Harpia harpyja) is the largest Neotropical bird of prey and is threatened by human persecution and habitat loss and fragmentation. Current conservation strategies include local education, captive rearing and reintroduction, and protection or creation of trans-national habitat blocks and corridors. Baseline genetic data prior to reintroduction of captive-bred stock is essential for guiding such efforts but has not been gathered previously.The harpy eagle  negative values of Fu and Li's FD and F, and Fu's FS. This expansion, estimated at approximately 60 000 years BP (99 000–36 000 years BP 95% CI), encompasses a transition from a warm and dry time period prior to 50 000 years BP to an interval of maximum precipitation (50 000–36 000 years BP). Notably, this time period precedes the climatic and habitat changes associated with the last glacial maximum. In contrast, a multimodal distribution of haplotypes was observed for Central America suggesting either population equilibrium or a recent decline.We assessed levels of genetic diversity, population structure and demographic history for harpy eagles using samples collected throughout a large portion of their geographic distribution in Central America (n = 32) and South America (n = 31). Based on 417 bp of mitochondrial control region sequence data, relatively high levels of haplotype and nucleotide diversity were estimated for both Central and South America, although haplotype diversity was significantly higher for South America. Historical restriction of gene flow across the Andes  is supported by coalescent analyses, the haplotype network and significant High levels of mitochondrial genetic diversity in combination with genetic differentiation among subgroups within regions and between regions highlight the importance of local population conservation in order to preserve maximal levels of genetic diversity in this species. Evidence of historically restricted female-mediated gene flow is an important consideration for captive-breeding programs. Harpia harpyja) are the largest extant birds of prey in the New World with females, the larger sex, weighing as much as 9.0 kg. They feed on sloths, monkeys and other arboreal mammals as well as large birds, such as guans, curassows, and macaws of lowland rainforests Morphnus guianensis), the two are highly divergent genetically Harpy eagles , ocelots  and margays  than Central America . Total nucleotide diversity was similar between the regions  between Central and South America reflects restriction of gene flow around the northern extreme of the Andean range as seen with other predators in Neotropical forests, including the jaguar STF values among subgroups within South America and among Central and South American subgroups  resulting from an unconstrained analysis recovered two main clades with low support  that did not correspond to geographical origin and a third clade comprised of three Peruvian haplotypes each represented by a single bird . The difference in likelihood scores between the unconstrained and constrained phylogenies was not significant (p>0.10). Our finding of non-reciprocal monophyly for mtDNA lineages based on geography supports current classification of p = 0.063, r = 0.03, p = 0.084) and significant (p<0.05) negative values of Fu and Li's FD and F and Fu's SF , but these statistics are conservative and use little information in the data SF has been shown to be more powerful than mismatch distributions in detecting demographic changes under a variety of conditions including both very recent and older population expansions F nor FD supported expansion within Central America. These two summary statistics use an outgroup sequence to identify recent intraspecific mutations and are less affected by small sample sizes than test statistics based on mismatch distributions or Fu's SFFor Central America, the null hypothesis of population demographic expansion was not rejected based on the mismatch distribution  and falls entirely within the last ice age and more specifically, well before the last glacial maximum (LGM) of 22 000–19 500 BP igrate estimated a smaller long-term female effective population size (Nfe) in Central America  corresponding to female effective population sizes of 9,406  for Central America and 111,787  for South America. These estimates are similar to, but higher than, the total effective population size of female harpy eagles estimated in MDIV . Higher rates of female gene flow from Central America into South America  were estimated by the mtDNA data. With MDIV, estimates of migration were relatively low, 1.08 (0.83–1.33 95% CI) but significantly different from zero .The degree of genetic isolation observed with significant FST values, however reciprocally monophyletic lineages do not correspond to geographical locations in maximum likelihood analyses. Captive breeding programs that wish to breed eagles in a manner consistent with historical patterns of gene flow may want to limit the introduction of South American mitochondrial haplotypes into Central America, although the recovery of three shared mitochondrial haplotypes between these broad regions suggests that total isolation of captive breeding stocks by geographic origin may not be necessary. Furthermore, it would be best to evaluate these trends with nuclear data and additional sampling of individuals before modifying existing captive-breeding and release programs.For species in intermediate stages of divergence it is often difficult to differentiate between the alternative possibilities of contemporary gene flow and recent isolation with retention of ancestral polymorphism due to incomplete lineage sorting. The data presented here are consistent with a pattern of intermediate polyphyly  in southeast Asia where the species has experienced >95% decline in the past fifteen years with a minimal decrease in levels of neutral genetic diversity observed prior to the initiation of a large-scale captive breeding program using birds originating over a wide geographic distribution in Pakistan, India and Nepal Such high levels of genetic diversity were not expected because recent habitat loss and evidence for local extinctions of harpy eagles throughout much of Central and South America Our analyses indicate that recent population declines for harpy eagles have not yet caused a reduction in the levels of mitochondrial genetic diversity below those reported for other Accipitridae populations. Future studies should compare these results with estimates from nuclear DNA , with 30 µl of 100 ng/ml dithiothreitol (DTT) added to the extraction buffer when working with feathers. DNA extraction from museum toe pads was performed as described in Lerner and Mindell We designed four primers to amplify 417 bp of domain I of the mitochondrial control region . PCR ampSamples were grouped by geographic regions see  to test h) and nucleotide diversity (π) using the program Arlequin v. 3.0.1 etwork, v. 4.5.1.0 (available at www.fluxus-engineering.com) with varying genetic distance parameter epsilon , and equal weights for transitions and transversions. The median-joining approach followed by the maximum parsimony (MP) option returns a network that corresponds most closely to the strict consensus of maximum parsimony trees found in phylogenetic analyses The level of genetic diversity within regions and subgroups was estimated by calculating the number of haplotypes, haplotype diversity  we converted the parameter τ, estimated from the mismatch distribution, using the equation τ = 2μtThe demographic history of each region was investigated by comparing the shape of their respective mismatch distributions calculated in Aθ = Nefμ, where Nef is the female effective population size and μ is the divergence rate per locus per year) were estimated with MIGRATE [v. 2.1], θ generated from default settings were used as initial starting points for final runs. Three final runs were conducted to verify convergence upon similar values using the following parameters: 10 short chains of 100 000 steps and two long chains of 20 000 000 steps with sampling every 100 steps and a burnin of 200 000 steps. Likelihood ratio tests were performed in each final run to evaluate the support for symmetric versus asymmetric migration.The migration rate between regions and relative effective population sizes , migration between the regions (M = 2Nefμ), time since divergence (T = t/2Nef where t is the generation time) and time to most recent common ancestor (TMRCA = tμ) were estimated using a Bayesian likelihood approach with the HKY finite sites model in the program MDIV M, θΤ, and T were set to ten. The posterior distribution of T approached but did not reach zero in the upper portion of the distribution, so additional analyses were performed with an upper bound of 20. The posterior distribution for runs with this larger prior remained level rather than converging upon zero, so runs using the smaller prior are reported here. The length of the Markov chain was set to 2.5 million generations with a burnin of 500,000 generations. Posterior distributions for the parameters were plotted and the mode of the posterior distribution was selected as the best estimate with the exception of the parameter T, where the point with the highest likelihood value was used.To evaluate the differing scenarios of recurrent gene flow and ancestral polymorphism we used two coalescent-based methods that simultaneously estimate gene flow and divergence times. Estimates of the female effective population sizes . A mutation rate has not been calibrated for any Accipitridae species, so we used a range of mutation rates originally calculated for the control region in grouse Table S1Sample information for harpy eagles (Harpia harpyja) and one outgroup (Morphnus guianensis) analyzed in this study(0.10 MB DOC)Click here for additional data file.Table S2Primer sequences used for the amplification of the mitochondrial control region in harpy eagles(0.03 MB DOC)Click here for additional data file.


