Radix balthica. This analysis was based on microsatellite variation and mitochondrial haplotypes using Generalised Linear Statistical Modelling in a Model Selection framework.Factors and processes shaping the population structure and spatial distribution of genetic diversity across a species' distribution range are important in determining the range limits. We comprehensively analysed the influence of recurrent and historic factors and processes on the population genetic structure, mating system and the distribution of genetic variability of the pulmonate freshwater snail R. balthica were found throughout North-Western Europe with range margins marked either by dispersal barriers or the presence of other Radix taxa. Overall, the population structure was characterised by distance independent passive dispersal mainly along a Southwest-Northeast axis, the absence of isolation-by-distance together with rather isolated and genetically depauperated populations compared to the variation present in the entire species due to strong local drift. A recent, climate driven range expansion explained most of the variance in genetic variation, reducing at least temporarily the genetic variability in this area. Other factors such as geographic marginality and dispersal barriers play only a minor role.Populations of To our knowledge, such a population structure has rarely been reported before. It might nevertheless be typical for passively dispersed, patchily distributed taxa (e.g. freshwater invertebrates). The strong local drift implied in such a structure is expected to erode genetic variation at both neutral and coding loci and thus probably diminish evolutionary potential. This study shows that the analysis of multiple factors is crucial for the inference of the processes shaping the distribution of genetic variation throughout species ranges. One of the major unsolved questions in evolutionary biology is why the vast majority of species fails to adapt to conditions outside their present niche and, as a consequence, usually exhibit geographically confined range limits -5. TheorOne of the most influential framework on the distribution of genetic variation across species' ranges is the Abundant-Centre Hypothesis (ACH) . It statAsymmetrical gene-flow from larger sized, more abundant central populations to the range margins can counteract the previously described setting. Such gene-flow may prevent local adaptation by constantly supplying 'maladapted' alleles from the core range into marginal populations . Under tA recent exhaustive review across different taxa showed that in about two out of three empirical studies genetic variability indeed decreased and population differentiation increased towards range margins, as expected under the ACH . HoweverGenetic variability across species ranges may also be influenced by local biotic interactions, in particular by competition with closely related, ecologically similar species or hybridisation with them in parapatric settings . While tAlso contingent historic events like presence of geological dispersal barriers, population fragmentations and range expansions e.g. due to Pleistocene glaciations in temperate regions may have exerted their lasting influence on the distribution of genetic variation within a species. Here, the expectations on the distribution depend on the actual population history and may include decrease in genetic variation due to founder effects and population bottlenecks or an increase e.g. in secondary contact areas of previously isolated lineages ,14-18. TWhile most factors act on genetic variability in a one-way direction, the mating system both influences genetic variability and its prevalence can be driven by at least some of the above described factors. On the one hand, a mixed mating system decreases the effective population size. Populations with a mixed mating system or purely selfing populations are therefore expected to experience increased drift . Local dThe factors described above provide alternative, but not necessarily mutually exclusive explanations for the distribution of genetic variation. Thus testing only a single factor at a time may lead to erroneous conclusions on the factors and processes governing the distribution of genetic variability over species' ranges . EmpiricRadix balthica as model organism. This species is one of several species in the morphologically cryptic species complex Radix Montfort 1810 [R. balthica is suspected to have a mixed mating system [In the present study, we tackled this issue using a pulmonate freshwater snail ort 1810 . It is dort 1810 ,24, R. bg system . Withoutg system ,26. Likeg system ,28. WithR. balthica using statistical modelling and model selection techniques.Since more than a single factor may contribute to the distribution of genetic variability, we analysed the population structure, mating system and simultaneously tested the influence of the various factors outlined above by assessing the geographic distribution of supposedly neutral nuclear and mitochondrial genetic variability across the species range of R. balthica with DNA barcoding. Together with previously identified R. balthica populations, this resulted in the first comprehensive molecularly confirmed estimate of the species range . The Bayesian cluster analysis indicated that the hypothesis of 20 clusters was most strongly supported by the data . The colour coded cluster memberships of each individual are depicted in Figure FST s against the geographical distance . It was thus not necessary to correct the following analyses for geographical distance among populations [The average overall ulations .EH) was 0.448 +/- 0.168 (mean +/- s.d.) with an observed minimum of 0.068 (sampling site SKJ) and maximum of 0.955 (FTO). The average number of alleles per locus (A) was 15.6, the overall rarefied value per sampling site and locus 3.207 +/- 1.312. The observed minimum value was 1.370 (SKJ), the maximum 7.734 (SSO). The correlation between E Hand A was very high .The average expected heterozygosity over all loci .At least partial selfing was inferred for 47 out of 81 populations (58%). The average population selfing rate was 0.20 +/- 0.25. The maximum value observed was a completely selfing population . The estimated degree of self-fertilisation was only poorly correlated to the genetic variability measures A total of 132 mitochondrial haplotypes was identified over the species range. After rarefaction, 4.085 +/- 2.193 haplotypes per sampling site were observed, ranging from a single haplotype  up to 11.765 different haplotypes (SKR).All measures of diversity per sampling site and a graphical representation of their spatial distribution can be found in Additional File It was possible to test the 34 non-selfing populations on signs of recent population bottlenecks. Nine 26%) of these showed a significant heterozygous excess. The populations with recent bottlenecks were widely distributed over the species range, but not in the recent expansion area  was above the overall average in the sampling sites grouped by the predictor variables dispersal barrier (bar), biotic interaction (bio), LGM refugia (ref), Holocene expansion (hol) and distance to range limit (lim). By contrast, it was reduced relative to the mean in the expansion sampling sites (exp) and environmentally marginal sites  was on average lower than the overall average in sites grouped by the predictor variables bar, bio, ref, hol and size, while it was higher in exp, marg and lim. However, the variance was very high in each group  was increased at sites with presumed biotic interaction (bio) and to a lesser extent in the Holocene expansion sites (hol). In all other groupings, the haplotype diversity was decreased with the strongest effect observed in the recent expansion sites .Almost all models explained a portion of variance significantly larger than zero at the 5% error probability level or less. The models highlighted below were all highly significant (EH) was explained by the additive effect of four models with two or three variables. It was best supported by the additive effect of dispersal barriers (bar) and expansion area (exp) .The distribution of expected heterozygosity (A was best explained by the additive effect of the factors biotic interaction (bio) and exp (Akaike weight 0.88), followed by bar, exp and environmental marginality (marg) .The rarefied number of alleles marg, exp, lim and size (Akaike weight 0.97). However, only 49% of the total variance was explained by this model  .The haplotype variability was best explained by the additive effect of the model with three variables exp), none of the other predictors yielded a significantly stronger or weaker structured grouping. In the recent expansion area, the average population pair-wise FST was 0.198 units higher than in the remaining range. This difference proved to be significantly different from zero with an error probability of less than 0.001 according to the randomisation test employed  that we will discuss them together hereafter . This means that high selfing rates are not predominantly responsible for the loss of genetic variability. A low correlation further allowed investigating whether the factors considered influenced the mating system. The predominant factor in all models with substantial support was the recent, climate driven range expansion (exp), which lowered the level of variability for all genetic markers considerably . Thus, the effect of drift in small populations may be difficult to estimate from habitat size alone, but depend rather on the mating system or the founding history. Second, freshwater snail populations are often subject to high population turnover or size fluctuations [Population size, as rather crudely estimated from the size of the water body, had no detectable effect on the distribution of genetic variability Figure , Table 2tuations ,52 whichThe size of a water body, however, did have an effect on the selfing rate Figure , Table 2lim) that received substantial support in the data  the larger part of the present species range and ii) that with the possible exception of the North-West, also the range margins were adequately sampled.In the case of  species . Therefoeography , it cannnt study . In SwedHowever, the ACH does not predict precisely, how variation should decrease towards range margins . By testmarg + exp + lim + size) explained not even half of the variance in selfing, indicating that probably additional, untested factors significantly shaped the mating system.The factors evaluated here had also an impact on the variability in the mating system. The common quality of the factors identified to trigger changes in mating system towards more self-fertilisation seemed to be increased population turn-over Table . ActuallR. balthica was found to be passive, probably bird-mediated, distance independent dispersal along a Southwest to Northeast axis. Apart from the expected effects of a recent range expansion, other processes or factors suggested in the literature had only a minor effect on the geographic distribution of genetic variability. This dispersal mode led to high overall genetic variability, but locally impoverished populations. Low local variability, along with high population turn-over  and range-wide dispersal dynamics argue against a high evolutionary potential [R. balthica on a regional scale [The process mainly responsible for the population structure and distribution of genetic variability measured as nuclear and mitochondrial across the species range of otential ,58, Thisotential . A recenal scale . HoweverR. balthica.The particular population structure observed is thus probably the main explanation for the previously inferred intraspecific climatic niche conservatism from the LGM to the present day . It is lAs in particular species inhabiting ephemeral habitats,  show similar population structures and