Coccinella septempunctata L. with those of two phenotypically differentiated populations considered as rear‐edge populations and subspecies based on phenotype (Algeria and Japan). According to the central‐marginal model and expected characteristics of rear‐edge populations, we hypothesize that these rear‐edge populations have (1) a reduced genetic diversity, resulting from their relative isolation over long periods of time, (2) a higher population genetic differentiation, explained by low contemporary gene flow levels, and (3) a relationship between genetic diversity characteristics and phenotypes, due to historical isolation and/or local adaptation. Based on genotyping of 28 populations for 18 microsatellite markers, several levels of regional genetic diversity and differentiation are observed between and within populations, according to their localization: low within‐population genetic diversity and higher genetic differentiation of rear‐edge populations. The genetic structuring clearly dissociates the Algerian and Eastern Asia populations from the others. Geographical patterns of genetic diversity and differentiation support the hypothesis of the central‐marginal model. The pattern observed is in agreement with the phenotypic structure across species range. A clear genetic break between populations of Algeria, the Eastern Asia, and the remaining populations is a dominant feature of the data. Differential local adaptations, absence of gene flow between marginal and central populations, and/or incapacity to mate after colonization, have contributed to their distinct genotypic and phenotypic characteristics.Population genetics and phenotypic structures are often predicted to vary along the geographic range of a species. This phenomenon would be accentuated for species with large range areas, with discontinuities and marginal populations. We herein compare the genetic patterns of central populations of  Processes such as genetic drift, gene flow, and natural selection impact the distribution of the genetic diversity and structuring across a species range. These processes may be strongly affected by both the species evolutionary history and its present demographic characteristics, such as population size, biotic and abiotic factors it might experience, or habitat fragmentation. A major paradigm explaining species distribution and population structure is the central‐marginal model. This model considers that core populations would exhibit increased abundance due to optimal conditions, whereas demographic parameters (reproduction and survivorship) should decline toward the edges , the seven‐spot ladybird, is an appropriate model to investigate the effect of location (core vs edges) on the genetic structure and diversity across a wide range. Indeed, being widely distributed across the Palearctic region, C. septempunctata experiences diverse biotic and abiotic environments. Its distribution expands from the Iberian Peninsula in the west to Japan in the east, to the Sahara in the south and the tundra in the north, but displays some discontinuities, especially in Siberia . Marin et al. (C. septempunctata by combining the molecular data (ISSR) and the patterns of spots on the elytra, together with the assessment of potential barriers by crossbreeding. Although they found a high variation in the size of spots for the Japanese population, they showed a monophyly of all populations, without a clear genetic structuring along the range of the species, even for the marginal populations. ISSR provide large information to assess the genetic variability at perispecific level, but they might be inappropriate in an evolutionary history study  and a further investigation, with codominant markers, is needed.In this context, not only the population genetic structure of widespread species is predicted to vary along the geographic range ” populations and therefore predict (1) marginal populations  less genetically diverse than the core ones, (2) a higher population structuring of the rear‐edge populations compared to the core, and potentially to the leading‐edge, populations, and (3) consistence between the phenotypic divergence observed for the rear‐edge Algerian and Japanese populations, and the genetic divergence due to historical isolation and/or local adaptation. Our objective is to characterize the patterns of genetic variability and structure in the rear‐edge populations by comparing populations across the entire distribution area. To uncover the factors involved in shaping the genetic structure over the range and to test whether its spatial structure is consistent with the central‐marginal model, we compared the genetic diversity of 28 sampled populations covering the native range, genotyping 407 individuals for 18 microsatellite markers developed from this species  and to the “rear” edge at the southern and eastern limits, including a priori the populations from Algeria in the south and west and from Japan in the east .The individuals were sampled by several collectors in sites covering the entire native range of st Table . The samTotal genomic DNA was extracted from entire individual (minus elytra) using DNeasy Blood and tissue Kit  with PBS protocol according to the manufacturer's instructions. The 407 individuals were genotyped for 18 microsatellite markers, previously developed for the seven‐spot ladybird  of populations were computed with GENETIX v. 4.04 , standard sample size consisted of the smallest population sample size with a complete genotype at all loci.The levels of genetic diversity  was tested using FSTAT (Goudet Tests for deviation from Hardy–Weinberg equilibrium (HWE) were conducted in GENEPOP Rousset , P‐value9 Goudet  and the T Goudet  by permuHs) were compared between several groups of populations using FSTAT .To test for population differentiation, pairwise T Goudet  were calFST/(1 − FST)) and log‐transformed geographic distances calculated as the linear distance between sampling sites in km. Mantel tests were conducted, with the ade4 package , analyzing correlation between the pairwise linearized genetic differentiation of populations  were conducted, K varying from 1 to 15. The optimum number of clusters was determined according to the delta K method  was used based on the data from all populations, as implemented in STRUCTURE version 2.2  is significantly higher or below the heterozygosity predicted at mutation – drift equilibrium (Heq) on the basis of the observed number of alleles (N < 20), that is, likely to arise from a population size reduction or expansion, respectively . For populations with more than seven individuals (21 populations), the average number of alleles per locus ranged from 4.17 to 8.33, with a mean of 6.14 (SD = 1.2). The average expected heterozygosity (He) was 0.656 (SD = 0.03) ranging from 0.598 (China) to 0.711 (Germany), and the mean observed heterozygosity (Ho) was 0.52 (SD = 0.05) varying from 0.428 (Spain) to 0.615 (Poland). Detailed data for the genetic parameters are given in Table A total of 407 individuals from 28 populations, covering the native range of the species, were genotyped and analyzed at 18 polymorphic microsatellite loci. A total of 300 alleles were detected in the complete dataset, with in average 16.5 (SD = 8.4) alleles/locus. No significant linkage disequilibrium was detected between any pair of loci across all populations  to 4.62 ; the mean expected heterozygosity (He) was 0.656 (SD = 0.03) ranging from 0.598  to 0.711 , Japan , and China . Populations from northern limits were highly diversified , as well as core populations . Among the other populations, no significant difference (P > 0.50) was observed between the leading‐edge populations  and populations from potential refugia that could have participated to the colonization of northern areas .The mean AR, standardized for seven individuals per population, was 4.30 (SD = 0.29), ranging from 3.60  among the 21 sampling sites, explaining 6.38% of the variation. A hierarchical structure was observed between the three groups , and all the other populations, FCT = 0.116, P < 0.001). Such partitioning explained 11.6% of the genetic variance and 86.5% was within populations  and all the others. More specifically, the highest genetic differentiations were observed between the Algerian (no significant differentiation between the two populations sampled) and the Japanese or Chinese populations , between Japanese and Chinese populations , and between Algerian, Japanese, or Chinese populations with all the other populations . With the exception of the pairwise comparisons including the Algerian, Chinese, or Japanese populations, pairwise FST‐estimates were generally low, and many of them were not statistically different from zero, except for some pairwise comparisons, including mainly comparisons with populations of India and Iran .Global analyses of genetic differentiation revealed highly variable Individuals from Algeria, Japan, and China, unlike most of the populations, showed some particularities in their genotyping supporting their genetic differentiation. First, in the standard conditions, the success of genotyping for two markers was lower than for the other populations (tetra118 for Algerian populations and tetra112 and tetra118 for the Japanese and Chinese populations), suggesting substitutions and/or indels in the adjacent region of the repeats where the primers were designed. Second, we detected a number of alleles in Japanese samples either with a high PCR product size or a very low size, suggesting a high number of repeats or no repeat at all (related to the observed size and the number of repeats in the reference sequence). These private alleles or shared for some of them with only the Chinese sample were sequenced and revealed deletions or insertions in the adjacent region of the repeat. For example, the allele “191” of the di208 locus, at high frequency in the Japanese population , revealed four AG repeats and a deletion of 14 bases; the allele “267” of the di282 locus, with a frequency of 0.472 in Japan, showed five AC repeats plus a deletion of 15 bases .P = 0.0036; r = 0.47): a cluster of extremely differentiated populations encompassing populations from Algeria, China, and Japan and a cluster of poorly differentiated populations . This pattern is not due to a stepping‐stone dispersal consistent with IBD, but to the high differentiation of the populations from Algeria, China, and Japan compared to the others. When we excluded these four populations, we observed a significant pattern of IBD  with coefficient of determination higher (r = 0.63) than for the complete dataset.The signal of IBD over all data is complex, with two discrete clusters of points , Japan and China (Eastern Asia), and each of the other populations Fig. . The firK = 3), discriminating Algerian populations from Eastern Asia populations (China and Japan) and from a cluster comprising all the 24 other localities . Only few individuals were admixed and this result suggested little gene flow, ancient or recent, between clusters . The DAPC inferred the optimal number of genetic clusters as five (K = 5). The first principal component axis  separated one cluster  from all the others , was attributed to the three clusters . The relative frequencies of the components of the three clusters in the populations showed no geographical pattern .The individual‐based clustering approaches  provided evidence of genetic substructuring among populations. The STRUCTURE analysis revealed an optimal number of genetic clusters of three  being more prone to yield significant signatures of expansion (Table S3). Conversely, a signature of bottleneck was detected in Japan and China under the TPM model of mutation, with a small proportion (5–30%) of stepwise mutations in the TPM model (Table S3). Moreover, when loci, with a significant NA frequency, were excluded, most of the results were similar, except for China (Table S3).C. septempunctata populations could be consistent with the central‐marginal model , (2) all the populations would potentially be impacted by their presence, and we identified a signature of either expansion or bottleneck according to the populations, and (3) the patterns were overall similar with or without loci with significant frequency of NA.Distinct levels of regional genetic diversity and differentiation were identified using nuclear polymorphic markers. More specifically, the rear‐edge populations from Algeria (southern limit) and Eastern Asia (Japan and China) show particularities, both in terms of differentiation and genetic diversity. Taken together, our results suggest the presence of three main clusters with the populations from Algeria and Eastern Asia (Japan and China) highly genetically separated from the rest of the populations sampled. Indeed, the level of genetic differentiation between the core populations and the southern (Algeria) and eastern (China + Japan) edge populations is significantly higher than between the core populations. This suggests a low gene flow, either contemporary or historical, between these populations. Our results differ from the study based on polymorphic nuclear markers (ISSR), where no geographic pattern was identified  and eastern edges  are consistent with the features expected for rear‐edge populations  suggests limitation of gene flow, likely due to ancient isolation. Indeed, during the Last Glacial Maximum (c.a. 18 ka and possibly earlier cold periods), a land bridge across the East China Sea used to connect the currently isolated region of Japan . This suggests either absence or limited gene flow between North Africa and Europe. However, some individuals attributed to C. algerica have been found in sympatry with the typical form in Gibraltar . A further phylogeographical study, confronting sequences from both nuclear and mitochondrial markers, could provide a better knowledge of the patterns of genetic structure and diversity in the native range and be useful to evaluate the historical influences (impact of glaciations on genetic diversity and structuring) on the contemporary genetic patterns.This study investigates the spatial genetic diversity of the None declared.Figure S1. Plots of population allele frequencies per locus represented by dots of varying size. The two populations from Algeria are clustered.Click here for additional data file.Figure S2. Results of the Bayesian structure analyses of Coccinella septempunctata populations across Palearctic, calculated with the program STRUCTURE.Click here for additional data file.Figure S3. DAPC assignment: posterior probability of assignment of each individual to the five DAPC clusters.Click here for additional data file.Table S1. FIS values, per population and per locus, and across all loci with the significant values indicated in bold.Click here for additional data file.Table S2. Null allele frequencies estimated per locus and per population. “–” means that null alleles were not encountered.Click here for additional data file.Table S3. Genetic signatures of demographic changes in Coccinella septempunctata identified with BOTTLENECK, significant deficiency of heterozygotes are indicated as “expansion” and excess as “bottleneck”.Click here for additional data file.


