that displays high levels of MHC class I variation [High diversity in the MHC suggests positive selection and indeed, there are abundant nonsynonymous polymorphisms throughout these Class I sequences Table . Our seqariation . We alsoariation , direct The MHC plays a vital role in antigen presentation to the immune system, so selection is expected to maintain diversity to broaden pathogen recognition capability . AlthougST = 0.073). Network analysis of MHC Class I alleles shows that the Timor alleles are distributed across multiple parts of the network, indicating that multiple colonizers carrying divergent alleles founded the island population. KST estimates between subspecies for MHC Class I are lower than from neutral markers. This pattern, however, is expected, as measures of population divergence are necessarily lower for highly polymorphic markers [ST [The two zebra finch subspecies appear to have been isolated since the early Pleistocene or late Pliocene . Thus, o markers . Balancikers [ST .ST = 0.665, p < 0.001, Table To provide a more complete picture of population genetic variation in this model system, we sequenced a portion of the mitochondrial ND2 gene. Mitochondrial genes have long been used as markers of population history , and thuND2 polymorphism in zebra finches is comparable to previous reports of ND2 nucleotide diversity in other passerines . These two populations also show statistically significant differences in ND2 allele frequencies (KST = 0.050). Together these finding provide evidence of genetic distinctiveness of wild populations in Australia and domesticated populations in the USA.Zebra finch populations were domesticated in the 1800s. As a result, domestic and wild populations have relatively low, but statistically significant, divergence in MHC Class I allele frequencies (KDomestication and artificial selection have important and predictable consequences –77. HoweAll sequences used in this study have been deposited in GenBank under accession numbers KT595736-KT595877 for MHC class I and KT595878-KT595935 for ND2.


Patterns of genetic variation in a population carry information about the prehistory of the population, and for the human Y chromosome an especially informative phylogenetic tree has previously been constructed from fully-sequenced chromosomes. This revealed contrasting bifurcating and starlike phylogenies for the major lineages associated with the Neolithic expansions in sub-Saharan Africa and Western Europe, respectively.We used coalescent simulations to investigate the range of demographic models most likely to produce the phylogenetic structures observed in Africa and Europe, assessing the starting and ending genetic effective population sizes, duration of the expansion, and time when expansion ended. The best-fitting models in Africa and Europe are very different. In Africa, the expansion took about 12 thousand years, ending very recently; it started from approximately 40 men and numbers expanded approximately 50-fold. In Europe, the expansion was much more rapid, taking only a few generations and occurring as soon as the major R1b lineage entered Europe; it started from just one to three men, whose numbers expanded more than a thousandfold.Although highly simplified, the demographic model we have used captures key elements of the differences between the male Neolithic expansions in Africa and Europe, and is consistent with archaeological findings. Around 50 to 70 thousand years ago (approximately 60 KYA), modern humans expanded out of Africa, and by approximately 15 KYA had colonized all inhabitable continents . In sub-Genetic evidence has contributed to our understanding of these events. There has been debate about the extent to which the genomes of present-day inhabitants of these areas have been derived from Neolithic farmers or from Paleolithic hunter-gatherers. The first large-scale molecular-genetic analyses in Europe were based on mitochondrial DNA (mtDNA) from present-day Europeans and were interpreted as favoring a Paleolithic entry for the majority of European mtDNAs . More diThe Y chromosome has several properties that make it potentially very informative about historical events, including the Neolithic transition. Its lack of recombination over most of its length means that it provides the most detailed and informative phylogenetic tree for any locus in the genome, while as a consequence of its strict father-to-son transmission it carries information specifically about male events . Y-chromIn the current study, we accept R1b and E1b1a as lineages that expanded during the Neolithic, and set out to explore, using coalescent simulations, the demographic conditions under which their different phylogenetic structures might be expected to arise. We found that these differ between the two continents, and link our conclusions to the available archaeological evidence.The samples consisted of 21 high-coverage Y-chromosomal sequences downloaded from the Complete Genomics website , eight f6 nucleotides, assuming a generation time of 30 years [-8 per nucleotide per generation [Simulations were performed using MaCS , a coale30 years , a mutatneration  and zeror) was calculated by summing the terminal branch lengths and dividing by the sum of the internal branch lengths multiplied by one plus the sum of each internal branch length beneath its node:Since we needed to compare the output from the simulations with the trees from the real data, as described below, we constructed statistics related to ones used previously  to compab is a tree branch of length lb, which has nBEN branches of length lbi beneath its node, nTER is the number of terminal branches and nINT is the number of internal branches.where The other two statistics were calculated by determining the branch length of the TMRCA of each combination of the individual haplotypes and computing the mean and standard deviation. The three statistics thus reflect both the time depth of the tree and how starlike its structure is.To identify the range of simulation parameter values that best fit the empirical trees, we created heat maps of a summary value of the three statistics, designated the average normalized delta (AND) value. The AND value was computed by dividing the difference of the simulated statistic and the empirical statistic by the empirical statistic and averaging these three distances:s indicates a simulated value, o an observed value, r a singleton/shared ratio statistic, m a mean TMRCA statistic and d a standard deviation of a TMRCA statistic.where the subscript A low AND value thus indicates a good fit to the empirical data. We completed 1,000 simulations for each demographic scenario and averaged each statistic to use as the simulated value.The ranges for the parameters on the first set of simulations and corresponding heat map were each chosen to be very wide, including all reasonable estimates for their values  of a single locus (the Y chromosome), and this may not be representative of the population. Nevertheless, there are several reasons to believe that our results should capture features of interest. First, the male history represented by the Y chromosome is of interest whether or not it corresponds to the history of other regions of the genome. Second, the single Y lineages we examined are the most frequent in their respective geographical regions, being found in >75% and >80% of males from many Western European and sub-Saharan African populations, respectively, so form a major constituent of the Y-chromosomal gene pool. Furthermore, the chromosomes sampled within each of the two lineages have diverse geographical origins: the R1b chromosomes come from the CEU (Northwestern Europe ), TSI (IWith these caveats, we can consider how the Y-chromosome-based genetic findings fit with other genetic and archaeological evidence. The Neolithic transition in Europe has been studied extensively by archaeologists. It appeared in Greece approximately 9 KYA and reached the extreme west by approximately 4 KYA ,2. The dStudies of this kind can be improved by considering more complex demographic models and larger