CPT1A, LRP5, THADA) and vascular smooth muscle contraction (PRKG1). By employing a new method that paints phased chromosome chunks by their ancestry we distinguish local Siberian-specific long-range haplotype signals from those introduced by admixture.Following the dispersal out of Africa, where hominins evolved in warm environments for millions of years, our species has colonised different climate zones of the world, including high latitudes and cold environments. The extent to which human habitation in (sub-)Arctic regions has been enabled by cultural buffering, short-term acclimatization and genetic adaptations is not clearly understood. Present day indigenous populations of Siberia show a number of phenotypic features, such as increased basal metabolic rate, low serum lipid levels and increased blood pressure that have been attributed to adaptation to the extreme cold climate. In this study we introduce a dataset of 200 individuals from ten indigenous Siberian populations that were genotyped for 730,525 SNPs across the genome to identify genes and non-coding regions that have undergone unusually rapid allele frequency and long-range haplotype homozygosity change in the recent past. At least three distinct population clusters could be identified among the Siberians, each of which showed a number of unique signals of selection. A region on chromosome 11 (chr11:66–69 Mb) contained the largest amount of clustering of significant signals and also the strongest signals in all the different selection tests performed. We present a list of candidate cold adaption genes that showed significant signals of positive selection with our strongest signals associated with genes involved in energy regulation and metabolism ( Hominins evolved in Africa for millions of years and adapted to survive in low latitudes and warm environments. After the dispersal out of Africa, over the past tens of thousands of years, however, our species has colonized almost all inhabitable climate zones of the world, including high latitudes and extremely cold environments. Siberia is one such region inhabited by humans today, being home to the coldest inhabited place in the world with a recorded minimum temperature of −71.2°C in winter. In present-day Siberia the climate varies dramatically over its different regions with an annual average temperature of −5°C. Archaeological evidence suggests that modern humans reached Southern Siberia by 45–40KYA Technological and cultural innovations have certainly facilitated territorial expansion of humans in Siberia. Besides these, a number of morphological and physiological adaptations have been proposed to have evolved in Siberian populations in response to their environment. Low serum lipid levels were observed in indigenous Siberian populations, which are thought to be a consequence of increased energy metabolism and their elevated basal metabolic rate, BMR Over the past few years, genome-wide analyses based on the HapMap In this study, we introduce genome-wide genotype data for 200 individuals from ten different indigenous Siberian populations. We set out to identify regions in the genome that have been targets of positive natural selection. Several physiological functions have been associated with cold exposure and adaptation. On mild cold exposure, the body will try to conserve its heat by energetically inexpensive means such as vasoconstriction, piloerection, and by changes in posture to decrease surface area We sampled 200 individuals from ten indigenous Siberian populations that form part of the DNA collection at the Genetics Laboratory, at the Institute of Biological Problems of the North, Magadan, Russia. The samples comprised 24 Buryats from Buryat Republic, 24 Evenks from the Krasnoyarsk region, 22 Yakuts from Sakha (Yakutia) Republic, 24 Shors from the Kemerovo region, 24 South Altaians , 25 Koryaks from Severo-Evensk District of the Magadan Region, 24 Evens from Severo-Evensk and Ola Districts of Magadan Region, 14 Chukchi from Anadyr, Chukotka Autonomous Okrug and 19 Eskimos from Novoe Chaplino, Chukotka Autonomous Okrug. We also genotyped 18 Vietnamese individuals sampled in Singapore as a Southeast Asian reference group for our study. The study was approved by the Ethics Committee of the Institute of Biological Problems of the North, Russian Academy of Sciences, Magadan, Russia  and Cambridge Ethics Committee (HBREC.2011.01). All subjects provided written informed consent for the collection of samples and subsequent analysis.Samples were genotyped using Illumina OmniExpress Bead Chips for 730,525 SNPs. The data genotyped in this study has been deposited to the NCBI GEO repository and is accessible with GEO accession number GSE55586. Data filtering, quality checks and merging with other available data were performed in PLINK 1.07 Following quality control we phased autosomal SNPs using SHAPEIT K) and assigns individuals the ancestry proportions using an unsupervised clustering method. We used European and Han Chinese populations from HapMap Phase 3 2r value greater than 0.1 within a 50-SNP sliding window which was advanced by 10 SNPs each time K at K = 2 to K = 10 and analysed the cross-validation errors and log-likelihood estimates for each value of K to estimate the optimum number of K clusters.To investigate population structure within the Siberian populations we used three commonly used methods: admixture analysis, model-based clustering, and Principal Component Analysis (PCA). ADMIXTURE 1.21 http://www.hapmap.org). We performed EM inference using 10 EM steps to estimate the effective population size from our data and then used this estimated parameter in ChromoPainter. To perform Markov Chain Monte Carlo (MCMC) analysis for fineSTRUCTURE we used 5,000,000 burn-in iterations and sample iterations with a thin interval of 5,000. Visualisation of the posterior distribution of clusters was then performed using the tree-building algorithm of fineSTRUCTURE. Finally, PCA was performed on the normalized version of the coancestry matrix output from ChromoPainter using the fineSTRUCTURE GUI, which is an LD corrected version of standard PCA.To complement ADMIXTURE results and perform clustering of individuals we employed ChromoPainter/fineSTRUCTURE The population structure analyses furthermore allowed us to filter out additional outliers that did not group with their respective groupings and inbred individuals  for use in subsequent analyses. For the haplotype-based tests we grouped Siberian populations into three groups such that the groups contain a reasonably homogenous set of individuals as implied by the population stratification analyses. The clustering of individuals followed, with the exception of a few outliers, the self-reported population and the geographic area of sampling: Southern Siberian group was composed exclusively of Altai-Kizhi, Teleuts, Shors and Buryats; the Central Siberian group of Evenks, Yakuts and Evens and the Northeast Siberian group of Koryaks, Chukchi and Eskimos. Simulations have shown that iHS and XP-EHH tests maintain power with sample sizes of ca. 40 chromosomes or above Q for the respective estimated ancestry) and which grouped in the same cluster in fineSTRUCTURE analysis were used for this purpose. This resulted in the identification of four different ancestral populations; Europeans, Vietnamese, unadmixed Central Siberian individuals (S1) and unadmixed Northeastern Siberian individuals (S2). These ancestral populations were used as donors in ChromoPainter From our population structure analyses results we selected groups of unadmixed individuals that showed maximum divergence from other groups and used these as a panel representing “ancestral populations” to paint the phased chromosomes of all Siberian individuals. Individuals that showed no admixture with other ancestries from the ADMIXTURE analysis (≥0.99 in the ancestry coefficient matrix f3) f3 test were considered as significantly admixed, and a subset of them were subsequently analysed with ALDER In order to evaluate the admixture scenarios suggested by the ADMIXTURE plots, we tested all possible sets of recipient and source populations with the three population test (We used the Integrated Haplotype Score (iHS) http://hgdp.uchicago.edu. HapMap GRCh37 genetic map was used to calculate genetic distances between markers. Ancestral states were retrieved from the Ensembl Variation database rel. 68. iHS and XP-EHH were computed for autosomal SNPs. SNPs with inferred ancestral states and a minor allele frequency of at least 5% were used for iHS. This setup yielded ca. 11,000 windows for iHS and ca. 13,000 windows for XP-EHH, for each Siberian population group. Vietnamese were used as an outgroup population for the haplotype homozygozity tests as no degree of admixture with a Siberian specific ancestral component was observed in the Vietnamese samples , which represents the amount of allele frequency change at a given locus in the history of the test population since its divergence from other populations Our top windows from the selection tests were mapped to genes using the Ensembl Genes Human database rel. 68. Manhattan, ADMIXTURE, PCA and regional paintings plots were drawn using the ggplot2 We performed window enrichment analysis looking for increased representation in the top 1% iHS and XP-EHH windows of biological functions that could potentially be involved in cold adaptation. We used Gene Ontologies (GO-terms) Since PBS is an allele frequency based test, we identified the protein-coding genes containing SNPs exhibiting the highest PBS scores from our top 1% PBS window data and used these genes as input to DAVID We generated a cold adaptation-related seed gene list by considering genes associated with phenotypic features that have been attributed to cold adaptation. These include basal metabolic rate, non-shivering thermogenesis, response to temperature, smooth muscle contraction, blood pressure and energy metabolism using relevant Gene Ontology terms, pathways and experimental evidence while defining the lists.http://genapps2.uchicago.edu:8081/dbcline/climate.tar.gz. Since we are interested in cold adaption selection signals we used the minimum winter temperature variable for comparison. We extracted the strongest signals with transformed ranked signals <5×10−4 and mapped these signals to our top 1% iHS, XP-EHH and PBS selection regions for all Siberian populations. Loci were mapped to genes using the Ensembl Genes Human dataset rel. 68.We used published results from a study that examined selection in response to 9 climatic variables in a panel of 61 worldwide populations, including three from Siberia  and Yakut) We genotyped 200 Siberian individuals from ten indigenous Siberian populations across the Siberian landscape  and callFirstly, we used PCA to reveal levels of genetic differentiation and population structure in the Siberian populations in the broader Eurasian context . The firK used in ADMIXTURE analysis, we ran ADMIXTURE 100 times using K values from 2 to 10 in each iteration. The minimum cross-validation error in the ADMIXTURE analysis was observed at K = 4. The difference between maximum and minimum log-likelihood scores over all 100 iterations was 0 at K = 4 suggesting that the log-likelihood score had reached its global maximum  and other Asian populations  as ancestor