dispersal dynamics , the conRadix populations were sampled in the presumed range throughout North-Western Europe. Because taxonomic identity cannot be deduced from morphologic features in this genus, all individuals used in this study were DNA barcoded, for which a COI sequence of less than 300 bp proved sufficient for unequivocal species identification [R. auricularia[habitat ad M Balthici littera") of Radix balthica . We associated therefore the biological entity MOTU2 with the taxonomic name R. balthica and will use this name hereafter.fication . In thisricularia), therefR. balthica . Additionally, we used stored DNA and published data, respectively, from individuals sampled for previous studies [To avoid potential Wahlund effects, individuals were sampled along a shoreline of max. 12 m, which is well within the estimated neighbourhood area of  studies ,33,46.et al. [DNA was extracted using glass fibre DNA extraction after a protocol developed by the Canadian Centre of DNA Barcoding . Cytochret al. . SequencENEMAPPER 4.0 software (Applied Biosystems).All snails were genotyped at eight highly polymorphic microsatellite loci . MultiplFST s and the overall FST for both nuclear and mitochondrial markers, calculated in Arlequin 3.1 [TRUCTURE[TRUCTURE implements the Markov Chain Monte Carlo (MCMC) algorithm for the generalized Bayesian clustering method to classify individuals into clusters using genotypic data of unlinked markers. We used the location prior option, implementing the assumption that individuals from the same location have a higher probability to stem from the same population than individuals sampled at different locations. A uniform prior for alpha was applied for all populations, with an initial value of 1. We used the admixture model assuming a number of clusters from K = 1 to K = 40. All MCMC runs were repeated five times for each value of K for 200,000 generations with 25,000 burn-in steps. We used the maximum LnP(D) value to infer the most likely number of clusters, given the data. LnP(D) is the log likelihood of the observed genotype distribution in K clusters. The analysis was repeated also without location prior, as recommended by Falush et al. [ST matrix using NTSYSpc version 2.0 and plotted it on a map. Populations pair-wise linearised FST s were calculated in Arlequin 3.1 [The population genetic structure was estimated by population pair-wise quin 3.1 . Additio[TRUCTURE. STRUCTUh et al. . To visuquin 3.1 .R. balthica, we plotted the population pair-wise linearised FST estimates for the microsatellite data against the geographic distances among the respective populations. Statistical significance was tested with the Mantel's test option in Arlequin 3.1 [Spatial autocorrelation between populations may bias tests for difference in genetic distance and genetic variation . To testquin 3.1  with 10,EH) and allelic richness (A), expressed as average number of alleles per locus. Although both measures are interrelated, A is more affected by stochastic drift than E Hand therefore the more sensitive measure [E Hfor each population using ARLEQUIN 3.1 [ENALEX[A to the minimum number of individuals sampled per site, applying a method of rarefaction recommended for standardisation of allelic richness [For each sampling site with at least seven genotyped individuals, we calculated two different measures of within-population nuclear diversity: arcsin transformed expected heterozygosity . To obtain more precise estimates, only individuals for which more than 400 bp congruent sequence information was available were included. The number of haplotypes per population was extracted from the data using DNASP[Within population diversity in the mitochondrial genome was estimated from the number of COI haplotypes   was then gained by calculating the Euclidian distance of each population from the origin of the coordinate system spanned by the significant PCA axes. To infer visually whether this and the previous predictor lim tended to result in higher or lower than average diversity measures, sample sites falling within the 85% quantile were contrasted to the remaining populations.To assess the environmental marginality, we extracted 35 climatic parameters  for each sampling point for the period from 1960 - 2000 from publicly available WorldClim data, incorporated in DIVA-GIS . We usedsize): small = drainage ditches, small streams and ponds smaller than approximately one hectare; large = lakes larger than one hectare. Besides the obvious influence of habitat size on the population size, the extension of a water body may also influence the probability to be the target of long range passive dispersal and thus of successful gene-flow, positively affecting genetic diversity. Additionally, larger water bodies are expected to be more stable than smaller ones, allowing potentially accumulation of genetic variation over a longer time period.The factor determining genetic drift experienced by a population is the effective population size . As direR. balthica in the eastern and south-western parts of the range. Here, predictive niche modelling suggested larger suitable ranges than are actually occupied by R. balthica [Radix species as identified by barcoding [bio).Biotic interactions may play a role in balthica . These aarcoding  (Additioref), potentially retaining the larger part of the genetic diversity [hol, Figure R. balthica has already expanded its range as a consequence of ongoing climate change as suggested for freshwater benthos [http://www.ma.slu.se) with canonical correspondence analysis .Based on the results from , we contiversity , against benthos , we analR. balthica is limited by the Alps, the Atlantic Ocean and the Mediterranean, we considered populations bordering these dispersal impeding structures in a categorical predictor (bar). The geographical distribution of the sampling sites and their grouping in predictor variables is illustrated in Figure As the range of EH, A, s, mtH) in turn. We used all possible predictor combinations in a Generalised Linear Model (GLM) approach to compute the residual sums of squares (SSR) in the software package STATISTICA[d.f.), the Akaike information criterion with correction for small sample size was then computed for each model [Statistical modelling was employed to evaluate the relative influence of the predictors described above on all measures of population diversity , we have therefore compared the average among population differentiation in this category with the differentiation among the respectively remaining populations. Because pairwise FST values are not independent data points, we have applied a simple randomisation scheme to assess the statistical significance of the observed differences. To test whether the observed average FST difference between the populations in the respective category and the rest was larger than expected by chance, we created a null distribution by randomly shuffling populations 1000 time among the contrasted categories.Declining population size and gene-flow among populations towards range margins should also result in increased differentiation among populations . For allMP conceived the study, analysed the data and drafted the manuscript, MS sampled most of the sites, performed parts of the molecular work, the initial analyses and contributed to drafting the manuscript, TH performed parts of the molecular work and commented on the manuscript, BF contributed to writing the manuscript.We thank Martin Plath, Mathilde Cordellier, Eugenia Zarza, Aline Depráz and three anonymous reviewers for constructive comments on a previous manuscript version. The work received financial support within the AQUASHIFT priority programme from the DFG (grant MP390/4-2) and was supported by the research funding programme "LOEWE - Landes-Offensive zur Entwicklung Wissenschaftlich-ökonomischer Exzellenz" of Hessen's Ministry of Higher Education, Research, and the Arts.Radix taxaDistribution of . Spatial distribution of the Radix MOTU as defined in Pfenninger et al. 2006 plus an additional, newly discovered taxon. This map is the basis for the inference of the species range of R. balthica.Click here for fileR. balthicaSampling site table and spatial distribution of diversity indices, selfing estimates and inferred population bottlenecks for . Table of sampling site code, geographical position in decimal degrees latitude and longitude, number of individuals analysed with microsatellites (Nnuc), expected heterozygosity (HE) and standard deviation across loci, mean rarefied number of alleles per microsatellite locus (A) and their standard deviation, number of individuals analysed for mitochondrial variation (Nmt), rarefied number of mitochondrial COI haplotypes (Hmt), number of individuals measured for body size (Nsize). Figures A1 - A3 show a graphical representation of the spatial distribution of He, Hmt and, s, respectively.Click here for fileAssessment of environmental marginality. PCA  on 35 climatic parameters for the period from 1960 - 2000 from publicly availableWorldClim data.Click here for fileR. balthicaInference of a recent climate driven range expansion in . Analysis of the freshwater benthos long term monitoring data of the Swedish national monitoring databases at the Swedish University of Agricultural Sciences SLU with canonical correspondence analysis.Click here for file


When grouping the samples into two alternative sets of two populations, W and E, vs. N and S, neither dataset exhibited a strong pattern of allelic endemism, suggesting that none of the considered regions consistently harboured older populations. Differentiation in dataset 1 was similarly strong between W and E as between N and S, with 3–5 significant FST tests out of 14 tests in each scenario. Coalescent simulations indicated that, given the power of the data, this result probably reflects idiosyncratic histories of the taxa, or a weak common differentiation pattern (possibly with population substructure) undetectable across taxa in dataset 1. Dataset 2 identified a common genetic break separating the northern and southern populations of Greenwayodendron suaveolens subsp. suaveolens var. suaveolens, Milicia excelsa, Symphonia globulifera and Trichoscypha acuminata in Lower Guinea, in agreement with differentiation across the N–S seasonal inversion. Our work suggests that currently recognized tree taxa or suspected species complexes can contain strongly differentiated genetic lineages, which could lead to misinterpretation of phylogeographic patterns. Therefore the evolutionary processes of such taxa require further study in African tropical rainforests.Comparative phylogeography is an effective approach to assess the evolutionary history of biological communities. We used comparative phylogeography in fourteen tree taxa from Lower Guinea  to test for congruence with two simple evolutionary scenarios based on physio-climatic features 1) the W-E environmental gradient and 2) the N-S seasonal inversion, which determine climatic and seasonality differences in the region. We sequenced the  The rich biodiversity of tropical rainforests results from the complex interaction of continental drift, past climate oscillations, dispersal events and adaptive processes. The rainforests of Atlantic Equatorial Africa, ranging from Liberia in the West to Gabon in the East, are among the World’s major biodiversity hotspots Comparative phylogeography, which examines the distribution of genetic lineages across co-distributed taxa, is an effective approach to assess the evolutionary history of biological communities. Genetic lineages bear the signal of past range dynamics and population size fluctuations , enabling researchers to test whether genetic data are compatible with defined evolutionary scenarios In this paper, we focus on the processes that may have shaped the genetic diversity of co-distributed rainforest tree taxa in Africa’s