Timema cristinae stick insects to test the effects of different factors on genomic divergence in a system undergoing ecological speciation. We find patterns consistent with effects of numerous factors, including geographical distance, gene flow, divergence in host plant use and climate, and selection against maladaptive hybridization (i.e. reinforcement). For example, the number of highly differentiated ‘outlier loci’, allele-frequency clines and the overall distribution of genomic differentiation were recognizably affected by these factors. Although host use has strong effects on phenotypic divergence and reproductive isolation, its effects on genomic divergence were subtler and other factors had pronounced effects. The results demonstrate how genomic data can provide new insights into speciation and how genomic divergence can be complex, yet predictable. Future work could adopt experimental, mapping and functional approaches to directly test which genetic regions are affected by selection and determine their physical location in the genome.Diverse geographical modes and mechanisms of speciation are known, and individual speciation genes have now been identified. Despite this progress, genome-wide outcomes of different evolutionary processes during speciation are less understood. Here, we integrate ecological and spatial information, mating trials, transplantation data and analysis of 86 130 single nucleotide polymorphisms (SNPs) in eight populations (28 pairwise comparisons) of  The fiea priori predictions can be made for the manner in which divergence will be affected by particular processes [FST values). In contrast, allopatric speciation might be characterized by divergence across more of the genome, leading to a different distribution of genomic differentiation than observed with gene flow [Although non-overlapping predictions concerning the specific causes of patterns of genomic divergence are still being developed, a number of rocesses . For exarocesses ,24, whilene flow . AdditioTimema cristinae stick insects. We collected genomic data from a mosaic of populations where past work clearly indicates that numerous factors, such as divergent host adaptation, geographical separation, gene flow and reinforcement all affect speciation [T. cristinae, rather than focusing in on specific genes that causally affect adaptation and speciation. Our Bayesian analyses incorporate genotype uncertainty, and thus account for the uneven coverage among gene regions and individuals inherent in NGS data.Our goal here is to assess these predictions and their applicability in population comparisons of eciation . The genThe results demonstrate interplay between different speciation processes, each of which is associated with different patterns of genomic divergence. Surprisingly, we find that the effects of host use on genomic divergence are subtle, despite the strong effects of host use on ecological speciation, and that factors other than host use have pronounced effects on genomic divergence. The results highlight how genomic data have great potential for advancing understanding of speciation.2.Timema cristinae is found feeding on two strikingly different host plant species in southern California . There is strong evidence that divergent host adaptation has contributed to phenotypic divergence  and reproductive isolation, and is thus causing ecological speciation [eciation . For exaFurthermore, populations can be geographically separated from one another (i.e. by regions containing unsuitable hosts) or in direct contact . Past work indicates that adjacent population pairs experience more gene flow than separated pairs, but that mating isolation between adjacent pairs has nonetheless been promoted by reinforcement –28. HybrTimema cristinae is therefore ideal for analysing the genomic consequences of multiple evolutionary processes.In contrast to mating isolation, divergence of other characters is constrained by gene flow. Morphological traits such as size, shape and coloration are less divergent between geographically adjacent than geographically separated populations ,27. For 3.(a)t-test was used to test whether the degree of mating isolation differed between separated versus adjacent population pairs.We quantified sexual isolation and morphological divergence for the specific pairs of populations examined for genomic divergence . Previously published protocols were used to conduct no-choice mating trials ,31 durint-test was used to test whether the degree of divergence between populations in the proportion of striped individuals (% striped on Adenostoma − % striped on Ceanothus) differed between geographically separated versus geographically adjacent population pairs. Labels for populations for which genomic data were not collected are as follows: R23A is 6A, R6C is 7C, LOGA is 8A and PC is 9C T. cristinae via sequencing of reduced complexity genomic libraries . DNA was extracted from the legs of T. cristinae using Qiagen DNeasy kits. We used genomic enrichment prior to high-throughput sequencing involving a restriction enzyme digestion step followed by PCR amplification to produce a pool of fragments for sequencing [EcoRI adaptor on two lanes of an Illumina GAIIx genetic analyser are described in the electronic supplementary material.To characterize genomic divergence, we generated genome-wide sequence data from eight populations of quencing ,35. Illu(c)eqMan Ngen v. 3.0 . The de novo assembly placed 10 852 133 reads into 293 614 contigs containing a minimum of eight reads for an average coverage depth per contig across all individuals of 36×. We used the consensus sequences from the contigs constructed during de novo assembly to construct an artificial reference, and assembled the complete set of 46 153 271 reads onto this reference using a template-guided assembly. This placed 34 357 655 reads onto the reference sequences, and resulted in an average coverage depth of 125× per genetic region across all individuals . After calling variants using bcftools in samtools and further quality trimming, 86 130 variant sites (i.e. SNPs) spanning 57 969 contigs were used in subsequent analyses. Details are in the electronic supplementary material.An average of 284 896 reads were generated per barcoded individual. We executed a de novo assembly on a set of 20 million reads using S(d)prcomp function (R Development Team).We used a Bayesian model to estimate population allele frequencies for each SNP based on the observed sequence data ,37. The αi relative to the estimated genome-wide distribution of α (i.e. logit(FST)) as described in earlier studies by Gompert and co-workers [α is ∫∫Normaldμdτ, where μ′ and τ′ are the posterior probability distributions of μ and τ. Specifically, α is a vector with one element for each locus. Each element of α is the logit FST for a locus, μ is the mean or genome level logit FST, τ describes variation in FST among loci, and is equal to the precision (one divided by the variance) of locus-specific logit FST.We used a hierarchical Bayesian implementation of the F-model to quantify genetic differentiation among populations . Statistical outlier loci  between individual population pairs were identified based on estimates of -workers ,37. The αi was not contained in the interval Nq, where Nq is the interval bounded by the 95th and (1–95th) quantiles of the genome-wide distribution of α. We tested for associations between allele frequencies and bioclimatic variables using methods described in the electronic supplementary material.We called loci statistical outliers if the posterior point estimate of Δ) for each pair of variable sites [Δ is a population-level measure, and per population coverage was relatively high even when per individual coverage was lower.We estimated Burrow's composite measure of Hardy–Weinberg and linkage disequilibrium We used approximate Bayesian computation (ABC) to model historical divergence and gene flow among the 28 population pairs. Specifically, we compared two alternative models for each pair of populations: (i) divergence without gene flow, and (ii) divergence with gene flow. See the electronic supplementary material for details.(f)We used simple and partial Mantel randomization tests to compare various matrices with one another while accounting for potential non-independence of matrix elements, as described in the electronic supplementary material . For exa(g)2 area surrounding N34 30.958 W119 48.050) from two populations that varied in their distance from the transplant site. The individuals used in this experiment were from populations that are phenotypically variable for traits involved in host adaptation (owing to gene flow) and are thus expected to survive, on average, equally well on each host species. Thus, these experiments examine the effects of geographical distance, rather than host, on survival. The first transplant involved the population R12C that is located approximately 25 km from the transplantation site. This corresponds to the maximum distance in our genomic sampling and therefore represents a ‘distant’ transplant. The second transplant involved the population FHA (N34 31.089 W119 48.166) located about 1 km from the transplant site, representing a ‘near’ transplant. We used ANOVA to test whether the proportion of survivors (i.e. % recaptured) in each transplant was dependent on the transplant (fixed factor), host (fixed factor) and block (random factor). We tested a full model and a reduced one containing only main effects.Insects were transplanted to one general area (approx. 1 km4.(a)T. cristinae [n = 503 trials) demonstrated that the specific adjacent population pairs examined here for genomic divergence exhibited twice the mating isolation of separated pairs n = 28). The results revealed highly variable genetic differentiation between populations. Specifically, average differentiation was relatively strong (mean FST = 0.111), but ranged among population pairs from very low (minimum FST = 0.007) to very high (maximum FST = 0.306). PCA based on genotypic state posterior probabilities revealed genetic clustering according to the geographical distances between populations .The genomic data yielded 86 130 SNPs. We used Bayesian models to estimate allele frequencies , genotype probabilities and genome-wide differentiation between all possible pairwise comparisons between the study populations (Δ) within populations were very low, indicating these SNPs were largely statistically independent from one another and did not generally exhibit pronounced departures from Hardy–Weinberg equilibrium  [Information about the genomic distribution of the SNP loci will require future linkage mapping studies or a whole genome reference sequence. However, estimates of Burrow's composite measure of Hardy–Weinberg and linkage disequilibrium (results) . SNPs wiInvestigations of genomic divergence often focus on the identification and enumeration of loci with unusually high levels of population differentiation. We used a Bayesian method developed for next-generation sequence data to identify such ‘outlier loci’ that are statistically diverged beyond background null expectations ,37. OutlΔ for outlier loci were low, and not markedly different from that of all loci . Thus, outliers appear to largely represent independent markers of population divergence and not tightly linked blocks of loci, but confirmation of this awaits mapping studies. The number of outlier loci varied drastically among population pairs , providing the requisite variation to test which factors affect this variability. Two factors clearly and significantly did so (p > 0.40 in Mantel tests).A total of 15 207 loci were categorized as statistical outliers. Absolute levels of These patterns raise four fundamental questions: (i) does host adaptation recognizably affect genomic differentiation? (ii) Is the enrichment of outliers between adjacent population pairs a consequence of reinforcement? (iii) Why do the number of outliers increase with increased geographic separation? (iv) Are the genomic effects of these factors restricted to outlier loci? We address each question in turn.