Tetrao urogallus) is a specific bird species, which, despite its very broad distribution and large global population size, is highly endangered in many Western and Central European countries. According to the species situation, in many countries (including Poland), breeding and reintroduction programmes have been started. One of the most complex and large-scale reintroduction programmes was started in Bory Dolnośląskie Forest, and the Capercaillie Breeding Centre in Wisła Forest District was used as one of the sources of individuals for reintroduction. As genetic tools provide essential knowledge about species biodiversity, which is crucially important during the breeding process and reintroduction, both captive and reintroduced grouse populations were genetically analysed. We were particularly interested in genetic diversity of the individuals in both populations and the genetic relationship between them, as well as between them and other capercaillie representatives from their current range. To fulfil these goals we determined nine microsatellite loci along with a fragment of the mitochondrial control region. Genetic diversity parameters were moderate to high compared to populations from other Central and Western European countries. Both populations were clustered into three distinct genetic clades based on microsatellites. Phylogenetic analysis placed all mitochondrial haplotypes we revealed in the Eurasian clade. The present results will play an important role as they will help to preserve and maximize genetic diversity in captive populations, and will provide a basis for future monitoring of the reintroduction process.The Western capercaillie ( Tetrao urogallus) is distributed among Western Palearctic boreal and mountain forests, but in spite of its wide distribution, in many central European countries it is critically endangered [T. urogallus population decline are degradation of habitats due to improper forest management, disturbance of capercaillie habitats and ecosystems, excessive hunting, industrial pollution and increased density of predators. In Poland, like many other central European countries, the Western capercaillie population decline has been severe. From over 2500 individuals at the beginning of the 20th century, currently the Polish T. urogallus population is estimated at 380–500 birds, which are located in four isolated populations: Augustowska Primeval Forest, Western Carpathian, Solska Primeval Forest and Bory Dolnośląskie Forest (BDF) [The Western capercaillie (dangered . The sizdangered . The maist (BDF)  as a criIS = 0.45 in BDF and Western Carpathian (FIS = 0.36)). Rutkowski et al. [T. urogallus restitution programmes were undertaken in several Polish forest districts, and in 2009 one was started in Ruszów in the BDF [Information about the genetic diversity within species, and in particular within critically endangered species, is important knowledge, crucial for proper management and population restoration . Westerni et al.  conclude the BDF .2), where detailed analyses of habitat quality were performed using the Habitat Suitability Index [The number of capercaillie in the BDF decreased from ~360 individuals in the mid-20th century  to 18 bity Index , and thety Index .The majority of individuals reintroduced in Bory Dolnośląskie Forest were reared in the CBC-WFD, which was founded in 2002 and is one of the biggest capercaillie breeding stations in Poland –24.The reintroduction process is complex and its success depends on many factors, including the understanding of species ecology, habitat preparation, the number of reintroduced individuals and the reintroduced population’s adaptive potential . AdaptatT. urogallus from other European countries.To meet the goals of the best practices in reintroduction, we analysed the genetic diversity in both the captive (which was one of the source populations for reintroduction) and the new wild populations. We were interested particularly in: (i) the level of microsatellite and mtDNA diversity in captive and reintroduced populations, (ii) the genetic relationship between capercaillie from the CBC-WFD and the BDF revealed by its individuals genotype clustering, and (iii) the phylogenetic relationships between captive and reintroduced individuals with Tetrao urogallus) and 150 juvenile birds in the CBC-WFD, Poland. The used protocol was approved by II Local Ethics Commission for Experiments Carried on Animals  and authors possess individual permission for carrying the experiments on animals, including capercaillie; Ewa Łukaszewicz—permit: No. 23/2009, Artur Kowalczyk—permit: No. 24/2009, both documents dated on June 19, 2009. During all procedures all efforts were made to minimize animals suffering.The National Forestry in Wisła District obtained permission  issued by the General Director of Environmental Protection, signed by Dr Michał Kiełsznia, for keeping, reproduction and collection of the biological materials for experimental purposes, every year up to 50 adult capercaillie  according to the producer’s manual. DNA from faeces was extracted with the DNA Stool Kit (Qiagen) according to the manufacturer’s instructions modified according to Segelbacher  and Regnwww.softgenetics.com). Then, the results were developed in the package Microsatellite Toolkit