Genetic evaluation models often include genetic groups to account for unequal genetic level of animals with unknown parentage. The definition of phantom parent groups usually includes a time component (e.g. years). Combining several time periods to ensure sufficiently large groups may create problems since all phantom parents in a group are considered contemporaries.To avoid the downside of such distinct classification, a fuzzy logic approach is suggested. A phantom parent can be assigned to several genetic groups, with proportions between zero and one that sum to one. Rules were presented for assigning coefficients to the inverse of the relationship matrix for fuzzy-classified genetic groups. This approach was illustrated with simulated data from ten generations of mass selection. Observations and pedigree records were randomly deleted. Phantom parent groups were defined on the basis of gender and generation number. In one scenario, uncertainty about generation of birth was simulated for some animals with unknown parents. In the distinct classification, one of the two possible generations of birth was randomly chosen to assign phantom parents to genetic groups for animals with simulated uncertainty, whereas the phantom parents were assigned to both possible genetic groups in the fuzzy classification.The empirical prediction error variance (PEV) was somewhat lower for fuzzy-classified genetic groups. The ranking of animals with unknown parents was more correct and less variable across replicates in comparison with distinct genetic groups. In another scenario, each phantom parent was assigned to three groups, one pertaining to its gender, and two pertaining to the first and last generation, with proportion depending on the (true) generation of birth. Due to the lower number of groups, the empirical PEV of breeding values was smaller when genetic groups were fuzzy-classified.Fuzzy-classification provides the potential to describe the genetic level of unknown parents in a more parsimonious and structured manner, and thereby increases the precision of predicted breeding values. Historically, genetic groups have been included in genetic evaluation models to account for selection not described by known genetic relationships. Introduction of animal models, which makes is possible to account for known relationships in the genetic evaluation, reduced the need for genetic groups ,2. HowevAn animal whose parent(s) are unknown can be assigned so-called phantom parents. These phantom parents are assumed to be unrelated, non-inbred and to have a single descendant. Phantom parents themselves are not of interest, but are considered only to facilitate modelling and computations .The strategy for assigning unknown parents to genetic groups should reflect the average genetic level of unknown parents . DiffereExcept for a few examples where genetic groups are clearly distinct, definitions of genetic groups are often based on arbitrary rules. An accurate modelling of the expected breeding values of unknown parents will lead to the creation of many groups, each one with only a few animals. However, the drawbacks of such a strategy are either confounding with other fixed effects in the model  or impreTo improve the adjustment for seasonal effects in the genetic evaluation, Strandberg and Grandinson  have sugThe aim of this study was to develop an algorithm for fuzzy classification of phantom parent groups in an animal model. This approach is illustrated with a simulation, where fuzzy-classified genetic groups are used to handle uncertainty about the time of birth. In addition, the possibility to model a linear time trend in the average genetic level of phantom parents with a small number of genetic groups will be illustrated.The infinitesimal model assumes that an animal's breeding value is the sum of the parent average and a Mendelian sampling deviation that resembles the random process of sampling parental genotypes (e.g. ):ui and Ni are the additive genetic value and Mendelian sampling deviation for animal i, respectively, and s and d are the sire and dam of animal i. In matrix representation:where where:ub = vector of additive genetic effects for phantom parents;u = vector of additive genetic effects for known animals;Pb P] = matrix that relates parents to progeny; each row contains two non-zero elements (0.5) in the columns pertaining to the sire and dam;, and D-1 = var(φ). For animals with known parents, each row of H contains three non-zero elements: a one and two negative halves. For base animals with phantom parents, each row of H usually contains three or more non-zero elements: a 1 and several negative values corresponding to the phantom parents' groups. These elements have a value of -0.5pij, where pij is the proportion of phantom parents of i assigned to group j. This structure leads directly to an algorithm for forming A* that is a simple extension of Henderson's A-1 algorithm. Let si (dk) be the number of groups the phantom sire (dam) of i is assigned to, or 1 if the sire (dam) of i is known; mj (fk) the equation number of the animal's sire (dam) or its group if the sire (dam) is unknown, and x = where A* resemble those for forming the inverse of an average numerator relationship matrix in case there are a finite number of potential parents .A* are in Table A*, for animal B and C 16,For the example pedigree Figure , the conA* areand for animal E 25. For animal C there are 16 contributions, but only 9 elements of A* can become very small if phantom parents are assigned to genetic groups with low proportions, for example 0.04 to element  for animal B.affected because the phantom parents of animal C are (in part) assigned to the same group. Observe that the values added to A* for this example is:The complete A population subject to mass selection was simulated for 10 non-overlapping generations, subsequent to a base population of unrelated and non-inbred animals (generation 0). Each generation, 50 males and 200 females were randomly mated. Each mating produced two offspring, one of each gender.For each animal a phenotypic record was simulated as the sum of an overall mean, the animal's breeding value and a random residual. An animal's breeding value was generated as the sum of the parent average and a Mendelian sampling deviation that considered inbreeding of the sire and dam. Genetic and residual variances were both 10, yielding a heritability of 0.50.For each replicate, a "real life", incomplete data set for genetic evaluation was created by randomly deleting data. When data were deleted, both phenotypic records and relationship were deleted. The probability of deletion decreased linearly with increasing generation number, and ranged between 0.30 (generation 10) and 0.70 (generation 0).-10.The model for genetic evaluation included an overall mean, random animal effect and genetic groups for phantom parents. The simulated variance components were used to predict breeding values. Solutions to the mixed model equations were obtained using the preconditioned gradient algorithm, which was assumed to be converged when the relative average difference between the right and left hand sides was smaller than 10Fuzzy classification was compared with distinct classification of genetic groups in two situations: 1) to handle the uncertainty about which group a phantom parent should be assigned to, and 2) to model the average genetic level of unknown parents with a small number of parameters.For the genetic evaluation and forming of genetic groups, uncertainty about the generation of birth was simulated for 25% of the animals with at least one unknown parent. The uncertainty was such that unknown parents could belong to two possible generations: the true generation of birth and the generation prior to that, each with equal probability (0.50). Phantom parents were grouped based on gender and generation number, which resulted in 20 different genetic groups. In the distinct classification, for animals with simulated uncertainty, phantom parents were randomly assigned to just one genetic group, either for the true generation of birth or the generation prior to that, with equal probability. In the fuzzy classification, these phantom parents were assigned to two genetic groups, for both possible generations, with equal proportions (0.50).The simulation was repeated 50 times. For each replicate the empirical mean and variance of prediction errors  were computed within generation for animals with simulated uncertainty. In addition, the rankings on predicted breeding values for distinct and fuzzy classification were compared with the rankings on true breeding values. Animals were grouped in deciles based on the predicted and true breeding value, and the percentage of animals classified in the correct decile was determined for animals with and without simulated uncertainty and for animals with both parents known.Q matrix is not of full rank, meaning that only 3 degrees of freedom are used to model the genetic groups.Two strategies for assigning phantom parents to genetic groups were compared: distinct and fuzzy classification. In the distinct classification, phantom parents were grouped on the basis of gender and generation number, which resulted in 20 different genetic groups. The average number of animals per genetic group was 107 and group size ranged between 59 and 146. In the fuzzy classification there were four groups: two groups for the parent's gender  and two groups to describe the average genetic level of parents of generation 1 animals and one for parents of generation 10 animals. Phantom parents of animals in intermediate generations were assigned to both groups, with proportions depending on generation number. For example, a phantom sire of an animal born in generation two was assigned with 50% and 0% to the male and female parent genetic group and with 45% to the generation 1 genetic group and with 5% to the generation 10 genetic group, for a phantom dam of an animal born in generation three the proportions were 0%, 50, 40% and 10%, respectively, etc. This way, the sum of proportions was always equal to 1. The resulting The simulation was repeated 50 times. For each replicate, the empirical mean and variance of prediction errors  were computed within generations, separately for animals with 0, 1 or 2 unknown parents. The across-replicate standard deviation of estimates for each genetic group  was also inspected as an indication of the SE of genetic group solutions. In addition, the across-replicate correlation between genetic group estimates was computed as an indication of sampling correlation of genetic group solutions.The empirical prediction error variance was 1.6% lower (P < 0.05) for animals with simulated uncertainty when genetic groups were fuzzy-classified. For animals with unknown parent groups and for which the generation of birth was accurately known there was no difference in empirical prediction error variance between both grouping strategies. For animals with simulated uncertainty, the percentage of correctly ranked animals was higher  lower when genetic groups were fuzzy-classified for animals with one unknown parent Figure . The impThe correlation between solutions for genetic groups for phantom sires and dams (for the same generation) was moderately negative (~-0.3) for the distinct classification of genetic groups, but slightly positive (~0.1) when groups were fuzzy-classified (results not shown). This result may explain why the prediction error variance differed between alternatives for animals with one phantom parent, but not for animals with two phantom parents. For an animal with two unknown parents, overestimation of the solution for the genetic group effect for one parent can be compensated by an underestimation of the solution for the genetic group effect for the other parent. When only one parent is unknown such compensation does not occur, and a more precise estimation of the genetic group effects, as in the fuzzy-classified alternative, is favourable.The rules for building the numerator relationship matrix in the case of fuzzy-classified groups are similar to those for building an average numerator relationship for cases where parenthood is uncertain, but limited to a small number of possible parents . InclusMembership functions should follow the pattern of uncertainty and accurately describe the average genetic level of phantom parents as much as possible. However, consideration should also be given to the precision and the estimability of genetic group effects when determining the proportions with which phantom parents are assigned to genetic groups. For example, very small values are added to the diagonal elements of the mixed model equations for a genetic group to which phantom parents are assigned with small proportions, resulting in a high standard error for that genetic group. Also, to avoid confounding among genetic groups it is necessary to have several combinations of different genetic groups and for the same combination of genetic groups to use several different sets of membership proportions. Therefore, in the evaluation of fuzzy-classified genetic groups it is important to carefully examine the precision of genetic group estimates and possible confounding of genetic groups.A linear trend in the average genetic level of phantom parents was modelled with a low number of parameters (groups) by fuzzy-classification in the simulation. More complex modelling of the average genetic level of phantom parents is possible by means of the fuzzy-classification approach. For example, other factors, like origin and selection path, could be incorporated in a similar way.The author declares that they have no competing interests.WFF conceived and designed the study, programmed and carried out the computer simulations, analyzed and interpreted the results, and wrote the manuscript.


