Garrulax elliotii) is largely restricted to high-elevation shrublands in eastern Himalayas. We used mitochondrial DNA and microsatellites to investigate how genetic diversity in this species was affected by Pleistocene glaciations.Pleistocene climate fluctuations have shaped the patterns of genetic diversity observed in many extant species. In montane habitats, species' ranges may have expanded and contracted along an altitudinal gradient in response to environmental fluctuations leading to alternating periods of genetic isolation and connectivity. Because species' responses to climate change are influenced by interactions between species-specific characteristics and local topography, diversification pattern differs between species and locations. The eastern Himalayas is one of the world's most prominent mountain ranges. Its complex topography and environmental heterogeneity present an ideal system in which to study how climatic changes during Pleistocene have influenced species distributions, genetic diversification, and demography. The Elliot's laughing thrush (Garrulax elliotii shows continuous population growth since late Pleistocene (about 0.125 mya).Mitochondrial data detected two partially sympatric north-eastern and southern lineages. Microsatellite data, however, identified three distinct lineages congruent with the geographically separated southern, northern and eastern eco-subregions of the eastern Himalayas. Geographic breaks occur in steep mountains and deep valleys of the Kangding-Muli-Baoxin Divide. Divergence time estimates and coalescent simulations indicate that lineage diversification occurred on two different geographic and temporal scales; recent divergence, associated with geographic isolation into individual subregions, and historical divergence, associated with displacement into multiple refugia. Despite long-term isolation, genetic admixture among these subregional populations was observed, indicating historic periods of connectivity. The demographic history of Garrulax elliotii. During the Pleistocene, these populations became isolated on subregions during interglacial periods but were connected when these expanded to low altitude during cooler periods. The resultant genetic admixture of lineages might obscure pattern of genetic variation. Our results provide new insights into sky island diversification in a previously unstudied region, and further demonstrate that Pleistocene climatic changes can have profound effects on lineage diversification and demography in montane species.While altitude-associated isolation is typical of many species in other montane regions, our results suggest that eco-subregions in the eastern Himalayas exhibiting island-like characteristics appear to have determined the diversification of  Climatic oscillations over the past few million years have had profound effects on the demography of, and patterns and levels of genetic diversity in, many extant species -4. TempeGarrulax elliotii) is endemic to the eastern Himalayan region [G. elliotii populations thus contributing to genetic diversity in this bird.The eastern Himalayan mountain range, which has perhaps the most complex topography on Earth, offers an unusual opportunity to study interactions among geography, climatic changes and diversification. These mountains rose rapidly with the uplift of the Tibetan Plateau during the past three million years -18. Mounn region -26, whern region ,20, it iG. elliotii is predicted to have expanded and contracted along an altitudinal gradient resulting in historic periods of connectivity and isolation. Given the potential for repeated contacts between populations on different mountains, it is possible that populations merged into a single gene pool during historic periods of connectivity [High altitude parts of the eastern Himalayas were subject to repeated glaciations during the Pleistocene . These gectivity ,2,6. Howectivity -14.G. elliotii's distribution. Third, we test whether the timing of diversification within G. elliotii is consistent with climatic shifts induced by Pleistocene glacial cycles. Fourth, we use coalescent simulations to test whether populations from different mountains have descended from a single ancestor (single refugium), or, alternatively, remained isolated on individual mountains during down-slope expansions (multiple refugia). Finally, given that the range of G. elliotii is predicted to have expanded and contracted in response to Pleistocene climatic fluctuations, we also examine historical demography of this species.We used mitochondrial DNA and microsatellites to address the effects of Pleistocene climatic changes on genetic diversification in the eastern Himalayas. We first evaluate whether Elliot's laughing thrush from each eco-subregion or cluster of mountains comprises a distinct evolutionary lineage. Second, we investigate the contribution of geographic and environmental barriers to Pseudopodoces humilis, Montifringilla adamsi, Pyrgilauda ruficollis, Urocynchramus pylzowi and Kozlowia roborowskii [Chrysolophus pictus, Spizixos semitorques, Garrulax sukatschewi, G. davidi, Yuhina diademata, Paradoxornis przewalskii, P. paradoxus and Parus davidi [The eastern Himalayan mountain region occupies western and north-western Yunnan, western Sichuan, south-eastern Tibet, southern Qinghai and south-western Gansu. Steep mountain ranges and major rivers in deep valleys divide this region into several distinct eco-subregions ,20 see . Tang 2 and Zhanorowskii ,31,32. Torowskii -26. The s davidi -26,31-33s davidi , ants [3s davidi , fishes s davidi , reptiles davidi  and mamms davidi .G. elliotii were obtained are shown in Figure These three subregions are further divided into thirteen biogeographic sectors corresponding to clusters of mountains . The sitb (cyt-b), cytochrome c oxidase subunit I and II (COI and COII), NADH dehydrogenase subunit 2 and 6 (ND2 and ND6), ATP synthetase subunit 8 (ATP8) and control region (CR) genes were amplified via the polymerase chain reaction (PCR) [HM601543-602022).DNA was extracted from tissue samples using the DNeasy Tissue kit (QIAGEN). The cytochrome on (PCR) -44. The on (PCR) . All seq2 and 5 pmol primers. These loci were originally developed for the following passerine species: Hirundo rustica (HrU5-A) [Loxia scotica (LOX1) [Catharus ustulatus  [Poecile atricapillus (Pat43) [Phylloscopus occipitalis (Pocc6 and Pocc8) [Dendroica petechia (Dpu16) [2, template DNA concentrations and annealing temperatures. Microsatellites were amplified with fluorescently labelled forward primers (FAM and HEX dyes). Fragment lengths were analyzed with the internal size marker GENESCAN-500 ROX (Applied Biosystems), and scored with GENEMARKER 3.7 (SoftGenetics).PCR amplifications were achieved in a 10 ul reaction volume containing 10-30 ng DNA, 0.5 U Taq DNA polymerase, 0.2 nmol dNTPs, 1-3 nmol MgCl(HrU5-A) , Loxia sa (LOX1) , Catharud Cum32) , Poecile (Pat43) , Phyllosd Pocc8)  and Dend (Dpu16) . OriginaFS [D were used to detect departures from the mutation-drift equilibrium that would be indicative of changes in historical demography and natural selection. All three tests were implemented in DnaSP.For mitochondrial data, we calculated the number of segregating sites, haplotype diversity and nucleotide diversity for each population and all populations combined using DnaSP 4.0 . McDonalFS  and Fu &FS D were usNA), average allelic richness (AR), observed heterozygosity (HO), heterozygosity expected from the Hardy-Weinberg assumption (HE) and exact tests of linkage disequilibrium (LD) between pairs of loci for each population were calculated with ARLEQUIN 3.5 [For microsatellite data, parameters such as the number of alleles per locus (QUIN 3.5  and FSTAQUIN 3.5 .G. affinis), exquisite laughing thrush (G. formosus) and streaked laughing thrush (G. lineatus), which are considered closely related to G. elliotii [The maximum-likelihood algorithm implemented in PHYML 2.4.4  was usedelliotii .7 and 5 × 108 iterations without prior population information and following the admixture model. We conducted ten replicate runs for each value of K (most likely number of populations) from 1 to 10. The most likely K was identified using the maximal values of Pr(X/K), typically used for STRUCTURE analysis [K based on the rate of change in the posterior probability of data between successive K-values [We used two methods to identify genetically distinct groups among microsatellite genotypes. First, genetic relationships between individual samples were quantified using Nei's standard genetic distance in GENETIX 4.05 . The resanalysis  and ΔK bK-values .G. elliotii were identified in order to investigate whether intraspecific lineages were separated by areas of unsuitable environmental conditions. The habitat-mapping model was developed in ARCGIS 9.0  using vegetation, topographic layers and the known distribution range of G. elliotii. Shrublands were identified as suitable vegetation types [G. elliotii to generate a map of suitable habitats. Gaps between patches of suitable habitat were considered habitat gaps. GENELAND 1.0.5 [G. elliotii populations and locate areas of genetic discontinuity. GENELAND used microsatellite genotype data together with geographic information  to estimate population structure. Areas of genetic discontinuity were detected as geographic areas with low posterior probability of membership. Following the approach of Coulon et al. [K to vary (from 1 to 10) and inferred the most probable K using five replicate runs with 5 × 105 Markov chain Monte Carlo (MCMC) iterations. For these analyses the maximum rate of the Poisson process was fixed at 100 with no uncertainty in the spatial coordinates, and the maximum number of nuclei in the Poisson-Voronoi tessellation was fixed at 300. The Dirichlet model was used to estimate allele frequencies.Suitable habitats for on types -26. We cND 1.0.5 , a compuND 1.0.5 , was impn et al. , we alloST as the measures of genetic differentiation. In the case of microsatellite data, genetic distance was measured as the FST and Nei's Ds. An array of predictor variables was grouped into six sets: (i) coordinate (latitude and longitude); (ii) vegetation ; (iii) subregion ; (iv) elevation; (v) temperature ; (vi) mean annual rainfall. All predictor variables except vegetation and subregion were continuous, therefore these were treated as categorical variables with two states; 1 if a sample was located in a given vegetation type or subregion, and 0 if it was located in another vegetation type or subregion. Thus, each vegetation type or subregion was regarded as a vector corresponding to five vegetation types and three subregions, respectively. Vegetation or subregion was analyzed as a set, i.e. these variables were combined in a single test.Additionally, a distance-based redundancy analysis ,72 was uhttp://www.worldclimate.com) databases. Subregions were defined according to Li [P values in these analyses were obtained using 9 999 simultaneous permutations of the rows and columns of the distance matrix. To examine which subset of predictor variables provided the best model explaining genetic differentiations among G. elliotii populations, the forward selection procedure in the program DISTLM forward [P values were obtained using 9 999 permutations of the rows and columns of the multivariate residual matrix under the reduced model.Environmental variables were taken from the WWF Terrestrial Ecoregion and WorldClimate  [8 steps with a burn-in of 5 × 107 steps and a linear heating scheme (g1 = 0.05). IM was run under the HKY substitution model (estimated by Modeltest 3.7). Convergence on stationary distributions was assessed by monitoring the similarity of posterior distributions from independent runs and by assessing the autocorrelation parameter values over these runs [b gene (1 × 10-8 per site per year) [b alone to deduce the mutation rate for all fragments combined. We used this modified mutation rate to convert the divergence