for Excel [IS), Genepop 4.1.4 was used [ID), to find matching genotypes and to estimate genotyping errors in faeces samples.To determine the genetic variation within nuclear DNA markers, a polymorphism of nine microsatellite loci was analysed among wild and captive populations. Selected loci were previously described by Segelbacher  and Pieror Excel , in whicwas used . As a cowas used . Furtherwas used . Gimlet was used  was usedAs we wanted to analyse the genetic relationship between CBC-WFD and BDF populations, the Bayesian approach implemented in STRUCTURE 2.3.4 –36 was aT. urogallus from our study and other parts of its Eurasian range, a fragment (757 bp) of the mitochondrial control region (CR) was amplified and sequenced. A modified light primer from Segelbacher et al. [5’-TTGTTCTCAACTACAGGAAC-3’) along with a newly designed primer (CR_R 5’-TCCGACAAGCATTCACTAAA-3’). PCR conditions were as follows: 95°C– 1 min  x 35, 72°C– 5 min. After amplification, DNA was sequenced in both directions using an ABI 3730 sequencer.To determine the genetic variability within the mitochondrial DNA and to infer phylogenetic relationships between r et al.  was usedD), nucleotide diversity (ND) and the number of polymorphic sites.Sequences from both primers were aligned using Bioedit  and a coT. urogallus along with two rooting sequences (Lyrurus tetrix and Tetrao parvirostris)   were carID) was low (4.64 ×10−8), as was the probability that siblings would share the same genotype (8.71 ×10−4). Thus, we treated samples with the same genotype as one individual and, after removing identical genotypes, the remaining 62 individuals were used for further analysis. All duplicates had the same mtDNA haplotypes and were collected in the same locality. The mean scoring error rate across PCRs of faeces samples was 0.07 for allele dropout and 0 for false allele. Across all loci, the mean error rate for faeces samples was estimated as 0.08 and for blood samples results in both replications were the same for all individuals, i.e. we found no evidence for genotyping errors in blood samples.Seventy-two out of 81 (88.9%) faeces samples, after DNA isolation, were of proper quality and sufficient quantity of DNA to reach ≥ 7 microsatellite loci per sample. The probability that two unrelated individuals would have the same genotype  are sex linked and as such should be eliminated from any population analyses of capercaillie. Furthermore, we checked all other loci used in our study for sex linkage but we found no evidence for sex linkage for any other microsatellite locus.In both wild and captive populations, deviations from HWE were found for several loci—four in the CBC-WFD and seven in the BDF . Both ofRealistic results in non-invasive samples are difficult to obtain as genotyping errors always more or less affect their quality. To avoid erroneous interpretation of results, one has to adapt laboratory procedures and quality control to low DNA samples . Even thA: 6.3 vs. 5.3; HE: 0.755 vs. 0.675). On the other hand, average observed heterozygosity was higher in the wild population (0.772 vs. 0.863). Both populations (with both sex-linked loci excluded) were characterized by excess of heterozygotes. What we found interesting, when we analysed both populations without eliminating BG10/BG12 loci, the wild population presented only slightly different diversity parameters, while in the breeding one observed heterozygosity and FIS differed widely . This is probably the result of different sex ratios in the two populations. In the CBC-WFD the male-to-female ratio is 1:2.2. In the reintroduced population the sex ratio is unknown, but in other wild capercaillie populations there is usually a majority of males: 1:1.6 [Genetic diversity parameters, revealed with microsatellites in populations from the CBC-WFD and the BDF, are presented in s: 1:1.6  and 1:1.s: 1:1.6 . That exE = 0.5, NA = 3.4 [E = 0.57, NA = 11.75 [A) and 0.45–0.66 (HE). Finally, a Polish T. urogallus population described by Rutkowski et al. [E equal to 0.585 and average allele number equal to 4.45, while the former BDF population had HE = 0.59 and NA = 5. Thus, diversity parameters presented by us (NA = 5.3 and HE = 0.675) in the newly created BDF population indicate high genetic variation, which may result in adaptive potential necessary for population restoration and survival.When compared to other wild capercaillie populations, the population newly reintroduced in the BDF was characterised by high values of diversity parameters revealed with nuclear DNA. An isolated, Cantabrian capercaillie population presented low genetic diversity . Selecti et al.  revealedControl region amplification revealed the presence of six mtDNA haplotypes in the BDF and seven among the captive founder population  we did . DespiteD = 0.684, ND = 0.003) had lower values when compared to average nucleotide and haplotype diversity indices in three groups of capercaillie analysed by Duriez et al. [Reintroduced population mtDNA diversity indices obtained in our study  and the lowest standard deviation for K = 3. The delta K method confirmed those results and also indicated K = 3 as the most probable number of distinct genetic clusters among all analysed individuals. In the CBC-WFD founder population, three clusters consisted