Many group–living species display strong sex biases in dispersal tendencies. However, gene flow mediated by apparently philopatric sex may still occur and potentially alters population structure. In our closest living evolutionary relatives, dispersal of adult males seems to be precluded by high levels of territoriality between males of different groups in chimpanzees, and has only been observed once in bonobos. Still, male–mediated gene flow might occur through rare events such as extra–group matings leading to extra–group paternity (EGP) and female secondary dispersal with offspring, but the extent of this gene flow has not yet been assessed.Pan troglodytes verus) and bonobos (Pan paniscus), we found low genetic differentiation among groups for both males and females. Characterization of Y–chromosome microsatellites revealed levels of genetic differentiation between groups in bonobos almost as high as those reported previously in eastern chimpanzees, but lower levels of differentiation in western chimpanzees. By using simulations to evaluate the patterns of Y–chromosomal variation expected under realistic assumptions of group size, mutation rate and reproductive skew, we demonstrate that the observed presence of multiple and highly divergent Y–haplotypes within western chimpanzee and bonobo groups is best explained by successful male–mediated gene flow.Using autosomal microsatellite genotyping of samples from multiple groups of wild western chimpanzees (The similarity of inferred rates of male–mediated gene flow and published rates of EGP in western chimpanzees suggests this is the most likely mechanism of male–mediated gene flow in this subspecies. In bonobos more data are needed to refine the estimated rate of gene flow. Our findings suggest that dispersal patterns in these closely related species, and particularly for the chimpanzee subspecies, are more variable than previously appreciated. This is consistent with growing recognition of extensive behavioral variation in chimpanzees and bonobos. Dispersal, the shift in residence of an individual from one area or social group to another, is a fundamental process affecting population structure. It is typically considered an inbreeding avoidance mechanism Dispersal is an intrinsically infrequent event that is seldom observed, particularly for species with slow life histories such as primates, and may be difficult to distinguish from the disappearance or death of individuals. Advances in the use of non–invasive samples to genetically characterize individuals and in the analysis of genetic data offer an opportunity to investigate this important, often cryptic aspect of life history in wild animal populations There are many mechanisms by which the putatively nondispersing sex may contribute to gene flow between groups. In most taxa, the dispersal bias between the sexes is observed to be strong but not absolute, and some adult individuals of the typically philopatric sex may successfully join new groups  and paternally–transmitted Y–chromosome in eastern chimpanzees and bonobos suggested that male–mediated gene flow is absent in eastern chimpanzees and potentially rare in bonobos Pan troglodytes verus) and bonobos (Pan paniscus). We used autosomal, as well as Y–chromosomal microsatellite markers to characterize genetic variation in multiple social groups. For investigation of genetic differentiation, in both species we applied a small scale sampling regime, and for the western chimpanzees also examined groups over a slightly larger geographical range to assess whether, as might be expected, genetic differentiation increases as geographic distance increases. Our goal was then to assess the evidence for male–mediated gene flow in these two male–philopatric primates. Potential sharing of Y–chromosomal variants is not necessarily evidence for male gene flow, but could also represent the retention of ancestral haplotypes in different social groups. We therefore employed a simulation approach to examine, under realistic estimates of relevant demographic factors , expected levels of within–group Y–chromosomal genetic variation and to identify potentially immigrant types. By comparing our simulation results to our empirical data, we derived a potential range of levels of male–mediated gene flow in both study populations.In this study, we investigated potential differences in the amount of genetic differentiation and of male–mediated gene flow between wild western chimpanzees  with slight modifications Sex was determined or confirmed using polymerase chain reaction pIDsib, the probability that two identical multi–locus genotypes do not come from the same individual but rather from siblings. Estimated average pIDsib values were <0.01 for our five most informative autosomal loci (chimpanzees and bonobos), or for the six (bonobo) or seven (chimpanzee) least variable loci, indicating that even partially complete genotypes from close relatives would be distinguishable (data not shown). Genotypes from different samples that were found to come from the same individual (pIDsib <0.01) were combined into a consensus genotype. In total we genotyped 203 individual chimpanzees (genotypes 97.0 % complete) and 101 bonobos (genotypes 92.8% complete) at 19 autosomal loci  and 97.2%  complete.Each male was then genotyped at 13 (chimpanzee) or 10 (bonobo) Y–chromosomal loci developed in humans and previously assayed in eastern Previous research on habituated bonobos and chimpanzees has shown that it is difficult to obtain samples from young individuals, particularly dependent infants . Therefore, we assumed that our sample of unhabituated chimpanzees and bonobos would primarily consist of adult, adolescent and some juvenile individuals. To obtain comparable, approximately single–generation data sets from the habituated research groups, we used only genotypes from individuals present in 2001 and 2009 for the chimpanzees and bonobos, respectively, and the samples from unhabituated individuals were collected over a span of several months. We thereby excluded individuals that were known or estimated to be younger than 5 years.ST between pairs of social groups at the autosomes, for males and females separately, and at Y–chromosomal loci using ARLEQUIN 3.11 p–values of group pairwise FST were obtained from the permutation procedure implemented in ARLEQUIN. We also examined overall genetic differentiation in an AMOVA framework. Here, inspection of the overlap in the 95% confidence limits generated by bootstrapping genetic differentiation values obtained in a locus–by–locus AMOVA was used to evaluate whether (a) autosomal genetic differentiation between males and females was different within species and when comparing bonobos and chimpanzees and (b) genetic differentiation of Y–chromosomal loci differed between bonobo and chimpanzee males.We measured genetic differentiation FST–based genetic differentiation measures are not entirely independent of the amount of genetic variation present within groups, with high levels of genetic variation potentially leading to lower FST estimates than low levels of variation ST can therefore be biased and should be corrected. To allow for a meaningful comparison of genetic differentiation at the haploid Y–chromosome, where levels of variation are relatively low and slight differences between the species might be consequential for FST estimation, we also used a standardized measure of genetic differentiation for the Y–chromosomal data. Standardized FST expresses genetic differentiation as the maximum amount of genetic differentiation possible given the amount of within–group variation We examined genetic differentiation at two hierarchical levels in the more widespread chimpanzee sample to (a) obtain a data set readily comparable to the more local bonobo sampling and (b) examine whether the amount of genetic differentiation increased at the larger scale as might be predicted To minimize stochasticity, for all analyses of genetic differentiation between communities we excluded social groups with fewer than four sampled individuals. We chose four because in some of the completely sampled habituated groups only four individuals of the respective category  were present.within empirical bonobo and chimpanzee groups could be generated through mutation or must originate externally through male–mediated gene flow. Starting groups of a fixed number of males, with all males possessing identical Y–haplotypes, were simulated over discrete generations in which the individuals of one generation were used as the fathers for the next generation. Starting the simulations with groups that contained only dissimilar, highly divergent haplotypes produced identical results, as most haplotypes were lost rapidly and new, similar ones were subsequently generated by mutation. Therefore, we only report results for starting groups with one haplotype. The following factors were included in the simulations:There are two ways to investigate gene flow between social groups; one is to focus on variation shared between different groups, and the other is to look at the number and types of variants present within groups. We performed simulations  to examine whether the Y–chromosomal variation present i) Mutation generates new haplotypes in groups. Individual Y–haplotypes mutate with a probability derived as the product of mutation rate and the number of Y–chromosomal loci  typed in our empirical data, following a stepwise mutation model. We used the most recent published human mutation rate . Analyses using both mutation