time estimate into calendar years.In order to overcome the limitations of conventional estimates of divergence time based on ne flow) -77, we ene flow) ,79. As rne flow)  we firstese runs . The peaese runs . Credibier year) ,83. The 7 and 5 × 108 iterations under the SSM model with initial short runs to provide prior estimates. A geometric heating scheme was employed and the program run four times with different random seed numbers to ensure convergence of parameter estimates. The IMa2 output included mean values of the parameters for two sets after the burn-in period, representing the first and second half of the total generations of the post burn-in run. We ran our analyses until the peak location set values were equal for both sets. This, along with the high effective sample size and low autocorrelation estimate, suggested the Markov chains reached convergence after a sufficiently long burn-in period and were sampled from the appropriate likelihood space. A generation time of 1 year [-5 to 10-4 per generation for birds [For microsatellite data, timing of population splits was estimated using the IMa2  with burf 1 year  and a muor birds -89 were To determine whether populations remained isolated in individual habitats (multiple refugia), or merged into a single gene pool (single refugium) during down-slope dispersal, we used coalescent simulations in MESQUITE 2.5  to test Ne for individual populations of G. elliotii using values for θ calculated in MIGRATE 2.3.2 [Ne using the equation for maternally inherited mitochondrial data θ = 2 Neμ, using same mutation rate in divergence time estimate. The estimates of Ne for all populations were summed to calculate Total Ne and scaled the branch widths of our hypothesized population trees using the proportion of Total Ne that each population comprised. The e Nof the refugial population was constrained to a size proportional to the relative e Nof the population sampled from the sites of the putative refugia. Thus, if samples from the site of the putative refugium had an effective population size of one-third that of the Total Ne, we would constrain the population size in the simulation prior to population expansion to one-third the total Ne. During coalescent simulations, both Total Ne and lower and upper bounds of the 95% CI for e Nwere used as model parameters in order to encompass a wide range of potential Ne values The divergence time specified for each model corresponded to the approximate age estimated using IM and IMa2 as follows: 0.1 mya for the southern and north-eastern populations (two-refugia model), 0.015 mya for northern and eastern populations (three-refugia model). In the single refugium model, we specified divergence time as 0, which assumed a panmixia population derived from a single refugium. For converting coalescent time (in generations) to absolute time, we assumed a generation length of 1 year [For coalescent simulations, we first estimated TE 2.3.2  under thf 1 year .S, the minimum number of sorting events required to produce the genealogy within a given model of divergence [S value is a measure of the number of parsimony steps in characters for a reconstructed gene tree, such that more discordance between the population model and gene tree leads to a higher S value. To obtain a distribution of S values, 10 000 gene trees were simulated, constrained within each model of population divergence under a neutral coalescent process, and the amount of discordance between the simulated genealogy and population model was determined. Overall, this produced a null distribution of 10 000 S values based on reconstructed genealogy for each model of population divergence. The S value for observed genealogy was calculated and compared to the distributions of S values from coalescent simulations to determine whether the observed genealogy could have been generated under a given model.The amount of discordance between a gene tree and population model was measured by vergence . The S vWe also examined the ancestral area distributions for deeper nodes of the mitochondrial haplotype phylogeny with an event-based method in DIVA 1.1 . Each inG. elliotii were estimated using the Bayesian skyline plot method implemented in BEAST 1.4.6 [The past population dynamics of mitochondrial lineages of ST 1.4.6 . This apST 1.4.6 .g) was also estimated for each mitochondrial lineage by FLUCTUATE 1.4 [g calculated from the results of these separate runs. Because this genealogical method yields estimates of g with an upward bias [g values following the conservative approach of Lessa et al. [g value indicative of population growth when g > 3 SD (g).The exponential growth rate  and expected heterozygosity (HE) ranged from 0.01 to 0.909 and 0.091 to 0.905, respectively. There was no evidence of linkage disequilibrium after adjusting the significance level for multiple comparisons (P < 0.001). Five locus-population combinations showed significant deviation from the Hardy-Weinberg expectation after Bonferroni correction, which involved four populations and all due to heterozygosity deficiency . The geographic distributions of the two clades appeared uneven, with the majority of first clade's haplotypes found mainly in populations in the northern and eastern subregions (north-eastern clade), whereas haplotypes of second clade were most common in southern subregion (southern clade)  was comprised of two populations from the southern subregion (DL and ZD), the second cluster (eastern lineage) consisted of the five eastern populations , and the third cluster (northern lineage) included the three northern populations  Figure  and 4. YTo assess the genetic admixture, we carried out an exclusion method implemented in the program GENECLASS 2.0 to identify potential admixture individuals. Using the previously determined three genetic clusters and geographic sampling locations as prior population information, GENECLASS identified a considerable proportion of admixture individuals (25%), most of which were assigned to the geographically intermediate populations MK, YA and BC   revealed that the three clusters are separated from adjacent clusters by areas where environmental conditions are unsuitable for G. elliotii. Comparing genetic divergence with spatial landscape pattern shows that gene flow barriers coincide with the spatial distribution of habitat gaps . For mitochondrial data, posterior probability for divergence time peaked at time t = 3.415 . When converted to a scale of years, the divergence time between north-eastern and southern lineages was estimated to be 0.109 (0.088-0.138) mya. The migration parameters estimated by IM (m1 and m2), which represented the number of migrants per mutation (m = m/μ), were converted to population migration rates (M = 2 Nm = θm/2). The migration rate per generation was estimated to be 0.43 (0.09-0.65) from the southern to north-eastern lineage, and 0.37 (0.07-0.63) from the north-eastern to southern lineage.As the four runs of IM and IMa2 gave similar results, we report below the estimates from the run with the highest effective sample size (ESS) for the parameter t peaked at 0.975 (0.185-2.325) for the southern and north-eastern lineages, and at 0.15 (0.045-0.315) for the northern and eastern lineages. When converted to a scale of years, the divergence time between the southern and north-eastern lineages was estimated from 0.097 (0.018-0.232) to 0.01 (0.002-0.023) mya. For the northern and eastern lineages, divergence time was estimated between 0.015 (0.004-0.032) and 0.0015 (0.0005-0.003 2) mya. The migration rate per generation (2 Nm) was estimated at 0.83  from the north-eastern to southern lineage, and 1.01 (0.38-2.2) from the southern to north-eastern lineage. From the eastern to northern lineage, the migration value was estimated to be 1.34 (0.56-1.8), and 0.33 (0.02-1.14) from the northern to eastern lineage.For microsatellite data, the posterior probability of parameter Ne = 2 994 038 (95% CI: 2752885 - 3235192), which equated to a MLE estimate of θtotal of 0.0467 with lower and upper bounds of 0.0429 and 0.0505, respectively. For the observed gene tree we computed Slatkin & Maddison's S = 40. The discordance predicted by coalescent simulations rejected the single refugium hypothesis  across a range of Ne. However, coalescent tests supported both the two-refugia  and the three-refugia divergence models  in three Ne values.All gene trees were simulated within population trees with an effective population size of Reconstruction of ancestral areas indicates that the north-eastern clade might have originated from YA and MK, while the southern clade probably derived from ZD and BC Figure . Relativg: southern clade: 1604; north-eastern clade: 3226).The historical population trend inferred by the Bayesian skyline plot seems a relatively good fit to the climate trend since the late Pleistocene glaciations Figure . Past poG. elliotii populations. Such a landscape effect is evident in the eastern Himalayan region. Although G. elliotii has a rather restricted geographic distribution, mitochondrial data support the separation of a southern and a north-eastern lineage with incomplete gene sorting, while microsatellites indicate a clear subdivision into three lineages, a southern, a northern and an eastern. Despite intermixing of mitochondrial haplotypes, none of the haplotypes are geographically widespread. All shared haplotypes occur within the same subregional population. In this case, the steep mountains and deep valleys of the Kangding-Muli-Baoxin Divide, recognized as a geographic barrier by Zhang et al. [Geographic complexity and environmental heterogeneity are likely to have shaped the genetic structure among g et al.  and Li [g et al. , appear G. elliotii is restricted to shrublands in the three subregions separated by deep valleys. The habitat-mapping model shows that these subregions or lineages are separated by areas where the environmental conditions are unsuitable for G. elliotii. Consistent with the expectation that these valleys prevent or restrict gene flow, each subregional population represents an evolutionary lineage. Thus, the contrast in environmental conditions at high and low elevations in the eastern Himalayas may have created a "sky island situation" for G. elliotii.Mountains, like sky islands, and are separated by areas of unsuitable habitats that act as barriers to gene flow ,15. SpecG. elliotii has occurred on a broader geographic scale, eco-subregions. G. elliotii is an alpine bird found between 2 000 and 4 000 m.a.s.l. [Although many sky island species in other regions show high levels of isolation in clusters of mountains -14, our m.a.s.l. -26. ThisWhile our study may demonstrate newly discovered sky island diversification in a previously unstudied region, sky islands also have the extra connotation that mountain ranges are equivalent ecologically and they share the same biogeographic history. Since three eco-subregions in the eastern Himalayas present different ecological systems, our result should be considered cautiously. Whether the eastern Himalayan region is a sky island situation ultimately depends on the ecology of the organisms under study. Further research on more organisms inhabiting this region, especial the same eco-climatic subregions is required to clarify this.G. elliotii populations on different subregions. Congruent with this, our divergence time estimates indicate that lineage diversification occurred during the late Pleistocene interglacial period (MIS5). Climatic fluctuations during the late Pleistocene resulted in several glacial-interglacial cycles in the eastern Himalayas [G. elliotii. It is plausible therefore that the glacial and interglacial cycles would allow G. elliotii to disperse among subregions during glacial periods, but isolate population in different subregions during the interglacial periods.The Pleistocene glacial cycles have been considered to induce environmental shifts in the eastern Himalayas resulting in the