of: i) cluster one—all individuals from the Belarusian lineage  and female 48, which was of Western Carpathian origin but was clustered with individuals from Belarus; ii) clusters two and three—individuals from the Western Carpathian lineage. In both populations (wild and captive) we found the presence of all three genetic clusters . In the The most frequent mtDNA haplotypes in the reintroduced population were haplotypes no. four (43%), eight (32%) and two (18%), while in the captive founder population they were haplotypes no. two (35%), four (17%) and one (12%) . In the Both analysed populations (reintroduced and captive) presented the same genetic structure revealed with microsatellites. Three distinct genetic clusters differed however with frequencies in both populations. Thus, based on microsatellites we may state that both analysed groups of grouse were genetically similar. When we compared both populations with mtDNA, we found much greater disparities. Thirty-four percent of individuals from the BDF had two mtDNA haplotypes (no. eight and nine) not present in the captive population, which suggests that at least 34% of wild individuals were reared or are offspring of females reared outside the CBC-WFD.Both ML and BA phylogenetic trees revealed the same topology showing two distinct clades, Eurasian and Cantabrian , as founst generation offspring derived from the Western Carpathian lineage turned out to have in part or in whole genotypes specific to capercaillie from Belarus. This may be the result of wrong assignment of founder individuals into genetic lineages or an inexact process of egg incubation and chick rearing. As all captive individuals belong to the same phylogenetic lineage, Eurasian, in this case crossing of individuals with different origin did not have negative consequences. But, once more it shows how important it is to implement genetic tools in captive breeding. Genetic based selection provides solid information which will ensure proper selection of animals for breeding and thereby ensure the preservation of a high level of biodiversity in the breeding flock.When we compared pedigree data with genetic based information, we found several inaccuracies. First, some founder individuals, which should (according to breeding documentation) derive from a certain genetic line (Belarusian or Western Carpathian), did not derive from those lines. Second, captive population 1In conclusion, the genetic characteristics of both reintroduced BDF and captive CBC-WFD populations of Western capercaillie showed a high level of diversity. The main objective of captive breeding is to create a self-sustainable population capable of producing progeny that can be further used for reintroduction . Thus, wS1 Table(PDF)Click here for additional data file.S2 TableNames, primers and fluorescent dyes with annealing temperatures are presented.(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S1 FigNumbers along nodes are their Bayesian posterior probability values  and aLRT values .(TIFF)Click here for additional data file.


This review explores the limitations of self-reported race, ethnicity, and genetic ancestry in biomedical research. Various terminologies are used to classify human differences in genomic research including race, ethnicity, and ancestry. Although race and ethnicity are related, race refers to a person’s physical appearance, such as skin color and eye color. Ethnicity, on the other hand, refers to communality in cultural heritage, language, social practice, traditions, and geopolitical factors. Genetic ancestry inferred using ancestry informative markers (AIMs) is based on genetic/genomic data. Phenotype-based race/ethnicity information and data computed using AIMs often disagree. For example, self-reporting African Americans can have drastically different levels of African or European ancestry. Genetic analysis of individual ancestry shows that some self-identified African Americans have up to 99% of European ancestry, whereas some self-identified European Americans have substantial admixture from African ancestry. Similarly, African ancestry in the Latino population varies between 3% in Mexican Americans to 16% in Puerto Ricans. The implication of this is that, in African American or Latino populations, self-reported ancestry may not be as accurate as direct assessment of individual genomic information in predicting treatment outcomes. To better understand human genetic variation in the context of health disparities, we suggest using “ancestry”  to describe actual genetic variation, “race” to describe health disparity in societies characterized by racial categories, and “ethnicity” to describe traditions, lifestyle, diet, and values. We also suggest using ancestry informative markers for precise characterization of individuals’ biological ancestry. Understanding the sources of human genetic variation and the causes of health disparities could lead to interventions that would improve the health of all individuals. International HapMap Project was the first multi-institutional effort to catalog variations and develop a haplotype map (HapMap) of the human genome. The HapMap project had identified over 5 million SNPs in the human genome including their distribution among people in different parts of the