Y-chromosomal datasets. While it may seem obvious that more complex and thus more realistic models should be preferable, models are only useful if the different scenarios they encompass can be discriminated between using the data available, so the simplest model that captures a relevant aspect of the data may still be the most appropriate one. Thus while future models in this context could incorporate spatial structure and phenomena such as surfing , a singlWe have identified demographic scenarios that can lead to the contrasting phylogenies observed for the major Y-chromosomal lineages that expanded during the distinct Neolithic transitions in Europe and Africa. These suggest that in Europe, the R1b lineage experienced an extremely rapid and extensive increase as soon as it entered the continent, expanding more than a thousandfold in a few generations. The expansion in Africa began from a larger population size, took thousands of years and ended only recently. While these conclusions are based on a simplified demographic model, they capture major differences between the continents and fit many aspects of the archaeological findings.aDNA: Ancient DNA; AND: Average normalized delta; KYA: Thousand years ago; mtDNA: Mitochondrial DNA; SNP: Single nucleotide polymorphism; TMRCA: Time to the most recent common ancestor.The authors declare that they have no competing interests.CTS and YX conceived the study. MJS, VC, YX and CTS designed the study. MJS performed the simulations. MJS, VC, YX and CTS interpreted the results. MJS and CTS drafted the manuscript. All authors read and approved the final manuscript.Examples of commands for MaCS. An example of a command for each of the R1b and E1b1a simulations is shown, along with the parameter set from which the command was derived.Click here for fileStarting parameter values for the simulations.Click here for fileHeat maps illustrating the AND values from sequential simulation runs.Click here for file


Cephalorhynchus eutropia, is the only endemic cetacean of Chile and is found in two different coastal habitats: a northern habitat with exposed coastlines, bays and estuaries from Valparaíso (33°02′S) to Chiloé (42°00′S), and a southern habitat with highly fragmented inshore coastline, channels and fjords between Chiloé and Navarino Island (55°14′S). With the aim of evaluating the potential existence of conservation units for this species, we analyzed the genetic diversity and population structure of the Chilean dolphin along its entire range. We genotyped 21 dinucleotide microsatellites for 53 skin samples collected between 1998 and 2012 . Bayesian clustering and spatial model analyses identified two genetically distinct populations corresponding to the northern and southern habitats. Genetic diversity levels were similar in the two populations , while effective size population was higher in the southern area (Ne: 101 v/s 39). Genetic differentiation between these two populations was high and significant (FST = 0.15 and RST = 0.19), indicating little or no current gene flow. Because of the absence of evident geographical barriers between the northern and southern populations, we propose that genetic differentiation may reflect ecological adaptation to the different habitat conditions and resource uses. Therefore, the two genetic populations of this endemic and Near Threatened species should be considered as different conservation units with independent management strategies.Understanding genetic differentiation and speciation processes in marine species with high dispersal capabilities is challenging. The Chilean dolphin,  The marine environment provides an opportunity to examine speciation processes and population structure in species with high dispersal capabilities, generally in the absence of obvious barriers to gene flow ,2. In ceTursiops truncatus, distributed across the contiguous range from the Black Sea to eastern North Atlantic Scotland, a population structure that coincided with transitions between habitat regions has been reported. These regions can be characterized by ocean floor topography, and oceanographic features such as surface salinity, productivity and temperature [Phocoena phocoena, in the south-eastern North Atlantic [Delphinus delphis from the Atlantic, Pacific and Indian Oceans [Populations of small cetaceans found in coastal habitats often show a population structure pattern related to geographic distance or environmental characteristics . For exaperature . SimilarAtlantic  and in an Oceans .A reliable understanding of the historic/contemporary genetic variation, demographic partitions throughout the geographical range and of the distribution of genetic diversity is crucial to devise effective and sustainable management plans for species , 13. GenCephalorhynchus are distributed in inshore waters of the Southern Hemisphere [Cephalorhynchus dolphins based on mtDNA analysis. Following the West Wind Drift, these dolphins would have colonized New Zealand and then South America [Cephalorhynchus genus . Studies on population genetic structuring have been conducted on three of these species. For Hector’s dolphin, endemic to New Zealand, two subspecies have been recognized based on genetic and morphological evidences: C. hectori maui for the North Island population and C. hectori hectori for the South Island populations [C. hectori subspecies independently [C. c. commersonii, found in shallow waters of the southeastern coast of South America, including the central and eastern Strait of Magellan and waters around the Falkland Islands/Islas Malvinas [C. c. kerguelenensis, distributed around the sub-Antarctic Kerguelen Islands in the southern Indian Ocean [C. c. commersonii showed differences among areas within Tierra del Fuego [Cephalorhynchus heavisidii, endemic to coastal waters of the western coast of South Africa and Namibia, shows no evidence of population structure based on mtDNA analyses along almost 1000 nmi of the South African/Namibian coast [Cephalorhynchus eutropia, a genetic study of mtDNA extracted from teeth of museum specimens focused on resolving uncertainty in species identification [Dolphins of the genus misphere  and appemisphere ,17. A co America . Four spulations ,19. Signulations . Based oendently . In CommMalvinas ,25 and Can Ocean ,27. Geneel Fuego  and alonel Fuego . Unlike an coast . Finallyfication ; howeverThe Chilean dolphin, is the only endemic cetacean species of Chile , althougC. eutropia during the last decade were its use as bait in southern king crab fisheries in southern Chile [Initially, the Chilean dolphin was classified as Data Deficient (DD) due to the insufficient information available to conduct a reliable assessment of its conservation status, in particular because of the lack of demographic information on population trends . Howeverrn Chile ,44,45 anrn Chile . Currentrn Chile .Considering that the identification of conservation and management units based on genetic evidence provide essential information for the development of effective and efficient conservation practices  this stuBetween 1998 and 2012, a total of 66 tissue samples were collected from Chilean dolphins at 8 localities along the Chilean coast ranging from 35°20′S to 52° 40'S . Skin saThe sex of each individual was identified using two sets of oligonucleotide primers simultaneously , which aSamples were genotyped at 22 dinucleotide microsatellite loci: EV1, EV14, EV37, EV94, EV104 ; KWM12 ; MK5, MKTaq DNA polymerase (Invitrogen Life Technologies). The thermal cycler profile consisted of a preliminary denaturing period of 3 min. at 94°C followed by 30 cycles of denaturation for 30 s at 94°C, primer annealing for 30 s with variable temperature (45°C to 55°C) depending of primer used, polymerase extension for 30 s at 72°C. A final extension period for 10 min. at 72°C was included. Following amplification and genotyping, allele peaks were visually verified using Gene Marker (Softgenetics). To minimize genotyping error each amplification and size run included a negative control to detect contamination and at least two internal control samples to ensure comparable allele sizing across all runs. Additionally three blind replicates were independently run for 10 loci.Amplification reactions contained 25–50 ng DNA, PCR buffer, 5 mM MgCl2, 0.4 μM of each primer, 0.2 mM deoxynucleoside triphosphate (dNTP) and 0.5 units Platinum Microchecker v. 2.2.3 [Cervus v. 3.0 [Pedigree v. 2.0 [The dataset was initially checked for genotyping or scoring errors caused by null alleles, stuttering and short allele dominance using v. 2.2.3 . Re-samps v. 3.0  and Pedie v. 2.0 . To avoiGeneland [Structure v. 2.3.1 [Structure Harvester program [Geneland and Structure programs was verified by the Bayesian method of Rannala and Mountain [et al. [Geneclass v. 2.0 [Genepop [ST [Genetix [ST value was calculated by FSTAT Software [To determine spatial population boundaries we used Geneland , a Bayesv. 