Tetranychus urticae Koch) coexist in China: a red (carmine) form, which is considered to be native and a green form which is considered to be invasive. The population genetic diversity and population genetic structure of this organism were unclear in China, and there is a controversy over whether they constitute distinct species. To address these issues, we genotyped a total of 1,055 individuals from 18 red populations and 7 green populations in China using eight microsatellite loci.Two colour forms of the two-spotted spider mite  and a low genetic diversity in each population. In addition, genetic diversity of the red form mites was found to be higher than the green form. Pearson correlations between statistics of variation (AR and HE) and geographic coordinates (latitude and longitude) showed that the genetic diversity of the red form was correlated with latitude. Using Bayesian clustering, we divided the Chinese mite populations into five clades which were well congruent with their geographic distributions.We identified 109 alleles. We found a highly significant genetic differentiation among the 25 populations  effect. These factors in turn contribute to the strong subdivision of genetic structure. In addition, population genetic structure results don't support the separation of the two forms of spider mite into two species. The morphological differences between the two forms of mites may be a result of epigenetic effects. Tetranychus urticae Koch is a serious pest of various agricultural plants including fruit trees, vegetables, ornamentals and agricultural crops. Owing to its rapid development and high reproductive capacities, and ability to feed on more than 900 plants [T. urticae has spread worldwide. Despite the importance of this pest, the population genetic structure of this mite has been unclear in China. A better understanding of population genetic structure could help to manage mite populations by providing more reliable estimates of population dynamics and the risk of spreading acaricide resistance genes. The population genetic structure of an organism is determined by various factors, such as geographical barriers, ecological difference, and historical processes, as well as the dispersal ability of the species. T. urticae has multiple dispersal mechanisms. Spider mites are wingless and usually rely on crawling for their dispersal [T. urticae, the population structure and diversity would be complex. Two studies have used molecular markers to investigate the genetic structure of T. urticae in China [T. urticae, a more systematic and wide scope study based on more microsatellite markers using more powerful Statistical analysis methods are needed.The phytophagous two-spotted spider mite 0 plants , T. urtiispersal . But theispersal ,4. Due tin China ,6. HowevT. urticae in China, there is a big question about its taxonomy. T. urticae has two forms: a green form that occurs in temperate and cold areas, and a red (carmine) non-diapausing form ) that occurs in warmer areas [T. urticae would help to clarify the longstanding issue of their species status.In addition to questions about the population structure and genetic diversity of er areas . Althouger areas ,9. Thereer areas . The carer areas ,11. TherT. urticae in China. Our main goals were to unveil the genetic structure across the known range of T. urticae in China, and clarify the taxonomic status of red and green spider mites from a population genetic structure perspective.In this study, two new microsatellite markers developed by ourselves together with six previously defined loci ,13 were GU068508 and GU068509, respectively) revealed polymorphism and were used.Of the 180 clones sequenced, 127 were discarded because the repetition pattern was too short. Of the 53 remaining microsatellites, 44 appeared to be either monomorphic or to have an unclear banding pattern in gels, and seven would not amplify in each of the 40 adult females. Only two microsatellites isolated here . MICRO-CHECKER software identified the presence of null alleles. The frequencies of null alleles per locus per population ranged from 0 to 0.434 (only four cases > 0.4). In 179 of 200 locus-population combinations, the frequencies of null alleles were lower than 0.200. There was a strong overall heterozygote deficit, with f = 0.272 . Nineteen of 25 populations showed a significant deficit of heterozygotes . Of 200 locus-population combinations, 192 were in Hardy-Weinberg equilibrium. Only three populations, 20 (R), 1 (G) and 4 (G), were not in Hardy-Weinberg due to heterozygote excess at 2-3 loci.The allelic richness based on a minimum population size of 34 diploid individuals ranged from 2.14 to 6.23 with an average value of 3.89 for every population  but that AR did not contribute significantly . There was no evidence that either AR or HE of the red form mite was related to longitude. However, for the green form mite, neither latitude nor longitude was correlated with AR or HE. This suggests that the intra-population genetic diversity of the red form mites was negatively correlated with latitude. However, the intra-population genetic diversity of the green form mites was clearly not correlated with latitude.A Pearson correlation analysis showed that FST value  indicated a high level of population differentiation. Pairwise estimates of FST calculated between pairs of populations showed that most tests for population differentiation were significant (Table FST = 0.042). Thirty-five pairwise populations showed moderate genetic differentiation (pairwise FST < 0.25). The rest of the pairwise populations displayed high genetic differentiation (pairwise FST ranged from 0.255 to 0.785). The presence of null alleles caused the level of pairwise population differentiation to be overestimated. But the level of population differentiation was also high with global FST ENA} {= 0.473 . In the unbiased FST ENA} {data set, 42 pairwise populations showed moderate genetic differentiation .The overall nt Table . The onlFST/(1-FST) with ln (distance), Mantel test after 1000 permutations,  was significant in the populations of the carmine mite [regression of T. urticae populations, and thus no clear clusters. When the date was corrected by the INA method, the NJ tree topology showed subtle differentiation. However, the DCE genetic distance was smaller when calculated with the corrected data than when calculated with the raw data. Moreover, the topology of the tree based on the corrected data , indicating that the gene flow between the two colour forms in China was limited. The genetic divergence of the mites among the different host plants  was lower than the genetic divergence between the red and green mites, but still significant. Similar results were obtained for the corrected genotype data  and no effect on the green form mite . The limitation of gene flow associated with geographical distance is in agreement with the results of previous studies of spider mites [The pairwise s Figure . This maressalis . Howeverressalis ,23. The er mites ,24,25.The declining genetic diversity of the red form mite with increasing latitude may be because the northern populations have fewer generations per year. In southern China, which has a hot-humid climate, the spider mite has more than 20 generations per year whereas in northern China, which has a cold-arid climate, the spider mite has 12-15 generations per year. This would result in a higher mutation rate in the southern populations. Mutation is one of the key sources of genetic diversity. So the southern populations would be expected in turn to have a higher genetic diversity than the northern populations. However, only seven populations of green mites were considered in this study, and all of the seven populations were located in the north of China. Therefore, scarce populations of green mites scattered over a much lower latitudinal gradient than the populations of red mites might lead to the statistical bias.T. urticae and T. cinnabarinus have significantly different body colours and are reproductively isolated in China, our genetic data don't support the separation of the two forms of spider mite into two species. The level of genetic differentiation between the two colour forms is similar to that examined between geographically separated populations. Bayesian clustering cannot separate the red form mite from the green form mite when K = 2. AMOVA analysis also revealed that there is more variations among the 5 groups inferred by STRUCTURE software than between the two colour forms of spider mite. An attempt to distinguish the two forms by phylogenetic analysis of their endosymbiont Wolbachia was unsuccessful, possibly because of a tendency of Wolbachia to co-evolve with their hosts [T. urticae and T. cinnabarinus are reproductively incompatible, there may be no intrinsic difference in their DNA compositions. The morphological difference between the two forms of mite may be due to epigenetics. In this case, different gene expressions between the two forms of spider mite caused by DNA methylation, histone deacylation and other epigenetic factors may lead to the different phenotypes. Thus, the two forms of T. urticae may be good candidates for epigenetic studies.Even though ir hosts . Althougir hosts . Based oir hosts . The comSpider mites possess low levels of genetic diversity, limit gene flow between populations and significant IBD (isolation by distance) effect. These factors in turn contribute to the strong subdivision of genetic structure. Due to the founder effect, the genetic diversity of the invasive green form of spider mite is lower than that of the red form. Fewer generations per year at higher latitude lead to the declining genetic diversity of the red form with increasing latitude. In addition, the population genetic structures of the two forms of mites do not support their separation into two species. The morphological difference between the two forms of mites may be the result of epigenetic effects.T. urticae mites were subjected to further analyses. Total DNA was extracted from adult female mites according to the method of Gomi et al. [During the summer of 2008, we collected a total of 1055 adult females of the two-spotted spider mite  at 25 sites in China. Figure i et al. .EcoRV, and the restricted fragments were then ligated with a specific blunt adaptor (consisting of a 48-mer: 5'GTAATACGATTCACTATAGGGCACGCGTGGTCGACGGCCCGGGCTGGT3' and 8-mer with the 3'-end capped by an amino residue: 5 ACCAGCCC-NH23') by use of a DNA ligation kit .Two of the eight loci used in this study (TECI 04 and TECI 08) were isolated by us from a genomic DNA library using a suppression - PCR procedure described by Lian et al. . BrieflyEcoRV DNA library using compound SSR primer (AC)6(AG)5 or (AG)6(AC)5 and an adaptor primer AP2 (5'CTATAGGGCACGCGTGGT3'). The amplified fragments were integrated into the plasmids using a pT7 Blue Perfectly Blunt Cloning Kit (Novagen) and the plasmids were transferred into Escherichia coli according to the manufacturer's instructions. The inserted fragment lengths were checked by 1.5% agarose gel electrophoresis. Amplified fragments between 300 and 800 base pairs were selectively sequenced directly using a Thermo Sequence Pre - mixed Cycle Sequencing Kit  with a Texas Red-labeled T7 primer  in an SQ-5500E sequencer (Hitachi). For each fragment flanking (AC)6(AG)n or (AG)6(AC)n compound SSR sequences at one end, a specific primer (IP1) was designed from the sequence using Primer 5.0 (http://www.premierbiosoft.com). The primer pairs of IP1 and compound SSR primer were used as a compound SSR marker.Fragments were amplified from an T. urticae adult females sampled from 21 provinces of China were used for the template DNA extraction according to protocols described by Gomi et al. [-1 of each dNTP, 1 × PCR buffer , 2.5 mmol L-1 Mg2+, 0.125 U of Ampli Taq Gold (Applied Biosystems), and 0.5 μmol L-1 of each IP1 and a Texas Red-labeled compound SSR primer ((AC)7(AG)3 or (AG)7(AC)3). Reactions were preceded by a 9-min denaturation step at 94°C and were cycled 40 times with 30s at 94°C, 30s at 55°C, and 1 min at 72°C, followed by a final 5 min extension step at 72°C. The reaction