world [http://www.1000genomes.org/). This haplotype resource at finer scales will facilitate the understanding of genetic variation at genomic and geographic levels [The human genome is composed of over three billion bases of DNA and contains between 25,000 and 30,000 protein-coding genes . On averc levels .alleles. Alleles with a frequency greater than 5% are called common variants, those with a frequency of 1%–5% are low frequent variants and those less than 1% are rare variants. Because rare variants might have arisen after populations diverged or occurred in recent human history, they are more likely to be population-specific and, therefore, they may not be shared with different populations. Thus, the overrepresentation of rare causal variants in certain population could explain the observed differences in disease prevalence, including asthma [Because of their sheer number, SNPs are the major sources of genetic and phenotypic diversity, accounting for 95% of all known sequence variations [g asthma .There are two potential reasons why some variants are relatively common in one population but absent (or nearly so) in another: a) a recent emergence of a variant that has not yet had time to spread to other populations and b) natural selection in a specific local environment. An example of the first scenario is a SNP that causes hereditary hemochromatosis, which is common in Europe but very rare elsewhere. Lactase persistence is an excellent example of the influence of natural selection on allelic frequency. Lactase persistence into adulthood is prevalent in Somali camel herders from Ethiopia where milk consumption continues beyond childhood . PositivAnatomically, modern humans first appeared in Africa some 150,000 to 200,000 years ago . About 6Race and ethnicity are widely used interchangeably in population research and incorporate cultural, linguistic, biological, and geopolitical factors . Althougr = 0.585, P < 0.001) than those in Fortaleza  [The classic mirror reflects skin color; but the DNA mirror reflects our common ancestors” [Although on average, populations that are geographically close to one another show stronger correlation and higher genetic similarity than geographically separated populations and substantial differences in allele frequencies are also observed within geographic regions . Several< 0.001) . Thus, scestors” .Table 1Empirically speaking, ancestry is estimated using ancestry informative markers (AIMs), which are a set of genetic variations for a particular DNA sequence that appear in different frequencies in populations from different regions of the world. The use of AIMs compares an individual’s polymorphisms at these markers with previously analyzed genomic reference sets from people whose ancestral history is fairly well known. AIMs are used to estimate the geographical origins of an individual’s ancestors, typically expressed as proportions of one’s ancestry that comes from different continental regions .Availability of genetic markers that are ancestry-informative and newly developed statistical methods may overcome concerns regarding race/ethnicity categorization . There iAutosomal DNA (testing both sexes) markers: autosomal DNA tests utilize DNA from the 22 pairs of autosomal chromosomes. Autosomal DNA is inherited from both parents. Autosomal testing provides percentages of ethnicity using autosomal DNA SNP test , and it is the most commonly used test to infer ancestry across diploid genome.Y-DNA or Y-SNPs  markers: a haploid Y-DNA is the paternally inherited non-recombining portion of the Y chromosome, and it tests only for males. The Y-DNA testing tests the Y chromosome which is passed intact from father to son with no DNA from the mother. Y-DNA testing can then be used to trace direct paternal line. Y-DNA remains the same in each generation, allowing us to compare surname from different regions to see if we are from the same family. Y-line testing does not indicate anything about the contributions of the other ancestors in a family tree. In other words, you could be 3/4th Native American, with only the direct paternal line being European, and this test would tell you nothing at all about those other three Native lines. When testing the Y-chromosome, there are two types of tests, short tandem repeat (STR) and SNP markers. STR tests are best for recent ancestry while SNP tests tell about more ancient ancestry.Mitochondrial DNA  markers: mitochondrial DNA or mtDNA haploid is the maternally inherited mitochondrial genome (mtDNA) . All chiX chromosome (X-DNA testing) markers: an X chromosome DNA test looks at markers on X chromosome(s). Males have one X chromosome that they inherit exclusively from their mother, and females have two X chromosomes that they inherit from both parents, one from their father and one from their mother. This creates a unique inheritance pattern that may provide many insights into one’s maternal heritage. STR markers on the X chromosome have been used in population genetic studies and forensics.Although autosomal SNPs are commonly used as genetic markers to infer ancestry or race/ethnicity membership, haploid such as mitochondria, Y-DNA, and X-lined markers are also important to provide separate stories of ancestry of individuals from paternal and maternal