The human population in Greenland is characterized by migration events of Paleo- and Neo-Eskimos, as well as admixture with Europeans. In this study, the Y-chromosomal variation in male Greenlanders was investigated in detail by typing 73 Y-chromosomal single nucleotide polymorphisms (Y-SNPs) and 17 Y-chromosomal short tandem repeats (Y-STRs). Approximately 40% of the analyzed Greenlandic Y chromosomes were of European origin . Y chromosomes of European origin were mainly found in individuals from the west and south coasts of Greenland, which is in agreement with the historic records of the geographic placements of European settlements in Greenland. Two Inuit Y-chromosomal lineages, Q-M3  and Q-NWT01 (xM265) were found in 23% and 31% of the male Greenlanders, respectively. The time to the most recent common ancestor (TMRCA) of the Q-M3 lineage of the Greenlanders was estimated to be between 4,400 and 10,900 years ago (y. a.) using two different methods. This is in agreement with the theory that the North Circumpolar Region was populated via a second expansion of humans in the North American continent. The TMRCA of the Q-NWT01 (xM265) lineage in Greenland was estimated to be between 7,000 and 14,300 y. a. using two different methods, which is older than the previously reported TMRCA of this lineage in other Inuit populations. Our results indicate that Inuit individuals carrying the Q-NWT01 (xM265) lineage may have their origin in the northeastern parts of North America and could be descendants of the Dorset culture. This in turn points to the possibility that the current Inuit population in Greenland is comprised of individuals of both Thule and Dorset descent. Information on the genetic diversity and structure of human populations is highly relevant in many fields of genetics. It is, for example, very important to control for population stratification in genetic association studies to exclude false-positive associations due to population specific characteristics . FurtherThe North Circumpolar Region has been colonized by humans for the last 5,000 years. The early Paleo-Eskimos began to expand from Beringia along the northern coastlines of Alaska and Canada approximately 4,500 years ago (y. a.), and the Saqqaq Paleo-Eskimos reached Greenland approximately 4,000–4,500 y. a. . HoweverThe first European settlement in Greenland, by the Norse from Norway and Iceland, is dated to 985 AD . HoweverIn accordance with the historical records, the contemporary Greenlandic population is admixed containing an Inuit component and a European component ,12,13. SAs a result of the non-recombinant nature of the human Y chromosome, all human Y chromosomes can be assigned to a Y-chromosomal lineage. The Q-M242 Y-chromosomal lineage is considered to be a major paternal lineage among Native American populations –19, althThe aim of this study was to perform a detailed investigation of the distributions of European and Inuit Y-HGs in the contemporary male population of Greenland. A total of 73 Y-chromosomal single nucleotide polymorphisms (Y-SNPs) were studied. To refine the male population of Greenland, 17 Y-chromosomal short tandem repeats (Y-STRs) were also investigated. Particular attention was given to the Inuit component of the population in Greenland to gain knowledge regarding the North Circumpolar Inuit groups. In addition, the European Y chromosomes in the male Greenlandic population were defined and compared to those of the Danish population.The results showed that 40% of the male Greenlandic population carries Y chromosomes of European origin. The European lineages were primarily found in south and west Greenland. In regards to the Inuit component, our results points to a gene flow between the Inuit populations of the Dorset and Thule cultures, which can help to elucidate the current debate on the Inuit founders of the Greenlandic population.Overall, 21 Y-HGs were found in the male Greenlandic population  Table. TA total of 11 (4.6%) Greenlandic and 13 (5.4%) Danish Y chromosomes could not be defined by the five multiplexes used in this study. Therefore, Y-STR profiles were used to predict the Y-HG of these 24 Y chromosomes . A singlFST), East Sermersooq was found to be significantly different (p<0.05) from the Kujalleq, West Sermersooq and Qeqqata populations  Inuit (Q-NWT01 (xM265) and Q-M3 ), (2) European  and (3) ‘other’ (Y chromosomes that were undefined in this study). The distribution of these three groups in the five geographic regions of Greenland can be seen in Figure B in Intra-population comparisons. The Y-chromosomal variation in Greenlanders was further refined by Y-STR analysis. The overall Y-STR diversity in the Greenlanders was 0.98 (Table C in RST) and the genetic distances based on the discrete Laplace method [RST, the population of East Sermersooq was found to be significantly different (p<0.05) from all of the other populations in Greenland  and visualized in an MDS plot (MDS plot . Data frMDS plot . With thMDS plot . The popMDS plot .The Y-STR diversities and mean pairwise differences (MPD) within the Y-HGs of Greenlanders are presented in The Inuit Y-chromosomal lineages in the Greenlanders, HG Q-NWT01 (xM265) and Q-M3 , had approximately half of the Y-STR diversity compared to the European lineages R1b-M343 and I-M170 . AlthougAll of the Y-STR haplotypes belonging to Q-M242 were connected in a median joining network . The InuThe Y-STR haplotypes of the Q-NWT01 (xM265) chromosomes from this study and from Dulik et al.  were conTo further compare the relative chronology between Q-NWT01 (xM265) and Q-M3  in the Greenlanders, the time to the most recent common ancestor (TMRCA) was estimated for the two haplogroups. The TMRCA for the Q-NWT01 chromosomes using ρ-statistics indicated a coalescent event that occurred approximately 7,000 y. a. . SimilarIn accordance with previous studies ,14, we fApproximately 40% of the Y-HGs in the male Greenlandic population were found to be of European origin. Only considering the European Y-HGs  in Greenland, the relative frequencies of these Y-HGs in the Greenlanders resembled those observed in the male Danish population examined in this study and other male Scandinavian –26 and IThe Y-STR haplotype diversities and the MPDs within the European Y-chromosomal lineages I-M170 and R1b-M343 in the Greenlanders were similar to each other and to the variations found in the same Y-HGs in the Danes. This can, for example, be explained by continuous gene flow from Scandinavia, primarily from Denmark. However, multiple founders of the European lineages in Greenland can also explain this observation. Some of the diversity could, for example, be attributed to the multiple settlements in Greenland by Europeans. Previous studies have tried to differentiate between gene flow from the Norse and from the Scandinavians . HoweverTwo Y-chromosomal lineages, Q-M3  and Q-NWT01 (xM265) were found to be prominent in Greenlanders. The Q-M3 lineage has previously been found among Native American populations throughout North and South America ,28,29, wThe Y-chromosomal lineages Q-MEH2 (xL54) and Q-L54  and Q-M3  lineages clustered to different regions of the island , which mThe estimated TMRCA  of the Q-M3  in Greenland was lower than that of the Q-NWT01 (xM265) . This indicates that in Greenland, the Q-M3  lineage has a more recent origin than the Q-NWT01 (xM265) lineage. However, the estimates are largely overlapping; therefore, caution should be taken when making comparative conclusions.The estimated TMRCA of the Q-M3  lineage in Greenlanders is lower than that estimated for the same lineage among the Inuit populations of the Canadian Northwest Territories . The samThe estimated TMRCA of the Q-NWT01 (xM265) lineages in Greenland  was approximately three-times older than that of the same lineage in the Canadian Northwest Territories, which was 2,900–5,700 y. a . HoweverThe presence of Y-HG Q-NWT01 (xM265) (as well as Y-HG C-P39 which was not studied in this work) in the Inuit populations was previously suggested to represent a second expansion of the human population within North America via a migration from the Northwest region . HoweverRecently, Raghavan et al  reportedOverall, our results are in agreement with previous studies, indicating that the populations of the North Circumpolar Region, including Greenland, share a common origin with Native American populations. However, the male Inuit population in Greenland appears to be a mixture of individuals belonging to two Y-chromosomal lineages within Y-HG Q-M242. Males carrying the derived state at the M3 locus appear to stem from an expansion of humans from the northwest region of North America. Most likely, these individuals are descendants of the Thule culture that reached Greenland approximately 800 y. a. Individuals carrying the derived state at the NWT01 locus may have been part of the same expansion . Howeverhttps://www.retsinformation.dk/Forms/R0710.aspx?id=137674), see the Supplemental materials and methods in http://www.uma.net/e/policy/b3.htm). The DNA was extracted from 200 μl blood using the Blood QiaAmpDNA Mini kit  according to manufacturer’s protocol. The DNA was eluted in 50 μl of AE buffer.A total of 468 randomly sampled male individuals were analyzed, 227 from Greenland and 241 from Denmark. The individuals from Greenland were confirmed to have a birthplace in Greenland. All of the samples involved in the study were anonymized DNA extracts from unrelated male individuals obtained from the biobank of the Department of Forensic Medicine, Copenhagen Denmark . The use of the samples are in accordance with the Danish Law, LOV nr 593 af 14/06/2011  were constructed using the Assay Design software for the Sequenom MassARRAY system  http://www.r-project.org/) as described previously [The mass spectra were viewed and analyzed in Typer 4.0 . The raw data were further analyzed using the open software R v. 2.15  according to the manufacturer’s protocols in a reaction volume of 10 μl. The amplification products were separated on an ABI 3130xl Genetic Analyzer  and visualized using Genmapper IDX v. 1.4  or a combination of GeneScan v. 3.7  and Genotyper v. 3.7 . An analytical cut-off signal of 50 RFU was used. The Y-STR profiles were used to predict (http://www.hprg.com/hapest5/) the Y-HGs of the individuals which could not be assigned a Y-HG based on the five multiplexes used in this study.All of the Greenlandic individuals were analyzed using the AmpFThe results of the Y-SNP and Y-STR typing were submitted to the Y chromosomal haplotype reference database (YHRD), accession number YA003999, YA004000, YA004001, YA004002, YA004003 and YC000109.The Greenlandic individuals were divided into groups based on their place of birth according to the four municipalities of Greenland  were estimated using Arlequin v. 3.5 (http://cmpg.unibe.ch/software/arlequin35/) [Vp) were calculated as described previously [The Y-SNP haplogroup frequencies were calculated by mere counting. The haplotype diversity, mean number of pairwise differences (MPD) and pairwise genetic distances (quin35/) . The refquin35/)  (see theeviously . In shorhttp://www.fluxus-engineering.com/) [The median joining networks of the haplotypes (15 Y-STRs) were constructed using Network v. 4.6.1.1 (ng.com/)  and the ng.com/)  using thng.com/) . The foung.com/) . The TMRng.com/)  assumingng.com/) .For all of the statistical analyses, DYS385a/b was excluded, because the physical order of the two alleles cannot be determined. Furthermore, DYS389b was calculated by subtraction of the repeat number of DYS389I from the repeat number of DYS389II. In addition, a single haplotype was excluded due to a duplication event in the Y GATA H4 locus.S1 FileFST values below the diagonal and the corresponding p-values above the diagonal based on the Y-chromosomal haplogroup frequencies between five sub-populations in Greenland. Table C, The diversity of the Y-chromosomal haplotypes in Greenlanders and within the sub-populations in Greenland. Table D, The genetic distances, pairwise RST values below the diagonal and the corresponding p-values above the diagonal based on the Y-chromosomal haplotype frequencies, between the five sub-populations in Greenland. Table E, The genetic distances obtained using the discrete Laplace method below the diagonal and the corresponding p-values above the diagonal based on the Y-chromosomal haplotype frequencies between the five sub-populations in Greenland. Table F, The references for the population data used for the comparisons. Table G, The PCR conditions for the five multiplexes. Table H, The SNPs for which modifications were made for analysis in R.Figure A, A simplified tree of the Q-M242 lineage and the sub-lineages in Q-M242 discussed in this study. Figure B, The distribution of the Y-HGs  in the five investigated regions of Greenland. Inuit: Q-NWT01 (xM265), Q-M3 . European: I-M170, R1a-M513 and R1b-M343. Figure C, A simplified Y-chromosomal tree, including information on the investigation strategy of the five multiplexes. Table A, The diversity of the Y-chromosomal haplogroups in Greenlanders and within the sub-populations in Greenland. Table B, The genetic distances, pairwise (DOCX)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 TableFor Y-SNPs, haplogroups according to International Society of Genetic Genealogy (ISOGG) and mutations in parentheses.(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