2.3.1 . The adm program ,68. AssiMountain  with an  [et al.  MCMC re-s v. 2.0 . Fisher [Genepop  was perfepop [ST  in Genet[Genetix , while tSoftware . The staBayesass Software [Contemporary gene flow between these populations was estimated by Bayesian inference of recent migration rate using multilocus genotype implemented in Software . The delGenetix [FSTAT Software [Arlequin v. 3.5 [Deviation from Hardy-Weinberg equilibrium and linkage disequilibrium were analyzed for each population identified through the Bayesian clustering methods. We performed a permutation test using 10,000 iterations using Genetix . GeneticSoftware ; expecten v. 3.5 . Effectin v. 3.5  which imn v. 3.5 .Cephalorynchus eutropia based on the diagnostic characteristics of the species as left premaxillary bone length, exposition of the frontal bone, size of the optic foramen and rostrum length [All entangled individuals were correctly assigned to m length .Microchecker, except for locus Sgui02, which exhibited null alleles, and was eliminated for further analyses.From the 66 dolphin samples, 55 samples were successfully genotyped for all 22 loci. Unsuccessful amplification was mainly associated with skin swabbing samples. Additionally, two samples were excluded from further analyses based on having identical genotypes and sex. Of the 53 individuals, 32 were males and 21 were females. Replicates for sex identification and genotyping did not shown any mismatches. The overall sex ratio did not differ from the expected 1:1 . Do not evidence for genotyping, scoring errors or null alleles was detected using Geneland identified two clusters, a ‘North area’ population including the four sampled locations from San Antonio to Maullín, and a ‘South area’ population including the four sampled locations from Aysén to Punta Arenas, with a boundary between Maullín  and Aysén   and strong population differentiation was also observed for both males  and females The spatial model of  . The ind . Resultsf groups . These tStructure . However, two individuals sampled by skin swabbing in Aysén could be considered as second or third generation migrants  and no linkage disequilibrium was found over 210 comparisons between loci per population after Bonferroni correction. No difference in heterozygosity values was detected between the North area and the South area . Seven out of 21 loci had greater allelic richness in the North area while 14 loci exhibited greater allelic richness in the South area (e) showed lower values in the North  area than in the South .No significant deviations from Hardy–Weinberg equilibrium across populations were detected (North area: Futh area . ThirteeST = 0.15 and RST = 0.19) were generally higher than those reported among populations of other dolphin species: at a large geographic scale, FST = 0.034 for a comparison between bottlenose dolphins of coastal waters of the Gulf of Mexico and those inhabiting the waters of the western North Atlantic [ST = 0.02–0.09 for a worldwide phylogeography of common dolphins, Delphinus sp.[ST = 0.05 was reported for shorth-beaked common dolphins along the East Australian Coast, [ST = 0.05 for inshore Indo-Pacific bottlenose dolphin, Tursiops aduncus, in Moreton Bay, Austalia, [ST = 0.14–0.314 between the Black Sea and eastern Atlantic, but much lower FST = 0.001–0.09 among continuous areas in the eastern North Atlantic [Two genetic populations of Chilean dolphin were identified by 21 microsatellite loci; the North area population from San Antonio to Maullín and the South area population from Aysén to Punta Arenas see . The difhinus sp.. At smaln Coast, ; FST = 0ustalia, . DiffereC. hectori hectori and C. hectori maui . However, in contrast to the Chilean dolphin, these populations are discontinuous, separated by the Cook Strait, which is likely acting as a barrier to dispersal and gene flow between North and South Islands [ST = 0.039–0.071, p<0.005; [The genetic differentiation reported between  Islands ,83,84. Op<0.005;  associatp<0.005; ,83,85 haThe pattern of genetic structure observed in the present study appears to coincide with two major marine spatial biogeographic units: (1) an area located from 30°S to 42°S  characterized by an open and exposed coast with the presence of river runoff  where thEngraulis ringens, and Pacific sardine, Sardinops sagax; large predators include the jack mackerel, Trachurus murphyi, hake, Merluccius gayi, and different cephalopod species [Strangomera bentincki, anchovy and róbalo/Chilean rock cod, Eleginops maclovinus [Macruronus magellanicus and southern hake, Merluccius australis [Geographic positions of biogeographic breaks are mainly determined by the coincidence in the limit of distribution of an unusual number of species . Many sp species . The Chiclovinus ,37,38. Iustralis . UnfortuPontoporia blainvillei in the Western South Atlantic [Habitat discontinuities associated with changes in oceanographic features, prey distribution and philopatric behavior have been previously proposed as influencing the spatial genetic structure of several delphinid species e.g. ,99,100).,100.99,1Atlantic . FurtherUnderstanding population structure is crucial for informing management actions ,103.The C. hectori, the North and South Island populations, exhibited a strong phylogeographic structure, evidencing the absence of gene flow between them [C. eutropia North area and South area populations, our study does not provide a historical perspective that would allow if these populations could be categorized as an ESU and consequently, as potential subspecies. The use of additional molecular markers, such as mtDNA control region, would certainly bring important information about the long-term evolutionary history of C.eutropia that can be used to discuss their current taxonomy.In the case of Hector’s dolphin een them ,20. Despeen them ), and theen them . In thisC. eutropia, it is necessary to recognize the current threats to which each of the Chilean dolphin populations are exposed, and design management strategies adjusted to each particular area if needed. This necessity is made more urgent by the apparently small effective size and the absence of any estimates for the census size of either population.Our study provides valuable biological and ecological information for the Chilean dolphin, a species among the least known members of the family Delphinidae  and clasS1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Varecia variegata), in particular, has recently experienced rapid population declines due to habitat loss, ecological sensitivities to habitat degradation, and extensive human hunting pressure. Despite this, a recent study indicates that ruffed lemurs retain among the highest levels of genetic diversity for primates. Identifying how this diversity is apportioned and whether gene flow is maintained among remnant populations will help to diagnose and target conservation priorities. We sampled 209 individuals from 19 sites throughout the remaining V. variegata range. We used 10 polymorphic microsatellite loci and ∼550 bp of mtDNA sequence data to evaluate genetic structure and population dynamics, including dispersal patterns and recent population declines. Bayesian cluster analyses identified two distinct genetic clusters, which optimally partitioned data