sides ,43. TherThere are two main benefits in using haploid (Y-DNA and mitDNA) markers over diploid  markers: 1) they lack recombination. This allows for more easily recoverable phylogenies than is possible for the autosomal markers, allowing for the easier identification of geographically restricted clades, which could be indicative of past historical migration. The second benefit in using the sex-specific systems is their 2) much small effective population size related to autosomal markers due to their haploid mode of inheritance through one sex only. Genetic diversity of present-day American populations is very complex due to the demographic events that resulted in extremely admixed populations . ThroughAlthough variation in humans reflect genetic differences at single allele as well as haplotype level, most local ancestry estimators use allele frequency data (locus-by-locus) between parental contributions along the chromosome, ignoring molecular information that is available in haplotype block structure. Individual mutations carry only weak signals about population ancestry. By adding information across the whole genome at haplotype level, we can reconstruct these admixture events more accurately. It has been described that less than 50% of admixture is hard to detect from single locus (or non-recombining genome) data. The power of detecting ancestry switch points between European and African ancestry per person becomes feasible as more and more loci are identified . This apIn a founder population, patients with a genetic disease are likely to share predisposing genes from a common ancestor. Depending on the distance of the relationship, patients are expected to share extended segments of DNA around the disease gene, thus the extent of linkage disequilibrium (LD) between the disease and the surrounding marker (about 1 cM) is small enough to be meaningful and large enough to be observed. Because of the size of the shared segment, a genomic search with DNA markers for such regions can efficiently locate the map position of genes using identity by descent (IBD) mapping . IBD mapLimitations related to ancestry markers include the reference sets, which are comprised of the genomes of relatively few sampled individuals who are themselves from a relatively few, geographically restricted regions. Thus, to what extent is a panel derived by contrasting a “Yoruban” sample with “Europeans” appropriate for use in African-American samples? How much is the Yoruban population represents Africa and hence African Americans are debatable . HoweverClinical asthma outcome variables such as pulmonary function tests (PFTs) include forced vital capacity , forced expiratory volume in 1 s , and FEV1/FVC ratios. The variation in ancestry in relation to these clinical predictive variables may help to explain differences in disease phenotypes among ethnic subgroups. Recent study showed that in Mexican Americans, European ancestry was associated with more severe asthma, as measured by FEV1, a quantitative measure of lung function. A decrease of 1.7% baseline FEV1 was observed per 10% increase in European ancestry . FEV1 isp = 0.007), FVC (p = 0.0003), and FEV1/FVC (p = 0.035) (Table p = 0.001) and an 11.8-ml decrease in FVC (p = 0.0001). Higher African ancestry was associated with a greater likelihood for an asthma-related physician visit (p = 0.004) and greater frequency of urgent or ED visits among asthmatics treated with an inhaled glucocorticoid (p = 0.01). In African Americans with more severe asthma, the magnitude of decreased lung function associated with African ancestry was twice that observed in the general population . These http://www.p3g.org/secretariat) promotes collaboration between researchers in the field of population genomics to ensure public access to population genomic data. These resources typically include biological samples  linked to structured clinical databases  data) [To increase sample sizes and power, several research groups are implementing meta-analysis to combine multiple consortia projects. The recently established Public Population Project in Genomics (P3G) (R) data) ,66 in a R) data) . As the R) data)  analyzedR) data) . Thus, aRecent advance in high-resolution genome-wide genotyping allow the inference of genetic or “biogeographical” ancestry using empirical description of individuals and populations . In detestructure [FST measure [FST analysis, one could reliably identify subpopulations within major geographic regions  that exhibit lower or higher pairwise FST . For populations of complicated admixture or unknown origins, a large number of loci with high resolution need to be genotyped, followed by principal component analysis (PCA) to individual-level genetic data. PCA can detect the presence of population mixture and admixture in a sample and thus can be used to determine the axis of variation in different dimensions based on biogeographical ancestry. Adjustment made using PCA approach increases investigator confidence that genetic association findings are not spurious due to stratification. Finally, characterization of culture, socioeconomic status, and environment should be made in disease genetic study, otherwise any or all “racial/ethnic” differences in disease risk factors can erroneously be attributed