Toxoplasma gondii, which affects warm-blooded animals including humans. Its prevalence rates usually vary in different regions of the planet.Toxoplasmosis is a zoonosis caused by an obligate intracellular parasite, Toxoplasma gondii antibodies by indirect fluorescent antibody test (IFAT) was also evaluated in order to compare it with enzyme-linked immunosorbent assay (ELISA) and to assess the use of 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) and o-phenylenediamine dihydrochloride chromogens.In this study, an analysis of the seroprevalence of toxoplasmosis among Brazilian students was proposed by means of IgG specific antibodies detection. The presence of anti-The IFAT method showed a seroprevalence of 22.3%. These results were similar to those obtained by ELISA (24.1%). The seroprevalence was directly estimated from the IgG avidity, which showed that in a sample of 112 students, three of them had acute infection, an incidence of 1.6% in the studied population.T. gondii-reagent serum. The extrapolation of results to this population must be carefully considered, since the investigation was conducted on a reduced sample. However, it allows us to emphasize the importance of careful and well prepared studies to identify risk factors for toxoplasmosis, to adopt preventive measures and to offer guidance to at-risk populations about the disease.In this study, the use of different chromogenic substrates in immunoenzymatic ELISA assays did not display different sensitivity in the detection of  Toxoplasma gondii is a protozoan parasite that is responsible for toxoplasmosis in warm-blooded animals [ animals –7. The i animals , 8–14. T animals .Human infection is usually asymptomatic. The main clinical signs after the onset of the disease include lymphadenitis, fever, asteny, and myalgia. Encephalitis, meningoencephalitis, ocular lesions, septic syndrome, myocarditis or hepatitis may be found occasionally , 16, 17.Recently the diagnosis of toxoplasmosis has been drawing close attention. Several serological techniques have been applied and have shown good sensitivity, specificity, and are quickly carried out. Among different diagnostic techniques, the following are noteworthy: Sabin and Feldman technique, indirect fluorescent antibody test (IFAT), complement fixation (CF), hemagglutination (HA), enzyme-linked immunosorbent assay (ELISA) and immunosorbent agglutination assay (ISAGA) –27.T. gondii antibodies carried out in the Republic of Benin revealed a seroprevalence of 87.7% [Seroprevalence indexes have varied in different countries and regions of the planet . Serologof 87.7% . In diffof 87.7% , 4.T. gondii.Three suspicious cases of toxoplasmosis were reported in São Paulo State University (UNESP), in Assis, São Paulo state, Brazil  stirring up a series of controversies and discussions. The first one refers to clinical likelihood of the disease in college students. However, no serological test was conducted to uphold the diagnosis. The second one is the relation between these cases was the increased cat population. And, finally, a student who had an eye affliction and tested positive for T. gondii antibodies in students by IFAT, in order to compare IFAT and ELISA techniques for the toxoplasmosis diagnosis, and to evaluate the use of 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) and o-phenylenediamine dihydrochloride (OPD) chromogens in the ELISA test for the toxoplasmosis diagnosis.Since these reports and suppositions were based on inconsistent diagnostic assertions and subjective causal links, without the due clinical evaluation and laboratory diagnostic evidence, a project was devised to find out the seroprevalence and incidence of toxoplasmosis in a sample of UNESP students, by means of serological tests for IgG specific antibodies detection. In addition, the study also verified the presence of anti-The project was carried out at the Laboratory of Human Parasitology and Immunology of São Paulo State University (UNESP), in Assis, with the cooperation of the Laboratory of Zoonoses and Public Health of the Department of Preventive Veterinary Medicine, State University of Londrina (UEL), in Londrina, Paraná state, Brazil  and of the Laboratory of Protozoonoses of the Institute of Tropical Medicine of São Paulo, University of São Paulo (USP), São Paulo, São Paulo state, Brazil .Assis is located in western São Paulo state, Brazil, 434 km away from the state capital and comprises a population of 100.911 inhabitants. The annual average temperature is 21.37°C  and the sample size was proportional to the number of students in each course  technique for the screening of anti- Camargo . Human aT. gondii IgG antibodies were determined by the methodology of Camargo [et al. [T. gondii soluble antigen extract were used for OPD ELISA; and those sensitized with 10 μg/mL of T. gondii soluble antigen extract were used for ABTS ELISA. Plates were sensitized with antigen by incubation at 4°C for 18 hours, with 100 μL of antigen solution per well. After washing, plates were blocked by incubation for one hour at 37°C with 250 μL of PBS-T per cavity  containing 3 g of skimmed milk powder . Afterward, plates were washed five times in PBS-T for five-minute periods. Plates containing serum with human-peroxydase-conjugated anti-IgG dilutions  were incubated for the test in stove for one hour. Following both incubations, plates were washed three times in PBS-T for five minutes. The color reaction was carried out subsequently in the dark for 15 minutes at room temperature with 100 μL of the OPD chromogenic solution  and was quenched by the addition of 25 μL of 2 M sulfuric acid solution. For the OPD chromogenic solution, the optical density (OD) was determined by the reading at 492 nm, and for the ABTS chromogenic solution, by the reading at 414 nm and 612 nm, in a plate spectrophotometer . For OPD ELISA and ABTS ELISA, in both the first analysis and the one six months after, the cutoff point was obtained from the mean of results from negative control sera evaluated by IFAT plus two standard deviations.Anti- Camargo  and Uchô [et al. . Plates et al.[T. gondii protein antigen to the wells at 1 μg/mL concentration, in an 8 M urea chaotropic (Sigma®) solution in PBS. After blocking possible free sites, the wells received 100 μL of PBS-T diluted serum samples . Plates were incubated for one hour at 37°C and washed five times in PBS-T. For each dilution, two wells received additional ten-minute incubation at 37°C with 6 M urea neutral chaotropic solution (Sigma®) in PBS-T. Wells containing the control solutions were kept in PBS-T. Afterwards, the antibody development was carried out with the use of human anti-IgG peroxydase conjugate (Sigma®).The same procedure was used for IgG analysis through in-house ELISA, according to the methodology described by Hedman et al.. The plaThe estimation of effective titer of each serum was carried out by means of the isolated dilutions of each sample in a log-log linear regression model. The titers of total or high avidity chaotropic-resistant antibodies were achieved by means of log-log regression, with the use of the values able to generate 1.0 absorbance in ELISA (Log = 0). Avidity (AVT) was determined by the percentage of chaotropic resistant titers. Samples with AVT values higher than 30% were considered as high avidity. The optical density (OD) was determined by the reading at 492 nm in a plate spectrophotometer.The correlation rate was used for the analysis among the interspecific titers and the analysis of association among the positive results and variables related to risk factors . Values were submitted to the chi-square analysis, with the help of the Epi Info version 6.04 statistics software . A reliaThe present study was approved by the Research Ethics Committee of Marília Medical School  under protocol n. 449/08. It included only patients who authorized the collection of blood samples through free informed consent according to the Brazilian National Committee of Ethics in Research (CONEP), resolution 236/96, National Health Council, Brazilian Ministry of Health.T. gondii IgG antibody screening by means of IFAT and ELISA with OPD or ABTS chromogen substrate. The correlation between the results obtained in the immunoenzymatic and immunofluorescence assays may be seen in Table Serum samples from 112 students were submitted to anti-The comparison between OPD and ABTS ELISA enzymatic assays showed high agreement (kappa = 0.9024), 92.5% sensitivity  and 97.6% specificity (95% CI: 93.2-97.8) and the positive predictive values were 92.5% (95% CI: 90.6-96.3) and negative predictive values 97.6% (95% CI: 93.2-97.8) , similar to the one found in two imunoenzymatic tests. The OPD ELISA test showed a seroprevalence of 24.1% (95% CI: 16.2-32.0), the same found in the ABTS ELISA.The confirmation and comparison of IgG serological test results by IFAT and ELISA with OPD and ABTS chromogen substrate methods are shown in Table T. gondii-reagent serum. The ELISA test with OPD chromogenic substrate with plates sensitized with 1 μg of antigen revealed 27 positive sera, the same result observed in the ELISA IgG test with ABTS chromogenic substrate and plates sensitized with 10 μg of antigen.The use of different chromogenic substrates in the immunoenzymatic ELISA assays did not show different sensitivity for detecting T. gondii antibodies. The analyses of 112 students showed that three who tested negative in the first time, six months after were seropositive by IFAT, OPD ELISA and ABTS ELISA. The other serum samples showed the same results of first analysis, with exception of four samples: two were negative by IFAT and OPD ELISA and positive by ABTS ELISA whereas the other two were negative by IFAT and ABTS ELISA and positive by OPD ELISA. The three seroconverted samples were submitted to IgG avidity test. Students showed low IgG avidity, indicating recently acquired infection , which allows projecting an annual incidence of 3.2% in the studied population.T. gondii infection frequency. No differences were observed when variables taken into account were: gender , contact with the cats of the university , consumption of raw vegetables , and the direct contact with soil . The variables contact with cats outside the university  and ingestion of raw or undercooked meat  were the statistically significant.Table T. gondii antibodies by IFAT showed a 22.3% prevalence in students. The seroprevalence data achieved by IFAT serological method were confirmed by the immunoenzymatic ELISA assay with OPD and ABTS chromogenic substrate (24.1%). The tests had highly coherent results, an unusual fact for assays with different antigens such as IFAT and hemagglutination [The screening of IgG anti-ubstrate .1%. The et al.[et al.[Toxoplasma gondii acute infection diagnosis, some patients have shown persistent low avidity antibodies against the lysate of T. gondii antigenic cells for several months [Therefore, as proposed by Tanyuksel et al. and Reis[et al., the avil months , 45. On l months , 47.Another aspect that may interfere in seroprevalence results of a given sample refers to the chromogen used in immunoenzymatic assays. In addition, when comparing OPD chromogen sensitivity with ABTS, it is possible to find that OPD chromogen is more sensitive and detects antibodies expressing higher optical densities as previously reported . Thus, wIn this study, ELISA test with IgG antibodies, ABTS or OPD chromogen, demonstrated the same sensitivity and specificity for toxoplasmosis reagent sera Table . HoweverA careful evaluation of serological tests may result in useful data for the prevention and institution of protocols for the detection of groups at risk, including the IgG avidity test. Transversal studies, as simple as this one, may help in a correct analysis of the results and, accordingly, collaborate for proposals and development of strategies to facilitate the interpretation of diagnosis, and this may result in more reasonable prophylactic measures for controlling this infection.T. gondii[T. gondii infection [Soil and consumption of food contaminated with oocysts are considered, in some regions, the major sources of infection by . gondii. Howevernfection –52.et al. [et al. [Concerning gender, no significant differences were noted, which is in agreement with Souza , Daguer et al.  and Shar [et al. .T. gondii antigen for each substrate showed the same sensitivity for detection of toxoplasmosis reagent sera  (ABTS) and O-phenylenediamine dihydrochloride (OPD) chromogens in immunoenzymatic ELISA assay with adequate quantities of T. gondii infection. Therefore, one may conclude that the most relevant risk factors for toxoplasmosis were contact with cats outside UNESP and eating raw or uncooked meat (Table According to the current findings, the cat population within the university is not a risk factor for at Table .The extrapolation of these results to the general population must be carefully considered, since the investigation was conducted on a reduced sample. However, it allows stating the importance of careful and well prepared studies, not only to identify risk factors for toxoplasmosis, but also to adopt preventive measures and offer guidance to at-risk populations. Thus, the implementation of simple cross-sectional studies may provide important epidemiological contribution and establish consistent causal associations, avoiding thereby the development of hypotheses that do not support rigorous questioning.


