Aphelocoma californica) to test for a signature of Haldane’s Rule: a narrower cline of introgression in mtDNA compared to nuclear markers.Haldane’s Rule, the tendency for the heterogametic sex to show reduced fertility in hybrid crosses, can obscure the signal of gene flow in mtDNA between species where females are heterogametic. Therefore, it is important when studying speciation and species limits in female-heterogametic species like birds to assess the signature of gene flow in the nuclear genome as well. We studied introgression of microsatellites and mtDNA across a secondary contact zone between coastal and interior lineages of Western Scrub-Jays , Northern Mexico, and Southern Mexico. Consistent with predictions from Haldane’s Rule, mtDNA showed a narrower cline than nuclear markers across a transect through the hybrid zone. This result is not being driven by female-biased dispersal because neutral diffusion analysis, which included estimates of sex-specific dispersal rates, also showed less diffusion of mtDNA. Lineage-specific plumage traits were associated with nuclear genetic profiles for individuals in the hybrid zone, indicating that these differences are under genetic control.A. californica (Pacific Slope); A. woodhouseii (Interior US plus Edwards Plateau plus Northern Mexico); A. sumichrasti (Southern Mexico). The distinctive Edwards Plateau population in Texas, which was monophyletic in mtDNA except for one individual, should be studied in greater detail given habitat threat.This study adds to a growing list of studies that support predictions of Haldane’s Rule using cline analysis of multiple loci of differing inheritance modes, although alternate hypotheses like selection on different mtDNA types cannot be ruled out. That Haldane’s Rule appears to be operating in this system suggests a measure of reproductive isolation between the Pacific Slope and interior lineages. Based on a variety of evidence from the phenotype, ecology, and genetics, we recommend elevating three lineages to species level:  The study of speciation has been influenced during the last decade by increasing awareness that different parts of the genome tell different evolutionary stories -3. PreviOne case in which mtDNA data can be particularly misleading occurs during gene flow between populations in secondary contact. Haldane was the first to note that when two differentiated lineages come back into reproductive contact, the heterogametic sex  often shows reduced fertility . The mecperceived importance of the BSC. As empirical evidence continues to mount demonstrating mito-nuclear discordance [The potential mismatch between gene flow estimated from mtDNA and gene flow estimated using nuclear markers is not just theoretical; it has been demonstrated empirically by studies of female-heterogametic organisms -18. Thiscordance ,22, the Aphelocoma californica) are an excellent model for testing the mismatch between mtDNA and nuclear markers across a hybrid zone predicted by Haldane’s Rule because previous work documented a zone of secondary contact and hybridization between two divergent lineages. Western Scrub-Jays are widely distributed in North America from British Columbia, Canada to Oaxaca, Mexico and east to the Great Plains  are thought to have diverged from each other anywhere from 1 to 4 million years ago (Ma) based on divergence times calibrated with a fossil and estimated molecular substitution rates . CoastalThe hybrid zone was originally described by Pitelka  who travThe purpose of this study was to test for a prediction of Haldane’s Rule that there will be a mismatch between mtDNA and nuclear gene flow across the contact zone. Specifically, we predict that if Haldane’s Rule is operating in this system, we will see a narrower  cline in the transition of mtDNA haplotypes compared to nuclear markers. Before addressing this hypothesis, however, we needed to determine the exact nature of the contact zone in western Nevada and whether there were any undescribed contact zones nearby that might confound our results. We therefore first assessed broad-scale patterns of divergence and gene flow among Western Scrub-Jays across their entire geographic range, using deep sampling of populations and a suite of nuclear markers. Previous molecular studies sampled only one or a few individuals of different populations, focusing largely on allozymes  or mtDNATo assess gene flow throughout the range of Western Scrub-Jays, we conducted simple mtDNA typing of all 689 individuals using restriction digest, which showed that most populations are composed entirely of coastal or interior mtDNA types, with very few populations showing mixing of mtDNA types. The four populations showing mixing were 1) the previously described contact zone in western Nevada, which featured several populations having mixed types 100 km east and southeast of Reno and Carson City  showed significant deviations from HWE across many populations following Bonferroni correction. We removed this locus from the data set. Four other loci showed deviations from HWE across some populations. We ran downstream analyses with and without these loci to investigate whether they were driving any of the patterns of genetic structure we observed. Results among analyses including and excluding loci were qualitatively similar, so we retained the larger group of 13 loci. No loci showed consistent signs of other microsatellite artifacts like large-allele drop-out or null alleles.K = 1–30 found increasing likelihood (LnL) values until K = 9, followed by a plateau in LnL until K = 13, followed by a decrease from K = 14–30. Structure runs on successively smaller clusters revealed five distinct geographic clusters of nuclear DNA variation , including sampling from throughout the genus. We chose samples to both capture the major genetic lineages within Western Scrub-Jays and outgroups, and also to sample deeply within certain microsatellite groups of interest . The larger phylogeny, including all Aphelocoma samples, is available through Dryad [A. insularis). In addition to the Island Scrub-Jay, phylogenetic analyses placed three other highly supported and divergent clades within Western Scrub-Jays: (1) a Pacific Slope clade that was sister to the Island Scrub-Jay (1.0 posterior probability or PP); (2) a clade from Mexico south of the Transvolcanic Belt ; (3) a clade of all other interior individuals (0.94 PP). Within this last clade, we recovered a weakly supported (0.87 PP) group from the Edwards Plateau in Texas, with the exception of one individual that grouped elsewhere in the interior clade  could also be found in a mtDNA phylogeny, we sequenced a subset of individuals at the gh Dryad . In Figuduals 0.9 PP. Withc) were similar and had overlapping confidence intervals. The center of the mtDNA cline was ~300 km (280 – 319), and the center of the nuclear DNA cline was ~315 km (271 – 377). The width of the mtDNA cline was ~131 km (76 – 270), and the width of the nuclear DNA cline was ~331 km (145 – 678). The estimated width of the mtDNA cline fell outside the 2 log likelihood confidence intervals for the nuclear DNA, and vice versa, therefore the mtDNA cline was significantly narrower than the microsatellite cline . Model 1 with no scaling was accepted as the most likely model for both datasets. Estimates of the center of the cline  from Structure and many phenotypic traits for the full data set of 123 individuals that included many pure coastal and interior individuals outside the hybrid zone, especially for plumage traits and principal components (PC) axes describing bill shape . All individuals in the Edwards Plateau population except one possess a unique mtDNA haplotype differing from other interior US haplotypes by two substitutions. Nuclear and mtDNA differentiation on the Edwards Plateau is interesting given the history of isolation of this region and its unique flora and fauna. The oak savanna and Ashe juniper woodland of the Edwards Plateau are separated from other scrub woodland to the west by an extension of the Chihuahuan Desert in the Pecos Valley. Fossil and pollen data suggest, however, that pygmy woodland was continuous across this region, and connected the Edwards Plateau to western forests throughout the last glacial maximum until about 11,000 years ago [Setophaga chrysoparia), and other organisms in this region have distinct populations or subspecies [This study is the first to show clear evidence of nuclear and mtDNA differentiation of Western Scrub-Jays on the Edwards Plateau in Texas  occur at lower elevations, seemingly occupying the Western Scrub-Jay niche [The nuclear genetic break between the Interior US and Northern Mexico populations, occurring roughly where the Sierra Madre Occidental and Oriental turn into a network of sky islands near the US border, could be explained either by a break in suitable habitat or a sampling artifact. Addressing the latter point, observations posted on eBird (ebird.org) confirm observations of Western Scrub-Jays in southeastern Arizona and northern Sonora (in the vicinity of the genetic break), but these records could represent wandering individuals. Likewise few observations exist in the vicinity of the genetic break in southwestern Texas, and virtually no observations exist from northern Coahuila, where congeneric Mexican Jays  high levels of dispersal and gene flow link coastal populations; or (2) recently expanded coastal populations have had insufficient time to accumulate genetic differences. Available evidence suggests that Western Scrub-Jays generally have low dispersal , notwithA. californica, including subspecies californica, oocleptica, caurina, obscura, hypoleuca, superciliosa, immanis, and cactophila) is phenotypically distinct [We recommend splitting the current concept of the Western Scrub-Jay into three species, as has already been suggested previously . The Pacdistinct , possessdistinct ,28, is mdistinct ,24. ThisA. sumichrasti, including subspecies sumichrasti and remota) also meet criteria for species recognition. They are phenotypically distinct [Southern Mexico populations  be elevated to species level, given their diagnostic phenotype [Finally, we recommend that the remaining interior populations ; A. woodhouseii (Interior US plus Edwards Plateau plus Northern Mexico); A. sumichrasti (Southern Mexico). The distinctive Edwards Plateau population in Texas, which was also monophyletic in mtDNA except for one individual, should be studied in greater detail given conservation concerns.We report a