Introductions of non-native tiger salamanders into the range of California tiger salamanders have provided a rare opportunity to study the early stages of secondary contact and hybridization. We produced first- and second-generation hybrid salamanders in the lab and measured viability among these early-generation hybrid crosses to determine the strength of the initial barrier to gene exchange. We also created contemporary-generation hybrids in the lab and evaluated the extent to which selection has affected fitness over approximately 20 generations of admixture. Additionally, we examined the inheritance of quantitative phenotypic variation to better understand how evolution has progressed since secondary contact.We found significant variation in the fitness of hybrids, with non-native backcrosses experiencing the highest survival and F2 hybrids the lowest. Contemporary-generation hybrids had similar survival to that of F2 families, contrary to our expectation that 20 generations of selection in the wild would eliminate unfit genotypes and increase survival. Hybrid survival clearly exhibited effects of epistasis, whereas size and growth showed mostly additive genetic variance, and time to metamorphosis showed substantial dominance.Based on first- and second- generation cross types, our results suggest that the initial barrier to gene flow between these two species was relatively weak, and subsequent evolution has been generally slow. The persistence of low-viability recombinant hybrid genotypes in some contemporary populations illustrates that while hybridization can provide a potent source of genetic variation upon which natural selection can act, the sorting of fit from unfit gene combinations might be inefficient in highly admixed populations. Spatio-temporal fluctuation in selection or complex genetics has perhaps stalled adaptive evolution in this system despite selection for admixed genotypes within generations. Whether natural or anthropogenic in origin, zones of secondary contact provide a powerful test of the compatibility of previously allopatric species, and lend insight into the mechanisms responsible for reproductive isolation and speciation. When reproductive isolation is incomplete and secondary contact results in the formation of a hybrid zone, the resulting dynamics also give us a glimpse into the genetics underlying phenotypic divergence and the evolution of reproductive isolating mechanisms. On those rare occasions when we observe the early generations of contact, we can also examine ecological and genetic changes as they unfold, providing a view of short-term dynamics that is not available in most well-established, natural hybrid zones.Hybridization between two species has many potential evolutionary consequences . At oneIn either natural or human-mediated hybrid zones, exceptionally fit individuals have the potential to establish new evolutionary lineages, either displacing one or both parental lineages or diverging into a new ecological zone ,11-13. TThe mode of gene action underlying particular phenotypes also plays an important role in the potential of hybrid populations to maintain genetic diversity and respond to selection . PhenotyFor decades, secondary contact between species mediated by incidental human transport or deliberate introduction has been recognized as potentially detrimental to native species -23. Whenprior to secondary contact ), and Tmet and growth were square-root-transformed As described above for the linear regressions, we performed the joint scaling test of the typical quantitative genetic series starting with the additive effect only and added dominance and epistasis terms up to the full second order polynomial in Eq. 3. For each survival model fit, we used restricted maximum likelihood to account for family membership as a random effect. We also tested for differences in survival among line crosses with pairwise Fisher's exact tests with a sequential Bonferroni adjustment.th generation hybrids have greater mean fitness than 1st and 2nd generation hybrids with the same admixture proportions, we compared our results from contemporary hybrid families to weighted averages corresponding to the expected frequencies of each line cross in the initial generations of contact. Our most recent estimate of the admixture proportion in the contemporary Pond H population based on 64 independent genetic markers is 62.1% introduced [To investigate whether contemporary, 20troduced . Therefotroduced .JRJ participated in the design of the study, collected and analyzed the data, and drafted the manuscript. BMF participated in the conception and design of the study, data analysis, and manuscript preparation. HBS participated in the conception and design of the study and manuscript preparation. All authors read and approved the final manuscript.Line-cross Mass plot. Observed mean Mass and standard errors for each line cross type plotted against expected values under alternative quantitative genetic modelsClick here for fileLine-cross Snout-Vent Length plot. Observed mean SVL and standard errors for each line cross type plotted against expected values under alternative quantitative genetic modelsClick here for fileLine-cross Growth plot. Observed mean Growth and standard errors for each line cross type plotted against expected values under alternative quantitative genetic modelsClick here for fileLine-cross Time-to-Metamorphosis plot. Observed mean Tmet and standard errors for each line cross type plotted against expected values under alternative quantitative genetic modelsClick here for fileLine-cross photographs. Images of a typical metamorphosed individual from each source index categoryClick here for file


Nsp), which facilitated the host shift of Pieridae butterflies onto Brassicales host plants ~80 Myr ago.A molecular population genetics understanding is central to the study of ecological and evolutionary functional genomics. Population genetics identifies genetic variation and its distribution within and among populations, it reveals the demographic history of the populations studied, and can provide indirect insights into historical selection dynamics. Here we use this approach to examine the demographic and selective dynamics acting of a candidate gene involved in plant-insect interactions. Previous work documents the macroevolutionary and historical ecological importance of the nitrile-specifier protein . Nsp exhibits unexpectedly high amounts of amino acid polymorphism, unequally distributed across the gene. The vast majority of genetic variation exists within populations, with little to no genetic differentiation among four populations on two continents. A comparison of synonymous and nonsynonymous substitutions in 70 randomly chosen genes among P. rapae and its close relative Pieris brassicae (Large Cabbage White) finds Nsp to have a significantly relaxed functional constraint compared to housekeeping genes. We find strong evidence for a recent population expansion and no role for strong purifying or directional selection upon the Nsp gene.Here we assess the microevolutionary dynamics of the Nsp gene in P. rapae are dominated by recent population expansion and variation in functional constraint across the repeated domains of the Nsp gene. While the high amounts of amino acid diversity suggest there may be significant functional differences among allelic variants segregating within populations, indirect tests of selection could not conclusively identify a signature of historical selection. The importance of using this information for planning future studies of potential performance and fitness consequences of the observed variation is discussed.The microevolutionary dynamics of the  Studying plant-insect interactions provides an opportunity to investigate the coevolution of species on a molecular, ecological, and evolutionary level. While ecologists are interested in the overall dynamics and interactions between plants and their insect herbivores, biochemical and molecular level studies focus on the genes and gene products that actually interact between these species groups . EcologiNsp) )Nsp protein will be necessary in order to understand the observed excess amino acid variation. In sum, while regions of relaxed constraint certainly harbor more variation, this does not mean such variation is neutral. Indeed, much of the known amino acid variation affecting the kinetics of metabolic enzymes is located upon the surface of enzymes .-64.Nsp pNsp gene appear to be a mixture of demographic effects (population expansion and high migration) coupled with variation in functional constraint across the gene. Patterns of nucleotide diversity and indirect molecular tests for historical selection reject strong local adaptation, as well as directional and strong purifying selection. Rather than taking the absence of clear signatures of historical selection upon the Nsp gene as conclusive evidence for no fitness variation among alleles, we recognize the limitation of such indirect approaches and remain curious as to the functional effects of the extremely high amount of amino acid diversity. Thus, this study provides a foundation for the design and insightful use of molecular markers for genetic variants whose ecological performance and fitness can be characterized in the field (e.g. [The microevolutionary dynamics at the ld (e.g. ).P. rapae. If this allelic variation has functional consequences, the effects are likely to be environmentally dependent and potentially small. As such, future studies will need very large sample sizes for many families across a range of potentially relevant conditions. Families can be sampled from the field as ovipositing females and will contain sufficient diversity for study. In addition, individuals will need to be sampled during larval stages in order to provide access to the cDNA of the Nsp gene, as the entire coding sequence must be sequenced for no single polymorphic site will suffice to characterize Nsp allelic variation. Only with functional study of the identified genetic variation can we begin to conclusively assess the extent to which the observed variation at Nsp plays an ongoing role in the microevolutionary dynamics of P. rapae and its interaction with the highly variable chemical defense system of their Brassicales hostplants.We now know that there is an exceptional amount of amino acid variation within nearly every population of HH-F carried out the molecular laboratory work, with the help of HV. HH-F and CW analyzed the raw data and performed the statistical analyses, CW conducted the ABC analysis. HH-F and CW designed the study and wrote the manuscript. HV and DH participated in its design and helped draft the manuscript. All authors participated in the writing and approved the final version.Additional Figures and Tables. Figure S1: Comparison of synonymous and nonsynonymous site changes in the NSP domains; Table S1: Summary statistics for molecular tests of selection; Table S2: Summary statistics for MK test; Tree file from GARD output; Pieris species cDNA comparison datatable for dN/dS analysis.Click here for file


Anolis sagrei) was interrupted by rat invasion. An island population that was devastated by rats recovered numerically following rat extermination. However, population genetic analyses at six microsatellite loci suggested a possible loss of genetic diversity due to invasion when compared to an uninvaded island studied over the same time frame. Our results provide partial support for the hypothesis that invasive predators can impact the genetic diversity of resident island populations.Invasive species can dramatically impact natural populations, especially those living on islands. Though numerous examples illustrate the ecological impact of invasive predators, no study has examined the genetic consequences for native populations subject to invasion. Here we capitalize on a natural experiment in which a long-term study of the brown anole lizard ( Rattus spp). Invasive rats have colonized more than 80% of the world's islands, and their extremely catholic diets impact plants, invertebrates, mammals, birds, and reptiles Invasive species are generally recognized as one of the most pervasive threats to natural populations Whereas the ecological impact of invasive species, including rats, has been well documented residents has hitherto gone untested. In part, this may reflect the obvious ethical impediments to performing experimental manipulations with invasive species in nature. Here we capitalize on a natural experiment Although a growing number of studies have demonstrated the genetic consequences for exotic species following invasion 2) islands in The Bahamas since 2005 Mimus polyglottos, and green herons, Butroides striatus). The islands used in our studies are located within 2 km of each other and are dominated by a mixture of small trees and native shrubs  that make up the majority of perching habitat used by anoles.All research carried out in this study was approved and carried out under IACUC protocol 07-02-03. We have studied natural selection on small  of tail tissue from each individual for genetic analyses, and introduce propagules of ca. 200 lizards to each of several offshore islands which we have previously denuded of resident lizards. Each fall, ca. 4 months following introduction, we recapture all surviving lizards  and remove them from the islands. During 2008, our fall census on one experimental island turned up a single surviving male lizard, and inspection of the island's vegetation showed clear signs of herbivory by rodents. Two independent observations of rats on the island (RC and RMC) confirmed invasion. Following our fall censuses, we exterminated the rats using standard poisoning protocols Each spring, we capture The spring following rat extermination, the lizard population on the invaded island showed signs of initial recovery, presumably from eggs laid prior to rat predation. We captured all (N = 13) lizards present on this island and sampled an equal number of lizards from a second study island that was not affected by rat invasion and which therefore served as a control.We genotyped tissue samples from both islands using a library of six polymorphic microsatellite markers  and amplification conditions modified from A. sagrei, we used approximate Bayesian computation .1,10 = 4.58 P = 0.058; 1,10 = 10.72 P = 0.008; As predicted, the lizard population that had been invaded by rats showed reduced levels of allelic richness compared to our control population, though this result was not significant probably owing in part to low statistical power . We performed 10 µL PCR reactions with 1 µL template DNA, 1X GeneAmp PCR Buffer II (Applied Biosystems), 1.5 or 2.0 mM MgCl2 (see below), 0.4 mM dNTPs, 0.25 µM of each primer (forward and reverse), and 0.3 U of Taq polymerase. PCR cycles consisted of an initial denaturation step at 94°C for 5 min followed by 29 or 35 cycles (below) of 45 sec at 94°C, 1 min at primer-specific annealing temperatures , and 1 min at 72°C, followed by a final extension for 5 min at 72°C. All PCRs were performed on a DNAEngine Thermal Cycler (Bio Rad).(0.04 MB DOC)Click here for additional data file.