products were electrophoresed on 6% Long Ranger sequencing gel  using an SQ - 5500E sequencer. Electrophoretic patterns were analysed by Fraglys v.3 (Hitachi).To examine the effectiveness of primer pairs designed as compound SSR markers, 40i et al. . PCR ampTaq from QIAGEN following the manufacturer's recommendations. Amplifications were conducted on Gene Amp PCR System 9700 (Applied Biosystems), and genotyping was performed on an ABI 3100 sequencer. Genotypes were then checked by eye with GeneMapper 3.0 software (Applied Biosystems).We scored eight microsatellite loci: TU1, TU11, TU35b , TUCA12,A), (2) the allelic richness [AR), and (3) the fixation index (FIS) at each locus and over all loci in each population. The deviation of FIS from zero was tested in each population by 1000 permutation tests with a sequential Bonferroni correction. The observed heterozygosity (HO) and expected heterozygosity (HE) were calculated at each population using GenALEX 6 software [P values were corrected for multiple comparisons by applying a sequential Bonferroni correction [http://www.montpellier.inra.fr/URLB/) [INA method) [HO, HE, FIS statistics and HWE test using the above methods.Micro-Checker software (Ver. 2.2.3)  was firsrichness  based onsoftware . Deviatisoftware  at each software . P valuerrection . To minirrection  implemenr/URLB/) . The est method) -40. The AR and HE) and geographic co-ordinates (latitude and longitude) for each population were analysed. Stepwise regression analysis of AR and HE in relation to the two independent variables (latitude and longitude) were further assessed separately. Both analyses were conducted using SPSS 13.0 for Windows [In order to understand whether genetic variation within populations is correlated with geographical gradients, Pearson correlations between the statistics of variation  using the FREENA program [FST/(1-FST) matrix and FST ENA}{/(1-FSTENA}{) matrix with a geographic distance matrix (ln Km) using the Mantel test, with significance tests performed over 1000 permutations [Pairwise  program . To deteutations . The tesutations .INA method implemented in FREENA was also used to constructing a NJ tree using the above methods.Phylogenetic relationships among populations were estimated by constructing a neighbour-joining (NJ) tree  based onK clusters. We took advantage of an admixture ancestry model under the correlated allele frequency model. The Markov chain Monte Carlo simulation was run 20 times for each value of K (1-11) for 106 iterations after a burn-in period of 105. All other parameters were set at their default values. We used the Δ K method of Evanno et al. [K. The proportional membership of each cluster was estimated for each individual and each population. Owing to the difficulty of correcting multilocus genotypes precisely, three loci, TUCA12, TUCT17, TUCA72 abounded with null alleles over the 18 population were deleted. The genotype data with the remaining five loci were also used for Bayesian clustering to exclude the effect of null alleles. After the three loci were deleted, 79 of 125 locus-population combinations were found to have no null alleles. The average frequency of null alleles for the remaining five loci was estimated at 0.083 by MICRO-CHECKER software.The Bayesian clustering method was also used to elucidate the genetic structure among populations using STRUCTURE v. 2.2 . The modo et al.  to choosINA method were analysed. Genetic variance was partitioned into three levels: (1) among different groups defined on the basis of phylogenetic clusters, host plants, and mite colour patterns, (2) among populations within groups, and (3) within populations. Significance of fixation indices was tested using a nonparametric permutation approach with 1000 permutations [Population genetic variance was further analysed by an Analysis of Molecular Variance (AMOVA)  performeutations , as perfXYH JTS participated in the design of the study. JTS CL performed the experiments. CL JTS participated in genetic data analysis. JTS MN XYH drafted the manuscript. All authors read and approved the final manuscript.Pearson correlations between CHE and geographic latitude. Expected heterozygosity calculated by the corrected data (CHE); R = -0.469, P < 0.05.Click here for fileFST ENA} {values between all populationsPairwise .Click here for fileFST ENA} {vs. geographical distance for pairwise population comparisonsScatter plots of .Click here for fileINA methodConsensus neighbour-joining tree based on DCE distances calculated on corrected data using the .Click here for fileClustering analysis by structure for five-loci dataset.Click here for fileINA methodAMOVA results of the corrected data set using the .Click here for file


Arbacia lixula, an amphi-Atlantic echinoid with potential to strongly impact shallow rocky ecosystems. Sequences of the mitochondrial cytochrome c oxidase gene of 604 specimens from 24 localities were obtained, covering most of the distribution area of the species, including the Mediterranean and both shores of the Atlantic. Genetic diversity measures, phylogeographic patterns, demographic parameters and population differentiation were analysed. We found high haplotype diversity but relatively low nucleotide diversity, with 176 haplotypes grouped within three haplogroups: one is shared between Eastern Atlantic (including Mediterranean) and Brazilian populations, the second is found in Eastern Atlantic and the Mediterranean and the third is exclusively from Brazil. Significant genetic differentiation was found between Brazilian, Eastern Atlantic and Mediterranean regions, but no differentiation was found among Mediterranean sub-basins or among Eastern Atlantic sub-regions. The star-shaped topology of the haplotype network and the unimodal mismatch distributions of Mediterranean and Eastern Atlantic samples suggest that these populations have suffered very recent demographic expansions. These expansions could be dated 94–205 kya in the Mediterranean, and 31–67 kya in the Eastern Atlantic. In contrast, Brazilian populations did not show any signature of population expansion. Our results indicate that all populations of A. lixula constitute a single species. The Brazilian populations probably diverged from an Eastern Atlantic stock. The present-day genetic structure of the species in Eastern Atlantic and the Mediterranean is shaped by very recent demographic processes. Our results support the view (backed by the lack of fossil record) that A. lixula is a recent thermophilous colonizer which spread throughout the Mediterranean during a warm period of the Pleistocene, probably during the last interglacial. Implications for the possible future impact of A. lixula on shallow Mediterranean ecosystems in the context of global warming trends must be considered.We present the global phylogeography of the black sea urchin  Arbacia lixula  is currently one of the most abundant echinoids in shallow rocky habitats of the Mediterranean A. lixula has a considerable trophic plasticity, ranging from omnivory to strict carnivory A. lixula broadly overlaps its habitat with the common edible sea urchin Paracentrotus lividus . Both species are traditionally thought to have the ability to trigger the development of subtidal barren zones of reduced benthic productivity and diversity A. lixula could actually be playing the principal role in producing and maintaining these barrens The European black sea urchin Arbacia lixula is commonly regarded as a typical native species in the Mediterranean fauna A. lixula (reported as A. pustulosa), probably originated at the Tropical Atlantic region, from where it spread into the Mediterranean. Kempf A. lixula was a thermophilous species.et al. reported that Arbacia lixula had become abundant in Marseilles during the previous 30 years et al. reported a 12–fold increase in the abundance of A. lixula in Corsica over a period of nine years (1983–1992) and speculated that a long term rise in the water temperature could have been the cause for this proliferation A. lixula densities was reported at the Port-Cros Marine Reserve (France) A. lixula and summer surface water temperature, suggesting increased reproductive potential with temperature In NW Mediterranean, increasing abundances over time have been reported for this species. In 1950, Petit Arbacia is an ancient genus with a fossil record that dates back to the Paleocene Arbacia has a history of latitudinal shifts A. dufresnii, is able to live in cold Subantarctic waters. A. lixula is the only species in the genus that lives in the Old World. Its present distribution includes Brazil, the African Atlantic coast from Morocco to Angola, the East Atlantic archipelagos of Cape Verde, Canaries, Madeira and Azores, and the whole Mediterranean basin, excluding the Black Sea. It has never been reported from the Atlantic European coast north of Gibraltar , probably due to the low sea surface temperature originated by the southward Portugal Current et al.Arbacia, using sequences of the mitochondrial COI (cytochrome c oxidase I) and the nuclear gamete recognition protein bindin, which has clarified many interesting questions on inter-specific relationships within this remarkable genus. Notably, the sequence of speciation events was consistently reconstructed and their divergence times were reliably estimated. Thus, the splitting between A. lixula and its sister species, the NW Atlantic A. punctulata, was estimated to have taken place some 2.2–3.0 Mya (millions years ago) based on COI sequences, or 1.9–3.3 Mya based on bindin sequences. The phylogeny of bindin sequences also allowed these authors to infer that Brazil populations separated from the rest of A. lixula some 1.8–3.4 Mya; i.e. very early in the evolution of this species .Recently, Lessios Arbacia lixula. Considering its unusually wide present distribution area, which ranges from equatorial waters to temperate Mediterranean, the great colonizing potential shown by this species, including the ability to cross trans-oceanic barriers to gene flow Arbacia lixula. Our goals were to answer relevant questions concerning the history and present-day distribution of the species: What are the relationships between the main geographic areas where the species is found? Do the main geographic barriers to gene flow, that are known to regulate the genetic structure of many other marine organisms, affect the present-day genetic structure of this species? Can recent geographic and/or population expansion events be traced and reconstructed by analysing the signature left in sequence data of this species?Yet, many questions remain open about the intra-specific relationships of Field sampling required for this work involved only invertebrate species which are neither endangered nor protected. All necessary permits for sampling at localities placed inside protected areas  were previously obtained from the competent authorities. Non-destructive sampling techniques  were used in these localities in order to minimize impact on the ecosystems.Arbacia lixula individuals (average: 25.2) per location were sampled. In all cases, tissue samples were stored in absolute ethanol at −20°C until processed.Between April 2009 and July 2011, we obtained samples from 24 localities belonging to three predefined regions: West Atlantic, East Atlantic and Mediterranean see . For morwww.sigma.com) from either one tube foot or a tiny portion (5–10 mg) of gonad. A fragment of the COI gene was amplified and sequenced using specific primers designed using the complete genome sequence of A. lixula mitochondrion primer 3.0 www.bio-rad.com). The PCR products were purified and both strands sequenced in Macrogen (www.macrogen.com) using the same primers for the sequencing reaction.Total DNA was extracted using REDExtract-N-Amp Tissue kit .All the sequences were edited in Bh), haplotype diversity (Hd) and nucleotide diversity (π) were computed with DnaSP v. 5.10 contrib v. 1.02 t-test was used for comparing its values between regions having more than two sampled locations .Number of haplotypes  We used network v. 4.6.0.0 (http://www.fluxus-engineering.com/sharenet.htm), which implements the median-joining method, in the absence of