Otaria flavescens) at the Falkland Islands  in the South Atlantic. We genotyped 259 animals from 23 colonies across the Falklands at 281 bp of the mitochondrial hypervariable region and 22 microsatellites. A weak signature of population structure was detected, genetic diversity was moderately high in comparison with other pinniped species, and no evidence was found for the decline being associated with a strong demographic bottleneck. By combining our mitochondrial data with published sequences from Argentina, Brazil, Chile and Peru, we also uncovered strong maternally directed population structure across the geographical range of the species. In particular, very few shared haplotypes were found between the Falklands and South America, and this was reflected in correspondingly low migration rate estimates. These findings do not support the prominent hypothesis that the decline was caused by migration to Argentina, where large-scale commercial harvesting operations claimed over half a million animals. Thus, our study not only provides baseline data for conservation management but also reveals the potential for genetic studies to shed light upon long-standing questions pertaining to the history and fate of natural populations.Understanding the causes of population decline is crucial for conservation management. We therefore used genetic analysis both to provide baseline data on population structure and to evaluate hypotheses for the catastrophic decline of the South American sea lion ( For eThe Pinnipedia, a group of 33 extant species of marine mammal comprising the true seals, eared seals and the walrus, have been subjected to numerous genetic studies. This is partly because they provide interesting case studies for understanding the effects of historical exploitation on contemporary levels of genetic diversity and population persistence. In particular, many populations of eared seal were reduced to low enough densities to be considered extinct , yet somOtaria flavescens) can be found in breeding colonies along the coasts and offshore islands of South America from Peru to Uruguay, as well as in the Falkland Islands  in the South Atlantic. At the Falklands, this species experienced a major decline, with pup production falling from over 80 000 in the 1930s [The South American sea lion  and Cent (5′-GAGCGAGAAGAGGTACACTTT-3′) following Wynen et al. . Each PCt v. 7.1 . Sites c [et al. . All of 2.3.t = −1°C) and 60 s at 68°C; 30 cycles of 30 s at 94°C, 45 s at 55°C and 60 s at 68°C and one final cycle of 30 min at 60°C. Fluorescently labelled PCR products were then resolved by electrophoresis on an ABI 3730xl capillary sequencer and allele sizes were scored using GeneMarker v. 1.95. To ensure high genotype quality, all traces were manually inspected and any incorrect calls were adjusted accordingly.After testing 48 microsatellite loci for PCR amplification in eight sea lion individuals, we selected 22 loci with polymorphic and unambiguous banding patterns for genotyping the full sample set .2.4.h) and nucleotide diversity (π) were calculated using DnaSP v. 5.1 [rlequin v. 2.0 [enepop on the Web [p-values were corrected table-wide for the false discovery rate (FDR) [value [stat v. 2.9.3.2 [eneDive v. 2.0b23 [reeNA [The number of mitochondrial haplotypes, the number of polymorphic sites, haplotype diversity (P v. 5.1 . Haplotyn v. 2.0 . Genepop the Web  was usedte (FDR)  using th) [value . To calc 2.9.3.2  and Gene. 2.0b23 . Finally3 [reeNA  to estim3 [reeNA .2.5.rlequin. These analyses were carried out separately for the mitochondrial and microsatellite data at two different hierarchical levels: (i) comparing West and East Falkland and (ii) among the 23 breeding colonies. For the mitochondrial data, we used the measures Fst [Φst [Fst [Rst [Population structure within the Falkland Islands was assessed using hierarchical analyses of molecular variance (AMOVA) conducted within Aures Fst  and Φst Fst [Φst , the forFst [Φst  while thΦst [Fst  and Rst Fst [Rst , the lattructure v. 2.3.3. [K populations. We ran five independent runs for K = 1 to 10 using 1 000 000 Markov chain Monte Carlo (MCMC) iterations after a burn-in of 100 000, the correlated allele frequencies model and assuming admixture. The most likely number of clusters was evaluated using the maximal average value of Ln P(D), a model-choice criterion that estimates the posterior probability of the data. As we have previously found that Structure results can be sensitive to the inclusion of individuals with substantial amounts of missing data, we restricted this analysis to 233 individuals genotyped at 20 or more loci.In order to test for population structure without knowledge of the sampling locations of individuals, we also conducted a Bayesian cluster analysis of the microsatellite dataset using S. 2.3.3. . This pr2.6.rlequin. This resembles a unimodal wave in samples drawn from recently expanded populations, whereas samples from static or bottlenecked populations tend to exhibit multimodal distributions [rlequin was also used to test for deviation of the observed dataset from a model of rapid population expansion assuming the same mean number of pairwise differences as the observed sample [naSP to test for deviations from neutrality with Tajima's D [Fs [The distribution of the observed number of differences between each pair of haplotypes (the ‘mismatch distribution’) was calculated within Aibutions ,51. Arlejima's D  and Fu's's D [Fs . Signifi2.7.ottleneck v. 1.2.02 [Heq) was determined using 10 000 simulations and then compared against observed heterozygosity (He). We then recorded the number of loci for which He was greater than Heq and determined whether the overall set of deviations was statistically significant using standardized differences and Wilcoxon's signed rank tests. Bottlenecked populations are also expected to exhibit a characteristic ‘mode shift’ in the frequency distribution of alleles away from the L-shaped distribution expected under mutation-drift equilibrium [ottleneck was also used to generate a qualitative descriptor of whether the observed allele frequencies at each locus deviate from such a distribution.In order to investigate whether the Falkland Islands population underwent a genetic bottleneck, the microsatellite dataset was tested for heterozygosity excess ,55 using. 1.2.02 . One cri. 1.2.02 . Althoug. 1.2.02 , multi-s. 1.2.02 ,60. We tilibrium . ConsequM-ratio for the microsatellite dataset using the program M_P_Val [Mc, a critical value below which bottlenecks are inferred, using the program Critical_M [gp), the average size of non-single-step mutations (Δg) and θ = 4Neµ (where Ne is the effective pre-bottleneck population size at equilibrium and µ is the mutation rate). We used the default settings of gp = 0.2 and Δg = 3.5 [θ between 1 and 1000, the latter corresponding to an effective pre-bottleneck population size of 500 000 assuming a commonly used estimate of the dinucleotide microsatellite mutation rate of 5 × 10−4 mutants per gamete per generation [We also calculated Garza and Williamson's  M_P_Val . The sigitical_M . This prΔg = 3.5  and varineration  as suggeneration .2.8.Ne were loosely based on knowledge of the demographic history of the species. For details of the models and priors used, please see electronic supplementary material, table S2. The microsatellite mutation rate was set between 5 × 10−4 and 5 × 10−3 substitutions per generation. The mitochondrial DNA mutation rate was bounded between 8.12 × 10−7 and 3.8 × 10−6 substitutions per site per generation [M) and five summary statistics for the mitochondrial control region . These statistics were chosen on the basis of their sensitivity to demographic change. For each scenario, we simulated 1 × 106 datasets separately for the microsatellite data, the mitochondrial data, and the combined microsatellite and mitochondrial data. After that, we used a polychotomous-weighted logistic regression on the 4 × 104 simulated datasets closest to the observed dataset to determine the posterior probability for each scenario. In order to evaluate confidence in each scenario, we also calculated the posterior predictive error.To further investigate the demographic history of the Falklands population, we used approximate Bayesian computation ,65 as imneration ,68. We u2.9.ioEdit. All the sequences were then adjusted to the length of the shortest sequence (254 bp). To provide a broad overview of the relationships among the mitochondrial haplotypes, we then constructed a median joining network using Network v. 4.516 [Fst and Φst values were calculated and their significance determined using 10 000 permutations of the dataset.In order to facilitate comparisons between the Falkland Islands and the South American mainland, we collated mitochondrial control region sequence data from published studies for which haplotype frequency data were available. Representative data were obtained for Argentina, Brazil, Chile and Peru . We used the recommended heating scheme  and estimated the number of migrants per generation by multiplying the estimated mutation-scaled migration rate by the Θ value.Finally, we used Mv. 3.6.4 ,72 to es3.Out of a total of 277 samples collected, we successfully sequenced a 281 bp region of the mitochondrial control region and genotyped 22 microsatellites in 259 sea lions from 23 different breeding colonies around the Falkland Islands .3.1.Fst and approached significance using Φst. For microsatellites, significant differences were found using Fst between West and East Falkland as well as among the 23 colonies. However, none of the variance components were significant using Rst suggesting that the nuclear signal of population structure is rather weak.AMOVA was used to determine the proportion of genetic variation attributable to each level of population substructure, separately for both mitochondrial DNA and microsatellites . Contrastructure [K) from 1 to 10. The highest average log-likelihood value was associated with K = 1  indicating that Structure could not detect any population structure within the Falkland Islands.Arguably, the most powerful tests of population structure need not rely on knowledge of the sampling locations of individuals. We therefore carried out a Bayesian clustering analysis of the microsatellite dataset using Structure . Five ru3.2.p = 0.42; raggedness index = 0.019, p = 0.77) meaning that we could not rule out a model of population expansion. On the other hand, however, we also could not reject the null hypothesis of neutrality because Tajima's D and Fu's Fs values were negative but not statistically significant .To provide insights into the recent demographic history of South