isolation of imalayas ,99-101. imalayas . Palaeocimalayas ,103; durimalayas ,105. BecG. elliotii appear to reflect historical differentiation in multiple refugia. Coalescent simulations and reconstruction ancestral areas of the mtDNA phylogeny suggested a divergence model involving the multiple refugia, while microsatellite data identify a distinct genetic structure concordant with the geographically separated southern, northern and eastern subregions. These results suggested that subregional populations of G. elliotii were isolated into separated refugia during down-slope expansions, although we could not distinguish between the two-refugia and three-refugia model. Furthermore, while coalescent simulation assumes that the discordance between the reconstructed gene tree and multi-refugia population trees reflects the retention of ancestral variation, the discordance could also result from migration among subregional populations (see below).Given the potential for repeated contacts of populations from subregions during glacial expansions, it is possible that subregional populations merged into a common gene pool. However, our analyses of diversification pattern in G. elliotii. This is consistent with genetic admixture among lineages occurring during glacial periods. The two partially sympatric mitochondrial lineages show shared ancestry, and microsatellite data indicate gene flow among the three subregional populations. Furthermore, GENECLASS identified a considerable proportion of admixed individuals, most of which were assigned to the geographically intermediate populations MK, YA and BC. Interestingly, despite the relatively high degree of genetic admixture, these locations were also identified as the potential refugia that most likely defined the boundary of each genetic lineage.In contrast to the deep phylogeographic partitions found in other sky island species -14, we oG. elliotii periodically expanded to lower elevation where they mixed. Thus, the three intraspecific lineages likely established a contact zone along their boundaries. Because these lineages are geographically relatively close and are likely to have experienced repeated glacial cycles, this has resulted in multiple episodes of genetic admixture. Over time, this could have defined a centre of gene flow, such as populations YA, MK and BC. This process might cause the mixing of genetic lineages thereby obscuring the pattern of genetic variation [Based on these observations, it is probable that isolated populations of ariation .Nevertheless, the rather large degree of genetic separation among lineages indicates that these have been isolated from each other for a relatively long period. Although the southern and north-eastern mitochondrial lineages are intermixed, no haplotypes are shared between them. This lack of sharing suggests that the two lineages are at an intermediate stage of divergence along the continuum from complete panmixia to paraphyly and ultimately to reciprocal monophyly ,107. In G. elliotii experienced population growth during the warmer MIS5 period.Climatic changes that resulted in population expansion and contraction are also expected to lead to demographic changes over time between lineages confined to different subregions. Congruent with this, the Bayesian skyline plot revealed an increasing population size in each genealogical lineage since 0.125 mya, i.e. during the warmer interglacial MIS5 period. During the Quaternary, the eastern Himalayas were uplifted to 4 000 - 4 500 m.a.s.l. Climatic conditions responsible for high rainfall moved away from the centre of mountains and mountain glaciers shrank ,99. The G. elliotii, the Bayesian skyline plot revealed a stable population during the cooler MIS4 and MIS2 periods. Maintenance of a rather stable population size can probably be attributed to the frequency and location of glaciations in this region. Unlike high latitude regions of Europe and North America that were covered by heavy ice during much of Pleistocene, glaciations in the eastern Himalayas were restricted to relatively high altitudes and did not affect the lower slopes or valleys [G. elliotii even during periods of climatic change, making it possible for this species to maintain a stable population size during the cooler MIS4 and MIS2.Contrary to the expectation that profound ecological upheavals during cooler periods would have reduced the population size of  valleys . It is l valleys ,107-109.G. elliotii indicates that lineages have been isolated on individual subregions since the late Pleistocene interglacial period (MIS 5). Despite being isolated in multiple refugia during glacial advance, gene flow periodically occurred when populations expanded their ranges to lower altitudes. The resultant genetic admixture might have caused the mixing of genetic lineages thereby obscuring the pattern of genetic variation. The Bayesian skyline plot shows a gradual increase in population size in each mitochondrial lineage since the late Pleistocene interglacial period (MIS 5). Our results provide new evidence that climatic changes during the Pleistocene glaciations had profound effects on lineage diversification and demography in a bird species in a previously unstudied montane region - the eastern Himalayas.The genetic structure observed in YHQ, XL and FML conceived and designed the study. RYZ performed lab work. XL, GS and FSZ collected most of the samples. All authors read, made significant comments and approved the final manuscript.Table S1. The geographic origins and GENECLASS assigned groups for the admixed individuals [ividuals ; 1 repreividuals . The proividuals .Click here for file


Mental imagery, or the ability to simulate in the mind information that is not currently perceived by the senses, has attracted considerable research interest in psychology since the early 1970's. Within the past two decades, research in this field—as in cognitive psychology more generally—has been dominated by neuroscientific methods that typically involve comparisons between imagery performance of participants from clinical populations with those who exhibit apparently normal cognitive functioning. Although this approach has been valuable in identifying key neural substrates of visual imagery, it has been less successful in understanding the possible mechanisms underlying another simulation process, namely, motor imagery or the mental rehearsal of actions without engaging in the actual movements involved. In order to address this oversight, a “strength-based” approach has been postulated which is concerned with understanding those on the high ability end of the imagery performance spectrum. Guided by the expert performance approach and principles of ecological validity, converging methods have the potential to enable imagery researchers to investigate the neural “signature” of elite performers, for example. Therefore, the purpose of this paper is to explain the origin, nature, and implications of the strength-based approach to mental imagery. Following a brief explanation of the background to this latter approach, we highlight some important theoretical advances yielded by recent research on mental practice, mental travel, and meta-imagery processes in expert athletes and dancers. Next, we consider the methodological implications of using a strength-based approach to investigate imagery processes. The implications for the field of motor cognition are outlined and specific research questions, in dynamic imagery, imagery perspective, measurement, multi-sensory imagery, and metacognition that may benefit from this approach in the future are sketched briefly. In pursuit of this objective, the paper is organized as follows. In the first section, we shall review the strengths and weaknesses of conventional approaches to sampling that have dominated research on mental imagery for over a century. Next, we shall present an alternative approach to mental imagery research that we call the “strength-based” paradigm. The significant factors that led to the emergence of this paradigm will also be summarized. In the final section, we shall outline some potentially fruitful new directions in imagery research that can be addressed by supplementing traditional approaches with this strength-based paradigm.Imagery, due to its ephemeral nature, has provided significant challenges for psychology since it first received formal research attention over a century ago  and its executed counterpart . The aforementioned factors, allied with the expertise paradigm, based on the theory of deliberate practice . To tap this expertise efficiently, it is proposed that the converging methods approach is applied and moreover, that ecological validity is considered in the experimental design Table .Two key issues integral to the application of the strength-based approach are now highlighted. These are quantifying expertise and applying the principle of ecological validity. Subsequently, recent examples of this approach in music, sport, and dance samples are discussed. And finally, we highlight where we foresee the “strength-based” approach can illuminate the interaction between cognition and action (motor cogntion), the integration of multi-sensory information in perception and simulation, and the role of conscious thought and knowledge in imagery (metacognition).Object-Spatial Imagery Questionnaire. Another example of the implicit expertise approach was the inclusion of US air force pilots in a study by Dror et al. . In other words, complex and dynamic tasks in which imagery processes are important should be subject to scientific scrutiny or aspects of the underlying processes may be overlooked. One persistent criticism of imagery research within the sport context is the use of tasks lacking ecological validity . The findings indicated that although the majority of participants predicted correctly that imagery would have beneficial effects on learning and reasoning, few subjects were able to predict accurately the results of mental rotation experiments (whereby more time is required to accomplish greater amounts of rotation of images) or mental scanning studies (whereby longer distances between points in an image take longer to scan than shorter distances). Furthermore, a majority of participants rejected the idea that mental imagery could enhance the performance of motor skills . This latter finding led Denis and Carfantan (Theoretically, a potentially valuable new route for imagery researchers in cognitive neuroscience concerns the investigation of the neglected topic of “meta-imagery processes”—“their beliefs about the nature and regulation of their own imagery skills” Moran, , p. 415.s Denis, . In ordearfantan  to conclarfantan ,b. Athlearfantan .In 2002, Moran suggested that it would be interesting to discover if top athletes have greater insight into and control over their use of imagery compared with their less successful counterparts. To date the preponderance of the evidence favors an expertise effect for meta-imagery. In fact, a model of meta-imagery in action suggests that there are three components-knowledge, monitoring and control, which opens up possibilities of developing a test of meta-imagery (MacIntyre and Moran, In this paper, we have argued that a strength-based approach to mental imagery can augment rather than replace the traditional approach to this construct. After all, this latter approach has been highly successful in, for example, illuminating both visual imagery and visual cognition (Kosslyn et al., Object-Spatial Imagery Questionnaire (Blajenkova et al., Despite the success of traditional approaches to imagery, however, a strength-based approach may contribute in the development and refinement of imagery inventories, as implicitly employed in the case of the Cognitive neuroscience has made impressive progress in the illumination of the nature, function, and neural basis of mental imagery Kosslyn, . NeverthThe 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.