Gasterosteus aculeatus). Plateless stickleback have been found in only a few lakes and streams across the world; they represent the far extreme of a phenotypic continuum (plate number) that has been studied for years, although plateless individuals have not yet been the subject of much investigation. We use a dense single nucleotide polymorphism dataset made using RADseq to study fish from three freshwater populations containing plateless and low plated individuals, as well as fish from full plated marine populations. Analyses were performed using FastStructure, sliding windows FST, Bayescan and latent factor mixed models to search for genomic differences between the low plated and plateless phenotypes both within and among the three lakes. At least 18 genomic regions which may contribute to within‐morph plate number variation were detected in our low plated stickleback populations. We see no evidence of a selective sweep between low and plateless fish; rather reduction of plate number within the low plated morph seems to be polygenic.Understanding the genetic basis of traits involved in adaptive divergence and speciation is one of the most fundamental objectives in evolutionary biology. Toward that end, we look for signatures of extreme plate loss in the genome of freshwater threespine sticklebacks ( Gasterosteus aculeatus).A fundamental objective in evolutionary biology is to understand the genetic basis of traits involved in adaptive divergence and speciation Ellegren . The stuThe threespine stickleback is a well‐known model organism for the study of adaptive evolution. Throughout the Northern Hemisphere this species colonized lakes and streams from its ancestral marine environment, following the glacial retreat at the end of the Pleistocene (Bell and Foster EDA (Ectodysplasin‐A) has been identified as the major locus controlling plate morph variation  along the Norwegian coastline in 2011 and 2012 . From the marine populations the individuals were sampled randomly, while we collected at least 10 low plated and 10 plateless individuals from each freshwater lake. The exact number of plates was counted for all fish included in the latent factor mixed models (LFMM) analyses . In addition, 305 fish from Melavatn, 215 fish from Mosvatn, and 194 fish from Bårdsrudtjern were scored for presence/absence of plates in order to determine the proportion of low plated and plateless fish in each lake. To count plate number, sticklebacks were removed from individual storage containers and dried for approximately 5 min before one of us carefully counted all the lateral plates on both sides of the stickleback using a microscope.PstI (NEB); (2) each sample was ligated to a unique barcoded P1 adapter prior to pooling in a single library; (3) libraries were sheared by sonication on a Bioruptor (Diagenode) where the target size range fraction (300–500 bp) was achieved after six cycles of sonication; (4) after concentration to 25 μL by DNA capture on magnetic beads (beads solution:DNA = 0.8:1), libraries were size selected by gel electrophoresis and manual excision; (5) capture on magnetic beads (beads solution:DNA = 0.8:1) was employed in all following purification steps ; (6) PCR amplification was performed in 8 × 12.5 μL aliquots pooled after the amplification in order to reduce amplification bias; (7) DNA concentration of libraries was quantified by a fluorometric‐based method  and molarity checked on an Agilent Bioanalyzer using an Agilent DNA 1000 Kit. A final volume of ca. 20 μL per library with a DNA concentration of 20–25 ng/μL was submitted for paired‐end 100 bp sequencing on the ILLUMINA HiSeq2000 sequencer at the Norwegian Sequencing Centre, University of Oslo. Libraries were sequenced in 12 lanes (each library on two lanes). Of the 144 individuals sequenced in these six libraries, 128 fish were included in the following downstream analyses, 68 freshwater (at least 20 from each population) and 60 marine (20 from each population) – 16 individuals from the total sequenced 144 were excluded due to poor sequencing quality.Genomic DNA was extracted using the DNeasy Tissue kit  following the manufacturer's guidelines. We created six RAD libraries of 24 individuals each, following the process outlined in Etter et al.  with theStacks  statistical model.Raw sequence reads were demultiplexed using python scripts (in‐house scripts available upon request). Filtering out individuals with more than 75% missing loci resulted in 107,240 loci in 74 individuals with an average of 35% missing data per locus. Plotting the positions of the SNPs along the sequence across all the loci, we observed an unexpected increase in the occurrence of SNPs in the last three base pairs. As SNPs should be evenly distributed along the sequences, we identified this accumulation as an artifact of the SNP calling process. Trimming the reads to a shorter length before the SNP calling didn't alleviate the problem; we therefore removed SNPs found in the last three base pairs of the sequence. We further filtered for a maximum of 65% heterozygosity. We set this arbitrary cut‐off to reduce the risk of including paralogs  in our dataset. GC content was normally distributed in our loci with an average value of 0.47, and SNPs were distributed equally across the length of the read until 92 bp  This final filtering resulted in a dataset containing 92,979 loci genotyped in 74 samples that were used for the subsequent analyses, with the exception of the pairwise FST analyses that was run using the script populations within Stacks, applying a different filtering strategy to our initial catalog (see below).The catalog of loci created by Stacks was exported, checked and filtered using custom Figtree . FastStructure performs inference for the simplest, independent‐loci, admixture model as an implementation of the software Structure, specifically suited for analyzing large datasets of bi‐allelic loci such as SNPs. It uses a variational Bayesian framework for inferring the structure in the population and heuristic scores to identify strong population structure in the data. As only independent biallelic loci are allowed, we selected one SNP at random from each RAD locus. In order to speed up the computations, the dataset was reduced to a random selection of 4850 bi‐allelic loci that were considered as suitable to provide evidence of any signature of genome‐wide population structure in the data. We tested a flat beta prior distribution over population‐specific allele frequencies at each locus (linear prior) using a range of k values  from 1 to 2. The script choose.py included in the fastStructure package provides two estimates of the best k: one that maximizes the marginal likelihood, and a second estimate that best explains even very weak structure in the data. Both values for k were stored and the analysis repeated 100 times. We further ran fastStructure on all samples, including the three marine populations, exploring k values between 2 and 8. As before, 100 independent replicates including the structure.py script (linear prior) on the selected values for k and the choose.py script were run and results checked for convergence.FastStructure  for the kernel smoothing, and filtered out any alleles with a frequency of <0.015. The smoothed FST was plotted against physical location across each chromosome for each within‐lake morph pair and for each freshwater versus marine population comparison using R v.2.10.1 . We visually layered all of our pairwise FST comparisons to detect areas of high FST that converge across the comparisons.Using the FST values that are then interpreted as signatures of local adaptation. It incorporates the uncertainty due to small sample size in the inference and it is therefore suited for the comparison between the two morphs within each lake separately. Bayescan was run on each freshwater lake sample separately to search for outliers between the plateless and lowplated fish. For each analysis, we ran two long chains of 50,000 iterations using prior odds of 100 and assessed the statistical significance of a locus being an outlier by the use of q‐values. As we expected to find outlier loci differentiating all marine fish versus all freshwater fish, we ran an additional analysis between these two groups to confirm the presence of these expected outliers.Two additional tests for detecting outlier loci were performed using the set of 92,979 loci resulting from the data filtering described above under “Sequence filtering and SNP calling.” The fully Bayesian approach developed by Foll and Gaggiotti  and implFST‐based search implemented in Bayescan. This method is particularly suited to detect small‐effect loci that are contributing to a chosen biological gradient. We did not use an environmental gradient but instead used as input a meristic trait, plate count, under the assumption that a phenotypic gradient should show a similar pattern to an environmental gradient so long as the trait in question is under selection. We therefore analyzed all freshwater individuals for which the plates had been counted , using the number of plates as the trait in question . As the number of latent factors should reflect the expected genetic structure in the sample and then the number of populations identified by the structure analysis , we set the latent factors to 3. In this case, one random SNP per locus was selected and invariant loci across analyzed individuals were carefully excluded returning an input dataset of 57,999 loci. Five replicates were checked for convergence. When using a cutoff for the −log10 of 5, ca. 600 putative outliers were detected in each run. To focus only on the most extreme outliers we applied two independent criteria. First, loci showing a −log10 above 7 were grouped along the chromosomes using a window size of 500,000 bp and only windows containing three (or more) significant loci were retained for further investigation. Additionally, the top 10 outlier loci  consistently returned by all five replicated runs  above 16) were directly retained for further investigation. Significant loci were investigated to ensure that missing data were not influencing the categorization of these regions as outliers in this analysis (Table S2), and subsequently annotated using the stickleback genome reference in the Ensemble database , consistent with the results from the ML analysis . However, the two clusters in this population did not segregate according to plate morph (plateless vs. low plate).Using the Bayesian clustering algorithm as implemented in fastStructure, the overall structure of the data was best explained with four clusters . Lower peaks on Chr V, IX, and XIX are also consistent with regions found in Jones' analysis. Our FST analysis comparing low and plateless fish showed no outlying peaks in FST, both when the freshwater populations were pooled together  and when each population was analyzed separately . By contrast, many outliers were detected when Bayescan was run to compare the freshwater and