rates produced similar results, so we report here the results based on the better–substantiated human mutation rate estimate.ii) Skewed reproduction removes haplotypes from groups. Reproduction among Pan males is expected to be skewed over the short term with males of higher social dominance rank enjoying a reproductive advantage . However, over an entire generation skew should be lower, as individual opportunities to reproduce change iii) Male–mediated gene flow adds new divergent haplotypes to groups. In our model, male–mediated gene flow adds unique, infinitely distant (divergent) Y–haplotypes to the group at a specified constant rate. We chose to make the immigrant haplotypes highly divergent so that they can be distinguished from new haplotypes that arise via mutation and are highly similar to those already present. Since we thus consider any highly similar haplotypes as arising via mutation and highly divergent haplotypes as products of immigration, we may underestimate the rate of male migration that contributes haplotypes to the group similar to ones already there.The habituated Taï chimpanzee groups contain nine reproductively active males on average , as does the one bonobo research group investigated here. We therefore considered simulations of groups containing 5, 9, 10 and 15 males as realistic for the examined populations.All simulations reached stability after less then 100 generations and we simulated 1000 identical starting groups of the above sizes for 1000 generations. In order to see how much haplotypic variation could arise and be maintained in these closed groups with no gene flow, we averaged generations 100–1000 to examine the proportions of groups containing variation (more than one Y–haplotype) and the maximum number of mutations typically (more than 1% of the observations) found between any different Y–haplotypes present in the same group. From this simulation we thus inferred how many different haplotypes might be found in a group and how different they would be from one another. We then used our empirical data to examine how frequently groups contained multiple haplotypes that were more different from one another than we would expect given these simulation results, representing potentially immigrant haplotypes. In a second step we incorporated male–mediated gene flow by adding unique, infinitely distant Y–haplotypes to the simulated groups at pre–defined rates and then examined which rates of male–mediated gene flow were compatible with the frequency with which we observed empirical groups to contain ‘immigrant’ haplotypes. The source code for the simulation is available as electronic ST was similarly low among male and female chimpanzees and bonobos , even though several groups had small sample sizes. Y–chromosomal variants did not reliably delineate social groups in either species and were shared not only between neighboring groups, but also over distances of ∼50 km in our chimpanzee sample  and over a sustained period in the absence of male–mediated gene flow. Using varied size groups of reproductively active males and realistic assumptions for the mutation rate and distribution of reproductive skew, we found that 20 – 78 and 17 – 69% of simulated chimpanzee and bonobo groups, respectively, contained multiple Y–haplotypes, overlapping with the empirical data in bonobos and approaching the levels observed in wild chimpanzee groups . As expeWe next examined how often highly divergent Y–haplotypes are likely to be observed within groups, and whether such divergent types are likely to be observed in the absence of male–mediated gene flow. We therefore determined the maximum number of mutational steps between Y–haplotypes arising within groups over time in the absence of gene flow, using the simulation conditions specified above. The simulations suggest that maximum numbers of mutations between Y–haplotypes observed within simulated groups at frequencies >1%, were 6 mutations in chimpanzees  and 5 mutations in bonobos  of groups in chimpanzees or 2/6 (33.3%) in bonobos, rates of male–mediated gene flow of 3.5 – 7% or 14.5 – 28.5% immigrant Y–haplotypes/generation, depending upon the group size, are necessary in chimpanzees and bonobos, respectively .Pan dispersal patterns We explored patterns of genetic differentiation and specifically the potential for male–mediated gene flow among social groups of western chimpanzees and bonobos. Autosomal genetic differentiation was similarly low in both species, and slightly but not significantly higher in males than in females. While this finding is consistent with Pan populations. Variation in the effective extent of male philopatry has been suggested for other primates exhibiting some form of male philopatry, such as hamadryas baboons We found significantly less genetic differentiation at the Y–chromosome in western chimpanzees than in bonobos and eastern chimpanzees ; 62. ThiOur observations of sharing of Y-chromosome haplotypes among nearby groups as well as those separated by ∼50 km, the lack of increased genetic differentiation at larger geographic scales , and theThe simulations also showed that when using realistic mutation rates and reproductive skew parameters, social groups would not be expected to contain Y–haplotypes as divergent as those we observed in our empirical data. Thus, we estimated male–mediated gene flow to be, on average, 4.8% immigrant Y–haplotypes/generation in chimpanzees and 19.8% immigrant Y–haplotypes/generation in bonobos. This lower estimate of gene flow among western chimpanzees as compared to bonobos seems, at first look, inconsistent with lower overall levels of Y–chromosomal differentiation among this chimpanzee population, and several explanations are possible. However, both estimates are based on a small number of observations and are associated with a large degree of uncertainty, and the bonobo gene flow estimation in particular was based on fewer social groups, thus potentially harboring a larger stochastic component due to sampling variance. An alternative explanation would point to differences in male effective population sizes as contributing to differences in these estimates of Y-chromosomal genetic differentiation or male-mediated gene flow. However, male effective population size is not expected to differ greatly between western chimpanzees and bonobos as the long-term effective population sizes of both taxa are similar Finally, we estimated gene flow by considering only highly dissimilar haplotypes as originating outside the group. It seems likely, however, that some of the highly similar haplotypes might also originate from outside the group. Our simulations showed that, particularly in smaller groups with 5 males, most often only one haplotype is present  because mutation does not suffice to generate new variants frequently enough to counteract the loss of haplotypes when not all males get to reproduce. Thus, our estimated rate of male–mediated gene flow in the western chimpanzee population might be considered a minimum estimate and further study is needed in both western chimpanzees and bonobos.Several mechanisms can facilitate male–mediated gene flow among groups. Despite years of cumulative study, quantitative data on population dynamic processes like group splits or takeovers that might redistribute genetic variation are limited in chimpanzees and bonobos . However, more information exists on individual behaviors that may allow male–mediated gene flow. Dispersal by adults of the more philopatric sex, as seen in many other mammals Click here for additional data file.Table S2Simulated distributions of reproductive skew among bonobo and chimpanzee males for groups of different sizes. Values represent probabilities of transmission of an individual haplotype to the next generation and were derived from the original data using a best-fit log-function adjusted to the respective group sizes. Data were derived from human hunter-gatherer populations (DOC)Click here for additional data file.Table S3ST) in western chimpanzee (A) and bonobo (B) groups.Pairwise autosomal genetic differentiation  are shown in bold. To minimize stochasticity, for all analyses of genetic differentiation between communities we excluded social groups with fewer than four individuals genotyped at the respective marker . Therefore, the number of pairwise comparisons differs between the autosomal and Y-chromosomal data Click here for additional data file.Table S4Y-chromosomal microsatellite haplotypes of bonobo and western chimpanzee males. Locality indicates sampling locations as shown in a, Y-chromosomal loci DYS; b, Indel, coded as highest repeat number due to insufficient knowledge of mutational pattern.(DOC)Click here for additional data file.Table S5ST) in western chimpanzee (A) and bonobo (B) groups.Pairwise Y–chromosomal genetic differentiation (F Significantly differentiated pairs (p<0.05) are shown in bold. Sample sizes are indicated in brackets. To minimize stochasticity, for all analyses of genetic differentiation between communities we excluded social groups with fewer than four individuals genotyped at the respective marker . Therefore, the number of pairwise comparisons differs between the Y-chromosomal and autosomal data Click here for additional data file.Information S1Supplementary analytical procedures. Two-step amplification of DNA from fecal samples.(DOC)Click here for additional data file.Information S2Simulation source code. The code is written in Java (Sun Microsystems Inc. 1994–2009).(DOC)Click here for additional data file.