recombination tcs v. 1.21 program Relationships and geographical distribution of the haplotypes were analysed in a haplotype network constructed with Strongylocentrotus purpuratus from GenBank  were calculated with arlequin v. 3.1 Fst values was performed with RStudioDspadeD were corrected to zero. We calculated a confidence interval around the obtained values by 1,000 bootstrap replicates. We set this confidence interval, using the normal approximation, at the appropriate P-value following the B-Y correction as explained above. Significant differentiation was inferred when this confidence interval excluded zero.Pairwise genetic distances between populations (F-statistics (i.e. only with haplotype frequencies), and their significances were tested running 90,000 permutations in arlequinAnalyses of molecular variance (AMOVA) were performed to assess population structure, using conventional Fst/1–Fst) oogle Earth 6 (http://www.google.com/earth). The significance of the correlation was tested by the Mantel test procedure arlequin, with 20,000 permutations for each analysis.The effect of isolation by geographical distance was assessed, for the whole dataset or separately for different populations sets, by the correlation of linearized genetic distances  between observed and expected distributions was used as a measure of fit, and the probability of obtaining a simulated SSD greater than or equal to the expected was computed by randomisation. If this probability was >0.05, the expansion model was accepted, and its parameters θ0, θ1 and τ were calculated. For those populations showing large values for the final effective population size θ1, this method does not usually converge and flawed results could be obtained. In this case, we kept the value of τ calculated by this method, which is consistently robust naSP to calculate the value of θ0 which minimized the SSD, letting θ1 have an arbitrary large value of 1000 Demographic history was inferred for the three studied regions and for each sub-basin by analysing the mismatch distributions. Populations that have recently experienced a sudden demographic growth show unimodal distributions, whereas those at demographic equilibrium show multimodal distributions To estimate the approximate time of a demographic expansion (t) from coalescence methods, the relationship τ = 2 µkt was used D test of neutrality FsR2naSP. The confidence limits of Tajima’s D were obtained assuming that it follows the beta distribution Fs and R2 were based on a coalescent simulation algorithm implemented in DnaSP, with 20,000 simulations. Harpending’s raggedness index rarlequin and its significance was tested using parametric bootstrapping . These indices were calculated for the three regions and the six predefined sub-regions.In order to add more statistical support for population expansions, Tajima’s Arbacia lixula individuals from 24 localities (P>0.95). A total of 161 haplotypes were obtained from all the sequences (d) and nucleotide diversity (π) calculated for the whole geographical range were 0.912 (±0.007 SD) and 0.00658 (±0.00026 SD), respectively , indicating that the Eastern Atlantic populations are more genetically diverse than their Mediterranean counterparts. The small number of samples available from Brazil prevented us from performing any diversity comparison of this area with other regions.We sequenced 635 bp of the mitochondrial gene COI from 604 calities . We founequences . Of themectively . All divbaps clustered the sampled haplotypes into three haplogroups . Haplogroup A is the most abundant in all Eastern Atlantic and Mediterranean populations, but it is absent from Brazil, haplogroup B can be found in all three regions and haplogroup C is exclusive from Brazilian populations , with the exception of Torremuelle  where Fst analysis showed significant differences with two other Mediterranean localities, though these differences were not significant when D measures were analysed. Between Eastern Atlantic and Mediterranean, however, 38 (D) and 31 (Fst) comparisons (out of 85) were significant. Remarkably, the localities of Carboneras (Western Mediterranean), Crete and Kos (Eastern Mediterranean) did not show any significant difference to any other Eastern Atlantic or Mediterranean population, despite the large geographical distances involved in the case of the two latter localities.The analyses of population pairwise genetic differentiation  reflected the same overall structure.The MDS analysis  graphicavs. Mediterranean regions were compared . Ramos-Onsins & Rozas’ R2 was significant for all cases except the Eastern Mediterranean sub-basin, and the raggedness value r was consistent with unimodal distributions, except for Brazilian and Cape Verdean populations.The neutrality and population expansion tests calculated for the different regions and sub-basins  were larCOI and other mitochondrial markers have proven to be the most useful tool for tracing both intraspecific and intrageneric genealogies of many echinoid species Arbacia lixula populations sampled showed high values of haplotype diversity and haplotype richness, but relatively low values of nucleotide diversity. The lowest diversity was found in Brazilian populations and, specifically, in the westernmost locality (Itaipu), which is close to the distribution limit of the species and separated from the other Brazilian locality by the Cabo Frio upwelling. In contrast, the highest diversity was found in the East Atlantic, as expected if this region is the geographical origin of the species A. lixula. One of them (Group A) seems to be ancestral and is found only in Eastern Atlantic and Mediterranean populations, while another (Group B) is present at both sides of the Atlantic. The third one (Group C) is derived from Group B and found only in Brazil.The et al.Arbacia lixula split from a common ancestor with A. punctulata ca. 2.6 Mya, and attributed this split to the mid-Atlantic barrier, separating the western A. punctulata from the eastern A. lixula, which would later have crossed back this barrier to establish itself, as an isolated clade, in the coast of Brazil. A problem with this view is that the mid-Atlantic barrier was fully in place long before the estimated date of the split, so the separation of the two species could not be a vicariance event but a range expansion event (on the part of the lineage that would become A. lixula), and two crossings of the barrier are required to fully explain the present-day distribution of the species  view, as the haplotypes from Brazil formed a derived monophyletic group nested within the amphi-Atlantic Group B, rather than the opposite. This indicates a derived lineage in Western Atlantic, old enough to have had time to evolve forming the haplotype Group C. A more thorough sampling of the whole range of the Western Atlantic distribution and the inclusion of more data from Western Africa, are necessary before firm evidence can be obtained about the historical whereabouts of the main lineages of A. lixula.In a recent work, Lessios Fst, Jost’s D, MDS and AMOVA analyses) showed three groups of populations that differed significantly from each other , while little structure could be found within these groups. It is remarkable that the Fst measures based on sequence distance metrics and the differentiation measure D based on haplotype frequencies yielded essentially the same results. This is attributable to the prevalence of close haplotypes separated by small number of mutations  that are widespread among populations. Thus, haplotype genetic differences had relatively little weight and most population structure derives from haplotype frequency differences.Overall, the pattern of distribution of genetic variability  detected significant genetic differentiation between these groups of populations  that populations in the different Mediterranean sub-basins have not had yet enough time to diverge from each other.Even if there is no phylogenetic break in the Mediterranean , which have been extensively sampled and thoroughly described The almost complete lack of fossil record for A. lixula, providing several tens of pieces of individuals, including the oldest known record of this species. These deposits are currently dated to 130–120 kya A. lixula fossil specimen known from the Pleistocene of Madeira As for the Atlantic archipelagos, recent work on the fossil echinoid fauna of Azores Islands Arbacia lixula in the Mediterranean, and somewhat more, but still scarce, evidence of the colonization of the Atlantic archipelagos of Azores and Madeira, which probably occurred during the last interglacial period of the Pleistocene (MIS 5e). These observations are in agreement with the genetic signatures we observed in the mismatch distributions, which clearly show that recent sudden expansions have occurred in the Mediterranean and Macaronesian populations . This period was also the longest of all interglacial warm periods of the Pleistocene. The minimum winter surface temperature of the Mediterranean Sea stayed warmer than 19°C for several thousands of years A. lixula to cross the Strait of Gibraltar and colonize the Mediterranean.Thus, there is scarce paleontological evidence of the occurrence of ulations . This isulations  and by tulations . Our tem1. This expansion probably occurred more recently than in the Mediterranean (31.3–68.4 kya). This estimation falls within the Late Pleistocene, an epoch generally dominated by the last glaciation (Würm), during which the mean sea level dropped down to 80 m below the present level A. lixula in the Eastern Atlantic. Contrary to what happens in the Mediterranean, the fossils available show that the species was present in Macaronesia before this expansion A. lixula seems to be more complex than that of the Mediterranean populations. To complete the picture of the colonization of Atlantic archipelagos, data from continental African shores would be highly valuable.In the case of Eastern Atlantic populations, the exponential demographic expansion is even more apparent, since the mismatch distribution follows a sharp unimodal curve which fits to a sudden expansion model with a very high value for θArbacia lixula, that have shaped contemporary communities as the result of a colonization event that took place many years ago, can be falsely viewed as native A. lixula has indeed colonized the Mediterranean recently and complies with the terms of the former definition, even if it is usually viewed as native because its colonization took place following natural climatic changes, without human intervention.An invasive species can be defined as a “species that threatens the diversity or abundance of native species, the ecological stability of infested ecosystems, economic activities  dependent on these ecosystems and/or human health” A. lixula in Mediterranean ecosystems, thus possibly turning a “natural” colonization into an ecological problem related  to human intervention. The ongoing warming A. lixula in Northern Mediterranean, by releasing the constraint to larval development due to low water temperature. Warnings have been issued about its potential population increase and the generation of barren grounds in sublittoral habitats Whether considered as an “old natural invader” or as native, the present trend of global warming can potentially boost the negative impact of Arbacia lixula as a thermophilous species that has recently colonised the Mediterranean and whose densities may increase in the foreseeable future. Monitoring of populations seems highly recommendable as a management tool in the near future for protecting the threatened Mediterranean shallow water ecosystems.Thus, genetic data are in agreement with the consideration of Table S1Arbacia lixula COI for all sampled localities. Haplotypes shared by two or more localities are represented in bold, while numbers not in bold correspond to private haplotypes. Background colours correspond to the three different haplogroups. Outgroups weights calculated by tcs are also displayed for each haplotype, and that with the highest outgroup weight (A2) is highlighted in green background.Haplotype frequencies of (XLS)Click here for additional data file.


Rhinanthus minor and Rhinanthus angustifolius (Orobanchaceae) are annual hemiparasites, which occur sympatrically in Europe and are known to hybridize. We studied chloroplast and nuclear (amplified fragment length polymorphism [AFLP]) diversity in R. minor and compared genetic structuring in this species with R. angustifolius by analyzing the AFLP data for both species simultaneously. The AFLP data revealed that populations in Italy, Greece, and southeast Russia initially identified as R. minor were so distant from the other R. minor populations that they probably belong to another, yet unidentified taxon, and we refer to them as Rhinanthus sp. R. minor s.s. showed a clear geographic genetic structure in both the chloroplast DNA (cpDNA) and nuclear genome. The simultaneous analysis of both species shed new light on the previously published findings for R. angustifolius, because some populations now turned out to belong to R. minor. The admixture analysis revealed very few individuals of mixed R. minor–R.angustifolius ancestry in the natural populations in the west of Europe, while admixture levels were higher in the east. The combined haplotype network showed that haplotype H1 was shared among all species and is likely to be ancestral. H2 was more abundant in R. angustifolius and H3 in R. minor, and the latter probably arose from H1 in this species in the east of Europe. The occurrence of H3 in R. angustifolius may be explained by introgression from R. minor, but without interspecific admixture, these are likely to have been old hybridization events. Our study underlines the importance of including related species in phylogeographic studies. Quercus: Betula: Fraxinus: A growing number of studies have demonstrated wide-ranging sharing of chloroplast DNA (cpDNA) haplotypes across species boundaries in plants, often trees  offers a good model system for the study of genetic interactions between closely related species. The genus comprises 25 annual hemiparasitic species in Europe  markers , where the species have grown together for at least 12 years (R. minor) to +4 (R. angustifolius). We used DNA of nine plants, two plants with index +4, two with +2, two with 0 and all markers present , two with –2, and one with –4.To examine whether interspecific hybrids could be detected in the natural populations, we used plants of known descent as reference. In a garden experiment conducted in 2007, a mixed population was created with potted plants grown from seed from a  markers . We used12 years . The hyb12 years , resultiR. angustifolius , 1 min at 94°C, 1 min at 55°C, 2 min at 72°C (30 cycles), and 7 min at 72°C (1×) using the Gene Amp PCR System 9700 thermocycler . The amplification products were checked on 1% agarose gels and samples were diluted 10 times before sequencing. Sequencing reactions were performed forward using 1 µL of 5 µM a or c primers and 4 µL of diluted PCR product. PCR products were dye-labeled using a Big Dye Terminator Kit  and reactions were run on an ABI 3100 automated sequencer . Sequences were manually aligned using BioEdit . The AFLP reactions and analyses of one randomly chosen individual for each of 24 R. minor populations  were duplicated to serve as a blind sample in order to test the reproducibility of the scoring and to calculate the error rate. Two other R. minor individuals were run on each plate to check for among-plate repeatability , the total gene diversity (hT), the coefficient of genetic differentiation over all populations (GST), and equivalent parameters computed by taking into account the similarities between haplotypes . In order to statistically compare the two parameters of genetic differentiation .Analyses were performed on the tifolius  was constifolius . Diversintiation , a permuntiation . IsolatiR. minor, R. angustifolius and known hybrids), we constructed a binary matrix by scoring bands with sizes between 50 and 500 base pairs (bp). Presence of unambiguous bands was scored as 1 and absence as 0. Bands that were not perfectly reproducible between replicates were eliminated from the matrix (23 out of 204 bands). We calculated a matrix of genetic distances between individuals in the full dataset using the module Restdist in PHYLIP 3.69 (http://tree.bio.ed.ac.uk/).For the entire dataset  using the admixture model, uncorrelated allele frequencies models, no prior information, and the following parameters: K from 1 to 12, 25 replicate runs for each K, a burn-in period of 2 × 105 and 106 iterations. The optimal number of clusters K in our dataset was selected using the methods described by FST computations for R. minor populations were conducted using ARLEQUIN version 3.0 , estimated by the Nei's unbiased diversity estimator were computed using AFLPSURV 1.0  using the Microsoft Excel add-on POPTOOLS (http://www.poptools.org/).Genetic structuring was further investigated using a Bayesian model-based clustering algorithm implemented in STRUCTURE . We analsion 3.0 . PairwisSURV 1.0  by pooliSURV 1.0 . The R sSURV 1.0  was usedpulation . To testtrnT-trnL spacer and trnL intron sequences for the 295 successfully sequenced individuals  among the western and northern populations, excluding the southeastern populations in Italy, Greece, and Russia. Differentiation was significantly higher when similarity between haplotypes was taken into account (NST = 0.77). The Mantel test was significant for western populations , showing that geographically distant populations were more differentiated than adjacent ones. As only two haplo-types were observed in the southeastern populations (H1 and H21), gene diversity within populations was low (mean: 0.07) and tests for differentiation and isolation by distance were not performed.We defined five informal haplotype groups: H1, close to H1, H3, close to H3, and H2 . Among tR. minor  previously detected in R. angustifolius , and an inquiry at the source of the seeds, the Botanical Garden of the University of Copenhagen, could not confirm the identity of the plants in the wild population where they had been sampled, but based on our results, it is highly likely that the seeds had been sampled on R. minor plants. The remaining populations in the G4 group, from Poland (PL1) and Romania (RO1), belonged to the R. minor Central cluster  are at the opposite end, close to the R. angustifolius AFLP cluster. The South cluster did not form a single branch; its members were spread out over the tree, often on very long branches . Among the southeastern Rhinanthus sp. populations, DW values were much higher, on average 0.850, with the highest score recorded in Russia (1.89). Genetic differentiation among Atlantic, West, Central, and Spanish R. minor populations was relatively high (FST = 0.581), and the Mantel test  showed an effect of isolation by distance. For the southeastern Rhinanthus sp. populations genetic differentiation was much stronger (FST = 0.835) and the Mantel test  demonstrated a strong effect of isolation by distance.On average, gene diversity within observed . The rarn Norway , with anR. minor. Group G1, the largest group, formed part of the R. angustifolius AFLP cluster in the current analysis, together with group G5 and one population in G3—the Romanian population RO2. The other population in G3, the Turkish population TR1, showed a mixture of the Russian, the R. angustifolius, and the South cluster in the current STRUCTURE analysis  had 2% and 16%. The plants with +4 (97% and 97%) and –4 (0.2%) were probably indeed pure R. angustifolius and R. minor, respectively, without signs of hybridization.The F mothers . The twoR. minor, 57 individuals had a maximum q-value of less than 0.95, corresponding to 23% of the 245 individuals in the four main clusters. For the individuals in the Atlantic cluster, most of the admixture was with West and Central, but some populations in Scotland and Ireland contained plants with high q-values for the Spanish cluster, between 0.06 and 0.28  or R. angustifolius and/or South .In the west, admixture with Rhinanthus sp. populations, admixture was much rarer. No admixture was found in the Greek and Italian clusters. One individual in the Russian cluster had a q-value of 0.29 for the Greek cluster. The Italian population in the South cluster showed no admixture at all, in contrast with all the other plants in this cluster.In the southeastern R. angustifolius AFLP groups. For the main group G1, q-values below 0.95 were only found in three individuals (5%) in BE1, SC3, and NL5. The Romanian population RO2 (AFLP group G3) clustered with the other R. angustifolius, with a q-value of 0.11 for South in one individual. Of the 14 individuals in the original G5 cluster, six (43%) had R. angustifolius q-values below 0.95, and showed admixture with West, South, and Italian. As mentioned earlier, TR1 (AFLP group G3) and FR5 (AFLP group G2) had R. angustifolius q-values well below 0.5 and were admixtured with Russian and South (TR1) or South only (FR5).Admixture levels varied considerably among R. minor AFLP clusters differed considerably in their frequencies of the main cpDNA haplotype groups  and de1 (n = 2).The e groups . Haploty network  shows thRhinanthus minor that were sampled in Italy, Greece, and Russia (Caucasus), although morphologically similar to this species, turned out to be strongly genetically differentiated from all other R. minor populations from central, western, and northern Europe. The fact that they turned out to be genetically more similar to but still distinct from R. angustifolius argues for treating these populations as a distinct taxon at the species level, here tentatively named Rhinanthus sp. At present, we only have data to show that they are closer to R. angustifolius, but detailed morphological examination of these populations in combination with a comprehensive phylogeny of the genus would be needed to determine the affinities of this taxon and name it appropriately. A possible candidate is Rhinanthus personatus (Behr.) Beg., a species described from Italy  Chabert, which only occurs in northern Europe above 60°N and in the mountains in Norway, plus on the Faroe Islands and Iceland (R. groenlandicus is considered a subspecies of R. minor (R. minor ssp. groenlandicus [Chabert] Neuman) in Scandinavia (http://linnaeus.nrm.se/flora/), and it is possible that our samples from Norway contained genetic material from this (sub)species.A third explanation would be hybridization with another  Iceland . Althoug Iceland , R. groeR. minor samples from the east of Europe, we cannot exclude that colonization of Scandinavia occurred from a separate, more eastern lineage, but the R. minor samples from Poland, Bulgaria, and Romania fell within the Central cluster and did not share any haplotypes with the Scandinavian populations  sequence data. We have detected haplotypes H1, H2, H13, and H31 in R. alectorolophus, along with three unique haplotypes derived from H1 and H2, and R. alectorolophus does not hybridize with R. minor . In two other species, Rhinanthus freynii from Italy and Rhinanthus rumelicus from Greece, we found H1 and H2, respectively. This suggests that H1 and H2 are ancient haplotypes in the genus, as they are shared by several species. However, given its abundance and the number of derived haplotypes in R. minor, it is likely that H3 has evolved later, and only in R. minor. If this is indeed the case, then the presence of H3 in five R. angustifolius individuals across Europe can only be explained by hybridization or convergent evolution. The former explanation is very likely in population BG1, which was a mixed population with two R. angustifolius and two R. minor individuals in our dataset. The two R. minor plants in this population carried H3, and one of the R. angustifolius plants had H3 as well. The other three R. angustifolius populations with H3 present in one or two individuals were found in southern Sweden, the North Sea coast of the Netherlands, and in the Czech Republic, all areas where hybridization with cpDNA introgression could have occurred given the