The role of dispersal potential on phylogeographic structure, evidenced by the degree of genetic structure and the presence of coincident genetic and biogeographic breaks, was evaluated in a macrogeographic comparative approach along the north-central coast of Chile, across the biogeographic transition zone at 30°S. Using 2,217 partial sequences of the mitochondrial Cytochrome Oxidase I gene of eight benthic invertebrate species along ca. 2,600 km of coast, we contrasted dispersal potential with genetic structure and determined the concordance between genetic divergence between biogeographic regions and the biogeographic transition zone at 30°S. Genetic diversity and differentiation highly differed between species with high and low dispersal potential. Dispersal potential, sometimes together with biogeographic region, was the factor that best explained the genetic structure of the eight species. The three low dispersal species, and one species assigned to the high dispersal category, had a phylogeographic discontinuity coincident with the biogeographic transition zone at 30°S. Furthermore, coalescent analyses based on the isolation-with-migration model validate that the split between biogeographic regions north and south of 30°S has a historic origin. The signatures of the historic break in high dispersers is parsimoniously explained by the homogenizing effects of gene flow that have erased the genetic signatures, if ever existed, in high dispersers. Of the four species with structure across the break, only two had significant albeit very low levels of asymmetric migration across the transition zone. Historic processes have led to the current biogeographic and phylogeographic structure of marine species with limited dispersal along the north-central coast of Chile, with a strong lasting impact in their genetic structure. It has long been established that the geographical structure of the genetic diversity of benthic species is strongly shaped by life history traits. Among the main life history attributes, mode of larval development, duration and characteristics of larval life, have been highlighted as features that influence the maximum distance of dispersal in many benthic marine species In the recent years however, the correlation between pelagic larval duration (PLD) and the spatial scale of genetic differentiation has been repeatedly questioned by meta-analyses and comparative studies of benthic and coastal marine species Among the different alternatives, biogeographic transitions have been considered as ideal situations to study the historical and contemporary processes that affect the distribution and abundance of species. Indeed, depending on the species ability to disperse and survive in different environmental conditions, biogeographic breaks often reflect species range limits Lessonia nigrescensMazzaella laminarioidesAcanthina monodonConcholepas concholepas, and the squat lobster Pleuroncondes monodon and the cephalopod Dosidicus gigas, did not show genetic structure at a macrogeographic scale across 30°S Limnoria by rafting on macroalgae Pyura chilensis on boat and ship hulls Lessonia nigrescens and A. monodon have planktonic dispersive stages that last a few hours to a few days, whereas C. concholepas, J. cirratus, P. monodon and D. gigas can spend weeks to several months in the water column. Thus, these studies strongly suggest that the biogeographic break is the result of a past discontinuity that is currently not perceivable in the macrogeographic genetic structure of species with high dispersal potential.This study further tests these predictions by undertaking a comparative phylogeographic analysis across a biogeographic boundary along the coast of Chile. The lineal coast of Chile, north of 42°S, encompasses approximately 2,600 km with a natural temperature gradient associated to latitude, but with neither apparent geographic barriers to dispersal nor any sharp environmental discontinuity. The cold and rich nutrient Humboldt Current influences the coastal area between 18 and 42°S, with upwelling as the dominant feature influencing coastal communities The current study aims at contributing to the knowledge of phylogeographic patterns of marine benthic organisms along the Chilean coast under the influence of the Humboldt Current focusing on eight species with high and low dispersal potential. More broadly, the role of dispersal potential on the population genetic diversity and differentiation is addressed. We predict that species with low dispersal potential will have greater genetic structure, lower genetic diversity than high dispersers and the presence of a historic phylogeographic break around 30°S, coincident with the biogeographic transition zone.Emerita analoga (Stimpson) (Hippidae) and Petrolisthes violaceus Stimpson (Porcellanidae); two starfishes of the class Asteroidea (order Forcipulatida), Heliaster helianthus (Lamarck) (Heliasteridae) and Stichaster striatus Müller and Troschel (Asteriidae); and the sea urchin Tetrapygus niger (Molina) . The remaining three species were considered as having low dispersal potential: the peracarid Orchestoidea tuberculata Nicolet  that broods its embryos, and the gastropods Scurria scurra (Lesson)  and Tegula atra (Lesson)   with reflects an upper bound of dispersal rather than a mean dispersal time Emerita analoga JX130229 - JX130344; Heliaster helianthus JX130054 - JX130098; Orchestoidea tuberculata JX129929 - JX129953; Petrolisthes violaceus KC543356 - KC543409; Scurria scurra JX130193 - JX130212; Stichaster striatus JX129954 - JX130053; Tegula atra JX130156 - JX130192; Tetrapygus niger JX130099 - JX130155.COI gene sequences were obtained from 2,217 individuals of eight species , coverinT. niger to 672 in S. scurra. Variable sites varied between 2.7% in S. scurra to 23.2% in O. tuberculata, with an average of 10.5% . The genetic structure detected for S. striatus was weaker than on the three low dispersers.St values . The lowerculata , and oveerculata . Mantel’ST values were significantly associated with the interaction of dispersal potential and biogeographic differentiation , suggesting that differences detected between low and high dispersers were not biased by the inclusion of O. tuberculata and S. striatus.Overall, GLMMs indicated that pairwise Φrsal see  on the rTegula atra and S. striatus showed a very similar pattern; although there was greater structure in T. atra both species had a significant and high portion of the total variance within populations , and a considerable amount explained by biogeographic region . Overall, GLMMs revealed significant differences between high and low dispersers for each of the AMOVA’s Φ-statistics Fig. 4), erculata , revealiad k = 4 . For botS. striatus  and divergence times were >0, indicating that not all shared haplotypes are a consequence of gene flow but there is also ancestral polymorphism  . In genestriatus ; Fig. 5Bt recent , confirmmorphism ; Table 7plitting . ScurriaOur results suggest that the phylogeographic patterns of marine benthic organisms of the Chilean intertidal have been strongly influenced by their dispersal potential. Compared to other regions of the world, such as the northeast Pacific et al.E. analoga and O. tuberculata that inhabit sandy beaches. All the rest are all found in the mid-intertidal rocky shore. The species with the most divergent habitat is O. tuberculata that inhabits the supralittoral (STF) Several studies have explored the determinants of the geographic structure of the genetic diversity of marine species. Among them, Weersing & Toonen littoral , howeverS. striatus, was assumed to have high dispersal potential based on the estimated PLD of its congener S. australisS. striatus did not change the implications of results, i.e. dispersal potential was still a strong predictor of genetic diversity, genetic differentiation, and of the presence of a phylogeographic break at 30°S. Of the four species with structure across 30°S, S. striatus had the least structure. For example, ΦCT (among groups) was non-significant for S. striatus while it was for all low dispersers. When analyzing genetic differentiation and isolation by distance of S. striatus at each side of the break, only the northern biogeographic region had significant differentiation and isolation by distance. The southern region showed non-significant differentiation and lack of an isolation by distance pattern. Contrastingly, all low dispersers had significant genetic differentiation and isolation by distance in both biogeographic regions. There was a tremendous difference between the genetic diversity (2 Ne) of the northern and southern biogeographic regions for S. striatus. Presumably the genus Stichaster has cold-water affinities S. striatus: (1) S. striatus is a true high disperser and its spatial genetic structure is the result of the effects of strong local genetic drift, or (2) it is a medium to low disperser, in which case observed results would be consistent with the marine connectivity paradigm. The effective size inferred from isolation-with-migration model is comparable to the two most structured species (O. tuberculata and S. scurra) and much smaller than the other low dispersal species, T. atra, suggesting that genetic drift may indeed have been strong in S. striatus. On the other hand, PLD is highly variable within the order Forcipulatidae, with a minimum of zero days . The main way in which populations and species loose genetic diversity at neutral markers is by genetic drift. However, the actual influence of genetic drift depends on its interaction with other evolutionary forces, such as gene flow: reduced dispersal may allow local populations to demographically fluctuate and increase extinction-recolonization rates Finally, because low dispersers have subdivided populations they are prone to metapopulation dynamics, i.e. extinction and recolonization S. scurra, T. atra and S. striatus) showed similar and relatively recent divergence times, suggesting that the genetic discontinuity originated either during the last glacial period (based on t 2%) or just after (when based in t 10%). However, O. tuberculata had a divergence time between biogeographic regions of an order of magnitude higher and overlaps those estimated for other reported genetic discontinuities in the same region, such as the kelp Lessonia nigrescens with estimated divergence at 3095 ky ±1200 ky Bayesian analyses based on the isolation-with-migration model take full advantage of the data by using a coalescent approach to jointly estimate migration, time of divergence and effective population size. Ideally, when using coalescent approaches such as those implemented in IMa2, several polymorphic and unlinked loci should be used for a robust estimation of parameters and to deal with phylogenetic uncertainty S. striatus and T. atra. Both species showed a greater contribution from the southern to the northern region. Likely these two species have been able to achieve a low yet significant connectivity between biogeographic regions (probably enhanced by the Humboldt Current), slightly eroding the historic signature, which is anyhow strong in both species. Concordantly, shared haplotypes of S. striatus and T. atra between biogeographic regions are best explained by a combination of migration, shared ancestral polymorphism and drift. For the northeast Pacific coast, Wares et al.Perumytilus purpuratusMazzaella laminarioidesMigration estimations suggested low yet significant northward migration across the 30°S break in two of the four species that had genetic structure among biogeographic regions, O. tuberculata and S. scurra) and biogeographic regions (the north for S. striatus and the south for T. atra) may have stronger effects of genetic drift. Concomitantly, biogeographic regions carry within their structure the signatures of historic effective population size, low migration and historic isolation, which probably leads to the significant interaction of dispersal potential and biogeographic region in models associated to nucleotide diversity and genetic differentiation between low and high dispersers.Consistently dispersal potential was the variable that always best explained the contrasting genetic structure of low and high dispersers. Genetic structuring measures that are more associated with genetic divergence , not only were determined by dispersal potential but also significantly by its interaction with biogeographic region. Based on isolation-with-migration analyses, the biogeographic regions have been completely or partially isolated in recent times. Isolation has led to differentiation, which in some cases is likely to have been enhanced by low effective population size. Genetic diversity was estimated to be unequal between biogeographic regions and species, such that some species  is based on reports that show that PLD is short and very conserved in the Patellogastropoda Tegula have conserved PLD’s of 6 to 7 days T. atra.Amphipods, as all peracarids, brood their embryos up to a crawl-away stage. Thus, E. analogaT. nigerH. helianthus can persist for at least 2 months without reaching metamorphosis. The New Zealand starfish Stichaster australis, the only congener of S. striatus from the southeast Pacific coast, has larvae that spend 38 days in the water column S. striatus; albeit it is possible this extrapolation is wrong given that PLD is highly variable in asteroids  to Chiloé (42°S) along