into populations occurring on either side of the Mangoro River. Localities north of the Mangoro were characterized by greater genetic diversity, greater gene flow (lower genetic differentiation) and higher mtDNA haplotype and nucleotide diversity than those in the south. Despite this, genetic differentiation across all sites was high, as indicated by high average FST (0.247) and ΦST (0.544), and followed a pattern of isolation-by-distance. We use these results to suggest future conservation strategies that include an effort to maintain genetic diversity in the north and restore connectivity in the south. We also note the discordance between patterns of genetic differentiation and current subspecies taxonomy, and encourage a re-evaluation of conservation management units moving forward.Lemurs are among the world's most threatened mammals. The critically endangered black-and-white ruffed lemur ( Lemurs are among the world's most endangered mammals IUCN 2013). Currently, 93 of 103 lemur taxa (90%) are classified as at least vulnerable and the number of species listed as Critically Endangered  has tripled since 2008  provides an ideal case study with which to investigate these relationships. Ruffed lemurs are medium-sized  suggests that both sexes disperse  as inferred from microsatellite markers and mitochondrial DNA sequence variation. Our analysis is unusually in that our samples were collected across the extent of the species’ range and thereby provides a species-level view of genetic apportionment for a critically endangered primate.Here, we describe the population genetic structure and dispersal patterns of black-and-white ruffed lemurs . Whole blood (1 mL/kg) samples were collected from the femoral vein and stored at room temperature in 5 mL of lysis buffer solution   from 19 localities from across the existing n = 20 individuals, and Vatovavy, n = 11 individuals). In these cases, SMH, MBP, and field assistants collected fecal samples from ruffed lemurs that had been previously collared for individual identification. Researchers and assistants collected 2–5 samples per lemur and removed seeds from fecal samples prior to preservation in RNAlater®  at a ratio of 1 mL feces to 5 mL RNAlater®. Samples were kept at room temperature for 15–105 days until transported to OHDZA, where they were stored at −20°C.Additional noninvasive sampling occurred in two sites , Stony Brook University IACUC (#2005-20081449), University of Calgary Life and Environmental Sciences Animal Care Committee (BI11R-15), Malagasy wildlife authorities, Convention on International Trade in Endangered Species regulations, and US Fish & Wildlife Service.Total genomic DNA was extracted from blood and fecal samples using standard nucleic acid extraction kits  following the manufacturer's protocols. Ten microsatellite loci , 12.5 μL QiagenHotStarTaq Master Mix and 10 μmol/L of each primer. Amplification conditions were as follows: initial denaturation at 95°C for 15 min; 35 cycles of 30 sec at 94°C, 40 sec at 54 to 60°C .Microsatellite loci were amplified in 25 μL volume with 4 μL template (20–50 ng), 12.5 μmol/L of each primer, 1.5 mmol/L MgCl2, 200 μmol/L dNTP, 10 mmol/L Tris (pH 8.3), 50 mmol/L KCl, and 0.5 U Taq DNA polymerase . Thermal cyclers profiles were as follows: 35 cycles of 30 sec at 95°C, 30 sec at 54 to 60°C, and 30 sec at 72°C with a 10 min final extension phase at 72°C , and alleles were sized relative to an internal size standard (ROX-500) using Gene Mapper software v. 4.0 . To detect and avoid allelic dropout, multiple PCR replicates were performed according to the concentration of DNA in each sample , we amplified the D-loop or control region of the mitochondrial DNA (mtDNA) using primers dLp5 . Final alignment of all sequences was performed using ClustalX software , the mean number of alleles per sampling locality (MNA), allelic richness per locality (AR), and observed (HO) and expected (HE) heterozygosities (Nei AR) was standardized to the smallest sample size in the dataset using rarefaction implemented in HP-Rare 1.0  according to Weir and Cockerham  as suggested by the microsatellite data. This method uses the Markov Chain Monte Carlo (MCMC) approach to group individuals into K populations based on their multilocus genotypes without prior information regarding their sampling localities . We also calculated the fractional membership of individuals within each population (Q). We evaluated the hypotheses K = 1–22, the number of sampling locations plus 3, following Evanno et al.  using the ΔK method . We then ran subsequent analyses within each of the K clusters from the original run following Evanno et al.  with a standard genetic distance matrix , among sampling localities within populations, among individuals within sampling localities, and within individuals. Distances were calculated using the Infinite Alleles Model .Results from the Bayesian cluster assignments and PCoA guided subsequent analyses. First, we examined microsatellite population genetic structure with a locus-by-locus Analysis of Molecular Variance  in GenoDive. Significance was calculated using 10,000 randomizations not assuming HWE and corrected for multiple comparisons (Bonferroni adjusted P = 0.0004).We also compared allelic diversity at each microsatellite marker between the inferred  Rousset . Signifi Rousset . FinallyenAlEx. We used the Geographic Distance Matrix Generator v.1.2.3 from the American Museum of Natural History (http://biodiversity-informatics.amnh.org/open_source/gdmg/) to calculate pairwise geographic distances (in km) between all sampling localities based on their decimal degree coordinates. This matrix was then compared with a matrix of Nei's genetic distances (described previously) using Mantel matrix correlations. Significance was evaluated based on 9,999 permutations.Dispersal or migration barriers can hinder gene flow, thereby increasing genetic distance, even in cases where populations are not geographically distant : among populations, among sampling localities within populations, and among individuals within sampling localities. We calculated nucleotide and haplotype diversity using DnaSP v5 . Epsilon (ε) was set equal to zero and variable sites were weighted equally. Finally, we performed pairwise tests for population differentiation (ΦST) in GenoDive. Significance was calculated using 10,000 randomizations not assuming HWE and corrected for multiple comparisons (Bonferroni adjusted P = 0.0004).To examine mtDNA population genetic structure, we used a standard AMOVA for haplotype data implemented in Gstat 2.9.3 (Goudet FIS); average relatedness (R); population differentiation (FST); mean Assignment Indices (mAIc); and variance in Assignment Indices (vAIc). FIS represents a measure of how well genotype frequencies within a population match expectations of Hardy–Weinberg Equilibrium (Hartl and Clark FIS) within the dispersing sex. The dispersing sex should also have lower average relatedness (R) among postdispersal aged members of a population than members of the more philopatric sex because dispersal reduces the likelihood that relatives are living in close association  (Hartl and Clark mAIc) and the variance (vAIc) of assignments between the sexes. These indices can then be standardized, subtracting the population mean AI from each individual's AI  lower average relatedness, (3) lower values of FST, (4) lower mean assignment scores (mAIc) and 5) greater variance in assignment (vAIc). We calculated two-tailed P-values using 10,000 randomizations, where sex was randomly assigned to genotypes while keeping the sex ratio and group identity constant thereby producing a null distribution  because of its robusticity to small sample sizes (<30) and small numbers of loci (<20) . Sites located between these localities  shared varying degrees of proportional membership with each of the K = 2 clusters. Furthermore, we observed a second, albeit small peak at K = 4  still clustered into a single population. However, with the exception of Nosy, northern sampling localities  exhibited substantial admixture and shared varying levels of proportional