populations. Furthermore, ADMIXTURE analysis revealed that Evens contain both k1 and k2 components consistent with the geographic area of their sampling since they are located in between the populations that are largely composed from the k1 and k2 components.To estimate the optimum number of clusters We also performed analyses with ChromoPainter/fineSTRUCTURE Long term habitation in an extremely cold environment can be expected to result in biological adaptations that may affect the distribution of allele frequencies and homozygosity patterns. To examine this hypothesis we employed haplotype-based tests - iHS THADA, ITPR3, GNGT1, PRKG1, RELN, CPT1A and LRP5) that were also present in the pre-defined cold adaptation seed gene list that was detected by at least one of the tests , none of which was significant after FDR correction . Since the region contains several other protein coding genes (which were not in our cold adaptation candidate gene list) which lie in the same block of high homozygosity and the same ancestry, it is not possible from the given data to distinguish which gene, in particular, drives this signal.The strongest selection signals over all tests map closely in a 3 Mb region (chr11:66–69 Mb) of chromosome 11  which sh66–69 Mb . We usedth ranking XP-EHH signal) was POLD3 that encodes a subunit of DNA polymerase delta which is involved in DNA damage response produced by UV irradiation POLD3 gene show high signals in all selection tests with the highest signals coming from the southern Siberian populations  has similarly the highest expression in the liver and plays a role in bone growth LCTCPT1A and LRP5 genes mapped to the same high haplotype homozygosity block along with several other genes further sequencing data from this genomic region will be required to determine which gene carries functionally significant variants that may be driving the selection signal.The strongest signals mapped to chromosome 11:66–69 Mb region that contains a cluster of seven top 10 ranking windows over all three tests which also included two cold adaptation candidate genes, and LRP5 . Moreoveand LRP5  showed tUCP1 and UCP2, suggesting activation of non-shivering thermogenesis which uncouples the mitochondrial respiration by impairing ATP production and dissipating energy as heat. Previously, Leonard et al. CPT1A is regulated by thyroid hormone and insulin THADA Click here for additional data file.File S2This file contains Table S1 through Table S13. Table S1, Sample sizes of Siberian populations that were genotyped in this study. Table S2, Sample sizes of Siberian populations used in PBS tests. Table S3, Admixture dating for Siberian populations/groups. Table S4, Empirical ranks of the top 1% iHS windows in three Siberian population groups. Table S5, Empirical ranks of the top 1% XP-EHH windows in three Siberian population groups. Table S6, Empirical ranks of the top 1% PBS windows in Siberian populations. Table S7, Top 10 windows present in more than one selection test. Table S8, Window enrichment analysis of the top 1% ranking genes by haplotype homozygosity tests in three Siberian population groups. Table S9, Gene enrichment analysis of the genes with maximum PBS present in the top 1% windows in all Siberian populations. Table S10, Definition of gene lists hypothesized to be involved in cold adaptation. Table S11, Cold-adaptation genes among the top 1% ranking windows by haplotype homozygozity tests. Table S12, Cold-adaptation genes found in top 1% ranking PBS windows. Table S13, Significant windows in the iHS, XP-EHH and PBS tests that intersect with the significant SNPs for minimum winter temperature variable in Hancock et al. 2011 study.(XLSX)Click here for additional data file.


Baylisascaris schroederi (Ascaridida: Nematoda), is one of the most important causes of death in giant pandas, and was responsible for half of deaths between 2001 and 2005. Mitochondrial (mt) DNA sequences of parasites can unveil their genetic diversity and depict their likely dynamic evolution and therefore may provide insights into parasite survival and responses to host changes, as well as parasite control.Infection with the parasitic nematode, B. schroederi populations by combining two different mtDNA markers, ATPase subunit 6 (atp6) and cytochrome c oxidase subunit I (cox1). Both sequences were completely amplified and genetically analyzed among 57 B. schroederi isolates, which were individually collected from ten geographical regions located in three important giant panda habitats in China .Based on previous studies, the present study further annotated the genetic variability and structure of atp6, 24 haplotypes of cox1, and 39 haplotypes of atp6 + cox1. Further haplotype network and phylogenetic analyses demonstrated that B. schroederi populations were predominantly driven by three common haplotypes, atp6 A1, cox1 C10, and atp6 + cox1 H11. However, due to low rates of gene differentiation between the three populations, both the atp6 and cox1 genes appeared not to be significantly associated with geographical divisions. In addition, high gene flow was detected among the B. schroederi populations, consistent with previous studies, suggesting that this parasite may be essentially homogenous across endemic areas. Finally, neutrality tests and mismatch analysis indicated that B. schroederi had undergone earlier demographic expansion.For the DNA dataset, we identified 20 haplotypes of B. schroederi populations do not follow a pattern of isolation by distance, further revealing the possible existence of physical connections before geographic separation. This study should also contribute to an improved understanding of the population genetics and evolutionary biology of B. schroederi and assist in the control of baylisascariasis in giant pandas.These results confirmed that The online version of this article (doi:10.1186/s13071-014-0606-3) contains supplementary material, which is available to authorized users. Howevlumnaris , Dictyocviparous , and Haeontortus ,16. MoreAiluropoda melanoleuca), a flagship species for wildlife conservation, is one of the world’s most recognized and threatened animals with a total wild population size estimated to be only 1,600; these individuals are restricted to six mountain ranges in China , of which 44.36% are in Minshan, 27.38% in Qionglai and 17.23% in Qinling [Baylisascaris schroederi (Ascaridida: Nematoda) is the most important cause of death in wild giant pandas and thus it poses a significant threat to these populations [B. schroederi infection follows ingestion and the life cycle is complete without the need for an intermediate host. Infective second-stage (L2) larvae hatch in the small intestine, migrate into the liver and the lung, and finally arrive at the small intestine where they mature, mate and produce eggs [B. schroederi infection rate among wild pandas is often over 50% and can even be 100%, thus making it a leading cause of death in wild populations [Ascaris schroederi [B. schroederi in 1968 [B. schroederi focused mainly on morphological characteristics, fundamental biology, life cycle, pathogenicity, vaccine development and prevalence [. Recent advancements in PCR and sequencing technologies have led to the publication of several gene fragment sequences from B. schroederi , as well as complete mtDNA and microRNA [B. schroederi remain scarce, with only one report publically available in which a single mt cytb was utilized for this purpose [The giant panda , to expand and improve our knowledge of the genetic diversity of B. schroederi. Our data analysis included plotting transition and transversion frequencies of these genes and determining the genetic distances between populations. These data provide more detailed information into the genetic diversity of B. schroederi populations in three endemic areas of China , and enable a reevaluation of the population structure. In addition, we compared the genetic diversity of the population and inferred the phylogenetic relationships between different isolates according to their geographical distribution.Considering the limitation of using just a single gene to define the genetic structure of a parasite -33, the Between two and three parasite specimens were collected per giant panda (10-15 pandas per mountain range), except for Qinling , eluted with 30 μL of elution buffer (pH 8.0) according to the manufacturers’ recommendations, and then stored at -20°C for PCR amplification. Two pairs of PCR primers were designed to amplify the complete mt atp6 and cox1 genes using Primer Premier 5.0 software  and the published mtDNA sequence of B. schroederi (GenBank Accession No.: HQ671081). The primer pairs were as follows: atp6, forward: 5′-CGCGGATCCTTCGATATTCGTGGCCT-3′, and reverse: 5′-CGCAAGCTTCTAATATGGTGTCTTCGG-3′; cox1, forward: 5′-TTTAGAGGTTGGAATGTAGGGT-3′, and reverse: 5′- CCATCCCCTTAATCTGCAAT-3′. All PCR reactions contained ~20 ng of genomic DNA and were carried out in 50-μL reaction volumes containing 25 μL 2× Phusion High-Fidelity PCR Master Mix , 2 μL of each primer, 2 μL DNA and 19 μL of ddH2O under the following cycling conditions: an initial denaturation at 95°C for 5 min; then for cox1, 35 cycles at 95°C for 1 min, 50°C for 1 min, and 72°C for 1 min; but for atp6, 35 cycles of 95°C for 45 s, 50°C for 45 s, and 72°C for 45 s; followed by a final step at 72°C for 10 min. Negative controls were included in each PCR to test for possible contamination. All PCR products were examined on agarose gels (1%) to verify that they represented the target bands. The corrected gel-isolated fragments were subsequently column-purified and sequenced. Sequencing was performed by Invitrogen  using terminator-based cycle sequencing with BigDye chemistry  on an ABI 3730 DNA sequencer (Applied Biosystems). A “primer-walking” strategy (in both directions) was used and, to ensure maximum accuracy, each amplicon was sequenced twice independently. A third PCR product was sequenced in case of any discrepancies. Consensus sequences were deposited in the GenBank database.Genomic DNA was extracted from 0.5 g of tissue from each individual atp6 and 1578 bp for cox1) were aligned using the ClustalW program in MEGA v.5.0 [atp6 and cox1 were concatenated into a single 2,178-bp fragment for each specimen for realignment according to the aforementioned method. During this procedure, any ambiguous regions within these alignments were filtered with Gblocks 0.91 b [atp6, cox1 and pooled datasets were constructed using the maximum parsimony (MP) method, with each node estimated using 1,000 bootstrap replicates. Data were plotted with MEGA v.5.0 [B. schroederi, a Bayesian statistical method [atp6 and cox1, and was directly applied in the following concatenated data). A total of 10 million generations were run and the trees were sampled every 10,000 generations. When the mean standard deviation of the split frequencies had reduced to less than 0.01, the first 250 trees were discarded as “burn-in” and the remaining trees were used to generate a 50% majority rule consensus tree for each data set. Homologous sequences obtained from Baylisascaris transfuga  were used as outgroups in all phylogenetic analyses due to the close relationship between B. schroederi and B. transfuga [. In addition, the Data Analysis in Molecular Biology and Evolution (DAMBE) program [Sequences with