Lower Guinea (LG) phytogeographic region using a comparative phylogeographic approach. LG is the part of the African rainforest that encompasses forests from Southern Nigeria, Cameroon, Equatorial Guinea and Gabon to the south-western part of the Republic of Congo. It is one of three sub-centres of plant endemism – together with the Upper Guinea  and the Congolia (C) – of the Guineo-Congolian tropical forest phytogeographic region ca. 2000 mm/year), lower insolation (ca. 1300 hours/year) and have less marked seasonality than inland forests, which receive ca. 1600–2000 mm precipitation per year and up to 2000 hours of sunlight vs. E regions, for instance in the case of old differential adaptation to water availability and/or past history, we would expect differentiation between Atlantic (W) and inland (E) forests. Allelic endemism patterns could then emerge in either the W or E depending on the age of the differentiated populations. For instance, the distribution of distinct sister species of Erythrophleum (Fabaceae) close to the Bight of Bonny (W) and inland (E) concurs with this expectation The LG rainforests reflect a west-east (W-E) environmental gradient, with generally evergreen forests located close to the coast of the Gulf of Guinea (W) and semi-evergreen forests inland (E) Greenwayodendron suaveolens (Annonaceae) displays N-S differentiation in LG The second feature that characterizes the LG region is the climate hinge, or latitude of N-S seasonal inversion, which runs roughly parallel to the equator between ca. 0° and 3° N. During the boreal summer (June-August), the northern part of the LG forest  receives increased rainfall while in the southern part (Gabon) an overcast sky limits evapotranspiration in the absence of rain. In the austral summer, southern Gabon receives heavy rainfall while dry continental winds produce a dry season in the northern fringes of the forest block In this study we examined two alternative evolutionary scenarios by characterizing the genetic diversity and its spatial structure at maternally-inherited plastid DNA markers in fourteen tree taxa in the LG. We asked the question whether one of the before-mentioned physio-climatic features, the W-E environmental gradient, or the N-S seasonal inversion, was strong enough to have imprinted a very general geographic pattern of genetic variation at maternally-inherited genomes in Central African trees. To capture genetic signals that may have resulted from Plio-Pleistocene or still on-going events, we focused on low-level taxa, usually taxonomic species. Early phylogeographical studies identified considerable levels of lineage divergence within taxonomic species in central Africa and other tropical regions Baillonella toxisperma Pierre (Sapotaceae) is listed as “vulnerable A1cd ver 2.3″, Milicia excelsa (Welw.) C.C. Berg (Moraceae) as “lower risk/near threatened ver 2.3″, and Greenwayodendron suaveolens (Engl. & Diels) Verdc. as “least concern”, while there is no data on the remaining taxa.We sampled fourteen common rainforest tree taxa typically occurring in evergreen and semi-evergreen rainforests of the Lower Guinea. Sampling sites included national parks and public land. Sampling permits were obtained from the CENAREST (Gabon) and the Ministry of Scientific Research and Innovation (Cameroon) and sample collection was facilitated by the Wildlife Conservation Society , the CIRMF , the logging company Pallisco (Cameroon), the Missouri Botanical Garden and the NGO Nature Plus. According to IUCN, Erythrophleum harbours two vegetatively similar and closely related sister species traded under the same timber name  CarapaStrombosiopsis (GreenwayodendronErythrophleumCarapaIn a first stage (dataset 1), we sampled fourteen common tree taxa typically occurring in evergreen and semi-evergreen rainforests of the LG . ThirteeGreenwayodendron suaveolens subsp. suaveolens var. suaveolens (Engl. & Diels) Verdc., totalling n = 169 individuals from 41 locations published by Dauby et al. Milicia excelsa (Welw.) C.C. Berg, n = 127, 19 locations, published by Daïnou et al. Symphonia globulifera Lf., n = 67, 20 locations and Trichoscypha acuminata Engl., n = 73, 28 locations. Complementary information indicated that these taxa most likely matched the biological species concept; specifically, in G. suaveolens subsp. suaveolens, we only included samples morphologically and genetically identified as var. suaveolens which likely represents a distinct biological species from G. suaveolens subsp. suaveolens var. gabonicaM. excelsa, polymorphism was low and no deep lineage divergence was previously identified in LG S. globulifera, the same samples included here did not display deep lineage divergence when sequenced at another marker  or the Herbier National du Gabon (LBV) and at the herbarium of the Université Libre de Bruxelles  supplemented with 0.1 µM of each primer  kit, Invitek, Berlin, Germany) and sequenced using Big Dye v.3.1 chemistry  and an ABI3100 sequencer (Applied Biosystems).Total DNA was extracted from silica-dried plant material using either a CTAB procedure S, the number of singletons, the number of insertion/deletion (indel) polymorphisms, inversions and microsatellite (SSR) length polymorphisms. Considering point mutations only, we used DnaSp v. 5.10.1 software Hd and two estimators of the population mutation parameter, θ = 4Neµ, namely, nucleotide diversity (π), which represents the average number of nucleotide differences per site between two sequences θWS. We then considered all polymorphism types  to define haplotypes and computed a haplotype distance matrix for each taxon, defining the distance between two haplotypes as the proportion of polymorphisms differing between them. Statistical parsimony networks among haplotypes were constructed based on these distance matrices using the program TCS 1.21 S. globulifera data set. We computed genetic diversity h and vSequence trace files corresponding to forward and reverse reads were aligned with CodonCode Aligner 3.0.1. . Within-taxon alignments of individual sequences were done with MUSCLE 3.6 ijF between pairs of individuals on the logarithm of geographic distance and compared the slope blog(ijF) to its null expectation from random permutations of the sampling locations using SPAGeDi <blog(ijF permuted)). We then tested whether nearby individuals were more likely to bear closely related haplotypes than individuals chosen at random, performing the same test using the coefficient Nij based on ordered haplotypes . Finally, we tested for phylogeographic structure by comparing blog(ijN) to its expectation from permuting rows and columns of the genetic distance matrix.To test for demographic or spatial genetic signals of population expansion, such as expected if population recovery and/or recolonization after Plio-Pleistocene fragmentation left a genetic footprint, we used the mismatch distribution with 1000 parametrical bootstraps in Arlequin 3.5.1.3 Nhaps), the rarefied haplotype richness , the number of private haplotypes (Npriv) and the rarefied private haplotype richness  using the program HP-Rare R software FST for unordered and NST for ordered alleles NST>NST(permuted), FST) in the candidate vs. alternative scenario (in dataset 1).To test for patterns of allelic endemism and genetic differentiation as expected under two contrasting evolutionary scenarios (see Introduction), we grouped samples into pairs of categories (hereafter regions) and examined levels of genetic diversity and endemism within regions and differentiation between them. For the W-E scenario (1), which emphasizes the response to environmental and climatic conditions , samples were classified as belonging to generally evergreen Atlantic forests or to semi-evergreen inland forests N throughout the genealogy. This model was motivated by the observation of substructure in some species of dataset 1 and could be interpreted as repeated divergence events triggered by recurrent climate oscillations  respectively in the divergence and constant scenarios and compared them to the observed data. For the differentiation scenario that most closely matched the observed dataset 1, we repeated the simulation recovering n = 4 samples for each of four demes at t0. We then used AMOVA in Arlequin to compute the differentiation between populations with simulated substructure, FCT[(01)(23)] (where subscript numbers stand for the four demes), and the differentiation between groups composed of samples from different populations, FCT[(02)(13)]. This mimicked grouping the samples into W-E vs. N-S, with one of the two groupings corresponding to the true divergence history. The fraction of simulations where FCT[(01)(23)]>FCT[(02)(13)] represents the power to identify the correct differentiation pattern between the two groupings considered.To assess the power of our approach to identify population subdivision, discriminate between the tested scenarios, and to assess the rate of false detection of population subdivision when it is absent (type I error), we applied a coalescent simulation approach using simulated sample sizes mimicking our initial (n = 8/population) and enhanced data sets (n = 30/population). We used the software fastsimcoal ions see , or by oions see , B, bothGST) between sampling locations was used together with the sampling coordinates to identify the three most important genetic barriers in each taxon. The co-location of barriers was then evaluated across taxa using the line density tool in ArcGis 10 software  in dataset 1 and the number of SNPs varied between 0 and 26 across taxa, resulting in πT values ranging from 0 to 0.0139 , on the logarithm of geographic distance . A phylogeographic structure was only observed in three taxa: Erythrophleum ivorense/suaveolens, Greenwayodendron suaveolens subsp. suaveolens var. suaveolens and Symphonia globulifera.The null model of demographic expansion was rejected in five, and the spatial expansion model was rejected in two out of 12 taxa with ataset 1 . In dataataset 1 . Isolatidistance . Isolatih, Npriv and 3Ar) were found to differ significantly among taxa . In dataset 2, private rarefied allelic richness Arpriv18 was numerically larger in all four species in the E than in the W, whereas no common trend was observed in the N-S scenario. Negative regression slopes with latitude were observed for within-population allelic richness (Ar3) in M. excelsa and for within-population genetic diversity h in S. globulifera, i.e. southern populations of these species had higher diversity statistics (FST or NST between regions in dataset 1 was significant (P<0.05) for four or five taxa in the W-E scenario and for three or four taxa in the N-S scenario ) was detected in Erythrophleum ivorense/suaveolens and Anthonotha macrophylla (W-E) and in G. suaveolens subsp. suaveolens var. suaveolens .In dataset 1, there was no significant difference of genetic diversity statistics between regions in any of the two evolutionary scenarios examined; however, some diversity statistics (23)]>FCT[(02)(13)], in 58% of the cases and FCT[(01)(23)] ≥ FCT[(02)(13)] in 98% of the cases. This suggested that the power of dataset 1 to distinguish differentiation patterns produced by alternative evolutionary scenarios was at least 58% . In simulations without genetic structure (B1–B4), only 0–4% of the tests detected a differentiation signal. This suggested a low level of false positive differentiation tests even for the small sample sizes of dataset 1.Simulations showed that a population divergence scenario with mean levels of diversity and differentiation similar to those in dataset 1 and sample sizes of n = 8 per deme, i.e. simulated scenario A3, would lead to 26% significant differentiation tests at a level of α = 0.05 based on FST . In dataThe barrier density analysis based on the four species of dataset 2 identified a region of barrier co-location separating