American sea lions at the Falkland Islands, we generated a mismatch distribution from the mitochondrial data  and testTo test the hypothesis that the population experienced a genetic bottleneck, we analysed the microsatellite data using the heterozygosity excess approach of Luikart & Cornuet . The resM-ratio of Garza & Williamson [M values obtained through simulation (0.63–0.81), implying a lack of support for a bottleneck.For populations with large pre-bottleneck sizes, the ratio of the number of alleles to the allelic size range has been suggested to be more informative about bottleneck history than heterozygosity excess . Consequlliamson . The reslliamson  as well Finally, we analysed the mitochondrial and microsatellite data within an approximate Bayesian computation (ABC) framework to evaluate statistical support for the following four demographic scenarios: (i) constant population size; (ii) population expansion; (iii) population reduction and (iv) a population bottleneck . The best supported scenarios were population expansion for the mitochondrial dataset, stable population size for the microsatellite dataset and a bottleneck for the combined mitochondrial and microsatellite dataset . However3.3.Fst and Φst values . From this figure it can also be seen that the Atlantic coast (Argentina and Brazil) and the Pacific coast (Chile and Peru) of South America are deeply divergent, with the Falkland Island haplotypes being more closely related to the former. In addition, two haplotypes were found to be shared between the Falkland Islands and Chile, and one between the Falkland Islands and Argentina, indicating a limited degree of maternally directed gene flow between the Falklands and the South American mainland.We analysed the relationship between sea lion colonies from the Falkland Islands and the South American mainland by comparing our mitochondrial data with 182 previously published mitochondrial sequences from Argentina, Brazil, Chile and Peru . This re3.4.igrate [figure 4a and b respectively) than Argentina and Brazil (figure 4c and d respectively). The two highest migration rate estimates were from Argentina to Brazil (green distribution in figure 4d) and from the Falklands to Brazil (pink distribution in figure 4d) possibly reflecting a combination of geographical proximity and prevailing currents (see Discussion).Finally, we used the program Migrate  to estim4.We conducted a genetic analysis of South American sea lion populations around the Falkland Islands and combined our data with previously published mitochondrial sequences to allow a comparison to be made between the Falklands and the South American mainland. Within the Falkland Islands, we found moderately high levels of genetic diversity at both types of marker and no evidence for a recent genetic bottleneck. Population structure was also rather weak in comparison to the strong maternally directed population structure observed across the broader species range. Finally, migration rate estimates were very low in all pairwise comparisons involving Argentina, Brazil, Chile and the Falklands, which is at odds with the hypothesis that the decline was caused by historical exploitation in Argentina.4.1.Fst but failed to reach significance using Φst, suggesting that the overall result may be driven by haplotype frequency differences.Tests for population structure within the Falkland Islands uncovered somewhat mixed results depending on the marker and genetic distance measure used. For this reason, and for consistency with previous studies of this species ,21,23, oFst and not Rst. Similarly, Bayesian cluster analysis of the microsatellite dataset failed to uncover any evidence for population structure within the Falklands. Taken together, these results are suggestive of the presence of very weak population structure within the Falkland Islands, as a simulation study found that Structure often struggles to find sub-populations when Fst is below around 0.02 [A similarly weak and rather inconsistent pattern was also obtained for the microsatellite data, despite our having used 22 loci. For this class of marker, a significant proportion of the genetic variance was attributable to differences between West and East Falkland as well as among colonies, but only using und 0.02 .et al. [et al. [et al. [These findings are in line with previous studies of this species on the South American mainland. For instance, Túnez et al.  found we [et al.  found no [et al.  found si [et al. –77. This [et al. .Our results are somewhat less straightforward to interpret, as genetic differences appear to be present among colonies but whether or not these achieve statistical significance depends on the genetic distance measure used. Moreover, taking our results at face value one might conclude that, although genetic differences among colonies are found at both class of marker, a difference between West and East Falkland is only apparent in the nuclear and not the mitochondrial genome. Such a pattern might be genuine, although this would be contrary to expectations based on strong female philopatry and male dispersal. Alternatively, it could be possible that different markers vary in their power to detect population structure . To distArctocephalus galapagoensis [Zalophus wollebaeki [Neophoca cinerea [Regardless of the exact pattern of population structure, South American sea lions at the Falkland Islands show much weaker structuring than is evident over similar spatial scales in several other related pinniped species, including the Galápagos fur seal, agoensis , the Galllebaeki  and the  cinerea . This co cinerea –84. This cinerea . However4.2.The first hypothesis we tested was that a historical bottleneck could have been involved in the failure of the sea lion population to recover from commercial exploitation. Although census data suggest that human exploitation may not have been severe enough to have depleted genetic diversity, these data are imperfect and effective population sizes are typically at least an order of magnitude smaller than census sizes, particularly in polygynous species such as sea lions where only a fraction of adults contribute towards successive generations . Moreoveottleneck only found support for a bottleneck with the IAM and TPM with 30% multi-step mutations, while the more conservative TPMs with 5% or fewer multi-step mutations did not detect a significant excess of heterozygosity. Although this is difficult to interpret because the true proportion of multi-step mutations is unknown, a clear contrast can be drawn with the Antarctic fur seal , which experienced a severe bottleneck that is reflected in a significant excess of heterozygosity with the TPM with 5% multi-step mutations [M-ratio was not consistent with a bottleneck, and fourth, ABC analysis also failed to find convincing support for a bottleneck scenario. In contrast again with the Antarctic fur seal study, where ABC analysis conclusively favoured a bottleneck, we found that none of the four scenarios were robustly supported, either by the mitochondrial or microsatellite datasets on their own or when the two datasets were combined. One explanation for this stems from a recent analysis of the numbers and sexes of sea lions hunted in the Falklands from 1928 to 1966 [To test for a bottleneck, we analysed genetic data from the Falkland Islands using three complementary approaches. Although the results are perhaps not as clear cut as one might hope, a number of lines of evidence suggest that sea lions at the Falklands probably did not experience an appreciable loss of genetic diversity. First, we detected moderately high levels of genetic diversity at both mitochondrial DNA and microsatellites in comparison with values reported for 18 different pinniped species (see table 7 in ). Secondutations . Third,  to 1966 . Althouget al. [A prominent hypothesis for the collapse of the sea lion population at the Falkland Islands is that large-scale commercial harvesting operations in Argentina could have been responsible ,16,18,91et al.  argued tArctocephalus australis [To explore patterns of genetic connectivity across the geographical range of the species, we analysed mitochondrial data from the Falklands, Argentina, Brazil, Chile and Peru. Only four shared haplotypes were found among these five regions, indicating very strong mitochondrial structuring over a continental scale. While this is consistent with a previous study of this species that compared Brazil with Peru , ours isustralis .We also found limited evidence of haplotype sharing between the Falkland Islands and the South American mainland, with only two haplotypes being common to the Falklands and Chile, and one being shared by the Falklands and Argentina. By implication, maternally directed gene flow appears to be restricted between the Falklands and South America. This clearly goes against the notion that migration to Argentina could have been responsible for the decline of the Falklands population, although our genetic data would be unable to detect migration if the majority of migrants had been culled prior to breeding. This, however, seems unlikely for two main reasons. First, if female migration was a previously unappreciated aspect of this species biology, one would expect this to be reflected in widespread haplotype sharing due to the migration that would have taken place prior to commercial harvesting in Argentina. Second, the sea lion population in Argentina was estimated to have been almost twice the size of the Falklands population in the 1930s .igrate yielded very low migration rate estimates for all of the pairwise regional comparisons, lending further support to the notion of restricted long-distance female dispersal. We also found some evidence to suggest that migration rates towards Chile and the Falkland Islands may be lower on average than migration rates towards Argentina and Brazil, although some degree of caution is warranted due to the fact that all of the estimates are very low. Interestingly, the two highest estimates were from Argentina to Brazil and from the Falklands to Brazil. One possible interpretation of this could be that the prevailing northwards flow of the Malvinas current [Consistent with the rarity of shared haplotypes, analysis of the global mitochondrial dataset within M current  may faciet al. [Finally, because we do not have any microsatellite data from the South American mainland we cannot exclude the possibility of male-mediated gene flow from the Falkland Islands towards Argentina. However, it is unlikely that the harvesting of adult males would significantly influence population growth as pinniped population dynamics are typically most sensitive to the survival of adult and subadult females ,96. Moreet al.  found noet al. . Furtheret al. [et al. [In conclusion, our study failed to find evidence of a strong bottleneck, consistent with the conclusion of Baylis et al.  based on [et al.  argued t