(c)FST for outliers specific to different-host pairs across all 28 population pairs (i.e. not just those in which they were outliers) was significantly greater for different-host than same-host-pairs . Although the number of outlier loci was not related to divergence in host use, some loci were associated with divergent host use. For example, 529 loci were observed as outliers in different-host pairs but never exhibited outlier status in any same-host pair (with 59 being outliers in multiple different-host comparisons). The outliers specific to different-host pairs are likely to reside in regions affected by host-specific selection. Their relatively low numbers could be due to the small fraction of the overall genome sequenced and due to gene flow preventing strong differentiation at loci subject to weak host-specific selection. Consistent with the latter hypothesis, mean st-pairs . Thus, h(d)As predicted by reinforcement, of the hundreds of outliers found between geographically adjacent pairs, almost all (493 of 554 = 89%) were restricted to being outliers between adjacent population pairs and were thus never outliers in the 25 comparisons between geographically separated pairs. The excess of outliers between adjacent pairs cannot be explained by accentuated climatic differences between such pairs, because adjacent pairs are the least climatically divergent .(e)T. cristinae. For example, mark–recapture data estimated that the average per-generation dispersal distance was only 12 m [The observed increase in genomic differentiation with increased geographical distance between populations could be explained by a number of processes, including stronger divergent selection, weaker gene flow, demographic variability and genetic drift, mutation rate variation or a combination of these factors . To testnly 12 m  and maninly 12 m . If the r = −0.75, −0.71, both p < 0.01, Mantel tests on raw and log-transformed data, respectively). Zero gene flow was observed at a scale below the maximum 25 km distance examined (Nem) fall to zero for populations separated by more than 10 km. Likewise, posterior probabilities for a model of zero gene flow increase abruptly from near zero to one at around 15 km of separation. Thus, factors other than reduced gene flow likely affect the number of outliers observed.We used ABC to estimate gene flow from the genomic data under a model of isolation with migration. The results revealed that gene flow decreased sharply with increased distance between populations . This pattern is consistent with stronger divergent selection between distant than nearby populations, but further data are required to confirm this  or near (1 km) the transplant area. When individuals were transplanted from a distant source population, their survival 8 days later was significantly lower compared with individuals transplanted a short distance . Thus, allele frequencies were not only affected by host plant use and geography, but also by climate. In sum, reduced gene flow likely contributes to an increased number of outliers with increasing geographical separation, but other factors such as increased selection or drift are likely involved as well, particularly at distances beyond 10–15 km.Finally, we tested for clinal variation in allele frequencies along environmental and spatial axes, as predicted if selection varies spatially. Specifically, we examined relationships between allele frequencies within populations and three continuous variables FST with reduced gene flow [FST distribution was more strongly ‘L-shaped’ for adjacent pairs than geographically separated pairs , with less density in the centre and a more pronounced tail of extreme values . Adjacent pairs, relative to separated pairs, exhibited significantly lower average FST values  and less overall variance in FST among loci , owing to adjacent pairs exhibiting many fewer loci with moderate FST values. Even among separated pairs, such patterns often became more accentuated as geographical distance between populations increased . These results demonstrate widespread effects of geography on genomic divergence. Indeed, it could be that geographical separation in widespread species is similar to speciation in a ring, where gradual reductions in gene flow with distance facilitate speciation [The effects of geography were not restricted to outlier loci. The overall distribution of differentiation across the genome was also affected by geographical distance, as expected under population genetic models that predict both increased mean and variance of ene flow . In parted pairs . As a reeciation .Figure 5.T. cristinae are an example of the process of ecological speciation via host adaptation [Our results have implications for understanding speciation. The ecotypes of aptation . The finaptation . For exaOur results highlight how the speciation process can have complex, but predictable, effects on genomic divergence, that reflect the consequences of geographical separation, gene flow, multifarious divergent adaptation, reinforcement and other evolutionary processes. Future work using experimental, mapping and functional genomics approaches to directly test which genetic regions are affected by selection, and to determine their physical distribution across the genome, will likely yield further insight into the speciation process.


ST statistics Northern and Southern Italy amounts approximately to 0.001 between, and around 0.002 between Northern Italy and Utah residents with Northern and Western European ancestry (CEU). The Italian population also revealed a fine genetic substructure underscoring by the genomic inflation , warning against confounding effects of hidden relatedness and population substructure in association studies.In spite of the common belief of Europe as reasonably homogeneous at genetic level, advances in high-throughput genotyping technology have resolved several gradients which define different geographical areas with good precision. When Northern and Southern European groups were considered separately, there were clear genetic distinctions. Intra-country genetic differences were also evident, especially in Finland and, to a lesser extent, within other European populations. Here, we present the first analysis using the 125,799 genome-wide Single Nucleotide Polymorphisms (SNPs) data of 1,014 Italians with wide geographical coverage. We showed by using Principal Component analysis and model-based individual ancestry analysis, that the current population of Sardinia can be clearly differentiated genetically from mainland Italy and Sicily, and that a certain degree of genetic differentiation is detectable within the current Italian peninsula population. Pair-wise F We estimate genetic differentiation among Italian samples and between Italian and other populations from the literature: 1) using a model free analysis like PC and a model-based analysis to infer individual ancestry components (ADMIXTURE software), 2) by calculating pair-wise FDNA samples were obtained from 49 unrelated volunteers from four different Italian macro-areas . Details of the affiliation of the municipalities within the macro-areas mentioned in this work are described in In addition we used existing genetic data relative to control subjects from both published and unpublished genome-wide association studies: a study on malignant pleural mesothelioma  DNA was purified from blood samples by a standard phenol/chloroform extraction method. DNA concentrations were determined by spectrometry . The Illumina HumanOmni 1–QUAD, v1.0 BeadChip Array  was used to genotype 1,140,419 SNPs on 49 unrelated volunteers. Genotyping, according to the instructions provided by the manufacturer, was carried out at the Human Genetics Foundation (HuGeF) in Turin. All the information about this data set is reported in linkage disequilibrium (LD) pruning, we used 125,799 SNPs in: 1,262 individuals in the European and Mediterranean dataset, 1,099 in the European data set, and 1,014 in the Italian dataset .Array-based SNP genotypes were subjected to stringent quality control, performed separately for each panel . Samples2 = 0.5).Principal Component Analysis was performed on a set of about 125,799 pruned markers. The pruning procedure was used to optimize the analysis of population structure, identifying a set of SNPs with low background LD (rprcomp of R (package mva) To perform the analysis we used the function stats) The identity-by-state (IBS) sharing analysis The software ADMIXTURE ST pairwise values between populations the Eigensoft program  was used. High FST values implie a high degree of genetic differentiation among populations.To estimate Fadegenetdist function of the R software, taking into account latitude and longitude of birth place).The Mantel test was calculated by using the R package We calculated the maximum possible inflation factor The eigenvectors for different subsets  were calculated in order to project the Italian data sets onto a two dimensional space  using 12The position of the Italian population samples suggests that genetic distances between these populations and other European and Middle East populations has a good correlation with geographic distances. At the same time, Sardinia was confirmed to be a genetic “outlier”.Our main goal was to investigate the genetic structure of the Italian population considering four main macro-areas . We carried out PC analysis on the Italian samples and plotted the eigenvectors 1 and 2 in *e−16 and PC2 R = 0.49 p-value = 2.2*e−16). Mantel test was also used to compare genetic distance identities by state (IBS) between individuals and geographic distance: it showed a good correlation with geographic distance between sampled individuals  pattern. Considering the state of this inversion, three different orientations of this region of DNA can be observed, i.e. inverted homozygous, heterozygous, or homozygous non-inverted. Using only the SNPs within this region (163 SNPs),those that contribute mainly to the PC4, we reiterated the PC Analysis  panel A.Using these individuals as a reference we roughly estimated the frequency of these genotypes by the K-means clustering  panel B.We used the ADMIXTURE software Distributions of IBS sharing between and within population are shown in Density estimates for empirical distributions of genome-wide mean proportions of alleles shared between Italian and Northern European populations , panel c*e−6, range 0.706–0.729), followed by Northern and Central Italy  and then Southern Italy (0.715 with a standard error 2.6*e−5 a range of 0.707–0.722). The distribution mode was similar and higher for the populations of the Italian Peninsula, and lower for the Sardinians.In the These results are consistent with the model-based ancestry analysis and with the position of these populations in the eigenvector PC space.ST statistics. Estimates are given in ST = 0.004) was slightly lower than for many other European population pairs separated by larger geographical distances, for example, Southern Italians and CEU (FST = 0.005). In general, FST was lower between population pairs in closer geographical proximity like Southern Italians vs Central Italians (FST = 0.001). Southern Italians showed a genetic affinity with Middle East populations, such as Palestinian and Druze; and Northern Italian populations were genetically closer to the French and CEU populations. However, it should be stressed that the estimates are less accurate for pairs involving a population with a small sample size.We quantified genetic differentiation between the Italian population and the subset of HGDP-CEPH populations from Northern Europe (French), the Middle East , Northern Africa  and from HapMap CEU, by calculating the pair-wise FGC) was calculated to evaluate the possible impact of population stratification inside the four defined Italian subpopulations, and other populations from the literature. GC = 1.247), and warns against the confounding effects of hidden relatedness and population substructure in association studies. As far as European and Middle Eastern or North African populations are concerned, results showed that a random Northern Italian population was well matched to the French population (λGC = 1.102), but this does not correspond to the comparison between Northern Italians and CEU from HapMap (λGC = 1.427).Population stratification refers to a situation in which subgroups of individuals within the population of interest are, on average, more closely related to each other than to other individuals of the wider population. These allele frequency differences, can bias testing results and lead to artifact associations in case control studies. The genomic control inflation factor  A finer view of the Italian substructure, can be seen in The genetic structure observed in our dataset is expected to be mainly a consequence of demographic processes such as internal migration within and between the macro-areas. Indeed, Italy remains characterized by a strong migratory movements of the population within its territory st distribution fits with the PC analysis of the first two component. The Fst among the Italian macro-areas is moderate (Fst≤0.001) when considering the Italian Peninsula, but is more pronounced between Sardinia and the other macro-areas (Fst = 