the N and the S of the LG study region between ca. 0 and 3° N, at the latitude of Equatorial Guinea, coinciding to some extent with the differentiation expected under the N-S scenario .This study shows that fourteen common Afrotropical rainforest tree taxa (dataset 1) exhibited variation in their patterns of genetic endemism and differentiation and did not show clear majority support for any of two simple evolutionary scenarios in response to physio-climatic features in the Lower Guinea, i.e., 1) the W-E environmental gradient or 2) the N-S seasonal inversion. This observation allows several possible interpretations: 1) absence of a common pattern, or non-detection of a common pattern because it was too weak to be detected with the available sample sizes; 2) existence of a common genetic structure pattern but only for a subset of the study taxa; or 3) a confounding effect due to sampling ill-defined taxonomic species. We applied coalescent simulations and complementary sampling in a sub-sample of four of the fourteen taxa in order to further examine these possible interpretations and to clarify our results. Interestingly, the complementary sampling distinguished an N-S differentiation pattern shared among the four species with larger samples.Erythrophleum ivorense/suaveolens) but some are most probably complexes of species that cannot be distinguished easily in the field  and, in any case, it cannot be taken for granted that any of the taxa considered in dataset 1 matches the biological species concept. Our study approach should generally remain valid for species complexes because we make no assumption on whether evolutionary forces affecting diversification led to differentiated populations or to distinct species. Indeed, the genealogy of a non-recombinant DNA fragment, as studied here, is not directly dependent on speciation processes One caveat of our study is that the tree flora of Central African rainforests is not sufficiently well known to allow unambiguous species identification. The taxa considered in dataset 1 are species-level taxa according to current taxonomic classifications  suggesting that further research is needed on species delimitation within these taxa, and also in Erythrophleum ivorense/suaveolens. Erythrophleum ivorense and E. suaveolens are sister species which cannot easily be distinguished based on vegetative characters and whose geographic ranges  have only recently been inferred based on a phylogeographic study that compared the distribution of genetic diversity of vegetative specimens with the reproductive morphology in a few fertile specimens Such confounding effects related to species discrimination problems are perhaps more likely for taxa with deep lineage divergence among haplotypes. We observed deep lineage divergence  more endemic alleles than western locations. This suggested that there was no general pattern in either region to harbour consistently older populations. If the W-E environmental gradient had enabled persistence of rainforest taxa in the moister Atlantic zone only during past climate oscillations FST tests) was ca. 26% in dataset 1 and ca. 73% in dataset 2, given the level of differentiation observed in our data. In dataset 1, three to five out of 14 taxa showed significant population differentiation compatible with the proposed W-E or N-S scenarios, which was close to the expected proportion from simulations. The comparison of FCT values between the two alternative groupings of simulated demes indicated that FCT was (strictly) larger in the true differentiation scenario in 58% of the cases vs. only 2% in the false scenario in samples analogous to those of dataset 1. This approach, i.e., the comparison of FST values between alternative groupings of our samples was therefore a more powerful approach to detect the correct scenario than FST tests. Our data displayed, respectively, six FST values larger in the W-E than in the N-S scenario, and six values larger in the reverse comparison. This suggested that, given the power of dataset 1, there was no majority support for neither of the two hypothesized scenarios. Simulations also showed that the strength of the differentiation signal had a strong effect on the power . Simulations also indicated that we could confidently discard the hypothesis that all taxa would exhibit panmixia in the LG since the rate of false positive differentiation tests was low in simulations (0–4%).Our simulation approach indicated that the power to detect a common differentiation pattern across taxa , in agreement with the expectation of the N-S evolutionary scenario. A similar genetic break zone in the LG region has been suggested based on nuclear markers in a review by Hardy et al. Santiria trimeraDistemonanthus benthamianusIrvingia gabonensisAucoumea klaineanaThe power to detect differentiation patterns was considerably improved in dataset 2 (see above) and a common genetic break zone was identified by the barrier density analysis separating the N from the S of the LG for these taxa Click here for additional data file.Table S2Life history traits of 14 African rainforest tree taxa.(DOCX)Click here for additional data file.Table S3Diversity gradients at the trnC-ycf6 region in four Afrotropical tree species.(DOCX)Click here for additional data file.Table S4Details on coalescent simulation results.(XLSX)Click here for additional data file.


Pistacia lentiscus across a highly disturbed landscape using microsatellite loci. Paternity analysis and the indirect KinDist and Mixed Effect Mating models were used to assess mating patterns, the pollen dispersal kernel, the effective number of males (epN) and their relative individual fertility, as well as the existence of fine-scale spatial genetic structure in adult plants. All methods showed extensive pollen movement, with high rates of pollen flow from outside the study site (up to 73–93%), fat-tailed dispersal kernels and large average pollination distances (δ = 229–412 m). However, they also agreed in detecting very few pollen donors (epN = 4.3–10.2) and a large variance in their reproductive success: 70% of males did not sire any offspring among the studied female plants and 5.5% of males were responsible for 50% of pollinations. Although we did not find reduced levels of genetic diversity, the adult population showed high levels of biparental inbreeding (14%) and strong spatial genetic structure (pS = 0.012), probably due to restricted seed dispersal and scarce safe sites for recruitment. Overall, limited seed dispersal and the scarcity of successful pollen donors can be contributing to generate local pedigrees and to increase inbreeding, the prelude of genetic impoverishment.Analysing pollen movement is a key to understanding the reproductive system of plant species and how it is influenced by the spatial distribution of potential mating partners in fragmented populations. Here we infer parameters related to levels of pollen movement and diversity of the effective pollen cloud for the wind-pollinated shrub  It is a general assertion that genetic drift compromises the evolutionary potential and long-term survival of fragmented populations throughout inbreeding depression and reduced response to selection Pollen flow is a main source of genetic variation among populations. Nevertheless, for small and fragmented populations not only the source of origin but the composition (i.e. diversity) of contributing pollen pool is equally important Pistacia lentiscus L. (Anacardiaceae) is a common shrub or (more rarely) a small tree that is abundant in the Mediterranean sclerophyllous vegetation. Life history traits of this species resemble, to some extent, those of larger trees . Furthermore, the species can be found from almost continuous monospecific stands in forest understories to small clumps in open habitats and even isolated individuals; hence P. lentiscus constitutes a suited case study to analyse pollination biology, mating system and fine-scale population genetic structure in a fragmentation context. In a previous study performed in a large, dense and continuous stand of P. lentiscus, Albaladejo et al. P. lentiscus embedded in an intensively managed matrix. Our main goal is to assess the pollination biology of P. lentiscus by studying the pollination connectivity both in quantity and diversity across this highly disturbed landscape and compare the results with those obtained in continuous populations. Specifically, we address (i) the spatial patterns of pollen flow in the study site, (ii) the shape of the effective pollen dispersal distribution  assessed by both direct and indirect methodologies, and (iii) the source diversity of successful mates . Further, we expect to find, (iv) lower levels of genetic diversity and a stronger pattern of spatial genetic structure of the adult population, and (v) higher levels of biparental inbreeding and within-mothers correlated paternity compared to continuous population due to noticeable long-term effects of fragmentation.In the present study, we focus on individual plants of Pistacia lentiscus is an evergreen long-lived shrub  representative of the woody plant species in the Mediterranean. The species is dioecious (i.e. obligate outcrosser) and wind-pollinated, with a narrow flowering period in the studied area which spans from mid-March to late April . Fruits, which are small black one-seeded drupes, ripe from September to December when they are actively consumed by a wide array of small or mid-size birds, many of them migratory sensu McIntyre & Hobbs Eucalyptus globulus Labill. The study site itself is also a highly disturbed site (Quercus suber L.) vegetation and scattered clumps of Pistacia lentiscus, Myrtus communis L., Quercus coccifera L. and Retama sphaerocarpa (L.) Boiss. We chose this site because (1) the area is representative of the current vegetation found in highly-humanized Mediterranean landscapes, (2) the number of bushes of P. lentiscus was large enough to guarantee the representativeness of the study, (3) every individual plant could be accessed and identified with ease and (4) outside the study site no dense clump or stand of P. lentiscus exists in several km around (only scattered bushes can be found elsewhere). Where necessary, the landowners authorized access to private areas.The lower catchment of the Guadalquivir river (southern Spain) is a ‘relictual’-type agricultural landscape . In October 2007, we selected 29 mother plants distributed throughout the site  according to the manufacturer’s protocol. We amplified seven unlinked polymorphic nuclear microsatellite loci following amplification conditions and PCR cycle profiles provided in Albaladejo et al. LOD scores) of the most-likely fathers of 50000 synthetic seeds with their father chosen among the 164 potential males, to the distribution of LOD scores of the most-likely father of 50000 seeds whose paternal genotype was randomly generated according to reference population allele frequencies. In wind-pollinated species, levels of pollen immigration can be high TF = 5.50  was set to 0.152, the value estimated for the adult population (see Results). After running the analysis we placed each analysed seed into one of the following three categories: (i) no compatible father in the study site (i.e. the minimum bound of incoming pollen flow rate), (ii) at least one compatible father but with a LOD score < TF, and (iii) one or multiple candidate fathers within the site with a LOD score > TF. In the latter case, we assigned paternity to the spatially closest male. We believe that the potential bias introduced by this procedure is negligible because most ties in our dataset occurred between genetically related neighbouring males.The pollen movement among individual plants within the study site was assessed using paternity analyses to assign each seed to its most-likely father by the maximum likelihood approach implemented