geographic distribution of R. minor and its haplotype H3. In none of these individuals, or in other individuals of the same populations, did we find increased levels of admixture, so if the presence of haplotype H3 is a trace of hybridization, it is likely not recent. Haplotype H12, derived from H3, was only found in a single individual in a German R. angustifolius population (DE2), and it may have arisen after an ancient hybridization event as well.Although DNA phylogenies have been published for the Orobanchaceae , and theR. angustifolius, was only found in two R. minor populations; one in Ireland, which was monomorphic for this haplotype, and one in southern Germany. This would suggest that H2 was not originally present in R. minor, but that was acquired through hybridization with R. angustifolius. While hybridization as a source for cpDNA haplotype introgression would be possible in Germany, R. angustifolius does not occur in Ireland, so hybridization in situ would be an unlikely explanation for the occurrence of H2 in an Irish population, leaving convergent evolution by a second occurrence of the single point mutation that separates H1 from H2, or an ancient polymorphism. Introgression of cpDNA from R. angustifolius to R. minor would require sufficiently extensive F1 hybrid formation to overcome the low germination rate of F1 hybrids with R. angustifolius cytoplasm (R. angustifolius (Haplotype H2, which is much more prevalent in ytoplasm , which mtifolius .R. angustifolius are caused by interspecific gene flow, then removing these from the cpDNA dataset might facilitate the detection of a phylogeographic signal in R. angustifolius (R. angustifolius, which is likely to have coincided with an increase in human populations and livestock grazing, which creates the open grassland habitat necessary for the species. The expansion of the species into northern Europe probably predates the formation of derived haplotypes, especially from H2, which are usually restricted to one or only a few populations. While the original haplotypes were similar in the different R. angustifolius lineages, H3 in R. minor probably arose early in central Europe and spread with the Central cluster. Both species have a low natural seed and pollen dispersal, so the lack of genetic structure within G1 in R. angustifolius, which covers most of central and western Europe, is difficult to explain. We may hypothesize that it occurred more often in hay meadows than R. minor, which is more abundant on poorer, dryer soils, and that R. angustifolius has thus profited more from human hay and livestock transport than R. minor.If all occurrences of H3 within tifolius . HoweverR. minor from R. angustifolius, and correctly identify hybrids between these species. Other taxa were also identified, in particular Rhinanthus sp. in southeastern Europe, and possibly the French population that clustered separately from R. angustifolius. This population formed a separate AFLP cluster, South, which also included highly divergent individuals from other populations that were on separate branches in the NJ tree, and this cluster should probably not be interpreted as a phylogenetic signal. It is possible that yet another Rhinanthus species is involved in the ancestry of the population from the French Pyrenees; five species are known to occur in France (Rhinanthus, where taxon limits are not clearly defined.In conclusion, the AFLP analysis was able to unequivocally distinguish n France . To corrn France  would ben France , which p


Aedes albopictus, is one of the 100 most invasive species in the world and a vector of human diseases. In the last 30 years, it has spread from its native range in East Asia to Africa, Europe, and the Americas. Although this modern invasion has been the focus of many studies, the history of the species’ native populations remains poorly understood. Here, we aimed to assess the role of Pleistocene climatic changes in shaping the current distribution of the species in its native range.The tiger mosquito, Ae. albopictus native populations at the Last Glacial Maximum (LGM). Individuals from 16 localities from East Asia were analyzed for sequence variation at two mitochondrial genes. No phylogeographic structure was observed across the study area. Demographic analyses showed a signature of population expansion that started roughly 70,000 years BP. The occurrence of a continuous and climatically suitable area comprising Southeast China, Indochinese Peninsula, and Sundaland during LGM was indicated by species distribution modelling.We investigated the phylogeography, historical demography, and species distribution of Ae. albopictus did not experience a fragmentation phase but rather persisted in interconnected populations and experienced demographic growth. The wide ecological flexibility of the species probably played a crucial role in its response to glacial-induced environmental changes. Currently, there is little information on the impact of Pleistocene climatic changes on animal species in East Asia. Most of the studies focused on forest-associated species and suggested cycles of glacial fragmentation and post-glacial expansion. The case of Ae. albopictus, which exhibits a pattern not previously observed in the study area, adds an important piece to our understanding of the Pleistocene history of East Asian biota.Our results suggest an evolutionary scenario in which, during the last glacial phase,  The Pleistocene was a time of intensive climatic fluctuations, during which glacial–interglacial cycles occurred at irregular intervals with varying durations In Far East Asian regions, Pleistocene climatic changes caused dramatic environmental changes, although the glacial advances were not as extensive as those in Europe or the Americas Most phylogeographic studies of animal species in these regions have focused on forest-associated species to investigate whether and how they were affected by ice-age-driven changes in forest distribution, both at mid-latitude and in the Indochinese Peninsula Aedes albopictus, a generalist species with wide ecological plasticity. It is adapted to both tropical and temperate zones and is distributed across East Asia, from Indonesia to Korea on the north–south axis and from Japan to India on the east–west axis. In temperate regions, Ae. albopictus overwinters by photoperiodic egg diapause  and nicotinamide adenine dinucleotide dehydrogenase subunit 5 (ND5) were obtained through Polymerase Chain Reaction (PCR). The primers TW-J-1305 and TK-N-3782 COI, tRNA-Leu and COII. The following primers were then designed to amplify and sequencing two regions of the COI gene: AealCOIa-f 5′- aaaaagatgtatttaaatttcggtctg-3′ and AealCOIa-r 5′-tgtaattgttactgctcatgctttt-3′; AealCOIb-f 5′-ttattacacaagaaagaggaaaaa-3′ and AealCOIb-r 5′-cattgcactaatctgccata- 3′. The ND5 gene region was amplified using the primers designed by Birungi & Munstermann 2, primers at 0.2 µM, 0.5 units of Taq DNA Polymerase , and 20 ng of genomic DNA extracted from single mosquito. The PCR cycling procedure was: 95°C for 5 min followed by 34 cycles at 93°C for 1 min, annealing temperature: 55°C for COI and COIb; 50°C for ND5 for 1 min, 72°C for 1 min 30s, and a single final step at 72°C for 10 min. PCR sequences were obtained using an ABI PRISM 3700 DNA sequencer by Macrogen Inc. (www.macrogen.com). All individuals analysed were sequenced using both forward and reverse primers, and all haplotypes that were found unique (see Result section) were re-amplified by using high fidelity Taq DNA Polymerase  and re-sequenced to check for consistency.Total genomic DNA was extracted from single mosquitoes following the standard CTAB (cetyltrimethyl ammonium bromide) protocol hromas 2.33  and Clustalx 2.0 Sequences were edited and aligned using the software CCOI and ND5 genes were assessed using the software Mega 5.0 aup* 4.0b10 h) and nucleotide diversity (π) were estimated for each locality  and for the overall dataset, using the software Arlequin 3.1 Nucleotide and amino-acidic polymorphisms of the etwork 4.6.1.0 software (Fluxus Technology Ltd). The loops in the resulting phylogenetic network were resolved by applying the criteria described by Pfenninger & Posada The genealogical relationships between haplotypes were investigated by constructing a phylogenetic network samova 1.0 a priori hypothesis of the expected structure. Given a user-defined number K of groups of populations, samova uses a simulated annealing procedure to define groups of populations that are as genetically homogenous as possible  and maximally differentiated from one another . We tested K values ranging from 2 to 15 and, to check for consistency, we conducted each analysis five times, with 10,000 independent annealing processes each.The possible occurrence of groups of genetically distinct populations was investigated by Spatial Analysis of Molecular Variance as implemented in the software rlequin 3.1. In populations that have undergone a sudden demographic expansion, the mismatch distributions are expected to be smooth and bell-shaped SF neutrality test SF indicate an excess of singleton mutations, which is expected both under departures from selective neutrality and in populations which have experienced a recent demographic expansion. It was computed on Arlequin 3.1, and its significance was assessed through 1000 replicates. Finally, historical demographic trend was investigated using the Bayesian skyline plot (BSP) method by Drummond et al. beast 1.6.1 beast in order to fine-tune MCMC parameters. The MCMCs were finally run for 20 million steps, and sampled every 1000 steps. The first two million steps were discarded as burn-in, after checking the likelihood history with tracer 1.4 To investigate signatures of past demographic changes we analysed the distribution of pairwise nucleotide differences between haplotypes (mismatch distribution) as implemented in the software AAe. albopictus was developed using the “maximum entropy model” as implemented in Maxent 3.3.3e Ae. albopictus, including records from literature, records from the “Global Biodiversity Information Facility” (GBIF) online database (http://data.gbif.org) and our sampled localities. We selected all georeferenced localities, while among those without geographical coordinates, we used only localities for which exhaustive geographical data are furnished by the authors. These localities were then georeferenced using the software Google Earth.The Species Distribution Model (SDM) for http://www.worldclim.org/). These 19 variables, derived from monthly temperature and rainfall values, were downloaded at spatial resolution of 2.5 arc-minutes for current and LGM conditions, and at spatial resolution of 30 arc-seconds for LIG conditions.The SDM was developed for Current, Last Glacial Maximum (LGM) and Last Inter-Glacial (LIG) conditions. To reconstruct the ecological niche of LGM conditions, we used two general circulation models (GCM): the Community Climate System Model 3 (CCSM 3) Pearson’s correlation matrix was generated for all 19 variables to quantify the correlation between them. For pairs that were highly correlated (correlation coefficient ≥0.75) we chose the variable more biologically meaningful for the species axent, each run was conducted using the default convergence threshold (10−5) and maximum number of iterations (500) values. For the regularization parameter β, as suggested by Warren & Seifert nmToolsc), was one for which β = 3. Twenty-five percent of record points were used for model testing, and the final SDMs performance was evaluated using the area under receiver operating characteristic (ROC) curve (AUC). An AUC score above 0.7 is considered good model performance In MND5 gene fragment, 405 and 689 base pairs respectively for AealCOIa and AealCOIb primer pairs) was obtained for 174 individuals. Sixty two haplotypes were found (GenBank Accession numbers: JQ436947-JQ437008) defined by 54 nucleotide substitutions (32 parsimony informative). Forty one nucleotide substitutions were located at