the coast of Chile. The intertidal areas are not privately owned in Chile, however, there are areas of restricted access (management or protected areas) where we did not sample. All sampling was performed in free access areas for which no permit was required. Also no permits were required for the sampling as species studied did not involve commercially important species or species endangered or protected.H. helianthus and S. striatus) and the urchin (T. niger), ambulacral feet and gonad tissue were sampled, respectively. Tissues were placed in absolute ethanol and stored at −20°C. Total DNA was extracted using standard phenol/chloroform protocol Organisms were processed a few hours after being extracted from nature, obtaining from them a piece of tissue. In most cases tissues corresponded to skeletal muscle. From starfishes  and ArbaR (5′TATACTTCAGGGTGTCCAAAGAATCA′3). These latter primers were designed for this study in GENEIOUS 5.5.4 2, 0.4 µM of each primer, 0.22 mM of each dNTP, 1.5 mg ml−1 of bovine serum albumin, and 1.5 U of Taq polymerase. Cycling conditions consisted of an initial denaturation at 94°C for 10 min followed by 35 cycles of 1 min denaturing, 1 min annealing at 50°C, and a 2 min extension at 72°C.A partial sequence of COI was obtained using the universal HCO and LCO primers nn statistic nn or the nearest neighbor statistic For each species, sequences were aligned and then truncated on both extremes using GENEIOUS. DnaSP 5.0 ST) were calculated in ARLEQUIN 3.5 nn and ΦST.Pairwise and global population genetic differentiation values based on sequence data (ΦST among and within regions). All models were constructed nesting the species as a random factor. The syntax of the diversity models was: Genetic Diversity = Dispersal X Biogeographic Region+Species . For the differentiation model the syntax was: Genetic Differentiation = Dispersal X Biogeographic Differentiation+Species .To test the influence of dispersal capacity and the 30°S transition zone on the genetic diversity and differentiation Generalized Lineal Mixed Models (GLMMs) were performed using the package lme4 in R. These models are appropriate for data influenced both fixed and random effects. In the models population-specific data of genetic diversity were grouped into biogeographic regions (north and south of 30°S). For models of genetic differentiation data was grouped according to biogeographic differentiation . To evaluate the presence of a genetic discontinuity coincident with the biogeographic break at 30°S, ARLEQUIN software was used to perform AMOVA by grouping individuals CT). The number of groups was denoted as k. In each species, statistical variance was evaluated with 1000 random permutations running repeatedly SAMOVA from 2 to 1-n populations, where n was the total number of sampled localities. The k value that maximized FCT was interpreted as the most likely geographic subdivision. A genetic break coincident with the biogeographic transition was inferred if when k = 2 the clustering FCT was significant and the groups of individuals were located north and south of 30°S.Two further analyses were conducted for species that displayed genetic structure with AMOVA, i.e., those that had less than 95% of the variation within populations. The clustering method implemented by SAMOVA 1.0 t (splitting time), effective population sizes (Θ = 2 Ne), and migration rates in each direction across the biogeographic break. For each analyzed species, we generated a random balanced subsample of individuals per biogeographic region with a double goal of having equal number of individuals per biogeographic region and reducing the number of sequences analyzed, which is recommended when using the model of isolation-with-migration with information from a single locus 3) algorithm The second analytical approach applied only on species with structure across 30°S, consisted in the evaluation of asymmetric migration and time of divergence across 30°S using the isolation-with-migration demographic model


The dualism of genetic predisposition and environmental influences, their interactions, and respective roles in shaping the phenotype have been a hot topic in biological sciences for more than two centuries. Heritable epigenetic variation mediates between relatively slowly accumulating mutations in the DNA sequence and ephemeral adaptive responses to stress, thereby providing mechanisms for achieving stable, but potentially rapidly evolving phenotypic diversity as a response to environmental stimuli. This suggests that heritable epigenetic signals can play an important role in evolutionary processes, but so far this hypothesis has not been rigorously tested. A promising new area of research focuses on the interaction between the different molecular levels that produce phenotypic variation in wild, closely-related taxa that lack genome-wide genetic differentiation. By pinpointing specific adaptive traits and investigating the mechanisms responsible for phenotypic differentiation, such study systems could allow profound insights into the role of epigenetics in the evolution and stabilization of phenotypic discontinuities, and could add to our understanding of adaptive strategies to diverse environmental conditions and their dynamics. Patterns and causes of biological variation have fascinated and challenged natural scientists for a long time. The Darwinian evolutionary theory highlights the importance of natural variation as raw material upon which selection processes can act, thereby increasing the fitness of locally adapted phenotypes . ConceptTherefore, current evolutionary investigations should consider several levels of biological variation . First, The molecular mechanisms underlying these components of phenotypic variation differ in their stability and in the time frames in which they confer phenotypic novelty. The genetic sequence is the most stable, evolving slowly through mutation and gradually accumulating changes over a large number of generations. In contrast, gene expression levels can be rapidly and continuously regulated within a very short time , much shIt has long been established that mutations in DNA sequence are the primary raw material for evolutionary change . The invEmpirical studies have demonstrated high levels of epigenetic variation within natural populations , 36–41. The epigenetic sources of variation can be stochastic epimutations, but a major part of the epigenetic variation is triggered by stress or changes in the environment , 22, 48,At the interface between genotype and environment, the overall rate of epimutations is often much higher than that of genetic mutations , resultiEpigenetic variation can have extensive consequences, even in the absence of genetic variability , 50, 51.Recently developed tools, in combination with traditional methods, can shed light on the complex interactions between genotype, epigenotype, and environment, and test for their individual contribution to phenotypic divergence and evolution. Evolutionary biologists could address the evolutionary relevance of heritable epigenetic polymorphisms by targeting closely related ecotypes or species (hereafter types) that show phenotypic differentiation without apparent genome-wide genetic divergence. Such types could be identified, for example, within asexual lineages or descendants of recent adaptive radiation events. We suggest a multifaceted research plan using an array of molecular techniques and field experiments to investigate whether epigenetics is involved in speciation by triggering phenotypic diversification.As speciation is facilitated by the process of divergence, the first question to be addressed should be whether phenotypic variation in the study group is discrete or continuous. Phenotypic variation is a common feature of populations and species, and only a discontinuity in this variation may indicate incipient divergence and the onset of isolating mechanisms. Therefore, various morphological, anatomical, and physiological traits among populations of different types should be compared to test whether the types form well defined, distinct groups or whether the extreme phenotypes are linked by individuals with intermediate traits or combinations of characters. In addition, measurements and observations of environmental characteristics  could identify limiting environmental factors, and relate them to anatomical, morphological, and physiological specializations.If main discontinuities in phenotypic variation separate populations along type boundaries , the uniformity within each group and constant difference between the groups might suggest a single origin of each type and subsequent dispersal . HoweverSingular versus multiple origin of each type should be tested by investigating the extent and structure of genome-wide genetic and epigenetic divergence within and among populations of both types. If populations cluster genetically in disagreement to the type , it may be hypothesized that their differentiation is underlaid by epigenetic mechanisms and that types have evolved several times in parallel. Alternatively, local high rates of gene flow combined with strong selection at a few adaptive genetic loci could hypothetically produce a similar pattern of highly porous genomes . In such0t filtration [As epigenetic variation is not detectable in genomic surveys of sequence variation, dedicated investigations have to be employed to address it. In recent years, a variety of genome-wide approaches, including techniques involving next-generation sequencing, have been developed to comparatively profile epigenetic patterns in nonmodel organisms , 63. Cosltration , 65 to eltration . Third-gltration  and thusltration , 69. Simltration , 44. ThiTo infer broad, genome-wide regulatory variation, in-depth quantitative gene expression analyses using next-generation sequencing  coulIf the molecular basis of phenotypic differentiation and/or adaptation to divergent environments is identified within epigenetic rather than DNA sequence divergence, the next research step would be to investigate how stable the phenotypic divergence is. This will also help to assess the stage of speciation in which the group is at present. While facilitating population divergence and speciation , nonheriReciprocal transplant experiments together with attempts to grow the different types under the same environment across several generations  should be installed to determine the extent of phenotypic plasticity, and the ability of the different types to cope with altered environmental conditions. Growing individuals of the alternative types in a uniform environment across several generations may reveal the heritability of morphological and ecological characteristics within each of the types (“nature versus nurture”) . ComparaAccording to the mechanisms underlying the observed differentiation, at least two possible outcomes can be anticipated. If the morphological and/or ecophysiological differences are triggered by continuous but nonheritable responses to local environments , there The result of these experiments could simultaneously allow for inferring evolutionary and population dynamics within the study group. If individuals of alternative types can adapt phenotypically to the habitat of the other and develop the habitat-specific syndromes following transplant experiments, the possibility of frequent gene flow between populations of both types should be considered. This might as well explain the lack of overall differentiation, as it prevents lineage sorting and hampers or slows down speciation. On the contrary, low fitness  of individuals in the native habitat of the alternative type may point to a differentiation that is strong enough to prevent gene flow between populations. In this case, we may be observing a process of ongoing speciation, where differentiation starts at the epigenetic level, triggering profound changes leading to segregation in terms of habitat, phenology, and/or biological interactions. Divergent selection may reinforce this environmentally induced specialization/niche segregation and bring about reproductive isolation. This will eventually result in virtual isolation of gene pools, and ultimately give way to stronger overall differentiation by accumulation of genetic differences due to the stochastic effects of drift.Heliosperma pusillum and allied taxa from the carnation family (Caryophyllaceae) contain a variety of morphologically different taxa  genomic and transcriptomic analyses, because all taxa have a relatively small genome (1C = 1.32 pg [n = 2x = 24). In addition, they can be easily grown from seeds and have short generation times, which make them optimally suitable for common garden and transplantation studies.Different authors , 81 havepapillae , 81. By papillae , 82. Mos studies   and so Although the possibility of epigenetic inheritance has now been established , 30, 84 The recently available possibility to profile the epigenome and transcriptome of nonmodel organisms in a high-throughput manner , 63, 85 Currently accumulating data will offer valuable clues on the establishment of broad regulatory determinants of functional diversity in natural populations. The early evidence we currently hold urges complementing our gene- and genome-centred evolutionary view with a substantial consideration of epigenetic factors when seeking to understand population processes that drive adaptation and divergence , 53, 87.