Hadramphus spinipennis Broun) and its host-plant Dieffenbach’s speargrass (Aciphylla dieffenbachii Kirk), which are both open habitat specialists with populations on Mangere and Rangatira Islands, Chathams, New Zealand. Both of these islands were heavily impacted by the introduction of livestock; the majority of the forest was removed and the weevil populations declined due to the palatability of their host-plant to livestock. An intensive reforestation program was established on both islands over 50 years ago but the potential impacts of this restoration project on the already endangered H. spinipennis are poorly understood. We combined genetic and population data from 1995 and 2010–2011 to determine the health and status of these species on both islands. There was some genetic variation between the weevil populations on each island but little variation within the species as a whole. The interactions between the weevil and its host-plant populations appear to remain intact on Mangere, despite forest regeneration. A decline in weevils and host-plant on Rangatira does not appear to be caused by canopy regrowth. We recommend that (1) these populations be monitored for ongoing effects of long-term reforestation, (2) the cause of the decline on Rangatira be investigated, and (3) the two populations of weevils be conserved as separate evolutionarily significant units.Human alteration of islands has made restoration a key part of conservation management. As islands are restored to their original state, species interactions change and some populations may be impacted. In this study we examine the coxella weevil, ( Island restoration is a key focus of conservation biology. Often islands are home to endemic flora and fauna which may be heavily impacted by human modification and introduced species . AlthougeN), can decrease fitness and lower ability to adapt to changing environments ] and invertebrates [e.g. Amychus spp. (Chatham’s giant click beetle)].Islands within the New Zealand archipelago have been actively restored for many years, using pest eradication, replanting, translocation, captive rearing and reintroductions . One groHadramphus spinipennis Broun 1911) is a large, flightless weevil endemic to the Chatham Islands. There are four species in the genus Hadramphus (H. spinipennis (Aciphylla dieffenbachii), which is found in open habitats on the islands. However, it has also been sighted on Pseudopanax chathamicum Kirk  several hundred meters away from A. dieffenbachii  Island is 219 ha and was heavily farmed until its purchase by the New Zealand government in 1953, after which all livestock were removed in 1961  Fig. 1)Fig. 1). A. dieffenbachii, started to regenerate with the removal of livestock in 1968 and an intensive forest planting program was implemented in 1974  Island (14–19 February 2010) and Mangere Island (17–23 February 2011) were surveyed for  to grow . Due to H. spinipennis  and cytochrome b (cytb), and one nuclear gene, internal transcribed spacer 2 (ITS2), were amplified by polymerase chain reaction (PCR). For all PCRs, 2.5 µl of the DNA extraction was added to the following: 2.5 µl of 0.25 mM of dNTPs, 0.2 µl of polymerase, 1 µl of 20 µM for each primer, 2.5 µl of 10× PCR buffer  and deionized water to bring the total reaction volume to 25 µl.Two mitochondrial genes, cytochrome COI was amplified using the primers LCO1490 and HCO2198 (656 base pair fragment) . The PCRDue to the difficulty in amplifying a large fragment of ITS2 using the original primer set, a new genus-specific primer set was developed for a 450 base pair fragment of ITS2   and forward and reverse sequences were manually aligned in Mega 5.05 . No inseFor Bayesian analysis, the COI data set was reduced to 16 sequences to match them with the cytb sequences. Sequences of the two genes were then concatenated in R 2.13.2. . The conThe mtDNA and ITS2 data were analyzed under a strict molecular clock with a coalescent (constant population size) tree prior. For each gene, four replicate runs of a chain run for 50 million generations were performed, sampling every 2000 generations. Convergence and effective sample size of each parameter was assessed in Tracer 1.5 and samples from the four runs were pooled using Log Combiner 1.7.2. After discarding the initial 10% as burn-in, a maximum clade credibility tree was compiled in TreeAnnotator 1.7.1 .For the mitochondrial data, two Bayesian analyses were conducted to provide estimates of divergence time using (i) the geological evidence and the timing of the volcanic emergence of Mangere, 3–2 million years ago , and ii. For geoPairwise genetic distances for mtDNA were taken from the Bayesian maximum clade credibility tree using PASSaGE 2.0 . A NeighA. dieffenbachii: East Clears, West Clears, and West Landing ; a chi-square test confirmed heterogeneity of base frequencies across all taxa . For cytb, a 432 bp fragment was obtained, which had one variable site, which was parsimony informative. Base frequency means for cytb were unequal and AT-rich ; a chi-square test confirmed heterogeneity of base frequencies across all taxa . The 30 COI sequences showed little variation with only two individuals sharing a single nucleotide difference between themselves and the rest of the samples. However, for cytb there was a clear difference at one nucleotide in all samples between individuals from each of the two islands. The pairwise genetic distances in the concatenated mitochondrial genes were low, with a mean of 0.002 (max 0.003) substitutions/site between the two island populations. The maximum P-distance within each island was 0.001 substitutions/site.In total, 30 specimens of After the removal of the AT variable repeat, a 428 bp fragment was obtained for ITS2 which contained 24 variable sites, 17 of which were parsimony informative. Four indels were found in the sequences: two from an individual on Rangatira and two from an individual on Mangere. The Neighbor-Net network does not show strong genetic structure within or between the islands . The resThe maximum clade credibility trees were very similar in topology to the NJ trees; due to the splitting of zero-length branches in the maximum clade credibility trees we opted to display the NJ trees. The mitochondrial tree shows a weakly-supported split between the two islands , and theThe genetic analysis shows a population of weevils that is highly similar across the islands with only a small amount of difference between the two islands in the mitochondrial genes. A genetic difference between the weevil populations of Mangere and Rangatira has been previously reported for a different section of the COI gene . The anaThe difference in the mitochondrial and nuclear gene trees can be explained by the four times faster coalescence time in mitochondrial genes compared to nuclear genes . The islHadramphus weevils  of A. dieffenbachii were found in 1995 and 2010, the plants were in decline in 2010 with no flowering and no large-sized plants. One population of A. dieffenbachii that was found in 1995 was no longer in existence, not even as seedlings, in 2010. The newly discovered population of plants on the West Clears contained mainly small plants and seedlings, with only one plant that flowered, and may not be able to support a large weevil population. The decline on Rangatira does not seem to be the result of forest regeneration, as the locations of all A. dieffenbachii populations are on open grasslands and cliffs where no forest has regenerated. Modeling suggests that if the distance between host-plant populations is great enough to not be easily traversed, but not so far that it is unreachable by the weevils, then the consumer-resource dynamic could persist for the long-term (H. spinipennis and A. dieffenbachii has been altered.On Rangatira, there has been a decline in both ong-term . The hosong-term . TherefoHadramphus spinipennis is listed as endangered by the New Zealand Department of Conservation and is considered a species of concern due to its very restricted range. Although spatial models have shown that the weevil population is in equilibrium, and could possibly be classified as “common” (H. spinipennis is common on both Mangere and Rangatira. In regards to the number of weevils and host-plant populations on Mangere, the weevil seems relatively abundant; however, on Rangatira lower weevil numbers were found. Genetic data indicates that both populations do not possess a large amount of genetic diversity and have most likely undergone a population bottleneck. Population bottlenecks are related to a loss of genetic diversity which can have significant consequences for long-term viability of small populations (A. dieffenbachii may decline due to a loss of open habitat. Although the population counts suggest no change in H. spinipennis on Mangere, the weevil may be unable to adapt to a possible decline in its host-plant resulting from the loss of open habitat. This is particularly a concern on Rangatira where a decline in weevil numbers was seen.“common” , combiniulations . As the H. spinipennis on Rangatira and Mangere Islands are shown to be genetically isolated from each other. The unique haplotypes from each island adds variation and allows for the possibility of cross-introduction of weevils to preserve genetic diversity. Our results strongly suggest that forest restoration on Rangatira and Mangere Islands has not had a negative impact on H. spinipennis or its host plant; however, a long-term survey, in particular accessing the area on Rangatira that could not be searched in this study, is recommended to confirm small population sizes. Although continued monitoring of the weevil populations is recommended, forest restoration should continue and is not impacting on the conservation of an open habitat specialist.A pattern of genetic structure separating a species by island haplotype is something that has been reported in other flightless beetles . Between10.7717/peerj.749/supp-1Appendix S1Click here for additional data file.10.7717/peerj.749/supp-2Supplemental Information 2The ITS2 alignment for Mangere and Rangatira; the STR is included in this alignment.Click here for additional data file.10.7717/peerj.749/supp-3Table S1Aciphylla dieffenbachii subpopulations surveyed on Rangatira and Mangere Islands.GPS coordinates in degrees, decimal minutes of the Click here for additional data file.10.7717/peerj.749/supp-4Table S2GenBank accession numbersClick here for additional data file.