0.004) (st = 0.004 between different countries). A certain degree of genetic homogeneity shown by the Fst analysis, and by the partial overlapping of the distribution of the pairwise IBS within each of the Italian subpopulations, can be possibly explained by serial historical events and shared ancestry. The Fst values presented here are lower than those published by Nelis et al. 2009 using 270K SNPs; they reported that the Southern Italian population sample showed an Fst value of 0.005 compared to the Northern Italy sample. The highest value of this pairwise Fst matrix was found between the Finns from Kuusamo and Southern Italy (Fst = 0.023).The overall F= 0.004) . This isADMIXTURE analysis confirms that there was no clear separation between Northern and Central Italy, at least as considered as macro-areas. Additional comparison of the distribution of pair-wise identity-by-state within each of the four populations and ADMIXTURE analysis clarified that this is not an artifact of the PC analysis. However, the PC and ADMIXTURE analysis results could be due to the sparse geographical coverage of our samples, especially for the Central and Northern macro-areas. In fact, many of the individuals (N = 413) in the North Italian sample analyzed in this study were from Piedmont- a North West Italian region- that has historically been affected by intense migration. At the same time, many individuals in the Central Italy macro-area (113 samples) are settled in Tuscany, an administrative region which is at the border with northern regions.ADMIXTURE analysis was the proportion of ancestry in Sardinia, an ancestry shared with all the European and Northern African populations included in this analysis but with the highest level in Sardinia .It is known that the major components are influenced by geographic clustering and secondly from areas with strong LD GC analysis a higher degree of fine-scale population structure can be revealed. Systematic differences in sampling and genotyping are potential confounders, and may introduce a bias in association studies. The degree of genetic substructure between population pairs has been also measured by inflation factor (λGC) statistics GC = 1.12, and in cases from Sardinia and controls from Northern Italy the genomic control inflation factor was 3.040 , Italian dataset (b) and Italian dataset without Sardinia (c).(TIF)Click here for additional data file.Figure S2Hidden population structure within the Italian dataset. Scatter plot of the first two eigenvectors based on 125,799 autosomal SNPs and 1,012 individuals. Colors represent the four different macro-areas; green- Southern Italy , red- Central Italy , black- Northern Italy , blue- Sardinia (these samples were labeled for the linguistic area). Subjects are symbol- labeled by municipality. Information on municipality was not used for calculations.(TIF)Click here for additional data file.Figure S3Italian population projected scatter plot of the PC3 and PC4. Panel A Analysis based on 125,799 autosomal SNPs and 1,012 individuals. Color code shows different Italian subpopulations; green: Southern Italy, red: Central Italy, black: Northern Italy, blue: Sardinia.Panel B K-mean (K = 3) of PC3/PC4. Different colors shows the three diffent clusters.(TIF)Click here for additional data file.Figure S4Variable contribution of each SNP to the first four PC against the genomic location. Manhattan plot done in the Italian data set, the top SNPs in panel d localize to chromosome 8 inside the 8p23 region.(TIF)Click here for additional data file.Figure S5Individuals predicted to be homozygous inverted or heterozygous or homozygous non-inverted for the 8p23.1. Panel A Scatter plot of the PC1 and PC2 done using only SNPs located inside the 8p23 region (163 markers). Panel B Individuals predicted to be homozygous inverted or heterozygous or homozygous non-inverted using K mean clustering, K = 3. The frequency of homozygous inverted (light blue triangles) was of 21%, of homozygous non-inverted (orange triangles) was of 31.19%, heterozygous of 47.9%. Individuals from HapMap used to confirm the predictions for the 8p23.1 were respectively NA12815 (homozygous inverted); NA11992 and NA12057 (homozygous non-inverted); NA11993 NA06993 and NA11994 (heterozygous).(TIF)Click here for additional data file.Figure S6Model-based ancestry analysis based on a subset from HGDP-CEPH and HapMap CEU data on 1260 individuals. Ancestry for each individual was inferred with ADMIXTURE (TIF)Click here for additional data file.Figure S7Cross-validation error plot from the ADMIXTURE program. Populations coming from the Italian dataset plus HGDP-CEPH  and some Hapmap populations. K = 1–10.(TIF)Click here for additional data file.Table S1Number of SNPs inside each panel before and after a filter for SNP call rate. SNPs intersection between the six studies was 163,355. After a filtering for minor allele frequency (MAF>0.01) the number of polymorphisms was reduced to 163,350. A subset of 163,095 SNPs passed Hardy-Weinberg equilibrium. A subsequent dataset of 125,799 SNPs after Linkage Disequilibrium pruning were used for PC analysis, Fst, genomic control estimation and IBS analysis.(DOC)Click here for additional data file.Table S2Correlation between PC's score and genetic/geographical values.Correlation between PC1 score, PC2 score, PC3 score PC4 score and genetic distance (IBS) and latitude, longitude and geographical distance (great circle distance) within the Italian dataset. All the correlation values were significative .(DOC)Click here for additional data file.


Rhododendron pseudochrysanthum s. l.A complex of incipient species with different degrees of morphological or ecological differentiation provides an ideal model for studying species divergence. We examined the phylogeography and the evolutionary history of the R. hyperythrum and R. formosana, both trees lacked reciprocal monophyly for all members of the complex. For R. pseudochrysanthum s.l., the spatial distribution of the cpDNA had a noteworthy pattern showing high genetic differentiation (FST = 0.56-0.72) between populations in the Yushan Mountain Range and populations of the other mountain ranges.Systematic inconsistency was detected between gene genealogies of the cpDNA and nrDNA. Rooted at R. hyperythrum, R. formosana and R. pseudochrysanthum s.l. Independent colonizations, plus low capabilities of seed dispersal in current environments, may have resulted in the genetic differentiation between populations of different mountain ranges. At the population level, the populations of Central, and Sheishan Mountains may have undergone postglacial demographic expansion, while populations of the Yushan Mountain Range are likely to have remained stable ever since the colonization. In contrast, the single population of the Alishan Mountain Range with a fixed cpDNA haplotype may have experienced bottleneck/founder's events.Both incomplete lineage sorting and interspecific hybridization/introgression may have contributed to the lack of monophyly among  The analysis of divergence patterns between species has been a major focus in systematics and molecular ecology -5. SpeciTaiwan, an island of the island-arc system along the western edge of the Pacific Ocean, first emerged from the waters via collision between Eurasian and Pacific plates about 9 million years before present (Proto-Taiwan Stage), but attained its modern shape only 5-6 million years ago (MYA) ,17. Its Phylogeographical patterns and genetic characteristics of populations/species are shaped by the interaction between historical vicariance and recurrent genetic exchanges. These evolutionary events would leave evolutionary footprints in the genetic polymorphisms within and between populations across the distributional range. Of the geological events, regular historical glacial cycles in the Eurasia Continent had prevalent influences on survival and recolonization of populations/species . PostglaRhododendron, one of the largest and most widespread woody plant genera, is distributed from the northern temperate zone, throughout tropical Southeast Asia, to northeastern Australia and consists of over 1000 species ) ) ,29,35.The power of molecular markers in elucidating plant phylogeography has been detected in many studies ,36-41. CR. pseudochrysanthum in Taiwan, revealing close relationships among these taxa  and at nrDNA[1.665 × θ/2U], where θ is a population mutation parameter obtained from IM, and U is the geometric mean of mutation rates, and compared them with the divergence times obtained from the BEAST analyses. According to the IM estimates of θother mtns = 95.43 and θYushan = 30.81, the expected time to coalescence of R. pseudochrysanthum s.l. ranged from 4.36 to 29.21 MYA based on cpDNA, and from 8.72 to 58.42 MYA based on nrDNA.To elucidate the possible evolutionary forces for the lack of species monophyly of R. formosanum. The ML tree for the cpDNA agreed with the results of Chamberlain et al. . Duringkonishii , Pinus lwanensis , Cycas tungensis , and Cycrevoluta . A potenutations .Abies kawakamii), a dominant species of subalpine forests at 2500-3700 m a.s.l. [R. pseudochrysanthum s.l. Given a stable, effective population size under diverse habitats, the populations of the Yushan Mountain Range fostered a great number of genetic polymorphisms. In contrast, genetic diversity can be lost by chance in fragmented populations, as occurred in the population of the Alishan Mountain Range. PH01, the interior dominant haplotype, is shared among the Alishan, Central, and Sheishan mountain ranges. A single population of the Alishan Mountain Range was fixed for the dominant haplotype PH01, indicating a possible genetic bottleneck due to a founder effect and/or genetic drift during a period when population sizes were small [The populations of the Yushan Mountain Range had the highest levels of genetic diversity (0.0079) in the cpDNA. A similar pattern was found in Taiwan fir   Table . The divf Taiwan . IndepenRhododendron populations of different mountain ranges represent some isolated patches. Given such geographical barriers, seeds are less likely to disperse across mountain ranges. Low capabilities of seed dispersal in current environments, plus possible independent colonization events followed by limited historical gene flow . In contrast, low levels of genetic differentiation at cpDNA detected among the populations of Alishan, Central and Sheishan Mountain Ranges (FST = 0.00-0.09) implied that there may exist historical gene flow during the glacial maxima, as the alpine populations migrated downwards and became connected to each other   Tables . Here, bistances ,64. LongR. pseudochrysanthum s.l. Combining cpDNA and nrDNA data, IM analysis was used to evaluate the ancestral population size and gene flow. Populations of the Alishan, Central, and Sheishan Mountain Ranges were characterized by a recent population expansion as θother mtns was approximately four times larger than ancestral θA [D of cpDNA and nrDNA  detected among populations (m = 0.05-13.45) likely represented historical gene flow across mountain ranges. Here, compared to the gene flow among populations of Alishan, Central, and Sheishan Mountain Ranges, with m = 0.25-13.45, much lower levels of migration rates were detected between populations of the Yushan Mountain Range and other mountain ranges . Even so, higher levels of migration rates detected from populations of the Yushan Mountain Range to those of the Alishan (m = 1.275) and Sheishan (m = 0.750) Mountain Range further indicated asymmetric gene flow among them. In contrast, historical dispersal may have occurred freely among other populations. That is, during glacial maxima, there may exist corridors among the mountain ranges. These corridors allowed frequent gene flow among populations of Alishan, Central, and Sheishan Mountain Ranges, and occasional gene flow between populations of the Yushan Mountain Range and the other mountain ranges. Altogether, populations of these mountain ranges may have been mediated with different levels of historical gene flow during the glacial maxima.Given low seed dispersal capabilities of R. pseudochrysanthum s.l. is phylogenetically more closely related to R. hyperythrum than to R. formosanum. In contrast, nrDNA failed to identify reciprocal monophyly of ingroup vs. outgroup, indicating possible incomplete lineage sorting and interspecific hybridization/introgression. For R. pseudochrysanthum s.l., the spatial distribution of cpDNA revealed a noteworthy pattern of high differentiation between the populations of the Yushan Mountain Range and the populations of the other mountain ranges. Molecular dating revealed that the split of the populations at