Koompassia malaccensis (Fabaceae). We tested hypotheses relevant to the genetic consequences of habitat loss: (1) that the K. malaccensis population in Singapore experienced a genetic bottleneck and a reduction in effective population size, and (2) K. malaccensis recruits would exhibit genetic erosion and inbreeding compared to adults. Contrary to expectations, we detected neither a population bottleneck nor a reduction in effective population size, and high genetic diversity in all age classes. Genetic diversity indices among age classes were not significantly different: we detected overall high expected heterozygosity (He = 0.843–0.854), high allelic richness (R = 16.7–19.5), low inbreeding co-efficients (FIS = 0.013–0.076), and a large proportion (30.1%) of rare alleles (i.e. frequency <1%). However, spatial genetic structure (SGS) analyses showed significant differences between the adults and the recruits. We detected significantly greater SGS intensity, as well as higher relatedness in the 0–10 m distance class, for seedlings and saplings compared to the adults. Demographic factors for this population (i.e. <200 adult trees) are a cause for concern, as rare alleles could be lost due to stochastic factors. The high outcrossing rate (tm = 0.961), calculated from seedlings, may be instrumental in maintaining genetic diversity and suggests that pollination by highly mobile bee species in the genus Apis may provide resilience to acute habitat loss.Over the last 150 years, Singapore’s primary forest has been reduced to less than 0.2% of its previous area, resulting in extinctions of native flora and fauna. Remaining species may be threatened by genetic erosion and inbreeding. We surveyed >95% of the remaining primary forest in Singapore and used eight highly polymorphic microsatellite loci to assess genetic diversity indices of 179 adults (>30 cm stem diameter), 193 saplings (>1 yr), and 1,822 seedlings (<1 yr) of the canopy tree  For example, some plant species that are naturally rare and/or endemic have strategies that allow for survival under conditions of self-fertilization or clonality, low genetic diversity, and high inbreeding Immigration and gene flow into small, isolated populations is generally considered to have positive genetic and demographic effects, as gene flow can increase effective population size and genetic diversity, and reduce deleterious inbreeding, e.g. Koompassia malaccensis Maingay ex Benth. (Fabaceae) is a long-lived , large , invertebrate-pollinated rainforest tree with wind-dispersed seeds. It is a canopy emergent tree of primary rainforest in Southeast Asia, ranging from Thailand to Borneo. Despite being classified as a species of low extinction risk over its entire range K. malaccensis is locally endangered within Singapore owing to severe habitat loss K. malaccensis has been widely harvested and therefore tends to be relegated to primary forest patches or occasionally found as isolated remnant adults in secondary forests. In some areas individual trees are left intact as K. malaccensis is frequently utilized by wild honey bees (Apis dorsata in particular) for nesting Koompassia is accomplished by A. dorsata, which is a large, migratory bee species, along with other Apis species . As a member of the Fabaceae, reproduction is hypothesized to occur yearly K. malaccensis is fast growing, and reaches reproductive maturity at around 30 cm stem diameter  K. malaccensis is almost exclusively in primary forest patches, it is fair to assume a high correlation between primary forest habitat loss and population loss. Thus we estimated that owing to habitat loss, K. malaccensis has sustained a population reduction of at least 90% over the last >150 years. It is important to note that whether actual population loss is exactly equivalent to the reduction of primary forest area is somewhat inconsequential given the scale of deforestation K. malaccenis through logging in southern peninsular Malaysia —a disturbance that typically removes a lower proportion of a tree population than deforestation—found fewer alleles per locus for seedling, sapling, and adult K. malaccensis individuals in logged versus unlogged plots, as well as a reduction in the number of polymorphic loci K. malaccensis, a study of the effects of extreme habitat loss in Singapore was well justified.Although no pre-deforestation abundance and distribution data for Koompassia malaccensis in Singapore is a putatively previously abundant, widespread and outcrossing species that underwent a severe decline in habitat starting ∼150 years ago and has a low likelihood of immigration and gene flow from outside populations. Hence, we tested two hypotheses relevant to the genetic consequences of habitat loss. First was that the K. malaccensis population in Singapore experienced a population bottleneck and a reduction in effective population size. Second was that recruit cohorts would show genetic erosion  and inbreeding compared to the adult cohort. To test these hypotheses, we assessed the genetic structure of comprehensively sampled adults, and randomly selected samples of saplings and seedlings, using eight highly polymorphic microsatellite loci.K. malaccensis adults with a dbh (stem diameter at 1.4 m above the base) of at least 30 cm in BTNR and MacRitchie Reservoir Park. For BTNR, we were provided access to an existing spatial database of all trees >30 cm dbh, from which we extracted the locations of all adult K. malaccensis. MacRitchie surveys were conducted by referring to existing vegetation maps of the central catchment K. malaccensis adults, all of which we sampled. Our data set was collected in >95% of the remaining primary forest area in Singapore, and was therefore expected to account for a roughly equivalent percentage of the remaining K. malaccensis population. However, given that we found a few isolated adults located in secondary forest, it is possible that we captured a somewhat smaller proportion of the remaining population than 95%.Our study area encompassed the central catchment and the Singapore Botanic Gardens . The cenK. malaccensis (dbh >30 cm) and leaves from 193 saplings  and 1,822 seedlings  from beneath 122 adults. Sampling was conducted under Singapore National Parks research permit NP/RP10-078. All necessary permits were obtained for the described study, which complied with all relevant regulations.We collected bark and/or leaves of 179 adult K. malaccensis) indicated that most recruits occur within 10 m of the parental tree. All recruits sampled had their compass bearing and distance measured to the nearest adult K. malaccensis, which was designated the putative mother. For each recruit, 3 to 5 leaflets were collected, placed in labelled bags and dehydrated with silica gel. We used one or both of the following sampling methods for adults: if no other adult K. malaccensis tree was within 50 m, a freshly fallen leaf was collected directly under the crown. If other adults were within 50 m, a 1 cm×1 cm bark scraping (<4 mm deep) was taken using sterilized equipment, placed in a 1.5 mL Eppendorf tube, and stored at −20°C the same day.Our field observations of isolated adults , 20 to 80 picomol of each F- and R- primer; 5 mM dNTPs, 2.5 mM MgCl2 and 1% DMSO final concentration in 5 µl total volume reactions. Cycling conditions: 95°C for 1.5 minutes; 34× 95°C for 30 seconds, 56°C for 30 seconds, 72°C for 30 seconds, and a final 5 minute 72°C extension. For each sample, the two multiplex PCR reactions were pooled for a single fragment analysis (ABI 3100 or 3730x) and run against a 600-LIZ internal size standard (ABI). Samples were scored using GeneMapper v. 4.1. One-third of adults and 0.7% of the recruit cohorts were re-extracted, PCRed and genotyped in their entirety, and 100% of adults and 24.9% of the recruit cohorts were re-PCRed and genotyped at 1 to 8 loci to confirm rare alleles as well as to estimate genotyping errors. Genotyping errors were calculated per locus as the percent of non-matching alleles in a second PCR , as the conclusions drawn would not be biologically accurate—i.e. a high FIS value at this locus would not reflect inbreeding. To evaluate the adequacy of our field sampling in capturing the full range of allelic diversity in Singapore, we produced curves for each locus depicting the accumulation of alleles as a function of the number of samples analyzed.Microchecker v. 2.2.3 e, Ho) and inbreeding co-efficients (FIS) were calculated with Fstat v. 2.9.3 e and R among age classes were compared with an Analysis of Variance at the locus level. For mating system calculations, seedlings or saplings were classified as the progeny of the nearest adult K. malaccensis. Mltr v. 3.4 m) and biparental inbreeding rates (tm-ts) under default parameters and with the possibility of null alleles; the putative maternal genotype for each set of putative offspring was included. Four chosen loci  were analysed for a random subsample of 9 adults in order to compare the mltr results from the Singapore population to the Malaysia population under more analogous parameters.Allelic richness (R), expected and observed heterozygosity  using the formula -b/(1-F1), where b is the slope of the regression between pairwise kinship coefficients 1 is the mean pairwise kinship coefficient within the first distance class  for the adults, saplings, and seedlings was analyzed with GenAlex v. 6.5.3 lass see . Identice = 0.843–0.854, p = 0.98; R = 16.72–19.45, p = 0.20) (IS = 0.013–0.076) and not significant (p = 0.18), although there was an upward trend in younger age classes  and the biparental inbreeding rate (tm-ts) was 0.111 (SD 0.011) (Genetic diversity was high and not significantly different between age classes (H = 0.20) . The inb classes . There w classes . Over thD 0.011) .K. malaccensis within Singapore.We detected neither a genetic bottleneck in any age class (no mode shift) nor a reduction in effective population size  . The larp was lowest in the adult cohort (0.0044), and increased with younger cohorts   . The SGS 0.0287) . The nonK. malaccensis in Malaysia K. malaccensis in Singapore is generally on par with populations studied in more intact Malaysian forests. While these unexpected results may not be representative of the majority of primary forest species (and demographic factors are still a cause for concern) they demonstrate lower than expected genetic vulnerability for a locally threatened, primary forest tree species in a highly urbanized landscape.Gene flow and intergenerational studies using exhaustively surveyed landscapes are crucially lacking in the tropical tree literature K. malaccensis in Singapore would be