mismatch in cline widths between mtDNA and nuclear markers across a hybrid zone in Western Scrub-Jays. This result is consistent with predictions based on Haldane’s Rule, where there is less mtDNA introgression between lineages in female-heterogametic species. This study adds to a growing list of studies that support predictions of Haldane’s Rule using cline analysis of multiple loci of differing inheritance modes. Structure results of microsatellite variation confirm that there is only one major area of contact between coastal and interior lineages. Furthermore, Structure identified at least five clusters of individuals with strong spatial structuring: Pacific Slope, Interior US, Edwards Plateau, Northern Mexico, and Southern Mexico. Based on a variety of evidence from the phenotype, ecology, and genetics, we recommend elevating three lineages to species level: We obtained tissues from Western Scrub-Jays representing all known subspecies, genetic lineages, and geographic regions from existing frozen tissue specimens in museums or from new specimens . Previously characterized microsatellite loci have been isolated from the Florida Scrub-Jay . From thhttp://genepop.curtin.edu.au). Strong population structure can lead to false positives for deviations from HWE when populations are pooled (Wahlund Effect), so we assessed HWE in each population at each locus individually, applying Bonferroni correction to control for false positives resulting from the effect of conducting multiple simultaneous tests. We also performed a test of linkage disequilibrium to confirm independent sorting of loci.After amplification, we determined fragment size using an ABI PRISM 3730 capillary sequencer and analyzed the resulting data using Geneious v. 6.0.4 (Biomatters). We called alleles objectively by creating bins based on known microsatellite repeat motifs , and assK) from a group of individuals based only on their genetic variation, without prior information on where the individuals originated. We did not use the method of Evanno et al. [K’ because this method is known to underestimate genetic structure in all but cases of very strong genetic differentiation [K = 2 as the best model for our data; however, analysis at higher K revealed strong structure that was highly justified based on biology and geography. Thus, because one of our goals was discovery of the basic units of spatially structured genetic diversity, we instead used Structure to analyze successively smaller clusters of individuals at K = 2 until Structure could no longer find any clustering within groups, as demonstrated previously [K = 2 revealed no remaining geographical clusters. After the first run, we excluded hybrid populations  from the analysis. We ran Structure with an admixture model, correlated allele frequencies, and a burn-in period set to 100,000 generations, followed by 500,000 generations, which was sufficient for each run to reach stationarity.We assessed genetic structure of microsatellite data using Structure v. 2.3.4 , which uo et al.  to detecntiation . Initialntiation  method seviously . For exaK = 2; their resulting Q-scores were recorded for later use in cline analysis. The Q-score values provide an overall estimate of nuclear variation that is expected to suffer less from random processes affecting individual loci. Moreover, cline analysis requires binary data or frequency values varying from 0 to 1. Due to large number of alleles at a given locus, individual microsatellite loci do not usually conform to this standard.To assess population assignment and potential hybrid ancestry of individuals across the hybrid zone, we delimited 13 populations along a transect running northwest to southeast from the Sierra Nevada in northeastern California into west-central Nevada Figure . This licyt b gene, using previous protocols [Aphelocoma and close outgroups  from GenBank, and we generated a phylogeny from these sequences using BEAST v. 1.7.5 [cyt b gene determined through Akaike Information Criterion (AIC), which was HKY + I + G with six gamma categories and base frequencies estimated as determined by MrModeltest, v2 [6 iterations, sampling every 1,000. The analysis converged quickly as determined by observing the plot of likelihood scores and ESS scores > 200 with Tracer. We discarded the first 1,000 of 10,000 trees as burn-in and produced a consensus tree from the remaining posterior trees using Geneious [Previous studies that generated mtDNA phylogenies of Western Scrub-Jays did not sample deeply within populations ,24. Thusv. 1.7.5 . We ran test, v2 . We usedGeneious . The maxw), center (c), delta , and tau . We also incorporated the possibility that Pmax and Pmin (the “top” and “bottom” of the cline) were either fixed or free to vary. We fit three sets of five cline models using the Metropolis-Hasting algorithm in R. Model set 1 has no scaling , model set 2 has fixed scaling , and model set 3 allows Pmin and Pmax to vary. Within each model set, scaling and tails are fixed or free to vary. We compared these models to a null model of no clinal transition.We used HZAR v2.5 , a stati6 generations. We ran three independent chains for 9.0 × 106 generations and assessed for convergence and stability by visualizing the MCMC traces. We performed model selection using corrected AIC (AICc). We estimated two log-likelihood confidence intervals around each parameter estimate. If the parameter estimate from one dataset did not overlap with the confidence interval of the parameter estimate from the other dataset, we considered them to be significantly different. We also used estimates of cline width to determine the time since contact under a neutral diffusion model solving for t in the equation w = 2.51σ√t[t is the time since contact and σ is the root mean square dispersal distance from estimates in [For the two genetic datasets, we set up a covariance matrix by running each model for 1.0 × 10= 2.51σ√t, where tmates in . BecauseWe collected phenotypic data on a subset of 124 coastal and interior Western Scrub-Jay specimens within and outside the hybrid zone. Unfortunately we could not perform a cline analysis along the transect because we did not have sufficient sample sizes from pure populations at both ends of the transect. However, to determine the extent to which phenotype is correlated with nuclear DNA variation, we conducted regression analysis of phenotypic traits (plumage and morphology) with Q-scores at several spatial scales. The set of 124 specimens includes the full ranges of coastal and interior phenotypes, many hybrids, and pure individuals of each lineage with no evidence for hybrid genetic ancestry (Q-score at or close to 1 or 0). Because environment can influence phenotype when considering allopatric lineages, a more robust test of the genotype-phenotype link occurs within the hybrid zone where individuals share the same environment and there is more continuous variation in Q-scores. Here, we looked specifically at the 68 specimens that are part of the hybrid transect, as well as a more restrictive sample of 50 individuals within the core hybrid zone defined as sites 35–45 in Figure To ensure consistent measurements, one author (TCW) measured wing, tail, tarsus, bill length, bill depth, and bill width to the nearest 0.1 mm with digital calipers on all 124 birds after first verifying high repeatability scores for all traits. On a subset of 66 of these birds, another author (JEM) assessed three qualitative plumage traits known to vary between coastal and interior lineages: the amount of blue collar (reduced in interior birds); size of the eyestripe (reduced in interior birds); and color of the undertail coverts . We determined variation in plumage traits by lining up all 66 specimens at the same time and arranging them in order of trait appearance . We then divided the arranged specimens into six even categories and assigned a value of 1 to 6. We repeated these steps for each trait. We analyzed morphological data with principal components analysis on the correlation matrix in Stata 10 to identify axes defining the most variation in the data. We assessed relationships between Q-scores and univariate and multivariate phenotypic traits with linear regression at each of the three spatial extents described above  using Stata.http://dx.doi.org/10.5061/dryad.57f48.New DNA sequences have been deposited in Genbank under accession numbers KJ835799-KJ835861. Museum catalog numbers, localities, raw microsatellite data, phenotypic data, and Q-scores for each individual are available through Dryad The authors declare that they have no competing interests.FCG collected samples and genetic data, conducted Structure analyses, and helped write the manuscript; JMM helped design the study, conducted cline analyses, and helped interpret results and write the manuscript; CC helped design the study, provided samples, and helped revise the manuscript; ATP helped design the study, provided samples, and helped revise the manuscript; BCF contributed to data acquisition and helped revise the manuscript; TCW helped collect genetic data, collected and analyzed phenotypic data, and helped revise the manuscript; JEM designed the study, collected samples and genetic and phenotypic data, analyzed genetic and phenotypic data, and helped write the manuscript. All authors read and approved the final manuscript.FCG conducted part of this research for her Master’s thesis at the Moore Laboratory of Zoology at Occidental College; JMM is the Collections Manager at the Moore Laboratory of Zoology; CC is Staff Curator of Birds at the Museum of Vertebrate Zoology at Berkeley; ATP is Distinguished Professor in the Ecology and Evolutionary Biology Department and Curator of Ornithology at the Biodiversity Institute at Kansas University; BCF is an Assistant Research Scientist at UCLA; TCW was an undergraduate at Louisiana State University while conducting this research; JEM is Curator of Birds and Mammals and Director of the Moore Laboratory of Zoology and an Assistant Professor in the Biology Department at Occidental College.Loadings for principal components analysis.Click here for file