Leishmania species of the subgenus Leishmania and especially L. donovani are responsible for a large proportion of visceral leishmaniasis cases. The debate on the mode of reproduction and population structure of Leishmania parasites remains opened. It has been suggested that Leishmania parasites could alternate different modes of reproduction, more particularly clonality and frequent recombinations either between related individuals (endogamy) or between unrelated individuals (outcrossing) within strongly isolated subpopulations. To determine whether this assumption is generalized to other species, a population genetics analysis within Leishmania donovani complex strains was conducted within a single village. The results suggest that a mixed-mating reproduction system exists, an important heterogeneity of subsamples and the coexistence of several genetic entities in Sudanese L. donovani. Indeed, results showed significant genetic differentiation between the three taxa  and between the human or canine strains of such taxa, suggesting that there may be different imbricated transmission cycles involving either dogs or humans. Results also are in agreement with an almost strict specificity of L. donovani stricto sensu to human hosts. This empirical study demonstrates the complexity of population structure in the genus Leishmania and the need to pursue such kind of analyses at the smallest possible spatio-temporal and ecological scales. Leishmania parasites and transmitted by sandfly bites. More information is needed on the population biology of these pathogens for diagnostic and epidemiological inquiries and for drug and vaccine elaboration. For studies dealing with the population genetics, exploring the genetic patterns of such organisms at microgeographic scales is fundamental. In this context, we made a population genetic study, based on 20 microsatellite loci, on 61 strains of Leishmania donovani complex collected in a Sudanese village, Babar El Fugara, during the epidemic of 1996–2000. Results showed that considering the whole sample as a single population was not adequate because of the coexistence of several genetic entities and a genetic differentiation between the human or canine strains. In addition, our findings suggested that clonality may have a strong impact on the L. donovani complex, unlike other Leishmania species. This study demonstrates the need to pursue population genetics studies in Leishmania species from sampling designs that control maximum possible confounding factors and to elaborate such kinds of analyses at the smallest possible spatio-temporal and ecological scales.Leishmaniases are a serious public health problem, especially in developing countries, caused by  Leishmania. These parasitic infections are a serious public health problem, with about 350 million persons at risk and 2,357,000 new cases per year Leishmania totals approximately 20 described species causing human infections , and L. archibaldi in Sudan and Ethiopia Leishmania donovani sensu lato was the only cause of visceral leishmaniasis in East Africa , including Sudan Leishmania infantum and L. donovani were the only recognized species of the L. donovani complex L. donovaniVisceral leishmaniasis is mainly caused by species from the Leishmania parasites have been recognized as presenting a basic clonal mode of reproduction associated with rare recombination events Leishmania species in different environments showed strong levels of homozygosity and little amount of multilocus repeated genotypes (MLGs) Leishmania parasites could alternate different modes of reproduction: clonality in both vertebrate host and insect vector and recombination  within the vector Leishmania donovani sensu lato within a sample collected in a single Sudanese village. We therefore analyzed the population structure of 61 L. donovani s.l. strains, collected in Barbar El Fugara, a village of the Atbara River region on the Sudan-Ethiopian border, at 20 polymorphic microsatellite loci. The results of this work suggest that L. donovani complex is a heterogeneous taxon, that dogs are not infected by the same entities as human hosts and that the different units that compose this complex are probably strongly subdivided with a significant impact of sexual recombination between related individuals. We discuss sampling strategy issues regarding further studies and insist on the need to narrow as much as possible the spatio-temporal and ecological sampling scales.Nowadays, with the development of elaborated experimental techniques and sophisticated statistical tools, our understanding of the evolutionary processes that govern the propagation of these parasites is continuously improving. Since 1990, Leishmania donovani complex were collected and then cultured. We obtained the samples for this study from the “the French National Reference Center of Leishmania”, under the agreement of Dr. Alain Dessein.A census of the village population was conducted by Bucheton et al. L. donovani, 17 strains as L. infantum and 11 strains as L. archibaldi , following the manufacturer's recommendations.The 61 strains from Sudan were isolated from dogs (ten strains) and humans (51 strains) and characterized using the MLEE technique by Dereure et al. L. chagasi outgroup) under study were amplified according to the following conditions. Every 30-µL reaction mix was composed of 1 µL of each primer (10 µM), the forward being labelled, 100 ng template DNA, 0.9 µL dNTP mix (5 mM), 3 µL buffer 10× and 0.3 µL Taq Polymerase . Amplifications were carried out in a thermal cycler using the following reaction conditions: 35 cycles of 94°C for 30 s, annealing temperature of each locus  and by Nei's unbiased estimate of genetic diversity within subsamples sHF statistics FIS measures the relative inbreeding of individuals due to the local non-random union of gametes in each subpopulation, and FST measures the relative inbreeding in subpopulations attributable to the subdivision of the total population into subpopulation of limited size. FST thus also measures genetic differentiation between subpopulations. FIS ranges between −1 and 1: a negative value corresponds to an excess of heterozygotes, a positive value to heterozygote deficiency; 0 is expected under panmixia. The significance of the departure from 0 was tested by 10,000 randomisations of alleles within subpopulations (to test random mating) and individuals across subsamples (for differentiation). The statistic used for random mating (Hardy-Weinberg Equilibrium) testing was simply Weir and Cockerham's estimator f (FIS and FST). For the genetic differentiation test, we used the log likelihood ratio G-based test of Goudet et al. Data were processed through Create V 1.1 sH, can lower the maximum possible value for FST. According to classical formulation (e.g. FST = (QS−QT)/(1−QT), where QS is the probability to sample twice the same allele in a subpopulation and QT is the probability to sample twice the same allele in different subpopulations. If a population was totally subdivided, then the probability to sample twice the same allele in two different subpopulations should be null and thus FST should be equal to the probability to sample twice the same allele in a subpopulation QS. sH being the probability to sample two alleles that are different hence QS = 1−sH. The maximum possible value for FST in a sample with a given sH can thus be estimated as 1−sH and a corrected version of FST as FST′ = FST/(1−sH) Genetic diversity, as measured by Nei's Leishmania species (as recognized by MLEE typing), year of sampling and host species. To assess the possible contribution of these factors to genetic partitioning (Wahlund effect), we compared FIS obtained with four different sampling strategies. The first sampling strategy considered each Leishmania species-year of sampling–host species combinations as different subsamples . The second strategy ignored the Leishmania species distinction . The third strategy ignored the year of sampling  and the fourth one ignored the host species . For significant difference testing, we undertook planned paired Wilcoxon signed rank tests between “All separated” and each of the other three strategies ordered as above with sequential Bonferroni correction . Unilateral  Wilcoxon signed rank tests were undertaken under R FST estimation and G-based randomisation test). When two values were obtained for the same type of differentiation , these values were combined with an unweighted mean for FST (e.g. over years) and Stouffer's Z test  for P-values as recommended Data were heterogeneous regarding G summed over all subpopulations, known to be more powerful than other combinatory procedures α = 0.05. Thus we used the unilateral (“greater”) exact binomial test to check if there was significantly more than 5% significant tests in the 190 tests series under R Linkage disequilibrium between pairs of loci  was assessed with a randomisation test (genotypes at two loci are associated at random a number of times) using Fstat software Version 2.9.3.2 software . FIS was recalculated in each best distribution identified by BAPS and noted FIS_C. Then, for the three