the marine fish Fig. .Using LFMM, we retrieved 10 outlying loci Fig. , Table 1The nearest genes for each significant outlier or outlier region were annotated Table , and GenGenome‐wide association studies (GWAS) linking phenotypic traits to their underlying genetic basis have been widely used in model and nonmodel species, although their power and reproducibility have been often questioned  every ~10 kbp after filtering, which is high coverage for RADseq, but less coverage than typically achieved with whole‐genome sequencing. Yet, using sliding window s et al.  when we s et al. , with frFST analysis, we used two other methods to detect outliers, one based on population differentiation (Bayescan) and the other based on the covariance between allelic frequencies and lateral plate counts (LFMM). LFMM has proven to be more effective in detecting the signature of polygenic selection , although it is characterized by a relatively high false discovery rate  to STN 211, a previously described microsatellite that is linked to a plate number modifier  , and also the very top of the plate number distribution (those being the fish best able to survive attacks). If the fish do not mate assortatively, this could preserve the pattern we see of lakes with a wide range of plate number within the low plated morph, including plateless individuals.Plate number evolution in the stickleback system is often used as a clear example of parallel evolution by natural selection acting in response to known selection pressures. Yet the debate continues about what the selection pressures acting on plate number actually are, why this evolution is so parallel and so rapid, and how much variation is explained by The absence of clear genetic differentiation between plateless stickleback and those that are low plated – as shown in our study – suggests plateless fish should not be considered a fourth plate morph category , but we may continue to place them within the low plated morph. Nevertheless, it remains that plateless fish are a unique phenomenon and a better understanding of the variation in selection pressures on completely plateless versus low‐plated fish is needed to fully understand why plateless fish can be maintained in relatively high proportions in some populations while they are absent in the majority of the stickleback distribution.http://www.ncbi.nlm.nih.gov/bioproject/314185.Raw reads have been uploaded to the NCBI Sequence Read Archive at this link: None declared.Appendix S1.Figure S1. (A) Average number of SNPs by position across loci for all reads in all libraries showing a very slight, nonsignificant increase after base 92. (B) Mean GC content across loci, showing a normal distribution.Figure S2. Genetic clustering analysis using FastStructure on the Melavatn population with K = 2.Table S1. Sample information: Fish # refers to the number of the fish within a Code.Table S2. Comma Separated Values containing 34 outlier loci (Table ci Table  and theiTable S3. Gene ontology keywords ranked by abundance for the nearest gene(s) listed in Table Click here for additional data file. Click here for additional data file.


The majority of studies to date have been of plants and some invertebrate parthenogens, particularly aphids, and have not necessarily caught invasion at very early stages. Here we describe the early stages of an invasion by a Northern Hemisphere Hymenopteran model in three different land masses in the Southern Hemisphere. Nematus oligospilus Förster (Hymenoptera: Tenthredinidae), a sawfly feeding on willows , was recently introduced to the Southern Hemisphere where it has become invasive and is strictly parthenogenetic. In this study, the number of N. oligospilus clones, their distribution in the landscape and on different willow hosts in South Africa, New Zealand and Australia were assessed using 25 microsatellite markers. Evidence is presented for the presence of two very common and widespread multilocus genotypes (MLGs) or ‘superclones’ dominating in the three countries. Rarer MLGs were closely related to the most widespread superclone; it is plausible that all N. oligospilus individuals were derived from a single clone. A few initial introductions to Australia and New Zealand seemed to have occurred. Our results point towards a separate introduction in Western Australia, potentially from South Africa. Rarer clones that were dominant locally putatively arose in situ, and might be locally favoured, or simply have not yet had time to spread. Data presented represent rare baseline data early in the invasion process for insights into the mechanisms that underlie the success of a global invader, and develop Nematus oligospilus as a valuable model to understand invasion genetics of clonal pests.Clonality is a common characteristic of successful invasive species, but general principles underpinning the success of clonal invaders are not established. A number of mechanisms could contribute to invasion success including clones with broad tolerances and preferences, specialist clones and adaptation  One of the major paradoxes of invasion biology is how species with very low genetic variation  can still be successful invaders One commonly observed feature of invasive species is the tendency to become clonal in the invasive range, which has been suggested to enable invasive organisms to thrive in the face of low diversity It is common for invasive organisms to transition to obligate or functional parthenogenesis; for example: Nematus oligospilus Förster (Hymenoptera: Tenthredinidae). This taxon has recently invaded the Southern Hemisphere N. oligospilus representing a group of species and the native range of the invasive species is unknown A high proportion of studies on these topics have been of cyclic parthenogens; in particular, Hemiptera, and more specifically aphids, are extremely well-represented in pest species and studies of them N. oligospilus in its invasive range in the Southern Hemisphere N. oligospilus as an exciting model taxon of parthenogenetic invasion. Here we assess the distribution, genetic relatedness and host associations of N. oligospilus MLGs in South Africa, New Zealand and Australia from widespread, substantial sampling very early in the invasion process. Specifically, we address the following questions: 1/What clones of N. oligospilus are detectable in the invaded range? 2/What are their geographic and host-based distributions? 3/How closely related are they, and can their mode of origin be inferred? 4/Are there candidate ‘superclones’ and newly-evolved variants that would be valuable to monitor in time and space?A recent study developed a high-resolution microsatellite assay and identified several multi-locus genotypes (MLGs) in In southeastern Australia, collections occurred in December 2007 and January 2008. The southern part of Western Australia was sampled in February 2010, while New Zealand was surveyed in February 2009. Individuals from South Africa were collected at one site in 2008 (n = 26). More than 1100 individuals were collected and genotyped from 21 sites in New Zealand and 69 sites in Australia . All sitet al. 1995).Sites were selected on the basis of presence of willow taxa, and distance from other sites . At each site, a single mature tree per willow taxon was sampled. Foliage was inspected visually for sawfly presence, and any sawfly stages were collected into 96% ethanol, to a maximum of 30 individuals per tree per site. Adults were excluded because of their high mobility, except for South Africa due to small sample size. Defoliation of willow trees (i.e. when foliage on any branch had been completely consumed irrespectively of the amount of defoliation) was noted. Willow species were identified using keys for southeastern Australia N. oligospilus individuals was extracted per site per willow taxon using a salting-out DNA extraction protocol The DNA of up to 10 N. oligospilus in locations with males as well as females, and in other tenthredinid taxa Twenty-five microsatellite loci were used to identify multilocus genotypes (MLGs) that differ by one or more allele N. oligospilus. The rationale for the choice of these geopolitical units is that their physical organization, economic activities and biosecurity implementation are likely to be dominant factors in the likelihood of introduction. Six regions were defined: two in New Zealand (the North and South Islands), three in Australia , and South Africa. Genotypic diversity (G/N) was calculated for each country and region, using the number of genotypes (G) divided by the number of individuals genotyped (N). Shannon-Weaver diversity index, allelic diversity, observed (Ho) and expected heterozygosity (He) were calculated in GenAlex 6.4 He to allow comparison based on the number of genotypes present in the populations. Departures from Hardy-Weinberg equilibrium (HWE), linkage disequilibrium and F-statistics were calculated and assessed with exact tests in Genepop 4.0 FST estimates between populations were calculated based on the 25 loci with combined statistical significance calculated according to Fisher's method as implemented in Genepop. HWE analyses were performed and expected and observed heterozygosity calculated initially for the full dataset, and then for a reduced dataset containing one individual per genotype to avoid distortions caused by multiple representatives of the same clonal genotype Genetic analyses were conducted according to sampling units based on country , and within countries, regions separated by major land or water barriers between populations of Unrooted neighbour-joining trees were constructed based on Cavalli-Sforza's chord measure of genetic distance  in Populations 1.2.32 . One tree was built to depict the relationships among MLGs , and one tree depicted the population network on the basis of one individual per MLG per region. To assess genetic variance within and between countries and regions, a nested Analysis of molecular variance (AMOVA) was carried out with regions nested within countries in GenAlex 6.4 The combination of 25 microsatellite markers discerned 16 different MLGs in the Australasian and South African invaded range sampled . Of the Other than ‘Main’ and ‘North’ clones, most MLGs were localized and had low frequencies. Three exceptions were clone Aus1, which was found in several sites in Western Australia around the port city of Albany and was unique to this region, and Aus2 and Aus3 which were common locally  and 3. CThe two most widespread MLGs, ‘Main’ and ‘North’ clones were found on all willow taxa sampled showing no obvious host specialization. Similarly, locally abundant MLGs Aus1 and Aus2 were found on all the willow taxa present at the sites where they occurred. Only the ‘Main’ ‘North’ and Aus1 MLGs have been found at sites where willows suffered defoliation.The 16 MLGs sampled were extremely similar at their 25-locus genotypes: they differed by a maximum of 3 alleles and 3 loci . AlthougHe and allelic diversity  did not differ significantly in allele frequencies (p>0.05). According to the AMOVA, most of the molecular variance, 88%, was attributed to within-region effect, while only 12% was attributed to between-region effects and less than 1% to between-country effects . For example, some of the rarer clones seem to have arisen N. oligospilus did not depend on association with specific willow taxa, indicating that this species may be a host generalist. However, in the field, when several willow hosts are present, population densities of N. oligospilus are constantly higher on some host taxa than others N. oligospilus and increase the chance for mutations to spread. In a study on Sitobion aphids, although no host specialization was found, rarer clones had enhanced performance on the most chemically defended host plants, and it was hypothesized that rarer clones were relatively favoured partly because of the presence of chemical defences Host plants can provide selection pressures that lead to specialization of herbivore populations N. oligospilus that have invaded the Southern Hemisphere has not yet been resolved, and genetic data can contribute to clarification of relationships of these insects around the world The native range source of the N. oligospilus flew from South Africa to Australasia, although we cannot rule out transport by wind. Similarly, it seems improbable that ‘North’ MLG found in southeastern Australia and Western Australia dispersed unassisted across 1100 km of the Nullarbor Plain (arid habitat lacking willow hosts), or that the single individual of the ‘North’ clone sampled in Tasmania self-dispersed to the location of its capture close to Launceston airport. Most of the dispersal seen between regions is more likely due to human-mediated jump dispersal, which is also a major cause of long-dispersal in many other invasive species; for example: Daktulosphaira vitifoliae Fitch), in which dispersal among localities is due solely to human-mediated movements of infected plants Sawflies in general are not strong flyers N. oligospilus occur in the Southern Hemisphere invasive range. Empirically these superclones are highly successful invaders, comprising most of the spread of N. oligospilus through the colonized range. Whether this success persists over longer periods remains to be tested. Clones with broader preferences and tolerances may be better at colonizing new areas, but may not be as good at competing N. oligospilus, new MLGs seem to have arisen in situ within the Southern Hemisphere. Although two main MLGs now dominate, new and rare MLGs may be favoured subsequently, following changes in environmental conditions and as non-equilibria such as founder effects dissipate N. oligospilus in the Southern Hemisphere highlights the importance of biosecurity. The introduction of a single individual capable of reproducing asexually and pre-adapted to the conditions of the new environment could lead to a successful invasion.Two dominant MLGs (or superclones) of Nematus oligospilus is a valuable model in invasion genetics. Data and tools have been established early during invasion of significant sections of the globe. We encourage repeated and broad sampling of the invaded range and potential native range to help infer the mode and location of generation of MLGs. Coupled with detailed studies of the attributes of tractable individual clones, this can greatly add to the understanding of invasion genetics of clonal pests.We propose that Table S1Nematus oligospilus genotyped.Sites and willow taxa surveyed. N: number of (DOCX)Click here for additional data file.


Myriophyllum spicatum populations distributed throughout China with 12 microsatellite loci, and we analyzed its association with geographic distance, geographic barriers, and environmental dissimilarity using causal modeling and multiple matrix regression with randomization (MMRR) analysis. Two genetic clusters were identified, and their geographic distribution suggested mountain ranges as a barrier to gene flow. The causal modeling revealed that both climate and geographic barriers significantly influenced genetic divergence among M. spicatum populations and that climate had the highest regression coefficient according to the MMRR analysis. This study showed that geography and environment together played roles in shaping the genetic structure of M. spicatum and that the influence of environment was greater. Our findings emphasized the potential importance of the environment in producing population genetic differentiation in aquatic plants at a large geographic scale.The effects of geographic and environmental variables on the pattern of genetic differentiation have been thoroughly studied, whereas empirical studies on aquatic plants are rare. We examined the spatial genetic differentiation of 58  Assessing the relative importance of environmental and geographic factors on the genetic structure of populations is one of the fundamental issues of population genetic studies. Isolation by distance (IBD) is the common model of gene flow and often serves as an explanation for the effect of geographic isolation on the pattern of genetic differentiation Slatkin . RecentlBoth patterns of IBD and IBE are often present simultaneously in nature  is a widespread submerged aquatic plant that is native to Europe, Asia, and northern Africa  in the spatial analysis of genetic data. We aimed to assess the influence of geography and environment on the pattern of gene flow and genetic differentiation in Eurasian watermilfoil populations in China.In China, Eurasian watermilfoil is present throughout the whole country, including the Qinghai‐Tibetan Plateau (QTP) region . Eight to 20 individuals from each population were sampled according to the size of habitat occupied by each population. The plants were collected randomly at intervals of at least 10 m to avoid collecting ramets from a single genet in the clonal Eurasian watermilfoil. Voucher specimens were deposited in the herbarium of Wuhan University (WH). A closely related species, M. sibiricum, co‐occurs in the QTP region and northeast China  following the manufacturer's protocol.M. spicatum , the effective number of alleles (ENA), Nei's  diversitM. spicatum due to dosage problems. Additionally, the statistics of microsatellite markers were developed for diploid organisms and not suitable for polyploid organisms   were obtained indicating that a sample size of no less than six can provide reliable evaluation of pairwise ΦPT‐values. Therefore, a total of 43 populations containing no less than six genets were included in the analysis of genetic differentiation.Because the allelic copies of microsatellites are ambiguous in polyploid species, we could not determine the exact genotypes of heterozygotes in K value (K = 1–20) with a burn‐in period of 20,000 iterations and 100,000 MCMC iterations under the admixture model. The best‐fit number of clusters was determined based on the ∆K method  was used to assess the number of clusters best fitting the data  for the studied sites from the BioClim layers Busby  using ArThe correlations between genetic differentiation and geographic/environmental factors were determined by a combination of Mantel tests and matrix regression analysis with a distance matrix. We performed simple Mantel tests for the correlation of genetic differentiation with geographic distance, barrier matrix, and environmental distance. When all of the three factors were significantly correlated to genetic differentiation see , partialΦPT) was 0.521 with the maximum of 0.907 between TB5 and GD and the minimum of 0.141 between JS and SD3. All pairwise ΦPT were significant (P < 0.05).The number of alleles (NA), the effective number of alleles (ENA), and genotype diversity (D) ranged from 22 to 84, 22 to 59.184, and 0 to 1 with a mean of 51.276, 42.054, and 0.756, respectively (Appendix S1). Five populations, namely XJ4, SHX, SX1, NM1, and LN4, were genetically monomorphic indicating that all samples were clones of a single genet within each population. A total of 496 genotypes were revealed in the 869 samples, and nearly all of the obtained genotypes were population‐specific with the exception of two genotypes shared by GX1 and GX2 as well as four genotypes shared by HEB2 and TJ (see multilocus genotype data in Dryad for details). The overall genetic differentiation . Cluster 1 (northwestern cluster) comprised mainly populations from the northern and western part of China, including northeast China, northwest China, and the QTP region, while Cluster 2 (southeastern cluster) was distributed across the southern and eastern part of China . When K = 3, the northwestern cluster was retained with the exception of two populations (QH2 and TB4), while the southeastern cluster was subdivided into two subclusters with no obvious intracluster genetic structure .Significantly positive correlations were found between genetic and geographic distances, between genetic differentiation and barrier matrices, and between genetic and environmental distances Table , Fig. 2.β1/β2 ≤ 5, Wang M. spicatum populations  feed on the seeds of Myriophyllum (Olney A. crecca) mainly breed north of China and migrate southward for the winter, and the waterfowl rarely travel from west to east or in the opposite direction  are needed to confirm the contributions of environmental factors to the pattern observed in M. spicatum, our study suggests the important role of ecological factors in the formation and maintenance of genetic differentiation in M. spicatum.An obvious IBE pattern was revealed in tum Fig.  and 2, stum Fig. . Many sptum Fig. . The clitum Fig. . The firh Patten . These eM. spicatum. As one of several case studies, our findings suggest the importance of climate in shaping patterns of genetic variation and imply a potential of local adaptation to different climatic conditions in aquatic plants at a large geographic scale.In conclusion, our study revealed both geographic barrier and climate could influence the pattern of gene flow in Eurasian watermilfoil populations and a potentially greater prediction of IBE on the genetic differentiation of None declared.http://dx.doi.org/10.5061/dryad.9v2g5).The microsatellite genotype data and the BioClim variables of the studied samples were deposited in Dryad , number of genets (Ng), number of alleles (NA), effective number of alleles (ENA) and Nei's genotypic diversity index (corrected for sample size) (D) for the 58 Myriophyllum spicatum populations.Click here for additional data file.Appendix S2. Causal model of the expected relationship between genetic distances and geographic/environmental variables.Click here for additional data file.Appendix S3. Modelling of the number of genetic clusters in Myriophyllum spicatum using STRUCTURE.Click here for additional data file.Appendix S4. Spatial genetic structure using DAPC analysis.Click here for additional data file.Appendix S5. Weighting of each climatic variable in the PCA analyses.Click here for additional data file.