in Famoz http://www.juntadeandalucia.es/agriculturaypesca/ifapa). Directional frequency histograms of the distribution of observed and potential mating, and prevailing wind direction were compared through Watson’s 2U-test using the R package circularTo check whether mating success was a mere function of the spatial position of males and females , we compared the observed frequency distribution of mating events with its potential distribution  using a Kolmogorov-Smirnov test. We also evaluated the directionality (i.e. isotropic vs. anisotropic) of this distribution a) and shape (b) parameters of the power-exponential dispersal function as well as the average effective pollination distance (δ) derived from it b controls the tail of the distribution so that b <1 provides fat-tailed dispersal functions  while b >1 provides thin-tailed functions. In our case, we first checked that among-mothers correlated paternity significantly decreased with (log) distance  and immigration rates (m), the ratio between observed and effective male density (obs/depd) and, remarkably, the relative individual fecundity of each male (see below) in the study site. We used the following minimum and maximum values as prior information: obs/depd = 1, 150; δ = 50, 1500; b = 0.1, 10; and m = 0.4, 0.95. Because P. lentiscus is dioecious, we set to zero the prior values for s. Since the analysis requires from reference outside allele frequencies for the calculation of the immigration rate  after the Famoz analysis. The analysis was run for 100000 iterations plus an initial burn-in period of 20000 iterations. We also included a covariate in the analysis which reports on the local environment where each male was located in the study site  was calculated with the software MLTR 3.3 mt) and single locus (st) outcrossing rates estimated through the Newton-Raphson algorithm. We assessed mt-st statistical significance by comparing 1000 bootstrap values of mt vs. st by means of Student’s paired t-tests. Bootstrap replicates were done with resampling of families. Within-mother correlated paternity (pr) was calculated as twice the average pairwise kinship coefficient ijFpr values against the distance to the nearest male and to the nearest three and five males, respectively. The effective number of pollen donors for the sampled mothers in the study site (epN) was deduced as pr−1. We compared this result with the epN value derived from the mean value of the obs/ded probability density function obtained from the MEMM analysis.Biparental inbreeding , the expected heterozygosity (eH) and the inbreeding coefficient (f). Departure from Hardy-Weinberg (HW) equilibrium was assessed by means of a Markov Chain-based exact test using Genepop v.4 For the adult plants , we also calculated single-locus descriptive diversity parameters using Fstat v.2.9.3.2 ijF) between individuals on the logarithm of the spatial distance as corresponding to a bidimensional space ijF values into distance classes. To allow a direct comparison with the continuous population studied by Albaladejo et al. ijF values as twice the standard error (SE) obtained by jackniffing over loci. The intensity of the SGS was also quantified by the pS statistic, a dimensionless parameter useful to perform comparisons between species and populations pS is calculated as b-log/(F(1)−1), where F(1) is the mean ijF among individuals pairs in the first distance class and b-log is the slope of the correlogram. All SGS analyses were performed with the software SPAGeDi v.1.2 We analysed the existence of SGS in the adults by regressing the pairwise kinship coefficients  had at least a compatible father but with a LOD score below the threshold required for a confident assignment. This result means that a minimum of 73.3% and a maximum of 82.4% of the seeds were sired by fathers located outside our study site.The cumulated exclusion probability (when the mother is known) for the seven microsatellite loci used was 0.995, supporting the suitability of our dataset to conduct a paternity analysis see . The estslen R05 . Out of d = 0.280; P<0.001). In spite of the large distance for the observed matings, these occurred at distances shorter than expected  and the one of prevailing winds during the flowering period . Interestingly, the observed distribution was a somewhat combined distribution considering both the spatial position and wind direction.Most pollination events took place in the S-N direction  accordina = 2.8 10−4 and b = 0.16, as well as an average effective dispersal distance of δ = 268.5 m. Normal, geometric or exponential functions showed a poorer fit based on the least-square residuals (results not shown).The KinDist analysis successfully fitted a power-exponential function to the data and provided point estimates for the scale and shape parameters of b = 0.19 (0.10–0.46) and δ = 229 m (52–1069 m). Additionally, the immigration rate estimated by MEMM was very high, m = 0.937 (0.915–0.949), which supports the results of the direct paternity analysis.The mean conditional log-likelihood for the two runs with MEMM, either assuming a log-normal or gamma distribution for individual male fecundity, were −8145 and −8138 respectively; an approximate Bayes factor, computed as the ratio of the estimated likelihoods, strongly supported the model with the gamma distribution , indicating that about 14% of matings occurred between genetically related plants. Individual within mother correlated paternity showed a significant negative correlation with the (log)distance to the nearest male  but this trend vanished and became non-significant when referred to the average distance to the nearest three and five males . The average within mother correlated paternity calculated from kinship coefficients was also high (pr = 0.231) indicating that in average about 23% of seeds in a given mother plant were sired by the same father. This value translates into an effective number of fathers epN = 4.3. The Mixed Effects Mating model provided a posterior mean value  for the ratio obs/ded = 16 (2.7–45) which corresponded to a epN value of 10.2 (3.6–60.7) according to the observed census of males (obsN = 164) in the study site.In the study site, we found a highly significant difference between multilocus and single-locus outcrossing rates  did not sire any seed in any of the 29 females sampled within the study area. Only nine fathers (5.5%) were responsible for nearly 50% of the assignments . However, most loci showed a significant excess of homozygotes  indicating that genetically related adult individuals were aggregated across the space. The correlogram revealed a sharp decline in the kinship coefficients with distance, with significant positive average ijF in the first three distance classes, which extended through a distance of approximately 85 m  distance was highly significant  of the sampled seeds were sired by incoming pollen flow. Admittedly, we ignore the actual location of the male plants that contributed so remarkably as pollen donors, but we believe that they must probably be located in privileged positions (see below) in the vicinity of the study site, despite only scattered bushes and very small clumps of In agreement, both the direct and indirect methods that we used indicated large average pollination distances, although the estimates varied somehow across the methods. The paternity analysis, which is constricted by the precise spatial distribution of sampled female plants , provided an estimate of about 400 m, well above the mean dispersal distance inferred through direct methods for wind-pollinated species Although we have shown extensive pollen flow in this fragmented landscape there is also an important component of restricted pollen dispersal at local scales. Below 500 m, the paternity analysis reveals more matings than expected at random see , which sAssessing the directional distribution of successful matings is also important because it may determinate the number of sires available for female plants. Our sampling scheme resulted in heterogeneity in the directional distribution of males and the sampled females (with northwards matings being clearly favoured), which surely is responsible for the high proportion of matings in the S-N direction we detected see . InteresOne aspect of the reproductive ecology of plants that becomes crucial in fragmented populations is correlated paternity (the diversity of fathers siring progeny in a mother) because where spatial connectivity and the availability of suitable places for seed arrival and seedlings establishment is reduced, progeny arrays composed of full-sibs can perform poorly compared to those composed of half- or unrelated sibs Besides high correlated paternity, we have also found a strong variance in male relative fecundity since just a few males have copped a high proportion of all mating events. To explain this unbalance in male contributions several reasons have been adduced, among them differential quantity or quality of the pollen released We found significant levels of inbreeding in all but one locus, which in this dioecious species can be explained by significant levels of biparental inbreeding. A relevant impact of null alleles was ruled out because their estimated frequency was in average low and because significant inbreeding was also detected in the same population with other nuclear markers Biparental inbreeding accounted for a moderate portion (14%) of the mating events, being probably a straight consequence of the existence of significant SGS (see below) and to the fact that mating were more likely at short distances than at random (discussed above). Further, the proportion of full-sibs within mother progeny arrays is significantly higher as the distance to the nearest male decreases which suggest some degree of monopolization of some females by their closest male pollen cloud. However, this influence is spatially very restricted since only the relationship with the distance to the nearest male was significant. This negative relationship  was also found in a previously-studied continuous population but only in one of the studied seasons P. lentiscus was relatively high and similar to that of other Mediterranean woody plants, either in fragmented populations or not Despite the extreme disturbance of the studied landscape, genetic diversity of pS value (0.0122) is indicative of a strong pattern of SGS, since it is roughly twice the typical for wind-pollinated (0.0064) or animal-dispersed (0.0088) plant species pS = 0.0022; recomputed from P. lentiscusFinally, the The real impact of anthropization on the viability and performance of natural plant populations is still controversial. So far, ideal experimental designs under a fragmentation genetics perspective are very difficult to meet in nature. Moreover, to assess the actual scale of gene dispersal (pollen and/or seed) within and among plant populations  become essential to reconcile the theoretical and empirical evidence Keeping this idea in mind, this is one of the scarce studies devoted to assess pollen dispersal and the shifts imposed by severe anthropization in the mating system of a long-lived woody plant species. Specifically, our study illustrates the paramount importance of small clumps or isolated individual plants in keeping genetic connectivity even in, at first glance, remote and isolated populations. Indeed, we have found genetic diversity  at levels that, in the light of the ecological characteristics of the study site, were unexpected.P. lentiscus was severely impacted by fragmentation: compared to a large and continuous population Nevertheless, we also found that most male plants did not sire a single seed from the studied female plant progenies and that the mating system of Figure S1Correlation between among mother correlated paternity  against the distance  among mothers pairs.(PDF)Click here for additional data file.