consensus lengths  and index of genetic differentiation (Fst) were also estimated, and the corresponding gene flows (Nm) between populations were indirectly calculated as follows: Nm = 0.5*(1-Gst)/Gst [Nm = (1-Fst)/4Fst [Gst statistic has been used widely to evaluate population subdivision on the basis of the divergence of allele frequencies from random mating [Aligned FASTA files were collapsed into variable sites and haplotypes for parsimony network reconstruction using Dna SP 5.0 . A statiGst)/Gst  or Nm = st)/4Fst . Of thesm mating . Finallym mating  was perfB. schroederi, 1,000 simulations in Arlequin [P values for each population. Moreover, to measure frequency distributions of pairwise differences between sample sequences, mismatch-distribution analysis was also performed using Dna SP 5.0 [Different approaches were used to infer historical demographic expansions, including those of Fu’s Fs, Tajima’s D and the mismatch-distribution tests -47. To aArlequin  were pera SP 5.0  for 1,00This study was reviewed and approved by the Research Ethics Committee in College of Veterinary Medicine, Sichuan Agricultural University . All parasite samples were collected from giant pandas after the permissions of the Forestry Department of Sichuan Province and Shaanxi Rare Wildlife Rescue Breeding Research Center, with no specific permits being required by the authority for the parasite collection.atp6 and 1,578-bp cox1 sequences from 57 B. schroederi isolates were deposited in GenBank under accession numbers KJ587749-KJ587805 and KJ587806-KJ587862, respectively. No insertion/deletions (indels) were detected. In total, 20 haplotypes (A1-20) were identified in atp6, 24 haplotypes (C1-24) in cox1 and 39 haplotypes (H1-39) in atp6 + cox1. Haplotypes A1 and A8 (inferred from atp6), C10 (inferred from cox1) and H11 (inferred from concatenated sequences) were shared by all samples collected from the three mountain ranges  and nucleotide diversity (0.00359 for atp6 and 0.00204 for cox1). However, the concatenated sequences seemed to have greater degree of haplotype diversity  when compared with atp6 and cox1  differed between atp6 and cox1, and the R-value between the three populations was lower for cox1 (22.10) than for of atp6 (50.48) (data not shown).The 600-bp B. schroederi isolates collected from different geographic regions, the atp6 and cox1 genes were analyzed, separately or in combination, to infer the phylogeny, and the corresponding constructed phylogenetic trees are shown in Figure To study the phylogenetic relationships between the cox1 haplotypes, the network map revealed a star-like pattern around haplotype C5, which contained seven individuals, with three in the Qionglai and four in the Minshan populations. A total of 12 other haplotypes were found in the Qionglai population (C1–12), while there were 5 haplotypes present in the Qinling population . Interestingly, the haplotype C10 was shared by samples derived from all three mountain ranges, while haplotypes C4, C5 and C11 were shared by only the Qionglai and Minshan populations and C14 by only the Minshan and Qinling populations  for all pairwise comparisons, which was in agreement with results from the phylogeny and network analyses. A similar result was also found for the concatenated sequence data , while the lowest Gst and Fst values were found between Qinling and Qionglai populations . Interestingly, a similar trend was also observed for Fst values of atp6, but this was contrasted with Gst values of atp6, where the highest Gst value was detected between the Qinling and Qionglai populations   were unimodal and had undergone at least one expansion event   were combined to further explore the genetic variation and population structure of B. schroederi.Parasites are a crucial component of natural communities, since they can directly or indirectly alter the structure of the communities by impacting the number of free-living species or their relative abundance . With adterparts -33,52,53terparts  and the ytb gene , in thisGst and Fst values indicated small genetic differentiation in the B. schroederi population  between the three populations was always observed for both atp6 and cox1, and it could be speculated that the effective number of B. schroederi individuals that exchanged genetic material was sufficient to overcome the effects of genetic drift. Thus, B. schroederi isolated from different geographical regions may be a homogenous species with strong population diffusion, little obstruction for gene flow, simple genetic structure and small differentiation. Moreover, the parasite is a specialized organism that parasitizes a specific host, and the genetics and behavior of the host can also have an important impact on parasite genomic variation. The population history of the giant panda, as characterized by Zhao et al. (2012), showed that pandas in the Qinling mountain range were distinct from other populations (including Minshan and Qionglai-Liangshan-Daxiangling-Xiaoxiangling) due to geographical isolation, human activities and global changes in climate [B. schroederi showed only weak population subdivisions  B. schroederi can be transmitted from one geographical origin to another because of the cross-regional reproductive behavior of pandas; (iii) thus, mating of B. schroederi could occur between individuals from different geographical areas. Alternatively, there could be another reason for possible mixed infections in the hosts, as B. schroederi specimens from the same individuals had low genetic differences and different haplotypes. Perhaps future complete mitochondrial genome-based population analysis involving a larger B. schroederi population would provide novel and/or further insights into this issue.In this study, low on Table , and thi climate . Howevero et al. 012, showmusculus . Additiomusculus ,58. Theree refs. ,23,59): B. schroederi populations, which was similar to a previous report [B. schroederi and this revealed a relative lack of genetic variation across the ascaridoid, regardless of geographical origin and population size. Interestingly, similar findings were also described for other Ascaris species, including Ascaris galli [Ascaris suum [Parascaris equorum [atp6 and cox1), which was probably due to the small sample size; however, the genetic diversity in the Qinling population was less clear. The analysis of atp6 and cox1 genes showed low geographic separation between the three mountain range populations, which was consistent with the findings of a previous report [atp6, C10 for cox1 and H11 for atp6 + cox1. The most frequent haplotypes were A1 of atp6, C10 of cox1 and H11 of atp6 + cox1 in the examined populations . Interestingly, haplotypes A2, C5 and H6 were shared only by Minshan and Qionglai mountain range populations. Thus, it could be speculated that these three haplotypes differentiated after from the ancient haplotype and would display a trend of widespread geographic distribution and may evolve to form a single haplotype across all three mountain ranges in the future. Taken together, our data indicated that the B. schroederi species was not genetically differentiated.The other important finding of this present study was the low level of genetic diversity found across all three s report . Low nucis galli , Ascarisris suum ,61 and P equorum . Togethes report . FurtherB. schroederi-panda co-evolutionary system, the host has recently experienced two population expansions, two bottlenecks and two divergences [B. schroederi. Encouragingly, negative Fu’s Fs (high) and Tajima’s D (low) values (Table B. schroederi had been subjected to a sudden demographic expansion in the past [B. schroederi infections is relatively high (50–100%) [B. schroederi, and its control relies largely on anthelmintic drugs. However, some studies have reported the occurrence of drug resistance in B. schroederi populations in China [B. schroederi populations and the potential of rare resistance alleles to spread, an investigation of drug resistance in B. schroederi is urgently needed.In general, certain aspects of parasites often closely track counterparts of their host’s biology due to long-term interplay between them ,64. In tergences . Thus, ies Table , 3 and 4the past , and thi50–100%) ,22. No cin China . Given hatp6 and cox1, to explore population genetic variability and structure of 57 B. schroederi isolates sampled from the three main giant panda habitats in China. The low levels of genetic diversity and high levels of gene flow indicated that B. schroederi was not genetically differentiated and had experienced lineage re-arrangement. Moreover, the negative Fu’s Fs and Tajima’s D values coupled with mismatch analysis consistently showed that the B. schroederi population could have been subjected to a sudden demographic expansion in the past. These results, together with previous studies, further suggest that B. schroederi populations did not follow a pattern of isolation by distance, revealing the possible existence of physical connections before the populations became geographically separated. This finding should contribute to a deeper understanding of population genetics and evolutionary biology of B. schroederi, a nematode parasite of the giant panda.This investigation initially combined two different mitochondrial markers,


Formicivora paludicola) is a recently-described and critically endangered bird from São Paulo State (Brazil) whose total estimated population is around 250–300 individuals, distributed in only 15 isolated marshes around São Paulo metropolitan region. We used microsatellite DNA markers to estimate the population genetic characteristics of the three largest remaining populations of this species all within 60 km of each other. We detected a high and significant genetic structure between all populations  which is comparable to the highest levels of differentiation ever documented for birds, , but also evidence for first-generation immigrants, likely from small local unsampled populations. Effective population sizes were small  yet there are high levels of genetic variability within populations and no evidence for inbreeding. Conservation implications of this work are that the high levels of genetic structure suggests that translocations between populations need to be carefully considered in light of possible local adaptation and that remaining populations of these birds should be managed as conservation units that contain both main populations studied here but also small outlying populations which may be a source of immigrants.Small populations of endangered species can be impacted by genetic processes such as drift and inbreeding that reduce population viability. As such, conservation genetic analyses that assess population levels of genetic variation and levels of gene flow can provide important information for managing threatened species. The São Paulo Marsh Antwren ( Endangered species often exist in small isolated populations, which can suffer increased risks of extinction due to inbreeding and loss of adaptive genetic variation through genetic drift –3. The dFormicivora paludicola, Thamnophilidae). This endangered species is a small insectivorous bird only recently described as a distinct species [One species where such an analyses would be especially useful is the São Paulo Marsh Antwren  Measure levels of genetic