samples corresponding to the three species of Leishmania, the FIS_C was compared with the initial FIS using a unilateral Wilcoxon signed-rank test for paired data (with the software R), the pairing units being the 20 loci. If FIS_C is significantly lower than FIS, it is probable that the initial subsamples were composed of several genetically distinct entities .The BAPS version 5.1 software identifies a hidden structure within populations (admixture) through a Bayesian analysis Leishmania species between humans and dogs (50 human strains and 20 dog strains), and the significance was tested using an exact Fisher test under the software R Since we got the data's prevalence from Dereure et al. study A Neighbor-Joining (NJ) tree Simulations where also handled with Easypop 2.0.1 . The genotypes obtained are presented in supplemental 75±0.148 .L. infantum and L. donovani. Cluster B (sustained by a bootstrap of 32%) corresponded to L. archibaldi taxon and three L. infantum from dogs. It has to be noticed that other studies have observed, using microsatellite method, small bootstrap for large clusters and important bootstrap values only for small clusters for L. braziliensisL. infantumThe dendrogram, based on 20 polymorphic microsatellite loci, represented in FIS comparisons between “All separated” strategy and the three others gave significant differences, as illustrated in Leishmania species, year of sampling and host species in order of importance, displays a significant signature on the apportioning of genetic information. Consequently, each Leishmania species of each year and each host species must be considered as separate subsamples. It has to be noticed that the significant results we obtained cannot come from an insufficient number of samples. Indeed, the significant differences evidenced are statistically valid and ignoring it might lead to overlook important ecological processes currently involved in the population biology of these Leishmania “lineages”. Moreover, these differentiations were confirmed by paired subsample differentiation tests, as indicated in Leishmania species are genetically different. Species differentiation seems very pronounced between L. donovani and L. archibaldi (FST′∼0.767) and smaller for the two other pairs (FST′∼0.2–0.3)  . Tempora species .L. donovani in 1997 and 1998 where no differentiation could be evidenced . Consequently, clinical forms were not considered further in our analyses.Clinical forms . The results, presented in L. donovani is clearly found in humans rather than in dogs , that L. infantum displays a tendency to infect dogs more often , while the difference is not significant for L. archibaldi . If Bonferroni adjusted, only L. donovani test stays significant .The data's prevalence from Dereure et al. Leishmania species, year of sampling and host species combination as a distinct subsample. This provided 19 locus pairs out of 190 tests in significant linkage. This is far above the 5% expected under the null hypothesis . These significant tests involved 18 of the 20 loci. Within each Leishmania species, small subsample sizes limited the power of the test. For L. archibaldi  only five tests out of 190 were significant . In L. donovani 22 tests were significant  and in L. infantum 19 tests were significant . There is thus a global linkage at a genome-wide scale in the three Leishmania species populations.This analysis was undertaken over all the data but considering each Leishmania species, a global and highly significant heterozygote deficit, highly variable across loci, was observed  in the best partitions found by BAPS for the two species for which such analyses could be done (L. donovani and L. infantum) (c = 0.99) and strong Wahlund effects (pooling one representative of each strongly isolated subpopulation into one subsample). Nevertheless, in each of these simulations, fairly numerous multilocus genotypes (MLGs) appeared, in contrast to the real data, where on the whole data set only two MLGs (2 observations of two samples presented the same multilocus genotypes) were observed. Consequently, something else is occurring. Finally, using the NJ Tree pattern of L. donovani strains belonging to most homogeneous clusters , no significant linkage disequilibrium and a reasonable proportion of MLGs (one repeated twice and a second repeated three times) but very small subsample sizes. It has to be noticed, that the global same topology of the NJ tree using Cavali Sforza distances has been obtained using shared allele distances, and also the Minimum Evolution tree using either Cavali Sforza and shared allele distances.For each observed . These hnfantum) . SimulatLeishmania reproductive mode is far from being settled and therefore deserves further investigation. Recent publications on different Leishmania species and in different environments seriously challenge the view that the species of the genus should display a predominantly clonal genetic signature because of important homozygosity levels and rarity of MLGs L. braziliensisTrypanosoma brucei s.l. Leishmania subsamples that probably results from Wahlund effects (mixture of differentiated true populations), because strains were collected at too large spatial and/or temporal scales. To prevent such possible biases, we selected a sample of L. donovani, collected at a village scale, reducing the risk of hidden substructuring.Despite the latest studies in this area, the debate on population structure and L. donovani sensu stricto, L. archibaldi and L. infantum, be it a true species, a subspecies or any other taxonomic level, is supported by our results, in contradiction with recent papers In this Sudanese village, the validity of the distinction between L. infantum and to a much lesser extent L. archibaldi. Gene flow (gene flow) appears much reduced between dogs and human hosts for L. infantum and two different kinds of cycles must be present here, involving probably different vector's species and reservoirs. For L. archibaldi the difference is much less obvious but may be as a result of modest sampling sizes. In L. donovani, the greater specificity of strains to human (L. donovani strain (LEM3785) genotyped from a dog did not show any originality as regard to its human counterparts , the second hypothesis appears more likely.Time also appeared as a significant subdividing factor but only for FIS distribution as illustrated by Supplementary Failing to consider all the above factors as relevant resulted in a very odd Our data, and especially the NJTree approach, also suggest that hybridization between the different taxa is not impossible, though rare enough to prevent homogenization, but frequent enough to enhance heterogeneity within each cluster that could be defined.L. donovani clinical forms . Indeed, this result could suggest that the development of PKDL in treated patients is more likely link to host's factors than to parasite's factors. This potential association between PKDL and host has already been suggested by Blackwell J.M.'s team. Indeed, results of this study proposed a genetic association between the polymorphism at IFNGR1 and the susceptibility of patients after treatments to PKDL  An interesting point to notice is the absence of genetic differentiation obtained between FIS loci, that displayed a much wider range than what was observed for the much more homozygous L. braziliensisFIS and its variance across loci with very few MLGs, as in the real data, was impossible to achieve. The existence of a strongly structured hierarchical meta-population, with for instance the individual hosts playing the role of micro-populations for the parasites, in combination with occasional gene flow between different genetically distant entities (species hybridizations) and/or different cycle types (zoonotic vs. anthroponotic), could explain the pattern observed on our microsatellite loci. However, as previously said, this requires further investigation. We cannot exclude the possible disturbing role played by gene conversion known to occur in LeishmaniaLeishmania (genomic conversion) we would have expected a much more homogeneous homozygosity across loci than what was observed. Some loci are indeed almost always homozygous while some others display substantial amounts of heterozygosity  was worth trying and indeed represents a too often neglected factor in Leishmania population studies in particular and in pathogenic microbes in general Leishmania species from sampling designs that control maximum possible confounding factors. These parasites indeed seem to be subdivided at very narrow spatio-temporal and ecological (host) scales.To conclude on this population genetics study within the Figure S1FIS for each of the loci in the entire population of L. donovani complex. There is a large heterozygote deficiency at each locus.(TIF)Click here for additional data file.Checklist S1STROBE checklist. Checklist of items included in this population genetic study.(PDF)Click here for additional data file.Table S1Description of data set and microsatellite genotypes. Each sample is detailed by sample code, species attribution by MLEE (XLS)Click here for additional data file.