Leptodactylus fallax) populations before, during and after the emergence of chytridiomycosis, we quantified the real-time species level impacts of this disease. We report a range-wide species decline amongst the fastest ever recorded, with a loss of over 85% of the population in fewer than 18 months on Dominica and near extinction on Montserrat. Genetic diversity declined in the wild, but emergency measures to establish a captive assurance population captured a representative sample of genetic diversity from Montserrat. If the Convention on Biological Diversity’s targets are to be met, it is important to evaluate the reasons why they appear consistently unattainable. The emergence of chytridiomycosis in the mountain chicken was predictable, but the decline could not be prevented. There is an urgent need to build mitigation capacity where amphibians are at risk from chytridiomycosis.Amphibian chytridiomycosis has caused precipitous declines in hundreds of species worldwide. By tracking mountain chicken ( These targets were driven by a generally perceived failure of the international community to achieve the CBD’s goal of halting biodiversity loss by 2010, yet it is becoming apparent that many of the 2020 targets will also be missedRecent studies indicate that the Earth has entered a sixth period of mass extinctionBatrachochytrium dendrobatidis (Bd), is thought to have caused the decline or extinction of over 200 species of amphibian world-wide in recent decades6With over 40% of species currently threatened with extinction35Chytridiomycosis-induced amphibian declines usually occur following the introduction of Bd to a naïve population. The precise timing of disease introduction, however, is rarely identified and often the first recognition of Bd emergence in an amphibian population is the loss or rapid decline of that species at a study location88910111213Alytes obstetricans)A lack of monitoring and the speed of chytridiomycosis-driven amphibian declines have resulted in few data being available for assessing the genetic impact of the disease. Despite the numerous population declines reported, only one study has investigated the associated genetic impact, providing evidence of a disease-induced population bottleneck in the common midwife toad  is the largest frog endemic to the Lesser Antilles which can live 12 years181920The mountain chicken (Eleutherodactylus spp.) within shipments of produce, most of which was imported directly from DominicaIn December 2002, reports of dead and sick mountain chickens were first received by forestry officers from members of the public in Dominica. Initially, only isolated reports were made but within weeks widespread mortality was apparent. A diagnosis of chytridiomycosis due to Bd infection was made on carcases shipped to the Zoological Society of London for pathological examinationOn both Dominica and Montserrat, long-term monitoring programmes produced mountain chicken population demographic data and genetic samples before, during and after the onset of epidemic chytridiomycosis. Here, we quantify the population and genetic impacts of the disease, discuss the management responses to this crisis and evaluate their effectiveness in terms of conserving biodiversity. This study of the predictable near-extinction of a culturally iconic species due to a process addressed by Aichi Target 9 (invasive species and pathways) is a useful model for understanding how failures in national and international governance and support mechanisms are impeding our ability to meet the CBD 2020 targets.th February and 28th March 2003 from three sites across Dominica. Histological examination of hind-leg skin and feet showed large numbers of intracellular sub-spherical, septate structures characteristic of BdDetailed pathological examinations were conducted on four adult frogs found dead between 19Dead mountain chickens were first reported on Montserrat in February 2009 on the eastern side of the island. Twelve transects were surveyed across the island during the following two weeks, and epidemic mortality was observed at three sites in the east. No mountain chickens were found at five long-term population monitoring sites in the north or west of the Centre Hills, and severely depleted populations at sites in the south and east. Only two sites, Fairy Walk and Corbett Spring, were found to contain apparently healthy mountain chicken populations. Both of these sites are in the extreme south-east of the Centre Hills. Using qPCR, we confirmed the presence of Bd DNA in skin swabs at five of the seven sites in which mountain chickens were found, including all five with observed mortality. Only skin swabs from Corbett Spring and Fairy Walk did not test positive for Bd DNA. On returning to Montserrat in August 2009, no mountain chickens were found at any site other than Fairy Walk and one other site nearby, where epidemic mortality was observed. Of 120 mountain chickens skin-swabbed during two weeks of monitoring at Fairy Walk in August 2009, 105 (87.5%) tested positive for Bd DNA. The temporo-spatial pattern of mortality and decline suggested a north-west to south-east epidemic wave with an active front in the remaining populations in the south-east.No explanatory variables other than time were retained in the final generalised linear mixed effects model (GLMM) of the Dominica decline . Mean visual encounter rates per 250 m declined by more than 50% from 2 (SE = 0.31) to 0.85 (SE = 0.18) within 12 months from August 2002 . By MarcOn Montserrat, season (Likelihood ratio test (LRT) Chi-sq = 20.997, df = 1, p < 0.0001) and time  were retained as fixed effects in the GLMM for the 1999–2007 pre-decline data. A greater number of individuals were encountered on transects during the dry than the wet season, and the visual encounter rate increased between 1999 and 2007 . HoweverTime was the only significant parameter retained in the generalised linear model (GLM) of the Fairy Walk decline , with the encounter rate per 250 m of transect declining from a maximum of 10 (week 5) individuals to 2 (week 18) a decrease of c. 80% in just 13 weeks . Continu2 to 38.08 km2) and the estimated area of occurrence (AOO) was reduced by 94.4% (from 35.25 km2 to 1.75 km2) between 2002 and 2014  of the mountain chicken was reduced by 95% (from 663.63 kmand 2014 . The mou2 to 1.53 km2) and the estimated AOO by 80%  there was a significant decline in diversity (p = 0.03). We present the results including all markers for the remainder of this section. No significant linkage disequilibrium was found between any pair of loci in each island’s population.Analysis of eight microsatellites showed a total of 52 alleles  with an average of 6.5 per locus and 34 shared between both islands . The exppre-decline showed no significant differences in expected heterozygosity .To avoid the confounding effects of rapid population decline on genetic diversity, the interisland genetic comparison was conducted using only samples collected prior to the arrival of Bd. The comparison between Dominica and Montserrat ntserrat . Consistpre-decline, n = 29) were both greater than those of the wild individuals sampled in 2014 . Expected heterozygosity declined from 0.61 to 0.49 and allelic richness reduced from 5.2 to 3.5, although these differences were not significant (pre-decline dataset (p = <0.05) and three loci in the post-decline dataset . While these results suggest a decrease in genetic diversity after the population crash, demographic analyses using Bottleneck did not find evidence for a genetic bottleneck in the post-decline population on Dominica (Heterozygote excess Wilcoxon’s Sum Rank test TPM p = 0.58 and SMM p = 0.88).Within Dominica, the expected heterozygosity and allelic richness (corrected for sample size) for samples collected from 2002 to 2006  were a genetically representative sample of the pre-epidemic wild Montserrat individuals . The Founders and Wild populations were both overall within HWE. The differences in average number of alleles per locus, allelic richness and heterozygosity between these populations (Wild population had a positive inbreeding coefficient (FIS = 0.18) that was significantly larger than zero (p < 0.001), but which did not statistically differ from that in the Founder population , 0.39–0.58 (observed heterozygosity), and 0.52–0.64 (expected heterozygosity). Thus, the resampling analysis showed that the genetic variation captured by the Founder animals was within the expected distribution of genetic variation in the Wild population , but the encounter rate halved every seven weeks until monitoring had to be suspended after five months due to volcanic activity.Detectability of mountain chickens on transects was likely not 100%. We attempted to fit an open population N-mixture model2829Saiga tatarica) declined by 95% over 15 years due to huntingGyps bengalensis) declined in India by 99.9% over 15 years as a result of secondary poisoningAmphibian chytridiomycosis has caused similarly rapid declines in other populations89101112Rana muscosa) populations were extirpated over a four year period by epidemic chytridiomycosis. The reduction in mountain chicken population connectivity, however, undoubtedly affects the level of gene flow between remaining individuals, increasing the risk of extinction16The range collapse of the mountain chicken was also precipitous, with a decline of over 80% in both AOO and EOO on Montserrat and of ~95% on Dominica. The species’ range on Dominica has collapsed to a small part of the west coast and has undergone severe fragmentation. On Montserrat, only four individuals at three sites have been discovered since 2012 despite repeated intensive surveys; since 2014 only two of these individuals have been recaptured, both at the same site. While the persistence of individuals after a chytridiomycosis epidemic has been recorded in other studies353637The optimal temperature for the growth of Bd is 17–25 °CPrevious analyses of mitochondrial DNA sequence data  for the mountain chicken , found aST ~ 0.2), it is likely that further population size reductions will exacerbate the differences between the relic populations, as well as further reducing the overall genetic variation of this species.While the inbreeding coefficient was significantly larger than zero on both islands, results suggest that, before the population crash, the Dominican population was larger than that of Montserrat, harbouring greater genetic variation. The island of Dominica is approximately eight times larger than Montserrat and both island biogeography theory and population genetics studies indicate that populations persisting on smaller and more-isolated islands tend to have lower genetic diversity than those living on larger and more-connected islands e.g.4445. It 4446pre- and post- decline heterozygosity and inbreeding coefficients were not significant, the difference between pre- and post- decline allelic richness was close to significant  in central Spainpost-decline population on Dominica using the program Bottleneck, despite seeing both a trend of loss of heterozygosity and marked differences in genetic diversity metrics for the pre- and post- decline populations. However, very recent bottlenecks often go undetected in genetic data using limited sample sizes, even when it is known that those bottlenecks did occurpost-decline dataset for the Dominican mountain chicken population was confined to 17 samples taken in 2014 and this sample size might not have been high enough to detect a genetic bottleneck signature.Within Dominica, a temporal trend in genetic diversity could be seen. Whilst comparisons of Founders) and the wild individuals sampled pre-decline (Wild) showed very little difference in expected heterozygosity or allelic richness, and genetic differentiation (FST) between the two populations was zero. Also, the resampling analysis showed that the genetic diversity captured by the founder stock adequately represented the wild population, while the FIS indicated no significant evidence of inbreeding. Captive breeding programs for wildlife conservation require