Natural hybridization plays a key role in the process of speciation. However, anthropogenic (human induced) hybridization of historically isolated taxa raises conservation issues. Due to weak barriers to gene flow and the presence of endangered taxa, the whitefish species complex is an excellent study system to investigate the consequences of hybridization in conservation. We focused on three naturally reproductively isolated whitefish taxa in Germany: the endangered, anadromous North Sea houting (NSH) and Baltic houting (BH), which were reintroduced after local extinction, and the commercially stocked European whitefish (EW). To evaluate the genetic integrity of each taxon, source and reintroduced populations of NSH and BH, and EW populations were characterized based on two mitochondrial and 17 microsatellite loci. Additionally, we investigated gill raker counts as an adaptive phenotypic trait. Even though clear genetic and phenotypic differentiation confirmed the houtings as separate evolutionarily significant units, admixture analyses revealed an extensive hybrid zone. Hybridizations were introgressive, positively correlated with genetic diversity, and were reflected in the gill raker counts. The BH distribution range showed higher heterogeneity and stronger admixture than the NSH range. Erroneous stocking with non-native genotypes best explained these patterns, which pose challenges for the conservation of the endangered NSH and BH. The present age has been coined ‘the sixth extinction’  and the Baltic houting (BH)  (EW), which is commercially stocked in lakes across the same geographic ranges.The recent postglacial origin of the whitefish species complex (Coregonidae) has resulted in porous barriers to gene flow  Anthropogenic translocations outside the historic ranges by erroneous stocking, which could then be followed by diffusion from short-range migrations, termed ‘stepping stone and diffusion’  and 17 microsatellite loci. We also assessed gill raker counts (GRCs), which represent a meristic trait related to feeding ecology  to the River Treene (NSH_TRE) in 1987  completed the dataset, as it is the most genetically distinct whitefish taxon in Germany but can occasionally hybridize with EW . Due to their high protection status, no NSH_VID samples for GRCs were available, and literature GRCs for indigenous NSH from Schöter . Primers were obtained from Østbye et al. , 1 μL of each primer (5 mm), 0.1 μL DreamTAQ taq polymerase , 1 μL 10 ×  buffer, and 5.4 μL HPLC water. The PCR cycles started with an initial denaturing phase of 3 min at 95°C, followed by 32 cycles , and ended with 10 min at 72°C final extension. PCR products were cleaned with ExoSAP-IT® (Thermo Fischer Scientific Inc.) followed by cycle sequencing using the Big Dye® Terminator v.3.1 kit , and a final purification using the Big Dye® XTerminator™ kit (Applied Biosystems®). Products were sequenced on an ABI 3130 XL Automated Genetic Analyzer (Applied Biosystems®).Two mtDNA loci with different mutation rates were used: the NADH dehydrogenase, subunit 3  region, and the cytochrome oxidase e et al. . PCR wasFst) . DnaSP v.5.10.01 (Librado and Rozas ) Wright  based on) Wright .As demographic events can blur evolutionary patterns . Presence of null alleles and large allele dropout was assessed with Microchecker v.2.2.3  was calculated as measure of genetic diversity harbored by populations using HP-RARE v.1.0  and test for linkage disequilibrium were assessed using Arlequin v.3.5.1.2. Finally, the mean allelic richness Fst calculated in Arlequin v.3.5.1.2, using 10 000 permutations. Last, an analysis of molecular variance (amova) was run with 1000 permutations to estimate the distribution of genetic variability among a priori taxa and populations.To evaluate the relationship between all NSH, BH, and EW populations, an unrooted neighbor-joining tree using Cavalli-Sforza distances, which are well suited to reveal topologies based on microsatellite frequency data ] from microsatellite data and log-transformed geographic distances between river mouths measured in Google Earth v.7.1.1.1888. Transformations followed considerations in Rousset . The most likely K was determined based on ΔK , BH (r), and EW (s) and their 90% credible intervals (CIs) to each individual's genotype. Two methods were then used to assign individuals as pure or hybrids. First, under ‘STRUCTURE relaxed’, based on a hybrid threshold of 0.10, individuals were assigned as pure NSH, BH, or EW if either q, r, or s ≥ 0.90, and as hybrids showing genotypic contributions of two different taxa if q, r, and s < 0.90 and either q and r, q and s, or r and q > 0.10. In addition, individuals showing genotypic contributions of all three taxa in this study  were defined as compound hybrids. With the number of microsatellite loci and the observed Fsts in our study, the applied thresholds should allow for efficient hybrid identification according to Vähä and Primmer  were assessed following Latch et al.  with mod Primmer . Second, Primmer . Here, iq; BH range: r), and (iii) the mean admixture proportion  in each of the two ranges with two-sample t-tests. The latter two parameters were then also compared among reintroduced populations within the distribution ranges, among the years 2004–2012 for the NSH_TRE, and among the three NSH_RHI sites with anovas and Tukey's post hoc tests.The extent of admixture was compared between the NSH and BH distribution ranges based on (i) the proportions of pure native, pure non-native and hybrid individuals using a chi-square test, (ii) the mean contribution of the native genotype  and of non-native houting genotype  within populations with distance from the canal, with the expectation of negative correlations if the canal serves as corridor.The two alternative explanations for secondary contact in the study system were migrations via Kiel Canal and translocations by erroneous stocking. To distinguish between them, we assessed the genotypes present in the canal population BH_NOK, with the expectation of a mixture of NSH and BH genotypes if the canal serves as migration corridor. We also analyzed the correlation of the mean proportion of non-native genotype . Secondly, GRC of indigenous BH and NSH populations were both significantly lower than EW GRC . The situation among reintroduced populations differed between the distribution ranges , the BH_SCH was comparable with the NSH populations NSH_TRE  and NSH_ELB , and the BH_NOK as well as BH_LAC with the EW population EW_BOR  and sex  were not associated with GRC.When comparing the source populations, the indigenous BH population BH_PEE was characterized by slightly but significantly lower GRC than indigenous NSH populations  than NSH (22.1%) and EW (43.9%), and Hap 2 more dominant in a priori NSH (63.6%) than BH (18.5%) and EW (14.0%). Mapping haplotypes revealed a gradient across the reintroduced populations . The remaining 11 haplotypes were dominated by two haplotypes differentiated by seven-point mutations with a combined coverage of 78.4% (Hap 1 – 36.18% and Hap 2 – 42.23%) over the dataset . On average, Hap 1 was more common in Fst values proved to be high and significant between the sources NSH_VID and BH_PEE  and between both of these sources and EW . Except between the NSH_RHI and NSH ELB , within the NSH distribution, no significant differentiation among reintroduced populations could be observed. This was not the case within the BH range where the BH_TRA was the only reintroduced population not significantly differentiated from the putative source population BH_PEE . We also found that BH_SCH, BH_LAC, and BH_NOK were strongly differentiated from their putative source BH_PEE, but not from NSH or EW populations, respectively . Investigating possible demographic events failed to detect any consistent sign of recent bottlenecks or expansions in any of the populations (Table S4).Fst = 0.15, P < 0.001; Fsts > 0.10 and P < 0.001 for all pairwise comparisons of NSH_VID and BH_PEE versus EW populations) , BH_SCH versus BH_LAC  and BH_TRA versus BH_PEE . Interestingly, the latter comparison is the only one showing no significant differentiation from the source population within a given distribution range, and all reintroduced populations showed intermediate differentiation between the putative source and other source populations . The general pattern of population clustering within the distribution ranges of NSH and BH was confirmed by the neighbor-joining tree  Table. ContempK = 3 as most likely number of clusters represented by the sampled individuals after the exclusion of vendace . The analysis confirmed the dominance of native NSH genotypes across the historic NSH range, but also revealed the additional influence of both EW and BH genotypes in reintroduced populations , suggesting that admixture occurred before 2004 or elsewhere than in the Treene River. Within the NSH_RHI system, no spatial structuring was detected .Overall, the STRUCTURE analysis revealed ions Fig.. No tempSTRUCTURE unravelled a more complex and heterogeneous situation across the historic BH range of distribution Fig.: While ta priori expected genotype   and of pure non-native individuals  and hybrids (P < 0.001) in the NSH distribution range compared with the BH range. Within the distribution ranges, for NSH, admixture proportions differed significantly and ranged from 11% in the NSH_TRE to 36% in the NSH_ELB . For the BH, the admixture proportion was 27% in the BH_TRA, but significantly higher  in the BH_LAC and BH_SCH . In addition, hybrids and non-native genotypes dominated in the BH_LAC and BH_SCH, and non-native EW genotype in the BH_NOK population  Fig., Table4.ely) Fig., Table4,The number of alleles in linkage disequilibrium in the reintroduced populations of both NSH and BH was comparable with that in the pure source populations, with the exception of elevated numbers in the NSH_ELB, BH_LAC, and BH_NOK Table.r = −0.28, P = 0.585) and non-native houting genotype  in reintroduced populations across the NSH and BH distribution ranges were not correlated with geographic distance from the canal. Furthermore, the most predominant genotype in the Kiel Canal, contrary to expectation, was the EW genotype and not a mix of NSH and BH  and BH  distribution ranges, IBD between reintroduced populations could not explain the observed genetic structuring.The proportions of non-native genotype  .The mean allelic richness 24) Table. The patr = 0.947, df = 6, P = 0.001), only the BH populations , or only BH without the highly admixed BH_NOK population  were included in the analysis  the homogenous structure of the source populations (with some exceptions for EW) revealed by STRUCTURE. Our results thus clearly support that for the ancestral/source populations of NSH and BH, the conservation classification as separate ESUs  confirmed that hybridizations were not due to historic admixture in the source populations, but must have occurred after the onset of reintroductions to Germany ∼20–25 years ago. The two possible explanations were anthropogenic translocations via erroneous stocking, or migrations via the potential invasion corridor Kiel Canal (Gollasch and Rosenthal Interviews with resource managers revealed that adults, fertilized eggs, and fry of NSH, BH, and EW have been periodically kept in the same facilities. Although official records did not indicate stocking of the historic houting ranges with non-native ESUs, stocking errors therefore appear possible. The clear range-specific differences observed here may relate to the fact that the same person has been responsible for NSH reintroductions from the onset Jäger , whereassensu Orr (The intensity of stocking with non-native genotypes strongly influences the outcome of hybridizations (Salminen et al. ensu Orr . Yet, thFrom the evolutionary perspective, following Hewitt , the newIn contrast to declines in genetic diversity frequently associated with stocking due to bottlenecks Pister , diversiThe confirmation of the previous classification of NSH and BH as separate ESUs (Hansen et al.