membership among the remaining K = 3 clusters. It is interesting to note, however, that animals from Nosy, an island population whose founders were introduced in the 1930s  grouped into K = 2 subpopulations. The two sampling localities from within Ranomafana National Park  clustered together with the northernmost (Fan) and southernmost localities (Mab) in Subcluster 2.1, while the fragmented  habitats of Kian, Vavy, and Laki clustered together in a second subcluster (Subcluster 2.2). Tol exhibited substantial admixture of the two.Similar patterns of substructure were detected among populations in the south. Localities within the original Cluster 2  that grouped sampling localities into two clusters. Clusters showed minimal overlap and appeared to separate according to geographic location. Sampling localities north of the Mangoro River clustered together (bottom right), as did sampling localities to the south of the river (top left). This component accounted for 35.2% of the total molecular variance. Another 17.9% of the variance was explained by Axis 2 (PCo2), which seemed to primarily separate sampling localities within the south. It is interesting to note that, again, the genetic clustering did not correspond to subspecies status , as well as for each of the 10 markers individually ; four to 11 alleles (mean = 8) in the north and three to 13 (mean = 6.6) in the south Table . Genotyply Table . ResultsFST comparisons suggest the same pattern of relationships as the Bayesian methods and are consistent with results from the locus-by-locus AMOVA suggesting significant levels of genetic differentiation both between northern and southern clusters and among sampling localities . Somewhat contrary to the individual-based analyses, most sampling localities show significant divergence from one another; however, this is more prevalent among southern sampling localities  than among localities in the north .Pairwise calities . Overall71 cases . Pairwisr2 = 0.501 P < 0.0001). This same pattern held true when looking among individuals within either of the two clusters .In addition, we found strong evidence of isolation-by-distance , we found 19 haplotypes and 44 polymorphic sites  and negative for females (mean = −0.216), this difference was not significant. Moreover, variance in AIc was very high for both sexes, and differences were not significant. Furthermore, males and females did not differ significantly in their number of haplotypes across sampling localities .We did not find evidence of sex-biased dispersal Table . Estimatottleneck, we found evidence of deviations from mutation-drift equilibrium at 5 of 9 sampling localities when either the IAM or TPM models were assumed  will help to better understand the patterns observed herein.Looking within each of the two genetic clusters, we found that FST for this species is the highest observed in any lemur study to date  under two  of the three models, but only two sites under the SMM. Earlier work has suggested that the TPM may be the most appropriate model for microsatellites given its intermediate status between the more conservative SMM and the rather unconstrained IAM  and genetic diversity among sampling localities; however, this is also where a majority of the illegal hunting and timber extraction has occurred due to recent political unrest , and there is some evidence that several have undergone recent population declines , an isolated V. variegata population originally introduced to the island in the 1930s (Petter and Peyreiras ara). Although, to the best of our knowledge, no known records exist regarding their true origins , our results suggest that the founder population for the Nosy Mangabe individuals might have come from Mananara Nord, a coastal mainland site located ∼312 km south of the island. Thus, sampling animals and localities from across the full species’ range – as we have done in this study – provides not only a comprehensive picture of genetic diversity, but also useful tools for wildlife forensics.Finally, our results raise the possibility of returning confiscated animals to their likely region of origin, and estimating the likely provenance of some captive animal populations. Nearly, all individuals (91%) within our study were assigned to their source localities with high statistical certainty across genetically differentiated sampling localities. Even in cases where individuals could not be assigned to their particular locality of origin, they could be successfully assigned to their appropriate genetic cluster (9%). Interestingly, S


Tetraclita serrata, a dominant species on rocky intertidal shores. Restricted gene flow in some geographical areas was hypothesized based on oceanic circulation patterns and known biogeographic regions. Barnacle population genetic structure was investigated using the mitochondrial cytochrome oxidase subunit 1 (COI) region for 410 individuals sampled from 20 localities spanning the South African coast. The mtDNA data were augmented by generating nuclear internal transcribed spacer 1 (ITS1) sequences from a subset of samples. Phylogenetic and population genetic analyses of mitochondrial DNA data reveal two distinct clades with mostly sympatric distributions, whereas nuclear analyses reveal only a single lineage. Shallow, but significant structure  was detected for the mtDNA data set, with the south-west African region identified as harbouring the highest levels of genetic diversity. Gene flow analyses on the mtDNA data show that individuals sampled in south-western localities experience gene flow primarily in the direction of the Benguela Current, while south and eastern localities experience bi-directional gene flow, suggesting an influence of both the inshore currents and the offshore Agulhas Current in the larval distribution of T. serrata. The mtDNA haplotype network, Bayesian Skyline Plots, mismatch distributions and time since expansion indicate that T. serrata population numbers were not severely affected by the Last Glacial Maximum (LGM), unlike other southern African marine species. The processes resulting in the two morphologically cryptic mtDNA lineages may be the result of a recent historical allopatric event followed by secondary contact or could reflect selective pressures due to differing environmental conditions.The evolutionary effects of glacial periods are poorly understood for Southern Hemisphere marine intertidal species, particularly obligatory sessile organisms. We examined this by assessing the phylogeographic patterns of the southern African volcano barnacle,  Past climatic changes in the form of glaciations, as well as associated changes in temperature and precipitation, have been suggested as drivers of demographic change in both terrestrial and marine species. Numerous marine species have been shown to be affected by sea level fluctuations that occurred during the last 100 000 years and especially around the Last Glacial Maximum  Such variance in patterns also applies to the study of demographic change; some marine species have been shown to maintain demographically stable populations throughout the LGM, without significant changes in population sizes, while others show population bottlenecks with post-LGM recolonization; both scenarios seem equally plausible As with a number of studies, the link between life-history characteristics and population genetic structure is tenuous at best. Evidence from direct and indirect estimates of dispersal suggest that larvae consistently disperse on smaller spatial scales than expected based on their pelagic larval duration (PLD) Tetraclita serrata, one of the dominant invertebrates of the mid intertidal zone with a wide distribution ranging from KwaZulu-Natal (east coast of South Africa), extending into Namibia , 0.2 µl Taq polymerase (Super-Therm) and 13.4 µl distilled water. PCR reactions were carried out in an ABI GeneAmp 2700 automated thermocycler with the following conditions: an initial denaturation step at 94°C for 3 min, followed by 35 cycles of denaturing at 94°C for 30 s, annealing (for 30 s) and an extension at 72°C for 45 s, ending with a