Galemys pyrenaicus, is a small semi-aquatic mammal that inhabits clean streams of the northern half of the Iberian Peninsula and is endangered in most of its geographic range, but its genetic structure is currently unknown. While the stringent ecological demands derived from its aquatic habitat might have caused a partition of the genetic diversity among river basins, Pleistocene glaciations would have generated a genetic pattern related to glacial refugia.Species with strict ecological requirements may provide new insights into the forces that shaped the geographic variation of genetic diversity. The Pyrenean desman, G. pyrenaicus, we used mitochondrial and intronic sequences of specimens covering most of the species range. We show, first, that the Pyrenean desman has very low levels of genetic diversity compared to other mammals. In addition, phylogenetic and dating analyses of the mitochondrial sequences reveal a strong phylogeographic structure of a Middle Pleistocene origin, suggesting that the main lineages arose during periods of glacial isolation. Furthermore, both the spatial distribution of nuclear and mitochondrial diversity and the results of species distribution modeling suggest the existence of a major glacial refugium in the northwestern part of the Iberian Peninsula. Finally, the main mitochondrial lineages show a striking parapatric distribution without any apparent exchange of mitochondrial haplotypes between the lineages that came into secondary contact , indicating incomplete mixing after the post-glacial recolonization. On the other hand, when we analyzed the partition of the genetic diversity among river basins, the Pyrenean desman showed a lower than expected genetic differentiation among main rivers.To study the relative importance of historical and ecological factors in the genetic structure of G. pyrenaicus showed the predominant effects of Pleistocene glaciations on the genetic structure of this species, while the distribution of the genetic diversity was not greatly influenced by the main river systems. These results and, particularly, the discovery of a marked phylogeographic structure, may have important implications for the conservation of the Pyrenean desman.The analysis of mitochondrial and intronic markers in  The genetic diversity patterns of species are a consequence of their evolutionary history  and of contemporary constraints to dispersal (e.g. habitat fragmentation). These processes are expected to give rise to specific phylogeographic patterns-3, the dGalemys pyrenaicus) is a small semi-aquatic mammal endemic to the northern half of the Iberian Peninsula. It occupies streams of clean and cold flowing waters with shallow but permanent water levels throughout the year, an habitat generally found in mountain areas. Its distribution is highly dependent on the presence of larvae of benthonic macroinvertebrates that the desman captures underwater. Adaptations to the aquatic life include a highly-mobile protracted snout, large hindfeet and a long tail with stiff hairs[The Pyrenean desman , and therefore they are the only two extant representatives of this group of semi-aquatic mammals. Fossil data indicate that desmanines were much more diverse in the past[The Pyrenean desman forms part of the family Talpidae, which is included in the mammalian order Eulipotyphla . Within Talpidae, the Pyrenean desman is placed within the subfamily Desmaninae together with the Russian desman , following the manufacturer’s instructions, in a final elution volume of 50 μl of water. These extractions were carried out in a separated UV-irradiated area with dedicated equipment. Tissue samples were processed with QIAGEN DNeasy Blood and Tissue Kit, according to the manufacturer’s instructions, and eluted in 75 μl of water. When necessary, to ensure maximum tissue lysis, samples were incubated in a lysis buffer with proteinase K at 56°C overnight.The extraction of the two museum bone samples was carried out in a dedicated ancient DNA laboratory. The samples were powdered and decalcified overnight in a 10 M EDTA solution at 37°C, followed by an overnight incubation in a lysis buffer with proteinase K and SDS at 56°C. The DNA was then extracted using a standard phenol-chloroform protocol and finab gene (1140 bp) and a fragment containing 342 bp of the 5′ distal part of the D-loop, using G. pyrenaicus specifically-designed primers , annealing (30 s at 54°C) and extension (30 s at 72°C). A 5-minute extension at 72°C was finally added. PCR products were revealed by electrophoresis in a 1% agarose SYBR-Safe (Invitrogen) stained gel.Museum samples may contain degraded DNA due to chemical damage during the preservation process, thus incorporating induced mutations into some DNA molecules,29. To pPreliminary tests revealed that the sample IBE-C3161 (a rib fragment) had DNA with a similar concentration and quality than fecal samples. Thus, the protocols used for this sample were the same ones used for feces, except that two independent PCR reactions per fragment were performed so that we obtained two independent sequences.b and the D-loop was achieved using a two-step multiplex approach[b, two independents sets (A and B), each consisting of 8 non overlapping PCR products, were used, while the D-loop fragment was amplified using two smaller A and B sets of 3 and 2 PCR products, respectively. The 42 primer sequences are available upon request. In the first, multiplex step, all the primers of each independent set were used in a PCR reaction containing 5 μl of DNA, 2 units of Promega GoTaq DNA polymerase and 0.15 μM of each primer. The reaction conditions were as follows: initial denaturation at 94°C for 9 min and 30 cycles comprising denaturation at 94°C for 20 s, annealing at 54°C for 30 s and extension at 72°C for 30 s. Then, the second-step simplex PCRs were carried out for each individual fragment using a 1:20 dilution of the corresponding multiplex PCR product as the template, a concentration of 1.5 μM for each primer and following the same cycling conditions as for the first reaction. The 21 PCR reactions were directly sequenced and we obtained a single sequence. To obtain a second estimate of the sequence we repeated this multiplex PCR approach. Due to some difficulties with directly sequencing of some bands in the first PCR experiment, in the second round the fragments were cloned into the pstBlue-1 vector (Invitrogen). Three insert-containing plasmids were sequenced for each PCR fragment, thus obtaining a consensus sequence in which PCR errors revealed by the cloning process were disregarded.The sample IBE-C3159 (a claw), on the other hand, was obtained from an individual captured in 1973 and had a much more degraded DNA. Therefore, the amplification of the cytochrome approach. Two oveFinally, for each sample, a comparison was made between the two sequences obtained via independent rounds of PCR reactions, and no differences were found. Thus, it seems that no extensive DNA damage had occurred during the preservation process of these two museum samples.ACOX2-3, COPS7A-4, DHRS3-3, LANCL1-4, PRPF31-3, ROGDI-7 and SMYD4-5, chosen from the set described in Igea et at.[ACPT-4, obtained during the filtering processes leading to this set[A subset of 29 tissue samples that represented all mitochondrial lineages and covered the whole geographic distribution of the species was selected, and eight nuclear single-copy introns were sequenced from them. The amplified introns were a et at., and an this set. The priDesmana moschata and a representative of Talpinae , following procedures similar to those described above.Sequences of all the intronic markers were also amplified from All PCR products were purified using ExoSAP-It (Affymetrix) and sequenced in both directions using the original PCR primers with BigDye v3.1 at different sequencing services. Sequences were inspected, trimmed and assembled using Geneious Pro (Biomatters Ltd.).b and D-loop sequences of the 134 G. pyrenaicus samples were concatenated for further analyses. The optimal model of sequence evolution was determined using the Akaike Information Criterion with jModeltest version 0.1[G. pyrenaicus mitochondrial sequences were also inferred using a Bayesian approach, as implemented in BEAST 1.6.2[The cytochrome sion 0.1. The ression 0.1. From thsion 0.1. The phyST 1.6.2 by estimST 1.6.2 indicateFor the heterozygous nuclear sequences, distinct haplotypes were manually obtained since the sequences contained only one heterozygous position. Haplotype genealogies were then generated for each marker using Haploviewer from the corresponding PhyML tree.s[2[Nucleotide and haplotype diversity parameters were estimated using DnaSP version 5. Signalss and R2[4s[2 statistis[2.G. pyrenaicus distribution area were determined with the Monmonier’s Maximum Difference algorithm[Correlation of genetic and geographical distances was assessed with a Mantel test using the program Alleles In Space 1.0. Geneticlgorithm, which ilgorithm. For thiMitochondrial genetic diversity was estimated at each sampling location by using all sequences collected within 1 degree (approximately 100 km) of the location. This area allowed the estimation of genetic diversity from a good number of samples at each point, yet the resolution was good enough to distinguish regional differences in genetic diversity. In addition, centering the measurements around each sampling location, rather than using a fixed grid, allowed the efficient grouping in less sampled areas. To avoid inflating genetic diversity due to lineages in secondary contact, only sequences belonging to the same lineage were used for each locality but, for comparison, additional analyses were performed with mixed lineages. For each subset of sequences around a location with more than two samples, nucleotide diversity (π) was estimated. A regularly spaced grid of π values was then interpolated and a contour map was constructed using Surfer 10.2 (Golden Software Inc.).G. pyrenaicus could be used to date the mitochondrial lineage splits, we had to rely on more external mammalian fossil data. However, in trees of divergent mammalian groups, mitochondrial genes are saturated whereas nuclear genes are more adequate[G. pyrenaicus mitochondrial haplotypes was done in two steps. First, we obtained an accurate calibration of the G. pyrenaicus– D. moschata split from a Bayesian nuclear tree of Laurasiatherian mammals with multiple fossil data. For this analysis, we used the eight introns sequenced in this study from G. pyrenaicus, D. moschata and T. occidentalis, as well as the corresponding orthologous sequences of the following Laurasiatherian mammals with genomes available in the Ensembl database[Felis catus, Canis familiaris, Pteropus vampyrus, Equus caballus, Bos taurus, Tursiops truncatus and Sus scrofa. For some of the species, not all the orthologous introns were available, resulting in 7.5% of missing data. Intron alignments were built with MAFFT using the L-INS-i accuracy-oriented method[et al.