to presumed population genetic differences. Methods such as mixed model regression could help investigate the genetic and non-genetic risk factors. The failure to account ancestral background can thus prevent proper characterization of the genetic structure of a given study population, leading to inaccurate prediction of outcome as well as incorrect inferences about the evolutionary factors driving patterns of diversity [Studies showed that extrapolation of genomics data from genetically homogeneous to genetically structured populations could generate large numbers of false positive and false negative results . Populattructure . Using atructure . To inve measure . From thiversity .There are two major questions to answer before applying race/ethnicity category in biomedical research. First, is race/ethnicity a valid and reliable approach to ascertain individual ancestry? If so, should race be considered by those who study diseases and patient responses to treatment? Second, how do we define  race/ethnicity in the context of biomedical research? In general, people self-report their population origin correctly in terms of major population descriptors . However, these descriptions are not good indicators of the genetic composition of individuals, since genetic makeup of individuals are highly heterogeneous, and can be captured only with large dimensional genomic data. Genetic ancestry estimation at the individual level is bringing us closer to more personalized or individualized genetic-based medicine . Genomichttp://www.genome.gov/gwastudies/). Since most GWAS studies are done in populations of European ancestry, we examined the allele frequency patterns of 78 GWAS SNPs associated with asthma and deposited at the GWAS Catalog site. We used 1000 Genomes Project (http://www.1000genomes.org) and AncestrySNPminer  to explore these variants among African American (ASW), African (YRI), and European American (CEU) populations. Although further studies are required to determine the extent to which this variation is responsible for differences in asthma prevalence, the admixed AA population (ASW) exhibited allele frequencies that appear intermediate in relation to the ancestral CEU and YRI populations  and pharmacogenetic studies have been primarily performed in cohorts of European descent . In Euros Figure .Figure 5Large consortium-based next-generation sequencing studies such as the NIH/HLBI GO Exome Sequencing Project, the Consortium on Asthma among African ancestry Populations in the Americas (CAAPA), and the 1000 Genomes Project are currently using next-generation whole exome and whole genome sequencing studies to provide diverse genomic information from different admixed populations . These lCYP2A6 gene in Maori compared to that in Caucasians [Unlike self-reported race-based health disparity studies, which represent a combination of both genetic and environmental background , ancestrucasians . An admiucasians .Figure 6As the world becomes multiethnic, and intermarriage between different racial/ethnic groups gets more and more common , it is ihttp://www.fda.gov/Drugs/DrugSafety/ucm123927.htm). Abacavir is used to treat human immunodeficiency virus (HIV) infection. Patients with the HLA-B*5701 allele have a higher risk of developing a hypersensitivity reaction. Furthermore, several medication dosing algorithms around the world are now being developed using the patient’s own genotype data [Although knowledge gained in genomics has advanced our understanding of biology, the promise of personalized medicine continues to appear far off for minority and admixed populations. For example, recently, pharmacogenomic information has been added to over 70 drug labels , but theype data ,102,103.Although conceptual distinction between race/ethnicity and ancestry is widely recognized -106, it Given higher genetic diversity within races than between races, the use of race/ethnicity as a dissimilarity marker is misleading ,108. Gen


A total of sixteen haplotypes were detected. There was a common haplotype in each basin, that was haplotype A in Tarim Basin, haplotype G in Junggar Basin, and haplotype M in Qaidam Basin. Genetic variance mainly occurred among populations, geographic regions, and eleven geographic groups subdivided by SAMOVA analysis. E. przewalskii likely had a smaller and more fragmented geographic range during the Last Glacial Maximum, which was determined based on ecological niche modelling. Three groups of E. przewalskii populations were detected to have experience range expansion, and this was based on significant values of Fu’s FS, Tajima’s D, and unimodel mismatch distributions. The cold and dry climate during the glacial period of the Quaternary is postulated to have been a driver for significant genetic isolation and divergence among populations or groups in E. przewalskii, whereas the warmer and wetter climate during the interglacial period is speculated to have provided favourable conditions for range expansion of the species.Based on two chloroplast DNA sequences,  Junggar Basin, which is the second biggest interior basin and covers approximately 380,000 km2, is surrounded by Tianshan Mountains on the south, Altai Mountains on the north, and Alatau and Tarbagatai Mountains on the west. Junggar Basin is separated