careful genetic management to avoid further reductions of genetic diversityOn Montserrat, a comparison between the founders of the captive population  and its incursion to Montserrat, amphibian populations on the latter island were shown to be free from Bd infection and a risk analysis identified the most likely routes of introduction. Unfortunately, no identified mitigation measures were implemented. Capacity for sanitary measures that remove amphibian stowaways and contaminated soil from produce prior to export was developed in Dominica in 2007 to enable exports to the Lesser Antillean islands of Guadeloupe and Martinique. Had this requirement been imposed for produce shipped to Montserrat, the risk of Bd incursion would likely have been much reduced.Eleutherodactylus coqui from Puerto RicoE. Johnstonei on Montserrat (authors’ unpublished data) indicating some tolerance to Bd infection. The intentional or accidental transport of amphibians such as these between islands is a likely route of transmission of Bd throughout the Caribbean (http://www.mountainchicken.org/wp-content/uploads/2010/11/Chytridiomycosis-Management-Plan.pdf). The World Organisation for Animal Health (OIE) listed Bd in May 2008, making the pathogen internationally notifiable and thus subject to OIE standards to assure the sanitary safety of international trade in live amphibians and their products. Despite this, few if any of the OIE’s 180 member states have imposed relevant sanitary requirements on traded amphibians. This might be because, although it is a huge industry involving tens of millions of animals annuallyThere is evidence of enzootic Bd infection in Caribbean tree frogs such as 2, has a central active volcano creating its highest peak at 915 m. The Centre Hills to the north, with a maximum altitude of 740 m, are characterised by deep ‘ghauts’  in a radial pattern from a central peak2, and more mountainous than Montserrat, with steep interior slopes, most of which are covered in native forest. It has a tropical climate with year-round rainfall. Dominica lies approximately 150 km South East of Montserrat with the island of Guadeloupe situated between them.Montserrat and Dominica are small volcanic islands in the Lesser Antilles chain in the Caribbean. Montserrat, approximately 102 kmoC) were recorded.A full description of transect monitoring techniques is provided in ref. On Dominica, nine transects were established in 2002 in areas with mountain chicken populations, typically in lowland coastal areas. These were surveyed on an approximately monthly basis from August 2002 to March 2004. Surveys were conducted on eight occasions from January 2006 to July 2007. From January to September 2014, the established transects and an additional 13  were surveyed on three occasions to search for any surviving mountain chickens. On Montserrat, mountain chickens inhabit upland areas in which 17 transects of 200 m were established in 1998; 13 were monitored regularly from 1999 to 2005: transects were surveyed four times in 1999, biannually (wet and dry seasons) from 2000 to 2005 and once per year in 2007, 2011 and 2012. From 2003, some transects were extended to a maximum of 400 m to capture a greater proportion of the available habitat at each site, with effort per transect-metre maintained.Following the onset of declines on Montserrat, an extended 800 m length of the transect with the last known intact population  was surveyed using VES on a thrice-weekly basis from August 2009 to January 2010 as part of an amphibian chytridiomycosis treatment trialhttp://www.arguk.org/advice-and-guidance/view-category) to minimise Bd contamination of samples or the spread of pathogens between study sites by researchers. DNA was extracted from fresh/frozen hind toe and ventral pelvic skin samples and examined for the presence of Bd DNA using a Bd-specific qPCRSystematic pathological examinations were conducted on frogs found dead during the initial stage of epidemic mortality on each island by examining formalin-fixed samples of hind toe and skin from the ventral pelvic area (drink patch) from each carcase. Following the emergence of chytridiomycosis on each island, the epidemic was tracked via skin-swabbing of frogs found sick or dead using rayon-tipped swabsTo ensure encounter rates were comparable across transects of different lengths and between islands, a nightly average encounter rate per 200 m of transect was calculated, excluding two Dominican transects where no visual encounters were made. Generalised linear mixed models in RThe environmental variables collected for each survey, the number of observers, the season (Wet/Dry), and the average rainfall in the month of, and in the month prior to, the survey were included as covariates in the models to account for mountain chicken detectability. Models were developed using stepwise elimination of the least significant variable and a likelihood ratio test used to assess whether the model fit was reduced by exclusion of the variable compared to the model containing that variable at an alpha value of 0.05. This was continued until only variables that significantly improved model fit were retained.As widespread systematic monitoring of the mountain chicken distribution was not available outside the transects, two standardised range metrics used in the red listing processWe used pre- and post- declines (including between wild and captive populations), genetic analyses were conducted using microsatellite markers. Blood, buccal swabs or tissue samples from live and dead wild mountain chickens were collected from Montserrat and Dominica (To assess if there were differences in genetic variation between islands or between populations Dominica  between ST) were estimated with MSA v4.05IS) was estimated by FSTAT v.2.9.4.Eight newly-developed polymorphic microsatellite markers were genotyped for each animal  and the 64pre- and post- decline genetic comparison of the Dominican samples we use a decline end-date of 2006 by which time no animals could be detected in the wild. The post-decline (recovering) population was defined as animals caught in 2014 when mountain chickens began to be detected on three of the historical transects in the absence of chytridiomycosis driven mortality. In the 2014 surveys, a large number of ~2 year old animals were detected alongside a small number of older animals, suggesting that breeding had occurred at increased levels in 2012, although no samples were collected in 2012 or 2013. Although 6 juveniles were found at one location in 2011, they were infected with Bd and disappeared shortly after (presumed to have died of chytridiomycosis) and so these animals were not considered part of a recovering population (authors’ unpublished data). As these animals were caught so long after the initial decline but before the apparent population recovery began, the animals were excluded from the pre-/post- decline comparison but included in the general interisland genetic diversity statistics. The pre-/post- decline groups of samples were compared using standard population genetic diversity statistics described above to assess if genetic diversity in the post-decline population was significantly smaller as a consequence of the Bd-induced population crash. Additionally, signatures of demographic contraction in the 17 wild Dominican individuals caught in 2014 were tested using the Wilcoxon test in the software Bottleneck v. 1.2.02For the Founders), were representative of the genetic diversity in the larger wild population (Wild). For this purpose, we compared the average number of alleles per locus, observed and expected heterozygosity of the Fairy Walk individuals against 100,000 random samples of eleven individuals from the allele frequency distribution of the 74 wild individuals from Montserrat. We selected these statistics as they are affected by dramatic changes in effective population sizeA resampling analysis was carried out to establish whether the Fairy Walk (Montserrat) individuals collected in 2009 used to establish the captive founder population .


Anopheles nuneztovari sensu lato comprises cryptic species in northern South America, and the Brazilian populations encompass distinct genetic lineages within the Brazilian Amazon region. This study investigated, based on two molecular markers, whether these lineages might actually deserve species status.COI gene (Barcode region) and 12 microsatellite loci. Phylogenetic analyses were performed using the maximum likelihood (ML) approach. Intra and inter samples genetic diversity were estimated using population genetics analyses, and the genetic groups were identified by means of the ML, Bayesian and factorial correspondence analyses and the Bayesian analysis of population structure.Specimens were collected in five localities of the Brazilian Amazon, including Manaus, Careiro Castanho and Autazes, in the State of Amazonas; Tucuruí, in the State of Pará; and Abacate da Pedreira, in the State of Amapá, and analysed for the A. nuneztovaris.s. and A. dunhami, strongly (98 %) and weakly (70 %) supported, respectively. In the second phylogenetic analysis, the sequences from GenBank, identified as A. goeldii, clustered to groups I and II, but not to group III. Genetic distances (Kimura-2 parameters) among the groups ranged from 1.60 % (between I and II) to 2.32 % (between I and III). Microsatellite data revealed very high intra-population genetic variability. Genetic distances showed the highest and significant values (P = 0.005) between Tucuruí and all the other samples, and between Abacate da Pedreira and all the other samples. Genetic distances, Bayesian (Structure and BAPS) analyses and FCA suggested three distinct biological groups, supporting the barcode region results.The Barcode region dataset (N = 103) generated 27 haplotypes. The haplotype network suggested three lineages. The ML tree retrieved five monophyletic groups. Group I clustered all specimens from Manaus and Careiro Castanho, the majority of Autazes and a few from Abacate da Pedreira. Group II clustered most of the specimens from Abacate da Pedreira and a few from Autazes and Tucuruí. Group III clustered only specimens from Tucuruí (lineage III), strongly supported (97 %). Groups IV and V clustered specimens of A. nuneztovaris.l. in the Brazilian Amazon region. Lineages I and II may represent genetically distinct groups or species within A. goeldii. Lineage III may represent a new species, distinct from the A. goeldii group, and may be the most ancestral in the Brazilian Amazon. They may have differences in Plasmodium susceptibility and should therefore be investigated further.The two markers revealed three genetic lineages for The online version of this article (doi:10.1186/s12936-016-1217-6) contains supplementary material, which is available to authorized users. Anopheles nuneztovari sensu lato was originally described in San Carlos, State of Cojedes, western Venezuela. It is geographically distributed from eastern Panama to northern South America   . For the program . The gen program . In thisP values in all the statistical analyses of this study that involved multiple comparisons.The Bonferroni correction  was applThe dataset consisted of 103 sequences with a fragment size of 663 bp  were shared among samples and 20 (74.07 %) were singletons and/or exclusive of each sample  constituted a sister group of the monophyletic clade formed by A. goeldii , A. nuneztovaris.s. (Group II) and group IV (lineage III of this study). Of the three A. goeldii groups, group V is likely the most basal one and may actually be a distinct species. This group is a sister group to a monophyletic clade formed by groups I and III (A. goeldii), and II (A. nuneztovaris.s.) and IV (lineage III). This topology suggests that A. goeldii does not have an exclusive ancestry and could be paraphyletic. Groups I and III (A. goeldii) and II (A. nuneztovaris.s.) formed a monophyletic clade that is a sister group of group IV. The latter was well supported (88 %) and may also represent a distinct species. In this context, A. goeldii (groups I and III) could be