D value for HLA-DRB1 exon2 and dN/dS analysis showed that the HLA-DRB1 genes in both Mongolian populations were under balancing selection. However, the sites under natural selection changed. We proposed that the dramatically change of HLA frequencies in southern Mongolian was caused by a combination of inter-population gene flow and natural selection. Certain diseases specific to the south of China, such as malaria, may be the driving force behind the enhanced DRB1*12:02:01 frequency.Pathogen-driven balancing selection determines the richness of human leukocyte antigen (HLA) alleles. Changes in the pathogen spectrum may cause corresponding changes in HLA loci. Approximately 700 years ago, a Mongolian population moved from the north of China to the Yunnan region in the south of China. The pathogen spectrum in the south of China differs from that in the north. In this study, changes in the HLA genes in the Yunnan Mongolian population, as well as the underlying mechanism, were investigated. A sequence-based typing method (SBT) was used to genotype HLA-DRB1 in 470 individuals from two Mongolian populations and another five ethnic groups. Meanwhile, 10 autosomal short tandem repeats (STRs) were genotyped to assess the influence of genetic background on HLA-DRB1 frequencies. The frequencies of certain alleles changed significantly in the Mongolian population that migrated to Yunnan. For example, DRB1*12:02:01 increased from 6.1% to 35.4%. STR analysis excluded the possibility of a recent bottleneck and indicated that 50% of the genetic consistency between northern and southern Mongolians; Tajima's  The LRT statistic for the comparison of M8 and M7 is 2Δl = 47.2, which is also much greater than the critical values from a χ2 distribution with d.f. = 2. This result showed that some codons in HLA-DRB1 exon 2 were all affected by selection in the seven populations.In the PAML program, the two-pair mutation models were used for the analysis. One pairp0) of the sites were under strong purifying selection (ω0 was approximately 0.02), whereas approximately12% of sites were under strong positive selection (ω was 4.1–4.4). These results demonstrate that diversified selection has acted on HLA-DRB1 in the two populations, and the percentage of sites that were under positive selection was not significantly different between the two populations. In the other five populations, the proportions of sites under positive selection were approximately2.4% to 11.8%.In Mongolian_YN and Mongolian_IM, the proportions of sites under selective pressure were similar. In the two populations, approximately 70%  in PAML nine α76., indicatFST between the populations was calculated based on the 10 STRs and the HLA-DRB1 gene frequencies in the seven populations; the results are shown in FST values are shown in the lower triangle of the tables, and P values are shown in the upper triangle. All the pairwise FST values based on STR data are significantly different (α = 0.002 after Bonferroni correction), meaning that all paired populations have different genetic backgrounds. The FST between Mongolian_YN and Mongolian_IM was 0.015, which is smaller than the FST between Mongolian_YN and the other southern populations (0.018–0.026). Therefore, in terms of genetic background, Mongolian_YN and Mongolian_IM were more similar to each other than Mongolian_YN was to the other southern populations of China.The genetic background of a population can help determine which action, selection or demographic events, has changed the gene frequency in the population. Therefore, the pairwise FST based on HLA-DRB1 was found between Mongolian_YN and Wa (0.011); this value is smaller than the FST between Mongolian_YN and the other populations (0.033–0.074). On the other hand, the FST between Mongolian_YN and Mongolian_IM was 0.054. All the paired FST values were significantly different (α = 0.002 after Bonferroni correction), except for the Mongolian_IM-Han FST  and the Mongolian_YN-Wa FST . These results suggest that the HLA-DRB1 frequencies are similar between Mongolian_YN and Wa, but different between Mongolian_YN and Mongolian_IM.However, the lowest FST values. An additional 30% of the genetic components of Mongolian_YN . The DRB1*12:02:01 allele was common in the southern populations, which contained large proportions of the Wa, Hani and Dai people, with a gene frequency in the range of 16.9–44.2%. However, in Mongolian_YN (people immigrated to Yunnan from Mongolia), the gene frequencies of DRB1*07:01:01 and DRB1*15:01:01 decreased by approximately half. The frequency of DRB1*07:01:01 decreased from 13.3% to 5.8% , whereas that of DRB1*15:01:01 decreased from 15.3% to 7.3% .HLA-DRB1 in Mongolian_YN was subjected to the influence of selection following the migration into Yunnan, and the details of this change merit study. Thus, the composition ratios of the HLA-DRB1 alleles of the three populations were compared with each other using the R × C chi-squared test. As shown in FST analysis. The FST value between these two populations is not significant . As seen in 2 = 17.8, P<0.0001).The result of the comparison between Mongolian_YN and the Wa people was consistent with the results of the FST analysis and R × C chi-squared test. The FST based on HLA-DRB1 between the Mongolian_YN and Wa populations was not statistically significant , whereas the FST based on HLA-DRB1 between Mongolian_YN and Mongolian_IM was significant . The R × C chi-squared test also showed a significant difference between the HLA frequencies in Mongolian_YN and Mongolian_IM , whereas the difference is not significant between Mongolian_YN and Wa .Previous studies have reported on HLA-DRB1 genotype in certain populations, such as Wa, Dai and Yao, using PCR-SSOP methods. SimilarWe are very interested in understanding which force was responsible for changing the HLA-DRB1 frequencies so dramatically. Both demographic events and natural selection can alter the gene frequencies in a population. First, the demographic events that the Mongolian_YN experienced were investigated. Genetic drift and gene flow are two important demographic forces that can remodel gene frequencies. Because population expansion and reduction were excluded by a bottleneck analysis based on neutral STRs, genetic drift appears not to be the force that we are looking for. Thus, 10 neutral STRs were chosen to analyze the genetic backgrounds of the seven populations to identify potential gene flow. Using the STRUCTURE program, we found that Mongolian_YN and Mongolian_IM shared 50% genetic similarity. However, approximately 30% of the genetic component of Mongolian_YN might originate from southern Tibeto-Burman speaking people such as the Hani. Thus, gene flow from the local population of southern China has clearly had an impact on the genetic background of Mongolian_YN. However, certain aspects of this gene frequency are difficult to account for by gene flow alone. One factor is the high frequency of DRB1*12:02:01 in Mongolian_YN. DRB1*12:02:01 is a common variant in the southern populations of China. Yet the frequencies of DRB1*12:02:01 in the populations with the potential to exchange genes with Mongolian_YN were lower than that in Mongolian_YN. The frequency of DRB1*12:02:01 is 20.9% in the Hani people, and in other Tibeto-Burman speakers, the DRB1*12:02:01 frequencies are approximately 30%. Because D, was calculated. In the seven populations, all values for Tajima's D based on the sequences of HLA-DRB1 exon 2were larger than 0. Excluding the impact of the bottleneck effect caused by demographic events, we believe that exon 2 of HLA-DRB1 in all seven populations was affected by balancing selection. There were no significant differences in Tajima's D between the northern and southern populations, and the Tajima's D for Mongolian_YN (2.96) and Tajima's D for Mongolian_IM (2.93) were also similar. Another analysis was performed based on dN/dS using the PAML program. The results indicated that approximately 12% of sites were subjected to positive selection in both Mongolian populations, and there was no significant difference between the two populations. From the above results, it appears that although Mongolian_YN was challenged with a new pathogen spectrum after immigration, the types of selection exerted on HLA-DRB1 and the number of sites that enabled the selective force in the population did not change.Thus, we can conclude that natural selection took place in Mongolian_YN after the Mongols came to southern China roughly 700 years ago. Most researchers believe that balancing selection maintains a high degree of diversity in MHC, whereas pathogenic drive is one of the major factors responsible for maintaining this selective pressure., 4 Giventh amino acid was positively selected in the Mongolian_YN but not in the Mongolian_IM. On the other hand, the 74th amino acid was positively selected in the Mongolian_IM but not in the Mongolian_YN. Furthermore, the 57th amino acid of DRB1*12:02:01 is valine, whereas it is aspartic acid, serineor alanine in the proteins which other HLA-DRB1 alleles code. The positive selection maybe impact on these two sites and push DRB1*12:02:01 to a higher frequency.However, these findings do not necessarily mean that HLA-DRB1 is under similar selective pressures in both the northern and the southern Mongolians and that there is no significant difference of pathogen spectrum between northern and southern China. If Mongolian_YN underwent a similar balancing selection as Mongolian_IM, it is hard to explain why the frequency spectrum of Mongolian_YN did not show an excess of intermediate-frequency alleles, as is the case in Mongolian_IM. Furthermore, the dramatic increase in the DRB1*12:02:01 frequency in Mongolian_YN is also hard to explain with respect to similar balancing selection in the south and north of China. Therefore, we investigated the sites under positive selection using EBE analysis in PAML software. We found that the sites under positive selection were different in the two Mongolian populations. The 57D or dN/dS) to test natural selection, we obtain information on selection over a long period of time. Southern Mongolians only separated from northern Mongolian less than 800 years ago. DNA mutations have not had enough time to occur and become fixed in these populations. Therefore, when we used analyses based on DNA mutations, we found that northern and southern Mongolians appear to be under similar selective pressure and that the proportions of sites under selection are similar. In fact, in response to changes in the pathogens spectrum over such a short time, natural selection has already been acting on the different sites of HLA-DRB1 exon 2. This has led to increased frequency for some allele that already have existed in the population, such as DRB1*12:02:01. Therefore, we believed that positive selection impacted on some sites of HLA-DRB1 and altered the gene frequency in Mongolian_YN. The differences in HLA-DRB1 allele frequencies between Mongolian populations are adaptive.Based on these data, we propose the following model. HLA-DRB1 is known to be under balancing selection. The balancing selection is a heterogeneity action in a period time. In a way, this selection encompasses a number of types of selection. MHC genePlasmodium falciparum.[Comparing the composition of the pathogen spectrum of southern and northern China, the prevalence of malaria differed significantly. Malaria was very rare in the north, with high rates in the south, particularly to the south of 25°Nlatitude. The frequency of malarial infection was approximately 0.1% in the past 50 years, and the high incidence of the disease was caused by lciparum. Mutationlciparum. ThereforPlasmodium falciparum asexual-stage apical membrane antigen 1 (AMA1).[Individuals carrying the DRB1*12:01 allele have been reported to produce a high level of immune antibodies against 1 (AMA1). However,S1 FigA.) Mean likelihood L(K) value and variance per K value were calculated from five runs of STRUCTURE. When K = 5, the likelihood value was highest. B.) The ΔK plot, which was suggested by Evanno et al. (2005), was used to detecting the number of K groups that best fit the data. When K = 5, ΔK was highest.(TIF)Click here for additional data file.S2 FigTo address the possibility that the allele distributions of HLA-DRB1 in Mongolian_YN may be only due to gene flow, three scenarios were constructed. Scenario 1: after the Mongolian people came into Yunnan, gene flow with other populations did not occur. Scenario 2: gene flow occurred between Mongolian_YN and Hani after Mongolian southern migration. Scenario 3: gene flow occurred between Mongolian_YN and Wa after Mongolian southern migration. Details of the parameters used in each scenario are provided in the Materials and Methods section.(TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S4 Table(DOC)Click here for additional data file.S5 Table(DOC)Click here for additional data file.