variability within and genetic differentiation between antwren populations located in Mogi das Cruzes, Salesópolis, and São José dos Campos, 2) Estimate genetically effective population sizes and assess if populations have experienced recent bottlenecks, and 3) Assess levels of population connectivity through the detection of first generation migrants. Our overall goal is to use genetic information to evaluate if this species potentially faces present-day or future genetic risks that may impact its long-term viability and which mechanisms operate to influence levels of variation in the few remaining populations of this highly endangered bird.São Paulo Marsh Antwren was first discovered in 2004 . It is a2. The third area is São José dos Campos (SJC) , located in Upper Paraíba do Sul basin. These marshes are surrounded by a mosaic composed mainly of Eucalyptus spp. silviculture, pasturelands, and Atlantic Forest fragments. The linear distances between these areas vary from 30 to 60 km. The other marshes in which this species occur are all smaller than 11 ha and probably contain small numbers of individuals .We sampled birds at the three major marshes in which this species occurs . Two of São Paulo Marsh Antwrens live in pairs that defend small territories all year round which are easily identifiable in the field, as these birds promptly respond to playbacks of their song. At each study site, we first used playbacks to locate the territories. Once a territory was identified, we set up a mist net of 12 × 2.5 m, and playbacks were used again to attract the birds to the net. All captured birds were banded using a unique combination of PVC colored rings for individual identification, and a 10–20 μL blood sample was obtained from each bird by cutting the tip of a nail, after which birds were released . Blood wUsing these techniques, we collected blood samples from 57 birds from 2012 to 2014 . AlthougMyrmeciza exsul) . Raw microsatellite genotypic data is presented in DNA was extracted from blood using a standard phenol-chloroform-isoamyl alcohol protocol . For eac Mex176 –, 34), us Mex176 –. AmplifiStructure 2.3.4. [K (from K = 1 to K = 6). The most appropriate K was defined using the Evanno method [Structure Harvester [FST) [F’ST of [F’ST is unaffected by this sampling bias and incorporates a correction for within-population genetic diversity bias. Its significance was tested using the AMOVA procedure in GenAlEx 6.5 [RST [Population structure was inferred using the Bayesian method implemented in e 2.3.4. . We usedo method , implemearvester . The levr [FST) , and assr [FST) , after 1[F’ST of . These a[F’ST of ). The coAlEx 6.5 , 42, witAlEx 6.5  to estim.5 [RST , with siRST allele size randomization test of [Finally, to gain insight into the degree to which levels of differentiation reflect long versus short-term isolation, we performed the  test of  as imple test of , 46.HO) and expected (HE) heterozygosities using Genepop 4.2 [AR) scaled for differences in sample size between populations [FIS) [Fstat [We estimated observed   for each) [Fstat . To evalGeneClass2 [To evaluate levels of contemporary immigration, we used assignment tests in neClass2  to detecneClass2 , 50. LikneClass2 , and proNeEstimator, Ver. 2 [F. acutirostris) [Bottleneck [Effective population size was accessed using the linkage disequilibrium method implemented by software , Ver. 2 , using trostris) –and a crrostris)  in Bottlttleneck , 56. Micttleneck . In TPM ttleneck . As abouttleneck , we usedttleneck   for the sample and these clusters mirror our three sample sites exactly, although there is evidence for a small number of admixed individuals consistent with the presence of immigrants (see below) . Consistrom zero . Allele GeneClass suggest the presence of first generation immigrants in each population: one first generation immigrant was detected in MC, two in SL, and two in SJC  and expected (HE) heterozygosities averaged respectively 0.70 (range: 0.35–0.89) and 0.71 (range: 0.36–0.87) for MC, 0.66 (range: 0.41–0.88) and 0.66 (range: 0.50–0.82) for SL, and 0.70 (range: 0.36–0.86) and 0.73 (range: 0.43–0.90) for SJC  was similar across populations: 5.35 alleles (range: 2.88–8.69) for MC, 4.55 alleles (range: 2.00–7.28) for SL, and 5.69 alleles (range: 3.00–9.85) for SJC. The estimated effective population size was 99.9 individuals (95% CI: 69.5–165.8) for MC, 28.8 individuals (95% CI: 21.5–40.7) for SL, and 34.4 individuals (95% CI: 25.3–50.4) for SJC.Even though they are isolated, levels of genetic variation in these populations are high and there is no evidence for inbreeding. Observed  across all of the variance values used in the distribution of mutational steps, suggesting that a recent bottleneck may have occurred in this population, but it did not show the occurrence of recent bottlenecks neither in Salesópolis (SL) nor in São José dos Campos (SJC) , os (SJC) .FST values were substantially higher than those documented for other populations of tropical forest-dependent passerine birds, from either continuous or fragmented habitats studied in similar geographic scale [FST values that were one order of magnitude lower than those observed here [Laterallus jamaicensis) showed microsatellite-based FST estimates of 0.03 between populations in marshes that were a comparable distance (160 km) apart, with FST values of 0.1 only observed for geographically widely-separated populations ≥ 1100 km apart [Coturnicops noveboracensis) FST values of 0.1 were found for populations that were 2,000 to 3,800 km apart [One of the most striking results of this study are the high levels of genetic structure that are detected over a limited geographic scale between the three largest remaining populations of this critically endangered bird. In particular, the observed ic scale , 21, 22.ved here –26. Howekm apart . Similarkm apart . Our conFST over small geographic scale are most comparable to differentiation documented by [Turdus helleri), Yellow-throated Woodland-warbler (Phylloscopus ruficapilla), and Stripe-cheeked Greenbul (Andropadus milanjensis) found in cloud forest patches < 20 km apart on the top of mountains which are isolated by lowland savannas and degraded habitats in Tanzania [Our high ented by  for popuTanzania . In partTanzania , 57, andTanzania .F′ST were expected as we have used highly polymorphic microsatellites, and at a local scale each of the São Paulo Marsh Antwren populations studied here is likely part of a set of metapopulations (see below). Our observed levels of F’ST were higher than those obtained for populations of Réunion Grey White-eye, Zosterops borbonicus (0.12 to 0.21) that were 8.8 to 25.2 km apart. This is a passerine bird endemic to Réunion Island for which gene flow between populations is extremely low, probably due to behavioral processes [RST estimates were high and comparable to populations of cloud forest specialist passerines isolated in mountain tops. For example, in the East African Mountain White-eye, Zosterops poliogaster silvanus, RST values varied from 0.098 to 0.243 between populations approximately 15 to 80 km apart [High values obtained for rocesses . Finallykm apart . Howeverkm apart  was not km apart , 46.One striking result that is inconsistent with our observation of high levels of genetic differentiation is the relatively large portion of first generation migrants (4–14%) in each population. This high level of immigration suggests the potential for high levels of gene flow leading to less genetic structure than is currently observed assuming that these individuals could successfully breed. The fact that we were unable to assign these immigrants to sampled populations suggests that in fact, these birds do not represent potential genetic migrants between the main populations but are rather from local sources near each of the main populations.We see three possible scenarios for the origin of these immigrants. Under the first scenario these individuals are birds that were relocated during the establishment of the reservoir who did not settle in the new areas but moved into our study populations. However, this is unlikely because all birds that were translocated were banded and all the individuals identified as immigrants were unmarked. Under the second scenario the immigrant birds represent an unusual one-time event in which individuals from unknown populations existing in the flooded regions were displaced and moved into the Salesópolis and Mogi study areas, which are located on different sides of the reservoir. However, given the limited dispersal ability of São Paulo Marsh Antwren, this mechanism cannot explain the presence of immigrants in São José dos Campos, which is in a separate river (Paraíba do Sul) basin, to which dispersal from the reservoir region would be unlikely. The third, and in our view most plausible scenario, is based on the fact that surveys have shown that there are additional small populations consisting of limited numbers of individuals around each of the three main populations  see Fig that werOur analyses of population characteristics argue that these antwrens have likely existed in small isolated populations for substantial periods of time. The lack of evolutionary signal in the allele sizes randomization test  indicateChiroxiphia caudata) [Current effective population sizes of the three studied populations were small (~ 50 birds) and similar to those obtained for the Chestnut-backed Antbird in small and isolated forest fragments in Costa Rica , but arecaudata) . SurprisFrankham  observedWe see several conservation implications to our study. First, when populations are small and isolated, translocations of individuals between them has been suggested as a management choice to increase population viability , 10, 66,Typha domingensis (Typhaceae), whereas in others variable proportions of Cyperaceae are present  [Second, the low overall population numbers of this species and the potential importance of peripheral populations as sources of novel genetic variation mean that protection of all the 15 areas where São Paulo Marsh Antwrens are found should be a priority. This is especially important given that all of these sites are currently unprotected and exposed to human impacts . As the ra spp.) . Most ofra spp.) , 69, andOur study demonstrates that populations of São Paulo Marsh Antwren distributed in marshes embedded within the Atlantic Forest can be highly isolated, generating genetic diversification in a level comparable to mountain-top bird populations in Africa (“marshes as mountain-tops”). But it is important to note that mountain tops may be more stable environments than marshlands. Marshes can naturally disappear soon , but theS1 FileGenotypic data for 17 microsatellite loci presented as a three-digits GENEPOP file. Missing data are reported as 000000.(TXT)Click here for additional data file.S1 TableN), number of alleles (NA), allelic richness (AR), number of private alleles (AP), allele range in base pairs (T), observed (HO) and expected (HE) heterozygosities, inbreeding coefficient (FIS), and its probability of being different from zero (P) found for each analyzed microsatellite locus in three populations of the São Paulo Marsh Antwren. Critical value after Bonferroni correction is 0.001.Number of analyzed individuals ((DOCX)Click here for additional data file.