The Qinghai-Tibetan Plateau (QTP) and its southern and southeastern mountain ranges, Himalaya-Hengduan Mountains (HHM), are one of the most extensive habitats for alpine plants in the world. How ferns occurring in QTP and HHM changed their distribution ranges in response to Quaternary climatic oscillations remains almost unknown.rps4-trnS and trnL-trnF, to reconstruct phylogeography of the Sino-Himalayan fern Lepisorus clathratus, occurring mainly in the QTP and HHM. Individuals of this species have either dehiscent or indehiscent sporangia with the latter evolved from the plesiomorphic dehiscent forms. Based on a range-wide sampling, we detected 27 cpDNA haplotypes that were divided into five groups by network analyses. Populations in the Hengduan Mountains possess the highest genetic diversity, while a single haplogroup is detected across the north-central region. A distinct phylogeographical subdivision was detected between the Hengduan Mountains and north-central region by AMOVA analysis. The haplogroup distribution pattern, coalescence and AMOVA analysis suggest that a long term survival area (refugia) of the species was located in the Hengduan Mountains during glaciations, with probable range expansions into north-central regions during interglacial periods. Populations with indehiscent sporangium can carry private haplotypes and are inclined to maintain genetic homogeneity. One group with indehiscent sporangia most likely survived in situ on the QTP during glaciations.We employed sequences of two chloroplast DNA regions, This study for the first time sheds light on the response of alpine ferns in the QTP and HHM to the Quaternary climatic oscillations. It is commonly accepted that the QTP uplift had a major impact on the development of the Asian climate system and on East Asian biodiversity The Himalaya-Hengduan Mountains region (HMM) is home to over 20,000 species of vascular plants and harbors the richest alpine flora on earth, with notable richness of endemic species Pedicularis longifloraJuniperus przewalskiiPotentilla fruticosaAconitum gymnandrumPhylogeographical analyses are now widely used to reconstruct the history of species using genealogical data within and between populations Lepisorus clathratus (C. B. Clarke) Ching (Polypodiacae) to explore the phylogeographical history of alpine ferns in the QTP - HHM mountain systems. This species is a common fern in the HHM, stretching eastward to Northern China (Beijing and Inner Mongolia) and Japan, northward to South Siberia (Russian Altai Mountains), and westward along the Indo-Himalaya through Bhutan, Sikkim, Darjeeling, Nepal to West Indo-Himalaya . The species is also one of the most widely distributed plant taxa in the QTP. The species grows in fissures and crevices of natural rock outcrops and shows a preference for alpine habitats, from ca. 2,000 m up to 5,000 m. The taxonomy in this monophyletic complex has been the subject of controversial discussions L. clathratusPlatygyria Ching LepisorusPlatygyria was nested within Lepisorous clathratus. The results are consistent with the interpretation of the morphological evidence by C. Fraser-Jenkins L. clathratus was considered as the correct species name for both specimens with regular and irregular sporangia. Here, we accept the broader concept of Lepisorus clathratus because the single-species concept appears to fit best with the observed data Here, we selected the alpine fern L. clathratus based on a range-wide sampling. Our study aims to address the following questions: (1) does the pattern of cpDNA variation of wide-spread entities in the north-central region relate to the genetic variation found in QTP or HHM regions? (2) Where were the glacial refugia for entities occurring on the QTP during glacitations? Was there a single eastern plateau edge refugium, or did multiple microrefugia exist, possibly including some on the QTP itself? Is there any difference between the phylogeographical pattern in ferns and that in studied angiosperms? (3) Do the sporangial types correlate with spatial genetic differentiation and affect population structure? Indehiscent sporangia restrict the dispersal capacity and thus populations with indehiscent sporangia may show significant different population structures compared with populations with dehiscent sporangia.In this study, we used sequences of chloroplast genome (cpDNA) to investigate the phylogeographical pattern of rps4-trnS and trnL-trnF, we detected 27 haplotypes (H1 - H27), which clustered into five main haplotype groups (G1 – G5). Variable sites among these haplotypes were observed as 29 nucleotide substitutions, of which 17 were parsimony informative.By examining 1,655 bp of the combined dataset including the chloroplast DNA region L. clathratus chloroplast haplotypes are shown as a statistical parsimony network in Relationships between the G1 haplogroup was mostly distributed in SW Sichuan, except for two individuals occurring in NW Yunnan (Shangri-La/Zhongdian) and the QTP (Mozhugongka) respectively . ConversThe frequencies and distributions of each haplogroup and molecular diversity indices in each region are summarized . Region The utilized ecological niche modeling module adequately predicted the distribution range of the species , except ST = 0.36399, P<0.001) as well as between A and C . However, the spatial differentiation was relatively weak between regions AN and AS , but after excluding the wide-spread haplogroup G5 to remove masking effect, we found a more pronounced spatial structure between AN and AS   .FCT began to plateau for four to five groups of populations . Furthermore, the missing/extinct haplotypes in G3 on the network  may be attributed mainly to the intricate topography resulting from the rapid uplift of the QTP L. clathratus along the mountain ranges in the north-central region from the Hengduan Mountains has been achieved through dispersal events (G5 blue haplogroup). Entities in north-central regions all have dehiscent sporangia leading to unimpaired dispersal ability, which accounts for the wide distribution of the haplogroup G5. It is likely that reduction of genetic diversity (bottlenecks) likely occurred along the migration route since only a single haplogroup was detected for the north-central region populations. Reduced allele diversity, together with genetic homogeneity, were expected for populations in the area occupied by interglacial expansion, because it would be much more difficult for those behind the pioneers to advance when the area was swept by the Quaternary glaciations and recolonized rapidly by leading edge populations from the borders of refugia It is very likely that the recolonization of the ST = 0.06787) but with a more marked pattern of population differentiation (FST = 0.13963) after excluding the masking effect of the wide-spread haplogroup G5. Between the two sub-regions, South Hengduan Mountains possesses a higher genetic diversity . Our study reveals the phylogeographical history of alpine ferns occurring in this area for the first time, improving our understanding of phylogeographical patterns of vascular plants in the QTP and HHM.Haplotype H1 and the rare haplotypes H2 – H13 are mostly distributed in the Hengduan Mountains, while the overwhelming majority of individuals with indehiscent sporangia bear H18 and its two derivatives H19 and H20, which are only found on the QTP — the sole exception occurring in the southern Himalaya (Yadong). At the same time, two representatives carrying ancestral haplotype H1 and the rare haplotype H2 were detected on the QTP. Moreover, the AMOVA analysis detected a strong phylogeographical structure between the QTP and the Hengduan Mountains. This evidence implies that the populations with non-shared haplogroup G4 most likely survived on the QTP in situ during the Quaternary glaciations and the low frequent haplotype H1 and H2 may have been the result of long-distance dispersal from individuals in the Hengduan Mountains during postglacial periods. The still existing glaciers between region A and B  might beL. clathratus on the QTP possess lower genetic diversity  haplotypes possess exclusively indehiscent sporangia, which is unusual in ferns. Previous studies L. clathratus. To understand the later pattern, we need to consider that plants with indehiscent sporangia are dominantly intergametophytic selfers, but still have the potential to contribute sperm cells to the reproduction of outcrossing gametophytes that are generated from spores of dehiscent sporangia Entities with indehiscent sporangia are scattered across the haplotype network . This paL. clathratus. This is congruent with the results of a recent phylogeographical study of Juniperus tibetica complex One may argue that the private haplogroup on the QTP could be the result of colonization of haplotypes from southern Himalaya. The result of SAMOVA analysis suggested the South Himalaya as a possible refugium for individuals with uncommon sporangial pattern in Further comparative studies with other regions and other species are required to confirm the history of alpine species occurring in the QTP-HHM. Extensive studies conducted in high-altitude regions will allow us to extract shared phenomena among species, although each species has its own unique history during glacial and interglacial periods This study did not require any special permits because all collecting was performed by researchers located at institutes with the permits required such as IBCAS  in Beijing.L. clathratus, extending from the QTP and the HHM, to northern China and the Altai mountain ranges in Russia and westward to Nepal and Kashmir along the southern slope of the Himalaya. The sampling of these alpine plants was difficult since individuals were scarce and scattered, and usually found on mountain tops or on the damper and cooler faces of steep and high mountain ranges. Our sampling is particularly limited along the southern edge of the Himalaya. Populations usually consist of a few individuals, and suitable areas for colonization are often separated by long distances. The long creeping rhizomes of these ferns may result in the formation of clones as a result of individuals multiplying through fragmentation of the rhizome. Therefore, at each site we collected as many leaves as possible from spatially separated individuals. The leaf material was dried in silica gel and stored at room temperature.A total of 147 individuals  were samL. clathratus, that is, treated the whole complex as one single species Platygyria was nested within the complex and the sporangial dehiscence/indehiscence was considered as an intraspecific trait. Currently, we do not have any evidence for hybridization in the complex. More exhaustive analyses concerning the species delimitation in the complex are currently being performed and will be reported elsewhere. Our sampling includes several segregates including those recognized in the most recent revision of the complex: Lepisorus albterii (Regel) Ching, L. crassipes Ching & Y. X. Lin, L. likiangensis Ching & S. K. Wu, and L. thaipaiensis Ching & S. K. Wu Lepisorus clathratus as suggested by some authors We employed a broad species concept for trnL-F region including the trnL intron + the trnL-trnF intergenic spacer (IGS), and the rps4-trnS region including rps4 + rps4-trnS IGS) were amplified separately with standard polymerase chain reaction (PCR) conditions and with previously published primer sets Total genomic DNA was extracted from silica gel dried leaves using the modified CTAB procedure of Doyle and Doyle Sequences were analyzed using MegaBACETM1000 DNA Analysis Systems, following the manufacture’s protocols. Sequence data were edited and assembled in ContigExpress program from the Vector NTI Suite 6.0 . The resulting sequences were aligned using CLUSTAL X Phylogenetic relationships of the recovered haplotypes were reconstructed using algorithms that create networks assuming the occurrence of ancestral haplotypes, such as minimum spanning trees, as implemented in the software Splitstree 4.10 http://www.worldclim.org). The area of the habitat potentially available for L. clathratus was modeled in the DIVA-GIS 7.1.7.2 To produce distribution maps of the studied species, we used ArcGIS 9.3 To date, there is no consensus regarding the glaciations of the QTP at the LGM. Based on the interpretation by Kuhle In order to investigate genetic variation in different regions, we tentatively divided the sampling into three genetically similar regions according to the distribution of haplotype groups : HengduaAnalyses of molecular variance (AMOVA) FCT) among those groups FCT began to plateau.To test whether the pre-defined regions are consistent with genetically differentiated population groups, we analyzed our data with a spatial analysis of molecular variance (SAMOVA). Populations with only one representative were deleted from this analysis. Geographically close populations were clustered into a user-defined number of groups (K) using a simulated annealing approach to maximize the variance (http://beast.bio.ed.ac.uk/). We initially used several mutation rates reported for land plants L. clathratus clade in an independent divergence time estimation for the whole genus . These analyses were performed with a dataset including a single representative of each haplotype without outgroup taxa. Several models of coalescence analyses were performed, including Bayesian Skyline under step-wise constant population sizes with two to 10 groups and the Extended Bayesian Skyline that does not require the number of groups to be specified, but allows to integration of the information in the inheritance mechanisms of the markers http://tree.bio.ed.ac.uk/software/tracer) and by visualizing the chronograms in Figtree 1.3.1. (http://tree.bio.ed.ac.uk/software/figtree). Bayesfactor analyses carried out using Tracer suggested a scenario of two or three lineages as likely to best fit the observed data. The coalescence analyses provided also estimates of the effective population size through time.The pre-LGM history of the species was inferred by reconstructing the phylogeny of the lineage using maximum likelihood optimization as implemented in GARLI 0.951 Table S1Locality, sample sizes (N), cpDNA haplotypes and its numbers, haplogroup and coordinate of the studied L. clathratus populations.(DOC)Click here for additional data file.Table S2GenBank accession numbers for haplotype sequences included in the phylogeographical analyses.(DOC)Click here for additional data file.