Xiphias gladius L.) revealed significant differentiation among Mediterranean, North Atlantic and South Atlantic populations using both mitochondrial and nuclear DNA data. However, limitations in geographic sampling coverage, and the use of single loci, precluded an accurate placement of boundaries and of estimates of admixture. In this study, we present multilocus analyses of 26 single nucleotide polymorphisms (SNPs) within 10 nuclear genes to estimate population differentiation and admixture based on the characterization of 774 individuals representing North Atlantic, South Atlantic, and Mediterranean swordfish populations. Pairwise FST values, AMOVA, PCoA, and Bayesian individual assignments support the differentiation of swordfish inhabiting these three basins, but not the current placement of the boundaries that separate them. Specifically, the range of the South Atlantic population extends beyond 5°N management boundary to 20°N-25°N from 45°W. Likewise the Mediterranean population extends beyond the current management boundary at the Strait of Gibraltar to approximately 10°W. Further, admixture zones, characterized by asymmetric contributions of adjacent populations within samples, are confined to the Northeast Atlantic. While South Atlantic and Mediterranean migrants were identified within these Northeast Atlantic admixture zones no North Atlantic migrants were identified respectively in these two neighboring basins. Owing to both, the characterization of larger number of loci and a more ample spatial sampling coverage, it was possible to provide a finer resolution of the boundaries separating Atlantic swordfish populations than previous studies. Finally, the patterns of population structure and admixture are discussed in the light of the reproductive biology, the known patterns of dispersal, and oceanographic features that may act as barriers to gene flow to Atlantic swordfish.Previous genetic studies of Atlantic swordfish ( Katsuwonus pelamis) displaying no genetic differentiation among-oceans [Acanthocybium solandri) showing only incipient levels inter-oceanic differentiation [Isurus oxyrinchus) [Makaira nigricans) and sailfish (Istiophorus platypterus) [Thunnus obesus) [Thunnus albacares) and skipjack tuna [Xiphias gladius L.) shows significant genetic differentiation within the Atlantic Ocean [The epipelagic realm of the world’s oceans is a relatively homogeneous and contiguous environment characterized by the near absence of physical barriers. These features when coupled with the high dispersal behavior and demographic characteristics of many marine species result in high levels of gene flow . Specifig-oceans , and othntiation . Signifintiation , Atlantintiation , 9, albantiation , 11, andntiation  and sworntiation . Similarntiation , white mntiation . Within rinchus) , blue maypterus) , bigeye  obesus) , and in ack tuna . By contic Ocean , 17, 18 ic Ocean .Swordfish is a large epipelagic monotypic cosmopolitan species capable of long distance movements, as documented using both conventional tags  and pop-In recent years there has been an increase in the adoption of single nucleotide polymorphisms (SNPs) as an alternative to microsatellite data in Bayesian analyses of population admixture. In addition to a comparable differentiation power, SNPs have lower associated genotyping errors, simpler mutation models, higher amenability to automation and high throughput technologies, and increased amplification success with low-quality and ancient DNA as compared to microsatellites –34. FurtA total of 774 swordfish specimens from 18 localities in the Atlantic and western Mediterranean were collected from 1991–2006 . Muscle, to Dr. J.R. Rooker. The management boundaries at 5°N, separating the North Atlantic stock from the South Atlantic stock, and at the Strait of Gibraltar, separating the North Atlantic stock from the Mediterranean stock, were initially used to assign localities to the corresponding three management units or stocks currently recognized by ICCAT .CaM); Golgi pH regulator (GpHr); lactose dehydrogenase A (ldhA); myosin light chain (Mlc2); and signal recognition particle 54 (SRP54). Details on loci and corresponding SNPs selection, the minimization of ascertainment bias as well as the criteria to design primers and probes that target these, are given in Smith et al. [aM, and SRP54 and unlabeled probe high-resolution melting analysis (UP-HRMA) of ANT, GpHR, ldhA, and Mlc2 are described in Smith et al. [Total genomic DNA was isolated from tissue with a modified TENS and Proteinase K (20 mg/μL) digestion followed by ethanol precipitation without organic extraction . Ten nuch et al. . Brieflyh et al.  for detah et al. ). Procedh et al. .Two additional loci, AldB and Act2α, were scored as size polymorphisms as follows. A simple sequence repeat (SSR) identified in AldB  was amplLocus Act2α contains an SNP that corresponds to a restriction site of endonuclease Hpy8I and was amplified using primers; 5’-GTCACCGGAGTCCAGGACG-3’ and 5’-ATCTGGCACCACACCTTCTACAA-3’ (JX042448) using the same chemistry and thermocycling profile used in AldB amplification. Prior to digestion, the quality and quantity of PCR products were visualized in 1% Tris-acetate (TA) agarose gels via gel electrophoresis prior to digests. Restriction digests were conducted in 10 μL volumes containing 2–4 μL PCR product, 1X Buffer TANGO (Fermentas), and 0.4 U of Hpy8I (Fermentas), that were incubated for 16 hours at 37°C, followed by thermal inactivation at 80°C for 5 min. Restriction Fragment Length Polymorphisms (RFLPs) were separated in 2% TA agarose gels run at 100V for 30 min, and were visualized and captured on a Gel Doc XR (Bio-Rad) UV transilluminator using the Quality One v.4.6 (Bio-Rad) software.Ho) and expected (He) heterozygosities under Hardy-Weinberg equilibrium (HWE), and inbreeding coefficients (FIS) were calculated using GENALEX version 6.41 [FST-outlier detection method, in which observed locus FST values are compared to calculated global FST values expected under neutrality using coalescent simulations [FST, the first simulation used the ‘neutral’ mean FST option with a 0.995 confidence interval, a false discovery rate set at 0.1, and by pooling the sampling localities into the corresponding three populations (K = 3) identified by Bayesian clustering analyses (see below) as follows, North Atlantic , South Atlantic (12–18), and Mediterranean (8–9). The assignment power of each locus was ranked a posteriori to Bayesian analysis using the critical population method in WHICHLOCI v1.0 [The number of alleles, observed . Slatkin’s linearized FST [A hierarchical analysis of molecular variance (AMOVA) was implST tests . Initialzed FST  was alsozed FST , based ozed FST .FST < 0.20) [K) was estimated using an ad hoc approach [K)) and the ΔK approach of Evanno et al. [K value (1–10) were performed using 100,000 Markov chain Monte Carlo (MCMC) iterations with a burn-in period of 100,000. Results from STRUCTURE were compared with the results from GENELAND [K value (1–8). After estimating K, 25 independent runs with the optimal K value were run in GENELAND, 50 independent runs in STRUCTURE, and the optimal alignment of replicates were completed using CLUMPP version 1.1.12 [Genetic population structure and patterns of inter-population gene flow were assessed by Bayesian inference implemented in STRUCTURE version 2.3 . Previou < 0.20) . Accordi < 0.20) . The numapproach  by obtaio et al.  using STo et al. . Twenty GENELAND , which iGENELAND , 23. Corn 1.1.12 .Ho) and expected (He) heterozygosities, and inbreeding coefficients (FIS) are given in P < 0.05) after Bonferroni correction. Genotypic linkage was not significant among the 44 possible pairs of nuclear loci across all samples with (P < 0.01) and without (P < 0.05) Bonferroni correction. Likewise exact tests for genotypic linkage disequilibrium within each sample were also non-significant after Bonferroni correction (P < 0.05) (not shown). Initial FST outlier analysis of the ten loci of all individuals from the 18 localities identified CaM as a potentially under selection (P < 0.05) (P < 0.05). This procedure also yielded higher FST values for all loci as well as higher He values for Act2α, AldB, ATPsβ, CaM, and Mlc2.  . Howevernd Mlc2. . The powFST values among the 18 populations ranged considerably (<0.001 to 0.121) (FST >0.081 – 0.121). Several sampling localities identified as admixture zones  were excluded when estimating gene flow among North Atlantic (1–5), Mediterranean (8–9) and South Atlantic (12–18) populations. The lowest level of gene flow of about 5 migrants per generation (Nm) was estimated between Mediterranean and South Atlantic populations, compared to about 9 migrants per generation between North Atlantic and South Atlantic swordfish  and to a lesser extent in the central North Atlantic (6) locality.Multilocus pairwise o 0.121) . The comwordfish . SlatkinT values  highlighFCT) differentiation was highly significant (P<0.01). However, the proportions of variation explained by among-groups were slightly larger in two-group AMOVAs, with 6.63% with all localities and 7.05%, excluding admixture areas , than in three-group arrangements that yielded 5.17% and 6.45% with and without admixture areas, respectively. In addition, the proportion of variance explained by differences among populations within groups (FSC) was four to ten times larger in the two-groups arrangements than in the three-groups arrangements, with the lowest proportion of variation (0.19%) for the three-groups arrangement without admixture areas .In all alternative hierarchical arrangements tested  with AMOVA  the majoThe PCoA of the standardized covariance of pairwise population genetic distances provide concordant evidence of locality assignment to a minimum of three separate swordfish populations . The firK = 1–10) revealed that the mean ln P(D) increased from K = 1–3 and then sharply decreased when K ≥ 4  and K = 3 (ΔK3 = 33.6548), followed by very low ΔK (< 5.0) values  congruent with the genetic heterogeneity between the North and South Atlantic populations of swordfish. While Evanno’s ΔK method seeks to detect the uppermost hierarchical level of population structure the method is less reliable at lower levels of genetic differentiation and may underestimate K [K using the ad hoc evaluation of mean posterior probabilities from multiple analyses of K [K approach [Evaluation of the mean posterior probabilities from multiple STRUCTURE analyses  was also identified as an area of mixing with individuals of North Atlantic and South Atlantic . Further, this ghost cluster was unstable, as it’s placing or presence differed among replicate runs. The existence of ‘ghost’ clusters has been reported as an artifact of GENELAND which tends to overestimate K in a comparison to other Bayesian genetic clustering algorithms [K = 3) identified by STRUCTURE. Localities 1–5 and part of 7 comprised the North Atlantic , the associated error in the calculation of global neutral FST could be large. Accordingly, future studies utilizing genomic technologies will certainly yield greater numbers of loci to conduct more conclusive outlier tests.Genetic population structure studies using nuclear DNA have traditionally excluded adaptive markers to avoid introducing bias to estimates of migration rates and effective population sizes . In thisCaM as the most informative locus for distinguishing North and South Atlantic swordfish populations, this locus alone fails to differentiate between North Atlantic and Mediterranean swordfish populations. Further, STRUCTURE results without CaM , precluding individual cluster assignment. By contrast, Kasapidis et al. [CaM, is strong adaptive selection. Such explanation, however, fails to explain the concordance between mtDNA and nuclear SNPs data , includThunnus obesus)[T. thynnus) and yellowfin tuna  [Carcharhinus plumbeus) [Isurus oxyrinchus) [Makaira nigricans) mitochondrial cytochrome b gene sequences [FST values among marker classes and among loci within a marker class were consistent with the same population level Nem, given differences in both mode of inheritance and genomic sampling variance among estimates. Further, one microsatellite locus (Mn8) yielded a pattern concordant with mtDNA regarding both the strong inter-oceanic differentiation, and inferred unidirectional gene flow of Indo-Pacific blue marlin into the South Atlantic. In swordfish, the similarity in levels of differentiation between North and South Atlantic populations using mtDNA , is not consistent with gender-biased gene flow. Considering that both swordfish and blue marlin females attain larger sizes than males and are more abundant at higher latitudes [The shallow levels of differentiation between North and South Atlantic using microsatellites could be due to allele size homoplasy in combination with large effective sizes  of swords obesus), bluefinbacares) , sandbarlumbeus) , and shorinchus) , withoutequences  and RFLPequences  revealedequences . Such diequences , indicat 0.0244; ) and mulatitudes , 91, 92 Previous studies of Atlantic swordfish population structure only broadly identified potential areas of admixture between the North Atlantic and Mediterranean populations , 31 and CaM. Our analyses with PCoA . This result supports the hypothesis that samples from feeding grounds in both the NW Atlantic and the South Atlantic Ocean  may be more prone to population admixture as suggested by Alvarado Bremer et al. [Strong natal fidelity, with admixture primarily confined to feeding grounds, has been proposed as an explanation for the unparalleled intra-oceanic differentiation separating North Atlantic and South Atlantic swordfish populations . By contr et al. . Similarr et al.  and nDNAr et al. . Such coCaM data revealed since swordfish are deep divers. Nutrient upwelling, caused by prevailing winds off the western coast of Africa, creates a large hypoxic zone in the eastern tropical Atlantic [While fidelity to spawning and feeding grounds may explain low levels of gene flow between North and South Atlantic swordfish, it doesn’t fully explain why admixture only seems to occur in the Northeast Atlantic waters east of about 50°W and south of 30°N. Chow et al.  suggesteAtlantic  that appAtlantic  and sworAtlantic , and thuOther oceanographic features are known to acts as barriers to dispersal in proximity to coastal areas. For instance while recognizing that the Amazon discharge would not be expected to affect the movements of adult highly migratory pelagic fish, Chow et al.  hypothesThe findings of this study can be summarized as follows: first, significant differentiation among Mediterranean, North Atlantic, and South Atlantic swordfish populations is confirmed with very low rates of gene flow, particularly when compared to other highly migratory fishes. Second, population admixture between the North Atlantic and the South Atlantic population, and to a much lesser extent with the Mediterranean, is primarily confined to the Northeast Atlantic and in both cases appears to be unidirectional, from the Mediterranean to the NE Atlantic and from the South Atlantic to the NE Atlantic. Third, the genetic partitioning of these populations differs from currently recognized management boundaries. Fourth, and lastly, while the oceanographic and/or behavioral barriers to gene flow are not yet identified, future tagging, genetic, oceanographic, or physiological studies should focus efforts to areas of admixture in the Northeast Atlantic and areas where individuals were assigned with high posterior probabilities, i.e. North and South Atlantic spawning and feeding grounds. Additionally, future genomic studies utilizing large numbers of SNPs may provide insight into whether adaptive genetic variation is responsible for the fine-scale population structure here reported.S1 FigThe estimation of the number of clusters (K) in the STRUCTURE v2.3  analysis(TIF)Click here for additional data file.S2 FigThe STRUCTURE v2.3 individual assignments of 774 swordfish from 18 localities using the no admixture and correlated allele model and the location prior with A) only the CaM locus, B) all loci except the CaM locus, and C) all loci including the CaM locus.(TIF)Click here for additional data file.S3 Fig-1 are from the World Ocean Atlas 2009 1. The OMZ depicts a similar boundary as the posterior probability of membership contour maps in 1Garcia HE, Locarnini RA, Boyer TP, Antonov JI, Baranova MM, et al., editors (2010) World Ocean Atlas 2009, volume 3: dissolved oxygen, apparent oxygen utilization, and oxygen saturation. Washington, D.C. 344 p.The oxygen minimum zone (OMZ) in the South Atlantic swordfish feeding grounds at 100 m depth. The annual mean dissolved oxygen (DO) at 100 m depths in mL L(TIF)Click here for additional data file.S1 TableThe allele frequency distribution within the eighteen sampled localities for each of the ten nuclear loci.(XLS)Click here for additional data file.S2 TableA consensus power of assignment for each locus using WHICHLOCI v.1.0 between North Atlantic (n = 207) and Mediterranean (n = 142) swordfish inferred in STRUCTURE v2.3.(XLS)Click here for additional data file.S3 TableA consensus critical population (South Atlantic) power of assignment for each locus using WHICHLOCI v.1.0 between North Atlantic (n = 207) and South Atlantic (n = 297) swordfish inferred in STRUCTURE v2.3.(XLS)Click here for additional data file.S4 TableThe average population posterior probability membership (Q) of 18 localities in the Atlantic and Mediterranean inferred in STRUCTURE v2.3.(XLS)Click here for additional data file.S5 TableThe alleles for the ten loci and all 774 swordfish analyzed in this study. Allelic numbering corresponds to reported GenBank alleles for all loci except ALDB. Allelic numbering for ALDB corresponds to STR amplicon size scoring.(XLSX)Click here for additional data file.