final extension at 72°C for 5 min. PCR annealing temperatures were set at 58°C and 56°C for the COI and ITS1 loci, respectively. The amplified products were gel separated and purified using the Wizard SV Gel PCR Clean-Up System (Promega). Purified products were cycle-sequenced using BigDye (Applied Biosystems [ABI]) and analyzed on a 3730 automated sequencer (ABI).Genomic DNA was extracted from cirral tissue using the Wizard SV genomic DNA purification system (Promega). PCR amplification and sequencing of a fragment of the COI gene was initially carried out using universal primers Sequences were manually inspected and edited using BioEdit v7.0.9.1 h) and nucleotide (π) diversities for both marker types.Haplotype designation of the nuclear DNA data was performed in PHASE, using a coalescent-based Bayesian method ST comparisons. Bayesian geographical structuring was investigated using BAPS v5.4 Population genetic structure was investigated for the COI data using analyses of molecular variance (AMOVA) in Arlequin 3.5 Tetraclita squamosa and Tetraclita pacifica were used as outgroup taxa .Statistical parsimony haplotype networks were constructed using TCS 1.21 T. serrata. Fu's Fs test T. squamosa and T. pacifica (GenBank accession numbers DQ363697–DQ363706 and DQ363686–DQ363695) as outgroups, respectively. This was followed by a mismatch distribution to test for population expansion and to estimate parameters needed to date possible population expansions kT. serrata are unknown, we used the generation times of various barnacle species, ranging from one to two years −8−8 substitutions per site per generation Arlequin 3.5 was used to test the influence of historical sea level changes on the demography of A Bayesian Skyline Plot (BSP) The program Migrate 3.1.6 T. serrata comprises two genetically distinct groups with strong nodal support. The two genetic assemblages occur sympatrically  indicate that trically . These htrically  and are trically .Within Clade A, haplotype diversity was 0.979 (±0.003), with 187 haplotypes detected, and within Clade B it was 0.976 (±0.019), with 26 haplotypes detected. The nucleotide diversity of the complete data set was 0.019 (±0.01); generally mtDNA genetic diversity was highest on the south-west and south coasts, with a progressive decrease towards the east coast and the lowest values on the west coast . With thWithin Clade A, the haplotype network  shows thST = 0.035, P<0.01); most of the variation was present within sampled localities (96.44% for the entire data set). When sampling localities were partitioned into their respective coastal regions . When the individuals belonging to Clade B . For the entire data set, pairwise ΦST analysis showed that Kommetjie was significantly differentiated from all localities east of Cape Agulhas (south coast), with ΦST values ranging from 0.09 to 0.23  and a low nucleotide diversity of 0.010 (±0.005), which is comparable with that of the mtDNA data. No significant structure was obtained and Clades A and B formed by the mitochondrial DNA analyses collapsed into a single haplogroup .T. serrata data set , revealing a larger Z-value of 14.176 (P>0.05), which was not significant, with the removal of Clade B. The Mantel test was found to be non-significant when isolation by distance (IBD) was tested within each coastal region . South-west coast localities were not included in the test because only two localities had been sampled in that region.A significant correlation between geographic distances and genetic differentiation was obtained for the entire T. serrata, the coalescent stepping-stone model revealed migration rates that were largely bi-directional in the south and east coastal regions, whereas a more asymmetrical gene flow pattern was evident in a westerly direction between Mossel Bay and Hondeklip Bay (with the exception between Betty's Bay and Cape Agulhas) (Within the sampling range of Agulhas) . InteresAgulhas) .s revealed negative values for the entire data set  and Clade B , respectively. The MK test was not significant for T. serrata when T. squamosa  was used as an outgroup. The more closely-related species T. pacifica could not be used for this test because of the lack of nonsynonymous fixed differences with T. serrata in the sequenced gene region. A pattern of population expansion was confirmed for both clades by the observed unimodal mismatch distributions (not shown), which were not significantly different from the Poisson distributions predicted by a sudden expansion model. This hypothesis of population expansion is supported by both the Sum of Squared Deviations (P>0.05) and Harpending's raggedness indices , although this remains to be tested further.In the absence of a clear allopatric, geographic explanation that could have given rise to the two clades in l region , Fig. 2bet al.Despite the clade structure present in the mtDNA data and a sequence divergence of ∼7.92%, the lack of geographic structure for Clades A and B and the overall lack of structure for the nuclear data suggest incipient speciation at best , 2. ThisT. serrata may give some insight into the lack of structure recovered in this study. Even though the shared haplotypes found for the entire data set indicate that this brooding barnacle is able to disperse over long distances, suggestive of a long PLD, T. serrata does not form a panmictic population as several sampled localities are differentiated, even if ΦST values are low  sampled localities experience gene flow primarily in the direction of the Benguela Current, while half of the localities sampled on the south-west coast (see below) experience gene flow in the direction of the Agulhas Current (Parechinus angulosusT. serrataThe strength and directionality of gene flow in  Current . Ocean c Current . Such res simulations (T. serrata has undergone recent demographic change. The mtDNA haplotype network, showing several common haplotypes (T. serrata are indicative of a growing population. The population expansion of T. serrata predates the LGM, taking place 164 000 to 31 000 years ago, depending on the mutation rate and generation time used (T. serrataT. serrata would still have predated the LGM. The two mutation rates that were used from barnacle genera were substantially greater than the 2% rate and still the population expansion of T. serrata predated the LGM. Therefore, despite the limitations of calculating the timing of expansion T. serrata.The combination of high haplotype diversity and low nucleotide diversity is a typical signature of population expansion following a bottleneck ulations  and the plotypes , mismatcplotypes  and the ime used . The BSPime used . Althougime used . This reet al.Broadly, our results support Marko


Catla catla, and a mastacembelid, Mastacembelus armatus, both of which are found in the Narmada. Our goal was to use DNA sequence information from the D-loop region of the mitochondrial DNA to explore how this fragmentation could impact the genetic structure of these fish populations. Our results clearly show that these barriers can contribute to the fragmentation of the genetic structure of these fish communities, Furthermore, these barriers enhance the effects of natural isolation by distance and the asymmetry of dispersal flows. This may be a slow process, but it can create significant isolation and result in genetic disparity. In particular, populations furthest upstream having low migration rates could be even more subject to genetic impoverishment. This study serves as a first report of its kind for a river system on the Indian subcontinent. The results of this study also emphasize the need for appropriate attention towards the creation of fish passages across the dams and weirs that could help in maintaining biodiversity.India’s third longest river, the Narmada, is studied here for the potential effects on native fish populations of river fragmentation due to various barriers including dams and a waterfall. The species we studied include a cyprinid fish, The online version of this article (doi:10.1186/2193-1801-3-385) contains supplementary material, which is available to