Mental imagery has been associated with many cognitive functions, both high and low-level. Despite recent scientific advances, the contextual and environmental conditions that most affect the mechanisms of visual imagery remain unclear. It has been previously shown that the greater the level of background luminance the weaker the effect of imagery on subsequent perception. However, in these experiments it was unclear whether the luminance was affecting imagery generation or storage of a memory trace. Here, we report that background luminance can attenuate both mental imagery generation and imagery storage during an unrelated cognitive task. However, imagery generation was more sensitive to the degree of luminance. In addition, we show that these findings were not due to differential dark adaptation. These results suggest that afferent visual signals can interfere with both the formation and priming-memory effects associated with visual imagery. It follows that background luminance may be a valuable tool for investigating imagery and its role in various cognitive and sensory processes. It has been proposed that mental imagery is akin to perception in the absence of external stimulation, a product of sensory information retrieved from memory An ongoing debate in the literature is whether mental imagery involves pictorial low-level mechanisms, or whether it is symbolic in a similar vein to language, and thus a more high-level mechanism Pylyshyn (1973) on the other hand, suggested that imagery is not pictorial in nature but rather represented and stored by propositions in the same way as semantic information is represented by its relationship with other concepts. In this scenario the observed similarities between imagery and perception might simply be due to our ability to compose imagery in a way that resembles perception, not because they are inherently similar processes Strong evidence that imagery involves mechanisms in early visual processing comes from an empirical method developed by Pearson, Clifford and Tong (2008) in which imagery was used to bias subsequent binocular rivalry. Binocular rivalry refers to the fluctuations in visual awareness that result from displaying two different patterns, one to each eye. When participants were cued to imagine one of the rivalry patterns, imagery tended to prime subsequent perception of rivalry. The pattern a subject saw during the rivlary presentation tended to be the pattern they had imagined a few seconds earlier. In addition, it was shown that this priming was orientation specific, that is, for this bias to occur, imagery had to be at the same angular orientation as the rivalry patterns. This work sugests that visual imagery involves low-level mechanisms that overlap with visual perception Pearson et al. (2008) also showed that the higher the level of background luminance during the binocular rivalry task, the weaker the effect of imagery on subsequent perception. This suggests that the general level of background luminance can attenuate imagery's effect on subsequent perception. When using binocular rivalry to investigate imagery, to avoid any confounding effects of visual attention on perception, the authors always separated imagery and perception in time. It is well known that visual attention can change the perceived characteristics of a visual stimulus Little is known about the nature of the memory trace formed between imagery and the rivalry task. It is known to last several seconds and storage, these effects were not observed.In the current study we report that background luminance can attenuate both mental imagery generation and storage of the imagery trace. However, imagery generation seems to be more sensitive to luminance, as lower levels of luminance had greater effects on generation compared to the memory trace. In addition, when luminance was presented between experimental trials outside of imagery generation Thirteen undergraduate psychology students completed all conditions of the first two experiments. An additional nine participants were screened who did not meet a criterion of imagery priming above 50% . The criterion was implemented in order to exclude participants with very weak imagery, indicated by a lack of priming in the binocular rivalry task. For this study we were interested in how background luminance would attenuate processes associated with imagery. Hence, we required subjects who displayed a moderate or high level of imagery in the dark no luminance condition. Fourteen participants took part in the third experiment, with four who did not meet the above criterion. Subjects gave informed written consent before participating in the experiment, and received payment for participation in the form of course credit. The local Human Research Ethics Advisory Panel (Psychology) at The University of New South Wales approved this study.2. These oriented patterns had an internal contrast of ∼60% in relation to the average luminance of the stimulus only, as the surrounding background was black. The red grating pattern comprised of a CIE of x = .277, y = 0.613 with a horizontal orientation, presented to the left eye. The green vertical pattern  was presented to the right eye. Both patterns were presented in an annulus around a bullseye fixation spot 0.5° in diameter.The stimuli were red and green sinusoid luminance modulated oriented gratings. The contrast of the stimulus was applied to a Gaussian-windowed mean luminance profile , which had a mean luminance of 4 cd/mIn order to minimize potential eye bias, an initial task was completed to determine the correct balance of stimulus contrasts between the two rivalry patterns. The contrast of the gratings had to be adjusted for each participant in order to account for individual differences in eye dominance and bias. In order to determine these contrasts, the same procedure was used as in previous studies see This eye bias procedure involved each subject being presented with the rivalry patterns, in isolation (just one pattern) and subsequently together undergoing rivalry. Each of the isolated presentations lasted 4 seconds. Not only did this procedure allow us to equalize the pattern strengths, it also provided an opportunity for subjects to familiarize themselves with each of the patterns they would subsequently be required to imagine. In addition, before experimental trials were allowed to continue subjects were shown hard copy color printouts of each grating pattern and verbally asked if they felt familiar enough with each pattern to perform mental imagery of that pattern. Participants were asked to imagine the patterns as best they could and if possible to imagine the color, spatial frequency, size and location in visual space of the pattern.2 reflectance off a white surface) with black walls, on a calibrated (19″ Philip Brilliance 109P4) monitor with a resolution of 1280×960 and a refresh rate of 75 Hz. The experiment was run in MatLab and the Psychophysics toolbox All conditions of the experiment were conducted in a darkened room (0 cd/m2). These conditions were run in blocked sets of experimental trials. The background luminance ramped on and off smoothly over 500 ms to avoid visual transients.In the first experiment participants were randomly cued to imagine one of the binocular rivalry patterns for 10 seconds. “R” was the cue for the red horizontal grating and “G” for the green vertical grating see . During Following imagery generation, participants completed a rapid serial visual presentation task (RSVP) in which random letters were presented on the screen for a five second period, displayed for 300 ms each. Behind each letter was a small Gaussian patch of luminance matching the background in the 100% background luminance condition, this remained the same in all conditions. This was to maintain consistency in the letter task throughout all conditions. Participants were required to press the “C” or “V” key as soon as either letter was presented on the screen. The RSVP task was included as a distracter task to ensure that participants stopped imagining the stimulus at the required time. This task also ensured that any influence on rivalry was not due to an attentional effect as the participants had to apply their attention to the RSVP task. This was lastly followed by a binocular rivalry display for 0.75 seconds. Participants indicated the dominant pattern using 3 different keys, either “1” green, “2” for a mixed percept or “3” for the red pattern. The trials in which participants reported seeing a mixed percept were discarded from the analysis. If a subject reported a mixed percept on more than 20% of trials they were dropped from further analysis.There were at least 60 trials for each level of background luminance for each subject. Each block of 60 trials lasted for ∼20 minutes. In addition, ten practice trials were completed before testing began.The second experiment was otherwise the same, however, the luminance backgrounds were presented during the RSVP letter task, rather than during imagery generation see . A final = 7.2, p = 0.001). The 25, 50 and 100% conditions did not significantly differ from the chance score of 50%, (lowest p = 0.178), but were all significantly different from the dark 0% condition (highest p = 0.01).Experiment 1, examined the effect of different levels of luminance during the imagery generation period.  = 6.698 p = 0.001).In experiment 2 we examined how imagery's ability to prime rivalry was influenced by different levels of background luminance during the storage period in which subjects performed the letter detection task. The results of experiment 2 are shown in p = 0.199), and was significantly different from chance 50% . Suggesting the memory trace of imagery is less sensitive to background luminance as compared to the imagery generation process.Unlike experiment one, here the 25% luminance condition was not significantly different from the dark 0% condition  = .761, p = .523). This data suggests that the effect of luminance in the previous experiments was immediate and local in time.p = 0.26). In addition, there was no significant effect of luminance on the RSVP task scores in either experiment one  or two .There was no significant correlation between the performance on the RSVP letter task and imagery priming , who showed that forming a mental image and storing it in the presence of an illuminated screen results in weaker effects on subsequent perception. Furthermore, imagery generation seems to be more sensitive to luminance than imagery storage, as even the lowest amount of background luminance during imagery generation caused a significant decrease in imagery priming. The lowest level of background luminance, on the other hand, did not significantly disrupt the imagery memory trace. In addition, these results do not appear to be due to the general presence of luminance outside the trial sequence.Various explanations could be proposed to account for the effect of luminance on imagery. As the luminance ramped on slowly and remained constant until the end of the designated time period, it seems unlikely that the presence of luminance simply distracted subjects from performing the imagery task or led to increased perceptual load Both afferent visual signals originating from the illuminated background and the neural response related to imagery can involve activity in early visual areas Experimental participants here, and in previous studies The affects of dynamic visual noise have been used to illustrate commonalities between visual spatial working memory systems and visual imagery The results of the present study may support the pictorial nature of imagery. Luminance was able to disrupt imagery generation, and since luminance and imagery both activate early visual areas, it could be suggested that this interaction is occurring in early visual areas known to process visual stimuli and imagery in a pictorial/retinotopic manner Given that imagery generation is more sensitive than storage to luminance, it is possible that the cognitive process implicated in storage is actually different to that during generation. Therefore, imagery may engage two separate mechanisms, one for imagery generation and another for imagery storage. The nature of this imagery memory trace is yet to be fully explored. However, one possibility is that the memory involved in imagery is similar to perceptual memory, particularly perceptual priming This study shows that visual imagery can be attenuated by afferent visual signals and therefore imagery can be most effectively utilized and examined in dark environments. This has implications for future studies that aim at investigating the nature of imagery and its roll in cognitive functioning. Furthermore, the current data suggest the existence of two partially separable mechanisms associated with imagery, one for generation and one for storage. This memory mechanism for imagery resembles the processes underlying perceptual memory. However, the full extent of any mechanistic overlap is an interesting topic for future studies. We hope that the manipulation of afferent visual signals could be a valuable investigative tool giving traction to future studies of mental imagery.


Resolving threats to widely distributed marine megafauna requires definition of the geographic distributions of both the threats as well as the population unit(s) of interest. In turn, because individual threats can operate on varying spatial scales, their impacts can affect different segments of a population of the same species. Therefore, integration of multiple tools and techniques — including site-based monitoring, genetic analyses, mark-recapture studies and telemetry — can facilitate robust definitions of population segments at multiple biological and spatial scales to address different management and research challenges.To address these issues for marine turtles, we collated all available studies on marine turtle biogeography, including nesting sites, population abundances and trends, population genetics, and satellite telemetry. We georeferenced this information to generate separate layers for nesting sites, genetic stocks, and core distributions of population segments of all marine turtle species. We then spatially integrated this information from fine- to coarse-spatial scales to develop nested envelope models, or Regional Management Units (RMUs), for marine turtles globally.The RMU framework is a solution to the challenge of how to organize marine turtles into units of protection above the level of nesting populations, but below the level of species, within regional entities that might be on independent evolutionary trajectories. Among many potential applications, RMUs provide a framework for identifying data gaps, assessing high diversity areas for multiple species and genetic stocks, and evaluating conservation status of marine turtles. Furthermore, RMUs allow for identification of geographic barriers to gene flow, and can provide valuable guidance to marine spatial planning initiatives that integrate spatial distributions of protected species and human activities. In addition, the RMU framework — including maps and supporting metadata — will be an iterative, user-driven tool made publicly available in an online application for comments, improvements, download and analysis. Geospatial characterization of commercially important or conservation-dependent marine species provides crucial input for resource management in multi-use situations, as is currently described by ecosystem-based marine spatial planning However, resolution of population units for conservation is not always straightforward, and requires clear understanding of conservation objectives, as well as the natural history of – and threats to – the species of interest IUCN Red List of Threatened SpeciesSix of the seven marine turtle species are categorized as Vulnerable, Endangered, or Critically Endangered globally by the Molecular genetic analyses are often used to describe population structures and, by extension, to define MUs Nesting females typically demonstrate philopatry to nesting areas, and both males and females can be philopatric to breeding areas adjacent to a nesting beach Genetic analyses clearly contribute to the resolution of MUs, but are not always sufficient for this purpose; such analyses can indicate that genetic population structure exists, but usually cannot resolve the boundaries of that structure In response to these issues, we compiled, collated, and georeferenced available information on marine turtle biogeography – including individual nesting sites, genetic stocks, and geographic distributions based on monitoring research – to develop multi-scale Regional Management Units (RMUs). These RMUs spatially integrate sufficient information to account for complexities in marine turtle population structures, and thus provide a flexible, dynamic framework for evaluating threats, identifying high diversity areas, highlighting data gaps, and assessing conservation status of marine turtles.The IUCN's Marine Turtle Specialist Group convened the Burning Issues Working Group (MTSG-BI) for two meetings (August 2008 and September 2009) of marine turtle experts from around the world who represented government agencies, non-governmental organizations, and academic institutions. The primary objective of these meetings was to develop a process for evaluating and prioritizing the conservation status of marine turtle populations worldwide. In this context, the MTSG-BI developed Regional Management Units (RMUs) as the framework for this evaluation and prioritization process.http://tinyurl.com/29w4kbf), extracting information on nesting sites, population genetics, tag returns, and satellite telemetry, as well as other relevant natural history and biogeography. We then spatially integrated this information in ArcMap 9.3  from fine (i.e. points with geographic coordinates for nesting sites) to coarse (i.e. polygon shapefiles for distributions) spatial scales. We constructed separate layers according to distinct biological/spatial scales, including a layer for nesting sites , and two layers for genetic stocks . Finally, all finer-scale levels were nested within the known core geographic distributions of these population units, according to satellite telemetry and tag return data. In this way, all nesting sites that were sampled and analyzed for genetic studies were represented within genetic stock layers, and defined genetic stocks were represented within RMU layers, such that information shared across scales was retained in each relevant layer. We also compiled available information on population sizes  and trends  at each spatial/biological scale. Although considerable uncertainty exists in estimates of population sizes and trends for marine turtles among sites globally, we did not attempt to standardize or improve estimates; we urge caution in interpretation of these metadata. This “nested envelope” approach allowed for metadata to be arranged within and across biologically defined spatial scales.To generate RMUs for marine turtles, we collated and georeferenced available data from more than 1,200 papers, reports, abstracts, and other sources (available for download at