Anopheles pseudopunctipennis is an important malaria vector in the Neotropical region and the only species involved in Plasmodium transmission in the Andean foothills. Its wide geographical distribution in America, high preference for biting humans and capacity to rest inside dwellings after feeding, are attributes contributing to its vector status. Previous reports have tried to elucidate its taxonomic status, distinguishing populations from North, Central and South America. In the present study we used a mitochondrial marker to examine the demographic history of An. pseudopunctipennis in northwestern Argentina.cytochrome oxidase I (COI) gene was sequenced and haplotype relationships were analyzed by a statistical parsimony network and a Neighbor-Joining (NJ) tree. Genetic differentiation was estimated with FST. Historical demographic processes were evaluated using diversity measures, neutrality tests and mismatch distribution.Twelve localities were selected across 550 km of the distribution of this species in Argentina, including two near the Bolivian border and several in South Tucumán, for sampling. A fragment of the DT and Fu & Li’s F and D neutrality tests and mismatch distribution supported a scenario of Holocene demographic expansion.Forty-one haplotypes were identified, of which haplotype A was the most common and widely distributed. Neither the network nor the NJ tree showed any geographic differentiation between northern and southern populations. Haplotype diversities, Tajima’s An. pseudopunctipennis has undergone a single colonization process, and the ancestral haplotype is shared by specimens from all localities, indicating mitochondrial gene flow. Genetic differentiation was minimal, observed only between one northern and one southern locality. The estimated time of the population expansion of this species was during the Holocene. These data suggest that regional vector control measures would be equally effective in both northern and southern localities sampled, but also that insecticide resistant genes may spread rapidly within this region.The demographic pattern suggests that  Malaria affects millions of people globally every year. In 2010, 216 million malaria cases were registered, 81% of them in Africa.Anopheles (Anopheles) pseudopunctipennis Theobald is involved in Andean foothills and coastal area malaria transmission[Plasmodium transmission. For instance, the appearance of An. pseudopunctipennis on the dry coast of Peru was directly related to land use change from desert to irrigation for sugar cane and rice, creating suitable new species habitat[In the Americas, different malaria vectors are associated with distinctive eco-regions. Specifismission. The eco habitat.An. pseudopunctipennis has an extensive distribution from the USA to northern Chile and northwestern Argentina, including the Caribbean Lesser Antilles, Trinidad and Tobago and Hispaniola Island[et al.[et al.[et al.[An. pseudopunctipennis populations: one from the southern United States throughout Mexico and Guatemala; another extending from South America through Central America and Belize, both sharing an area of overlap in eastern Guatemala and southern Belize; and a third including only populations from the island of Grenada. Currently, An. pseudopunctipennis is considered to be a complex of at least 2 species[a Island–8. Sincea Island until it[et al., several[et al.–15. Estr[et al., 14 dete species. HoweverAn. pseudopunctipennis. Until the 40’s, the northern area of the yungas was a preserved rainforest, whereas in the southern area cultivation included sugar cane, citrus and soybean crops. However, after the 40’s, the dynamics in the yungas were altered and the northern area began to exhibit major landscape modifications with severe forest exploitation, recurrent occurrence of fires and the increased pressure of farming[th century, could explain the current distribution and abundance of An. pseudopunctipennis mosquitoes and the regional malaria endemicity[In Argentina, the historically wide geographical distribution of malaria appears to be reduced to the northwest, where it is still an important endemic parasitic disease–18. Land farming that reqcytochrome oxidase I gene (COI) for population demographic analyses of Anophelinae species has been well-documented, i.e. Fairley et al.[Anopheles (Nyssorhynchus) aquasalis Curry and Anopheles (Anopheles) punctipennis Say, and Mirabello & Conn[Anopheles (Nyssorhynchus) darlingi Root, among others. The effects of geographical barriers and latitude can show not only differentiation among populations but also possible barriers to gene flow[An. darlingi from Central and South America appear to be separate from Amazonia and southern Brazil specimens, with the southern ones considered more ancestral[The use of the y et al., 21 in Ao & Conn and Pedro & Conn for Anopncestral, and barncestral.An. pseudopunctipennis from two areas of the yungas ecoregion of Argentina by COI to elucidate the history of this species, as a first attempt to compare populations throughout its range in the Americas.In the present study, we analyzed the demographic history and population structure of An. pseudopunctipennis were collected from twelve localities in two areas, northern and southern yungas, situated in Salta, Jujuy and Tucumán provinces, northwestern Argentina  and Phyllostylon rhamnoides (Poiss.) Taubert), respectively, still remain. The southern area, namely the “tipa and pacará” forest, Tipuana tipu (Benth.) Kuntze and Enterolobium contortisiliquum (Vell.) Morong, respectively, has been modified by intensive sugar cane, soybean and citrus plantations, displacing more traditional and sustainable land use[One hundred and sixty-five adult female  Figure . These l authors–26. The  authors, 28. Theland use, 28.FiguCollections were made by CDC light traps baited with carbon dioxide in 2005, 2007 and 2008 from 16:00 h-12:00 h in the yungas covering a latitudinal transect of ~550 km. Adult females were identified using the taxonomic key of Wilkerson & Strickman and wereCOI gene was amplified by polymerase chain reaction (PCR) using the UEA3 and UEA10 primer pairs[DNA extractions were carried out from whole individual mosquitoes following the standard DNeasy Blood & Tissue Handbook protocol . A 1200 bp fragment of er pairs. Each PCSequences for each individual sample were automatically aligned using Sequencher 3.0  and corrected manually. The contig sequences were grouped together and aligned using MEGA version 3.1. In addiFew specimens were collected from the three most southern localities , sequence (K) and nucleotide diversity (π) indices using Arlequin 3.11[Statistical parsimony networks were constructed to assess relatedness among the software with a 9uin 3.11.D[D and F tests proposed by Fu & Li[FS test[R2 statistic[Statistical neutrality tests were performed to detect departures of DNA sequence variability from the expectations of the neutral theory of evolution. Tajima’D is based Fu & Li require [FS test is basedtatistic is basedtatistic was usedr) statistic was calculated and its significance was assessed using 10,000 replicates[A mismatch analysis for each partition (north–south) and overall was carried out using Arlequin 3.11. The analysis compares the frequency distribution of pairwise differences between haplotypes with that expected under a model of population expansion–42. To qplicates.An. pseudopunctipennis was estimated by FST using Arlequin 3.11[FST values were used as distance measures to create a NJ tree by DNAsp 4.50.3[GST values were calculated to estimate population differentiation based on differences in allele frequencies and Nei’s Nm, the mean per generation estimate of the absolute number of migrants exchanged among populations[Genetic differentiation between populations of uin 3.11. The FSTp 4.50.3. Nei’s Gulations. The populations. The hypulations. In addiulations.COI gene, from nucleotides 615–1269 was obtained. No stop codons were detected, indicating the mitochondrial origin of the DNA. All COI sequences are available at GenBank under accession numbers KC110039-KC110079. Forty-one haplotypes were identified, seven of which  were shared between all northern and southern localities, accounting for 76.9% of the sequences. The dominant haplotype, A, represented 61.21% of the specimens, and was found in all localities  between the FY (Jujuy Province) and EC (Tucumán Province) localities, which are 66 km apart.Nucleotide and haplotype diversity values are depicted in Table GST and Nm values detected low pairwise genetic differentiation and moderate gene flow between the north and south. The highest value (GST = 0.14210) was between Potrero Las Tablas (PT) and Vipos (VP), both in the south, separated by 50.5 km. Estimates of gene flow (Nm) varied widely between populations, ranging from 1.51 to 614.50; the lowest was between Potrero Las Tablas (PT) and Vipos (VP) (Nm ~ 1.51). Negative values were related to small population sizes and lack of gene flow[Nei’s ene flow, 47. In An. pseudopunctipennis in the yungas. Variation among the northern and southern groups was almost negligible (ΦSC = -0.00429). The populations differed significantly from each other (ΦCT = 0.442) and were responsible for 97.4% of the variance, although the value was non-significant. The remaining difference (3.54%) was explained by non-significant differentiation of haplotypes within populations (ΦST = 0.016). SAMOVA did not show any geographical limit within populations, and k = 3 was the partition providing the highest significant FCT (0.27), although it did not separate the northern-southern populations. Instead, TUCSUR (southern) and RF (northern) group separately from the other localities (grouping together). The Mantel test was not significant  showing that there was no geographic association with the genetic differentiation.The statistical parsimony network revealed a star-like topology , which was consistent with a model of sudden expansion for each partition.The mismatch distribution for the complete data set is presented in Figure t = τ/2 μ was used, where μ is the mutation rate per site per generation[Drosophila was used: 10-8/site/year[To calculate the time of population expansion, neration, 40. Theite/year and 10 gite/year. The anaAnopheles pseudopunctipennis in the yungas ecoregion of Argentina based on one mitochondrial marker. Therefore, the hypothesis of genetic differentiation or restriction of gene flow between the northern and southern yungas areas of Argentina is rejected, and there appears to be a single metapopulation in this region of northwestern Argentina. The AMOVA analysis did not reveal significant genetic differentiation when populations were grouped by distinct ecotype, the north and south divisions. Similarly, no boundaries were defined by SAMOVA for the complete data set. Previous findings in An. pseudopunctipennis in North, Central and South America showed deep population structure within this group using other molecular markers[An. pseudopunctipennis from the yungas is a unique population with COI mitochondrial gene flow among localities with similar demographic history. However, to test whether the current degradation of the natural ecosystem and landscape change is affecting the population further studies focused on markers with a faster mutation rate would be desirable[The present study shows very little genetic structure of the malaria vector  markers, 13–15. esirable, 13–15.An. albimanus from Colombia showed similar results[et al.[An. albimanus populations from southern Central America founded significant genetic structure between populations from Costa Rica and western Panama compared with those from central-eastern Panama, whereas in our study, divergence within groups was shallow and statistically insignificant.When compared to other neotropical malaria vectors, the genetic differentiation between two populations of the malaria vector  results; in spit[et al. studyingAn. pseudopunctipennis is similar to results obtained for An. darlingi and An. albimanus by Mirabello & Conn[et al.[et al.[An. pseudopunctipennis nucleotide diversity was low, but not haplotype diversity, can be explained by rapid population growth from ancestral populations.In addition, the low nucleotide diversity and the absence of isolation by distance observed for o & Conn, by De M[et al. and by M[et al., linkingm values between northern and southern populations showed not only high gene flow between them (Nm > 1) but also negative values (Nm <1) related to low gene flow, where significant population differentiation could occur through genetic drift. The more ancestral and diverse haplotypes of An. pseudopunctipennis were observed in both the north and south. As reported by Mirabello & Conn[et al.[An. pseudopunctipennis has undergone an extensive expansion population process. In fact, the presence of a dominant haplotype represents an ancestral lineage, because older haplotypes have had more time to spread, leading to a higher frequency and geographic distribution[An. darlingi (Amazonian region of Brazil)[Anopheles marajoara showed a more recent population expansion in Brazil, when it was compared to An. darlingi[An. pseudopunctipennis are commonly found in riverside pools colonized by filamentous algae of the genus Spirogyra in the foothills of the mountainous Mesoamerica region[The No & Conn, Molina-[et al. and Kamb[et al., older pribution. Further Brazil), 56, Anodarlingi. This didarlingi, 59. Unfa region–62. TherThe development of alternative breeding sites, or the increase of new suitable breeding sites because of the anthropic alterations of natural habitats, such as development of crops, may be COI gene to study the population demography of An. pseudopunctipennis in America, although the study was restricted to the Argentinian distribution. Results do not support the existence of northern and southern An. pseudopunctipennis population differentiation. Instead, this metapopulation seems to have undergone a single colonization process, without differentiation between northern and southern localities, with fairly high gene flow among them and evidence of a Holocene expansion.This is the first report of the use of the mitochondrial An. pseudopunctipennis. This study provides important baseline data that suggest that similar vector control measures should work in both the north and south, and also, if insecticide resistance evolves, it would likely spread fairly rapidly in this area.In summary, this research reports the pattern of the genetic variability and gene flow among northwestern Argentinian localities of