Cobitis) whose extinction risk estimations were based solely on their extent of occurrence (and/or area of occupancy) and its fragmentation, and conservation proposals do not consider diversity below species level. In this investigation we employed molecular genetic methods to describe present genetic structure of the Adriatic spined loaches and reveal their demographic history. The divergence of the Adriatic lineages inside the genus Cobitis started in Miocene and lasted until Pleistocene epoch. Geological events responsible for shaping recent diversity of spined loaches in the Adriatic basin are: the Dinarid Mountains upwelling, the evolution of Dinaric Lake system, local tectonic activity, river connections during glaciations and differences in sea level. Even though all the investigated species inhabit karstic rivers located in the same geographic area and that were subject of similar geological events, the results obtained reveal great differences in their genetic diversity and structure and point out the necessity of different conservation measures to ensure their future viability. High level of genetic polymorphism is characteristic for species located more to the south. Two species comprised of more than one population have completely different intraspecific structure; populations of C. illyrica are genetically distinct and represent separate evolutionary significant units, whereas intraspecific structure of C. narentana corresponds to metapopulational pattern. Without population genetic data, evolutionary significant units could be easily misidentified. Furthermore, the obtained results affirm that population genetic measurements are able to detect differences among closely located and related species and estimate extinction risk even more accurately than currently applied IUCN criteria.The region of Balkans is often considered as an ichthyologic “hot spot”, with a great number of species and high portion of endemics living in fresh waters in a relatively small area. The Adriatic watershed in Croatia and Herzegovina is inhabited by six spined loach species (genus  N) from effective (Ne) population size for criterion C . Furthermore, practical conservation efforts still focus almost exclusively on species  im im21] im 0.1–0.2 . Since epolation . Data onb sequences are: EF605302-EF605306, EF605312-EF605316, KJ487435-KJ487484, KP208162-208173; of RAG1 haplotypes: KJ487485-KJ487519; and of S7 first intron: KJ487520-KJ487554.GenBank accession numbers of cyt Cobitis started in Miocene and lasted until Pleistocene epoch. Mutation rates of two investigated nuclear genes were similar and about six times slower than of the cyt b gene .Phylogenetic tree obtained by Bayesian inference with timing of the splitting events is presented on the b to be the most reliable in elucidating intraspecific and intrapopulational genetic diversity due to its faster mutation rate) high level of genetic polymorphism is characteristic for three species distributed more to the south . On the other hand, especially C. jadovaensis, but then also C. bilineata express much lower level of genetic polymorphism. Cobitis herzegoviniensis, even though geographically belonging to the “southern group”, is characterized by lower level of genetic diversity. The frequency of haplotypes is also quite different among species. In C. herzegoviniensis, and even more pronounced in C. jadovaensis and C. bilineata, one cyt b haplotype predominates greatly, whereas the remaining haplotypes are found only individually. Frequencies of cyt b haplotypes were more equally distributed in the remaining species. Frequency of nuclear haplotypes mostly follows the same pattern, but differences among species are less pronounced and some exceptions are present. The problem with nuclear haplotypes is also that closely related species C. illyrica and C. herzegoviniensis even share some same nuclear haplotypes, most likely due to incomplete lineage sorting . I. IC. eloric belt , 27]. F. FC. eloric belt . Even thric belt . In accooptychia , it is lsiccated . Geologisiccated , 31]. B. BC. elosiccated . High seGlaciations, that started in the final stage of Pliocene  furtherPronounced differences among geographically closely located and phylogenetically related species in present pattern of DNA polymorphism imply different demographic history of each species after their separation from the common ancestor, but also points out different probabilities of viability and future survival.C. jadovaensis contains extremely low genetic diversity, which together with skewed allele frequency distribution (great domination of one haplotype) is an indication of a strong evolutionary bottleneck. Therefore, even though C. jadovaensis is an old species (probably of the middle Pliocene origin), its intraspecific genetic diversity, that serves as a reservoir for coping with potential future environmental changes, was significantly reduced during an evolutionary bottleneck, which also reduces its viability potential and points out necessity of effective protection. Maximum likelihood estimation of its effective population size is even smaller than previously proposed Ne of 500 (50/500 rule  do doNe/N


Coryphopterus personatus. We find that populations of this species show tell-tale signs of chaotic genetic patchiness including: small, but significant, differences in genetic structure over short distances; a non-equilibrium or “chaotic” pattern of differentiation among locations in space; and within locus, within population deviations from the expectations of Hardy-Weinberg equilibrium (HWE). We show that despite having a pelagic larval stage, and a wide distribution across Caribbean coral reefs, this species forms groups of highly related individuals at small spatial scales (<10 metres). These spatially clustered family groups cause the observed deviations from HWE and local population differentiation, a finding that is rarely demonstrated, but could be more common than previously thought.The phenomenon of chaotic genetic patchiness is a pattern commonly seen in marine organisms, particularly those with demersal adults and pelagic larvae. This pattern is usually associated with sweepstakes recruitment and variable reproductive success. Here we investigate the biological underpinnings of this pattern in a species of marine goby  Marine organisms with dispersive pelagic larvae are expected to be characterized by little to no genetic differentiation among populations over large geographic areas due to high gene flow. However, many marine species exhibit slight, yet significant, levels of genetic heterogeneity over various local spatial scales ,2 which There are several mechanisms that could create CGP across populations of marine organisms  includinCoryphopterus personatus. Despite being wide-spread throughout the Caribbean little information exists about its life history, particularly information about larval duration, dispersal, and population structure. We seek to use observed patterns of genetic structure to test the kin-aggregation hypothesis and discuss the consequences of this mechanism for the ecology of this and similar species. We then propose competing alternative hypotheses for the mechanism leading to the formation of kin-aggregations.Here we investigate the population genetic structure of a shoaling marine goby, C. personatus were collected by divers from a 1-ha area on the fore-reef and pooled for storage in 95% ethanol. All animals were humanely euthanized in the field using a standard fish sedative, Tricaine mesylate (MS222). Live fish were placed in a seawater bath with MS222 added for 5 minutes or until breathing ceased. All collections were approved by and performed in accordance with the ethical guidelines of the University of Windsor and the Belize fisheries department, the Mexican Secretaría de Medio Ambiente y Recursos Naturales, and the Department of Natural Resources and Environment of Honduras. Genomic DNA was extracted from pectoral fin tissue of each individual following the silica-based 96-well plate protocol of Elphinstone et al.[et al. [et al. [2 and bovine serum albumin  and pairwise FST between sites were calculated across all loci using adegenet [ade4 to test for isolation-by-distance among sites using PD and FST [One of the common hallmarks of chaotic genetic patchiness is many deviations from Hardy-Weinberg equilibrium combined with weak but significant genetic differentiation between samples. To test for deviations from HWE exact tests for goodness of fit to the expectations of HWE were performed in adegenet . To test[HWxtest . Inbreed[gstudio . Both inadegenet . A Mante and FST . Signifir) was calculated between all pairs of individuals using the triadic likelihood estimator, which has been shown to be the least biased relatedness estimator in many circumstances, using the R package related [To test the hypothesis that increased levels of relatedness within sites explains the pattern of chaotic genetic patchiness observed here pairwise relatedness  and if those groups were pooled during collection, then the mean pairwise relatedness at the site level would be artificially deflated. We performed a Discriminant Analysis of Principal Components (DAPC) to identify genetic clusters of individuals within sites as implemented in adegenet ,24. All As further evidence of elevated relatedness and to minimize error associated with continuous metrics of relatedness, COLONY was used to determine the number of full- and half-sib dyads within clusters using the pair-likelihood score algorithm allowing for inbreeding . We assuC. personatus represented as nodes and sibling relationships  represented as edges [igraph [In order to determine if elevated relatedness observed in clusters is the result of multiple dyads of related individuals or a few large groups of relatives within clusters undirected networks were created for each cluster identified using DAPC with individual as edges . The meaas edges ,28. Netw [igraph . = 0.0018, p = 0.97, is for six or more loci  .Significant differences in : ~5 km) . Mantel p < 0.05, p <0.05, Within sample mean pairwise relatedness was 34–60% higher than the random expectation in four sites  when using a random combination of five loci . The proportion of full- and half-sibs within clusters was calculated to be 22.4±13.8%  and was significantly greater than clusters of random individuals generated by permutation p <0.05; ; Fig 3B.Coryphopterus personatus Click here for additional data file.S2 FigSignificant deviations at a particular sample by locus comparison indicated with a black box.(TIFF)Click here for additional data file.S3 FigThe red points indicate the minimum BIC which was then used as the most likely number of clusters present within the site.(TIFF)Click here for additional data file.S1 File(CSV)Click here for additional data file.S2 File(CSV)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.