a synonym of A. nuneztovaris.s. (group II), or A. goeldii (group I) could be a synonym of A. nuneztovaris.s. and A. goeldii (group III), a distinct species, or even each group could be a distinct species. Both trees were rooted using A. oswaldoi species A as outgroup.In the second phylogenetic analysis , indicating balancing selection, recent bottleneck or cryptic population structure.Table ΦST = −0.01162 to −0.01975) and extensive gene flow (Nm = infinity) were observed among the three samples from the State of Amazonas, indicating genetic homogeneity (data not shown). In contrast, great and significant genetic differentiation was found between MN/CS/AU and TU  and between MN/CS/AU/TU and AP . In all comparisons, the Nm values were <1, suggesting absence of gene flow. AMOVA analysis including all samples also revealed significant structure between groups . High genetic structure was found between two groups (ΦCT = 0.3093), but it was not statistically significant (P = 0.09677 ± 0.01008) . The genetic distances among the three lineages  and then compared with the samples from TU and AP Table . The genDxy) between lineages were also calculated: I versus II, I versus III and II versus III were 1.60 % ± 0.00212, 2.30 % ± 0.00454 and 1.71 % ± 0.00273, respectively. Lineage I was separated from lineages II and III by zero and five fixed mutations, and two and zero shared mutations, respectively, whereas lineages II and III were separated by seven fixed mutations and 1 shared mutation  to 25 (Anu6), except the Anu14 locus that was monomorphic in the samples from TU and AP. The Anu6 locus was the most polymorphic one, varying from 12 (TU) to 25 (AU) alleles. The samples from MN, CS and AU had the largest allele numbers  and allele richness  compared with the samples of TU  and AP . Of the 12 loci analysed, Anu4, Anu10, Anu14 and Anu28 were in HWE for all samples, whereas the other loci were in disequilibrium in at least one sample. Nineteen (31.67 %) out of the 60 comparisons exhibited Hardy–Weinberg disequilibrium, most of them suggesting heterozygote deficits. LD analysis was carried out to infer whether these deviations were due to the Wahlund effect, inbreeding, selection, genetic drift, gene flow or null alleles. Forty-one (12.42 %) out of 330 exact tests showed significant results for LD (P = 0.00076), after Bonferroni correction. Tucuruí had the greatest number of pair-loci (18) with significant test results, followed by AU (9) and CS (8). The other two samples showed three pair-loci, each of them with significant test results. A total of 55 private alleles distributed among the five samples were observed .A total of 160 individuals encompassing the five samples were genotyped for 12 microsatellite loci , although the comparisons with AU showed statistically significant differences (P = 0.005). Higher and significant values (P = 0.005) were observed between the samples from Amazonas and TU (FST = 0.110-0.133), between the samples from Amazonas and AP (FST = 0.076–0.103) and between TU and AP (FST = 0.094), with a lower level of gene flow (Nm = 6.05–3.25). AMOVA analysis including all samples revealed highly significant structures among them  . There was isolation-by-distance (IBD) among the five samples , covering a range of ~100–1211 km, indicating that ~90 % of the genetic differentiation observed is explained by geographic distance.Table K = 3), based on the highest mean value estimated log probability of data −6119.90 (SD = 7.20) and on the value close to the initial “plateau” of the curve, when K was plotted versus the mean posterior probability. These groups corresponded to the three samples from the State of Amazonas (blue), samples from AP (green) and from TU (red). However, five specimens from AU (blue) exhibited a larger proportion of their genomes (q > 0.980 average of five specimens) assigned to the green group instead of the blue group  and so can those from TU (Q > 0.95 of belonging to the red group). Similarly, FCA analysis clearly separated three groups  were represented by the five samples of this study. Group I was represented by the three samples from the central Amazon region and few specimens from AP. Group II comprised most of the specimens from the northeastern region (AP). Group III consisted of specimens from the eastern region (TU). These groups were confirmed by the Bayesian analyses (structure and BAPS) and FCA with microsatellite data.A. goeldii) were not well resolved  and VI (A. dunhami)  and II (A. nuneztovaris.s.) may be same species or incipient species. Supporting these findings, they also presented low genetic distances (1.60–2.0 %). A similar topology (BI tree) was obtained by Scarpassa and Conn [Comparing the dataset of this study with the sequences from GenBank, the ML tree retrieved six groups, four of which  were represented by specimens of the five localities of this study plus groups II , suggesting that it does not belong to A. goeldii. Furthermore, although all haplotypes were connected in the haplotype network , and in the FCA analysis  between the samples of the three regions. In contrast, genetic homogeneity was observed among the three samples from the State of Amazonas (lineage I). Therefore, specimens of these locations may belong to a unique species that is undergoing a differentiation process from lineage II, and distinct from lineage III.In the present study, the barcode region data indicated very high genetic structure and absence of gene flow  of the A. albitarsis complex [A. dunhami and A. nuneztovaris.s. (2.55–2.61 %) in this study. Ruiz-Lopes et al. [A. albitarsis H from A. marajoara, two sister taxa of the A. albitarsis complex, whereas the genetic distance between A. triannulatuss.s. and A. halophylus/A. triannulatus C varied from 1.7 to 2.30 % [Nyssorhynchus subgenus [A. nuneztovari complex are of recent evolutionary origin, confirming previous studies [Lutzomyia umbratilis, that likely consists of two cryptic species which showed genetic distances from 0.8 to 1.4 % and moderately supported clades [The genetic distances (K-2P) among the three lineages were small (1.60–2.32 %) and very similar to those reported by Calado et al. , who als complex  and betws et al.  observedo 2.30 % . As obsesubgenus , 69–71,  studies , 31. An d clades , suggestwhite gene [A. goeldii group (lineage II) and a new species (lineage III). The occurrence of two distinct genetic pools in TU could explain the highest number of pair-loci (18) in LD for the microsatellites data, which remained significant after the Bonferroni correction.Most of the lineages observed in the present study were undetected in the isozymes study , except ite gene . Lineageite gene , whereasite gene . This liite gene  reportedite gene . In the 2 and precipitation, occurred during the Pleistocene and which may have influenced the isolation of these populations in refuge areas, causing the differentiation between them by allopatry. Therefore, the two lineages or species sympatric observed in TU could represent secondary contact zones.The diversification time estimated among the lineages falls in the Pleistocene Epoch (ranging from 0.34 to 0.50 myr), as previously observed , implyinHowever, previous reports have suggested the Amazon river to be a significant barrier to dispersal for several species –75, inclA. darlingi [A. marajoara [white gene [Appreciable and significant mitochondrial and microsatellite differentiation was also observed between AP and TU, situated on opposite sides of the Amazon river delta (mouth). In this region, the Amazon river is widest and can reach up to 50 km in the rainy season. The Amazon river together with the Xingu, Araguaia and Tocantins and other smaller rivers form a large network in this region. This network may act as a dispersal barrier, even porous, restricting the contact between anophelines from the north and south banks, promoting genetic differentiation. Previous studies have reported differentiation between populations of darlingi , 77 and arajoara  in this ite gene . The locA. goeldii, whereas lineage III may represent a new species and could be the most ancestral one in the Brazilian Amazon region.Taken together, the data clearly show that the three genetic lineages studied may be evolving independently in the Brazilian Amazon region. Lineages I and II may represent genetically distinct groups or species within Higher levels of intra-population genetic variability, estimated by the microsatellite loci, were detected for the central region (samples from the State of Amazonas) as compared to the samples from TU and AP, supporting previous findings of Scarpassa and Conn . Based oA. nuneztovaris.l. in the Brazilian Amazon region, confirming previous reports. Lineages I and II may consist of genetically distinct groups (likely species) within A. goeldii. Lineage III could represent a new species of the A. nuneztovari complex and may be the most ancestral one in the Brazilian Amazon region. Since A. nuneztovaris.l. has been incriminated as a major local malaria vector in the State of Amapá [A. goeldii) is probably a malaria vector in that area. Furthermore, based on the previous reports [A. goeldii) likely may be involved in the malaria transmission in the central region from the Brazilian Amazon. The involvement of lineage III in the malaria transmission remains to be clarified. This study highlights the importance of utilizing integrative approaches for separate lineages and species of this complex, in order to achieve accurate results.The two markers used in this study were concordant; both revealed three distinct genetic lineages for of Amapá , the lin reports , 9, 13 t


There is now a widespread recognition of the importance of mental imagery in a range of clinical disorders . This prAs papers in this issue highlight, imagery is a central feature across many clinical disorders, but has been ascribed varying roles. For example, the involuntary occurrence of traumatic memories is a diagnostic criterion for PTSD , and it In this opinion piece, we would like to put forward the proposition that the motivating role that imagery plays in behavior may be a key reason for its presence in this wide range of psychopathologies, and that understanding its motivating role can help us identify common features in disorders and their treatments. It has already been recognized that the link between imagery and affect gives imagery a causative role in affective disorders , with thElaborated Intrusion Theory was initially developed to provide a cognitive account of processes involved in the maintenance of substance dependence, and to demonstrate that dependence for different substances shared a common functional basis, despite gross differences in their psychopharmacology. More provocatively, EI theory saw substance-related craving as just an extreme example of a continuum of desire, and argued that it recruited the normal cognitive processes involved in everyday desires. Since its publication ten years ago, the key premises of EI Theory and hypotheses drawn from it have been supported in a substantial body of research , 12–14.According to EI Theory, environmental cues, physiological symptoms , affect, and automatic or overlearned associations that operate outside of awareness do not constitute desires, because the individual is not aware of them. Instead, they generate cognitive activity that, depending upon the task context and other cognitive activity, can become sufficiently strong to become the focus of attention, and be subjectively experienced as an apparently spontaneous thought. When this thought is about a target that is strongly associated with pleasure or relief, it is highly salient, and generates a cascade of cognitive activity, which draws on further associations to elaborate the thought. Sensory imagery is often involved in the intrusion and is a key component of the elaboration, leading to anticipatory responses and augmenting its positive affective impact. However, the sensory focus also elicits an increased awareness of current deficit states, which is increasingly aversive as acquisition of the