from Tarim Basin by Tianshan Mountains. Hami Basin locates in the east of Tianshan Mountains, and connects with Tarim Basin, Junggar Basin, and Hexi Corridor. Qaidam Basin, the third biggest interior basin in northwestern China, covers ca. 250,000 km2 and is relatively closed and surrounded by Kunlun Mountains in the south, Altun Mountains in the northwest, and Qilian Mountains in the north.The vast arid northwestern China comprises the entire Xinjiang Province, Hexi Corridor, Qaidam Basin, and western parts of Helan Mountains in Inner Mongolia (Alxa Dersert) . In thisAmmopiptanthus mongolicus (Masxim. ex Kom.) S.H. Cheng, Tetraena mongolica Maxim., and Ephedra przewalskii Stapf, are typical elements of the flora. In the Quaternary, pollen records provide evidence that glaciation was not present in deserts of the region, except in the intervening mountain ranges [In arid northwestern China, most plants groups are relicts, ancient, single-species or few-species, and endemic to the gobi (stony deserts) and deserts. Desert species such as n ranges ,3. Howevn ranges ,5. This n ranges ,7, region ranges ,9, and cEphedra przewalskii is a drought-tolerant shrub inhabiting extremely dry semi-deserts, piedmont, and gravelly saline soil, usually forming a large area within the plant community. It is usually an indicator species in plant communities of the deserts in northwestern China, and due to its powerful resistance to drought and sand burial, it is commonly used as a sand binder [d binder . It is pd binder .Ephedra species distributed in Qinghai-Tibet-Plateau (QTP) and adjacent arid regions has been studied, and three main lineages were found and systematic positions of some species were confirmed among members of the genus [E. przewalskii, an ancient species with an extensive geographic range, the information concerning genetic structure and phylogeography patterns across the whole distribution range in China was still unclear, and the following questions concerning this species have not been investigated: 1) genetic differences among the populations, 2) allopatric divergence among the basins, 3) and detection of range expansion throughout the species. These questions will provide a greater better knowledge to out understanding of the evolutionary history of plants in arid northwestern China.The phylogeographical history of he genus . HoweverChloroplast DNA (cpDNA) in plants is inherited maternally, and evolves slowly with low recombination and mutation rates ,13, so iEphedra przewalskii has woody stems, and is usually 0.5–2.4 meters high. The cone is usually sessile, and crowded into a group, opposite or whorled on the knot. The male cone is hazel, nearly spherical, with 7–8 stamens. The unique morphological feature of E. przewalskii, by which it differs from the other species of Ephedra in China, is that, when female cone mature, the bracts are increscent but not fleshy, dry, colourless, semi-lucent, and membranous. We sampled a total of 469 individuals from 45 populations, covering almost the entire geographic range of the species in China. Of the sampled populations, one (population 1) was from Urumqi, one (population 2) was from Turpan Basin, nineteen (populations 3–21) were from Tarim Basin, eight (populations 22–29) were from Junggar Basin, one (population 30) was from Hami Basin, seven  were from Hexi Corridor, two (populations 34 and 35) were from Alxa Desert of Inner Mongolia, and six (populations 39–44) were from Qaidam Basin  and Taberlet et al. (1991) respectively [2, 200 mM dNTP, 1 pmol primer, and 0.025 U mL-1 Taq polymerase (Takara), and the temperature profile of the amplification process is: 95°C for 1 min; 30 cycles of 94°C for 30 s; 52°C for 30 s; and 72°C for 1 min 30 s linked to 72°C for 10 min. Forward and reverse primers of the PCR amplification were also used in sequencing, conducted by the DYEnamic ET Terminator Kit (Amersham Pharmacia Biotech) at the Shanghai Sangon Biological Engineering Technology & Services Co., Ltd  with the use of an ABI-PRISM 3730 automatic DNA analyzer. Electropherograms were edited and assembled using SEQUENCHER, version 4.8. Sequences were aligned with CLUSTAL W [Total genomic DNA was extracted from dried leaf tissue using a modified 2 × CTAB method ,22. Afteectively ,24. Poly−9 substitutions per site per year (s/s/y) [−9 and a SD of 6.080×10−10 within the 95% range of the distribution to estimate the divergence times [Ephedra monosperma and E. major as the outgroups of haplotypes of E. przewalskii, and for the network, we used E. monosperma as the outgroup.Phylogenetic analysis of the resulting alignments was carried out by maximum-likelihood (ML) in PAUP* version 4.0b10 , to inve (s/s/y) . Becausece times . All parce times , and thece times  implemence times . For theHS), total gene diversity (HT), genetic differentiation (GST), and population subdivision of phylogenetically ordered alleles (NST) were estimated using HAPLONST. Spatial analysis of molecular variance was performed using SAMOVA, version 1.0, to test the spatial genetic structure, defining groups that are geographically homogeneous and genetically differentiated [K = 2 to 44, with 100 random initial conditions for each run. Analyses of molecular variance (AMOVA) at different levels were performed to study