considered high even for common, widespread species in pristine habitats The initial decline in genetic diversity during habitat loss is due to demographic factors, i.e. the removal of individuals. Subsequently, genetic drift and reduced genetic diversity generally results from only a subset of individuals contributing gametes to the next generation, e.g. −4 mutations/generation/locus The majority (∼99%) of alleles present in the adult population were also present in the recruit cohort, in addition to twelve private alleles in the seedling cohort . Given tLow inbreeding and high heterozygosity are positive indicators for short-term population viability; high allelic richness and a large proportion of rare alleles indicate an adequately large effective population size for long-term viability p = 0.0044) similar to other tropical tree species  for the whole dataset as well as the subset was as high as in the Malaysian populations m-ts) was much higher overall in Singapore than in Malaysia . In addition, the presence of a small number of large, adult K. malaccensis outside of primary forest habitat suggests persistence of pre-deforestation individuals within the degraded secondary forest and urban matrix.The higher than expected genetic diversity we detected may be explained by demographic as well as ecological factors. In terms of demographics, a larger number of pre-fragmentation trees than expected may remain in the extant adult population. Hence, a high proportion of Singapore’s pre-deforestation genetic diversity may have been retained to the present day. For example, Singapore’s largest and presumably oldest primary forest trees are protected from lightning by conductors, leading to increased longevity. Conductors have been installed on many trees in our study area, and several mature Apis has been identified as a pollinator of Koompassia  K. malaccensis, two key ecological aspects of pollination may be contributing to the high outcrossing rates of K. malaccensis. First is that A. dorsata forms colonies that may nest preferentially in K. malaccensis; secondly, A. dorsata is a highly mobile insect, capable of pollen transfer during foraging bouts or colony migrations of several km (R. Corlett pers. comm.). Other tree species with less mobile pollinators are unlikely to exhibit the same level of resilience to habitat loss as we have found with K. malaccensis.Ecologically, high outcrossing rates also likely contribute to genetic diversity detected. cited in  and the Our study stands in contrast to predictions of genetic erosion in formerly abundant species that have sustained rapid and dramatic population declines. However, long-lived species such as trees may need centuries for the accumulated effects of habitat loss and reduced population size to reach a critical threshold, and the century-old extinction debt in Singapore is likely ongoing. Stochastic factors  could eliminate rare alleles , and demographic factors such as limited primary forest habitat, lack of recruitment in secondary forest, and a high seedling mortality rate may contribute to a potential extinction debt in the future.Koompassia malaccensis in Singapore despite their demographic vulnerability suggest an “Apis rescue effect”: pollination provides a mechanism through which populations may retain resilience in the increasingly degraded and urbanized landscapes of Southeast Asia. However, the higher relatedness at short distances as well as the increasing SGS intensity in the recruit cohorts could indicate that Singaporean K. malaccensis may be undergoing a shift towards higher mating between (related) neighbours and a contracting neighbourhood size. Hence, the direction, frequency, and distance of pollen and seed dispersal within and between Singapore’s remaining primary forest fragments is crucial to determine the trends in population genetic dynamics resulting from habitat fragmentation.Conversely, the continued functionality of insect pollinators implied by the data may help maintain viability of this small population by increasing the likelihood that the majority of alleles present in the adults are passed via sexual reproduction to the next generation (simultaneously maintaining genetic diversity while reducing genetic drift and inbreeding). Hence, the positive genetic results for


Episyrphus balteatus (DeGeer) (Diptera: Syrphidae) is one of the most important natural predators of crop aphids in Europe. Three different overwintering strategies coexist in this species which makes it a good model in order to study ecologically-based speciation processes. The purpose of this study was to determine whether E. balteatus populations with alternative overwintering strategies are genetically differentiated. To that aim, we developed 12 specific microsatellite markers and evaluated the level of neutral genetic differentiation between E. balteatus field populations that overwinter in the three different ways described in this species . Results showed a lack of neutral genetic differentiation between individuals with different overwintering strategies although there are strong ecological differences between them. All pair-wise FST values are below 0.025 and non-significant, and Bayesian clustering showed K = 1 was the most likely number of genetic clusters throughout our sample. The three overwintering strategies form one unique panmictic population. This suggests that all the individuals may have genetic material for the expression of different overwintering phenotypes, and that their commitment in one particular overwintering strategy may depend on environmental and individual factors. Consequently, the prevalence of the different overwintering strategies would be potentially modified by landscape engineering and habitat management which could have major implications for biological control.Winter ecology of natural enemies has a great influence on the level and efficiency of biological control at spring. The hoverfly  Insect body temperature varies in relation with the environmental temperature. This results in insect life cycles being highly dependent on climatic conditions. Therefore, in temperate regions, adaptation to winter conditions is an important life history trait that may influence the ecological and evolutionary success of insects. To cope with adverse winter conditions, insects have developed a great variety of ecological strategies including migration Oceanodroma castro (Harcourt) (the Madeiran storm-petrel) exhibits very reduced or even completely disrupted gene flow between populations that breed in the same places but in different seasons Various processes may lead to reproductive isolation and genetic divergence between populations adopting distinct overwintering strategy. First, differences in overwintering strategy can lead to temporal segregation in adults spring appearance and consequently to assortative mating within each strategy Episyrphus balteatus (DeGeer) (Diptera: Syrphidae), at its larval stage, is one of the most important aphid feeding predators in Europe E. balteatus. Some individuals overwinter as adults in a facultative reproductive diapause The hoverfly E. balteatus adopting alternative overwintering strategies are genetically differentiated. To achieve this objective, 12 E. balteatus specific microsatellite markers were developed and specific sampling protocols were adopted to collect individuals belonging to the three overwintering strategies in four different study sites. Because of allochrony and different environmental pressures, we expected to observe a genetic differentiation between individuals adopting alternative overwintering strategies. Finally, we found no genetic divergence between the different strategies and sampling sites, which may have several implications for aphid biological control.The purpose of this study was to assess whether E. balteatus field populations that overwinter through different strategies. Individuals overwintering locally at a pre-imaginal or adult stage were collected during two years (2011 and 2012) in two French sites distant from each other 400 km  and is part of the Long Term Ecological Research network (LTER_EU_FR_003). The study site “Plaine et Val de Sèvre” (PVS) is located in west France  and covers a 450 km2 area of intensive agriculture. The choice of the sampling protocol allowed distinguishing unambiguously populations according to their overwintering strategy. Individuals overwintering as adults were caught by Malaise traps  during the winters 2011 and 2012. Traps were preferentially placed along south facing forest edges or along south facing hedges. Twelve Malaise traps were used in the VCG study site between 12-Jan-2011 and 08-Mar-2011 and ten between 22-Dec-2011 and 16-Mar-2012. Ten Malaise traps were used in the PVS study site between 22-Dec-2011 and 16-Mar-2012. Individuals overwintering at a pre-imaginal stage were caught in 2011 with emergence traps between the end of winter and the beginning of summer which is the period of adult emergence 2, Soil Emergence trap 96×26 mesh, Black, MegaView Science Co., Ltd, Taichung, Taiwan) in the PVS study site, and 30 large traps  in the VCG study site. The use of different size emergence trap was due to material availability in the two study sites. Collecting bottles of Malaise traps and emergence traps were filled two thirds full with 70° ethanol. In addition, migratory individuals were caught in the Pyrenees, on their migratory route towards Southern Europe. Two mountain passes  were chosen as sampling sites as they have been previously described as migration passes E. balteatus due to their high altitude. Migratory individuals were caught between 01-Sep-2011 and 02-Oct-2011 using an interception trap with a 4 m triangular opening described in Aubert E. balteatus individuals were stored individually in Eppendorf tubes in 90° ethanol, and kept at 4°C prior to molecular analyses.We used r 400 km . The stuThe emergence traps and Malaise traps were installed on private lands whose owners had given permission to conduct the study on these sites. Authorizations for sampling at Boucharo pass and Puymorens pass were respectively obtained from the Parc National de Pyrénées and from the Parc Naturel Régional des Pyrénées Catalanes. Sampling for this study did not involve endangered or protected species.VCG), overwintering pre-imaginal individuals caught in site PVS (PPVS), overwintering adults caught in site VCG (AVCG), overwintering adults caught in site PVS (APVS), migratory individuals caught at Boucharo pass (MBou), migratory individuals caught at Puymorens pass (MPuy). We also aggregated the groups from different sampling