authorized users. India is endowed with diverse aquatic habitats due in part to a unique geological history, highly diverse physiography, a monsoon climate and high biotic diversity. The aquatic habitats include an extensive network of rivers and streams made up of medium and minor river systems Rao . The majThe fragmentation of rivers due to dams or barriers may adversely affect fish populations by diminishing natural habitats required for all life stages and interfering with migration between populations. This in turn may lead to reduced population size, loss of genetic diversity, inbreeding and possible species extinctions. Dams may diminish natural habitats in rivers , surface dwelling habitat and its swimming ability, it can also be considered to be representative of many other cyprinids inhabiting Indian rivers , the Indira Sagar hydroelectric dam (92 m) and Sardar Sarovar hydroelectric dam (136.5 m). In addition a series of six major natural waterfalls in the Bhedaghat area (~30 m height) occur along this part of the river  and MADR (5′-TAGGGCCCATTTTAACATCT-3′). For C. catla the Carp-Pro and Carp-Phe primers were as described by Thai et al.  followed by cleanup and bi-directional sequencing using an ABI 3130 Genetic analyzer .Genomic DNA was extracted using the genomic DNA isolation kit (Promega wizard) from a total 43 i et al. . AmplifiSequences were aligned using Codon code aligner v4.0.3  and ArleM. armatus and C. catla (NCBI Accession numbers KF468051 to KF468110). The sequences of M. armatus (n = 60) showed 39 polymorphic sites, accounting for almost 4% of total number of sites examined. Among the polymorphic sites, 7 were singleton variable sites and 2 were parsimony informative sites. Similarly among the individuals representing the C. catla population (n = 42), there were 15 variable sites (~2%) including 6 singletons and one parsimony informative site.Mitochondrial DNA control region (D loop) sequences of 800 bp were obtained and aligned for the individuals from the Narmada river from to two fish species, M. armatus populations and thirteen were found in the C. catla populations. The distribution and frequency of the haplotypes in each population is shown in Table M. armatus. Five unique haplotypes, represented by only single individuals from Jabalpur, Mortakka and Rajpipla populations and three others were found in more than one population  whereas these values were lowest (0.154 and 0.0003 respectively) in the Hoshangabad population , but for this species the nucleotide diversity was highest (0.2404) in the population from Mortakka  of nt Table . For thens Table .Table 4M. armatus, the low pairwise Fst value were noted (-0.0410) between Mortakka and Rajpipla may indicate that these populations undergo genetic exchange events. Among the C. catla populations, the low Fst values for the Mortakka and Bharuch comparison (-0.00143) may also suggest high levels of genetic exchange . RMA regression analysis also revealed a negative isolation-by-distance relationship in case of M. armatus    Figure A. For thM. armatus population at Jabalpur . In C. catla there is no significant support for either sudden expansion or spatial expansion in any of the populations. The values for Harpending’s raggedness index were non-significant in all populations belonging to both species , with up to 39 nucleotide substitutions in some cases. This suggests that this population is evolving independently and may become further genetically differentiated. Similar observations were made for populations of P. squamosissimus species from the Parniba and Tocantinus river basins in China , overall levels of genetic variation of C. catla populations in the Narmada river basin appears to be weaker as overall levels of genetic diversity were only moderate to high. Most of the dams constructed across the river basin are almost 30 years old, and the Bedaghat fall is a natural barrier that is much older. The MtDNA data described here uphold the common assumption that analyzed populations of two native fish species existed in this river before the construction of the dams.In contrast, genetic differentiation among the M. armatus vs. thirteen haplotypes among 42 individuals of C. catla. The range of haplotype diversity values  and nucleotide diversity values (π = 0.0003 to 0.0240 and 0.1404 to 0.2044 respectively) .From samples across the entire river, we found eight haplotypes among 60 individuals of y) Table  on Kessly) Table  on P. sqM. armatus populations, which are invasive to the Narmada basin, originated from Jabalpur. This region was naturally isolated from the rest of downstream regions by the Bedaghat falls a few million years ago, and this would naturally limit genetic exchange for this population on the upstream side. The Jabalpur population has relatively moderate genetic and haplotype diversity, and this may also suggest a recent population expansion after a founder event . This result is consistent with the results based on pairwise Fst values as described in the previous section.The potential for isolation is also considered in the analysis of M values. Here, values of M below 2 imply isolation in the population structure Mallet . The paing Table . The popM. armatus  individuals are stopped or slowed in their upstream dispersion by a barrier 40 cm high weir


Pontoporia blainvillei, is the most threatened small cetacean on the Atlantic coast of South America. Four Franciscana Management Areas have been proposed: Espiritu Santo to Rio de Janeiro (FMA I), São Paulo to Santa Catarina (FMA II), Rio Grande do Sul to Uruguay (FMA III), and Argentina (FMA IV). Further genetic studies distinguished additional populations within these FMAs. We analyzed the population structure, phylogeography, and demographic history in the southernmost portion of the species range. From the analysis of mitochondrial DNA control region sequences, 5 novel haplotypes were found, totalizing 60 haplotypes for the entire distribution range. The haplotype network did not show an apparent phylogeographical signal for the southern FMAs. Two populations were identified: Monte Hermoso (MH) and Necochea (NC)+Claromecó (CL)+Río Negro (RN). The low levels of genetic variability, the relative constant size over time, and the low levels of gene flow may indicate that MH has been colonized by a few maternal lineages and became isolated from geographically close populations. The apparent increase in NC+CL+RN size would be consistent with the higher genetic variability found, since genetic diversity is generally higher in older and expanding populations. Additionally, RN may have experienced a recent split from CL and NC; current high levels of gene flow may be occurring between the latter ones. FMA IV would comprise four franciscana dolphin populations: Samborombón West+Samborombón South, Cabo San Antonio+Buenos Aires East, NC+CL+Buenos Aires Southwest+RN and MH. Results achieved in this study need to be taken into account in order to ensure the long-term survival of the species.Due to anthropogenic factors, the franciscana dolphin,  Knowledge of population structure and patterns of gene flow are key components of management efforts, as they contribute to a better understanding of the ecology and the adaptive potential of species –4. The dPontoporia blainvillei, is endemic to the Atlantic coast of South America. Its distribution extends from Itanúas (18°25’S) in Espíritu Santo (ES), Brazil, to Golfo San Matías (41°10’S) in northern Patagonia, Argentina  and the logarithm (Log) of geographic distance between sampling sites.Results of the Mantel test for correlation between the genetic distance [F(TIF)Click here for additional data file.S1 TableAsterisks show the novel haplotypes found in this study. Dashes indicate unknown frequency. AA and AB: haplotypes with unknown exact sampling site and/or frequency, collected from Argentina ,30 and B(DOCX)Click here for additional data file.S2 TableEstimates of migration rates, time since divergence and θ between locations. NC: Necochea; CL: Claromecó; MH: Monte Hermoso; RN: Río Negro.(DOCX)Click here for additional data file.