the third codon position, five at the second codon position and eight at the first one. Thirteen non-synonymous nucleotide substitutions were found. Overall haplotype and nucleotide diversity estimates were 0.945 (±0.009) and 0.0021 (±0.0012), respectively.A final sequence alignment including 1458 nucleotides , were found in populations spanning the entire east-west and north-south extension of the study area .Phylogenetic relationships among the 62 haplotypes found are shown in K value showed no significant increment in the CTF values. They showed a very narrow range  and the highest value (CTF = 0.40) was observed for K = 15, when all populations minus one were separated  no groups of genetically distinct populations were evidenced. All partitions of the sampling areas for each eparated .The haplotype diversity and nucleotide diversity estimates for each population are shown in COI and ND5 genes (P = 0.46) (Fs and the Bayesian skyline plot method (BSP). The former was large, negative and highly significant , showing the occurrence of a larger than expected number of singleton mutations, as expected for populations that underwent a population expansion. The demographic trend inferred by the BSP also indicated the advent of a phase of demographic growth, which roughly started about 70,000 years BP until recently  and concatenated D5 genes . The mis = 0.46) . The pasrecently .axent models predicted under LIG, LGM and current conditions of Ae. albopictus are shown on The MAe. albopictus with respect to the LIG of 42% . At the higher latitudes, in the northernmost part of its current range of distribution (Northern China and Japan), areas of predicted suitable habitats were mainly absent  and with lesser degree in temperate regions (i.e. China and Japan), similarly to the current conditions . The proy absent .Ae. albopictus mitochondrial diversity have shown that native populations of this species harbour little or no mitochondrial genetic diversity  that did not represent the genetic diversity in natural populations. This explanation was supported by subsequent studies in which individuals from natural populations were used wAlbA and wAlbB strains of Wolbachia , which we did not observe.The genetic pattern observed in In contrast, the genetic pattern observed best fits with a scenario in which the species never experienced a prolonged allopatric divergence but rather persisted in a wide area, most likely across Southeast China and the Indochinese Peninsula. In these regions, we found not only a lack of phylogeographic structure but also higher levels of genetic diversity , as wellAe. albopictus could have exploited and maintained large interconnected populations.This scenario is also consistent with the reconstruction of the species distribution at the LGM . AlthougAe. albopictus populations. The demographic trend inferred from the BSP is that of a gradual expansion starting about 70,000 years BP, that is, during the last glacial phase including the LGM. Although the lack of a calibrated molecular clock demands caution, paleoenvironmental reconstructions of the region and SDM at the LGM support the hypothesis that populations of the species expanded during this period.In the genetic pattern observed, we also found the signature for past demographic changes in Ae. albopictus populations and could account for the expansion inferred from genetic data. The effect of glacial-induced lowland emergence on population dynamics has recently been investigated in the Sardo-Corso region in a study by Bisconti et al. Hyla sarda.During the last glacial phase in Southeast Asian regions, the glacio-eustatic lowering of the sea level fully exposed the Sunda shelf , which connected the major islands of Sumatra, Java, and Borneo to mainland Asia by land bridges Ae. albopictus populations also encompasses the post-glacial phase  and suitable breeding sites; this could have favoured the inferred population expansion.The inferred demographic expansion of Ae. albopictus populations in the temperate regions of East Asia and in the Indochinese Peninsula. The results allow us to propose some hypotheses concerning the southernmost part of the range of the species, to be tested in future studies. SDM data showed the occurrence of climatically suitable areas across all of Sundaland. Therefore, it could be hypothesized that the emergence of Sundaland allowed population connectivity across the Indonesian islands and between these islands and mainland Asia. This scenario has recently been inferred for other mosquito species (such as Anopheles vagus s.l.) that can exploit open habitats Cynopterus brachyotisAe. albopictus. Our preliminary data show high genetic similarity in mitochondrial DNA among the populations from East Asia and 2 populations from Sulawesi Island  In this study, we focused on Ae. albopictus populations that were conducted using nuclear markers have shown a lack of genetic discontinuity across most of the geographic range of the species Previous genetic studies on Ae. albopictus experienced a demographic expansion that started in the last glacial phase and lasted until recently. The population expansion following the LGM, as expected, was driven by amelioration of climatic conditions at mid-latitudes and perhaps also by environmental changes attributable to human activities. On the contrary, the population expansion that occurred before the LGM  is an interesting exception. SDM, along with paleoenvironmental reconstructions and life traits of the species, suggest a positive role of Sundaland in the dynamics of Ae. albopictus populations. Also in this case, the ecological flexibility and adaptability of the species most likely allowed it to exploit the wide range of habitats offered by the emergence of Sundaland. Therefore, the case study of Ae. albopictus contributes to our understanding of East Asian phylogeography and supports the findings of those studies that showed the positive role of the glacial-induced emergence of lowlands in maintaining populations during glacial phases Historical demographic analyses also showed that Figure S1Spatial analysis of molecular variance. Fixation indices, obtained using the software SAMOVA, for the best clustering option for each pre-defined values of K. FCT: variation among groups of populations; FSC: variation among populations within groups; FST: variation among populations among groups.(TIF)Click here for additional data file.


Juniperus communis in a fragmented landscape across northwestern Europe by using amplified fragment length polymorphism (AFLP) markers. Furthermore we investigated the genetic diversity and structure on two spatial scales: across northwestern Europe and across Flanders (northern Belgium). We also studied whether seed viability and populations size were correlated with genetic diversity.Population extinction risk in a fragmented landscape is related to the differential ability of the species to spread its genes across the landscape. The impact of landscape fragmentation on plant population dynamics will therefore vary across different spatial scales. We quantified successful seed-mediated dispersal of the dioecious shrub Unexpectedly, estimated seed-mediated dispersal rates were quite high and ranged between 3% and 14%. No population differentiation and no spatial genetic structure were detected on the local, Flemish scale. A significant low to moderate genetic differentiation between populations was detected at the regional, northwest European scale (PhiPT = 0.10). In general, geographically nearby populations were also genetically related. High levels of within-population genetic diversity were detected but no correlation was found between any genetic diversity parameter and population size or seed viability.Juniperus communis to naturally colonize suitable habitats. However, it is not clear whether the observed levels of migration will suffice to counterbalance the effects of genetic drift in small populations on the long run.In northwestern Europe, landscape fragmentation has lead to a weak isolation-by-distance pattern but not to genetic impoverishment of common juniper. Substantial rates of successful migration by seed-mediated gene flow indicate a high dispersal ability which could enable  Habitat fragmentation and destruction eventually lead to a reduction in the genetic diversity of plant populations. The consequences of habitat fragmentation are related to the differential ability of plant species to spread their genes across the landscape . DioecioJuniperus communis L.). Common juniper is a wind-pollinated, coniferous shrub producing seeds that are primarily dispersed by birds. In northwestern Europe, common juniper occurs on heathlands and calcareous grasslands, which are among the most highly fragmented semi-natural ecosystems in western Europe )In this study, we used assignment tests to investigate seed-mediated dispersal of common juniper in a fragmented landscape across northwestern Europe. If seed-mediated gene flow is restricted, we expect practically all the sampled individuals to be assigned to the population in which they were sampled. Furthermore, we determined the genetic structure and the genetic diversity of common juniper at two spatial scales: at the regional scale across northwestern Europe and at the local scale across Flanders (northern Belgium) Figure , Table 1After scoring 101 AFLP markers for the total dataset of the Flemish samples, 7 markers were discarded which resulted in a final dataset of 94 polymorphic markers. Based on the replicates, 274 differences were observed of 2162 phenotypic comparisons , giving an error rate of 12.6% . The error rate at the allele level within a gel run was 3.2%, the other 9.4% of the error rate was due to variability between gels. For the assignment of replicates in the cluster analysis, 44 out of 46 fingerprints from the 23 replicated samples were correctly assigned as 'sisters'. Two fingerprints belonging to the same replicate pair were not positioned adjacent to each other but showed a higher genetic similarity to an other sample (collected in an other population) compared to the genetic similarity with its 'sister'-individual. The number of band difference between these two, not correctly assigned fingerprints was 15 and both replicate pairs were grouped in the same cluster (results not shown). The mean pairwise inter-individual genetic distance was considerably higher than the error rate; 35.1% (standard deviation S.D.: 0.05%) and 35.6% (S.D.: 0.02%) for the samples collected on the Flemish scale and on the northwestern European scale, respectively. The simulation and re-assignment procedures implemented in AFLPOP, resulted in an assignment success of the simulated genotypes above 91% (mean 95%) in all the 11 analysed populations, indicating that the probability of misassignments was low (< 9%).Juniperus communis is 1%. Within a 30 km radius buffer zone surrounding each sampled population, the estimated proportion of suitable habitat for Juniperus communis ranged from 0.27% (population Hühnermoor) to 6.75% (population Kootwijkerzand) with a mean of 2.18%  were at least 10 times more likely to originate from a population different from the sampling site. Whereas 11 shrubs 3%) seemed at least 100 times more likely to originate from a population different from the sampling population, indicating the presence of migrant genotypes in the set of analysed populations. From the latter 11 plants defined as outlier genotypes (minimum log-likelihood difference (MLD) = 2), six, two and three assumed immigrant genotypes belong to height classes 1 - 2 m, 2 - 3 m and > 3 m, respectively. Assignment tests conducted on the AFLP genotypes of the sampled 292 individuals confidently allocated each of 191 (65%) and 106 (36%) individuals to a population when MLD was set to 1 and 2, respectively , with a large amount of genetic variation found within populations. Pairwise values for population differentiation  were also significant  except for the German populations Meenser Heide and Weinberg  . Although, for some populations, the genetic data did not cluster consistently according to their geographical locations. This was the case for the French populations Grattepanche and Cocquerel and the German population Hühnermoor.Clustering on the population level based on a Principal Co-ordinate Analysis (PCoA) and on a Bayesian modelling approach is presented in Figure PT = 0.00, p > 0.5). Pairwise values for population differentiation were not significant . Also, when considering all 180 Flemish genotypes analysed , no structure according to the population of origin or geographic location was apparent on a PCoA (results not shown). With the Bayesian approach, highest posterior probabilities were obtained with all 180 genotypes located in the same cluster. On the local, Flemish scale, we found no evidence for the isolation-by-distance hypothesis with the Mantel test . Consistently, no spatial genetic structure was found with SPAGeDi 1.3 by regressing pairwise kinship coefficients against pairwise geographical distances (slope of the regression with ln (distance) b = 0.0012, p = 0.61).Genetic structure on the local, Flemish scale was inferred from 94 polymorphic AFLP markers and 181 shrubs representing 180 unique genotypes (Table Hj) of 0.391 and 0.368 for the European and the Flemish populations, respectively. Genetic diversity statistics based on the AFLP markers for the northwestern European as well as for the three Flemish populations are given in Table Br) or gene diversity (Hj) in these younger individuals compared to the older ones within each population (results not shown), nor for the AFLP data pooled over all populations  but lowet al.  (19%) an [et al.  . Ho [et al. ,27). Fur [et al. ,28,29).  [et al.  concludeJ. communis in northwestern Europe. Habitat fragmentation and habitat loss usually decrease the probability that migrant seeds will find suitable sites for establishment. By providing insight into seed-mediated dispersal rates as a direct estimate of patterns of gene flow, our results expand on previous work (e.g. [Banksia hookeriana (6.8%) [Myrtus communis (20% - 22%) [Fagus sylvatica (27%) [The analysis of the CORINE 2006 land cover data indicate a highly fragmented habitat for rk e.g. ,12-14). -14. J. crk (e.g. ). The rerk . Therk e.g. ,19). It . It J. c% - 22%)  and Faguca (27%) . In a stca (27%)  found thca (27%) . It is sca (27%) . AlthougPT = 0.103). Our results are in agreement with those reported for outcrossing, wind-pollinated gymnosperm species [ST = 0.026). This is probably linked to the fact that allozymes show lower levels of polymorphism compared to AFLP markers and may, in contrast to neutral AFLP markers, experience the selective force of the environment. Similar to this study, Provan et al. [PT = 0.0957) but a much higher degree of differentiation between the same populations based on chloroplast markers (ΦPT = 0.249). The latter is explained by the action of genetic drift on the smaller effective population size of the uniparentally  transmitted, haploid chloroplast genome [Population genetics theory predicts that habitat fragmentation increases genetic differentiation among populations because of increasing spatial isolation between patches and individuals (e.g. ). At the species ,34, usua species  found a n et al.  also fout genome .et al. [et al. [In general, we found a clear but weak association between the genetic clustering and the geographical location of the common juniper populations studied; neighbouring populations were generally also genetically related. Exceptions to this general pattern were the French populations Cocquerel and Grattepanche and the German population Hühnermoor, which did not cluster consistently to their geographical location. The weak geographic structuring of genetic diversity on the northwestern European scale was confirmed by a weak isolation-by-distance signal. In contrast, Michalczyk et al.  found no [et al.  found a  [et al. .et al. [et al. [Juniperus has an extremely long history in Europe [Juniperus may already have been distributed across Europe during the earliest Tertiary period (65 million years ago), at the start of its history, and appears to have colonized Asia, Africa and America from Europe via intercontinental land bridges [The geographic patters of genetic diversity observed in this study may be explained by the patterns of genetic diversity in the original metapopulation prior to fragmentation. Like Michalczyk et al.  hypotheset al. . This hyet al. ,37. Base [et al. . Fossil  [et al. ,38 in co [et al. ,40. Thern Europe . Juniper bridges .We observed high levels of within-population genetic diversity (in terms of band richness and percentage polymorphic loci) in all common juniper populations studied. This result is in line with previous genetic studies of common juniper employing different marker systems  ,12-14. Wth century [Although it is suggested in previous studies that the extremely low seed viability is likely not linked to neutral genetic diversity , this is century ,44. At p century ,14,44. H century ). Howeveex situ conservation of the present high levels of genetic diversity by the establishment of gene banks, and secondly, rejuvenation of existing, small populations skewed to old plants by restoration planting with young individuals grown from cuttings which should enhance the seed production of the populations [Our study contributes new data to the growing evidence that seed-mediated dispersal of dioecious shrubs and trees in fragmented landscapes is substantially higher than previously thought. The potential for seed-mediated dispersal among populations in a fragmented landscape could enable common juniper to naturally colonize suitable habitats. Nevertheless, management strategies that exclusively focus on seed dispersal would not guarantee recruitment in common juniper because of the extremely low levels of seed viability. These seems to be currently the main threat to common juniper populations in northwestern Europe (e.g. ). The loulations . Given tJuniperus communis L. (Cupressaceae) is a diploid, dioecious, wind-pollinated, woody shrub or small tree. The distribution range of common juniper basically covers the entire Northern Hemisphere [Turdus spp.) and common juniper does not produce a long-term persistent seed bank [et al. [misphere . It is teed bank . Lifespaeed bank . Genets eed bank , the UK eed bank  and Belgeed bank ,52). For [et al. .In 2005, we sampled 13 natural common juniper populations along a north-south transect from north-Germany and northern Netherlands to north France, and along an east-west transect from northwest France to northwest Germany  populations in Flanders that contain more than 100 individuals: the populations Heiderbos, Kattevennen and Hesselberg. The populations occurred on heathlands, a habitat that has become highly fragmented in Flanders . MoreoveFlanders . Again, Per shrub, 10 ripe cones were opened, the number of seeds was counted and filled seeds were exposed to 1% 2,3,5 triphenyltetrazolium chloride (TTC) solution in order to determine the viability of the embryos. Initially colourless, TTC is converted to formazan-red in the presence of living tissue , according to manufacturer's instructions and followed by an additional treatment with 0.4 μg RNAse (Fermentas) at room temperature for 2 min. DNA concentrations were estimated and standardised against known concentrations of λDNA (Fermentas) on 1.5% agarose gels.et al. [et al. [PstI (Fermentas) /MseI (Fermentas) and ligated to the PstI and MseI adaptors. Primer combinations used for the generation of fingerprints were PstI-ACT + MseI-ACA, PstI-ACT + MseI-ACC, PstI-AGT + MseI-ACC and PstI-AGT + MseI-ACA. Fragment separation and detection took place on a NEN IR2 DNA analyzer (Li-Cor Biosciences) using 36 cm denaturing gels with 6.5% polyacrylamide. IRDye size standards (50 to 700 bp) were included for sizing of the fragments. Fragments within the size range of 75 bp to 677 bp were scored with Saga Generation 2 (Li-Cor Biosciences) as present or absent. Prior to data analysis, monomorphic loci were discarded. Due to the long time period between the analysis of the northwestern European samples and of the Flemish samples, different PCR-machines and fabrication batches of products were used. Hence, following Coart et al. [AFLP analysis was performed on the northwestern European and the Flemish samples according to Vos et al.  and Van  [et al.  with folt et al. , the AFLet al. [et al. [Reproducibility was evaluated on the dataset obtained from the individuals sampled on the Flemish scale using intra- and intergel replicates. 23 samples  between [et al. . Secondl [et al. . We calc [et al. ). We als [et al.  to invesJuniperus communis with the programme ArcView version 3.1. (ESRI) and based on CORINE Land Cover 2006 vector data (CLC06) . CLC06 classifies the European land cover into 44 categories derived from Landsat and SPOT satellite images at a 1:100,000 scale and with a minimum mapping unit of 25 ha [Juniperus communis: natural grasslands (class 3.2.1), moors and heathlands (class 3.2.2), sclerophyllous vegetation (class 3.2.3) and transitional woodland-shrub (class 3.2.4). The percentage of suitable habitat was calculated for northwestern Europe within an area of 1.4E+7 ha, and within a 30 km radius buffer zone surrounding each sampled population.To estimate the degree of habitat fragmentation in the study area, we mapped the natural habitats suitable for of 25 ha . The folSeed-mediated dispersal events were estimated by individual-based population assignment tests using the computer program AFLPOP 1.1 . BecausePT-pairwise values. This reduces the risk of misassignment due to similar allele frequencies between population pairs.Prior to the allocation test, we assessed the power of our dataset for accurate assignment of the real genotypes with the population assignment simulator of AFLPOP 1.1 . The simPT analog for FST [Population genetic structure was analysed based on AFLP data on both spatial scales. Total genetic diversity was partitioned among and within populations by carrying out a hierarchical analysis of molecular variance (AMOVA) on Euclidian pairwise genetic distances . The ΦPT for FST  was calc for FST . To furt for FST  was used for FST , a Mante for FST  against  for FST .IS = 0.1) from Hardy-Weinberg genotypic proportions according to the outcrossing nature of the species. However, the results based on the different FIS-values were very similar and therefore, only those based on FIS = 0 are presented. Allele frequencies were then used to calculate Nei's gene diversity (Hj) and the percentage of polymorphic loci at the 5% level corrected for the sample bias (PPL). Furthermore, band richness corrected for the sample bias (Br) was computed on the AFLP data with AFLPDIV . This mribed in .IS) was estimated from AFLP fragment frequencies using FAFLPcalc [IS values only among our sample populations and within a sampling year and not to those from other studies.The level of inbreeding  were calculated for pooled population samples per height class within each spatial scale. Average inbreeding coefficients (FIS) were compared between young plants (height < 1 m) and older individuals (> 1 m height) of the common juniper populations sampled on the northwestern European scale by a t-test. Finally, we used Spearman rank correlations to identify possible relationships between AFLP-based genetic diversity measures  and population characteristics .In order to identify whether habitat fragmentation resulted in a decrease of genetic diversity in younger (height < 1 m) compared to older individuals (height > 1 m) of the common juniper populations, values of ΦAV, RG, SA, KV designed the research. RG, SA collected field data. AV, KC participated in the molecular genotyping. AV, KC, KV analyzed the data. AV drafted the manuscript. IM helped to draft the manuscript. All authors discussed the results and commented on the manuscript. All authors read and approved the manuscript.