Neosalanx taihuensis  is an economically important fish, which is endemic to China, restricted to large freshwater systems  and typically exhibit low vagility. The continuous distribution ranges from the temperate region of the Huai and Yellow River basins to the subtropical region of the Pearl River basin. This wide ranging distribution makes the species an ideal model for the study of palaeoclimatic effects on population genetic structure and phylogeography. Here, we aim to analyze population genetic differentiation within and between river basins and demographic history in order to understand how this species responded to severe climatic oscillations, decline of the sea levels during the Pleistocene ice ages and tectonic activity.The Salangid icefish b sequences (1141 bp) of 354 individuals from 13 populations in the Pearl River, the Yangze River and the Huai River basin. Thirty-six haplotypes were detected. Haplotype frequency distributions were strongly skewed, with most haplotypes (n = 24) represented only in single samples each and thus restricted to a single population. The most common haplotype (H36) was found in 49.15% of all individuals. Analysis of molecular variance (AMOVA) revealed a random pattern in the distribution of genetic diversity, which is inconsistent with contemporary hydrological structure. Significant levels of genetic subdivision were detected among populations within basins rather than between the three basins. Demographic analysis revealed that the population size in the Pearl River basin has remained relatively constant whereas the populations in the Yangze River and the Huai River basins expanded about 221 and 190 kyr ago, respectively, with the majority of mutations occurring after the last glacial maximum (LGM).We obtained the complete mtDNA cytochrome N. taihuensis is coherent with a scenario of multiple unrelated founding events by long-distance colonization and dispersal combined with contiguous population expansion and locally restricted gene flow. We also found that this species was likely severely impacted by past glaciations. More favourable climate and the formation of large suitable habitations together facilitated population expansion after the late Quaternary . We proposed that all populations should be managed and conserved separately, especially for habitat protection.The observed complex genetic pattern of  Population genetic structure is dependent on the interaction of the biology of a species and the environment in which it resides. Marine organisms generally show low levels of genetic differentiation over large geographic distances ,2. In opDue to periodic climatic oscillations during the Pleistocene, range contractions, range expansions and changes in interconnectivity within and between drainages during the periodic climatic oscillations of the Pleistocene have greatly influenced the distribution of many fish species, have significantly altered the amount and distribution of intraspecific genetic variation in many species and have remodelled the population structures of temperate fish faunas ,5. The mNeosalanx taihuensis, is one of the most economically important species in this Family. This species is endemic to China and is restricted to large bodied freshwater systems such as inland lakes, out-flowing rivers and estuaries, covering a continuous distribution from the temperate (Yellow River basin) to the subtropical zone (Pearl River basin) [N. taihuensis has undergone the lost of appropriate habitat and the decrease in population size [N. taihuensis, the habitat detection has to be implemented immediately, and the study on the N. taihuensis population structure and its phylogeoraphic pattern should be enhanced since the failure to detect population units will lead to local overfishing and ultimately to severe declines [Opsariichthys bidens  and Brachymystax lenok Pallas , both had genetic patterns coinciding with contemporary drainage structure [N. taihuensis is not conducted till now.The Family Salangidae, which is classified as Protacanthopterygii, Order Osmeriformes, Suborder Osmeroidei ,18, compr basin) . Also, tr basin) ,22. The r basin) . In receion size ,25. In odeclines . The Saldeclines ,28, bioldeclines , and moldeclines . Previoutructure , but theN. taihuensis mainly distributes, are important river systems in China. Although a strong uplifting of the Tibetan plateau occurred during the Pleistocene in southwest China [N. taihuensis to waters with some salt content, members of freshwater fish division cannot disperse by river connections [N. taihuensis. In addition, the glaciations during the Pleistocene, especially the last glacial maximum (LGM) with sea levels lowered (120–140 m below present sea level), should have severely affected the genetic structure of this species. In order to detect the population structure and phylogeographic pattern of N. taihuensis at large geographical scales, samples of N. taihuensis were collected from 13 populations among the Pearl River basin, the Yangtze River basin, and the Huai River basin  sequence was used to analyze population genetic differentiation of this species, population dynamics and demographic history, and to show how the glaciations influenced the phylogeographic patterns of freshwater fish, and how this species responded to the severe climatic oscillations and decline of the sea levels during the Pleistocene ice ages.The basins of Yangtze River, Huai River and Pearl River, in which st China , there ist China . The Yanst China . Given tnections . Therefocyt b gene analyzed in 354 individuals, there were 38 variable nucleotide sites, and no insertions or deletions in any of the sequences. The average nucleotide composition for all individuals was highest for C (0.334), followed by T (0.277), A (0.214), and G (0.175). Nucleotide composition showed an anti-G bias (G = 17.5%), which is characteristic for the mitochondrial genome [cytb sequence was translated into amino acid sequences, and based on the criteria of Zhang & Hewitt [cyt b sequences in N. taihuensis, which indicated that there was no signs of recombination (P = 0.925) and the neutral hypothesis (no selection) could not be rejected (P = 0.172) for these cyt b sequences. Thus, the cyt b was suitable marker for analyzing the evolutionary history of this species.For 1141 bp of the complete & Hewitt , we founThirty-six different haplotypes were identified in the 354 samples analyzed Table . The numh and π , are summarized in Table h was 0.713 ± 0.022 ranging from 0.473 ± 0.057 to 0.781 ± 0.017, and π was 0.0022 ± 0.0001 ranging from 0.0010 ± 0.0002 to 0.0027 ± 0.0001 among there basins. There are no evidences that heterogeneous levels of genetic variability were produced by unequal sample size for each population through Pearson's correlation test between diversity indices and sample size . These results showed a medium/high haplotype diversity and a low nucleotide diversity, and that the genetic diversity for three near-coastal populations  were higher than others (Table Diversity indices (average ± standard deviation), N. taihuensis.Unrooted ML tree revealed that there were no distinct haplotype groups with high bootstrap support (<60 %) and there was no geographical structure among haplotypes Figure. , which wΦST test among 13 populations showed that the differences among the most of populations were irrelevant to their distributions . Clade 1–2, clade 1–4, and two second cladistic levels of clade 2-1, clade 2-2 indicated restricted gene flow with isolation by distance, and the overall picture of NCA indicated historical contiguous range expansion.The NCA analysis  for these populations, suggesting that there were no historical reduction in effective population size in these regions. The mtDNA mismatch analyses for the entire region and the Yangze River basin showed bimodal profile which might result from constant population size among an old population or an admixture population  and Ramos-Onsins and Rozas's R2 (P < 0.05) test, as well as high growth rate (g = 2083.14 ± 529.35) confirmed population growth in Huai River basin, which was also supported by the test of Hri (P > 0.95), although the bimodal mismatch distribution  indicated a poor fit for the stepwise growth model (Table SSD (P < 0.05) values indicated a relative constant population size. Fu and Li's D* test, Fu's Fs and R2 test for Pearl River basin were not significant, which also rejected population expansion/bottleneck model , the estimated expansion time was found to be 245.5 kyr ago for the entire region and 221.9 kyr ago for the Yangze River basin. Using the coalescent method, an estimate of TMRCA was 190.0 kyr for this species in the Huai River basin and 73.53 kyr in the Pearl River basin, respectively  and the mutation rates for the N. taihuensis within the Yangze River basin and the Huai River basin, GENETREE [To get a more detailed picture of the mutational pattern for population expansion of GENETREE  was usedGENETREE , but theπ = 0.0022 ± 0.0001) and medium/high haplotype diversity (h = 0.713 ± 0.022) in N. taihuensis  and individuals from three basins did not partition into distinct clades Figure. , which iN. taihuensis. Our results were in contrast to the 'Stream Hierarchy Model' [Opsariichthys bidens  conducted in the Pearl River and the Yangze River in China [Beyond the prediction that vicariance among basins should play an important role in shaping population genetic structure, the results of AMOVA showed that the contemporary drainage structure served as a poor model in explaining the high levels of genetic differentiation, with only 8.43% variance among basins, it was suggested that there must be other factors shaping the genetic structure of y Model' , which sy Model' ,9,49,50,in China ,11,16 haΦST and AMOVA analysis confirmed the existence of some degree of restricted gene flow, which could be one of the important factors determining the genetic structure among populations of N. taihuensis. However, the Mantel test indicated no significant relationship between genetic