different mountain ranges approximated or predated the formation of the Central Mountain Range of Taiwan island, likely suggesting independent colonizations via individuals from previously differentiated populations on the mainland. At the population level, the populations of Central, and Sheishan Mountain Ranges may have undergone postglacial demographic expansion, while populations of the Yushan Mountain Range are likely to have remained stable ever since the colonization. In contrast, the single population of the Alishan Mountain Range with a fixed cpDNA haplotype may have experienced bottleneck/founder's events.In this study, inconsistent gene genealogies of cpDNA and nrDNA were detected. In the cpDNA tree, agreeing with Chamberlain et al. , R. pseuatpB-rbcL intergenic spacer) and nrDNA ITS sequences among populations of Rhododendron pseudochrysanthum s.l. (Table R. formosanum Hemsl. (subgenus Hymenanthes subsection Argyrophylla [R. hyperythrum (subgenus Pontica subsection Maculifera) were selected to serve as outgroups. In the field, R. hyperythrum is sympatric with R. pseudochrysanthum s.l., while R. formosanum is allopatric from two other species. Morphologically, R. hyperythrum and R. formosanum are distinguishable from R. pseudochrysanthum s.l. According to Goetsch et al. [R. pseudochrysanthum s.l. is phylogenetically more related to R. hyperythrum than to R. formosanum. A total of 70 R. pseudochrysanthum s.l. individuals were collected from 14 populations throughout their natural range, mostly from four major mountain ranges in Taiwan, i.e., Alishan, Central, Sheishan and Yushan Mountain Ranges , and R.h et al. , R. pseu2, NP-40 and ddH2O for 4 min (first cycle), and cooled on ice immediately. Primers, for the ITS (ITS5: 5'-GGAAGTAAAAGTCGTAACAAGG-3' and ITS4: 5'-TCCTCCGCTATATGATATGC-3') [atpB-rbcL intergenic spacer (atpB-1: 5'-ACATCKARTACKGGACC AATAA-3' and rbcL-1: 5'-AACACCAGCTTTRAATCCAA-3') [Taq polymerase  were added to the above ice-cold mix. The reaction was restarted at the first annealing at 52°C.Leaf tissue was ground to powder in liquid nitrogen and stored at -70°C. Total genomic DNA was extracted from the powdered tissue using a cetyltrimethyl ammonium bromide (CTAB) procedure . PCR ampCCAA-3') , dNTP anPCR products were purified by electrophoresis in 1.0% agarose gel using 1X TAE buffer. The gel was stained with ethidium bromide, and the desired DNA band was cut and eluted using agarose gel purification . Both direct and subcloning sequencing was conducted to check PCR error. Chloroplast DNA sequences were determined by direct sequencing, while nrDNA was sequenced via subcloning. For subcloning, the desired DNA fragments were cloned into the pGEM-T vector system . Seven clones of the plasmid DNA were selected randomly and purified using a plasmid mini kit (Qiagen). DNA sequencing in both directions was conducted with an Applied Biosystems Model 377A automated sequencer . No within-individual variation was detected for either DNA marker.Nucleotide sequences were aligned with the program MAFFT version 6  and lateTo reconstruct the phylogeny of populations, the combined data of both cp- and nrDNA sequences were analyzed by the program *BEAST v1.5.4 . This meD were also conducted [Levels of genetic diversity within populations and species were quantified with measures of nucleotide diversity (π)  using Dnonducted .R. pseudochrysanthum s.l. that were maximally differentiated based on sequence data [FCT values) when the program was assigned to identify K = 2-6 partitions of populations.SAMOVA  was applied to identify groups of geographically adjacent populations of nce data . We perft = tμ), scaled migration rates (m = m/μ), and scaled effective population size (θ = 4Neμ) for the ancestral and two current populations. The posterior probability densities of these parameters were generated by Markov chain Monte Carlo (MCMC) simulations, and three of these simulations were run with individual simulations being updated 50 million times. Within each simulation, we used a procedure to swap among 10 heated chains (Metropolis coupling). Each simulation yielded a marginal density histogram for the six parameters of interest, and the peaks of the resulting distributions were considered as the maximum likelihood estimates (MLE) of the parameter with credibility intervals equaling the 90% highest posterior density (HPD) intervals. Here we compared populations of the Yushan Mountain Range with other populations of three different mountain ranges based on the results of FST analyses. Parameters in the IM model are scaled by the mutation rates per generation for the loci, i.e., μ = UG, where U is the geometric mean of mutation rates of all the loci and G is the generation time.We used the simulation program IM  to invesR. pseudochrysanthum s.l. split for both cp- and nrDNAs using a Bayesian relaxed clock method with BEAST. We excluded sequences of 18S, 5.8S and 26S RNA regions of nrDNA from the calculation of the divergent time. To estimate the divergence between lineages a well-supported mutation rate is required. In this study, applying evolutionary rates of 1.0-3.0 × 10-9 [-9 [R. hyperythrum and R. pseudochrysanthum s.l. Additionally, Bayesian estimates of the divergence time of the MRCA of the R. pseudochrysanthum s.l. clusters were obtained using BEAST v. 1.3; available from http://beast.bio.ed.ac.uk/Main_Page[In the present study, we estimated the divergence time of the sister lineages of 0 × 10-9  and 5.0-10-9 [-9  substituMain_Page. FollowiMain_Page, the HKYMain_Page.G and 1.665 × 2NeG for cp- and nuclear DNAs, respectively [UG). Therefore, the former is then equal to 1.665 × θ/4U, while the latter is 1.665 × θ/2U. Here, θ values were obtained from IM analyses, and U is the geometric mean of mutation rates of cp- and nrDNAs.There are two primary processes that may cause a locus to show non-monophyly for a group of closely related species, i.e., incomplete lineage sorting and hybridization/introgression. Distinguishing between these two processes is difficult, nevertheless, estimates of divergence time can provide a means of assessing the probability that incomplete lineage sorting alone could account for the observed non-monophyly. The expected time for a locus to become monophyletic following divergence from a sister lineage is 1.665 × Neectively ,88. Accoectively , the effCCH, KHH, JCH, HDL and WKW carried out the molecular genetic studies and the statistical analysis, and participated in the sequence alignment. CCH, TWH and PYW took part in material sampling. CCH, TWH and TYC designed the research and drafted the manuscript. All authors read and approved the final manuscriptRhododendron pseudochrysanthum s.lTable S1. Absolute frequency of chloroplast DNA haplotypes of . Numbers of chloroplast haplotypes distributions from 14 populutions of Rhododendron pseudochrysanthum s.l. Cluster I and II are indicated. N: number of samples. Alishan Mountain Range: AA; Central Mountain Range: CB, CK, CY, CS, CN, CH; Sheishan Mountain Range: SS, SL, SB, SC; Yushan Mountain Range: YT, YY, YP. See Table 1 for the detailed information of populations.Click here for fileRhododendron pseudochrysanthum s.lTable S2. Absolute frequency of nuclear DNA haplotypes of . Numbers of nuclear haplotypes distributions from 14 populutions of Rhododendron pseudochrysanthum s.l. Cluster A, B and C are indicated. N: number of samples. Alishan Mountain Range: AA; Central Mountain Range: CB, CK, CY, CS, CN, CH; Sheishan Mountain Range: SS, SL, SB, SC; Yushan Mountain Range: YT, YY, YP. See Table 1 for the detailed information of populations.Click here for file


Fucus ceranoides provides a good example of highly differentiated populations along its most persistent distributional range at the present rear edge of the species distribution, in NW Iberia. Intrinsic dispersal restrictions are obvious in this species, but have not prevented F. ceranoides from vastly expanding its range northwards following the last glaciation, implying that additional factors are responsible for the lack of connectivity between neighbouring southern populations. In this study we analyze 22 consecutive populations of F. ceranoides along NW Iberia to investigate the processes generating and maintaining the observed high levels of regional genetic divergence.Factors promoting the emergence of sharp phylogeographic breaks include restricted dispersal, habitat discontinuity, physical barriers, disruptive selection, mating incompatibility, genetic surfing and secondary contact. Disentangling the role of each in any particular system can be difficult, especially when species are evenly distributed across transition zones and dispersal barriers are not evident. The estuarine seaweed F. ceranoides is composed of three divergent genetic clusters displaying nearly disjunct geographical distributions. Structure and AFC analyses detected two populations with an admixed nuclear background. Haplotypic diversity was high in the W sector and very low in the N sector. Within each genetic cluster, population structure was also pervasive, although shallower.Variation at seven microsatellite loci and at mtDNA spacer sequences was concordant in revealing that Iberian F. ceranoides, but possibly low-dispersal species in general, appear to be unified to a large extent by historical, non-equilibrium processes of extinction and colonization, rather than by contemporary patterns of gene flow.The deep divergence between sectors coupled with the lack of support for a role of oceanographic barriers in defining the location of breaks suggested 1) that the parapatric genetic sectors result from the regional reassembly of formerly vicariant sub-populations, and 2) that the genetic discontinuities at secondary contact zones (and elsewhere) are maintained despite normal migration rates. We conclude that colonization and immigration, as sources of gene-flow, have very different genetic effects. Migration between established populations is effectively too low to prevent their differentiation by drift or to smooth historical differences inherited from the colonization process.  Marine ecosystems have historically been considered to be relatively open, with populations demographically and genetically connected over broad spatial scales. In a range of coastal taxa, however, recent molecular surveys have consistently revealed considerable phylogeographical and population genetic structure, often at seemingly small spatial scales, indicating that connectivity is frequently much lower than previously assumed. Examples include a variety of species lacking planktonic dispersive stages and/or exhibiting particularly strict ecological requirements, such as intertidal fucalean and kelp seaweeds -5, seagrVicariance is usually invoked as the main driver of  genetic divergence. Extrinsic barriers to gene-flow are generally less obvious (or absolute) in marine compared to terrestrial landscapes ,20, but Inferring the existence of a particular dispersal barrier from molecular data may not be straightforward though . In specDisentangling historical from ongoing constraints to dispersal may also be problematic. Phylogeographical breaks and contemporary oceanographic barriers  are often mismatched in marine restricted dispersers . Historia priori be rare and have low probability of random increase due to drift alone , dispersal restrictions cannot account, at least as the sole factor, for the apparent lack of population connectivity along the much narrower NW Iberian coastline.In this study, we report a remarkable case of non-equilibrium divergence in the estuarine seaweed  markers ,52. Despividuals ,54. Such Iceland ,52. If FF. ceranoides from NW Iberia. This region was sampled at the finest scale of resolution achievable—a complete set of neighbouring estuaries—which was the scale over which gene-flow was more likely to be detected. We were particularly interested in the biogeographic context and the demographic processes contributing to the formation and integrity of stable genetic sectors in NW Iberian F. ceranoides.This study aims to understand this fundamental issue in the evolutionary ecology of populations, the apparently contradicting evidence for large scale dispersal mediating vast (re)colonisations concurrently with persistent, fine scale genetic discontinuities in older refugial regions. The specific question is whether such discontinuities arise and persist due to long-lasting dispersal barriers, or simply reflect resilient non-equilibrium conditions inherited from a complex demographic past. To address this question, in this study both mtDNA sequence and microsatellite genotypic data are employed to investigate the fine-scale distribution