Acer saccharum Marshall). Genetic diversity among seedlings, saplings, and mature trees was compared between selection cut and old‐growth forest stands in Québec, Canada. We found higher observed heterozygosity and a lower inbreeding coefficient in mature trees than in younger regeneration cohorts of both forest types. We detected a recent bottleneck in all stands undergoing selection cutting. Other genetic indices of diversity  were similar between forest types. We concluded that the effect of selection cutting on the genetic diversity of sugar maple was recent and no evidence of genetic erosion was detectable in Québec stands after one harvest. However, the cumulative effect of recurring applications of selection cutting in bottlenecked stands could lead to fixation of deleterious alleles, and this highlights the need for adopting better forest management practices.Selection cutting is a treatment that emulates tree‐by‐tree replacement for forests with uneven‐age structures. It creates small openings in large areas and often generates a more homogenous forest structure (fewer large leaving trees and defective trees) that differs from old‐growth forest. In this study, we evaluated whether this type of harvesting has an impact on genetic diversity of sugar maple ( Picea glauca [Moench] Voss; Rajora Pinus strobus L.; Buchert et al. Picea mariana [Miller] BSP; Perry and Bousquet Juglans nigra L.; Robichaud et al. Forest ecosystems are exposed to natural  and human  disturbances. The ongoing effects of climate change are being superimposed upon these disturbances in boreal  is a long‐lived deciduous tree that forms uneven‐aged stands. In addition to the syrup that it produces, this species has major economic value as saw timber in northeastern North America  and yellow birch  saplings were higher than those within control plots  are promoting sustainable forest management practices that include the protection of genetic diversity of forest trees. Using highly polymorphic microsatellite markers  and sugar maple–basswood (A. saccharum–Tilia americana L.) bioclimatic domains, where sugar maple is abundant . The selection cutting (SC) stands were part of a commercial harvest that consisted of the removal of 25–35% of tree basal area every 15–25 years MRNFPQ . The oldes; MRNQ . The loges; MRNQ , and OG2es; MRNQ . The resn = 20–22), saplings , and seedlings . Seedling age was determined as described by Graignic et al.  from 360 sugar maple individuals were collected in 2008. Three cohorts were sampled randomly per site: mature trees . All samples were genotyped for 18 variable microsatellite loci using PCR and genotyping protocols as previously reported  37 cycles in the PCR amplification procedure.DNA was extracted using Extract‐N‐AmpAT), mean number of alleles per locus (A), mean observed (HO) and expected (HE) heterozygosity, and the inbreeding coefficient (FIS) were estimated using fstat 2.9.3.2 , mean allelic richness (AR), mean observed heterozygosity (HO), mean expected heterozygosity (HE), pairwise FST, mean pairwise FST, and inbreeding coefficients (FIS) were estimated using fstat. Tests for heterozygote deficiency were performed using genepop . Tests were calculated for each stand using data for pooled individuals (PI), mature trees (M), saplings (Sa), and seedlings (S), separately. The genetic analysis was conducted using all the markers and also markers with < 10 alleles per locus. According to Balloux et al.  and between cohorts , we performed a linear mixed‐model analysis  were also compared between forest types and cohorts using fstat (1000 permutations).To compare amova) that is implemented in genalex 6.5b3  between pairs , between forest types (OG and SC) within pairs and within stands, and between pairs and between cohorts  within pairs and within cohorts for OG and SC.The structure of the genetic variation was determined using the hierarchical analysis of molecular variance (f ≥ 0.75), intermediate (0.75 >  f ≥ 0.25), low (0.25 >  f ≥ 0.01), and rare (f < 0.01). We also added two classes, low (0.25 >  f ≥ 0.05) and rare (f < 0.05), using a level of 0.05, as suggested by Marshall and Brown  and cohort within forest types.The effect of selection cutting on allele frequencies was tested using four classes: common . Four other markers also showed heterozygote deficiencies . These loci exhibited higher frequencies of null alleles (≥0.10) (Table S4). Their genetic structures (pairwise FST) were similar before and after correction for the presence of null alleles . All loci were considered independent because no significant linkage disequilibrium between pairs of loci within forest types was detected after Bonferroni correction. All loci (18) were used in further analyses.The total number of alleles per locus ranged from 3 to 28, while the mean number of alleles per locus ranged from 2.8 to 17.7 (Table S2). Five of the 18 loci  failed to meet HWE in three populations (Table S3). These deviations from HWE were due to heterozygote deficiencies (A) ranged from 9.2 (SC3) to 9.5  for stands and from 6.5 (M in SC2 and M in SC3) to 7.6 (M in OG3) for cohorts (Table AR) and expected heterozygosity (HE), using both LMM (in R) and fstat (Tables S5 and S6).The mean number of alleles per locus (HO) and the inbreeding coefficient (FIS) ranged from 0.440 (Sa in OG1) to 0.629 (M in OG3) and from 0.095 (M in OG3) to 0.348 (Sa in OG1), respectively , suggesting a departure from random mating in both forest types  compared to saplings  and seedlings  .Observed heterozygosity , but their respective values were very low .Means of FST values were detected using all markers (Table S7), subsequent analyses were performed with all markers included.Similar results were obtained when only the less variable markers  were included in the analysis . However, as more significant pairwise amova revealed that most of the variation  Tables  and S9.Recent bottlenecks were detected in the three SC stands, while none were apparent in the OG stands using IAM Table . In SC sA = 2.3, range: 1.10–5.00; HE = 0.131, range: 0.015–0.275), which is typical of Acer species and other tree species in northeastern North America (Table Acer platanoides L.), bigleaf maple (A. macrophyllum Pursh), American chestnut (Castanea dentata [Marsh.] Borkh.), and red oak (Quercus rubra L.), while lower levels have been observed in sycamore maple (A. pseudoplatanus L.) (Table A and AR than have been previously reported using the same 18 microsatellites (Table n = 60 vs n = 40). Diversity was found principally within stands in both studies . The presence of heterozygote deficiency is common in trees and has been reported for Acer species and trembling aspen (Populus tremuloides Michaux) using microsatellite markers  has been reported for trembling aspen and Korean maple (A. takesimense Nakai), across their natural geographic ranges  and higher FIS was observed in the younger cohorts  compared to mature sugar maple trees  of the regeneration cohorts  were observed in the selection cutting stands. Similar results have been obtained in other tree species. For example, fragmentation (clear‐cutting) did not change the level of genetic diversity in seedling populations of mountain hemlock (Tsuga mertensiana [Bong.] Carrière), relative to both adults and seedlings in old‐growth forests (Ally and Ritland Quercus robur L.)  in sugar maple (both for adult and seedling cohorts) were very low , which were indicative of unrelated individuals. It is plausible that extensive gene flow occurring between sugar maple populations had compensated for reductions in the number of potential parent trees (mature trees) within selection cutting stands and helped maintain a high level of diversity in seedling cohorts that have regenerated after harvesting. Khodwekar et al.   and regional scales  a high level of genetic diversity in sugar maple stands, (ii) low genetic differentiation between stands  of HO (a) and FIS (b) for cohorts  using markers with < 10 alleles per locus.Figure S2. Allele frequency distributions from old‐growth stands (black bars) and selection cut stands (open bars).Figure S3. Allele frequency distributions from old‐growth stands (black bars) and selection cut stands (open bars).Table S1. Summary of sample site coordinates and protection types.Table S2. Genetic variability estimates of microsatellite markers used in the Québec study of sugar maple (Acer saccharum).Table S3. Summary of P‐values for Hardy–Weinberg equilibrium using genepop.Table S4. Summary of null allele frequencies for each pair of loci and stands using freena.Table S5. Comparison of mean genetic variability estimates  between old‐growth (OG) and selection cut stands (SC) of sugar maple (Acer saccharum) in Québec for pooled individuals (PI), mature trees (M), saplings (Sa), seedlings (S) separately, between cohorts  for PI, OG and SC separately, and using all markers and markers with ≤ 10 alleles per locus (A).Table S6. Results of linear mixed‐effects models for genetic variability estimates in Québec, prior to model simplification, and using all markers (n = 324) and markers with ≤ 10 alleles per locus .Table S7. Pairwise comparisons of population FST  of pooled individuals, mature trees, saplings and seedlings of sugar maple (Acer saccharum) for the 6 stands, in Québec using all markers and markers with ≤ 10 alleles per locus (A). P‐values are above the diagonal.Table S8. Genetic variability estimates of sugar maple (Acer saccharum) stands in Outaouais, Québec, for mature trees (M), saplings (Sa), seedlings (S) and pooled individuals (PI) using markers with less than ten alleles per locus.Table S9. Number of alleles per frequency class, per cohorts of the stands.Table S10. Bottleneck results based on heterozygosity excess and mode shift, for mature trees (M), saplings (Sa), seedlings (S) and pooled individuals (PI).Click here for additional data file.