Procyon lotor) is often considered as a typical example for such a successful invasion resulting from a small number of founders. The species’ largest non-native population in Germany is commonly assumed to stem from a small number of founders and two separate founding events in the 1930s and 1940s. In the present study we analyzed 407 raccoons at 20 microsatellite loci sampled from the invasive range in Western Europe to test if these assumptions are correct. Contrary to the expectations, different genetic clustering methods detected evidence for at least four independent introduction events that gave rise to genetically differentiated subpopulations. Further smaller clusters were either artifacts or resulted from founder events at the range margin and recent release of captive individuals. We also found genetic evidence for on-going introductions of individuals. Furthermore a novel randomization process was used to determine the potential range of founder population size that would suffice to capture all the alleles present in a cluster. Our results falsify the assumption that this species has become widespread and abundant despite being genetically depauperate and show that historical records of species introductions may be misleading.Biological invasions provide excellent study systems to understand evolutionary, genetic and ecological processes during range expansions. There is strong evidence for positive effects of high propagule pressure and the associated higher genetic diversity on invasion success, but some species have become invasive despite small founder numbers. The raccoon ( While introduced populations are normally —but not invaders , 4. Indeinvaders  or if phinvaders . Generalinvaders , which hinvaders , 9. Besiinvaders , 3.Procyon lotor) is a medium-sized Central and North American carnivore that has colonized different parts of the world due to deliberate or accidental releases [The raccoon . However, North American-based studies frequently report more than ten alleles and values of HO>0.70 for many loci [However, accidental or deliberate releases of raccoons kept as household pets are fairly common , 12 and any loci –28.We genotyped 20 microsatellite loci to analyse the genetic diversity and population structure of German raccoons. Our overall objective was to test whether raccoons became established and reached high densities in Germany despite being genetically impoverished, or whether a larger pool of founders contributed to a genetically diverse population. Specifically, we wanted to reconstruct the number and geographic locations of founder populations and assess the degree of admixture between them. We used genetic assignment and exclusion methods to test for the presence of recently escaped or released individuals. Furthermore, we inferred the number of founder individuals in each population by estimating the number of individuals required to introduce all the alleles identified.A total of 407 raccoon samples  was collected from the core distribution in Germany (near the introduction sites of Edersee and Wolfshagen) as well as from the expanding range margins in Germany, Belgium and Luxembourg . SamplesBuccal swabbing was performed with sterile diagnostic rayon dry swabs . Swabs were stored in sterile tubes at -20°C. Tissue samples were stored frozen or in absolute ethanol. Hair samples were stored in filter paper with silica gel  at room temperature. Genomic DNA from tissue or swab samples was extracted using the DNeasy Blood and Tissue kit  following the manufacturer’s protocol (replacing ATL buffer with 200 μl PBS buffer for the swab samples). We extracted the hair DNA with a modified Chelex100 protocol, using 250 μl of a 10% Chelex100 Resin solution  with addition of 4 μl Proteinase K (18 mg/ml) per sample and an overnight lysis step .PLOT-01, PLOT-02, PLOT-03, PLOT-04, PLOT-05, PLOT-06, PLOT-07, PLOT-08, PLOT-10, PLOT-11, PLOT-13 [PLO-M15, PLO2-117, PLO-M3, PLO-M20, PLO2-14, PLO-M2, PLO-M17, PLO3–86 [PLM01 and PLM03 [PLOT-01, PLOT-02, PLOT-03, PLOT-04, multiplex 2 loci PLOT-05, PLOT-06, PLOT-07, PLOT-08 and multiplex 3 loci PLOT-10, PLOT-11, PLOT-13. Multiplex 4 contained loci PLO-M15, PLO2-117, multiplex 5 loci PLO-M3, PLO-M20, PLO2-14, PLO-M2 and multiplex 6 loci PLO-M17, PLO3–86. Multiplex 7 contained loci PLM01, PLM03. Reactions were performed in a final volume of 10 μl containing 1–10 ng DNA, 5 μl Type-it Microsatellite PCR master mix  and 0.3 μM of each primer. PCRs were performed in a Multigene Gradient Thermal Cycler  using the following program: one cycle of 15 min at 95°C, 32 cycles at 94°C for 30s, 90s at the multiplex-specific annealing temperature , followed by 40s at 72°C and a final extension for 30 min at 72°C. Hair and swab samples were amplified and scored twice to minimalize the risk of genotyping errors. The fluorescently labelled PCR products were analysed on a MEGABACE 1000 automated sequencer  and fragment lengths of the alleles were scored by eye using the Fragment Profiler 1.2 . Preliminary data analysis (results not shown) provided evidence for linkage disequilibrium between PLOT-04 and PLO-M17. We therefore excluded PLO-M17 from all analyses.All individuals were genotyped at 21 microsatellite loci:  PLOT-13 , PLO-M15 PLO3–86 , PLM01 and PLM03 . All locK) by performing ten independent runs of K = 1–12 with 106 Markov chain Monte Carlo (MCMC) iterations after a burn-in period of 105 iterations, using the model with correlated allele frequencies and assuming admixture. ALPHA, the Dirichlet parameter for the degree of admixture, was allowed to vary between runs. After deciding on the most probable number of sub-populations based on the log-likelihood values (and their convergence) associated with each K, as well as on the ΔK method by Evanno et al. [q), averaging q over different runs of the same K. In order to facilitate geographical representation, the average q values for each administrative district (‘Landkreis’) were calculated and mapped using ArcGIS 10.1 . Second, we also analysed our data using the ‘clustering of individuals’ algorithm implemented in BAPS v.6.0 [K = 2–12.We analysed the population genetic structure using two Bayesian genetic clustering algorithms. First, we analysed the data in STRUCTURE 2.3.4 . We estio et al. , we calcPS v.6.0 , which iq). We represented the results from K = 7, averaging q over eight runs with the highest log-likelihood values  in Genetix 4.05.2  and perfFST  in GenAlFST  and by aFij) [We tested the data set for isolation-by-distance (IBD) by analysing genetic relatedness between pairs of individuals as a function of geographical distance, using program SPAGeDi 1.2 . The sloFij)  was chosA), observed heterozygosity (HO) and unbiased expected heterozygosity (uHE) for each STRUCTURE cluster and the number of private alleles (pA) in a cluster. Allelic richness (AR) was calculated using Fstat 2.9.3.2 [r) by the use of a third individual as a reference, thus reducing the chance of genes identical in state being mistakenly inferred as identical by descent. We estimated effective population sizes (Ne) of each genetic cluster using the linkage disequilibrium method in program NeEstimator v.2.01 [We used GenAlEx to estimate the number of alleles . Furthermore, we did not observe any systematic deviations from Hardy-Weinberg expectations (HWE) at any of the remaining 20 loci. Between zero and five loci deviated from Hardy-Weinberg expectations (HWE) at α = 0.05-level in the seven clusters before multiple-test corrections. While no locus caused a problem in the HA, HE and SN clusters after correcting for multiple tests, three loci in the BB cluster  and one locus in both the LU  and RP  clusters exhibited a significant deficit of heterozygotes. In the urban KA cluster one locus exhibited a significant excess of heterozygotes After excluding -M17 see , we did The FCA essentially separated BB and SN from all other populations along its first axis and SN and HA from all other populations along its second axis . While tP < 0.001) and another three individuals at the P < 0.01 level . These results suggested that cluster RP (mostly) contained separately introduced individuals, thus all RP individuals were excluded from further analysis. No sample of the other six clusters could be excluded in a leave-one-out approach at the P < 0.001 level.In GENECLASS, seven of the 14 RP individuals were excluded with high probability from the other six clusters  were obtained between HE and the three western-most clusters  as well as between BB and SA. The AMOVA revealed that a significant part of genetic variation  was explained by the genetic clusters. According to the STRUCTURE results, all clusters had a relatively large degree of admixture . This proportion was even smaller in the remaining four clusters . The whole data set  was characterized by a strong and significant IBD pattern . However, when the analysis was limited to individuals within the same STRUCTURE cluster, the IBD pattern was still significant, but much weaker .The six STRUCTURE clusters (excluding RP) were strongly differentiated, with nd 0.166 . The lowdmixture . Only abAR ≥ 4.3 . In all other clusters, HO varied from 0.56 to 0.64 and uHE from 0.58 to 0.65  all having estimates of R ≥ 4.3 . The 13  to 0.65 . BB and  to 0.65 , with thThe relatedness estimates TrioML calculated in COANCESTRY showed a high relatedness in the urban population KA as well as in the population at the range margin (LU) . The effAs was to be expected, the total number of alleles included in the analysis affected the estimate of the minimum number of founders . The estThe majority of the information on the large-scale genetic structure of North American raccoons is available for the Eastern United States . When anOur results contradict the common assumption that the whole German raccoon population descended from two separate introduction events in central and eastern Germany –16, 22. FST-values) between the HA and HE clusters probably resulted from recent admixture.The fourth major genetic cluster was located in the Harz Mountains and in north-central Germany (HA). It has, indeed, been reported that up to 60 raccoons escaped from fur farms in 1945 in the Harz Mountains , howeverFST-based genetic differentiation estimates suggested that these KA and LU clusters were founded by individuals from the HE cluster. Since the first raccoons were sighted in the city of Kassel in the 1960s [2) in Europe [Both clustering methods also inferred the presence of two additional, smaller genetic clusters that were geographically coherent—the city of Kassel and Luxembourg/neighbouring regions—and therefore appeared to represent biologically meaningful units. The pattern of overlap in the FCA and the n Europe . A reducn Europe  and the However, there are alternative explanations for the inference of the clusters observed in our data set. For example, the pattern of IBD observed when considering the whole data set has the potential to cause the inference of artificial partitions by the clustering methods . HoweverP<0.01-level, had one of the five mitochondrial haplotypes typical for Germany [Finally, the results of the FCA and the GENECLASS exclusion tests provided substantial support for the hypothesis that the seventh STRUCTURE cluster (RP) essentially consisted of recently introduced individuals. These individuals formed five distinct clusters in the BAPS analysis, but were assigned to a single cluster by STRUCTURE. Even though three individuals excluded (at least) at the  Germany , our micWe found strong evidence for recent admixture of the separate founder populations. It is perhaps surprising that some 70 years after the last major introduction event (not considering Saxony) the founder populations are still genetically distinct. While the inner-German border fortifications might have contributed to a reduced admixture between east and west, the core distribution areas of the eastern HA and BB clusters were relatively isolated until an increase in population density was detected since 1990 . Given tA = 7.0; BB: A = 6.8) as well as the average heterozygosity values reported here (0.56 ≤ HO ≤ 0.61 and 0.60 ≤ uHE ≤ 0.62) were lower than comparable estimates from the raccoon’s native range [Even though not directly comparable to the present study due to differences in the suite of microsatellite used for genotyping, the average number of alleles observed in the two largest clusters  were too high and very likely uninformative. In the subsequent discussion, we therefore focus the method based on re-sampling of the empirical data set . The highest estimates of founder individuals were obtained when all individuals  assigned to a cluster were considered in the analysis. Because of the recent admixture between the different founder populations, these estimates might well be adequate representations of the minimum number of individuals that have contributed to the overall genetic composition of the current clusters.In order to attempt to infer the number of original founders, we removed all the individuals from a cluster whose genetic profiles contained alleles occurring at low frequency (< 0.02) . The corThe advantage of our new method is that it is very simple and does not require information on the allele frequencies in the source population(s). However, the estimates are affected by sample size and resampling from the allele frequency distribution clearly leads to overestimating founder numbers. Also the method cannot adequately account for genetic drift and does not allow for mutations creating new alleles. At the very least, the method provides some useful information regarding relative estimates of founder numbers (if sample sizes are broadly comparable). When we assumed all alleles in a cluster to have equal frequencies (to account for genetic drift), the BB estimate was in line with the historic information, while the HE cluster was estimated to have been founded by 21 individuals. In other words, it is likely that population HE at least had a larger number of founders than suggested by the historic record, even when considering the most conservative analysis.Our study illustrates that it is precarious to fully trust the historical record of founder events in invasion biology. We proved the existence of at least four independent founding events with probably a substantially higher number of founders than commonly assumed.S1 FigK did not converge on the same solution (for K = 4 to K = 7). The percentage above each bar plot shows the proportion of the runs that converge on the presented clustering solution.Each individual is represented by a single vertical line, representing its estimated proportion of membership to the different genetic clusters. Different independent STRUCTURE runs for the same value of (TIF)Click here for additional data file.S1 Script(R)Click here for additional data file.S2 Script(R)Click here for additional data file.S3 Script(R)Click here for additional data file.


Nanorana parkeri, a frog endemic to the Tibetan Plateau, across its distribution range in the southern plateau. Mitochondrial cytochrome b (Cytb) and three nuclear genes  revealed two distinct lineages (i.e. the lineages East and West), which were strongly geographically structured. The split of the two divergent lineages was dated back earlier than the Middle Pleistocene, probably being associated with climatic and ecological factors. Species distribution modeling, together with the phylogeographic structuring, supported the hypothesis of multiple refugia for N. parkeri on the Tibetan Plateau during the Pleistocene glaciations, and suggested the Yarlung Zangbo valley and the Kyichu catchment to be the potential refugia. Our findings indicate that Pleistocene climatic changes have had a great impact on the evolution and demographic history of N. parkeri. Our study has important implications for conservation of this and other frog species in the Tibetan Plateau.Quaternary climatic changes have been recognized to influence the distribution patterns and evolutionary histories of extant organisms, but their effects on alpine species are not well understood. To investigate the Pleistocene climatic oscillations on the genetic structure of amphibians, we sequenced one mitochondrial and three nuclear DNA fragments in  Quaternary climatic oscillations greatly influenced the current distribution patterns and evolutionary histories of many organisms, which could facilitate population divergence and consequent speciation135672 and an average elevation of 4500 meters above sea level (m.a.s.l.). It comprises broad planation surfaces and basins in its central part, and most of the highest mountains along the margins of the plateau10118913The Tibetan Plateau is the highest, largest and one of the youngest plateaus on Earth, with an area of 2.5 million kmEffects of Pleistocene climatic changes on the distribution and evolution have been investigated in many species endemic to the Tibetan Plateau and surrounding regions1516171819201516182217241521161719Nanorana parkeri occupies an extensive altitudinal range from 2850 to 4700 m.a.s.l.1719Amphibian species are sensitive to environmental changes because of their physiological constraints, and therefore are regarded as good indicators of geological and climatic changes over time2627N. parkeri in the Tibetan Plateau by analyzing the sequences of one mitochondrial  and three nuclear DNA  fragments. By collecting samples of N. parkeri across its range in the Tibetan Plateau, we aimed to (i) explore the genetic diversity and phylogeographic structure of the N. parkeri populations; (ii) examine whether the focal species survived in the plateau platform during the Pleistocene glaciations, or followed the pattern of glacial retreat to the low-altitude periphery and recolonization through postglacial expansion. The combination of phylogeographic analyses and species distribution modeling was used, which allow us to obtain a comprehensive picture of the phylogeography and historical demography of N. parkeri.In this study, we investigated the phylogeographic pattern and genetic population structure of N. parkeri from 30 localities  segments were obtained from 530, 531 and 530 samples, respectively. A total of 24 haplotypes were identified in c-Myc2, and 12 sites were variable with seven parsimony-informative. Two haplotypes were found in Rhod with one parsimony-informative site. In Tyr, five haplotypes were identified, and four sites were variable with one parsimony-informative site. All the haplotypes were submitted to GenBank with accession numbers KJ434188-KJ434292 and KJ810612  and maximum likelihood (ML) approaches. Two main lineages (East and West) were identified, and the lineage East was strongly supported  was observed between W1 and W2. Divergence between lineages East and West ranged from 4.24% to 4.35% (The phylogenetic relationships deduced from Cytto 4.35%  online.N. parkeri within its range (b lineage East (i.e. E1–E4) occupied the eastern region of the distribution area, while all the individuals (excluding one from CD) from the lineage West (i.e. W1 and W2) were confined to the western region. Sublineages E1 and W1 were most prevalent, while E2, E3, E4 and W2 occurred mostly in one or several populations. E2 was detected in four neighbouring populations . Similarly, E3 was restricted to three neighbouring populations . All individuals from the population LU belonged to E4, and W2 was only found in the population NM.Strong phylogeographic structure was observed for ts range . All theb haplotype diversity (h) was moderate to high (h = 0.52–0.85) in five of the six Cytb sublineages (except W1), while the nucleotide diversity was quite low .The CytST values ranged from 0.69 to 0.98 between the six Cytb sublineages, and all the values were significant . The AMOVA results showed strong genetic differentiation of Cytb sequences, with 93.2% of the total variance detected between the eastern and western regions  were determined in the eight western populations, and Hap21 and Hap22 derived from populations NM and PL were very different from Hap19 and Hap20 from the rest western populations  and a much faster-8/site/year) mutation rate, respectively. With the slower mutation rate, divergence time for the deep split between eastern and western populations was dated back to 3.72 million years ago (Mya) with 95% highest posterior density (95% HPD) of 2.72–4.74 Mya  in lineages East and West, as well as in sublineages E1 and W1 (P < 0.01) Fu’s SFwere detected in lineage East, sublineages E1, E2 and W1. The demographic trend for the sublineage W2 was not investigated in the mismatch and BSP analyses because of the small sample size (n = 9). In the analysis of mismatch distribution, SSD and Hrag values supported the population expansion model in all the Cytb lineages and sublineages (excluding lineage East). Sublineages E1– E4 and W1 each exhibited an observed unimodal mismatch frequency distribution  conditions. The Maximum Entropy (Maxent) model worked well in predicting occurrences under current conditions. The average AUC (area under