of genetic variation in F. ceranoides belonging to the 22 “core” populations. The median-joining network revealed three mtIGS lineages displaying nearly disjunct geographic distributions , and further south in VIA. Phylogroup B, composed by B1 and 10 related haplotypes, was distributed from ANL to CED , although a few B haplotypes were also detected in ORT and BAR. Finally, phylogroup C, composed by C1 and 8 related haplotypes, was exclusively found from ORT eastwards to NAV , and further east in the populations of VIL, SAN and BAY. Several peripheral populations were geographically closer to populations across the phylogeographic breaks than they were to their nearest population within the same sector. For instance, ANL (NW sector) is geographically closer to CAM  than to RCO , and the distance between CED (NW sector) and ORT  is smaller than between CED and FER .A total of 51 mtIGS haplotypes (GenBank: JN084346-96) were identified in the 352 individuals of A1, B1 and C1) plus two derived ones  were shared among at least two populations. The remaining 46 haplotypes were population-specific and among these, 12 represented non-singleton variants. Many W and NW populations harboured private haplotypes in relatively high frequencies. This pattern was apparent even in populations located inside the same drainage systems, such as UMI and ULL , or RCO, BET, ARE and FER . Hhap was high in the W (Hhap = 0.717) and NW (Hhap = 0.671) sectors due to the presence of most of the local haplotype radiations, but πhap was considerably higher in the former. Contrastingly, Hhap was very low in the N sector (Hhap = 0.166), but further east the populations of VIL and SAN possessed private, C1-derived haplotypes in relatively high frequencies.Globally, only the three dominant haplotypes (F. ceranoides (Table P = 0.957) and C (P = 0.750), but failed to support the expansion of phylogroup B  to 0.563   and the highest Hhap (0.717), whereas the N sector had the highest HE (0.553) despite very low Hhap (0.166). The NW sector showed intermediate levels of diversity for both markers. FST ranged from 0.021 (VIG vs. UMI) to 0.685 (PON vs. CED), whereas Dest ranged from <0.001 (VIG vs. PON) to 0.879 (PON vs. ANL) , although 42% of these had global frequencies below 0.01. F. ceranoides occurred between the W sector and the NW and N sectors  genetic clusters (K = 12), corresponding to smaller, less resolved geographic regions. Some admixture (or mixed-ancestry) between neighbouring clusters was pervasive, but also apparent in a few well separated population pairs .The microsatellite genotypic clusters recovered with the FCA showed a remarkable correspondence with the mtIGS phylogroups Figure b. The moP = 0.001), as well as in the W (P = 0.009) and N (P = 0.009) sectors .A significant IBD pattern was detected in the whole NW Iberian region  phylogeographic discontinuities (between CAM and ANL and between CED and ORT). There was a remarkable congruence between the nuclear (microsatellite) and the mitochondrial (mtIGS) data. Indeed, the multi-locus genotypes of F. ceranoides were broadly grouped  in two main higher-level clusters, one of which was further subdivided, resulting in 3 clusters matching the mtIGS-based phylogroups.The unprecedented spatial resolution here employed revealed a nearly perfect parapatric distribution of mtDNA lineages in the evenly distributed F. ceranoides is genome-wide, which excludes stochastic or selective sweeps as the drivers for the drastic mtDNA shifts. Also, the depth of genomic differentiation between phylogroups is high and characteristic of vicariant sub-populations that have long been diverging independently through accumulation of de novo mutations, drift and lineage sorting. Currently there are no large, estuarine-free regions along this shoreline and therefore distance per se cannot be invoked as a major contemporary factor accounting for the much deeper genomic differentiation among than within NW Iberian genetic sectors. The mean distance separating any two neighbouring populations (~32 km) and neighbouring populations across phylogeographic breaks (<40 km) are of the same order of magnitude, and several peripheral populations are geographically closer to populations across the phylogeographic breaks than they are to their nearest population within the same sector.The historical isolation, divergence and enduring integrity of three distinct and disjunct genetic pools at such a narrow spatial scale are as remarkable as they are puzzling. The congruence between the mtDNA matrilines and nuclear background shows that genetic differentiation in NW Iberian F. ceranoides seems rather unlikely. The divergence between interior mtIGS haplotypes  is about one fifth that between F. ceranoides and F. vesiculosus , which are estimated to have diverged between 0.73 and 3.77 million years ago , the subsequent expansion of these vicariant phylogroups along contiguous shorelines, and very limited gene-flow across meanwhile established secondary contact zones. The mismatch analyses and the distribution of the mtIGS variation are compatible with this scenario of independent range expansions. Within each sector, the only widespread haplotypes are the interior  haplotypes, whereas derived haplotypes (presumably younger) are typically restricted to single populations. In the terrestrial realm, similar expansion/contraction cycles have been invoked to explain the mostly parapatric distribution of lineages and sister-species in a range of Iberian taxa currently displaying relatively continuous distributions (reviewed in [The contemporary genetic sectors in Iberian iewed in -64).F. ceranoides, but it is impossible with the present data to establish the specific drivers and its spatio-temporal contexts with detail. Our data nevertheless permit us to speculate on the temporal sequence of colonization of NW Iberia if haplotypic diversity within each sector is assumed to represent a good proxy for the time since colonization. The arrival of phylogroup C to Northern Galicia is presumed to post-date the establishment of phylogroup A in Western Iberia. Indeed, most populations of phylogroup A harbour private haplotypes in relatively high frequencies and some even local haplotype radiations. Remarkably, the number of haplotypes found in these 8 estuaries sampled along a coastline sector as small as 150 km far exceeds the number found in central and northern Europe  remains highly speculative.The regular climatic changes and the dynamic shoreline/drainage geography across the Pleistocene glacial/interglacial cycles -59 have F. ceranoides can only be explained by very limited gene-flow across phylogeographic breaks. Migration may be particularly depressed there due to the presence of contemporary oceanographic barriers to dispersal, or be as low as elsewhere and simply reflect the inherent low vagility of the species.In this contraction/expansion scenario, the enduring integrity of the fine-scale phylogeographic structure within F. ceranoides matches these subdivisions remarkably well, although establishing a link between geographic and genetic subdivisions remains difficult, as their circumscriptions are not based on distinctive climatic, hydrological or oceanographical features that could be relevant in terms of species ecology or dispersal.The most distinctive feature of NW Iberia coastline is its “rias”, drowned river valleys formed during the marine transgression that followed the last glaciation. These rias are generally divided, based on their orientation, size and main geomorphological elements, into “Lower Rias” (between VIG and XAL), “Middle Rias” (between LIR and CED), and “Higher Rias” (eastwards of ORT), with the transitions at Cape Fisterra and Cape Ortegal, respectively. The distribution of mtDNA lineages of The movement of buoyant, surface-drifting seaweed rafts is constrained by near-shore circulation patterns, wind, coastline morphology, tidal currents and river plumes, although the prevailing shelf/slope circulation patterns also play a role in offshore transport. It is highly challenging at the study scale to track the movements and fate of reproductive drifters leaving/arriving estuaries and therefore to directly estimate migration rates between populations within and across sectors. However, several lines of evidence suggest the absence of any specific seascape feature generating persistent (year-round) physical discontinuities matching the location of the observed genetic breaks. In NW Iberia, circulation patterns are complex and seasonally variable ,66. DuriLittorina saxatilis, which showed mild subdivision north and south of Cape Fisterra [Sargassum muticum throughout Galicia, Cantabria and Portugal, shortly after its first detection in Asturias (1980) and in Galicia  also faThe absence of prominent seascape or ecological barriers matching the phylogeographic breaks implies two things. First, that their positions may be rather contingent and simply reflect the idiosyncratic sequence of (re)colonization of NW Iberian estuaries by the three phylogroups during past range expansions; and second, that the unusually sharp genetic discontinuities at secondary contact zones are maintained despite normal migration rates.F. ceranoides is dioecious and therefore effective estuarine colonization requires at least one male and one female fertile frond to be in close contact after dispersal (while synchronously releasing gametes) to produce in situ the foundational zygotes that will eventually initiate a new population. Entangled mats of drifting viable male and female F. ceranoides are more likely to form near established populations that may export significant amounts of freshly dislodged drifters. Successful colonization across intermediate and larger distances surely occurs, as exemplified by the haplotype sharing of ANL and CED, or the colonization of Norway (across the North Sea) and Iceland, but it is probably much less frequent. Anyway, the process will self-reinforce: if populations in the interior of a sector go extinct, favoured recolonization from nearby sources will preserve the pre-existing genetic pattern.A pattern of approximate stepping stone expansions originating in different refugia could promote the formation of genetic sectors even in the absence of dispersal barriers. This possibly reflects a relatively short viability of reproductive structures after frond dislodgement, or a density-dependent effect. F. ceranoides exhibits considerably more diversity [Rare effective inter-estuarine dispersal, while allowing the colonization of unoccupied estuaries, implies that gene-flow has little effect in counteracting differentiation between fully established populations, slowing or preventing any progress towards migration-drift equilibrium. The remarkable genetic homogeneity of northern Europe, which was colonized post-glacially, clearly demonstrates the lack of gene-flow from the interior of the species range, where iversity ,52. ThisThe primacy of colonization history over ongoing gene-flow reflects a poorly connected metapopulation system regulated by dispersal processes that are only effective within very restricted spatial and temporal windows. The apparent paradox of extremely limited population connectivity (here reported within and between sectors) despite an evident colonization potential (at least in the long-term) suggests extreme density-barrier effects. While the initial colonizers may reproduce relatively free of competition and contribute disproportionately to the genetic make-up of establishing populations, once populations become fully established the large disparity in the number of residents (descending from the colonists) and subsequent immigrants act as a demographic buffer against changes in allele frequencies ,51.F. ceranoides that inhabit patchy habitats and possess the capacity for rapid population growth and habitat saturation (compared to immigration rates) may be particularly prone to density-barrier effects. Fucus spp. are fecund, fertilization success is typically very high and recruitment in the vicinity of parental plants can be very efficient [Fucus species can be fast compared to their spread along unoccupied discontinuous shores [F. ceranoides may occupy vacant estuaries even faster and to a larger extent. The enclosed and sheltered nature of its habitat should improve the number and success of spawning events [F. ceranoides is a structural species that frequently forms monospecific (and often compact) belts within its particular tidal/salinity range. Contrary to the open shore where saturated communities compete for space, its estuarine habitat is free of similar competitors, potentially contributing to