Plasmodium vivax is the most widely distributed human parasite and the main cause of human malaria outside the African continent. However, the knowledge about the genetic variability of P. vivax is limited when compared to the information available for P. falciparum. We present the results of a study aimed at characterizing the genetic structure of P. vivax populations obtained from pregnant women from different malaria endemic settings. Between June 2008 and October 2011 nearly 2000 pregnant women were recruited during routine antenatal care at each site and followed up until delivery. A capillary blood sample from the study participants was collected for genotyping at different time points. Seven P. vivax microsatellite markers were used for genotypic characterization on a total of 229 P. vivax isolates obtained from Brazil, Colombia, India and Papua New Guinea. In each population, the number of alleles per locus, the expected heterozygosity and the levels of multilocus linkage disequilibrium were assessed. The extent of genetic differentiation among populations was also estimated. Six microsatellite loci on 137 P. falciparum isolates from three countries were screened for comparison. The mean value of expected heterozygosity per country ranged from 0.839 to 0.874 for P. vivax and from 0.578 to 0.758 for P. falciparum. P. vivax populations were more diverse than those of P. falciparum. In some of the studied countries, the diversity of P. vivax population was very high compared to the respective level of endemicity. The level of inter-population differentiation was moderate to high in all P. vivax and P. falciparum populations studied. Plasmodium vivax predominates, malaria in pregnancy is associated with detrimental effects on the health of the affected mothers and their infants (1 − ∑pi2), where n is the number of isolates analyzed and p represents the frequency of each different allele at a locus. The potential range is from HE = 0  to HE = 1 . For isolates that were fully genotyped at all P. vivax and P. falciparum loci, analysis of multilocus linkage disequilibrium (LD) (non-random associations among loci) was assessed using the standardized index of association (ISA). Differences of the estimated mean HE for each population and ISA were calculated using the LIAN 3.5 software [FST) was estimated using Wright's F statistics, and calculations were computed by Arlequin 3.5 [HE of any two groups for each country was assessed using the Mann-Whitney U test. Differences were considered significant if the calculated p value was ≤0.05 (two-tailed test).Genetic variation for each MS locus in each population was assessed by calculating both the number of alleles per locus (A) and the expected heterozygosity  whereas the lowest one was observed in Papua New Guinea (mean HE = 0.834) (HE = 0.841) displayed a mean Rs value of 8.94, whereas isolates from pregnant group  showed an Rs value of 6.28  . The iso of 6.28 .HE values from Brazilian pregnant and non-pregnant groups but no significant difference was found ; similarly, no significant difference (p = 0.126) was found comparing Rs values from the same subgroups.We compared the mean P. vivax populations showed a significant LD  at multiple loci in each population except for the case of Colombia (p = 0.062). A moderate degree of LD was observed in Brazil, India and Papua New Guinea , whereas India and Brazil displayed the lowest value (FST = 0.045) of the analyzed vivax populations.Levels of genetic differentiation between each possible pair of the four parasite populations are indicated by P. falciparum populations were also investigated in three of the study sites for comparison at six MS loci. The genotyping was performed in 137 P. falciparum positive blood samples obtained from pregnant women in Brazil (n = 15), Colombia (n = 50) and Papua New Guinea (n = 72)  while a significantly high LD value was found both in Brazil and Colombia   and, amolculated , Fig 2. Colombia , howeverFST = 0.252, p< 0.05) was found between Brazil and Colombia. In Papua New Guinea isolates displayed a moderate level of significant differentiation, about 14.7% and 12.9% of the variation found in the Brazil and Colombia populations, respectively  and in 1 from Brazil the same haplotype was identified in subsequent episodes occurring at 3, 11 and 17 weeks.Complete genotyping data were also obtained for isolates collected from successive samples during the follow up of 9 pregnant women with P. falciparum infection. In all cases, parasites from maternal and placental blood had an identical MS profile.In addition, parasite haplotypes from maternal peripheral blood at delivery and the placenta were compared in 4 women (2 from Colombia and 2 from Papua New Guinea) infected with HE value among pregnant women infected with P. vivax compared to those with P. falciparum infection . Additionally, the genetic diversity of Plasmodium isolates according to age, parity, parasitaemia (microscopic or submicroscopic) and presence of fever (≥37.5°C) among the infected women were compared (sample size for each compartment is shown in P. vivax parasites infecting multigravidae (p = 0.003), those ≥ 25 years old (p = 0.009) and those who were afebrile (p = 0.015), compared to primigravidae, those aged 11–20 years old and febrile women, respectively. In contrast, P. falciparum parasites infecting ≥ 25 years old (p = 0.003) and those who were afebrile (p = 0.032), showed a significantly lower genetic diversity, while significant difference was not observed in the genetic diversity level for other analyzed subgroups and countries. In Colombia, the situation was exactly the opposite of what was observed in Brazil, but the differences in HE values were not statistically significant. In Papua New Guinea, the genetic diversity was very similar in each age class for both vivax and falciparum, and the slight differences observed were not significant in Mann-Whitney U test.Only in Colombia we detected a significantly higher mean P. vivax populations in blood samples obtained from pregnant women across different endemic areas by MS analysis. Overall, a wide range of short tandem repeat variants and high level of diversity in P. vivax and also P. falciparum were observed. In particular, for each of the countries included in the study, the diversity of P. vivax populations was slightly higher than for P. falciparum populations, though a significant difference between HE values for the two species was only found in Colombian isolates.This study was aimed at characterizing the genetic diversity of P. falciparum populations show a direct association between the genetic diversity and transmission intensity. However, the situation for P. vivax is more complicated with observations showing high genetic diversity in parasite populations from low transmission settings [falciparum population from Papua New Guinea (hyperendemic country) showed the highest value of HE in comparison with the other populations studied, while high values of HE were found in vivax populations from India and Colombia (hypoendemic) and Brazil (mesoendemic), confirming that this parasite is able to maintain a high degree of genetic variability irrespective of the transmission situation of its endemic area. Actually, the lowest HE value among the vivax population included in this study was found in the parasite population from Papua New Guinea, which happens to be the country with a high malaria transmission intensity.By evaluating these data according to the level of malaria endemicity, we know that the investigated areas in Colombia and India show a low level of endemicity; Manaus, in Brazil, is considered mesoendemic whereas Madang (Papua New Guinea) is the only area with a high level of endemicity. settings –16. FindP. vivax populations examined showed moderate level of differentiation ranging from about 6.4% to 10.2% . On the contrary, falciparum populations showed to be highly differentiated from each other in the pairwise comparison between countries ; parasites from the two geographically closer Southern American countries, Brazil and Colombia, appeared to be the most divergent according to the FST analysis (25.2%). Our result is consistent with the independent origins of these two parasite populations, as shown by Yalcindag et al (2012) [P. falciparum populations are subdivided into two main genetic clusters: northern (including Colombia) and southern , due to two different introduction events during European colonization.Based on previously defined range of genetic differentiation –40, almol (2012) . Indeed,P. vivax and P. falciparum populations from Brazil, Colombia and Papua New Guinea. Interestingly, for the P. vivax population from Brazil, we have noticed that older age was associated with a significant increase in HE values, whereas for the Brazilian P. falciparum population an inverse relationship to age was observed. A possible explanation for this opposite trend in the two Plasmodium species might be that the different level of endemicity for P. vivax and P. falciparum populations could result in differences in reaching acquired immunity  and thiRepublic , 44. In Plasmodium isolates detected in pregnant infected women could show different genotypic characteristics compared to non-pregnant women. High HE values were observed in the Brazilian P. vivax populations both from pregnant (0.805) and non-pregnant (0.841) infected women and the differences between the two HE values were not statistically significant. Similar findings have been recently described in Colombia [vivax populations from pregnant women included a small number of samples (n = 11), which may have affected the evaluation of LD in this subpopulation.Malaria infection in pregnancy is associated with high risk of both maternal and perinatal morbidity and mortality. It is recognized that pregnant women have a reduced immune response and therefore clear malaria parasites with difficulty and are more exposed to the severe form of the diseases . In our Colombia . The obsP. vivax episodes, we observed that the genotype of the parasites of the admission episode can differ or can be identical to the genotype of the subsequent episode and that these results are in line to what was reported in previous studies [By genotyping nine patients with subsequent  studies –49. Henc studies . In our P. vivax  and a cP. vivax and P. falciparum population structures. As already reported in previous similar studies [P. vivax populations were more diverse than those of P. falciparum and in some of the studied countries, the diversity of P. vivax populations was striking compared to the respective level of endemicity. Molecular surveillance of the Plasmodium genetic variability is of great importance in the context of malaria containment efforts, since a correct and extensive appraisal of the genetic makeup of plasmodial populations is crucial in the identification of the most adequate control strategies, particularly in countries that are in the pathway towards malaria elimination. In the last few years, many studies have included the investigation of the genetic variability of P. vivax isolates through the analysis of the MS diversity, however, those including these analysis in pregnancy are scarce [The findings of this study confirm the usefulness of MS analysis as a genetic tool for investigating  studies , 29, 51,e scarce ,32. For S1 Dataset(XLSX)Click here for additional data file.S1 Table(TIF)Click here for additional data file.S2 Table(TIF)Click here for additional data file.S3 Table(TIF)Click here for additional data file.S4 Table(TIF)Click here for additional data file.S5 Table(TIF)Click here for additional data file.