Gila robusta complex) to better resolve patterns of genetic variation among local populations in the lower Colorado River basin. This group has had a complex taxonomic history and previous molecular analyses failed to identify species diagnostic molecular markers. Our results supported previous molecular studies based on allozymes and DNA sequences, which found that most genetic variance was explained by differences among local populations. Samples from most localities were so divergent species-level diagnostic markers were not found. Some geographic samples were discordant with current taxonomy due to admixture or misidentification; therefore, additional morphological studies are necessary. Differences in spatial genetic structure were consistent with differences in connectivity of stream habitats, with the typically mainstem species, G. robusta, exhibiting greater genetic connectedness within the Gila River drainage. No species exhibited strong isolation by distance over the entire stream network, but the two species typically found in headwaters, G. nigra and G. intermedia, exhibited greater than expected genetic similarity between geographically proximate populations, and usually clustered with individuals from the same geographic location and/or sub-basin. These results highlight the significance of microevolutionary processes and importance of maintaining local populations to maximize evolutionary potential for this complex. Augmentation stocking as a conservation management strategy should only occur under extreme circumstances, and potential source populations should be geographically proximate stocks of the same species, especially for the headwater forms.Ten microsatellite loci were characterized for 34 locations from roundtail chub ( In deserts of western North America, long periods of aridity have been punctuated by occasional wet interludes , leadingGila  that are endemic to the Gila River basin in the southwestern United States. Like most other fishes of the region, the geographic distribution of populations has been reduced dramatically by human impacts; numbers are dwindling throughout their ranges, and remaining populations face myriad threats to their persistence  to 0.075 [locus 36]), consistent with Hardy-Weinberg equilibrium results discussed above . Therefore, the majority of variation was partitioned among populations: Θ (≈ FST) ranged from 0.227 (locus 300) to 0.384 (locus C2) with a significant jackknife average of 0.278 .Partitioning of genetic variation into within and among population components identified significant population structure. Jackknife estimates of total genetic variation (FST) was partitioned by either taxonomy (three species) or river drainage  instead of among species (FCT = 0.016). A similar result was obtained when samples were partitioned by drainage, with considerably more variation attributable to samples within drainages (FSC = 0.245) than among drainages (FCT = 0.052).To further examine the role of historical factors and geography, among population variation .When all sample populations from the FST for G. robusta, G. nigra, and G. intermedia were comparable, and not significantly different among species . However, when samples from the Bill Williams River drainage  were excluded, the average for G. robusta dropped dramatically (FST = 0.071) and there were significant differences among the three species (P = 0.009). This is reflected in the neighbor joining network of pairwise FST values  were shorter. Most nodes were not supported by bootstrap analysis with the exception of some pairs of samples in relatively close proximity.Analysis of population structure independently for each species provides a different picture. Estimates of T values , where mG. robusta, pairwise FST ranged from 0.03–0.42. When pairwise FST was plotted against stream distance, there were three main clusters of points and one outlier in the scatter diagram  between pairwise FST and stream distance that was statistically significant (P = 0.027). When BOL and TRT (from the Bill Williams River drainage) were excluded, the correlation became negative and was not statistically significant . Differences in connectivity between major drainages are also supported by the Mantel correlogram  was weak and not statistically significant. The Mantel correlogram  over all sample populations and not statistically significant. In the Mantel correlogram, the standardized Mantel statistic was significant only for the first distance class .For relogram  displayeot shape  resembletermedia . The corK = 28 with each identified group represented by single samples except for two, one containing samples EFE and UEG from G. intermedia and the other comprised of most G. robusta samples  and NMFKS from G. nigra. STRUCTURE was used to characterize assignment probability for all K from 2–34. There was inconsistency across replicates for more divergent samples , as indicated by their consistent assignment to different groups for each value of K and reduced h’ values for these replicates (K = 20 (ΔK = 16.0), and ln likelihood values also reached a plateau at K = 20, supporting that conclusion [BAPS and STRUCTURE were used to estimate the number of groups encompassed by the 34 samples. BAPS determined that plicates . The metnclusion .K = 20 and K = 28 ; G. nigra from Tonto Creek (ROC and SPRSA); and three separate groups of G. intermedia samples .Evaluation of assignment probability plots from STRUCTURE is difficult due to variation among replicates and the large number of distinct samples. Analyses from K , BON and SPRVE , and NMFKS and TON  are routinely difficult to assign to specific groups, especially at low values of K (≤ 10). In addition, individuals from three of these locations  exhibit signs of admixture at lower levels of K (≤ 10) as there is considerable probability of assignment to a group that includes most samples of G. robusta. Similar perspective of two sites assigned to G. nigra  indicates that individuals from these locations may actually belong to G. robusta.While it is difficult to obtain much information from examination of assignment plots for each K , severalG. robusta complex [Results of the present study were consistent with previous molecular genetic studies of the  complex , 22. MosFSTs yields a topology with short internodal and long terminal branches, with limited support for grouping of pairs of most populations, let alone those in the same taxonomic group . It is important to emphasize here that these patterns are probably not an artifact of Bayesian methods [FST values among populations; nor are they an artifact of microsatellite markers, as Schwemm [Characterization of microsatellite variation for 10 loci failed to group samples by recognized species, a result consistent with the allozyme study of DeMarais  and Schwic group . High digroup . Resolving hierarchy may require a substantially larger data set, with additional markers capable of resolving deeper evolutionary events.Despite the high level of divergence among populations for mtDNA and nuclear sequence data and microsatellites, diagnostic markers were not identified for species. There are a few potential explanations for our inability to identify diagnostic molecular markers. When genetic divergence among subpopulations is large, it is possible that differences among individual subpopulations can obscure differences at deeper hierarchical levels , reducing the effectiveness of such markers for identifying these higher categories. For example, Hedrick  noted thGila robusta complex may represent insularization of a historically panmictic population due to natural and artificial habitat fragmentation, with observed patterns of morphological variation due to convergent selection for a common habitat-based morphotype , analogous to convergent selection observed in sticklebacks [The observed pattern could also reflect how different evolutionary forces have shaped this complex. Genetic structure within the klebacks . AssessmGila nigra was a taxon of hybrid origin, resulting from introgression between G. intermedia and G. robusta. During dry periods, G. intermedia and G. robusta are expected to be geographically isolated in headwater and mainstem reaches, respectively; however, during wetter times these species could co-occur and interbreed, producing local hybrid swarms. As streams again became desiccated, these hybrid populations would become isolated in headwater reaches, allowing for divergence through local adaptation. It is such populations that Minckley and DeMarais [Gila nigra, which is morphologically intermediate to G. intermedia and G. robusta. Because the present analysis of microsatellite DNA loci did not identify diagnostic markers for each species, it is impossible to specifically test the introgression hypothesis here. Regardless of the reason, the lack of diagnostic molecular characters to date does not inform the status of G. intermedia, G. nigra, and G. robusta relative to their recognition as distinct species. Instead these results highlight the role that local evolution has played in shaping patterns of variation in these taxa and the importance of accounting for this variation when managing the complex.Past introgressive hybridization could also be partly responsible for the observed pattern. These species have never been reported to be sympatric naturally despite occurring in close proximity  . DeMaraiDeMarais  hypothesGila robusta complex. Similar patterns were identified with allozymes [G. robusta (the mainstem species) from the Gila River basin exhibiting increased variability and lower levels of divergence and hierarchical structure than G. nigra and G. intermedia, which are typically found in smaller, more isolated streams.The high level of genetic subdivision detected in the present study indicates that forces acting on location populations  are driving patterns of genetic variation within the llozymes  and nuclllozymes , indicatllozymes , with saF-statistic analyses of G. robusta identified considerable variation in allele frequencies among samples (FST = 0.191); however, removal of samples from the Bill Williams River reduced structure considerably (FST = 0.071). This inference was also supported by scatterplots of pairwise FST versus stream distance  coming from the same relatively small tributary network of the Salt River  from the Gila River basin, noting that patterns of genetic variation reflected expectations derived from consideration of life history and predicted levels of movement among locations. Studies of genetic variation in other cyprinids yielded variable results. In their study of mtDNA variation in Richardsonius, Houston et al. [Rhinichthys osculus–[Lepidomeda–[Gila atraria but also noted additional divergence associated with evolved life history differences within this species.Results from our study of the ene flow , which pene flow  contrastn et al.  found mo osculus–, 58 and pidomeda–), where pidomeda– also ideProsopium williamsoni) where variation was hierarchically arrayed into five distinct assemblages corresponding to major drainage basins, but with no differentiation within major drainage basins. This contrasts to other salmonids from the same region, which exhibit more divergence among locations than within drainages [Catostomus discobolus), an endemic to the Colorado River basin. They identified three evolutionarily significant units and seven management units within this species, with each group defined by a geomorphological barrier and/or isolation due to aridity. Together, these