differentiation and geographic distance among populations. The discrepancy between these results indicated that the pattern of genetic differentiation for N. taihuensis was complex and could not be interpreted simply using the IBD model.Pair-wise comparisons between Dn and small Dc suggests long distance dispersal [Dc and large Dn, were indicative of range expansion and long-distance colonization (and/or past fragmentation) [In contrast, NCA analysis revealed that multiple unrelated long-distance founding events could be important in determining the patterns of genetic diversity. In NCA, the large ispersal . The NCAntation) . The obsntation) . Also, tntation) . In addintation) ,16. Althntation) , there intation) . It may Fs and R2 over other statistics [R2 test is more powerful for a small sample while Fs is more powerful for a large sample [Fs tests were significant for the entire region, Yangze River basin and Huai River basin. However, R2 tests were significant for the entire region and Huai River basin, but not significant for Yangze River basin  using FLUCTUATE 1.4 [However, the methods of mismatch distribution analysis and neutrality test did not make full use of the data . The coaUATE 1.4  and the UATE 1.4 , which pN. taihuensis was almost completely exposed and eradicated, and this species should have been severely impacted by the past glaciations. Our estimate of TMRCA in the Pearl River and the Huai River basin, and the population expansion time for N. taihuensis in the Yangze River basin were much older than that of LGM. It was difficult to link the population demographic history to any particular Pleistocene paleo-climatic event. However, the gene tree  [h) and nucleotide diversity (π) , with sea levels lowered (120–140 m below present sea level) [ Figure.  providedthe LGM) . Furtherer study ,25. No ser study . This inion time , which aistocene . There wNeosalanx taihuensis, is one of the most important commercial species in this family. The dry or frozen salangids are good materials in Chinese food, and they are also exported to Japan and other southern-east Asia countries. But, it has to be noticed that the population size of N. taihuensis and its fishing yield have decreased rapidly in recent years [5 kg in 1960s to 104 kg in 1987 [N. taihuensis was completely collapsed in Poyanghu lake thereafter [3 kg occurred in 1990 [et al. suggested that over-fishing was not the most important threat to this species [N. taihuensis [N. taihuensis has been polluted because of the waste water from urban, industry and agriculture [N. taihuensis is also from the habitat fragmentation. Since mid 1950s, the aquatic networks have been fragmented dramatically with the construction of dykes and floodgates across the river-lake connections [N. taihuensis, so it is urgent to make an effective plan to protect this economically important species.The Salangid, nt years ,25. For  in 1987 , and theereafter . The yie in 1990 . Accordi in 1990 , but You species . On the ihuensis ,66. The nections . Up to tN. taihuensis, suggesting that there was not a genetically distinct and independent population that could be considered as an ESU. However, AMOVA analysis indicated that significant genetic variance resulted from genetic differences among populations and within basins. In addition, based on low vagility, geographic isolation among most populations and rapidly population decline over the recent decades for this species [The study on population structure and its phylogeoraphic pattern could provide particularly information for us to more broadly consider conservation options and more accurately assign appropriate fishery management . Accordi species ,25,63, wN. taihuensis, but populations in the Yangze River basin and the Huai River basin expanded about 221.9 and 190.0 kyr ago, respectively, with the majority of mutations occurring after the LGM. We concluded that the more favourable climate and the formation of appropriate large-scale habitations facilitated this expansion. We suggested that it was necessary to develop a suitable management plan for N. taihuensis based on our results.Our results showed that this species presented a very random genetic pattern and was not compatible with the present hydrological structure. This genetic pattern might be formed mainly by multiple unrelated long distance founding events, combining with population expansion and the instream barriers restricting gene flow. Demographic analysis revealed that the populations in the Pearl River basin (TMRCA approximately 73.53 kyr) has kept a relatively constant population size of N. taihuensis from 13 populations in the Pearl River, Yangtze River, and Huai River basins from March in 2004 to September in 2005, 44 came from two populations of the Pearl River basin in 2005 (P1–P2), 196 from seven populations of the Yangtze River basin in 2004 (Y1–Y7), and 114 from four populations of the Huai River basin in 2005 (H1–H4)  and L15634 (5'-CTTAGCTTTGGGAGTTAAGGGT-3') in a 50 μl reaction volume mixture containing 25 μl Premix Taq , 1.0 μM of each primer, approximately 50–100 ng total genomic DNA template and 18–22 μl of sterile distilled water. A PE 9700 Thermal Cycler (Applied Biosystems) was used for the following cycles: pre-denaturation at 94°C for 5 min; 30 cycles of denaturation at 94°C for 30 s, annealing at 54°C for 45 s, and extension at 72°C for 1 min 10 s, plus a final extension at 72°C for 10 min. Purified PCR products were directly sequenced using primers L14321 and L15634 from both ends using the Big Dye Terminator Sequencing Kit  in a semi-automated DNA analyzer . To avoid the errors in amplification and sequencing, all singletons among polymorphism sites were verified by an additional amplification and sequencing. Sequences were aligned using the Clustal X program [cytb haplotype sequences of N. taihuensis were deposited in GenBank (EU376454–EU376489).Total DNA was extracted from the muscle tissue using standard phenol/chloroform procedures  or folloet al. . PCR was [et al. . 1141 bacyt b gene, linkage disequilibrium (LD) test was conducted using DnaSP 4.10 [cyt b was conducted in MEGA4.0 [N = dS [S and dN is the average number of synonymous substitutions per synonymous site and the average number of nonsynonymous substitutions per nonsynonymous site, respectively.In order to test whether there is recombination within aSP 4.10  and the aSP 4.10 , which c MEGA4.0  to check [N = dS ,77, wherh) and nucleotide diversity (π) [H), polymorphic sites (S) and the mean numbers of pair-wise differences among sequences (K) were also estimated. These diversity indices were computed using the software DnaSP 4.10 [H), haplotype diversity (h) and nucleotide diversity (π) against the number of individuals (N). Population structure and genetic variation of N. taihuensis were characterized and compared with Arlequin version 3.11 [Φ-statistics [ΦCT, the degree of differentiation among all basins, ΦSC, the degree of differentiation among populations within basins, and ΦST, the degree of differentiation among all populations. We tested whether the derived indices were significantly different from zero using a nonparametric permutation method . Moreover, we estimated pair-wise genetics differentiation between populations with ΦST [Genetic diversity was measured for all samples and for each basin grouping using haplotype diversity (sity (π) . Values aSP 4.10 . As uneqion 3.11 . Analysiatistics . Three hwith ΦST ,81 that with ΦST  for multwith ΦST  with 10,For phylogenetic analysis, we constructed unrooted maximum-likelihood (ML) tree using the program PAUP* 4.0 . The TrNcytb gene, a conservative class of substitutions, was used to resolve all cases in which assignment to a nested series was ambiguous [Intraspecific data typically consists of many similar sequences, some of which may be ancestral and its phylogenetic relationship are often more clearly and accurately represented by a network . In the mbiguous . The curmbiguous , under tmbiguous . It is ambiguous -94, but mbiguous ,95. Howembiguous .To detect mechanisms responsible for observed mtDNA patterns on various spatial and time scales, we developed different data analysis methods. Due to the limits and pitfalls of each method, the combined results might provide the best approximation of the population structure and history.N. taihuensis were evaluated with the skyline plots [P. chinensis (DQ191115) [Hri) [SSD) [Hri and SSD were assessed by parametric bootstraps , and the significant value was taken as evidence for departure from the estimated demographic model of sudden population expansion. Third, Fu and Li's D* [Fs [R2 [D* is designed to detect an excess of old mutations, characteristic of a population that has experienced a historical reduction in effective population size [FS and R2 are sensitive to an excess of recent mutations [g) for this species. All runs employed the following strategy: 10 short chains of 4,000 steps and five long chains of 400,000 steps, sampling every 20th step; random starting trees; empirical nucleotide frequencies; initial g value of 0.0, starting θ-value from Watterson's estimate [g are biased upwards [SD) of g regarded as significant.First, changes over time in nonparametric estimates of the effective population size of ne plots ,39 usingne plots . This mene plots , the genne plots . As the ne plots ,55, we cne plots . Theoretne plots . Occasione plots , so the ne plots  was usedQ191115)  before pQ191115)  under a 5) [Hri)  and the i) [SSD) . Signifi Li's D* , Fu's Fss D* [Fs  and Ramo [Fs [R2  tests fo [Fs [R2  and Arle [Fs [R2  with 10,ion size ,102. In utations ,41. Finautations  to estimestimate ; and a 2estimate  under thestimate  using on upwards , a conseτ to calculate the expansion time. The mismatch distribution analysis provided a rough estimate of τ, with the starting time of the expansion in units of 1/(2 ut) generations, where u is the mutation rate per locus per generation. The relationship with the absolute time in years (t), is t = τ/2 uT. The value of T is generation time (1 year for