increased growth rates, densities and space monopolization. The sheltered and densely occupied habitat of F. ceranoides together with the short range of gamete dispersal of Fucus spp. can also explain the observed heterozygote deficiencies as reflecting local inbreeding. Other possible causes for such deviations are unlikely, as small sample sizes appear unimportant when comparing with previously analysed larger samples [Organisms such as fficient ,79. Monos shores . Compareg events . Further samples , no recuF. ceranoides has relatively short temporal windows of opportunity between estuarine colonization and saturation during which rare immigration can potentially result in detectable gene-flow. Incipient reproductive isolation (pre- or post-zygotic) can add to density effects and further depress gene flow between divergent phylogroups . The. TheF. c species ,83. ImpoFucus ceranoides are to a large extent bounded by extinctions and (re)colonisations, our results challenge earlier assertions that marine species are mainly unified by gene-flow. In this seaweed, connectivity estimates based on allele frequency divergence (e.g. FST and related measures) are inflated (within sectors) or depressed (across secondary contact zones) to a great extent by historical colonization processes. Despite its evident parapatric structure, an IBD correlation was unexpectedly recovered in NW Iberian F. ceranoides, showing that IBD patterns can arise where a causal relationship between distance and gene-flow is missing. Such spurious correlations have been noted in other highly structured species [In revealing that populations of  species ,85, and Fucus ceranoides. The levels of differentiation and the absence of habitat discontinuities or prominent ecological/oceanographic barriers to dispersal indicate that its remarkable genetic structure is the product of past range dynamics  coupled with very strong density-barrier effects. These conclusions are highly relevant to other organisms with rare and spatially restricted dispersal, helping explain the apparent paradox of extensive genetic subdivision in geographically restricted refugial regions (indicating very limited connectivity) despite obvious colonization abilities of these same species at larger spatio-temporal scales . These This study also supports the view that the patterns of genetic structure and differentiation in marine-restricted dispersers often reflect persistent non-equilibrium conditions . In partF. ceranoides used in this study were collected in the estuaries of all major rivers between Vigo  and Navia , in NW Iberia , according to the manufacturer’s protocol. Individuals were sequenced for the mitochondrial 23 S/trnK intergenic spacer , and geeranoides. Primer es[et al.,52. Amples[et al.. Microsaes[et al. using thFucus vesiculosus as the outgroup (GenBank no GQ385125). Among the 88 models evaluated in jModeltest [6 generations, sampling every 100 generations. The number of substitution rates (Nst = 2) and among-site rate variation (Rates = Gamma) were set according to the substitution model selected, leaving the remaining options as default. 105 generations (1000 trees) were discarded as burn-in, and the remaining 38000 used to produce 50% majority-rule consensus trees and to calculate branch posterior probabilities.The geographic distribution of the mtDNA variation was mapped and the genealogic relationships of haplotypes were inferred using the median-joining algorithm implemented in Network 4.5 . A phyloodeltest ,95, the πhap) and haplotypic (Hhap) diversity within populations and inferred mtIGS phylogroups (see RESULTS) were calculated with DNASP 5.10 [A), Nei’s gene diversity (HE), observed heterozygosity (HO) and inbreeding coefficients (FIS), were calculated with GENETIX 4.05 [P > 0.05) of the fixation indices was calculated after 1000 permutations of individuals within sectors. For each phylogroup, the occurrence of recent spatial expansions (assuming constant deme size) was tested with ARLEQUIN 3.1 [Nucleotide  was ran five times using a burn-in of 200000 iterations and a run-length of 1000000 iterations, which was determined to be sufficient to have consistent results. The “true” number of K was inferred both from the posterior probability of the data, hereafter referred to as L(K), and following the ΔK choice criterion of Evanno et al.[The microsatellite population structure was assessed with both individual (genotype based) and population  approaches. First, the degree of congruence between the mtIGS structure/phylogeny and its nuclear background was visually inspected with the factorial correspondence analysis (FCA) implemented in GENETIX 4.05 . Populatno et al., better FST  in IBDWS [Pairwise FST  was estFST  was est; [Dest; . Isolatiin IBDWS .JN conceived the study, collected samples and molecular data, performed analysis and drafted the manuscript. GAP participated in the design of the study, performed some statistical analysis and revised the manuscript. MV participated in the design of the study and revised the manuscript. EAS coordinated the study and helped draft and revise the manuscript. All authors read and approved the final manuscript.Figure S1. Genetic diversity and differentiation of populations of Fucus ceranoides within W, NW and N sectors. (a) Haplotype diversity (Hhap) at population (box-plots) and sector (stars) levels. (b) Nei’s gene diversity (HE) at population (box-plots) and sector (stars) levels. (c) Box-plot of pairwise differentiation of populations (Dest) within regions. Box-plots depict the median  and the 25th and 75th percentiles (bottom and top of the box). Click here for fileTable S1. Estimates of pairwise differentiation between the 26 populations of Fucus ceranoides. FST (θ) values are given above diagonal and Jost’s Dest below diagonal. Non-significant FST values (1000 permutations) are depicted in bold. Population codes are given in Table Click here for fileFigure S2.Most probable number of genetic clusters of Iberian Fucus ceranoides according to STRUCTURE. Five iterations were run for each number of genetic clusters assumed (K). The most probable K (open symbols) were inferred with Pritchard et al.  and Evanno et al.  choice criteria. Click here for file


Here we report, for the first time, how a parasitic B chromosome  expands its geographical range aided by high gene flow in the host species. For six years we analyzed B frequency in several populations to the east and west of the original population and found extensive spatial variation, but only a slight temporal trend. The highest B24 frequency was found in its original population (Torrox) and it decreased closer to both the eastern and the western populations. The analysis of Inter Simple Sequence Repeat (ISSR) markers showed the existence of a low but significant degree of population subdivision, as well as significant isolation by distance (IBD). Pairwise emN estimates suggested the existence of high gene flow between the four populations located in the Torrox area, with higher values towards the east. No significant barriers to gene flow were found among these four populations, and we conclude that high gene flow is facilitating B24 diffusion both eastward and westward, with minor role for B24 drive due to the arrival of drive suppressor genes which are also frequent in the donor population.The B B (supernumerary or accessory) chromosomes are dispensable elements found within the chromosome complement of about 15% of eukaryotes Eyprepocnemis ploransProchilodus lineatusTrypoxylon albitarseE. plorans, B chromosomes have been found in almost all the populations hitherto analyzed within the subspecies E. plorans plorans around the Mediterranean Sea and the Caucasus B chromosomes are dynamic genome elements and their frequency in populations varies as a result of factors such as the intensity of drive, effects on the host genome and even historical factors E. plorans is that new B chromosome variants emerge regularly and replacements of one variant for another is common 2 by a larger new variant (B24) could be witnessed directly and was based on significant drive for B2424 frequency has remained very high in later samples of this population, the average transmission ratio of this B chromosome variant declined to 0.51 in only six years 24 in the Algarrobo population, located about 9 km west of Torrox, suggested that this new B variant is currently expanding from its original nucleus 24 chromosome, but recent samples shown in this paper have shown that it has also arrived to this population. In order to follow this spreading process, we documented spatial and temporal patterns of B chromosome variants from the Torrox and surrounding populations over a period of six years. This may be important, not only for disentangling the evolutionary history of these genomic elements but also to understand their evolutionary role as genome parasites.One of the most remarkable features of the B chromosome polymorphism in E. plorans. In a previous study, ISSR markers revealed the existence of high gene flow among E. plorans populations 24), and perform laboratory crosses to tract the inheritance of these elements, in the populations surrounding Torrox, i.e. its putative center of origin. The analysis of ISSR markers provided an estimate of gene flow and population structure that help us to understand the ongoing spread of new B-chromosome variants. Whereas B chromosome drive explains the rapid B invasion in a given population, reaching other populations by B chromosomes depends, as in other strictly vertically transmitted parasites, on the existence of gene flow in the host species. Here we show that gene flow in E. plorans is high enough to explain the spread of the newly arisen B24 variant.To obtain indirect estimates of gene flow between these populations, we analyzed Inter Simple Sequence Repeat markers (ISSR) in five populations. As ISSR markers evolve quickly, they provide a large number of polymorphic markers 2 and B24 variants previously described in this species 2 and three in B24  during this six-year period in which B24 prevalence passed from 0.087 to 0.205 . B24, the invading B variant, showed a contagious distribution pattern , but this association was significant for the 2006 sample . This result is consistent with the existence of isolation by distance.Significant spatial variation was found among populations for B pattern , with th pattern . Spearma2 chromosome and 9 for the B24 variant, which included the cytological analysis of 32 parents and 332 embryo offspring. As a whole, most crosses showed transmission ratios for B chromosomes not significantly different from the Mendelian one (KB = 0.5). The only exceptions were two crosses from Algarrobo (f13 × m20 and f26 × m21) that showed a significant deficit of B2 chromosomes transmitted via the male parent, one cross from Algarrobo (f4 × m19) showing significant B24 drive via the female parent, and one cross from Nerja-0 (f2 × m4) showing significant B24 elimination  being very close to the Mendelian one (0.5). However, B24 transmission rate showed higher variation between individuals (SD = 0.15) than B2 (SD = 0.11).To ascertain whether geographical patterns of B chromosome frequency variation could be explained by differences in B chromosome transmission between populations, we performed 16 controlled crosses in the laboratory. In total, we performed 12 progeny analyses for the Bmination . As a whIn the sample as a whole , 97% of (I) = 0.291±0.026 (mean ± SD) , θ(II) = 0.058±0.007 , and GST-B = 0.049±0.004 (which is a Bayesian estimate of GST). The expected heterozygosity per population (hs) was 0.235±0.005 in Algarrobo, Torrox and Nerja-2, 0.225±0.005 in Nerja-0 and 0.236±0.005 in Salobreña. The average expected heterozygosity (Hs) for the five populations was 0.233±0.003, and that for the five populations calculated from mean allele frequencies was Ht = 0.245±0.003. Only one private allele was found, i.e. 39-1000 in the Algarrobo population.A significant population structure was evident, as shown by θK) deduced from the Evanno et al. K = 4, the Structure software showed that Salobreña and, to a lesser extent, Algarrobo were the most differentiated populations, in accordance with their geographical distance. The three remaining populations  showed more mixed patterns, in keeping with their greater proximity  between population pairs , thus indicating that the effective number of migrants decreases with geographical distance. The IBD analysis was also performed by a regression analysis of log-transformed emN values on log-transformed kilometers The effective number of migrants per generation , showed that B24 is most frequent in Torrox (its