Reaumuria soongarica (Tamaricaceae) based on species wide investigation of sequence variation of chloroplast DNA and nuclear ribosomal ITS. Phylogenetic analysis uncovered a deep divergence occurring at ca. 2.96 Mya between the western and eastern lineages of R. soongarica, and ecological niche modeling analysis strongly supported that the monsoonal climate could have fragmented its habitats in both glacial and interglacial periods and impelled its intraspecific divergence. Additionally, the population from the east monsoonal zone expanded rapidly, suggesting that the local monsoonal climate significantly impacted its population dynamics. The isolation by distance tests supported strong maternal gene flow along the direction of the East Asian winter monsoon, whose intensification induced the genetic admixture along the latitudinal populations of R. soongarica. Our results presented a new case that the development of EAMS had prominently impacted the intraspecific divergence and population dynamics of this desert plant.Both of the uplift of Qinghai-Tibet Plateau (QTP) and the development of East Asian monsoon system (EAMS) could have comprehensively impacted the formation and evolution of Arid Central Asia (ACA). To understand how desert plants endemic to ACA responded to these two factors, we profiled the historical population dynamics and distribution range shift of a constructive desert shrub  Global climate fluctuations and geological events were major forces in shaping the current vegetation distribution and geographical structure of genetic diversity of species in the Northern Hemisphere3Arid Central Asia , adjoining to the QTP and accounting for one third of the arid regions in the world, had greatly attracted geological and ecological concerns with its formation and spatial and temporal evolution, especially the processes of aridification789101178131617Reaumuria soongarica (Tamaricaceae), a Tertiary relic shrub and typical xerophytewww.eflora.org). The geographical distribution range of R. soongarica is covering the whole ACA and also in situ fragmented by the QTP, which could provide an ideal model for understanding the demographic history of desert plants in response to the uplift of QTP and monsoonal climate oscillation. Previous phylogeographical investigation on one chloroplast(cp) DNA fragment of R. soongarica has revealed a significant intraspecific divergence that occurred from 1.1 to 3.5 Mya between two regional groups adjoining to QTPR. soongarica. Here, based on the species wide variations on five cpDNA fragments 2223R. soongarica? 2) Whether the population dynamics among the regional groups were affected by local monsoonal or global climate change? 3) How the EAMS affected the gene flow among the regional groups of R. soongarica? This work would shed light on our understanding on the demographic history of constructive species in the fragile desert ecosystems in response to the monsoonal climate change, and also supported empirical phylogeographic data to understand the spatial and temporal evolution of ACA and EAMS.Five cpDNA fragments and one nrITS fragments for a total of 272 in-group and 3 out-group individuals were sequenced. The sequences obtained from five cpDNA fragments were concatenated into a total length of 3,836 bps, with 41 chlorotypes defined by 44 polymorphic sites, including two indels . A totalChlorotypes C1 and C9, which coalesced to chlorotype C39 connecting with the out-group, could be the most recent common ancestral chlorotypes for the eastern and western clades, respectively . ChlorotSimilar to the chlorotypes, most of the ribotypes could be divided into two lineages, except for the ancestral ribotype R4 being shared among all populations from the eastern and western regions , which sR. soongarica recently colonized into the GuD region from two directions.As noted above, all chlorotypes and ribotypes found in the group GuD were derived from both lineages . InteresReaumuria, we carefully chose an average mutation rate of 4.87 × 10–10 substitutions per site per year (s/s/y) for chloroplast genome, which is suitable for desert shrub taxaR. soongarica between the two regions (Badain Jaran and Tengger deserts and Taklamakan desert) before the Middle Pleistocene. Interestingly, in the group GuD, the presence of the west-like chlorotype C12 and the east-like C10 and C11 could be dated to later than ca. 0.37 Mya (point “f” in R. soongarica could have occurred in the Gurbantunggut desert during the Late Pleistocene.Due to insufficiency of pollen or fossil records of HT = 0.934) of R. soongarica on cpDNA was considerably higher than the average diversity within populations (HS = 0.527), which was concordant with the significant phylogeographical structure found on cpDNA  was found between the group TaD and the other four groups  = 0.005, ΦST  = 0.084, ΦST  = 0.108; R. soongarica.The overall genetic diversity  and the lowest genetic differentiation (FST = 0.191) were found within the group QB, supporting the frequent maternal gene flow among the populations within this group , and along the longitudinal direction on cpDNA , Mantel tests were conducted along the latitudinal and longitudinal directions both on maternally inherited cpDNA and bi-parentally inherited nrITS. Significant genetic isolation by physical distance was found along both latitudinal and longitudinal directions on nrITS  and Fu’s Fs  strongly confirmed that the eastern group BJ-TD underwent a population expansion, whereas the western group TaD remained comparatively stable, as only Tajima’s D was significant negative . These findings suggest that geological events common to all populations could have impacted the genetic differentiation of the plants endemic to ACA.The cpDNA genealogy network and phyML tree  suggeste−10 s/s/y for the chloroplast genomeTamarixReaumuria and the out-groups could be dated to ca. 66 Mya  and LIG (EASM dominated), both the glacial and interglacial climates could have greatly contracted the habitat of this species and finally fragmented its distribution range  among the groups GuD, Ku-GG and QB, which suggested that the latitudinal gene flow had been markedly enhanced  and one nrITS fragment were selected for full phylogeographic surveys  with the thermal program referred to KM242133-KM242261).The total genomic DNA was extracted using DNeasy Plant Kits  and then stored at −30 °C in a refrigerator. Six individuals from populations separated by long distances were sequenced to verify the polymorphic sites from 23 pairs of cpDNA fragments . Due to  surveys , and alln), segregating sites (s), total haplotype diversity (hd) and nucleotide diversity (pi) for each population were also estimated with DnaSP Version 5.0. The genealogical topologies of chlorotypes and ribotypes were constructed using the program NETWORK Version 4.2.0.1 with a median-joining model (available at http://www.fluxus-engineering.com/)Sequences from each cpDNA fragment were concatenated into a supergene matrix by the program DnaSP Version 5.0HS, HT) and between-population divergence  were estimated for each region based on the cpDNA using the program PERMUT with 1,000 permutation tests (available at http://www.pierroton.inra. fr/genetics/labo/Software/PermutCpSSR)K) that were geographically homogeneous and maximally differentiated from each other. According to the various geographical locations and SAMOVA results, all populations of R. soongarica were divided into five groups as follows: QB, BJ-TD, TaD, GuD and KD-GG. Notably, the populations from the KD-GG group were considered one group due to the geological “junction” location of the GuD, TaD, QB and BJ-TD groups. The populations collected from the Hexi Corridor and from the Badain Jaran-Tengger deserts were combined as the BJ-TD group according to the analysis of the initial polymorphism survey and geographical locations (FCT), among populations within groups (FSC), and within populations (FST) was calculated with a hierarchical analyses of molecular variance (AMOVA) using Arlequin version 3.11ST) between all pairs of groups were also estimated with 1,000 permutations.Population gene diversity (ocations . Furtherhttp://www.poptools.org/). Under ideal circumstance, the pairwise genetic differentiation between the populations could not reject the Mantel test supporting isolation by distancehttp://ibdws.sdsu.edu/~ibdws/) based on the pairwise FST between populations using 1,000 randomizationsWe used the software BARRIER version 2.2The phylogenetic relationships among chlorotypes and ribotypes were reconstructed by PhyML version 3.0 with maximum likelihood (ML)61−10 s/s/y, which is suitable for shrub speciesTamarix and Myricaria) within Tamaraceae to calibrate the molecular clock. Based on this mutation rate and its assumed standard deviation (10%), the divergence time and coalescence time of lineages were estimated using a Bayesian approach implemented in BEAST Version 1.7.5 with the criteria set by BEAUti (available at http://BEAST.bio.ed.ac.uk)http://BEAST.bio.ed.ac.uk/TreeAnnotator/).To estimate the divergence time between the genealogical lineages, we carefully chose a mutation rate for cpDNA of 4.87 × 10D and Fu’s Fs and mismatch distribution analysis . The pairwise correlation for all 19 biological factors were tested, and 12 independent biological factors were used for further predictions. The distribution sites consisted of those examined in the field investigation together with the specimen records from the Chinese Virtual Herbarium and with coordination from the Global Positioning System (http://www.cvh.org.cn/); 75% of the sites were randomly selected to design a prediction scheme on consideration of the commission error, and the remaining 25% of the sites were used for evaluations of the goodness of the prediction.To predict the distribution of R. soongarica.The contemporary species-climate relationships were then projected to past climate layers to predict the potential range of the species during LGM  and LIG  periods. The project bioclimatic layers for LIG (resolution of 30 arc-second) and LGM (resolution of 2.5 arc-minute) were obtained from the WorldClim database version 1.4How to cite this article: Yin, H. et al. The role of East Asian monsoon system in shaping population divergence and dynamics of a constructive desert shrub Reaumuria soongarica. Sci. Rep.5, 15823; doi: 10.1038/srep15823 (2015).