N. taihuensis), and the value of u is derived from u = μk, where μ is the mutation rate per nucleotide per generation, and k is the number of nucleotides in the sequence. For other populations, however, we used GENETREE [θ following the method of Joy et al. [N. taihuensis.For stepwise expansion population, we used GENETREE  to estimGENETREE  of θ foly et al. . Each ruN. taihuensis to analyze their cartilage character, the genetic time clock is difficult to calibrate. However, it has been convincingly shown that most groups of fish have slower evolutionary rates compared with those of other species, for example primates [Since there is no fossil record for primates . A calibprimates ,107,108.AMOVA: Analysis of molecular variance; ML: maximum likelihood; NCA: nested clade analysis; IBD: isolation by distance; LGM: last glacial maximum; TMRCA: the most recent common ancestor.b gene. He collected, analyzed and summarized the data, and drafted the manuscript; JZ collected some samples, contributed some sequencing and contributed some to its design; ZL participated in analysis and interpretation of data, and helped draft the manuscript; SMF revised the manuscript and conducted part of analysis; FW and MX revised the draft manuscript and gave their some comments; ML conceived the study and participated in its design and data interpretation, and preparing the manuscript. All authors read and approved the final manuscript.LZ collected samples, isolated genomic DNA, and conducted the amplification and sequencing of the complete Cyt Nested clade analysis. Statistical analysis of the current and historic patterns of phylogenetic and geographic associations.Click here for file


Poecilia mexicana, Poeciliidae) in the Cueva del Azufre system in southern Mexico, where two strong environmental selection factors (darkness within caves and/or presence of toxic H2S in sulfidic springs) drive the diversification of P. mexicana. Nine nuclear microsatellites as well as heritable female life history traits (both as a proxy for quantitative genetics and for trait divergence) were used as markers to compare genetic differentiation, genetic diversity, and especially population mixing (immigration and emigration) before and after the flood.Local adaptation to divergent environmental conditions can promote population genetic differentiation even in the absence of geographic barriers and hence, lead to speciation. Perturbations by catastrophic events, however, can distort such parapatric ecological speciation processes. Here, we asked whether an exceptionally strong flood led to homogenization of gene pools among locally adapted populations of the Atlantic molly , but not geographic distance was the major predictor of genetic differentiation. Before and after the flood, each habitat type harbored a genetically distinct population. Only a weak signal of individual dislocation among ecologically divergent habitat types was uncovered . By contrast, several lines of evidence are indicative of increased flood-induced dislocation within the same habitat type, e.g., between different cave chambers of the Cueva del Azufre.The virtual absence of individual dislocation among ecologically different habitat types indicates strong natural selection against migrants. Thus, our current study exemplifies that ecological speciation in this and other systems, in which extreme environmental factors drive speciation, may be little affected by temporary perturbations, as adaptations to physico-chemical stressors may directly affect the survival probability in divergent habitat types. Poecilia mexicana, Poeciliidae)  suffered a strong decrease in female condition (od Table . Furtherod Table .Classification success for a separation by habitat type did not vary greatly before the flood (89.8%) and after the flood 87.0%), while the cross-validation DFA success was 66.2%  was Mechanisms of selection against immigrants).Our major question was whether the flood of 2007 has led to a homogenization of locally adapted populations, for which we found little evidence. A potential exception is the immigration of cave fish into the sulfur creek. It remains to be seen whether these immigrants are able to significantly contribute to the gene pool of the sulfur creek population or whether "cave alleles" will eventually vanish from the surface population with time . Hence, chamber XIII apparently is less separated from the other cave chambers than previously thought  genetic structure was lost after the flood in January 2008. While the small rearmost chamber XIII is usually separated by a 1.5 m waterfall from adjacent cave chambers, the flood in fall 2007 probably flooded the waterfall. Our current study provides several lines of evidence for the immigration of individuals into chamber XIII t e.g., ,77). The. Thee.g.. The. Tht  scale of the life history analyses, we observed several shifts from pre- to post-flood samples within habitat types . At first sight, this might be interpreted as an indication of flood-induced migration/displacement between the habitat patches. Still, there was no breakdown of trait divergence between habitat types, since the extreme ecotypes (Cueva del Azufre and El Azufre) clearly remained distinct with respect to female life history traits even after the flood (post-flood DFA). In particular, highly plastic traits like female and offspring body condition (fat content) were decreased after the flood in almost all populations. This could either be a signal of the disturbance of the ecosystems due to the rising water levels and increased flow forces or regular seasonal variation of body condition . Most imP. reticulata) populations from river versus creek habitats [In the surface habitats, however, the cross-validation classification success was by far the weakest. Potentially, this could be due to a plastic response to the flood, but it more likely represents general life history differences between the two surface habitats: RA is a large river, and fish were sampled in stagnant pools on a sandbank in the river, while AB is a small, fast flowing creek. Similar differences have been documented between guppy , as an indication of a genetic bottleneck. It needs to be recalled, however, that our post-flood sampling was done only two months after the flooding, while any mollies born after the flood would have needed more time to grow to adulthood . Hence, our samples originated from adult fish, and are probably not offspring of the few(er) remaining individuals in such  populations. Reduced values of Ho are, therefore, not as straightforward to explain as it may first seem. A possible scenario is that a big proportion of the population from our collection site in the Río Oxolotán was indeed swept away during the flood and was replaced almost entirely by fish from further upstream (the mountainous regions of Chiapas), and we hypothesize that fish in those upstream regions are characterized by lower genetic diversity. We plan to test this idea in the future by analyzing genetic diversity in more distant populations from all along the Río Grijalva drainage system.We found a drastic impoverishment of genetic diversity in the Río Oxolotán after the flood. How can this pattern be explained? In the Río Oxolotán, water levels rose up to eight meters. Massive currents during the flooding probably washed away a considerable proportion of the total population. At first sight one might be tempted to interpret the observed reduction in measures of genetic diversity, above all observed heterozygosity  habitats generally coincide with a reduction in overall genetic variability . Surefer to -117.i.e., the question of how exactly divergent natural and sexual selection translate into reproductive isolation [e.g., due to down-regulated expression of antioxidant enzymes under hypoxia [Particularly interesting in the study of ecological speciation are the mechanisms leading to and maintaining genetic differentiation, solation ,118-120.solation . A heritsolation ; reanalysolation ). Furthesolation ,123 and solation . On the  hypoxia -126) may hypoxia  for discBelostoma [i.e., cave fish at the surface and surface fish inside the cave [But what about genetic differentiation between surface and cave habitats? This question is of particular importance because even before the flood some migrants/dislocated individuals from inside the cave into the sulfidic creek (El Azufre) were detected, and dislocation into El Azufre tended to increase due to the flood Fig. . Given telostoma ,80,130, the cave . This mathe cave ,110,121.the cave .i.e., reinforcement, [In traditional models of ecological speciation, reproductive isolation evolves incidentally as a by-product of divergent natural selection -3,131. Brcement, ,132). Inrcement, . Altogeti.e., loss of small-scale genetic structuring within the cave). Overall, this supports the hypothesis that natural selection between habitat types in the Cueva del Azufre system is so strong that even severe ecosystem disturbances do not lead to a breakdown of the documented population differentiation. Hence, speciation processes in this and similar systems with presence of abiotic stressors  found the same pattern: increased flood-induced migration/displacement was found within the same habitat type, but not among ecologically divergent habitats (with the exception of slightly increased dislocation of cave fish into the sulfur creek). However, we did find some interesting patterns of flood-induced within-population homogenization (e system ,133 may e system ,73,74.The authors declare that they have no competing interests.BH and CS carried out the microsatellite analyses, and RR conducted the life history analyses. MP, BH, CS, RR, MT and RT conducted the statistical analyses and wrote the first draft of the manuscript. MP, RR, MT, FJGL and IS were equally involved in field work and sample collection.MP, RR, MT, FJGL, IS and RT were equally involved in implementing the project, and developing later drafts of the manuscript. All authors read and approved the final manuscript.