center of origin), and that this chromosome has reached the adjacent populations of Algarrobo (to the west) and Nerja (to the east), but it was not found in more distant populations such as Nerja-2 and Salobreña  emerged recently in the Torrox population as a derivative from the other variant (B2) 24 and thus witnessing evolution in action. We previously witnessed the increase of B24 frequency at the expense of B2 one, due to significant drive for the former 2 chromosome in 22 out of 43 males analyzed, with no traces of B24. We can therefore assume that B24 arrived in Nerja after 1984.The presence of two B chromosome variants in the 24 frequency, or else that it is too slow to capture in the six year time span conducted here. The spatial analysis, however, revealed very significant differences between populations, with B24 being most frequent in Torrox, i.e. its putative center of origin, and progressively less frequent to the east and west, thus suggesting that this new B variant is expanding its geographical range by diffusion toward nearby populations. The absence of significant temporal changes in B24 frequency is expected from the absence of net drive in the nine crosses performed for this variant, despite the extensive variation observed between crosses. It is also consistent with the idea that the diffusion of genes through a metapopulation is a very slow process Myrmeleotettix maculatus. In E. plorans, the B24 variant appears to be rather young (only a few decades) and its spread might have been somewhat influenced by the recent agricultural and tourist development of the Torrox area.Our present results show little temporal variation for B chromosome frequency. This suggests that changes in B frequency are mostly stochastic and do not show a clear tendency to increases in BE. plorans.The analysis of ISSR markers has provided valuable information on genetic variation, population structure, gene flow and isolation by distance (IBD) in (II) and GST-B values (both about 0.05) indicated some population subdivision. This was reinforced by the Structure analysis, which showed the existence of four genetic groups, although only the one including most individuals from Salobreña was well defined, the remaining showing much admixture due to high gene flow , suggesting a greater intensity of gene flow than of genetic drift emN>4, a metapopulation is usually considered to be behaving like a panmictic population STF, which is dependent on sample size and allele frequency STF values below 0.02  lead to emN estimates that may be influenced by sampling noise STF values makes it difficult to get good estimates of emN in species with a high dispersal capability. However, as pointed out by Palumbi E. plorans, the STF estimates can be considered more robust. The significant IBD shown by the Mantel test thus allows concluding, with greater confidence, that genetic differentiation in these E. plorans populations is the result of biological characteristics. Furthermore, emN values were calculated on the basis of the islands model emN estimates under the island model are of the correct order of magnitude but depend on the geographical distance between populations. In sum, the ISSR marker analysis supports the existence of enough population connectivity to facilitate the spread of these chromosome variants to nearby populations.The low but significant θene flow . Nem valE. plorans, since the transmission ratio of the B24 chromosome in the Torrox population decreased from 0.7 to 0.5 in only six years Prochilodus lineatusTrypoxylon albitarse24 in Torrox 24B chromosome invasions have occasionally been witnessed, and they always appeared to be very rapid 24 invasion so fast in Torrox while its diffusion to nearby populations is so slow?Why was B24 frequency in Torrox reached a maximum in 1994  24 chromosomes and the gene variants suppressing its drive, which had already reached high frequency in Torrox. This explains why the average transmission rate for B24 found in Algarrobo in 2004 (0.6) , suggesting the existence of polymorphism for drive suppression, a fact that is also apparent in the present crosses , namely Torrox and five other populations, one to the west (Algarrobo) and four  to the east  was investigated by means of Spearman rank correlation analysis. The map in Distance among populations and terrain topography were analyzed with the help of Google Maps. The existence of a possible inverse relationship between B. A total of 94 ISSR fragment sizes were obtained, from six different markers, in 29 individuals from Algarrobo, 27 from Torrox, 30 from Nerja-0, 30 from Nerja-2 and 23 from Salobreña , and a population furthest to the east , all sampled in 2006STF) and endogamy coefficient  were different from zero; ii) f = 0 model, which assumes the absence of intra-population endogamy; iii) θ = 0 model, assuming the absence of population subdivision, and iv) free model, where no previous information is assumed. The best model was chosen by the Bayesian DIC parameter  f = 0 model. Since the markers are dominant, however, the full model was discarded because it suggested unrealistic values for inbreeding (f = 0.95), which are very improbable for a polygynandric animal like E. ploransPopulation subdivision and inbreeding analyses were performed with Hickory v1.1 admixture model with a run of 50,000 steps for burn-in and 100,000 Markov Chain Monte Carlo iterations after burnin. We determined the number of population groups (K) best explaining the data, following the procedure suggested by Evanno et al.Population structure was analyzed by the Structure v2.3.1 software emN values obtained from pairwise STF values provided by the AFLP-surv software emN) was calculated by the equation: emN = 0.25(1− STF)/STF. The Mantel test was performed by the Zt-win software STF values. To overcome this drawback, we used the method described by Dice emN on log-transformed geographical distance values, as suggested by Slatkin Isolation by distance (IBD) was investigated by means of the Mantel test between geographical distances and the STF obtained with AFLPsurv software as the genetic distance matrix. The robustness of the results was estimated from 100 bootstrapped matrices.To determine whether geographical barriers are reducing gene flow, we used the Barrier software version 2.2 Figure S1Delta K values with respect to K, according to the calculation method posited by Evanno et al., . These results were obtained by using all the 94 ISSR markers analyzed. Note the highest peak for K = 4.(TIF)Click here for additional data file.Table S1Number of individuals analyzed for each ISSR primer.(DOC)Click here for additional data file.Table S2Proportion of individuals from each population assigned to each of the four groups (K = 4). N =  Number of individuals analyzed. Colors refer to groups in (DOC)Click here for additional data file.Table S3Pairwise matrices of geographical distance in meters  and emN10. values , both transformed to log(DOC)Click here for additional data file.


Zaprionus indianus was first recorded in Brazil in 1999 and rapidly spread throughout the country. We have obtained data on esterase loci polymorphisms (Est2 and Est3), and analyzed them, using Landscape Shape Interpolation and the Monmonier Maximum Difference Algorithm to discover how regional invasion occurred. Hence, it was apparent that Z. indianus, after first arriving in São Paulo state, spread throughout the country, probably together with the transportation of commercial fruits by way of the two main Brazilian freeways, BR 153, to the south and the surrounding countryside, and the BR 116 along the coast and throughout the north-east. Zaprionus indianus is an African species, that is now widespread throughout several tropical areas worldwide, probably as a result of the intense commerce of agricultural goods. In Brazil , and more recently, in Central America and the United States , the second with four . Furthermore, they proposed that maritime introduction was more probably a result of an increase in the commerce of fruits between Africa and Brazil. Nevertheless, how Z. indianus was capable of spreading so rapidly countrywide remains a mystery.Various tools have been employed for characterizing the species introduced into Brazil, such as alloenzyme polymorphisms , quantitWe resorted to a landscape genetics approach as a tool to answer this question. This requires constructing a framework for testing the relative influence of landscape and the environmental features of gene flow and genetic discontinuities . It alsoZ. indianus populations , whereupon the homogenate was applied to a 10% polyacrylamide gel. Electrophoresis was carried out in a Tris-glycine buffer pH 8.8 at 200 V for 3 h. A random sample of 20 individuals  from each population was used. In the case of the EST2 system, which is restricted to males (O) and expected (HE) heterozygosity, and Hardy-Weinberg equilibrium, were all estimated by TFPGA. The estimation of genetic distances  and the Monmonier Maximum Difference Algorithm (MMDA), was performed to evaluate inter-individual patterns of genetic and geographical variation. The calculated surface for LSI was based on the midpoints of edges derived from Delaunay triangulation , and that of Est2F in Onda Verde and Rio de Janeiro (0.08). The frequency of locus Est3 alleles (3. Est31 frequency varied from 0 (Ilhabela) to 0.94 (Santa Maria), Est34 from 0.05 (Rio Claro and Porto Alegre) to 0.89 (Ilhabela), and Est33 from 0  to 0.30 (Onda Verde). The frequency of Est32, although not detected in Santa Maria, Onda Verde and Ilhabela, was the highest in Brasília (0.69).The analysis of Est2 allele frequency distribution in Brazilian populations of indianus  shows fi alleles  varied cO) and expected (HE) was greater in Est3 than in Est2 (O ranged from 0 (Ilhabela) to 0.80 (Onda Verde) and HE from 0.20 (Ilhabela) to 0.70 (Mirassol and Ibirá). The average Est3 HO in populations from São Paulo state (0.54 ± 0.07), and the southern (0.47 ± 0.08) and northern (0.49 ± 0.11) states did not differ significantly (X2 = 0.10). On the other hand, Est2 HO ranged from 0 (11 populations) to 0.45 (Brasilia), and HE from 0 (11 populations) and 0.50 (Porto Alegre). The average Est2 HO in populations from São Paulo state (0.08 ± 0.03) and southern regions (0.19 ± 0.06) were not significantly different. Almost all the populations, except those from Ibirá and Maresias, were estimated to be under Hardy-Weinberg equilibrium, with p < 0.05.The average observed , and the pairwise estimates of FST ranged from 0.003 (Sud Menucci versus Paraibuna) to 1.000 .Pairwise genetic distance  and FST ST  indices Genetic boundaries depicted in Est2 and Est3 data are shown in Z. indianus arrived in Brazil in 1998  were almost the same as the three esterase HO of Indian population loci, each of which harboring 5 alleles, i.e., 0.54 and 0.56  were smaller than an esterase locus with two alleles in Indian populations, viz., 0.17  throughout the sampled regions, with the surface edges corresponding to the highest ratios. All the edges were located in southeastern Brazil, specifically São Paulo state, thereby indicating the higher genetic structuring of these populations, possibly due to both early origin and low gene flow. Historical data reinforce the idea of the earlier arrival of Z. indianus in Brazil can be visualized , whereas the second is coastal . A similar manner of diffusion, due to the fruit trade, may have occurred in the Palearctic region (Z. indianus to spread from India to Egypt. The great difference in the pace of spread between Brazil/USA and India/Egypt can be attributed to the more developed freeway networks in Brazil than in the Palearctic region.By identifying 3 boundaries for gene flow through MMDA analysis, a putative scenario of the spread of sualized . BoundarZ. indianus occurred from São Paulo, the state where commercial highway traffic is the heaviest, to the north and south of Brazil by way of both the BR153 and the BR116 highways. The landscape genetics approach hereby applied for characterizing the genetic structure of populations from an initial colonizer species soon after its introduction, as well as its relevance in offering the possibility of determining the source of invasion, and demographic parameters of the species, also offers a unique opportunity for accompanying the evolutionary dynamics of the invader species over time.These findings suggest that the spreading of The following online material is available for this article:ST and genetic distance between the Brazilian populations of Zaprionus indianus.Pairwise values of FThis material is available as part of the online article from http://www.scielo.br/gmb.