Serapias lingua.Fragmentation of habitats by roads, railroads, fields, buildings and other human activities can affect population size, pollination success, sexual and asexual reproduction specially in plants showing pollinator limitation, such as Mediterranean orchids. In this study, we assessed pollen flow, selfing rates, vegetative reproduction and female reproductive success and their correlations with habitat characters in nine fragmented subpopulations of To improve understanding of population structure effects on plant biology, we examined genetic differentiation among populations, pollen flow, selfing rates and clonal reproduction using nuclear microsatellite markers.ST was significantly correlated with the geographical distance between populations. Paternity analysis of seeds showed that most pollen flow occurred within a population and there was a positive correlation between percentage of received pollen and distance between populations.Smaller populations showed a significant heterozygote deficit occurred at all five nuclear microsatellite loci, the coefficient of genetic differentiation among populations was 0.053 and pairwise FThe fruit production rate varied between 5.10 % and 20.30 % and increased with increasing population size, while the percentage of viable seeds (78-85 %) did not differ significantly among populations. The extent of clonality together with the clonal and sexual reproductive strategies varied greatly among the nine populations and correlated with the habitats where they occur. The small, isolated populations tended to have high clonal diversity and low fruit production, whereas the large populations with little disturbance were prone to have reductions in clonal growth and increased sexual reproduction.S. lingua, where clones may have a greater ability to persist than sexually reproducing individuals.We found that clonality offers an advantage in small and isolated populations of  Fragmentation of plant populations, the process by which formerly continuous populations turn into patches of different sizes, isolated from each other, may have distinctive effects on populations: 1) affecting reproductive success, 2) altering patterns of pollen-mediated gene flow (pollen flow) and (3) affecting self-pollination and vegetative propagation. Although many plant populations are naturally isolated and small, populations of numerous plant species have become more isolated as a result of the recent anthropogenic fragmentation of habitats by roads, railroads, fields, buildings and other human activities  were calculated for every fruit.In addition, in each population five individuals with unopened flowers were bagged with a fine-meshed cloth to exclude pollinators to test for spontaneous autogamy. In June, the number of produced fruits was counted, and the ratio between the number of fruits/treated flowers was determined.One leaf from each individual in the three smaller populations and from each individual in the five selected areas of other six populations was sampled and stored in silica gel for subsequent DNA extraction and microsatellite  genotyping. Genomic DNA was extracted using a slight modification of the CTAB (cetyltrimethyl ammonium bromide) protocol of Doyle and Doyle . Approx.Serapias sp. [2 and 10 μl of reaction buffer. The amplification conditions were: 1 cycle of 94 °C for 3 min;30 cycles 30 s at 94 °C, 45 s at the locus specific annealing temperature (55 or 58 °C), and 30 s at 72 °C using a Perkin Elmer thermal cycler. One of the PCR primers for each locus was labeled with fluorescent dye . Labelled PCR products were run together with the internal size standard GeneScan ROX400 on an ABI 3110 , and individuals were genotyped using Genescan Analysis software and Genotyper software .To characterize the genetic structure of each population and genotype, we performed microsatellite genotyping on all the adult plants using five nuclear microsatellite loci previously isolated and tested on pias sp. , 30. AllMultilocus genotypes (MLGs) were assigned manually. Because individuals with the same MLG found in populations with both sexual and vegetative reproduction can be either ramets of the same genet or derive by chance from distinct events of sexual reproduction, we used the program GIMLET 1.3.2  to estimG) [G ranges from zero indicating that there is only one dominant clone, to one suggesting that every individual has a different genotype.Two different genotypic diversity indexes were calculated. The first measure was G/N, the ratio between the number of MLGs and the total number of individuals in a population . Values G)  which mea) and per population (Nap) [O), gene diversity (HE) [IS = 1 – HO/HE) were calculated for each locus and each population using FSTAT version 2.9.3.2 [T and HS [ST [T is the gene diversity in the total population, HS is the average gene diversity within populations, and FST is the coefficient of genetic differentiation among populations under an infinite allele model. Pairwise FST values were tested for significance by permuting genotypes among populations. To test for the presence of isolation by distance, a Mantel test between population-pairwise geographic distance and FST/(1 – FST) was applied [Population genetic analyses were based on a ‘corrected’ dataset in which all individuals with the same MLG were considered as ramets of a single genet. For nSSRs, the number of alleles, number of alleles per locus (Non (Nap) , observeity (HE) , and fix 2.9.3.2 . Departu 2.9.3.2 , with BoT and HS , and FSTd HS [ST  were est applied . Null al applied . Based o applied , employi applied  was used applied .Microsatellite profiles for each fruit were also determined to ascertain if fruit developed by plants in each population could have been produced by pollen transferred by individuals of the same population or different donors. In June, capsules were collected and seeds in the central part were used for molecular analysis. Seeds were observed under a binocular microscope and approx. 50 viable seeds (which means seeds with an embryo) from each capsule were collected and transferred into single 2 ml-Eppendorfs to extract their DNA. Nuclear microsatellite loci were amplified and analyzed following the protocol described above. Paternity analysis was performed by a likelihood-based approach based on multilocus genotypes for all adult genets and offspring using CERVUS version 2.0 . In thisAccording to the assigned paternity data, we categorized the fruit as derived from selfing, outcrossing within the study area, and outcrossing with a paternal parent that was not present in the study area. We defined the selfing rate as the number of selfed fruits divided by the number of examined fruits from each population.2) (Table S. lingua populations ranged from 300 m to 2.5 km). Three populations  showed significantly lower values of population size and higher values of population density than the other six populations, such as they had lower population areas  Table  and degras Table .Significant differences were detected among the populations in their fruit production rate. Indeed, the populations differed significantly in their fruit sets, which varied from 5.10 % to 20.30 % and was 14.53 % for the nine populations on average. More specifically, the three smallest populations in term of population size  showed lower values than the other populations, which showed values four times higher  bagged with a fine-meshed cloth to exclude pollinators showed any spontaneous autogamy.G), which ranged from close to zero (population C) to 0.794 (population H), with a mean value of 0.215 , and slightly higher values were shown by the other two (F and G) small populations Table . Higher PCR products were successfully obtained from all examined individuals, their fragment lengths fit into the predicted size ranges, and all examined loci were polymorphic across the nine populations. No significant linkage disequilibrium between loci was observed for any population, so all loci were used for further analyses.O = 0.38-0.42;HE = 0.52-0.60), while the other populations possessed higher heterozygosity (HO ranging from 0.77 to 0.80) that was close to expected values (HE ranging from 0.75 to 0.79) (Table IS) calculated at each nSSR locus in each population  varied among populations. Six populations showed a low heterozygote excess ranging from FIS = −0.02 (pop E) to FIS = −0.12 (pop A), while three others showed a significant heterozygote deficit (FIS = 0.22-0.28) at all five loci (Table ST) was estimated to be 0.053 for nSSRs. Pairwise FST/(1 – FST) was significantly correlated with the geographical distance between populations for nSSRs  and the number of alleles per locus ranged between 8 and 20 (data not shown). Three populations  had a lower mean allele number per population than the other populations, and possessed all alleles exhibited by natural populations. Moreover, in anthropic populations the observed heterozygosity was much less than expected , there is an approximately equal probability that a locus shows either a gene diversity excess or a gene diversity deficit. Populations that have experienced a recent reduction in their effective population size exhibit a correlative reduction in the number of alleles and gene diversity at polymorphic loci. But the number of alleles is reduced faster than the gene diversity. Thus, in a recently bottlenecked population, the observed gene diversity is higher than the expected equilibrium gene diversity computed from the observed number of alleles, under the assumption of a constant-size (equilibrium) population .In the paternity assignment experiments, 4967 fruits were obtained from 5176 plants in nine populations . But these values are similar to or slightly lower than those reported to date for other Mediterranean orchid genera, Dactylorhiza [Gymnadenia [Ophrys [In our investigations the number of alleles per locus (8–18) and the mean of 9.6 alleles per population are higher values than the alleles per locus (4–10) and alleles per population (3.6-5.6) detected by Pellegrino et al. , 43 in pylorhiza , Gymnademnadenia , 46, and [Ophrys , 48.E ranging from 0.69 to 0.79, average 0.694) compared with other microsatellite studies on orchids [The five markers included in this study showed medium levels of genetic variation (H orchids .ST=0.053) is due to the small geographic range of the S. lingua populations studied. Indeed, similar genetic differentiation values based on microsatellites have been reported in other small orchid populations of Caladenia huegelii [Gastrodia elata [The low value of genetic differentiation among populations  showed a substantial deficit in genetic diversity, the largest difference between observed and expected heterozygosity, and higher values of inbreeding coefficients (FIS), while the second group formed by the other populations possessed observed heterozygosity close to expected heterozygosity values and lower values of inbreeding coefficients , while the rate of gene flow decreased in populations farther from each other (distance from 1000 to 1500 m) and there was little or no inter-population gene flow between the three smallest and most isolated populations Fig. . In addiion size . In addiion size  showed tdividual , suggestdividual .S. lingua equally. Six larger S. lingua populations showed higher levels of clonality (DG = 0.71-0.79), for example, similar to those found in the endangered species Cypripedium calceolus  and reduced heterozygosity (HO = 0.38-0.42) in smaller populations, similar to those found in polish Epipactis atrorubens [Cephalantera rubra populations [S. lingua can persist in small, isolated populations where conditions are not favourable for sexual reproduction, providing a form of reproductive assurance by guaranteeing the survival of the species in case of limited pollinator service [S. lingua can explain the different levels of clonal propagation we found in different populations. In particular, a higher rate of asexual reproduction was found in C, F, and G than in other populations, the former consisting of a few hundred individuals located in a restricted area (about 70 m2) closed to a crossroads, the latter comprising a thousand individuals in a larger area (~0.5 ha). Populations subjected to more environmental stress and fragmentation by roads, railroads, fields, buildings and other human activities show higher levels of clonality [The results reported here showed that clonality represents a common reproductive strategy in all analysed populations, but clonality did not affect the different populations of  = 0.97; ), while rorubens  and Cephulations , was a culations . Althougulations , species service . Thus, tlonality , 67.S. lingua, whereby clones may have a greater ability to persist than sexually reproducing individuals [This study represents one of the few analyses of the effects of population structure on the pollen flow and clonal growth of a deceptive Mediterranean orchid. Population fragmentation is likely to reduce reproductive success due to reductions in population sizes and increases in the geographic distance between populations. We found that clonality offers an advantage in small and isolated populations of ividuals . Since c