studies show that levels of genetic connectivity within drainages can vary among taxa based on hydrogeographical patterns and the life history of a species.Diversity of pattern is not restricted to cyprinids. Whiteley et al.  quantifirainages . Hopken rainages  also exaMolecular and morphological variation provides critical information for management of this complex. In such situations, it is critical to understand evolutionary processes that generated the underlying genetic diversity, allowing for preservation of the evolutionary legacy and adaptive potential of the complex. To maximize preservation of evolutionary potential, we advocate an approach that preserves available genetic diversity as identified by morphological and molecular analyses. Conservation units should be defined in a hierarchical manner, with genetically distinct units identified within each morphologically recognized species and each subbasin. Importance of local adaptation, drift, and gene flow makes it advisable to consider hydrogeography as well as divergence when developing conservation plans. Note, however, that we found some discrepancies between assignments based on microsatellite data and putative taxonomic status based on morphological traits as defined by Minckley and DeMarais . BecauseG. nigra may result from admixture of G. robusta and G. intermedia. Connectedness among populations is difficult to envision in today’s environment that includes both physical and biological barriers to exchange; however, there is no obvious resolution of those issues. Instead, we must overcome the general need of placing specific populations into categories and acknowledge that conservation should focus on preserving processes that generate observed patterns as well as the patterns themselves, thus requiring preservation of the entire complex and not just individual species.In addition to maintaining discreteness associated with geographic isolation and evolutionary independence, it is possible that Gila robusta complex only G. intermedia is listed under the Endangered Species Act, and it thus is the only one to receive protection. There currently is no accommodation for integrated conservation of the complex and little likelihood this will change. Given this restriction, we advocate managing each species independently by sub-drainage, with efforts to avoid mixing stocks from different sub-basins to avert negative consequences associated with outbreeding depression. This requires genetic characterization to match donor and recipient populations prior to translocation. Because of high levels of local differentiation, augmentation should only occur under extreme circumstances , with special care to preserve local stocks. Efforts to establish new populations should utilize the nearest geographic population as a source, while avoiding transfer across different subdrainages; this is especially important for headwater forms.Among members of the S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


LGM), many plant and animal taxa have expanded their ranges by migration from glacial refugia. Weeds of cultivation may have followed this trend or spread globally following the expansion of agriculture or ruderal habitats associated with human‐mediated disturbance. We tested whether the range expansion of the weed Silene vulgaris across Europe fit the classical model of postglacial expansion from southern refugia, or followed known routes of the expansion of human agricultural practices. We used species distribution modeling to predict spatial patterns of postglacial expansion and contrasted these with the patterns of human agricultural expansion. A population genetic analysis using microsatellite loci was then used to test which scenario was better supported by spatial patterns of genetic diversity and structure. Genetic diversity was highest in southern Europe and declined with increasing latitude. Locations of ancestral demes from genetic cluster analysis were consistent with areas of predicted refugia. Species distribution models showed the most suitable habitat in the LGM on the southern coasts of Europe. These results support the typical postglacial northward colonization from southern refugia while refuting the east‐to‐west agricultural spread as the main mode of expansion for S. vulgaris. We know that S. vulgaris has recently colonized many regions (including North America and other continents) through human‐mediated dispersal, but there is no evidence for a direct link between the Neolithic expansion of agriculture and current patterns of genetic diversity of S. vulgaris in Europe. Therefore, the history of range expansion of S. vulgaris likely began with postglacial expansion after the LGM, followed by more recent global dispersal by humans.Since the last glacial maximum ( Determining the limits of species geographic ranges and their movements over time has been a fundamental goal for ecology  whose distributions were correlated with historical agricultural routes  to reconstruct past species range dynamics, especially for taxa lacking fossil records. SDMs can also be used to hypothesize about the location of glacial refugia, augmenting findings based on population genetic data. SDMs use current species locations and environmental variables to fit habitat suitability models. These models can be trained on current climatic data and projected onto different climatic datasets to predict species ranges in different regions or periods of time. For past range dynamics, current climate suitability models can be projected onto the reconstructed climate data for the LGM to determine putative locations of glacial refugia  Garcke has become widespread during historical postglacial expansion or through more recent agricultural expansion. Past genetic studies of S. vulgaris have found phylogeographic signatures of ancestral demes dispersed throughout Europe, making it difficult to predict past range dynamics  followed the spread of agriculture, westward from the Middle East as humans created disturbed land and transported seeds.In this study, we used the combined application of phylogeographic analysis and SDM to examine whether the weedy plant S. vulgaris in Europe, with one to 10 individuals sampled per population. Samples were collected as seeds from maternal families or as leaf tissue dried on silica gel  heterozygosity estimates for the 45 populations of n > 1 were calculated in GenoDive  with the default program settings and 1,000,000 MCMC iterations after a burn‐in period of 500,000 iterations. The optimal number of clusters (K) was determined based on the  across the 10 replicates. Results were visualized using pie charts of population‐averaged ancestry coefficients and mapped using ArcGIS 10.1 .To assess geographic patterns of genetic ancestry in different historically isolated groups, we used Bayesian clustering to assign multilocus genotypes into clusters using the program STRUCTURE version 2.3 , we created the SDM using both climate as well as present‐day environmental variables that are not available for the LGM. For the “climate‐only” model (model C), we created a second SDM that only included climate variables that were also available for projecting into the LGM.To identify locations of putative glacial refugia, we used MaxEnt to create SDMs to predict areas of suitable habitat for http://www.gbif.org). Our initial dataset included 219 seed collection locations and 14,238 post‐1950 records available from GBIF. The reported and observed spatial resolution (where available) for the majority of these points was 10 km2 or less. However, inspection of the GBIF dataset revealed obvious reporting biases among different locations, which could result in spatial autocorrelation. Overall, GBIF occurrence locations were biased toward western Europe, with many points covering western Europe and extremely few in eastern Europe, where S. vulgaris is also widespread based on the Atlas Florae Europeae . The first two principal components accounted for 74.3% of the variation in the 19 bioclimatic variables (Appendix S2 in Supporting information). In model C + E, we also included data on soil type, land use, and human influence, as these variables could be important for describing the niche of a widespread weed of cultivation. For soil type, we used multiple datasets from the European Soil Database version 2 at 10 km2 resolution: full soil code of the soil typological unit (STU) from the World Reference Base (WRB) for Soil Resources (WRB‐FULL), dominant parent material of the STU (PAR‐MAT‐DOM), full soil code of the STU from the 1974 (modified CEC 1985) FAO‐UNESCO Soil Legend (FAO85‐FULL), and dominant land use (USE‐DOM). We also included a more detailed land‐use dataset from the European Environment Agency, the Corine Land Cover 2006 database, version 16, downloaded at 250‐m resolution (Copyright © European Environment Agency). For another measure of human disturbance, we used the Last of the Wild version 2 Human Influence Index dataset at 1‐km resolution . The first two principal components explain 92.8% of the variation in the datasets (Appendix S2). The first two bioclimatic and two environment‐based principal components were used as environmental variables for the C + E model  output maps provided by MaxEnt  to test for associations between standardized heterozygosity and three predictors: latitude, longitude, and LGM climate suitability. Based on our two competing hypotheses, we predicted a significant correlation between diversity and longitude would support the agricultural expansion hypothesis, whereas a significant correlation between diversity and latitude or LGM suitability would support the postglacial expansion hypothesis.S. vulgaris across Europe  was an adequate fit based on test AUC  and the known current distribution of the species. An AUC value above 0.7 indicates “fair” model performance Swets , and thees Table . A similThe LGM prediction from the CCSM model showed moderately suitable habitat in most of the regions of Europe not covered by Eurasian ice sheets Fig.  with higS. vulgaris, the MESS map (Appendix S5) showed that the variables in our model were only extrapolated from their training range into northern Europe, predominantly in the location of the Eurasian ice sheet, above 52°N. Even with the extrapolation, the MaxEnt models correctly predicted very low suitability in this area, except farther north near Finland and Russia where there was the largest degree of extrapolation.A common concern with the SDM approach is the extent that predictions of suitable habitat can be biased when extrapolating into environmental space outside the known area of occurrence. For Q < 0.75 for K = 2) were removed from the analysis. In addition, when admixture is removed, there was a significant (P = 0.034) positive relationship between LGM suitability and heterozygosity, consistent with high genetic diversity in putative southern refugia.Spearman's rank correlation showed that standardized heterozygosity significantly declined with latitude Table . GeneticS. vulgaris throughout Europe in order to reconstruct past range dynamics and test two competing hypotheses for range expansion: postglacial expansion from southern refugia versus westward agricultural expansion. The results support the hypothesis of postglacial expansion from southern refugia and are not consistent with predictions of agricultural spread from the east. These findings are in contrast with the only other study that has contrasted the same hypotheses for two weeds of cultivation, L. perenne