the receiver operating characteristic curve) value for the test data across 4 runs was 0.92 with a standard deviation of 0.02. Averaged distributions under present and the LGM conditions are shown in N. parkeri on the Tibetan Plateau (We used species distribution models (SDMs) to predict the potential habitat distribution of  Plateau . During  Plateau .b lineages (East and West) were resolved in N. parkeri populations. Generally there were no genetic and geographic overlaps between these two lineages. The maternal split was confirmed by three nuclear genes in that eastern and western populations had distinct nuclear haplotypes. The estimated split time indicated an old divergence (1.4–3.7 Mya) between the two lineages. Therefore, this deep divergence suggests a long history of population isolation, wherein each group could represent a distinct lineage.In this study, two major Cytb E1 and W1 lineages  might have contributed to this deep divergence. However, there are no apparent geographic barriers between the two regions. Furthermore, widespread distribution of the Cytlineages  suggesteb haplotype diversity  and several following less extensive glaciations on the plateau. If they survived in one glacial refugia, then the genetic lineages would have mixed together in terms of nuclear genes. On the contrary, the two lineages had distinct nuclear haplotypes, implying that they survived in different refugia during the glacial periods. Second, refugia always provided suitable stable habitats for extant organisms through geological time scales . Together with the current occurrence, the SDMs indicated that the Yarlung Zangbo valley and Kyichu River catchment might be the most suitable habitats for N. parkeri  and animals  during glaciations. Third, this scenario fits well with the Pleistocene glacier conditions on the Tibetan Plateau where extensive ice caps and valley glaciers formed during the glaciations . A recent study with sedimentological, geochronological and palaeobotanical analyses revealed that the Kyichu River was free of ice in the Last Glacial (~0.034 Mya), before LGMInstead, several lines of evidence support the scenario of multiple glacial refugia on the plateau platform. First, two deep genetic lineages were revealed, dating back to the Middle Pleistocene or earlier, long before the LGM. Although the divergence time should be interpreted with caution in absence of fossil records, these genetic lineages probably survived the maximum glaciation11 parkeri  and S1 dN. parkeri. In particular, the Cytb sublineages E1 and W1 showed very strong signals of postglacial expansion in the neutrality tests, mismatch distribution and BSP analyses. It is, therefore, plausible to postulate that the boundary region was probably a second contact zone. A finer analysis based on more specimens should be performed to better understand the microevolutionary processes in this boundary region.Two distinct genetic lineages were detected, but several exceptions were also observed from the populations in the boundary region  and 3. IN. parkeri. These two distinct genetic lineages, which diverged for a long time and had limited gene flow between them, could be considered as two evolutionarily significant units (ESUs), and each needs to be protected. The Yarlung Zangbo valley and the Kyichu catchment were thought to be the potential refugia for this frog. These regions, in particular the Lhasa area (within the Kyichu River catchment), have been strongly affected by human activity as early as four thousand years agoThe apparent genetic structure and limited gene flow between the eastern and western populations has important conservation implications for In summary, our data provide comprehensive genetic evidence of anurans on the interior Tibetan Plateau that paleoclimatic events during the Quaternary have a great influence on the distribution and evolution of alpine organisms. Results of phylogeographic structure and ecological modeling analyses supported the hypothesis of multiple refugia on the plateau platform, with potential refugia in the Yarlung Zangbo valley and Kyichu catchment. Our study also provides useful genetic information essential for the conservation of this endemic frog.The methods were carried out in accordance with the approved guidelines of the Good Experimental Practices adopted by the Institute of Zoology, Chinese Academy of Sciences. All experimental procedures and animal collection were conducted under the permits (No. IOZ13015) approved by the Committee for Animal Experiments of the Institute of Zoology, Chinese Academy of Sciences, China.N. parkeri adults were collected by hand from shallow ponds on the Tibetan Plateau during the breeding season (July-August) in 2012. Tissue samples (toe clips from back legs) were taken and stored in 95–100% ethanol. All frogs were released back into the wild at the capture sites immediately after sampling without harming. Total genomic DNA was extracted from the tissues using a standard phenol-chloroform extraction procedure after digestion with proteinase Kb, Cytb) and three nuclear fragments  were sequenced for molecular analyses. The partial Cytb sequences were amplified with primers Cytb-F and Cytb-R, and the fragments of c-Myc2 with CYMC5 and CYMC6Rhod with Rhod1A and Rhod1D, and Tyr with Tyr1G and Tyr1Bm) in polymerase chain reactions (PCRs) are detailed in b fragments were sequenced in both directions.One mitochondrial (cytochrome http://seqphase.mpg.de/seqphase/).Mitochondrial sequences were viewed and edited with the program DNAStar SeqMan v7.21 . All sequences were corrected manually, aligned and trimmed to uniform lengths using BioEdit v7.1.847b haplotypes were reconstructed using Bayesian inference (BI) and maximum likelihood (ML) methods. A homologous fragment from the complete mitochondrial genome of N. pleskei (NC_016119), a closely related species to N. parkeri, was used as the outgroup. The Cytb sequences were partitioned by codon position. The best-fit model of nucleotide substitutions for each partition was estimated from 88 models using the ML topology optimization approach under the Bayesian information criteria (BIC) as implemented in jModelTest v0.1.1http://tree.bio.ed.ac.uk/software/tracer/) to assess the stationarity and convergence. The first 25% of the sampled trees were discarded as burn-in, and the remaining trees were used to construct a majority rule consensus tree and assess the posterior probabilities. The default values were used for the other parameters. The ML method based on the partitioned data was performed in RaxML v7.2.6Phylogenetic topologies of Cytc-Myc2 by using the BI method. JC+G model was chosen as the best-fitting model under BIC by jModelTest. MCMC chains were run for 5,000,000 generations, and the rest parameters were set as default.For nuclear genes, because of the limited number of the parsimony informative sites, phylogenetic topologies were only constructed in h) for each Cytb lineages (see Results) and population were calculated using DNAsp v5.10.00p-distance) between Cytb lineages were computed in Mega v5.1ST was estimated between Cytb sublineages. Statistical significance was tested by 10,000 non-parametric permutations at the 5% significance level. Both analyses were implemented in Arlequin v3.5http://www.fluxus-engineering.com/) for the mitochondrial (Cytb) and nuclear  haploytpes, respectively.Nucleotide (π) and haplotype diversity . An evolutionary rate of 6.9 × 10-9/site/year in mitochondrial genes has been estimated in bufonid species60616263-8/site/year was used in Cytb of Ranid speciesN. parkeri, we conducted the analyses by using both the mutation rates, respectively. The strict molecular clock with the HKY+G substitution model selected by jModelTest was used and the constant population size coalescent was set as the tree prior. The MCMC chains were run for 30,000,000 steps and the parameters were logged every 3,000 steps. Three independent runs were performed. The results were combined with LogCombiner v1.7.5 from the BEAST package, and then visualized in Tracer v1.6 with burning in 10% of the original chain length.The time to the most recent common ancestor (TMRCA) for the key nodes in CytDSFP < 0.05) negative values for these statistics indicate past population expansions. Secondly, a pairwise mismatch distribution analysis, which considers the distribution of pairwise differences between haplotypes, was conducted in Arlequin with 10,000 permutations. For each Cytb lineage, theoretical distribution under the sudden expansion model was compared to the observed data. Goodness-of-fit was tested between the observed and the expected distribution of pairwise differences for each lineage using the sum of squared deviation (SSD) and Harpending’s raggedness index (Hrag)b ranging from 6.9 × 10-9/site/year-8/site/yearDemographic histories were assessed in three different ways. We first calculated Tajima’s N. parkeri from the GBIF database , literatures6970The species distribution models (SDMs) were generated using the maximum entropy machine-learning algorithm as implemented in the program Maxent v3.3.3kwww.worldclim.org) with a resolution of 2.5 arc-minutes. These variables describe temperature and precipitationr). When two variables were strongly correlated (r > 0.9), we chose the one being more biologically meaningful for the speciesA set of 19 bioclimatic variables for both current (~1950–2000) and the Last Glacial Maximum  conditions were downloaded from the WorldClim v1.4 database  values for training and test data. An AUC of 0.5 indicates a random prediction of presence and absence, while an AUC of 1 represents a perfect discrimination. The mean maximum training sensitivity plus specificity was used as the threshold to discriminate suitable from unsuitable habitats, which has been proved recently an accurate approachM.-H.L. and J.L. designed the study. J.L. analysed and interpreted the data, and wrote the manuscript. X.H., X.X., P.L. and Q.Z. participated in the sample collection. C.W. and D.F. conducted the laboratory work. M.-H.L. and J.L. revised the manuscript. All the authors read and approved this paper.How to cite this article: Liu, J. et al.Phylogeography of Nanorana parkeri (Anura: Ranidae) and multiple refugia on the Tibetan Plateau revealed by mitochondrial and nuclear DNA. Sci. Rep.5, 9857; doi: 10.1038/srep09857 (2015).Supplementary Information