Garra barreimiae is a cyprinid fish from the southeastern Arabian Peninsula, which inhabits regularly desiccating wadis and survives in isolated ponds or underground. In 1984 a cave-dwelling population was found in the Al Hoota cave system and previous genetic analyses revealed some differentiation with limited gene flow between the surface populations and the cave population. Since no suitable markers are available for evaluation of gene flow between the cave population and the adjacent surface populations, we focused on designing and establishing novel microsatellite markers from next generation sequencing data.G. barreimiae  were used for analyses and characterization of the loci. On average, the number of alleles per locus is 7.6 (range: 2–20). Two markers displayed indication of linkage disequilibrium in both populations . Significant deviation from Hardy-Weinberg equilibrium was observed at four loci in the Misfat Al Abriyeen population  and at two loci in the Wadi Al Falahi population . Three of the microsatellite loci were significant for null alleles in one of the two populations . Expected and observed heterozygosities ranged from 0 to 95.0% respectively from 0 to 95.8%  and from 0 to 89.1% respectively from 0 to 95.0% (Misfat Al Abriyeen). Fourteen of these markers were successfully cross-amplified in G. rufa.19 microsatellite markers containing di- and tetranucleotide simple sequence repeats were developed from 454 sequences. Forty-four individuals from two surface populations  of G. barreimiae and potentially even in related species.This 19 microsatellite loci provide a useful tool to understand the structure and genetic differences of populations. Moreover, these markers will help to evaluate species delimitation in  Garra barreimiae Fowler & Steinitz (1956) is endemic to the southeastern Arabian Peninsula, where it is common in ponds and rivulets of the Hajar mountains in the northern Oman and the United Arab Emirates[G. barreimiae. Previous genetic analyses on the basis of the mitochondrial control region revealed a differentiation between cave and surface populations and possible gene flow as indicated by shared haplotypes[Garra barreimiae barreimiae, which can be found throughout the northern Oman, G. b. gallagheri Krupp[G. b. shawkahensis Banister & Clarke[Garra barreimiae may be comprised of more than one species. In the present study, we analysed individuals of G. barreimiae from two surface populations: Misfat Al Abriyeen is a locality near the mountain village Misfat, north of Bahla. The fish were collected in the Falaj drainage system, which channels the water through the villages’ gardens. About 15 km southwards from Misfat the small water body Wadi Al Falahi, a natural drain from the surrounding mountains, is located. Both localities hold numerous individuals of G. barreimiae. The individuals of G. rufa, which were used to test whether cross-species amplification works in another species of the genus, were commercially acquired.The cyprinid Steinitz 956 is enEmirates. Most ofEmirates. All aduEmirates. Until nplotypes. The sepplotypes. AccordiSteinitz 956 is en& Clarke revealedG. barreimiae[G. barreimiae using the new technological advantages.To estimate gene flow between the different morphs and populations for this – in terms of evolutionary biology – highly interesting fish species specific genetic markers are needed. The method of choice for this purpose are microsatellite markers, which however, were up to now not available for rreimiae. Next gerreimiae and multFor DNA extraction, we used tissue from a finclip taken from one specimen of the cave population. We followed the DNA-extraction protocol  and isolated genomic DNA. Approximate 500 ng DNA was used for 1/20 of a plate for 454 sequencing on a Roche/454 GSFLX sequencer at the Genomics Core Facility University of Leicester, UK. Sequencing generated a total of 36126 reads. The sequence reads were assembled into 193 contigs with size ranging from 500 – 3547 bp by using Newbler . Of the remaining 35503 non-assembled reads, we also considered 29418 high quality reads  with sizes between 100 and 731 bp.We designed primers with the program QDD version 2.1, which iWe selected primers with a high GC-content displaying no primer dimers and similar annealing temperature (comparing forward- and reverse primers of each primer pair). We tested the potential primer sequences against the original sequence with BioEdit version 7.2.5. In caseFinally 36 different microsatellite loci were left, which were first screened for consistent amplification via PCR. Those primers amplifying a product and working in different samples were chosen for microsatellite analyses of individuals from two surface populations . These individuals were collected in accordance with the local legislation of the Sultanate of Oman .Since we wanted to perform multiplex PCRs, we made use of the program Multiplex Manager version 1.0, a tool Multiplex PCR was performed either using the multiplex kit in 10 μl reaction volumes , or using the TopTaq kit also with a reaction volume of 10 μl . According to the three different multiplex reactions, those PCRs were named MPR1/2/3 for multiplex kit reaction 1/2/3 or TTR1/2/3 for TopTaq kit reaction 1/2/3. We followed a two-step cycling protocol for each reaction as follows: initial denaturation at 95°C for 15 min, 2 cycles denaturation at 94°C for 30 s, annealing at 65°C (R1), 64°C (R2), 60°C (R3) for 90 s, extension at 72°C for 60 s. This was followed by 35 cycles denaturation at 94°C for 30 s, annealing at 60°C (R1), 59°C (R2), 54°C (R3) for 90 s, extension at 72°C for 60 s and a final extension at 60°C for 30 min. PCR products were diluted 1:20 with AD, multiplexed according to the reactions and added to a mixture of Hi-Di formamide and size standard . After a denaturation at 94°C for 4 min, the products were run on a 3130xl Sequence Analyzer .o) and expected (He) heterozygosities and tested for linkage disequilibria and deviations from Hardy-Weinberg equilibria using Genepop v 4.2.2[ST values).After fragment analyses and testing for polymorphic loci, 19 different microsatellite markers proved to be suitable for genotyping. Primer sequences, PCR conditions, and their respective GenBank accession numbers are listed in Table  v 4.2.2. Subsequ v 4.2.2 to test  v 4.2.2. All locG. rufa . Out of the 19 loci, 14 were positive in G. rufa and proved to be polymorphic in at least 75% of the individuals (Table G. rufa.All markers were tested for cross-species amplification in 22 individuals of G. barreimiae and might also be used in other species of this genus. The results from using these microsatellite markers will provide deeper insights into the species’ population structure and its genetic diversity. Furthermore, it will be of utmost interest to use these loci in the cave population of G. barreimiae and estimate gene flow between the cave and the surface population. This will help to evaluate species delimitation of G. barreimiae and to assess whether any cryptic species exist besides G. barreimiae.The set of microsatellite loci isolated and tested in the present study will be useful DNA markers for further population genetic studies of


Acromyrmex striatus , a leaf-cutting ant species restricted to the open plains of South America. Additionally, we modeled the distribution of this species to predict its contemporary and historic habitat. From the partial sequences of the mitochondrial gene cytochrome oxidase I of 128 A. striatus workers from 38 locations we estimated genetic diversity and inferred historical demography, divergence time, and population structure. The potential distribution areas of A. striatus for current and quaternary weather conditions were modeled using the maximum entropy algorithm. We identified a total of 58 haplotypes, divided into five main haplogroups. The analysis of molecular variance (AMOVA) revealed that the largest proportion of genetic variation is found among the groups of populations. Paleodistribution models suggest that the potential habitat of A. striatus may have decreased during the Last Interglacial Period (LIG) and expanded during the Last Maximum Glacial (LGM). Overall, the past potential distribution recovered by the model comprises the current potential distribution of the species. The general structuring pattern observed was consistent with isolation by distance, suggesting a balance between gene flow and drift. Analysis of historical demography showed that populations of A. striatus had remained constant throughout its evolutionary history. Although fluctuations in the area of their potential historic habitat occurred during quaternary climate changes, populations of A. striatus are strongly structured geographically. However, explicit barriers to gene flow have not been identified. These findings closely match those in Mycetophylax simplex, another ant species that in some areas occurs in sympatry with A. striatus. Ecophysiological traits of this species and isolation by distance may together have shaped the phylogeographic pattern.Past climate changes often have influenced the present distribution and intraspecific genetic diversity of organisms. The objective of this study was to investigate the phylogeography and historical demography of populations of  Climatic oscillations during the Quaternary Period have a strong effect on the genetic diversity and distribution of extant species –4. IncreHowever, for southern Brazil, several studies have pointed out ambiguities in the predictions from these models , 13. It Acromyrmex striatus  is restricted to grassland habitats in the temperate zones of South America (above 30°). This makes it an excellent model to evaluate the influence of historical climate fluctuations on the origin and evolutionary dynamics of plains associated with open vegetation in southern South America. A. striatus is common in coastal sandbanks (restinga) and sandy soils throughout the Pampas (including the campos sulinos), the Brazilian southern coast in the states of Santa Catarina and Rio Grande do Sul, parts of the Chaco in Argentina, and