and L. rigidum  is consistent with range expansion out of southern refugia, in which founder effects (genetic drift during population establishment) reduced diversity along the pathways of expansion. Climate suitability in the LGM model was also significantly negatively correlated with latitude, providing further evidence that S. vulgaris likely formed southern refugial populations. Taken together, the reconstruction of the climate during the LGM and current patterns of genetic diversity are consistent with expectations of a postglacial expansion from southern European refugia. Further, when admixed individuals were removed from the analysis, there was a significant positive correlation between LGM suitability and heterozygosity. By contrast, there was not a significant correlation between heterozygosity and longitude, and the sign of this correlation was negative, contrary to hypotheses of S. vulgaris migrating westward during agricultural expansion. There was also low habitat suitability in the Middle East in the current and LGM models, further supporting that the center of origin was not located in this area. These findings exemplify the benefit of combining genetic, spatial, and suitability data to understand range dynamics and discriminate between competing hypotheses for the historical spread of weedy plants.Genetic diversity of In western Europe, west of Germany, and Italy, the STRUCTURE analysis found evidence for two demes consistent with expectations for divergent groups of descendants from different glacial refugia  in this study, but the results did not differ when removing these individuals from the analysis.Another possible explanation for the unexpected similarity between eastern and western regions of the Mediterranean is that there are three refugial groups originating in the three southern peninsulas, but STRUCTURE was unable to separate a third cluster based on the data. The STRUCTURE analysis showed moderate support for S. vulgaris, making it possible for refugia to exist on all three southern peninsulas as seen in many other species (Taberlet et al. S. vulgaris within the Central and eastern European regions based on the SDMs presented here, the genetic data do not allow a determination of the specific number and locations of which of these refugia were likely to be occupied. However, the models suggest locations where S. vulgaris could exist based on SDM habitat suitability within the broad regions predicted by the genetic data. Further genetic sampling using more markers could clarify the results and make it possible to match predicted refugia locations with areas of suitable habitat.The CCSM model hindcasted suitable habitat on the entire coastline of the Iberian Peninsula, western coast of France, Italy and surrounding islands, southern Greece, north of Greece around Serbia, coastal areas around the Black Sea, and the northern coast of Africa. These areas could all have potentially served as glacial refugia for S. vulgaris distribution showed that climate was by far the primary explanatory factor compared to other land‐based attributes which contributed little predictive value. Therefore, S. vulgaris' strong dependence on climate strengthens the findings of the LGM suitability models, and further suggests that S. vulgaris tracked suitable climates since the LGM, supporting the postglacial expansion hypothesis.Many studies make the assumption that climate variables alone are sufficient for projecting species distributions back into the LGM, as many other environmental predictors are unavailable for this period. However, it is important to check this assumption before projecting LGM distributions, as the LGM prediction would most likely become biased if climate were not the primary environmental driver determining the species' distribution. The model of current Lolium species (Balfourier et al. Arabidopsis thaliana (Beck et al. S. vulgaris support the hypothesis of postglacial expansion from southern refugia in Europe. This finding builds upon previous genetic studies on S. vulgaris with the addition of SDM analyses and uses the pairing of SDMs and population genetics to directly test these two competing hypotheses. As with past phylogeographic studies of weedy plants, our results did not show the exact expected pattern of postglacial expansion seen in other species, but still adequately support many aspects of this route, especially when compared with the alternative agriculture expansion. Predictions of refugial locations in the future would benefit from further sampling in Italy and eastern Europe, as well as expanded sampling across the genome. Finally, we found that including data from environmental variables other than climate and the known distribution of ice sheets and permafrost can significantly enhance confidence in the distribution of species during the LGM, and should be considered in future SDM studies.Postglacial expansion from glacial refugia since the LGM has been supported for a variety of taxa on many different parts of the planet. Many of these studies use genetic analyses and are beginning to use SDM as a powerful complement. However, one type of species that may not adhere to typical climate‐tracking trends is a widespread weed dispersed by humans and capable of growing in a variety of climates. Weeds of cultivation may have spread primarily by agriculture rather than by postglacial expansion, as was recently found for two None declared.Appendix S1. Standardized heterozygosity and observed heterozygosity interpolated using the inverse distance weighting method.Appendix S2. Principal component loadings for the climatic and land‐based variables used in the species distribution models.Appendix S3. Silene vulgaris climate suitability in Europe during the last glacial maximum based on the MIROC climate scenario.Appendix S4. Current climate suitability model results for Silene vulgaris in Europe.Appendix S5. MESS map for the climate‐only SDM showing the extrapolation of variables in the CCSM climate scenario.Click here for additional data file.


Panulirus penicillatus is a highly valuable species which is widely distributed in Indo-West Pacific and Eastern Pacific regions. Mitochondrial DNA control region sequences (566–571 bp) were determined to investigate the population genetic structure of this species in the Indian Ocean. In total, 236 adult individuals of Panulirus penicillatus were collected from five locations in the Indian Ocean region. Almost all individuals had a unique haplotype. Intrapopulation haplotype (h) and nucleotide (π) diversities were high for each locality, ranging from h = 0.9986–1.0000 and π = 0.031593–0.043441. We observed distinct genetic isolation of population located at the northwestern and southwestern edge of the species range. Gene flow was found within localities in the central and eastern region of the Indian Ocean, probably resulting from an extended planktonic larval stage and prevailing ocean currents.The pronghorn spiny lobster  Panulirus penicillatus is widely distributed in the Indo-West Pacific and Eastern Pacific regions. It is found in tropical and adjacent regions from South-Eastern Africa, the Red Sea, Southern India, the Southeast Asian Archipelago, Japan, Northern Australia, and the Southern and Western Pacific Islands, to Hawaii, the Galápagos Islands, and other islands of the Eastern Pacific [P. penicillatus supports considerable fisheries in Indo-Pacific regions. The species is fished wherever it occurs. Much attention has been paid to biological investigation of young and adult stages. Recognition of stocks is complicated by the life cycle of pronghorn spiny lobster, primarily because of the potential for long-range dispersal during its planktonic larvae. The early life history of spiny lobsters consists of a drifting larval period adapted for a relatively long-term stay in the open ocean, extending from several months to more than a year, with many possibilities for dispersal through ocean currents [The pronghorn spiny lobster  Pacific ,2,3. P. currents ,5.Several molecular techniques, including allozymes, restriction fragment length polymorphism (RFLP), mitochondrial DNA sequencing, and microsatellites, have been used in studying genetic variability in marine organisms at the population level ,7. MitocP. penicillatus, East-West Pacific differentiation using 16s and COI (cytochrome oxidase I) marker was described by Chow et al. [P. penicillatus in the Pacific region using highly polymorphic marker, mitochondrial control region by Abdullah et al. [P. penicillatus.There recently been studies of w et al. , and thih et al. . In thisP. penicillatus in the Indian Ocean region. The aim of the present study is to investigate population genetic structuring in a widely distributed P. penicillatus in the Indian Ocean region. We address this question using mitochondrial control region sequences data from Red Sea, Jeddah, Saudi Arabia; Fort Dauphin, Madagascar; Nilandhoo atoll, Maldives; Aceh and Java Sea, Indonesia  were found: 172 were parsimony informative and 74 were singletons. In total, 231 haplotypes were identified, and nearly all sequenced individuals had a unique haplotype  theP. penicillatus. The values generated in this study are comparable with those derived from the mtDNA control region sequences for the crustaceans Farfantepenaeus aztecus, Litopenaeus setiferus and Sergia lucens [h) within the geographical populations was high, ranging from 0.9986 in Nilandhoo atoll, Maldives to 1.0000 in Fort Dauphin, Madagascar. Nucleotide diversity (π) was generally high, ranging from 0.031593 in Red Sea, Jeddah, Saudi Arabia to 0.043441 in Fort Dauphin, Madagascar , which are relatively high values compared with the control region diversities of other Panulirus spp. (P. inflatus: h = 0.957–1.0000 and π = 0.019–0.028 [P. argus: h = 0.667–1.0000 and π = 0.003–0.066 [P. homarus: h = 0.985–1.0000 and π = 0.031–0.041 [P. penicillatus (h = 0.9996–1.0000 and π = 0.0255–0.0448) [In the present study, the mtDNA control region exhibited high genetic variability  and sim–0.0448) .P. penicillatus may be maintained by its large population size. In fact, because nearly all individuals represented a unique haplotype, an investigation based on sequencing the control region would require a very large sample size to detect common haplotypes and associate them with particular geographic areas [The high genetic variability of the control region in ic areas . The evoic areas .P. penicillatus with other localities. This suggests that the geographic isolation may be due to contemporary isolation/gene flow or prior isolation with recent gene flow, although the overall level of genetic exchange must be below that required to homogenize the populations.Genetic analysis of mitochondrial control region revealed distinct population at the northwestern and southwestern margin of geographic range of P. penicillatus, which explained high genetic variations (51%) between the regions. High genetic variation (85%) was also observed in previous study between eastern Pacific and west-central Pacific population of P. penicillatus [Congruent with the pattern observed in the network haplotypes , we founcillatus . To analSTΦ comparisons of each data set found no heterogeneity as observed within samples from the central-eastern region  (data not shown), although it was a limited CTΦ  samples were pooled, suggested by population tree, haplotype network and ocean division. In addition, our preliminary analyses of pairwise P. penicillatus in the Indian Ocean region do not