Several recent papers illustrate the importance of migration and gene flow in molding the patterns of genetic variation observed in humans today. We place the varied demographic processes covered by these terms into a more general framework, and discuss some of the challenges facing attempts to reconstruct past human mobility and determine its influence on our genetic heritage.http://www.biomedcentral.com/1741-7007/8/15 and http://www.biomedcentral.com/1471-2156/11/18See research articles:  Migration is a major force shaping patterns of genetic variation in humans . The hisA major aim in contemporary molecular anthropology is to determine when gene flow has occurred  and over what geographical range its effects were felt  Figure . ConsideIn practice, it remains difficult to determine the timing and geographic extent of specific episodes of historical human migration. Spatial control largely reduces to an issue of appropriate sampling . Increased geographical sampling is currently an expensive proposition, but advances in genotyping technologies are likely to reduce experimental costs to more reasonable levels in the near future. Attaining fine temporal control is a far more difficult prospect. To date, tight temporal resolution of human mobility has largely been the sole purview of ancient DNA studies. However, new analytical methods are currently being developed that can place chronological bounds on human gene flow using genetic data from individuals living today. The choice of genetic marker is also a major influence on what researchers can reveal about historical movements. Until recently, mitochondrial DNA (mtDNA) and the Y chromosome were favored as the most informative genetic markers available for studying human history. mtDNA is passed only from a mother to her offspring, while the Y chromosome is passed only from a father to his sons. This simple mode of transmission makes mtDNA and the Y chromosome easy model systems for studying human ancestry. In particular, these loci can be used to detect whether migration has occurred between populations in the past.BMC Biology adopted exactly this approach. Li and colleagues [The authors of a recent article in lleagues  report mBMC Genetics. They report approximately 50,000 single nucleotide polymorphisms  drawn from the nuclear genome. These markers were genotyped in individuals from two indigenous communities in Thailand that show linguistic and anthropological evidence of prehistoric connections. Using a suite of clustering methods, Xu and colleagues demonstrate that the Mlabri and Htin share more nuclear variants with each other than either does with surrounding populations.However, mtDNA and Y chromosome records are necessarily biased towards the sex-specific movements of women and men, respectively. Further, demographic reconstructions made from mtDNA and the Y chromosome have considerable uncertainty due to the substantially higher rates of stochastic genetic drift in these uniparentally inherited systems compared to autosomal loci. Ultimately, mtDNA and the Y chromosome are just two small loci in a vast ocean of human genetic variation. In contrast, the autosomes consist of millions of independently evolving regions, any number of which can be surveyed to characterize gene flow in humans. Today, the rapid advent of new genotyping technologies is allowing massive numbers of markers to be screened across the human genome. Xu and colleagues  adopted BMC Genetics, we adopted exactly this sort of strategy to quantify rates of gene flow on a global scale [m) were approximately five-fold higher among non-African populations relative to African groups [N) and migration rates (m) are inversely proportional in African and Eurasian groups - although migration rates are approximately five-fold lower in Africa, effective population sizes are approximately five-fold higher. Consequently, population migration rates (Nm) are globally very similar (Nm = 2.4). In other words, we found that approximately two migrants per generation have moved between these globally distributed populations on average through time. Some of these movements may have been recent, despite the large geographical distances between populations, but most would have occurred when these populations were geographically much closer . Developing statistical methods to determine exactly when this gene flow occurred remains an important outstanding task.While determining whether two populations share genetic variation is a relatively simple exercise, identifying and quantifying the amount of gene flow between them requires more advanced modeling and inferential statistics. This is usually applied within a framework of coalescent theory. Large quantities of genetic information are required to infer rates of gene flow, and data sets of this size have only recently become feasible. In a 2008 article in al scale . Insteadn groups . InteresToday, sequencing and comparing entire human genome sequences is the surveying method of choice. For example, the complete genomes of many individuals have now been sequenced, including such famous names as Archbishop Desmond Tutu  and NobeOne recent study has compared entire genomes to provide a surprising new insight into human history. Just this year, Green and colleagues  sequenceAlthough this particular episode of gene flow has been characterized with good spatial and temporal resolution, similar detail is rare in other studies of human gene flow. Indeed, many questions still remain about how gene flow has helped shape patterns of genetic variation in humans. mtDNA and the Y chromosome are just two small genetic regions within a very large genome, and as the Neandertal study shows, they are not necessarily representative of much of our genetic diversity largely due to their increased rates of genetic drift. Furthermore, sampling ancient DNA becomes increasingly difficult at ever greater time depths - at 4,000 years old, the Xiaohe cohort stretches back less than one-tenth of the continental history of Eurasia, a region first settled after 50,000 years ago. The Neandertal study does reach back this far, but reflects a contact event that was probably over quickly; by approximately 40,000 years ago, modern humans were dominant across Eurasia, and Neandertals were on the wane. For most other questions involving human movements, reconstructing an inclusive spatial and temporal history will depend on the vast genomic datasets now being produced for a wide range of populations. In tandem, improved inference methods must be developed to attain this goal. With the exception of ancient DNA studies, current rates of global gene flow are all long-term estimates . Our inability to resolve the timeframes over which specific demographic processes occurred is a major limitation that requires further attention.versus autosomes) is a prerequisite for detecting such patterns. Third, gene flow can be directional  or non-directional . Directional mobility along the Silk Road will produce quite different genetic patterns than intermarriage between adjacent communities. Improved regional sampling will be necessary to detect such patterns. Fourth, rates of gene flow may change over time. Varying rates of gene flow strongly affect patterns of genetic diversity [Finally, we note that human migration cannot simply be characterized by the timing and geographical extent of gene flow. Human mobility is far more nuanced, and at least four additional axes would seem to warrant further consideration. First, rates of gene flow are hard to determine with certainty. Although the presence (or absence) of migration can often be inferred relatively easily, estimates of actual rates frequently have considerable uncertainty . Methodsiversity , althougST F= 0) or very different (ST F= 0.3) even when they have exchanged the same number of migrants (that is, graph lines with the same color in figure We expand on just one of these points for illustration Figure . Even wh FST = 0. even wheThe series of migration vignettes presented in this commentary illustrates just how little we understand about the role of migration and gene flow in human history. Recent studies have shed some light on the processes affecting movement among communities, between continents and across the globe, but more sophisticated methods are needed. We look forward to meeting these methodological challenges and the clarity that a more nuanced understanding of human mobility will bring.


Alcippe morrisonia, has been reported to show deep genetic divergences among its seven subspecies. In the present study, we investigated the phylogeography of A. morrisonia to explore its population structure and evolutionary history, in order to gain insight into the effect of geological events on the speciation and diversity of birds endemic in south China.The role of Pleistocene glacial oscillations in current biodiversity and distribution patterns varies with latitude, physical topology and population life history and has long been a topic of discussion. However, there had been little phylogeographical research in south China, where the geophysical complexity is associated with great biodiversity. A bird endemic in Southeast Asia, the Grey-cheeked Fulvetta, Mitochondrial genes cytochrome b (Cytb) and cytochrome c oxidase I (COI) were represented by 1236 nucleotide sites from 151 individuals from 29 localities. Phylogenetic analysis showed seven monophyletic clades congruent with the geographically separated groups, which were identified as major sources of molecular variance (90.92%) by AMOVA. TCS analysis revealed four disconnected networks, and that no haplotype was shared among the geographical groups. The common ancestor of these populations was dated to 11.6 Mya and several divergence events were estimated along the population evolutionary history. Isolation by distance was inferred by NCPA to be responsible for the current intra-population genetic pattern and gene flow among geographical groups was interrupted. A late Pleistocene demographic expansion was detected in the eastern geographical groups, while the expansion time (0.2–0.4 Mya) was earlier than the Last Glacial Maximum.A. morrisonia in China mainland and its two major islands, and restricts gene exchange during climate oscillations. Isolation by distance seems to be an important factor of genetic structure formation within geographical populations. Although glacial influence to population fluctuation was observed in late Pleistocene, it seems that populations in eastern China were more susceptible to climate change, and all geographical groups were growing stably through the Last Glacial Maximum. Coalescence analysis suggested that the ancestor of A. morrisonia might be traced back to the late Miocene, and the current phylogeographical structure of A. morrisonia is more likely to be attributable to a series geological events than to Pleistocene glacial cycles.It is proposed that the complicated topology preserves high genetic diversity and ancient lineages for geographical groups of  The glacial cycles of the past two million years have traditionally been considered to have had profound effects on the genetic patterns of most extant species -3. HowevAs one of the species richness hotspots in Southeast Asia, south China has high biodiversity -16, reflAlcippe morrisonia is a small babbler widely distributed in tropical and subtropical habitats from Burma to Taiwan [A. morrisonia and its relatives have shown that the genus Alcippe is a polyphyletic group [A. morrisonia, in which Alcippe peracensis annamensis was grouped within A. morrisonia [A. morrisonia using the complete ND2 gene confirmed this taxonomic complexity and found deep genetic divergences among subspecies [A. morrisonia and explore its possible mechanisms responsible for the current genetic pattern to provide an insight