Bemisia tabaci, we selected two Chinese populations—one from Harbin, N China, and one from Turpan, S China—that experience substantial heat and cold stress and conducted knockdown tests under static high- and low-temperature conditions. ANOVAs indicated significant effects of populations and sex on heat knockdown time and chill coma recovery time. The narrow-sense heritability (h2) estimates of heat tolerance based on a parental half-sibling breeding design ranged from 0.47±0.03 to 0.51±0.06, and the estimates of cold tolerance varied from 0.33±0.07 to 0.36±0.06. Additive genetic variances were significantly different from zero for both heat and cold tolerance. These results suggest that invasive B. tabaci Mediterranean cryptic species possesses a strong ability to respond to thermal selection and develops rapid resistance to climate change.With advancing global climate change, the analysis of thermal tolerance and evolutionary potential is important in explaining the ecological adaptation and changes in the distribution of invasive species. To reveal the variation of heat resistance and evolutionary potential in the invasive Mediterranean cryptic species of  The invasion of exotic species has accelerated along with the intensification of global climate change, the invasion of exotic species has accelerated. With respect to ecology, as one of the effects of global climate change, bioinvasion has deeply changed the structure and function of global ecosystems The ability of an organism to remain active under extreme conditions is a critical component of fitness Bemisia tabaci (Gennadius) is a complex species, containing at least 30 morphologically indistinguishable cryptic species, and undergoes rapid evolution B. tabaci Mediterranean cryptic species (MED) can cause rapid and widespread invasions and is considered particularly dangerous B. tabaci MED has become the dominant pest species, expanding its range from tropical areas to frigid areas of China within ten years Bemisia tabaci (Gennadius) Middle East-Asia Minor1 (MEAM1) and indigenous species to become the new “super bug” B. tabaci MED is one of the potential mechanisms underlying its invasive success The whitefly B. tabaci MED to evaluate the evolutionary potential of these fitness components. By estimating the heritability of thermal tolerance, we show that B. tabaci MED exhibits high variance in its response to heat stress and has displayed modest evolvability after its successful invasion in China. As a first survey of the quantitative genetics of thermal resistance traits in the whitefly, we studied two introduced populations of the invasive B. tabaci MED whitefly distributed across China. We hypothesized that the broad distribution and ecological success of this invasive species could be explained by the high additive genetic variation in the physiological traits related to functional capacities.The present study reports the trait means and variations in heat and cold resistance of B. tabaci is not a protected species in China; thus, no specific permissions were required for these locations and activities .B. tabaci can be found from July to November in the open field and overwinters in greenhouses in the winter months in Harbin B. tabaci MED experiences a daily mean air temperature (DMAT) range from 18°C to 26°C in July, and the DMAT decreases slowly in August and September in Harbin. Field populations of B. tabaci MED suddenly collapse when the temperature drops rapidly in mid- or late October, and field populations then move into greenhouses in Harbin in December; however, adult Bemisia can also be found in the open field during this time Whitefly eggs, nymphs and pseudopupae were collected from cotton, tomato and eggplant crops in fields near Harbin , Heilongjiang Province, northern China, and Turpan , Xinjiang Uighur Autonomous Region, western China. Harbin has the coldest weather and longest winters among the major Chinese cities. Due to the Siberian high-pressure system and its location above 45°N, the 24 h average temperature in January is only −18.4°C, and the annual mean temperature is 4.25°C, with the lowest temperatures dropping down to −42.6°C. B. tabaci MED can be found in cotton fields from April to December in Turpan. As shown in B. tabaci MED also experiences DMATs ranging from 18°C to 26°C in April, and the DMAT increases gradually in May and June. The DMAT can exceed 30°C in July and August, and B. tabaci MED expands its population distribution in the field during this period in Turpan. Field populations in Turpan decrease gradually in September, October and December with the decreasing DMAT.Turpan, one of China's four “oven cities”, is located at the other climatic extreme where summertime temperatures can soar to 49.6°C. Turpan has a unique temperate continental arid desert climate, with bright sunshine, high temperatures, and large day-night differences in temperature. The average maximum temperature is above 38°C from June to August, with more than 100 days experiencing a daily maximum above 35°C and 35–40 days with a daily maximum temperature above 40°C. Therefore, we expected that whiteflies collected at these locations would be suitable to test our hypotheses regarding adaptation through thermal tolerance in an invasive species.B. tabaci MED. This technique has proven to be a highly reliable method for distinguishing MED from other complex species The Harbin and Turpan field populations (G0) were reared separately. Fifty field-inseminated females were collected from each population and used to establish iso-female lines in 100 specially designed rearing cages in the laboratory; the whiteflies were maintained at 26°C under a 14:10 hour light:dark cycle at 55–60% humidity. The cylindrical rearing cage was 13.5 cm long, with an internal fly-proof plastic screen (11.0 cm in diameter) made of transparent polypropylene to aid in ventilation. Each cage contained a single cotton seedling planted in a pot, which was filled with water. Species/cryptic species identification was confirmed in the G1 generation using the RAPD-PCR method To compare the heat and cold resistance between the populations and sexes, after three generations of mass rearing, four hundred individuals were randomly selected from each mass-bred population (G5) and scored for knockdown resistance  on the third day after emergence.At the same time, we used a standard paternal half-sibling breeding design to estimate the genetic parameters for heat and cold tolerance of both of the Harbin and Turpan MED populations. For both populations (G5), one hundred virgins of parental males (sires) were randomly collected from each mass-bred population immediately after emergence using an air suction insect collector (described below). Each sire was placed in a rearing cage containing a single cotton seedling (with two cotyledons) and five virgin females (dams); the flies were allowed to mate and oviposit for 3 days before the adults were tested individually for knockdown resistance. When their offspring (G6) emerged, 4 females of each dam were collected, reared on cotton plants, mated with males and scored for knockdown resistance  when they were 3 days old. The breeding experiment was performed in the laboratory at 26°C under a 14:10 hour light:dark cycle at 55–60% humidity.http://johnwhock.com/products/aspirators/mouth-aspirators).To test heat knockdown resistance, female individuals were carefully collected using an air suction insect collector , which was set at a constant temperature of 45°C. We measured the interval between when the tube was placed in the water bath and when the whitefly lost control of its body and fell to the bottom of the tube.To test cold resistance, we measured the time to recovery following a chill coma induced by cold shock. One adult, confined as described above, was exposed to −5°C through submersion into a 50% glycol solution cooled by a refrigeration bath circulator  for a period of 10 minutes, and recovery time was measured at 26°C. Whiteflies assessed for heat and cold stress were tested on the same day.X is the design matrix for the fixed effect of run B and Zs and Zd are the design matrices for the random effects of sire and dam, respectively. The total phenotypic variance (σ2P) for the breeding design for the purpose of estimating genetic parameters was represented by2S, σ2D, and σ2W, are the sire, dam and within-group level variance components, respectively. Variance and covariance components were estimated using the restricted maximum likelihood technique via the MIXED procedure in SAS . As we used a half-sib full-sib breeding design, the sire variance, σ2S, is one-fourth of the additive genetic variance (VA) VA, we multiplied the sire variance by four.Our data were generated from a standard paternal half-sibling breeding design . The animal model used to analyze the data was2S was set to zero to determine whether the levels of additive genetic variance for each trait were significantly different from zero VP) in knockdown resistance was computed using all known relationships among individuals. Then, we estimated the narrow-sense heritability for both traits. Narrow-sense heritability for each trait was estimated as the additive genetic variance (VA) divided by the total phenotypic variance (VP) IA, and the additive genetic coefficient of variance,The additive genetic variance for each trait was first estimated using a univariate model. Log likelihood ratio tests were performed, where the final model for each trait was compared to a model in which σP<0.05 was considered to be statistically significant.For the population and sexual comparisons, ANOVAs were performed to evaluate the differences in the knockdown and recovery times, followed by the Tukey-Kramer multiple comparison test. The population (Harbin and Turpan) and sex  were used as the two factors. Means, standard errors, and coefficients of variation (%) for heat knockdown time and chill coma recovery time of the Harbin and Turpan populations are listed in F1, 466 = 20.50, P<0.001) and sex  on heat knockdown time  and sex  on chill coma recovery time  on heat knockdown time or chill coma recovery time . Females showed a significantly higher heat resistance than males  and a significantly lower cold resistance .own time , Table 2ery time , Table 2The estimates of genetic variance were similar between the two protocols, as well as between the two populations . Narrow-B. tabaci populations. The results indicated that genetic differences in thermal tolerance existed between the populations, with an increasing heat tolerance and a decreasing cold tolerance in the Turpan population compared with the Harbin population. Because Turpan experiences a very specific climate, characterized by a long-term, extremely high temperature, the Turpan population may be acclimated to the heat due to its exposure to a generally higher temperature compared with the Harbin population. By contrast, Harbin has the coldest