[Since no reliable multiple fossil calibrations close to adequate. Therefodatabase: Felis cd method. Gblocksd method. These ed method, which id method. Using td[et al., we evalG. pyrenaicus – D. moschata split was used in a subsequent analysis using only talpid cytochrome b sequences to estimate the time to the MRCA of the G. pyrenaicus mitochondrial sequences. In addition to all sequenced cytochrome b haplotypes of G. pyrenaicus (35 unique haplotypes) and D. moschata, we obtained from GenBank complete cytochrome b sequences of representative Talpinae species (75 haplotypes belonging to 18 species). Talpinae is a sister group to the G. pyrenaicus – D. moschata group (Desmaninae) and therefore it is the most adequate outgroup. In the BEAST analysis of these 111 sequences, the TrN + Γ + I model of sequence evolution was chosen following jModeltest, and a UCLN clock was assumed . Due to the divergence of cytochrome b within the talpids family, particularly in third-codon positions, the sites were partitioned according to the three codon positions, so that each partition had its own model parameters. For the tree prior, a coalescent constant population size model was used since the node of main interest was intra-specific. The desmanines split was calibrated using a normal distribution with the mean and standard deviation taken from the results of the previous analysis of Laurasiatherians. Running conditions were the same as above.In a second step, the resulting posterior distribution of the age of the G. pyrenaicus, occurrence data were taken from the species distribution atlases of Spain[http://www.gbif.org/). For each record, the center of the corresponding 10×10 km UTM square was taken, resulting in a total of 680 unique data points. The study area was defined between 39 and 44° latitude, and −10 and 4° longitude, encompassing the whole distribution area of G. pyrenaicus plus additional areas of potential dispersal, suitable for the selection of background data[In order to develop a distribution model of of Spain, Portugaof Spain and Franof Spain. Coordinund data.http://www.worldclim.org). Climatic variables were downloaded for present conditions and for the Last Glacial Maximum (LGM). For the latter, both the Community Climate System Model (CCSM) and the Model for Interdisciplinary Research on Climate (MIROC) were used. Colinearity among the climatic variables for present conditions was analyzed by means of pairwise correlations using 1000 randomly selected points from the area of interest. After removing variables with correlation coefficients greater than 0.9, we retained the following 11 variables: BIO1 , BIO2 , BIO3 , BIO4 , BIO5 (Max Temperature of Warmest Month), BIO6 (Min Temperature of Coldest Month), BIO8 (Mean Temperature of Wettest Quarter), BIO9 (Mean Temperature of Driest Quarter), BIO12 , BIO14 (Precipitation of Driest Month), and BIO15 .The 19 BioClim climatic variables, which rTo predict the potential distribution of the species in current conditions and in the LGM we used Maxent version 3.3.3, which oG. pyrenaicus from 115 different localities covering a large part of the species distribution range and all important river basins  revealed that one of the few amino acid changes that occurred along this phylogeny is shared by all individuals of lineages A1 and A2  between groups A and B and 0.8% mean differences in the comparisons of both A1 with A2 and B1 with B2.b gene alone). When this value was calculated for each lineage, important differences were found among them, with one order of magnitude difference between the lineages with the highest (A1: 0.0036) and lowest (B2: 0.0004) nucleotide diversity values. In addition, a significant signal of population expansion was found only for the lineage A1 with the R2 and Fu’s Fs statistics but not with Tajima’s D. As previously shown[2 and Fu’s Fs have been shown to detect deviations from a constant population size in a wide variety of situations.The overall genetic diversity of the Pyrenean desman measured with the concatenated mitochondrial sequences is relatively low Table , with a ly shown, while TThe use of feces to obtain genetic data could lead to an underestimation of genetic diversity values if several samples of the same individual are used. Our choice of using only feces separated at least 1 km should prevent this problem but, for completeness in the phylogeographic analyses, we also included a few samples within that distance when haplotypes were different (see Methods). To test if this approach of selecting feces generated an unbiased collection of samples, we calculated nucleotide diversity for the 69 fecal samples and the 65 tissues separately , one of them (position 39 of intron DHRS3-3) was present in at least three of these lineages: A1, B1 and B2  interval of 0.15 - 0.56 Myr, clearly within the Middle Pleistocene  that have a marked parapatric distribution. However, this structure is not by itself proof of the effects of glaciations as it may predate the Pleistocene. Therefore it is important to obtain an accurate dating of the splits of these lineages. Our dating approach allowed us to estimate that the split of the two most divergent mitochondrial phylogroups occurred 0.32 Myr ago and that the subsequent divergence of the two pairs of lineages concomitantly took place at around 0.23 Myr ago. Since sequence coalescence must be older than the population split, these dates represent the upper limit at which the desman populations started to diverge. Therefore, given these Middle Pleistocene lineage split times, it is very likely that the four desman populations evolved in four isolated glacial refugia, supporting the importance of the Pleistocene glaciations in the population structure of this species. Most probably, the populations started to diverge during earlier phases of the glacial periods and not necessarily in the last glaciation, explaining the deep mitochondrial divergences observed[We have been able to gather a number of solid pieces of evidence that show that the evolutionary history of observed,60.Galemys and Desmana. The obtained date at 13.9 Myr was quite adequate to calibrate, in a subsequent step, the mitochondrial gene tree. On the one hand, this date is not as old as to present problems of saturation. On the other hand, it is not as recent as to suffer from the problems of coalescence, which can be exacerbated when dating very recent nodes of a gene tree (< 10 Myr)[b of talpids and, from this calibrated tree, we estimated the divergence time of the main Galemys mitochondrial lineages at 0.32 Myr. The evolutionary rate resulting with this approach for cytochrome b was 0.0224 substitutions/position/Myr. Although this evolutionary rate is line with those obtained for other mammalian groups[Galemys would only reduce, in the equivalent proportion, the split time of the mitochondrial lineages. Since our main purpose in this part of the work was to test if the separation of the mitochondrial lineages occurred in the Pleistocene, any increase in this rate would still support the Pleistocene split of the G. pyrenaicus mitochondrial lineages.Since we did not have reliable fossils in desmanines, we had to use more external calibrations of mammals for our dating analysis. This analysis benefited from the nuclear introns, which allowed us to reconstruct a Bayesian tree of mammals calibrated with multiple fossils and to estimate the divergence date of  10 Myr). This can groups,63, diffn groups-66. Alson groups. It has n groups may causn groups,69. Actun groups,70 or thn groups may leadn groups and we uGalemys-Desmana split time in the first step of our dating analysis, the low variability of these sequences  would have been recently colonized. However, the dispersal, at least of females, would not have progressed towards the northwestern parts of the Iberian Range (Demanda Mountains).Lineage B1 could have evolved in a refugium in the Cantabrian Mountains, where the maximum genetic diversity for this lineage was found. From this refugium, the species would have colonized the northwestern part of the Iberian Range (Demanda Mountains). However, at least the females would not have continued the dispersal towards more southeastern parts of the Iberian Range . Dispersal of lineage B1 from its putative refugium towards the western parts of the Cantabrian Mountains would have also been limited, and, at least the females, would not have crossed to the areas occupied by lineage A1.Despite potentially suitable refugia within the Pyrenees, the PyrThe distribution of mitochondrial lineages and their variability allowed us to infer a clear postglacial expansion of the desman populations from the refugial areas. This range expansion should be in principle accompanied by an increase in population size, but the population expansion parameters of the mitochondrial sequences did not show statistically significant support in all lineages Table . This isIt is also worth noting that the range expansions hypothesized here could have taken place during particular periods of very favorable conditions, as shown for other species. In the Thus, the evolutionary history of the Pyrenean desman supports the “refugia within refugia” hypothesis, which hContrary to the expectation that the genetic structure of a species with a semi-aquatic lifestyle and a strong dependence for clean waters, such as the Pyrenean desman, would be highly related to rivers and drainage basins, we found that only a small proportion of its genetic variation can be attributed to the grouping of populations by major river systems. In fact, identical mitochondrial haplotypes can be found at both sides of different mountain ranges, explaining the lack of strong differentiation among basins. Thus, these data allow us to infer that gene flow between basins exists or existed in a not so distant past. In conclusion, the genetic structure of the Pyrenean desman has been more influenced by the history of the Pleistocene glaciations than by its current aquatic habitat distribution, in spite of the strong fragmentation of such specialized habitat. This situation is intermediate between strictly aquatic organisms, whose genetic diversity has been more conditioned by river basins, and higThe most unexpected finding in the genetic structure of the Pyrenean desman was the existence of narrow contact zones between the mitochondrial lineages that came into secondary contact after the post-glacial recolonization, with no apparent mixing among them. Actually, dispersal of the four lineages in different directions from the peripheral glacial refugia and the lack of suitable areas in the central parts  have created an interesting circular distribution of the Pyrenean desman Figure . In addiThe strongest genetic gap was found in the middle of the Iberian Mountain Range, one of the places where the A and B groups meet Figure A. The 23G. pyrenaicus to analyze these aspects in depth. However, three variants of the obtained SNPs  allowed us to study the phylogeography of this species and provided evidence for an evolutionary history deeply influenced by the Pleistocene glaciations. One of the most striking findings of this work was the existence of a strong phylogeographic structure in the Pyrenean desman, in which two large groups, A and B, were subdivided into two further groups to give a total of four mitochondrial lineages with parapatric distribution . Two