represent a genetically homogenous assemblage. Pairwise STΦ comparisons detected genetic structure between the central-eastern region  and other populations (Madagascar and Red Sea)  . A relat berndti  and amphhium lar . The prehium lar ,22.STΦ values  with supported by significant and high pairwise T values , UPGMA tulations  this resulations , other s serrata ,24, have serrata ,26,27. M regions .P. penicillatus phyllosoma larvae may be necessary since genetic distinction in Red Sea populations may indicate the potential self-recruitment of phyllosoma larvae in this region. Although phyllosoma larvae are weak swimmers, they have the ability to maintain their vertical position in the water column [et al. [P. cygnus was a self-recruiting species, restricted to Western Australia. A more recent simulation study of P. argus found the average dispersal of P. argus in the Caribbean Sea to be possibly only 200–400 km, suggesting that larval behavior such as vertical migration, together with a retentive oceanographic environment, may increase the potential for self-recruitment [Further analysis on recruitment of r column . Larval  [et al. , discoveruitment .P. penicillatus inhabiting the central-eastern region of Indian Ocean: Nilandhoo atoll, Maldives; Aceh and Java Sea, Indonesia. The early life history of spiny lobsters consists of a drifting larval period adapted for a relatively long-term stay in the open ocean, extending from several months to more than a year, with many possibilities for dispersal through ocean currents [P. penicillatus phyllosoma [No population structure was found in the localites of currents ,5. Long yllosoma , coupledyllosoma , while tyllosoma ,34.D and Fu’ F tests. The significant negative values obtained in these P. penicillatus are often associated which experienced a population expansion during their geographic history [The neutrality of mutations in the mtDNA control region was rejected on the basis of Tajima’s  history . This pa history ,36,37.Although mtDNA has been proven to be a very useful marker, its use is not without complications. Due to the exclusively maternal inheritance mtDNA represents a marker strictly for historical process in females; they can potentially introgress between species ,39, and Panulirus penicillatus were collected from five localities in the Indian Ocean region including Red Sea, Jeddah, Saudi Arabia; Fort Dauphin, Madagascar; Nilandhoo atoll, Maldives; Aceh and Java Sea, Indonesia , the proportion of variation among population within groups (ScΦ), and the proportion of variation within populations (STΦ). We ran preliminary analysis of pairwise STΦ comparisons on each data sheet. The statistical significance of STΦ value was tested by 1000 permutations in Arlequin (ver. 3.5) software [STΦ statistics did not differ from zero. An UPGMA tree based on ΦST values was constructed using Neighbor in Phylip (ver. 3.6) [D [F [Sequence data were aligned using ClustalX , with desoftware . The Bonsoftware  was useder. 3.6) ,48. The er. 3.6) , for theer. 3.6) . Past de 3.6) [D , and Fu’6) [D [F , estimatP. penicillatus populations located at the northwestern and southwestern edge of the Indian Ocean region with other populations. Relatively small structure was found between southwestern population and central-eastern, while vast majority structures were observed between the northwestern edge population and the other populations. Despite a lengthy larval period, oceanographic currents are likely to create barriers and impact routes and directions of larval dispersal in the northwestern edge population that may have limited genetic exchange with other regions. These results have implications for fisheries management in the region; this should essentially provide information for ascertaining stock boundaries to better evaluate the long-term needs of spiny lobster management for this valuable species.The present study detected genetic structure in P. penicillatus was belonging to a single unique panmictic population or at least several breeding grounds with significant exchange of genetic material. Long larval life (8.3–9.4 months) of the P. penicillatus coupled with the considerable impact of oceanic current systems or gyres  may explain the observed patterns. Furthermore, transportation of larvae likely occurred indirectly via stepping stones by the currents, and a small amount of larval dispersal over some years might result in genetic homogeneity.At the scale of the central-eastern Indian Ocean, results suggested that


Cuvierina in relation to their biogeographic distribution across the world’s oceans. We aimed to assess species boundaries and to reconstruct their evolutionary history.Shelled pteropods are planktonic gastropods that are potentially good indicators of the effects of ocean acidification. They also have high potential for the study of zooplankton evolution because they are metazoan plankton with a good fossil record. We investigated phenotypic and genetic variation in pteropods belonging to the genus We distinguished six morphotypes based on geometric morphometric analyses of shells from 926 museum and 113 fresh specimens. These morphotypes have distinct geographic distributions across the Atlantic, Pacific and Indian oceans, and belong to three major genetic clades based on COI and 28S DNA sequence data. Using a fossil-calibrated phylogeny, we estimated that these clades separated in the Late Oligocene and Early to Middle Miocene. We found evidence for ecological differentiation among all morphotypes based on ecological niche modelling with sea surface temperature, salinity and phytoplankton biomass as primary determinants. Across all analyses, we found highly congruent patterns of differentiation suggesting species level divergences between morphotypes. However, we also found distinct morphotypes (e.g. in the Atlantic Ocean) that were ecologically, but not genetically differentiated.Cuvierina taxa, they may not respond equally to future ocean changes and may not be equally sensitive to ocean acidification. Our findings support the view that ecological differentiation may be an important driving force in the speciation of zooplankton.Given the distinct ecological and phenotypic specializations found among both described and undescribed The online version of this article (doi:10.1186/s12862-015-0310-8) contains supplementary material, which is available to authorized users. Pteropods are a group of heterobranch gastropods  that are2 (e.g. ), and he2 e.g. -8). Shel. Shel2 ..2 . A r. A r13])trations ,12. HencDiacavolinia, and emphasized the inadequacy of current systematic understanding of this genus )is genus . The taxiewed by .Cuvierina, an excellent model group for an integrative study of zooplankton because it has a worldwide distribution, it is abundantly present in museum collections, and has a well-described fossil record Cuvierinag shells . RWs wera priori classification of reference museum specimens (those identified by )r waters . Furtherm waters , suggestof 0.51, ) and Lit.35–0.7, ).C. urceolaris , C. columnella and C. pacifica N  morphotypes. The shell of C. urceolaris is bottle-shaped with pronounced micro-ornamentation similar to C. cancapae. This may be explained by the same hypothesis as for C. cancapae in the Atlantic Ocean, because C. urceolaris also occurs in the warmest waters with lowest viscosities in the Indo-Pacific. Similar to our findings, other studies have reported much stronger phenotypic divergence than  genetic divergence in marine populations , in parns e.g. ,95-97).-97.C. urCuvierina are found in several other open ocean plankton taxa, such as Diacavolinia pteropods, foraminifers, krill and copepods. Diacavolinia flexipes, D. angulosa and D. grayi show a zonation of equatorial and subtropical distribution patterns in the Indian Ocean similar to C. urceolaris and C. columnella distributions [C. pacifica S, the krill species Euphausia gibba is endemic to the large South-Pacific gyre [C. atlantica in the Atlantic Ocean is also observed for the pelagic copepod genus Pleuromamma [Haloptilus longicornis [H. longicornis, the C. atlantica morphotype is unable to reach the Indian Ocean around South Africa, where a warm current from the Indian Ocean impinges a cold current from the Southern Ocean. Numerous clades of planktonic foraminifers also have distribution patterns that are predominantly based on latitudinal zonation of water masses .Cuvierina  and the more bottle-shaped Urceolarica (C. urceolaris and C. cancapae) as proposed by [C. pacifica N and C. pacifica S are distinct species because they belong to different genetic clades (COI and 28S) and are morphologically disjunct. Because the holotype of C. pacifica belongs to the C. pacifica S morphotype, we propose a limitation of the description of C. pacifica to C. pacifica S and a new taxonomic description for the C. pacifica N morphotype . However, because we found no molecular evidence to support the species status of morphotypes within the Atlantic and Indo-Pacific clades, we consider it possible that they represent ecophenotypic varieties or incipient species.Based on results from this study, we do not find support for a subdivision into the subgenera posed by ,38. We fCuvierina morphotypes that is influenced by decreasing connectivity between the three world oceans since the Miocene  mya (Late Oligocene), but it is unknown for how long the South Pacific clade has been endemic to the large South Pacific gyre. The divergence between the Atlantic and Indo-Pacific clades of 16.1 (24.5–7) mya (Miocene) coincides with the Tethys Sea closure, suggesting a vicariant model of divergence, with the Indo-Pacific clade diverging from the Atlantic clade in the Indian Ocean and later dispersing to the Pacific basin. Until the Terminal Tethyan Event –18 mya, ), the At–18 mya, ,106. Dis–18 mya, ). HoweveC. columnella and C. urceolaris during glacial periods when sea levels dropped  does not seem to represent a physical barrier today, but could have functioned as such between Indian and Pacific populations of ed (e.g. ). This icopepods ,108. In  xiphias .C. atlantica suggest that there is a significant degree of structuring between populations in the northern and southern subtropical gyre systems in the Atlantic Ocean. If genetic structure is interpreted in light of gene flow, this implies a more limited dispersal than expected for open ocean holoplankton. Combined with the disjunct distribution of C. atlantica populations, this suggests that the equatorial upwelling waters in the Atlantic represent a significant barrier to dispersal. This equatorial dispersal barrier was also found for the mesopelagic copepod Haloptilus longicornis, for which a genetic break was observed between populations in the northern and southern oligotrophic Atlantic gyres [C. cancapae.Physical and ecosystem properties of different Atlantic water masses may be key to incipient speciation within the Atlantic clade. Our population genetic analyses of ic gyres . By contCuvierina taxa, they may not respond equally to future ocean changes and may not be equally sensitive to ocean acidification. Because the presence and strength of open ocean dispersal barriers depends on the ecological niche of a species, the capacity of a species to adapt to or to track a suitable habitat may vary across closely-related taxa [Given the distinct ecological and phenotypic specializations found among both described and undescribed ted taxa . AlthougThe data set supporting the results of this article is available at the Dryad repository, , and DNA