temperatures and longest winter months compared with other major Chinese cities; thus, the Harbin population can survive a colder climate compared with the Turpan population. Our study demonstrated that field populations of B. tabaci MED cryptic species possessed a high heat tolerance in response to thermal stresses. This finding implies that there is a significant difference in the adaptive strategies to temperature between the Harbin and Turpan populations as a result of their different experiences with air temperature. Thus, we inferred that high, narrow-sense heritability for both heat and cold resistance is the direct driving force for the adaptive microevolution of B. tabaci.In our study, we compared the thermal tolerances of the Harbin and Turpan Abutilon theophrasti) and green amaranth (Amaranthus retroflexus), significant genetic shifts were observed in just four generations of selection under treatments involving different soil moisture levels, competitor diversity regimes, and intraspecific densities B. tabaci were revealed using the static knockdown time method, which has been widely used in Drosophila experiments Founder effects, genetic drift, and natural selection can all lead to genetic differentiation in populations of an invading species relative to the invader's source population Hsp70, which plays an important role in heat tolerance, might cause the observed differences between females and males. The optimum mRNA expression of HSP genes in females promotes a higher survival rate under heat shock conditions, and there are large between-species differences in HSP gene expression levels Differences in heat shock proteins (HSPs), especially D. melanogaster based on knockdown time experiments at 39°C suggest values ranging from 0.03 to 0.11 B. tabaci MED based on chill coma recovery time at −5°C (0.33 to 0.36) also indicated that a substantial proportion of the total phenotypic variation was caused by additive genetic variation.Given adequate genetic variability, invading species are often able to make rapid evolutionary adjustments. With regard to heat resistance, estimates of heritability in A, which reflects the potential shift in a trait relative to the trait mean, were 0.285–0.322 and 0.263–0.294 for heat knockdown time and chill coma recovery time, respectively. Consequently, the evolvability of cold resistance indicated a stronger evolutionary response than that of heat resistance. For heat resistance, due to the influence of the experimental protocol on estimates, previous estimates of evolvability for D. melanogaster based on knockdown time at 38°C suggest broad values, ranging from 0.02 to 14.53. Estimates of additive genetic variance and measures of evolvability were higher when D. melanogaster flies were exposed to a static stress rather than a ramping (when temperature was increased to an upper limit) stress when genetic variances were standardized according to the trait means The values of the evolvability measure, CVB. tabaci), which is now widely distributed across China. By measuring the narrow-sense heritability (h2) of thermal tolerance traits and because of the high CVA value of knockdown time, we show that natural B. tabaci MED cryptic species exhibit a high adaptive potential against both heat and cold stress. The h2 and CVA values of heat tolerance traits were higher than the cold tolerance traits in natural MED, which indicated the whiteflies were more able to respond to heat selection in field MED populations than cold selection. We suggest that MED expansion is likely to be favored by climate warming and that the high evolutionary potential of heat tolerance has enabled its invasion into mid- and high-latitude areas.Several studies have investigated the heritability and evolutionary potential of thermal tolerance traits in invasive species. In the current paper, we provide data for the invasive Mediterranean cryptic whitefly species (


Hyla arborea group). We investigated a natural hybrid zone between two lineages  of Mio-Pliocene divergence (~5 My) for comparison with other hybrid systems from this group.Hybridization between incipient species is expected to become progressively limited as their genetic divergence increases and reproductive isolation proceeds. Amphibian radiations and their secondary contact zones are useful models to infer the timeframes of speciation, but empirical data from natural systems remains extremely scarce. Here we follow this approach in the European radiation of tree frogs  across two independent transects, indicating an advanced state of reproductive isolation and potential local barriers to dispersal. This result parallels the situation between Our findings corroborate the negative relationship between hybridizability and divergence time in European tree frogs, where 5 My are necessary to achieve almost complete reproductive isolation. Speciation seems to progress homogeneously in this radiation, and might thus be driven by gradual genome-wide changes rather than single speciation genes. However, the timescale differs greatly from that of other well-studied amphibians. General assumptions on the time necessary for speciation based on evidence from unrelated taxa may thus be unreliable. In contrast, comparative hybrid zone analyses within single radiations such as our case study are useful to appreciate the advance of speciation in space and time.The online version of this article (doi:10.1186/s12862-015-0385-2) contains supplementary material, which is available to authorized users. Reproductive isolation between nascent species is expected to gradually increase with their divergence time (up to a threshold ), as a rBombina toads ) solation , whereassolation . In anurtermedia  (~2-3 Mytermedia . These r molleri ), the EuHyla tree frogs, Palearctic green toads (Bufo viridis subgroup) lineages with only ~2.6 My divergence have already reached complete isolation [In contrast, the speed of speciation may greatly differ between related taxonomic groups: while we still observe limited gene flow after ~5 My of divergence in solation , with yosolation . The timsolation . TherefoHyla and few other amphibian systems, rather argues for the role of progressive, multiple genome-wide changes. For instance, the accumulation of single-nucleotide substitutions, each with a subtle phenotypic effect, might account for large interspecies differences, as shown in Drosophila [Hyla will help to comprehend the process of speciation.Documenting the relationship between hybridizability and divergence time sheds light on the mechanisms leading to reproductive isolation. In the case of speciation with interspecific gene flow, the action of few genes with major effects (i.e. “speciation” genes) was proposed to accelerate the speciation process , such asosophila . Future In the light of previous work, our data suggest that natural hybridizability decreases with divergence time in European tree frogs, between which reproductive isolation is nearly complete after ~5 My. Speciation thus seems to advance progressively, and at similar rates within this radiation, and might thus be driven by gradual genome-wide changes rather than a few changes with major effects. This timeframe, although generally in line with scattered amphibian studies, seems to vary greatly between species groups (e.g. different families of anurans), and broad-scale assumptions on the time necessary for speciation should be made with caution. Whereas comprehensive comparisons of hybrid zones involving related lineages of varying divergence, such as ours, remain scarce, this approach proves to be informative to accurately characterize the progression of speciation.Tree frogs were sampled from 97 localities  distributed across northern Greece, southern Serbia (incl. Kosovo), Bulgaria and western Turkey, corresponding to the southern parapatric ranges of the two species . To enabcytochrome-b (cyt-b) based on published sequences from both species  screened for restriction sites with the NEB cutter online tool (http://tools.neb.com/NEBcutter2/index.php). We selected enzyme MseI which distinctively cuts cyt-b haplotypes in a species-specific way, yielding four segments for H. arborea  and three for H. orientalis . A total of 578 individuals were mitotyped as follows: (1) cyt-b (~950 bp) was amplified in 10 μL PCRs (methods: [MseI (New England Biolabs), 0.06 μL of BSA and 0.4 μL of NEB buffer #4, following the manufacturer’s recommendations; (3) mitotyping profiles were visualized and scored on an 1.5 % agarose gel after ~45’ of migration at 110 V.We designed a mitotyping procedure by restriction digest of the mitochondrial methods: ); (2) PCH. arborea showed that 19 of these loci represent at least 10 unlinked genomic regions (seven linkage groups plus three unlinked loci), potentially covering 10 out of the 12 chromosome pairs of Hyla [We genotyped 582 individuals for 23 microsatellite loci . This multivariate analysis summarizes both geographic  and genetic  information into principal components to detect and test for spatial genetic structure. We conducted the sPCA using an edited Gabriel graph as a spatial connection network, and interpolated the obtained population scores to the entire study area.Second, we decomposed genetic variation by a Principal Component Analysis (PCA) of microsatellite genotypes ( package ). To get package  . Following the software’s documentation, we further specified distant Turkish H. orientalis populations (loc. 87–97) as sampled apart from the area of admixture (using the s option).Third, we inferred the nature of hybrid tree frogs in parapatric areas from our microsatellite dataset with NewHybrids , which cintrogress R package [H. orientalis composite alleles for each locus in each population using a custom R script. For each population on the two transects , we calculated the geographic distance between that population and the westernmost population of the transect (sites 27 and 54 for Serbia and Greece respectively) using Google Earth (https://earth.google.com). We then fitted four-parameter sigmoid clines to the population allele frequencies using the hzar R package [hzar. The fitting method accounts for differences in sampling sizes among localities along transects. For each transect, we inferred coincidence and concordance by comparing the confidence intervals of each cline’s width and center to the corresponding confidence interval obtained for the genome-average cline.To conduct cline analyses, we first converted each microsatellite locus to a two-allele system using the  package , and cal package , in whicThe data set supporting the results of this article is archived in Dryad, doi:10.5061/dryad.q50m1.