narrow contact zones between the major groups (A and B), one in the Iberian Range and the other in the Cantabrian Mountains, indicate incomplete mixing after the post-glacial recolonization, at least for females. Nuclear data seem to indicate some degree of gene flow in these contact zones but more data will be necessary to further study the dispersal patterns of the desman. It is interesting to note that the presence of two major and parapatric mitochondrial groups parallels the existence of the two described subspecies, G. p. pyrenaicus and G. p. rufulus, whose distributions roughly correspond to groups B and A, respectively.Mitochondrial and nuclear data in the Pyrenean desman  and a species distribution model projected to the LGM coincided in indicating that the most important glacial refugium was in the NW of the Iberian Peninsula. Other minor refugia can be postulated in other parts of the distribution areas of the present mitochondrial lineages. A Holocene expansion from these refugia, but interrupted at the contact zones, led to the current parapatric distribution of the mitochondrial lineages.The Pyrenean desman is an endangered species and its situation has worsened during the last few years in part of its distribution range, particularly in the most southern populations. In order to undertake the most favorable actions for the long-term survival of this species, conservation programs should keep in mind the peculiar genetic patterns found in this work. Most importantly, artificial mixing of desmans and, particularly, of individuals belonging to different lineages should be avoided. At the moment, almost no natural exchange between the lineages with different glacial origins has been observed and therefore no artificial translocations between them should be carried out until further studies establish the exact degree of genetic exchange between these populations. Although only following these criteria in management plans does not guarantee the conservation of the species, it would be essential to take this information into account in order to prevent an aggravation of the status of this singular species.Galemys, Laurasiatherians and Talpids have been deposited in TreeBASE under accession number S14084 (http://purl.org/phylo/treebase/phylows/study/TB2:S14084).All sequences obtained in this study have been deposited in GenBank under accession numbers JX290581 - JX291096  used as priors in the BEAST analysis of Laurasiatherian mammals. Figure S1. Contour plot of genetic diversity (π) of Galemys pyrenaicus in which samples of populations in secondary contact were mixed in the calculations.Click here for file


Ciona intestinalis (Ascidiacea: Enterogona: Cionidae), a model species complex, of which spA and spB are rapidly spreading worldwide. We intensively sampled 26 sites (N = 873) from both coasts of North America, and performed phylogenetic and population genetics analyses based on one mitochondrial fragment  and eight nuclear microsatellites. Our analyses revealed extremely complex genetic patterns in both species on both coasts. We detected a contrasting pattern based on the mitochondrial marker: two major genetic groups in C. intestinalis spA on the west coast versus no significant geographic structure in C. intestinalis spB on the east coast. For both species, geo-graphically distant populations often showed high microsatellite-based genetic affinities whereas neighboring ones often did not. In addition, mitochondrial and nuclear markers provided largely inconsistent genetic patterns. Multiple factors, including random genetic drift associated with demographic changes, rapid selection due to strong local adaptation, and varying propensity for human-mediated propagule dispersal could be responsible for the observed genetic complexities.Anthropogenic activities frequently result in both rapidly changing environments and translocation of species from their native ranges . Empirical studies suggest that many factors associated with these changes can lead to complex genetic patterns, particularly among invasive populations. However, genetic complexities and factors responsible for them remain uncharacterized in many cases. Here, we explore these issues in the vase tunicate  Identifying factors responsible for microevolutionary processes over contemporary timescales is important for understanding how species evolve in response to rapidly changing environments associated with recent anthropogenic activities . BiologiTo better understand these evolutionary mechanisms, it is necessary to identify potential generalities and inconsistencies within and among multiple invasive species across different geographic scales, and then identify the evolutionary/ecological factors associated with common and unique genetic patterns. For aquatic species, many factors including life-history characteristics , environmental heterogeneity , anthropogenic activities , coupled with varying propensity for human-mediated propagule dispersal, can lead to extremely complex genetic patterns . Recent Ciona intestinalis, is a well-known model species for developmental and evolutionary biology  sequences and nuclear microsatellites to characterize genetic patterns of 26 populations of Ciona intestinalis specimens were collected from the invaded ranges on both coasts of North America. On the west coast, sampling was conducted along the coast of California between July and November 2006, spanning from Tomales Bay to San Diego estuary, while on the east coast sampling was performed between October 2007 and March 2009 in Prince Edward Island, Nova Scotia, and Connecticut  and 15 . Bayesian inference (BI) and neighbor-joining (NJ) phylogenetic analyses were conducted using outgroup . The besoutgroup  with Akaoutgroup . Trees woutgroup  based onoutgroup . Clade soutgroup .n), haplotype diversity (h), and nucleotide diversity (π) were assessed using DNASP v5  substitution model and 10,000 permutations in ARLEQUIN v3.1 (ST/(1–ΦST)] and geographic distance, we performed isolation by distance (IBD) analysis for spA and spB using a Mantel test with 10,000 permutations implemented in GENEPOP v3.4 , allelic frequency (F), allelic richness (Ar), as well as the observed heterozygosity (HO) and expected heterozygosity (HE) using FSTAT v2.9.3.2 (U test), implemented in STATISTICA v6 , was used to test the difference in allelic richness (Ar) and expected heterozygosity (HE) among populations. Markov chain method  in GENETIX v4.05 . Ten independent runs were performed for each specified K-value, and for each run, 1,000,000 generations were used after discarding 100,000 generations as burn-in. The measure of ΔK (FST/(1–FST)] and geographic distances in each species. A hierarchical AMOVA was performed in ARLEQUIN based on microsatellite genetic distances between populations, and partitioning variance as for mtDNA.Population differentiation was determined by FST  for all IX v4.05  and a BaIX v4.05 . For there of ΔK  was usedA total of 663 individuals derived from 26 populations from both coasts were successfully sequenced for the COX3-ND1 fragment . Speciesh) varied from 0.429 to 0.861 for spA, and 0.191 to 0.821 for spB (h = 0.429) in spA was found in the population MO (Monterey Bay), sampled from the open sea shore on the west coast, while the highest was detected in major ports including SF , PH , and SD  to that in other populations  for spA and 27 haplotypes  for spB ; Fig. 2. for spB . The lowulations .ST values  to the among-group component  ranged from 3.4 to 7.0 (mean 5.3) for spA and from 3.7 to 9.3 (mean 6.8) for spB (HE) values were observed for all populations, ranging from 0.563 to 0.703 for spA and from 0.510 to 0.875 for spB  had the highest number of mtDNA haplotypes n = 8;  but a reFST values ranged from zero to 0.123 with an average of 0.059. In total, 36 of 55 (65.5%) population pairs were significantly different after Bonferroni correction (FST = 0.123) was detected between two neighboring populations, CI (Channel Islands) and PH  comparisons of population pairs remained significantly different after Bonferroni correction  and all other sites surveyed, with an average of pairwise FST values greater than 0.1  as the most parsimonious possibility , which is inconsistent with mtDNA results obtained from pairwise ΦST  and AMOVST . Bayesiasibility . Most insibility . Howeversibility . This pasibility . For spBsibility  and 6B, sibility  and pairT values . Since lT values . This fiT values . SimilarT values .C. intestinalis spA and spB. A contrasting geographic pattern was detected in the two species based on the mitochondrial marker: two major genetic groups in spA on the west coast versus no significant geographic structure in spB on the east coast  and NB (Newport Bay), located in two different geographic groups as defined by mtDNA  decrease. Moreover, compared to nuclear markers, mtDNA has smaller effective population size (Ne mtDNA≍ 1/4 Ne nuclear), likely leading to stronger signatures of genetic drift on mtDNA. For broadcast spawning marine invasive species including Ciona, two possible processes—sweepstakes reproductive success and dramatic demographic changes during translocation—can lead to strong genetic differentiation between populations. Marine species with broadcast spawning exhibit sweepstakes reproductive success, in which a subset of the population contributes the majority of offspring to subsequent generations  was observed not only between geographically distant populations, but also between adjacent ones  were divergent from all other populations , sampled from open sea shore, had lower genetic diversity than other populations . Compareulations  and 6. Aulations . The obsulations  and 6B, ulations . Bayesiaulations . Consequulations  suggestsulations ; Table 1ulations , and theulations .n = 3, h = 0.191), though the pattern was not repeated with microsatellite loci (Interestingly, we observed inconsistent patterns of genetic diversity when using mtDNA and nuclear microsatellites in both species . The twoite loci . In addiite loci ; Fig. 2.ite loci . Given tOur genetic surveys add to growing evidence that complex genetic patterns can emerge in invasive species, including closely related species having similar biological characteristics. In addition, inconsistent genetic patterns also can be detected when using two different types of markers, that is, mtDNA versus nuclear microsatellites. Detailed analyses suggest that multiple factors, including random genetic drift associated with population demography change, rapid selection due to strong local adaptation, varying propensity for human-mediated propagule dispersal, as well as their interactions, could be responsible for such genetic complexities. In addition, in order to avoid possible biased results caused by one type of molecular marker, our study highlights the necessity of use of both mtDNA and nuclear markers to perform genetic analysis in invasive species. The complex genetic patterns observed here, coupled with multiple putative factors causing such complexities, open a window for us to deeply investigate mechanisms of rapid evolution associated with fast environmental changes due to frequent anthropogenic activities.