Austropotamobius italicus, the white-clawed crayfish, are threatened. This species was once widely distributed in Spain, but its populations have shown a very strong decline over the last thirty years, due to different factors. Three Spanish populations of this crayfish—from different scenarios—were analysed with nuclear (microsatellites) and mitochondrial markers (COI and 16S rDNA). Data analyses reveal the existence of four haplotypes at mitochondrial level and polymorphism for four microsatellite loci. Despite this genetic variability, bottlenecks were detected in the two natural Spanish populations tested. In addition, the distribution of the mitochondrial haplotypes and SSR alleles show a similar geographic pattern and the genetic differentiation between these samples is mainly due to genetic drift. Given the current risk status of the species across its range, this diversity offers some hope for the species from a management point of view.Spanish freshwater ecosystems are suffering great modification and some macroinvertebrates like  Spain is the country with the greatest biodiversity of Europe, with around 80,000 catalogued taxa. The maritime barrier of the Mediterranean, the land barrier of the Pyrenees in the North, and the country's orographic and climate peculiarities, invest it with unique biogeographic characteristics. Therefore, the country's large number of endemic—specially freshwater—species makes it a biodiversity hot spot .At present, Spanish freshwater ecosystems are suffering great modification at the hands of climate change, environmental degradation, habitat fragmentation, the rise in human demand for water, and a range of human activities. Together, these factors have contributed to a notable increase in the size of Spain's arid and semiarid regions, and to changes in its biodiversity .Austropotamobius italicus was listed as vulnerable in 2008, but in 2010 it has been categorized as endangered [In 2008 more than 80% of Spanish endemisms were reported to suffer some level of threat in the IUCN Red List. At present, the assessments for some of these species have got worse. Among the macroinvertebrates, the crayfish dangered .Austropotamobius italicus was once a cornerstone of Iberian freshwater ecosystems with large populations widely distributed throughout most of the country's limestone basins. Indeed, it was absent in the more western areas, the highest mountain ranges, and the subdesert areas of the southeast and River Ebro valley. The dramatic decline in its numbers all over its Spanish range is the result of a combination of the factors mentioned above, as well as of the introduction of exotic crayfish species and the related spread of crayfish plague (caused by the fungus Aphanomyces astaci). As a consequence, only around 1000 small populations now remain in Spain  occupying marginal areas or short stretches of watercourses usually isolated from the main river systems [ systems .At present, restoration programs are based mainly on translocation of individuals from other natural or farmed populations and are limited by the low number and abundance of existing populations. Besides, it should be taken into account that availability of individuals for restocking purposes needs to be substantially increased by either traditional hatcheries or extensive ponds . These aNotwithstanding, a major goal of such programs should also preserve genetic variability—the basis for viability and future evolution of populations , 8. IndeAustropotamobius italicus populations from Spain. Our previous survey, by random amplified polymorphic DNA (RAPD) fingerprinting, detected a certain degree of polymorphism in some of the populations tested [In this context, our group is conducting a comprehensive study on the genetic variation and its distribution in s tested .Three molecular markers were used in the present study, two of them mitochondrial and the other one, nuclear. This approach combines the advantages of both methods. It is clear that the mitochondrial genome of animals is an excellent target for genetic analysis because of its lack of introns, its limited exposure to recombination, and its haploid mode of inheritance . MitochoTaking into account all above, our aim was to study the genetic variability of three Spanish populations of white-clawed crayfish belonging to three different scenarios—protected area, crashed population, and hatchery.Austropotamobius italicus were collected from three different populations  and 16S rDNA gene. For SSR analysis fifteen individuals from each of the three populations sampled were used.For each population ten individuals were studied employing two mitochondrial markers: cytochrome oxidase subunit I  using the DNeasy Blood and Tissue Kit from Qiagen  and resuspended in Tris-EDTA .μg/mL) and visualized with UV light transilluminator. All the polymerase chain reactions  spectrophotometer. Its integrity was verified by 0.8% agarose gels in Tris-EDTA buffer . Gels were stained with ethidium bromide  were caCOI gene was amplified in a final volume of 50 μL with 25 ng of total DNA, 1x reaction buffer, 2 mM MgSO4, 200 μM of each dNTP, 15 μg of BSA, 1 μM of each primer, and 1 U of Vent DNA polymerase . The primers used were COI Scylla [A fragment from the mtDNA I Scylla  and LCO I Scylla . The opt16S rDNA was performed with the primers 1472 and Tor12sc [μL, contained 25 ng of total DNA, 1x reaction buffer, 2.2 mM MgCl2, 200 μM of each dNTP, 330 μg of BSA, 0.44 μM of each primer, and 1 U of AmpliTaq DNA polymerase .The selective amplification of a segment of the mtDNA  Tor12sc , applyinDouble-stranded amplified products for both mitochondrial markers were purified with the High Pure PCR Product Purification Kit (Boehringer-Manheim) and used as templates for sequencing reactions. These reactions were carried out with the “BIG Dye Terminator Cycle Sequencing Ready Reaction Kit”  on a 3730 DNA Analyzer , using the primers employed for the amplification step, at the Genomic Unit of The Complutense University of Madrid.The SSR study included five loci. All primers used were developed by Gouin et al.: Ap1, Ap2, Ap3, Ap5 reverse, Ap6 , and Ap5μL contained 5 ng of total DNA, 3.25 μL of 2x QIAGEN Multiplex PCR Master Mix, 0.1 μM of Ap1 and Ap6 primers, and 1 μM of Ap2 and Ap3 primers. Amplification conditions were 95°C for 10 min followed by 36 cycles of 94°C for 30 s, 62°C for 60 s, and 72°C for 60 s, with an extension final step of 72°C for 10 min. Ap5 locus was amplified in a final volume of 6.5 μL with 35 ng of total DNA, 1x reaction buffer, 1.9 mM MgCl2, 8 μM of each dNTP, 0.2 μM of forward labelled primer, 0.4 μM of reverse primer, and 0.5 U of AmpliTaq DNA polymerase . The optimal PCR programme for this locus include an initial denaturation step of 94°C for 10 min followed by 15 cycles of 94°C for 30 s, 55°C for 35 s, and 72°C for 50 s, then 25 cycles of 94°C for 30 s, 60°C for 35 s and 72°C for 50 s, finally an extension step of 72°C for 10 min.QIAGEN Multiplex PCR kit  was used to amplify the Ap1, Ap2, Ap3, and Ap6 SSR loci. Reaction with a final volume of 6.5 PCR products were run with the internal size standard GeneScan 500 LIZ  on a 3730 DNA Analyser . Allele size was determined through Peak Scanner Software v1.0 .16S (1317 bp) and COI (1184 bp), were also used together to obtain a single sequence of 2501 bp length. The genetic diversity estimates  were calculated using DnaSP v 5.10.01 programme [The nucleotide sequences of the mitochondrial DNA were aligned using CLUSTAL W software  and editrogramme .FST pairwise genetic distances [istances , which qistances . Principistances  was perfistances  to visuaistances .A), effective number of alleles per locus (ne), the observed heterozygosity (Ho) and the Hardy-Weinberg expected heterozygosity (He) [FIS within each population by the Weir and Cockerham [Genetic diversity was quantified as the mean number of observed alleles per locus (ty (He)  through ty (He) . Genepopty (He)  was emplockerham  method.FST [ST [R-statistics were expected to be larger than F-statistics when stepwise-like mutations have contributed to population differentiation [FST and RhoST estimates.To assess the effects of genetic drift and mutation in the structure of these populations, two statistics of genetic differentiation, FST  and RhoSFST [ST , were cantiation . OtherwiGenetic structure of populations was inferred using the model-based clustering algorithms implemented in STRUCTURE v2.0 . For parIn order to analyse the homogeneity of samples, a correspondence analysis (CA)—on the matrix of allele counts per sample, both at the population and the individual levels—was conducted using Genetix v4.05.2 software . This teCOI gene and 1317 bp from 16S rDNA gene) fragment was obtained from 30 individuals. Sequence analysis revealed four single nucleotide polymorphisms (SNPs), three of them informative under parsimony . The highest haplotype and nucleotide diversity were found in the farmed population (RIL) .FST value revealed significant population differentiation at the mtDNA level (FST = 0.8547). The inferred Nm value was 0.09 that reduces to 0.03 when only the two natural populations are taking into account. The highest FST genetic distances were found between comparisons of NAV population and the other two samples .As a whole, Relationships among these three populations were visualized by the principal component analysis . The firCOI level, GRA and RIL samples shared one of the haplotypes found, although GRA was monomorphic whereas RIL also presented a private haplotype. In addition, NAV population held two different and exclusive haplotypes. Nonetheless, only two different groups were observed at rDNA 16S gene, since a single mutation separated the two haplotypes found. Thus, GRA and RIL shared the same haplotype while NAV sample held the other one.As shown in A total of 45 individuals were analysed through five SSR loci. Four of which were polymorphic—Ap1, Ap2, Ap3, and Ap6—whereas locus Ap5 was monomorphic.The total number of alleles detected for the three Spanish populations was 33. All samples had private alleles—GRA: Ap2: 190, 196, and 200; Ap3: 126, and 192; Ap6: 352, 354, 356 and 368. NAV: Ap3: 178, and 188. RIL: Ap2: 108; Ap3: 150; Ap6: 362 and 380—usually at low frequencies. The number of alleles per locus ranged from 1 (locus Ap5) to 8 (locus Ap6) and their frequencies are shown in A) and the effective number of alleles (ne) while NAV sample showed the lowest ones. In all populations, the average observed heterozygosity (Ho) was lower than average expected heterozygosity (He), mainly in NAV sample where a large homozygote excess was observed. The FIS values, as expected, confirm the results . The highest FST genetic distance was found between GRA and NAV samples (FST = 0.1030) and the lowest between GRA and RIL populations (FST = 0.0518).According to Wilcoxon's signed ranks test, RhoAustropotamobius [The main goal of the present work was the study of the genetic variability of three Spanish samples —located tamobius –39. On ttamobius . In thistamobius , includitamobius .COI gene resulted more sensitive for detecting genetic variability than 16S rDNA. COI is a powerful marker for the study of the genetic variation at the intraspecific level in crayfish [16S rDNA gene [COI and 16S—are discussed together in the present work.With respect to both analysed mtDNA sequences, crayfish , 44 and crayfish –47 becauDNA gene . NotwithDNA gene , the two16S rDNA and COI gene) and nuclear (SSR loci) level, despite the limited number of samples—from different scenarios—analysed in this study.Results indicate that the species exhibits in Spain a certain degree of genetic diversity at both mtDNA , although located in a protected area since 1996, showed the lowest variability at number of alleles per locus and exclusive alleles . These rs astaci . Althouginhabits , 65 so, The hatchery population (RIL) exhibits the highest genetic diversity at mitochondrial level and moderate values for all the SSR variability parameters Tables  and 3. TA. italicus in Spain are affected by successive and drastic bottlenecks and consequently, by the action of the genetic drift, enhanced in these small and fragmented populations. However, ancient historical events such as population fragmentation, recolonizations from refugia during the ice ages [At present, Spanish populations of white-clawed crayfish are in regression due to environmental changes among other factors , 5, 67. ice ages , 50, 68,ice ages –71.Our results underscore the usefulness of employing both mitochondrial and nuclear markers to assess current levels of genetic variability of the populations analysed as well as their genetic structure. Genetic information of the present study should be taken into account for future conservation plans. Though Spanish populations are in decline, a certain degree of genetic diversity has been detected. Given the pattern of the genetic variability found, it would be advisable an increase of within-population heterozygosity without eroding the differentiations that characterize the genetic structure of these Spanish samples. In this way, future works with more samples are needed in order to confirm these results and to provide guidelines about restocking purposes in each area. Hence, the crayfish hatchery analysed in this study (RIL) could be suitable for restocking the Southern population (GRA) but it is not fitted for the Northern one (NAV) that has to be considered as a different management unit , given i