The data on Hoarding, OCS, and tic symptoms were simultaneously analyzed in Mplus. A liability threshold model was fitted to the twin data, analyzing heritability of phenotypes and of their comorbidity. Following the criteria for a probable clinical diagnosis in all phenotypes, 6.8% of participants had a diagnosis of probable hoarding disorder (HD), 6.3% of OCS, and 12.8% of any probable lifetime tic disorder. Genetic factors explained 50.4, 70.1, and 61.1% of the phenotypic covariance between hoarding-OCS, hoarding-tics, and OCS-tics, respectively. Substantial genetic correlations were observed between hoarding and OCS (0.41), hoarding and tics (0.35), and between OCS and tics (0.37). These results support the contribution of genetic factors in the development of these disorders and their comorbidity. Furthermore, tics were mostly influenced by specific environmental factors unshared with OCS and HD.Hoarding, obsessive–compulsive disorder (OCD), and Tourette’s disorder (TD) are psychiatric disorders that share symptom overlap, which might partly be the result of shared genetic variation. Population-based twin studies have found significant genetic correlations between hoarding and OCD symptoms, with genetic correlations varying between 0.1 and 0.45. For tic disorders, studies examining these correlations are lacking. Other lines of research, including clinical samples and GWAS or CNV data to explore genetic relationships between tic disorders and OCD, have only found very modest if any shared genetic variation. Our aim was to extend current knowledge on the genetic structure underlying hoarding, OC symptoms (OCS), and lifetime tic symptoms and, in a trivariate analysis, assess the degree of common and unique genetic factors contributing to the etiology of these disorders. Data have been gathered from participants in the Netherlands Twin Register comprising a total of 5293 individuals from a sample of adult monozygotic ( Current classification systems of psychiatric disorders are primarily based on consensus statements with respect to clinical symptom diagnostics by physicians. These classification systems, i.e., the International Classification of Diseases (ICD)  and the More specifically, OCD, HD, and tic disorders/TD are complex neuropsychiatric disorders; all characterized by repetitive behaviors that show substantial comorbidity, i.e., co-occurring more often than expected by chance –6. OCD iPrevalence rates for these disorders range between 0.1 and 0.8% for TD –19, 2 anWith respect to comorbidity rates between HD and OCD, in clinical and epidemiological studies of OCD, between 18 and 42% of patients report hoarding behaviors, depending on phenotypic definition –26, and Family studies and genetic epidemiological twin studies on each separate disorder have shown substantial genetic contribution to each separate phenotype, with heritability estimates from twin studies ranging between 0.30 and 0.58 (OCD) –35, 0.35n = 20.821) specifically addressed the proportion of shared genetic and environmental factors underlying the liability to chronic tics, ADHD, and OCS . A substantial correlation of 0.45 between tics and OCS was found.Despite recent advances in psychiatric genetics, twin studies specifically investigating shared genetic and environmental influences between OCS, hoarding behavior, and tics are scarce. Two studies by Iervolino et al. in a sample consisting predominantly of female twins from the TwinsUk twin registry  have specifically examined the genetic and environmental overlap between OCS and HD behavior , 33. It  and OCS . Tics weFrom another line of research, Genome-wide association study (GWAS) data from samples of TD and OCD patients were analyzed to find a genetic correlation between OCS and TD of 0.41 , which wSo far, genetic epidemiological twin family studies to estimate the shared respective unique contributions of genetic and environmental factors between tic-HD symptoms and between tic-HD–OCS are lacking, as are molecular genetic studies to estimate shared genetic contributions from SNPs across TD, OCS, and HD phenotypes.Therefore, the main aim of this study was to extend the available data so far with respect to shared etiology between OCS and hoarding behavior  by expanParticipants included in this study are registered with the NTR. Since 1991, twins and their family members receive surveys by mail and are assessed with questionnaires about health, personality, and lifestyle , 47. ForThe assessment instruments used were the Hoarding Rating Scale-Self-Report (HRS-SR) for hoarding, the Padua Inventory Abbreviated Revised (PI-ABBR) for OCS, and an abbreviated self-report questionnaire  based on the Schedule for Tourette and Other Behavioral Syndromes (STOBS) for tics. The HRS-SR questionnaire consists of five items, each scoring on a 0–8 scale, that assess cluttering, difficulty in discarding items, excessive acquisition or collecting, distress derived from hoarding symptoms, and functional impairment . The disThe STOBS consists of a semi-structured assessment on tics and has been widely used in data collections by the Tourette Syndrome Association International Consortium for Genetics (TSAICG). It consists of 36 tic items , generating lifetime tic information . For theWe fitted a liability threshold model, using, for each phenotype, a categorical variable derived from several cut points applied to the full distribution of sum scores (for OCS and HD) and defining the presence/absence of a tic disorder (for tics). The liability threshold model assumes an unobserved (and not measured) liability (or risk) to disease, normally distributed in the population , 54. ThePrevalences, means, and distributions for the three phenotypes were calculated in the entire sample of 16,930 individuals. Performing these analyses on clinically defined significant symptoms has the advantage of increasing the generalizability of the results. Polychoric correlations  were calculated in Mplus  for the Using the Mplus software, we then fitted a trivariate genetic model to the data with the weighted least square mean and variance adjusted estimation option (WLSMV) , using tLastly, we performed a single factor analysis on the covariance matrices partitioned between the phenotypes. This analysis gives a representation in terms of the components shared by the three phenotypes.The mean age of the entire sample was 33.61 years (SD = 14.56); for males the mean age was 33.11 years (SD = 14.66) and for females 33.84 years (SD = 14.51). The mean average score for HRS-SF was 5.74 (SD = 5.6) and for the PI-ABBR was 6.89 (SD = 5.2). Males had on average higher scores than females on both the HRS-SF and the PI-ABBR. Also for tics, the prevalence rates were higher in males (13.0%) than in females (12.6%). Table n = 25) of individuals had the co-occurrence of all three disorders.Table Table Specific gender/twin environments were tested univariately for each phenotype. As expected from the twin correlations across all zygosities, the fit statistics results show that correlations could be equated across twins and sex, with no twin-specific or sex-specific environments observed Table .p < 0.001) for clinically significant HD, 0.38  for OCS, and 0.37 for any tic disorder   for clinically significant HD, 0.62 for OCS , and 0.63  for tics. No evidence was found for an effect of common environment.The total heritability estimates were 0.33  again suggests that the genetic factors are involved in the correlations between traits Table . The MZ 2 (6) = 0.876, p = 0.99 and AE vs. ADE model: χ2 (6) = 2.994, p = 0.81]. Hence, the best-fitting model to the data was one in which the covariation between the three phenotypes can be explained by a set of common A and E factors. Table A trivariate ACE model was fitted to the data in order to examine the relative contributions from shared genetic and environmental contributions to the covariance among the traits. Again, as suggested by patterns of twin correlations, no evidence for common environment was found, and the C parameter could be dropped when compared to the more parsimonious AE model [AE vs. ACE model: χLastly, single factor analysis for the A and E component revealed the degree of genetic and environmental overlap shared by the three phenotypes Figure . As showIn this study, we sought to examine the extent to which shared genetic and environmental factors contribute to clinically significant OCS, HD, and tic symptomatology. We had at our disposal the largest twin pair sample available to date in which these three phenotypes were measured at the same wave of data collection. The present results extend previous work in the same NTR sample on shared genetic contributions to OCS and HD .Our univariate prevalence rates for clinical significant HD symptoms and OCS are in the expected range when compared to the literature , 57. ForOur comorbidity prevalence rates  are within the expected range when compared with the epidemiological literature –31. For Previous results with data from the NTR, using by and large the same sample, have yielded heritabilities of 0.40–0.50 for OCS , 58, 0.3Second, our results provide evidence for shared genetic variation between the phenotypes. The phenotypic correlation between OCS and HD was of 0.30. As expected, we observed a higher phenotypic correlation between OCS and tics (0.25) than between HD and tics (0.15). The genotypic correlations also mirrored this – there was higher shared genetic variance between OCS and HD (0.41) than both OCS and HD with tics . Interestingly, a relatively high proportion of the phenotypic correlations were attributable to genetic factors. In other words, although the genetic overlap (expression of same genes) between tics and both OCS and HD is moderate, a substantial proportion of the phenotypic correlation is mediated by their shared genetic variance .Importantly, Iervolino et al. recently reported a genetic correlation between OCS and HD of 0.45, combined with their data suggesting that HD was mostly influenced by specific genetic effects (54.5% specific) . The autOur estimates of genetic correlations between OCS and tics (0.37) are somewhat lower than the genetic correlations (0.45) as found by Pinto et al. . The difWith respect to the shared genetic and environmental contributions to HD and tics, to our knowledge, this is the first twin-family study partitioning the covariance between tics and HD in its relative genetic and environmental components. Our moderate correlation estimate (0.35) supports the argument of viewing TD as distinct from HD.Third, the common factor model further supports the view of shared genetic etiology between the three phenotypes. Neuroimaging studies have reported structural and functional dysfunctions in the cortico–striato–thalamo–cortical (CSTC) circuitries across all three disorders that have negative implications for motor response inhibition and interference control in these disorders, which might underlie the phenotypic behaviors of all these three disorders –65. Our Finally, our results are relevant for the field of molecular genetics. The lack of power to detect specific genetic risk variants is a recurrent issue in genome-wide studies. One way to overcome this limitation is to combine related phenotypes therefore increasing sample sizes, with consequent power gains. A crucial point here is the balance between power gains from increased sample sizes and power losses from increased heterogeneity , 66. OurThese results should be considered in the light of some limitations, mainly considering the phenotypes. Because this is a population-based study, the data collected are based on self-report measures, rather than on clinician-administered structural interviews. The cutoffs have been empirically derived, and are therefore somewhat arbitrary. The cutoffs to determine symptom thresholds (in the case of OCS and HD), by considering the entire range of age available, may have rendered different prevalence estimates, which might have affected estimations of genetic and environmental effects. However, we note that although these threshold cut-offs do not represent definite clinical diagnoses, they do correspond to clinical significant symptoms. Moreover, investigation of dimensions rather than true/false categorical diagnosis is consistent with the ideas forwarded in the NIMH Research Domain Criteria (RDoC) .To conclude, OCS, HD, and tics share etiologic variance that can be explained by substantial genetic correlations. Tics are mostly influenced by specific environmental effects unshared with neither OCS or HD, suggesting that specific environmental stressors might cause the development of tics separate from OCS and HD. Our results are in line with the literature supporting the current definition in DSM-5 of separating these disorders into different, although related, entities.All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.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.