the extreme south of Paraguay [A. striatus is an intriguing species with regards to taxonomic, phylogenetic, and cytogenetic aspects. Cristiano et al. (2013) indicated that A. striatus has the same chromosome number as the genus Atta, but in a phylogeny it clusters with neither its congeners nor Atta, but forms a sister group to the other leaf-cutting ants evaluated [Ants are an important component of all terrestrial ecosystems, but so far little attention has been given to the phylogeography of South American taxa . The leaParaguay . Its nesParaguay , 27. In valuated .A. striatus  and to get insights into the history of South American plains we analyzed the phylogeography and population genetics of this species across much of its distribution. Our goal was to determine the genetic structure of its populations and the geographical patterns of genetic variation and to use these data to investigate how the glacial and interglacial periods have influenced the distribution in this species. In addition, we estimated the current potential distribution area and the historical potential area of A. striatus using paleoclimate models to allow a more detailed evaluation of the demographic history and phylogeographical patterns.To better understand the evolutionary history of Acromyrmex striatus were collected at 38 sites in Brazil and Argentina  [cytochrome oxidase subunit I gene (COI) and part of the nuclear gene wingless (wg). The primer pair CO11-3F and CO12-4R [Samples of . (2005) . The ext CO12-4R  was used CO12-4R  and Wg10 CO12-4R  for the Taq polymerase (Promega), dNTPs (0.25 mM each), MgCl2 (2.5 mM), 1X Taq buffer (Promega), primers (0.48 mM each) and 1 μL of DNA (50 ng) in a final volume of 25 μL. The amplification conditions included an initial denaturation step at 94°C for 3 min, followed by 35 cycles of 1 min at 94°C for DNA denaturation, 1 min at 53.5°C (COI) or 55°C (wg) for annealing of the primers and 72°C for 2 min (COI) or 1 min (wg) for primer extension followed by a final extension step at 72°C for 7 min.PCR was performed using 1U of www.macrogen.com), purified and sequenced directly in both directions (forward and reverse) using the same primers as in the amplification reactions.The amplicons were sent to Macrogen Inc., South Korea . The highest and statistically significant value of FCT for a determined K was identified among the groups analyzed.The population structure was evaluated by spatial analysis of molecular variance (SAMOVA) implemented in the SAMOVA 1.0 program . This ana priori population group, to test possible bias due our population grouping.An analysis of molecular variance (AMOVA) was estimated by the genetic variation and fixation indices implemented in Arlequin 3.5 . AMOVA wTo assess whether genetic variation could be explained by isolation by distance, a correlation was performed between the logarithm of genetic distance and geographic distance by Mantel tests  using thwww.fluxus-engineering.com) using the median-joining algorithm to verify the existence of a relationship between the distribution of haplotypes and the geographic distribution of the analyzed samples of A. striatus.A gene genealogy was reconstructed in the program Network 4.6.1.1. . Treating this as an intraspecific analysis, the identical sequences were removed and the coalescence of constant size model was used . Analyzes were conducted using the SRD06 nucleotide substitution model [To determine when major clades diverged and if the time of divergence corresponds to any of the known events in the Quaternary Period, we estimated the divergence times between clades following Seal et al. 2011) . The tim . The tion model , which aon model , to veriA. striatus was determined using the maximum entropy algorithm in Maxent [A. striatus under current and paleoclimates, we used 311 geo-referenced point locality data obtained from our own fieldwork (http:\\www.worldclim.org) and with a spatial resolution of 2.5 arc-min [A. striatus and its symbiotic fungus [A. striatus using 75% of the species data for training and 25% for testing the models, setting Maxent to run with random seed, a convergence threshold of 0.00001 with 500 interactions and 10,000 maximum number of background points. We selected the logistic output format to generate response curves jackknife results. In order to avoid spurious predictions, we defined a minimum training presence threshold, which is equal to the prediction of a model with a minimum value of presence for any occurrence. The modeling performance was evaluated using the average values of the 25% training gains through 10 bootstrap replicates and the mean of the area under the curve (AUC) of the receiver operating characteristic curve (ROC).The potential current and quaternary distribution of n Maxent . This moieldwork . The cli arc-min . These vc fungus , and to c fungus . Considehttp:\\www.worldclim.org) [A. striatus during the Quaternary.The model of potential current species distribution was trained and projected to three paleoclimate scenarios, one for Last Interglacial Period  and two for the Last Glacial Maximum period (LGM – 21 thousand years B.P.), the MIROC (Model of Interdisciplinary Research on Climate) and CCSM3 (Community Climate System Model) from WorldClim database (lim.org) , in ordeA. striatus, and no stop codon or indel was observed. For the nuclear gene wingless, 338 base pairs were sequenced (KU162879-KU162939); however, this gene did not show any intraspecific polymorphism. Thus, all following analyses were only performed with the COI gene. All COI sequences were deposited in GenBank under the accession numbers KR605508–KR605635. Of the 66 variable sites in COI, 58 were informative for parsimony and eight were singletons. We identified 58 haplotypes. The difference in the number of nucleotides between sequences averaged 12.96 nucleotides (min—max = 0–37), and the maximum divergence between two sequences was 4.01%. Although, the definition of what is a natural population is always an arbitrary issue. The population evaluations that follow inhere should be viewed with thoughtfulness since they could bear possible bias introduced by our population grouping strategy. Haplotype diversity (Hd) was 0.908 ± 0.021 and total nucleotide diversity (π) was 0.01406 ± 0.00094 across all populations. The populations presenting the highest and lowest haplotype diversity were ARGE and FLOR, respectively. The population FLOR also showed the lowest nucleotide diversity, while the highest was observed in NORT  being the most abundant . In general, the lowest values of FST were observed between neighboring populations. FST values between populations of Santa Catarina were not significant, suggesting that there is no significant lack of gene flow between these populations , indicating that the genetic differentiation between A. striatus populations may be partially attributed to isolation by distance. An analysis after exclusion of the most distant population from Argentina still yielded a significant result .The logarithm of genetic distance and the geographical distance were significantly correlated  and Tajima’s D (p < 0.05) tests. These results indicate that haplotype frequencies for MOTD differ from those expected by the neutral evolution hypothesis and suggest a recent population expansion. The unimodal distribution of pairwise haplotype differences in the mismatch distribution analysis of MOTD gave a similar result  and the demographic expansion models .Neutrality tests were conducted for each of the 13 populations of r result , and theA. striatus, although it included a few areas that are not suitable for this ant . The models achieved over 2.5 regularized gain values indicating good fit to presence data. The AUC was high, with a mean value of 0.981 and a standard deviation of 0.009, showing no over-fitting around the presence data. Projection of the model for the potential occurrence of A. striatus on the paleoclimatic parameters of the Last Glacial Maximum (LGM) and Last Interglacial Period (LIG) recovered appropriate potential areas for the occurrence of A. striatus  shared haplotypes with coastal populations, matching the observation that a few kilometers of sea do not represent an impassable barrier for flying insects [Across its range, populations are genetically highly structured, presumably reflecting long-lasting isolation of population groups in the past. Populations within the same geographical region are genetically similar, suggesting restricted gene flow between geographically separated groups of populations. AMOVA confirmed the existence of five previously defined population groups see , and SAM insects , 43.Mycetophylax simplex [A. striatus in parts of its coastal range. This congruency may suggest that open sandy habitats, free of obvious geographical barriers, facilitate ant dispersion over long distances. However, A. striatus populations in the southern part of the coastal plain were genetically structured in contrast to those of M. simplex. This discordance might be due to differences between these two species in dispersal capabilities and habit requirements. M. simplex is thought to disperse over several kilometers whereas A. striatus was suggested to disperse only over short distances [M. simplex nests always in open areas, which are abundant on the coast. In contrast, A. striatus nests on open, well-drained sites close to shrubby vegetation, which are patchily distributed on the coast.A similar pattern of unrestricted gene flow among coastal populations was also observed in the endemic sand dune ant  simplex , which oistances . FurtherA. striatus have low dispersion capability [A. striatus occurs. Both plains are characterized by sandy, permeable soils, and are influenced by strong winds. The coastal plains are geologically more recent and formed mainly of sandy soils. The Pampas are older environments with several different types of soil [A. striatus and matches similar findings in subterranean rodents of the genus Ctenomys [Petunia [Calibrachoa plants from the same habitat [Our study did not identify explicit physical barriers to gene flow. Although able to fly, queens of pability  and succpability . Selectipability . Thus, t of soil , 55 and Ctenomys , 21, and[Petunia , and Cal habitat .ST values, mainly between Argentine populations, were quite extreme . The reA. striatus are geographically structured as a result of stable historical demography during the evolutionary time and low dispersion capacity across sandy soil patches. Isolation by distance appears to have a strong influence and suggests equilibrium between migration and genetic drift in this ant species. These results are consistent with the hypothesis that the Pampas and the coastal region of southern Brazil were not strongly affected by the expansion of forests during the interglacial periods. Due to the diversity of responses observed in species inhabiting open vegetation environments, more species from such environments need to be studied to better understand the general processes that governed the diversification of these ecosystems.In conclusion, our results suggest that populations of S1 FigA. striatus is superimposed on the map (The haplotype network  of the m the map  approxim(TIF)Click here for additional data file.S2 FigHistograms represent the observed frequencies of the pair-to-pair differences between haplotypes and lines represent the simulated curve for the population MOTD under the demographic expansion (solid line) and spatial expansion models (dashed line).(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 TableValues shown in bold are significant (p < 0.05).(PDF)Click here for additional data file.