Sarcoptes mite populations from Pyrenean chamois (Rupicapra pyrenaica) in Asturias (Spain), using one multiplex of 9 microsatellite markers and Sarcoptes samples from sympatric Pyrenean chamois, red deer (Cervus elaphus), roe deer (Capreolus capreolus) and red fox (Vulpes vulpes).Implicitly, parasite molecular studies assume temporal genetic stability. In this study we tested, for the first time to our knowledge, the extent of changes in genetic diversity and structure of Sarcoptes mite under the host-taxon law (herbivore derived- and carnivore derived-Sarcoptes mite) among the sympatric wild animals from Asturias.The analysis of an 11-years interval period found little change in the genetic diversity . The temporal stability in the genetic diversity was confirmed by population structure analysis, which was not significantly variable over time. Population structure analysis revealed temporal stability in the genetic diversity of Sarcoptes mite. This could eventually be applied to other parasite species.The confirmation of parasite temporal genetic stability is of vital interest to allow generalizations to be made, which have further implications regarding the genetic structure, epidemiology and monitoring protocols of the ubiquitous  In the field of parasitology, different molecular markers have been used for parasite genetic characterization and genetic population studies. All molecular studies assume that genetic structure and diversity is relatively stable over time ,2. SinceSarcoptes mite over time.Here we describe, for the first time to our knowledge, a temporal analysis of microsatellite alleles and genetic structure at nine polymorphic loci to examine changes in genetic diversity of Sarcoptes mite continues to affect humans and a wide range of mammalian hosts worldwide  (P > 0.05), and no deviation from HWE [Hardy-Weinberg equilibrium] was detected from all loci in all the studied population except Sarms34 and Sarms38 in Pyrenean chamois collected in 1997, and Sarms33, Sarms35-38, and Sarms44 in red fox population.Sarcoptes population (red fox). Sarms38 was monomorphic with only 215 bp allele present in Pyrenean chamois from 1997, while Pyrenean chamois from 2008 has two alleles, 213 bp and 215 bp. Again, the new allele (213 bp) is present in all the other herbivore sympatric populations, but not in the carnivore red fox population.Allele diversity was identical in all loci from both Pyrenean chamois populations from 1997 and 2008, with the exception of Sarms34 and Sarms38: Sarms34 was monomorphic with only 176 bp allele present in Pyrenean chamois from 1997, while Pyrenean chamois from 2008 has two alleles, 176 bp and 198 bp. The new allele (198 bp) is present in all the other herbivore sympatric populations (red deer and roe deer), but not in the carnivore-derived Intra-host variation was detected in six individuals: one Pyrenean chamois from 1997 (variation in Sarms34), three red deer (variation in Sarms34), and two red foxes (variations in Sarms35 and Sarms40).ST F= 0.74808; P < 0.001), which indicates that the mite component populations differed greatly. ST Fvalue between both chamois-derived Sarcoptes mite populations was not statically supported , while red fox-derived Sarcoptes mite population was statistically (P < 0.001) different from all other herbivore-derived Sarcoptes mite populations -derived Sarcoptes mite population, while the highest value of the average number of pairwise differences was detected within V. vulpes-derived Sarcoptes mite population.AMOVA analysis showed differentiation among populations  formed another well-supported cluster , and illustrates the subpopulations within R. pyrenaica-2008- and V. vulpes-derived Sarcoptes mite populations , and well-differentiated from the carnivore (red fox)-derived Sarcoptes population.The scatter plot of the FCA, for individuals (data not shown) and populations Figure  of the mSarcoptes scabiei few loci and low sample sizes are sufficient to find strong population differentiation between host species [Sarcoptes mite from this carnivore animal and the sympatric herbivores , which is in concordance with the host-taxon effect among Sarcoptes populations from different sympatric wild animals [As with other highly divergent taxa, with  species ,29. The  animals .Sarcoptes mites from this host [Sarcoptes mites lack free-living stages, and individual hosts, depending on their susceptibility and behaviour, are essentially ephemeral habitats providing patchy environments that hamper random mating [The few detected cases of infra-host variations could be attributed to the skin-scale phenomenon , while this host ,31. Sarcm mating ,33.Sarcoptes population during the 11-years interval period, which could be attributed, simply to non-random sampling, or to little gene flow from the other sympatric herbivore(red deer and roe deer)-derived Sarcoptes mite,  but never from the carnivore sympatric host (red fox), following the host-taxon law [Two new alleles were detected in two different loci from the 2008-Pyrenean chamois population comparing with the 1997-population. Both new alleles are present in all the other herbivore sympatric populations, but not in the carnivore red fox population. This could be understood as small change in allele diversity within chamois-derived axon law .Sarcoptes mite populations collected in 11-year interval period. On the other hand, the results corroborate the presence of a host-taxon phenomenon and lack of gene flow or recent admixture between carnivore- and herbivore-derived Sarcoptes populations, among Sarcoptes mites from wild animals in Asturias, in concordance with other European wild hosts [All AMOVA analysis (showing differentiation among populations), the Bayesian assignment test (between mites), and the scatter plot of the FCA  confirmed the absence of genetic differentiation between the two chamois-derived ld hosts .Sarcoptes mite population , but it origin; .Sarcoptes mite population under the host-taxon law. The understanding of this factor is crucial, if generalizations are to be made concerning temporal genetic stability. Besides the genetic implications of our results, this study could have further ramification in the epidemiological studies and the monitoring protocols of the neglected Sarcoptes mite, and could have further applications in other parasite species.The analysis of 11-year interval period found little change in the genetic diversity and showed clear temporal stability in the genetic structure of Sarcoptes mite were collected from the skin crust of ten infected Pyrenean chamois, and (ii) in 2008, fourteen parasites were collected from the skin crust of seven infected Pyrenean chamois, two mites from two mangy roe deer, thirteen from eight infected red deer, and twelve from six red fox.Using postponed isolation and direct isolation (with aqueous potassium hydroxide digestion) techniques  sixty reS. scabiei on the basis of known morphological criteria [Sarcoptes mites was extracted using the HotSHOT Plus ThermalSHOCK technique [Sarcoptes mite DNA extraction by three cycles of thermal shock , followed by a short incubation (30 min at 95°C) and pH adjustment with 25 μl of a neutralizing reagent . Two blanks (reagents only) were included in each extraction to monitor for contamination.All mites were identified as criteria . The DNAechnique , as follSarcoptes mite microsatellites  were used with one 9× multiplex PCR. One primer from each set was 5' labelled with 6-FAM, VIC, NED or PET® fluorescent dye tag . Each 15 μl PCR reaction mixture consisted of 3 μl of the single mite DNA, together with the PCR mixture containing all primer pairs (ranged from 0.04 to 0.1 μM per primer), 200 μM of each dNTP, 1.5 μl of 10× PCR buffer , 1.5 mM MgCl2 and 0.15 μl (0.5 U/reaction) HotStar Taq . The thermal profile in a 2720 thermal cycler  was as following: 15 min at 95°C , followed by 37 cycles of three steps of 30 s at 94°C (denaturation), 45 s at 55°C  and 1.5 min at 72°C (extension), before a final elongation of 7 min at 72°C. Fluorescent PCR amplification products were analyzed using formamide with Size Standard 500 Liz  by ABI PRISM 310 Genetic Analyzer with pop4. Allele calling was performed using the GeneMapper v. 4.0 software .As described by Alasaad et al. , nine spEH) and observed (OH) heterozygosity, linkage disequilibria (LD), and Hardy-Weinberg equilibrium (HWE) tests were calculated using GENEPOP . DeviatOCHECKER .Sarcoptes mite populations was estimated by the partition of variance components (AMOVA) applying conventional ST Fstatistics using allele frequencies as implemented in Arlequin 3.11 [TRUCTURE . Burn-iv.2.3.3; . FinallyThe degree of genetic relationship among populations was further investigated with FCA  as implemented in Genetix v.4.05.2 .The authors declare that they have no competing interests.CG, RC, AO, LR and RCS conceived and designed the experiments. AO, RC and CG performed the field work experiments. AMM, AO and SA performed the molecular work. Manuscript was written by all co-authors. All authors read and approved the final manuscript.