into the ice age legacy in south China.The o Taiwan . It is ao Taiwan ,23. As ao Taiwan . Recent ic group . The comrrisonia . Anotherbspecies . The pecFJ472657–FJ472744), and the COI sequences contained 99 variable sites of which 81 were parsimony informative, generating 71 haplotypes (GenBank Access Number FJ472745–FJ472815). Modeltest indicated that the best substitution models were HKY+G and TIM+I for Cytb and COI respectively. For the combined sequence data, a total of 151 sequences of 1236 bp were obtained, as some individuals could not be sequenced for both partial genes. For the combined sequence data set, there were 239 variable sites of which 199 were parsimony informative, generating 116 haplotypes. The best model for the combined dataset was TrN+I+G. Phylogenetic trees estimated by three methods  for haplotypes of Cytb, COI and the combined dataset were generally compatible, the differences being the relative positions and the statistical support possibilities of some branches, then the topology based on the combined dataset is presented in Figure We obtained 642 bp of the partial Cytb gene from 156 individuals and 594 bp of the partial COI gene from 153 individuals. The Cytb sequences yielded 141 variable sites (118 were parsimony informative), identifying 88 haplotypes   and multiplied it by a factor of 0.90 to reflect the mutation rate of the combined sequence. The times of divergence between Fujian and Hainan, WYunnan and SWSichuan, Centre and Guangxi were 1.15, 0.87 and 1.12 million years ago, respectively. The Taiwan group separated from the Fujian and Hainan groups at 3.51 Mya, and the divergence time of the western (WYunnan and SWSichuan) and eastern  groups was 6.06 Mya. The divergence time between the Middle and Peripheral clades was earlier, dating back to 9.82 Mya  for all haplotypes of A. morrisonia was estimated back to 11.66 Mya  simulated the fluctuation of populations over time. Recent population increases were observed in the Fujian, Hainan, WYunnan and Centre groups; the times of growth were estimated at 0.20, 0.15, 0.30, and 0.23 Mya respectively  [A. morrisonia might be the result of various geological events.Phylogenetic reconstruction shows that populations from the middle of south China (Centre & Guangxi) are rooted at the base of the tree. Although geographically separated, the eastern populations  are sister groups of the western populations (WYunnan and SWSichuan), forming the Peripheral clade. The estimated T Miocene . The fir Miocene ,30. Alth Miocene ,32. Envi Miocene . It is rgetation , coincidGarrulax , which a1.0 Mya) , consist1.0 Mya) . Taking A. morrisonia is quite different from another phylogeographical study based on Chinese Hwamei (Leucodioptron canorum canorum) [A. morrisonia's in China continent. Putative gene flow was observed among geographical groups of L. c. canorum while it was hardly detected in A. morrisonia. We consider that some of the causes might be related to the limited gene exchange among geographical groups. Firstly, geographical barriers seem to be important effects on population divergences. Great mountains and deep valleys in south China possibly shaped multi-refugia for A. morrisonia in the cold weather extension, and then blocked secondary contact during the postglacial recolonization. It can be imagined that the complicated topology of this region played an important role in initiating phylogeographic differentiation and further sculpting pre-existing phylogeographic variety during the glacial oscillations. Secondly, as a sedentary bird in the forest understory and shrubs, small size and poor dispersal capability [A. morrisonia might impede gene flow among populations with a distant range, especially in the complicated topology of south China.There is no obvious trend of genetic diversity for geographical groups along the lat/longitude gradient. High haplotype and nucleotide diversity was observed both in western and in eastern geographical groups in the China continent. Deep genetic gaps and restricted gene flow were identified among the geographical groups. The interruption of gene flow along geographical groups of canorum) , which hpability  of A. moA. morrisonia [Within the geographical groups, association of restricted gene flow with isolation by distance was inferred from NCPA analysis, supposing that geographical distance was an important factor in forming the current genetic structure. This is consistent with the moderate mobility and limited foraging range of rrisonia  and the rrisonia ,42. WithTwo insular populations, Hainan and Taiwan, were separated from the mainland by the strait barriers, while divergence times of the two insular groups from their continental relatives are discordant (1.15 and 3.51 Mya respectively). This is similar to the genetic variance of Chinese mainland Hwamei with their two island relatives . A possiA. morrisonia mostly inhabits shrub, the understory of the tropical and sub-tropical evergreen leaf forests, so its demographical fluctuation might reflect changes in the flora of south China. Significant negative Tajima's D and Fu's Fs values were detected in the Fujian, Hainan, Taiwan and Centre groups, and mismatch distribution showed that most groups fit population expansion except for SWSichuan. Recent demographic expansions were also found in the eastern geographical groups by Bayesian skyline plot reconstruction, while the western groups WYunnan, SWSichuan and Guangxi seemed stable in the late Pleistocene. It seems that expansions occurred in the eastern groups rather than in the west, suggesting intense changes of flora in the eastern part of south China. Compared with the continuous high mountains in the western part of south China, the mountains in the east are isolated and low, so the vegetation communities might have changed more drastically in the eastern part of south China during the glacial oscillation [Paleovegetation based on pollen data implied that large areas of the exposed continental shelf in eastern China might have been dominated by grasslands, while the uplands of South China were occupied by less dense coniferous or temperate forests during glacier extension ,48. Therillation ,51; demo18 O value and pollen data evidenced that the most substantial glacial extension occurred MIS 16-MIS 18 in China [The time since demographical expansion was estimated mostly around 0.2–0.4 Mya, earlier than the Last Glacial Maximum (LGM), which was regarded as a vital event inducing population divergence -54. Incoin China . Since tA. morrisonia. The genetic distinction among geographical groups is associated with the complicated topology of south China, where high genetic diversity might be conserved and gene flows be blocked. Ecological barriers might result in variant divergence time between two insular groups (Taiwan and Hainan) from China mainland. Isolation by distance seems to be an important factor for genetic structure formation within the geographical populations. Recent demographical expansions corresponding to vegetation changes may have occurred during the largest glacial extension stages rather than the LGM, and more extensive in the eastern part of south China. However, the results suggest a long evolutionary history for A. morrisonia, the common ancestor of which could be dated to the late Miocene, and the population differentiations correspond to a series of geological events beyond the Pleistocene ice ages.The present study shows deep geographical differentiations of A. morrisonia , Alcippe dubia , Alcippe brunnea , Stachyris ruficepes  and Garrulax sannio  were used as outgroups.Sequences were assembled using Seqman II (DNASTAR) and proofread against the original chromatograms. The presence of stop codons or indels, which could reveal pseudogene sequences, was checked in MEGA3.1 . Sequenc6 generations or more until to the average standard deviation of split frequency below 0.01. The first 10% of the generations were discarded as 'burn-in', and posterior probabilities were estimated for the remaining saved generations.Haplotypes for Cytb, COI and the combined sequence were generated in Dnasp, version 4.0 . MaximumH), and values of haplotype diversity (h) [.The numbers of haplotypes (sity (h)  and nuclsity (h)  for eachsity (h) . The corsity (h)  in Arleqsity (h) ., updated November 2005).A maximum parsimony network was constructed using TCS 1.21  with a 9M and from 0 to 5 for T. The divergence times of splits between phylogroup pairs were estimated using the Formula tdivergent time = Tpop *(Theta/2 μk) with mutation rate μ and a generation time of 2 years.MDIV  was usedD [F [r) between observed and expected mismatch distributions were used as a test statistic; their P values represented the probability of obtaining a simulated sum of squared deviation greater than or equal to the one observed. Estimation and testing were done by bootstrap resampling  using Arlequin 3.1. The relationship Tau = 2 μkt [A. morrisonia or its relatives, we cautiously applied "2%"rule of molecular clock for avian Cytb gene [-8 per site per year for the avian mitochondrial Cytb gene was assumed [vs. that for Cytb alone for the geographical group pairs [A. morrisonia according to Sibley & Ahlquist [Values of Tajima's D  and Fu'sD [F  were cal = 2 μkt  was usedytb gene -82. An a assumed ,38. ThisAhlquist  and ZhouMRCA) of the seven geographical groups and the whole population were estimated using the same mutation rate as above. The results were summarized through TRACER 1.4 .In order to estimate the dynamics of population size fluctuations over time, we used the Bayesian Skyline Plot (BSP)  method iA. morrisonia. FL conceived and elaborated the design of the whole study, including sample collection, preparation, analysis and revision of the manuscript. All authors read and approved the final manuscript.This work is part of GS's Ph.D. thesis. GS is a Ph.D. graduate student of FL. He carried out and designed the study. He accomplished the laboratory work and statistical analyses with guidance and help from YQ, and drafted the manuscript. ZY participated in most of the field sample collecting. SL coordinated the study in sequencing samples from Taiwan and revised the draft of the manuscript. NL improved the organization of the discussion and corrected the choice of generation time for Alcippe morrisoniaGenetic variability and haplotypes based on 1236 bp of combined mitochondrial sequences of . A summary of genetic variability in each sample location based on combined mitochondrial data. Sample size (n), number of haplotypes (H), haplotype diversity (h), nucleotide diversity (π). Haplotype label in bold indicate it was shared in different samples and bold with * indicates it was shared between different locations.Click here for fileA. morrisoniaAMOVA analysis of . AMOVA result shows that most of the variance came from differences among groups, and the seven groups were best recognized with the maximum value of among-group variance.Click here for fileGenetic diversity and mismatch distribution analysis of the geographical groups. The genetic diversity and mismatch distribution for seven geographical groups were summarized in the table. N, group size, H and π is the genetic diversity index. PSSD and PH-R are parameters of the goodness-of-fit test to the sudden expansion model. Tau is the time in number of generations elapsed since the sudden expansion. T is the expansion time transformed by Tau = 2 μkt.Click here for fileA. morrisoniaNested clade phylogeographical analysis with IBD tests for . NCPA results show Allopatric fragmentation in network A. For low level clades, restricted gene flow with isolation by distance was revealed. However these associations were not confirmed by the Mantel Test or IBDWS; only clade 4-1 in network A showed a tendency towards a significant IBD pattern.Click here for file