sites sharing the same overwintering strategy , individuals overwintering locally at adult stage (A), individuals overwintering by migrating (M)) in order to test for genetic differentiation between overwintering strategies independently from sampling site.We defined six groups on the basis of the overwintering strategy and the sampling site: overwintering pre-imaginal individuals caught in site VCG  (eneMapper® 4.0 (AB-life technologies).Genomic DNA was extracted from insect head and thorax by a “salting out” protocol  . Microsaicrochecker 2.2.3 rlequin 3.5.1.3 E. balteatus sampling groups . The significance of differences between expected and observed heterozygosities (α = 0.05) was determined using Arlequin 3.5.1.3 after a sequential Bonferonni correction carried out on the 12 statistical tests in each group. We also used Arlequin 3.5.1.3 to calculate linkage disequilibrium between all pairs of loci in the total population and its significance by an exact Fisher test on 20000 permutations (α = 0.05) after sequential Bonferroni correction.First, we checked for the presence of null alleles for each locus and sampling group by calculating the proportion of missing data  and testOne locus (Ba13) displayed evidences for the presence of null alleles and was thus removed for subsequent analyses.e and Ho) were calculated using Arlequin 3.5.1.3, the mean number of alleles per locus (AN), the multilocus inbreeding coefficient (FIS), and the allelic richness (RS) were calculated using Fstat 2.9.3.2 IS were obtained after 1320 randomizations of alleles among individuals within sampling groups and after 660 randomizations of alleles among individuals within overwintering strategies, as implemented in Fstat 2.9.3.2 The genetic structure and diversity among the six sampling groups and the three overwintering strategies were assessed using genotypes across 11 microsatellite loci. For each sampling group and each overwintering strategy, the mean expected and observed heterozygosity  The level of neutral genetic differentiation between sampling groups and overwintering strategies was quantified using the Ftructure 2.3.3 tructure, the number of clusters (denoted by K hereafter) representing the data was explored by performing 20 replicates of each simulation from K = 1 to K = 9, with a burn-in of 50 000 steps followed by 500 000 Markov chain Monte Carlo (MCMC) iterations under the admixture model and the assumption of correlated allele frequencies among populations. Individuals were assigned to clusters based on their highest membership coefficient to a particular cluster averaged over the twenty independent runs. The K value which better fitted our genetic data was detected using the highest likelihood method Finally, population structure was inferred using a Bayesian clustering algorithm implemented in the computer program Sicrochecker 2.2.3 PUY, and loci Ba13, Ba30, Ba33, Ba35 and Ba9 in the sampling group MBOU). Particularly, the locus Ba13 showed evidence for null alleles in four of the six sampling groups . We considered that homozygote excess was due to chance or was an effect of sample size when it was significant only in one sampling group. In this case, we conserve the involved loci for population structure analyses. When a locus displayed homozygote excess in more than three of the six sampling groups, we considered that it could be due to the presence of null allele and removed this locus for subsequent analyses. In this way, the locus Ba13 was discarded. However, we note that the presence of this locus does not influence the results and conclusions of this study.A total of 376 individuals (sample sizes given in P = 0.99) neither between overwintering strategies . Among sampling groups, the mean observed heterozygosity ranged from 0.53 to 0.74 and the mean expected heterozygosity ranged from 0.67 to 0.74. Among strategies, the mean observed heterozygosity ranged from 0.65 to 0.69 and the mean expected heterozygosity ranged from 0.70 to 0.71. Except for the sampling group PPVS (FIS = 0.23), multilocus inbreeding coefficient (FIS) among sampling groups and overwintering strategies displayed low values (between 0.01 and 0.07). Permutation test revealed that none of FIS value was significant. The high but non-significant FIS value of the sampling group PPVS is probably an effect of the small size of this group (n = 5).We found 171 alleles over the 11 selected loci and the six populations . Loci BaST was very low. Pair-wise FST values varied from 0 to 0.022 and none was significant. A global test of differentiation among samples showed no differentiation (exact P = 1 on 30 000 Markov chains). The analysis of molecular variance (AMOVA) confirmed a lack of differentiation between sampling groups and between overwintering strategies: more than 99% of the molecular variance was attributed to the within-group variance although less than 1% was attributable to a differentiation between groups within strategies or between strategies  (The Bayesian assignment performed with the software SP>0. 99) .E. balteatus individuals overwintering with different strategies led us to hypothesize reproductive isolation among them. According to the classification proposed by Rundle and Nosil E. balteatus winter ecology is likely to produce three forms of reproductive isolation involved in ecological speciation: habitat isolation, temporal isolation and natural selection against immigrants. With (i) larvae or pupae overwintering in soil or litter of cultivated fields and grassy margins, (ii) fertilized females overwintering actively in forest edges and (iii) migrating adults overwintering at few hundred kilometres further south, E. balteatus exhibits both divergent habitat and developmental schedules that may induce strong differences between individuals in abiotic conditions and in the suit of predators and competitors they face. These environmental differences are potential ecological causes of divergent selection because of the divergent pressures they induce on individuals E. balteatus probably has a continuously breeding migration pattern, with each individual carrying out only a single part of the migratory circuit. Owing to local adaptation to origin region conditions, this migration pattern may cause differences between individuals coming from southern Europe at spring to colonize middle and northern Europe and individuals overwintering locally in these regions. These differences may induce selection against immigrants which is another potential cause of reproductive isolation.Ecological differences between Rhagoletis pomonella  (the apple maggot fly) Dalbulus maidis (Delong & Wolcott)  E. balteatus. Low FST values between sampling groups and overwintering strategies, extremely low contribution of between-sampling groups and between-overwintering strategies differentiation to the total amount of molecular variance and absence of genetic structure identified by genetic clustering are consistent with the fact that E. balteatus individuals belong to a unique panmictic population at large geographic scale and irrespective to their overwintering strategies. The genetic mixing between individuals performing different overwintering strategies results in the absence of genetic differentiation between sampling sites. Indeed, the long range migratory individuals should allow gene flow between distinct geographical zones and act as a genetic homogenization force. Genetic mixing is also facilitated by the multivoltin ecological cycle of E. balteatus. The several overlapping generations during the summer period would allow erasing the ecological adaptive differences observed during the winter period.Ecological speciation was previously shown in relation to differences in spring emergence time in et al. Danaus plexippus L.) are not genetically differentiated on neutral markers despite different migration patterns and distinct overwintering sites E. balteatus all individuals may have the genetic material for the expression of all three phenotypes, and their commitment in one particular overwintering strategy may be due to differential gene expression in response to environmental conditions, level of energetic resources, and age. Transcriptomic approaches could allow validating this hypothesis in the future.Although our results demonstrate there is no reproductive isolation between the overwintering strategies, we cannot conclude on the absence of divergent selection on them. Differences in overwintering strategy may result from selection on relatively few genomic regions and is not necessarily picked up by neutral genomic markers, as mentioned by Liedvogel E. balteatus can be understood as an ecological opportunity. Given that the females lay eggs continuously during more than one month E. balteatus is certainly a cause of the ecological and evolutionary success of this species which can be found in many different habitats from Palaeartic to Afrotropical, Oriental or Australian regions over-WinterE. balteatus adult overwintering population from landscape and climatic input data.Adoption of a particular overwintering strategy in E. balteatus which is a major beneficial insect in agriculture. Our results clearly state the lack of neutral genetic differentiation in this species at a large spatial scale, irrespective to the overwintering strategies of individuals. The lack of genetic differentiation could help improve biological control against aphids, as it would be possible to increase the proportion of the population locally overwintering by landscape engineering. Future studies that could focus on the factors responsible for the commitment to a particular overwintering strategy, based on next-generation sequencing technologies could confirm this hypothesis and determine precisely the necessary conditions for the local overwintering and therefore a more effective biological control.This study is the first population genetics study based on microsatellite markers on Figure S1Expected (He) and observed (Ho) heterozygosity at each locus in the six groups. Significance differences between these values ) are indicated by asterisks. Loci for which at least two groups showed significant departure from Hardy–Weinberg equilibrium were discard for subsequent analyses and are indicated in grey shading.(DOCX)Click here for additional data file.Figure S2Bayesian assignment probabilities for k = 2, K = 3 and K = 4. Each vertical line represents an individual, and colours indicate the proportion of an individual’s genotype assigned to a particular lineage, individuals are sorted by overwintering strategy and sampling site.(DOCX)Click here for additional data file.