www.seaturtlestatus.org) database, which relies on a global network of researchers who voluntarily contribute annual nesting data. We augmented the SWOT database with published information. We filtered all nesting sites to distinguish sites with confirmed, quantified nesting activity (i.e. counts) from those without quantified counts since 2000. Across species globally, we compiled more than 4,200 nesting sites, ranging from 30 for Kemp's ridleys (Lepidochelys kempii) to 1,337 for hawksbill turtles (Eretmochelys imbricata) . We acknowledge that this distinction means that RMU designations might not address threats occurring outside RMU boundaries but within the distribution of a species, but RMUs nonetheless encompass known core habitats and life stages. However, the important point is that whereas global distributions simply display broad geographic ranges for each species (including RMUs), the RMUs themselves provide refined spatial guidance for conservation strategies.Specifically, we defined the boundaries of RMU polygons by generating termini directly from published satellite tracks, tag returns, or other data sources. These polygons were then digitized or imported into ArcGIS 9.3 either using the georeference tool in the ArcMap Toolbox, or based on text descriptions. In the absence of sufficient information, boundaries were further refined by MTSG experts. To allow clear distinctions between RMUs and complete global distributions, we also generated global distribution polygons for all species . These gLepidochelys olivacea) , are unique among marine turtles for their polymorphic nesting behavior; i.e. synchronous mass nesting at particular beaches (termed arribadas) and disperse, asynchronous or solitary nesting at the rest of the species' nesting beaches. Therefore, we established separate RMUs for arribadas and solitary nesters in regions where both behaviors occur . Although the geographic boundaries of these RMUs are identical within a region, this dichotomy accurately reflects differences in population abundances and trends between the two behaviors Olive ridley turtles  was available, we developed ‘putative RMUs’ so that no region-species combination was excluded. As with all RMUs, these putative RMUs will require modification as new information becomes available, but in the meantime, they represent obvious research and reporting priorities.Chelonia mydas)  . The RMUhttp://seamap.env.duke.edu/swot). Thus, the applicability of RMUs to marine turtle conservation and research will be user-dependent, relying on collaboration among users in the community to maintain up-to-date, accurate files.Along these lines, because the metadata we compiled is derived from publicly available sources, the RMUs themselves will change with new, refined data. To facilitate iterative improvement of RMUs, we have made all map files , 5, 6, 7An important caveat inherent in the RMUs is that we are largely unable to differentiate between true absences (i.e. a species is not identified or is no longer present in an area despite thorough search effort) and absence due to lack of monitoring or reporting. Clearly, distributions of nesting sites and other types of information are biased by areas of high monitoring effort and reporting  to the particular marine turtle nesting sites and stocks that would be impacted, but also threats across a wider ocean area (e.g. fisheries bycatch) that could impact several nesting stocks and broader population units simultaneously Characterization of heavily-utilized areas for marine turtles at different spatial scales is fundamental for creating effective conservation strategies Likewise, areas of overlap among genetic stocks or geographic distributions, as well as regional variation in population trends at various spatial or biological scales could also be identified using RMUs. Although reproduction areas are relatively discrete geographically and genetically, foraging areas for marine turtles of all life stages host con-specific individuals from multiple stocks and geographic locations With this in mind, one next step for refining RMUs would be to expand on the genetic stocks based on nesting sites by spatially characterizing at-sea mixed-stock foraging or developmental areas Despite the emphasis on genetic resolution of MUs, reliance on population structure derived from such analyses can be inadequate for management Dermochelys coriacea) genetic stocks span thousands of kilometers of coastlines within geographic regions Along these lines, several studies have proposed or identified geographic or environmental barriers to migration of marine turtles among nesting sites or foraging areas that appear to result in significant population (but not necessarily genetic) differentiation. The distance between nesting sites that results in isolated nesting stocks appears to vary within Although barriers to gene flow have not been defined in most cases, these studies illustrate geographic and environmental influences on marine turtle population structures that can be tested within the RMU framework, because nesting sites as well as known genetic stocks are georeferenced. Thus, by applying oceanographic or other physical information as well as relatively simple distance buffering analyses to the RMU files, researchers can test hypotheses about spatial distributions of genetic stocks within and across geographic regions. Resulting distributions of population segments would allow more flexibility in defining MUs than those derived solely from quantitative analyses of genetic differentiation, and would also provide clear objectives for further analyses.Evaluation of species or population status, including population sizes and trends, as well as threats and relevant biological information, is a prerequisite for prioritizing conservation resources. However, because defining the relevant biological or geographic scale at which to conduct assessments and make recommendations is always a fundamental question in these processes, RMUs are a valuable resource to guide how populations of marine turtle species are assessed.IUCN Red List of Threatened Species represents the only globally accepted system for evaluating extinction risk for species, but the IUCN/SSC Marine Turtle Specialist Group (MTSG) has debated the utility and validity of a global classification system for marine turtles, and has advocated for regional assessments  for purposes of evaluating the species' current Threatened listing status on ESA, nine DPSs were defined, with distinct recommendations for each DPS Another example of incorporating population differences in conservation status assessments is the listing process under the U.S. Endangered Species Act (ESA). The ESA provides for designation and listing of Distinct Population Segments (DPS) of a species based on ‘discreteness’ and ‘significance’ of that segment It is important to note that in geographic regions where there are existing systems for effectively identifying marine turtle population segments to which to target conservation efforts, such as well-defined breeding populations in eastern Australia To optimize ecosystem function, especially in areas where multiple activities by multiple nations occur, ecosystem-based marine management approaches should be designed to preserve marine biodiversity, keystone species, and biological connectivity among marine habitats RMUs provide explicit, ecologically based spatial and demographic information about geographic distributions of marine turtle populations that could be integrated with other georeferenced layers, including human activities subject to management . Furthermore, the RMU concept could be applied easily to multi-scale biogeographical characterization of other marine megafauna species with similar life history traits and broad, complex distributions. As with marine turtles, identification of high-use habitats, connectivity between breeding and feeding areas, as well as overlapping distributions of distinct populations is extremely important for designing appropriate management schemes for these species The novel RMU framework synthesizes available biogeographical information on globally distributed and imperiled marine turtles into a multi-scaled, geospatial tool for which we envision numerous pertinent applications for marine turtle conservation and research. For example, the MTSG Burning Issues Working Group is utilizing RMUs as the basis for status assessments in a developing process of global conservation priority setting for marine turtles. The RMU classification system is consistent with endangered species laws in the United States and elsewhere, and could provide the IUCN-MTSG with a way forward for regional evaluations of extinction risk.IUCN Red List, Alliance for Zero Extinction, Conservation ‘Hotspots’), in the case of globally distributed species like marine turtles, regional population segments occupy distinct ecological roles and thus merit conservation attention, because extinction of an RMU would represent the loss of the species' ecological role within an entire region and ecosystems therein Although species are predominantly used as the currency for evaluating and prioritizing conservation efforts  for marine turtles worldwide, including number of nesting sites and genetic stocks contained within each RMU.(DOC)Click here for additional data file.Appendix S1Complete list of SWOT – The State of the World's Sea Turtles data providers.(XLS)Click here for additional data file.Appendix S2Metadata associated with each layer synthesized to generate Regional Management Units.(XLS)Click here for additional data file.