target is impeded. If the person is trying to withstand the temptation, they may also experience guilt about having the thought, or worry that it means they are losing control. While the affective consequences of the imagery lead to its continued elaboration and perpetuate the craving episode, its functional role is to motivate behavior toward satisfying the desire: it sets up a behavioral goal that competes with and can displace current goals.Conceptualizing desire in this way has allowed us to devise therapeutic and behavior change techniques that target the two key components of desire: the initial intrusion and its subsequent elaboration. As the intrusion follows automatic processing of triggers external and internal to the individual, the frequency of intrusions can be reduced by either reducing those triggers or impairing their automated processing. Many existing approaches can be seen to operate through their effect upon intrusions: medications that ameliorate withdrawal symptoms are an obvious way of reducing physiological cues; mood enhancement can target the contribution of negative affect; and attentional retraining can lower an individual’s preconscious processing of environmental cues associated with substance use. Once an intrusion has occurred, training in acceptance and a non-judgmental response style can help in letting the thought fade away and be displaced in focal awareness by mindful meditation or other cognitive tasks, without its elaboration dominating thought. If the thought does remain in focal awareness, a variety of imagery-based attentional control tasks can be employed to compete for imagery resources, blocking the sensory imagery of the target that maintains the elaborative cycle, shortening the duration, and weakening the affective intensity of the craving episode .There are clear parallels here with the ways in which imagery is being addressed within therapeutic developments in clinical psychology. For example, imagery rescripting  was initDesire does not only lead us to pursue illicit or unhealthy targets, of course: the strength of hockey players’ cravings to play hockey is also determined by the vividness of the sensory imagery that they experience during the episode . ImageryMotivation and goal fulfillment in behavior change may seem a long way from the issue of imagery within clinical disorders, but it is worth considering whether the experience of vivid mental images within psychopathologies might not also be linked to disorders of goal formation . Holmes We suggest that EI theory could provide a road map for effectively tackling imagery of maladaptive goals across clinical disorders: helping to identify risky situations for temptation and loss of control; reducing attention to intrusive thoughts through mindfulness or acceptance training; reducing the elaboration of dysfunctional desires through modality-specific interference , or resThis interpretation of imagery research within clinical psychology is speculative, and glosses over details and differences between the particular ways in which imagery may be critical to particular disorders. Further argument and empirical work are needed to explore the value of linking clinical conceptualizations of imagery to the rapidly developing work in cognitive psychology and behavior change. Imagery of course has a long history in clinical psychology, from systematic desensitization to covert practice, and has been extensively used to enhance sporting performance: the conceptualization of its role and application in the current paper needs to recognize and accommodate these phenomena, as well as expanding its explanatory power. Reappraising imagery as one part of a larger set of processes that act in consort to guide people in their everyday behavior raises the possibility of engaging clinically with each part of that mechanism, understanding why and how imagery is being distorted or populated by maladaptive content in different ways in a range of disorders. Understanding even part of the relationship would, we believe, justify the effort.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Recent studies suggest that alpine and arctic organisms may have distinctly different phylogeographic histories from temperate or tropical taxa, with recent range contraction into interglacial refugia as opposed to post-glacial expansion out of refugia. We use a combination of phylogeographic inference, demographic reconstructions, and hierarchical Approximate Bayesian Computation to test for phylodemographic concordance among five species of alpine-adapted small mammals in eastern Beringia. These species  vary in specificity to alpine and boreal-tundra habitat but share commonalities  that might result in concordant responses to Pleistocene glaciations. All five species contain a similar phylogeographic disjunction separating eastern and Beringian lineages, which we show to be the result of simultaneous divergence. Genetic diversity is similar within each haplogroup for each species, and there is no support for a post-Pleistocene population expansion in eastern lineages relative to those from Beringia. Bayesian skyline plots for four of the five species do not support Pleistocene population contraction. Brown Lemmings show evidence of late Quaternary demographic expansion without subsequent population decline. The Wrangell-St. Elias region of eastern Alaska appears to be an important zone of recent secondary contact for nearctic alpine mammals. Despite differences in natural history and ecology, similar phylogeographic histories are supported for all species, suggesting that these, and likely other, alpine- and arctic-adapted taxa are already experiencing population and/or range declines that are likely to synergistically accelerate in the face of rapid climate change. Climate change may therefore be acting as a double-edged sword that erodes genetic diversity within populations but promotes divergence and the generation of biodiversity. Pleistocene glacial cycles have dramatically shaped the spatial distribution of genetic variation in many species . These mDuring previous glacial intervals the Laurentide and Cordilleran ice sheets blanketed much of North America, isolating plant and animal species in multiple refugia . North oIn addition to the Beringian refugium, several cryptic northern refugia appear to have played an important role in the recolonization of previously glaciated regions for some cold-tolerant species ,25. ThesPhylogeographic studies suggest that the majority of the recently colonizing species in eastern Beringia (present-day Alaska) arrived via North America ,21. Pattet al. ) fell into two major phylogeographic groups: a well-supported Beringian clade (hereafter B) currently found in Alaska and northeastern Russia and a Canadian Arctic/eastern Alaska clade to the east (E). Taking into consideration fossil records, they concluded that the eastern lineage was previously isolated south of the Laurentide ice sheet , identified three distinct mtDNA lineages that may have diversified in Beringia . In. Inet alBeringia ,34, suggBeringia ). Phylogd, 1901)  and Colln, 1893)  are more /E[t]) should approach zero. For this dataset the mode of the distribution was close to zero . Analyses with differing priors on tau or theta confirmed the robustness of this pattern, with the mode of the distribution for tau centered on 1. When the N-SE+B split in Arctic Ground Squirrels was used instead of the E-W split, simultaneous divergence was even more strongly supported . The ratio of expected divergence times was similarly close to zero . While the modes of these distributions are similar, analyses including the N-EE-B split in Arctic Ground Squirrels resulted a slightly longer tail on parameter distributions, encompassing greater variation in the range of Ψ, Ω, and the expected time to divergence.Despite differences in divergence depth between eastern and Beringian phylogroups , hABC anOur results support simultaneous intraspecific divergence between central Beringian (B) and eastern (E) populations. Hoary Marmots and Brown Lemmings contained approximately equal genetic diversity and equivalent expansion signals (or lack thereof in Hoary Marmots) between Beringian and eastern phylogroups (B ≅ E). These results refute a history of recent northern expansion in the eastern lineage  and instH and Tajima’s D in Singing Voles. This isolation in multiple microrefugia—small favorable regions of unglaciated habitat such as easternmost Beringia [Pleistocene population centers within Beringia (Alaska) and northwestern Canada likely differed at fine scales by species and over time , resulting in differential genetic diversity but broadly similar phylogeographic patterns  across all five species). Spatial heterogeneity over time may explain differences in ancestral population size among phylogroups, such as the greater genetic diversity in eastern Arctic Ground Squirrels (E>B), which was not predicted under any of the models . PopulatBeringia  or nunatBeringia —would haBeringia  and ArctBeringia . SuitablBeringia .The hypothesis of simultaneous isolation of Beringian and sub-Laurentide populations and subsequent northward expansion of a small colonizing lineage  receivedθ), the degree of genetic divergence between lineages separated by a single event can be highly variable , the fact that a single simultaneous divergence in all five species was strongly supported may seem surprising. However, given sufficient differences in population size and mutation rate between species . In facble (see . This ovble (see ).E+B split for Arctic Ground Squirrels was used. This may reflect simultaneous divergence between all three groups, or it may instead indicate that many coalescent histories can fit the data. A recent multi-locus study of Arctic Ground Squirrels reached a similar conclusion that several hypotheses provide a partial fit to the observed data [It may seem counterintuitive that hABC analyses returned similarly strong support for simultaneous divergence whether the E-B split or the N-Sved data . Given tved data ), arcticved data ,20). If ved data ,82); thiWhile ancestral areas tend to harbor older genotypes, areas with the greatest genetic diversity tend to be regions of secondary contact between previously disjunct groups . The Wrasensu [The cold, arid, windswept “mammoth-steppe” of Beringia during the Pleistocene  is thougsensu ), then nThe demographic consequences of range shifts from the Pleistocene to the present are influenced by the degree of geographic shift as well as population structure. Small habitat patches at the LGM, especially in easternmost Beringia e.g., , may havMarmota olympus) [Ochotona princeps) [For species that have experienced glacial population expansion and interglacial population contraction, it might be more appropriate to consider their current areas of occupation “interglacial refugia” . While tolympus)  and Amerrinceps)  may presIn contrast, there is evidence of demographic expansion without subsequent population decline in Brown Lemmings . The modDifferences in the intraspecific divergence among the taxa in our study also highlight the role that fecundity, vagility, and population fluctuation play in preserving genetic diversity and increasing effective population size . The twoDespite differences in ecology and demography, broadly similar phylogeographic histories are supported for all species, suggesting that these, and likely other, alpine- and arctic-adapted taxa in the region have been shaped by the same historical phenomena. This includes strong support for contemporaneous population divergences across all five species and support for more recent demographic declines in three species. Such declines may be synergistically accelerating due to ongoing rapid climate change. Thus, the habitat affinities that promoted divergence in these species during past episodes of climate change may be leading to the erosion of genetic diversity as populations are confined to increasingly fragmented and isolated alpine habitats.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.