the genetic structure of the populations [Within-population diversity  [τ is the total number of mutations, and u is the mutation rate per generation for the whole analyzed sequence. The value of u was calculated as u = 2 μ·k·g, where μ is the substitution rate per nucleotide site per year (s /s/y), k is the mean sequence length of the analyzed DNA region, and g is the generation time in years. We again used a mean substitution rate of 2.0 × 10−9 to estimate the expansion times of SAMOVA groups. According to our initial observations, we assumed the generation time of this desert species to be 4 years.Tajima’s pansions ,38,39,40xpansion ,42,43,44Ephedra przewalskii populations included in this study , and for the trnT–trnF spacer, the length was 656 bp. A total of 12 informative characters were detected in the aligned sequence data: 7 nucleotide substitutions  and 5 indels . From the 469 sampled individuals of 45 populations, a total of 16 haplotypes (A–P) were identified  was raised from 2 to 11. The subdivision pattern of populations corresponding to K = 11 was represented in In the spatial analysis of molecular variance, HS) was 0.161 (SE 0.0335), and total gene diversity (HT) was 0.800 (SE 0.0342). Differentiation among populations was high , indicating populations were highly structured. NST  was significantly higher than GST as shown by the U-test , demonstrating a significant phylogeographic structure. AMOVA analysis showed that 86.21% (P<0.001) of the total variation occurred among populations, 46.05% (P<0.001) of that occurred among geographical regions, and 87.99% (P<0.001) of that occurred among SAMOVA groups , that was sister to the second clade including all other haplotypes except E and F, with a moderate support value (64%). The second clade consisted of an inner monophyletic clade  and two paraphyletic clades, haplotype D and P. In the inner monophyletic clade, haplotypes A, B, C, G, H, I, J, K, and L clustered into a clade with a moderate support value 0.67, and haplotypes M, N, and O clustered into a clade with a moderate support value 0.78. Divergence times at node 1 was estimated at 0.73 Mya (early-middle Pleistocene).Trees produced by ML and Bayesian analyses had the same topology, and thus only the ML tree was presented . In thisThe test AUC value for the ENM was very high (0.997), and the predicted range  represenEphedra przewalskii was high compared with other desert shrubs in northwestern China, such as Reaumuria soongarica (HT = 0.607) and Ammopiptanthus mongolicus (HT = 0.434) [E. przewalskii in Qin et al. (2013) [E. przewalskii probably originated in the late Tertiary [E. przewalskii spread across almost the entire arid zone in northwestern China. The habitats varied in geology and topography, might harbour locally adapted genetic variations of the species. All these might help account for the high level of genetic diversity observed in the species.Total gene diversity of = 0.434) , 51, and. (2013) . E. przeTertiary , and theTertiary ,54. E. pHigh total genetic diversity but low within-population genetic diversity resulted in significant genetic differentiation among populations, which was also supported by AMOVA analyses. Several factors might account for the high level of genetic differentiation among populations and geographic groups. First, numerous geographic barriers within the distribution range probably promote vicariant processes of the species. Populations in Tarim Basin were separated from those in Qaidam Basin by Kunlun Mountains and Altun Mountains, and separated from those in Junggar Basin by the extensive Tianshan Mountains. Thoughout Tarim Basin, populations and groups can be far from each other and separated by a vast desert. The high mountains and large desert expanse might obstruct gene flow and increase heterogeneity among populations and geographic groups. Second, we detected range expansion occurred in some subdivided geographic groups of the species. Repeated founder effect during range expansion could make some alleles fixed in populations, consequently reduced heterozygosity within populations and increased heterogeneity between populations ,56.As shown by the genetic variation distribution in Ephedra could be dated to the Middle or Late Miocene, and this was very likely linked to the uplift of the QTP and Asian aridification. The climate in northwestern China became cold during the Quaternary [In northwestern China, the generally dry climate could date back to the late-Miocene, and aridification promoted the formation of salt mineral resources in Qaidam Basin ,58. Sincaternary , and duraternary ,63. Fromaternary . When itHippophae tibetana Schltdl. and Aconitum gymnandrum Maxim. distributed in the QTP, with the major lineages diverging in the early Pleistocene as the cooling climate drove speciation [Reaumuria soongarica  Maxim. and Lagochilus Bunge ex Benth., distributed in northwestern China [Ephedra przewalskii , (b) group (6) of E. przewalskii that includes populations 22–24, 27–29 , (c) group (10) of E. przewalskii that includes populations 36–44 , and (d) all samples of N. roborowskii .Mismatch distribution analysis for chloroplast DNA data for (a) group (1) of (EPS)Click here for additional data file.S1 TableFigures in the parenthesis represent the number of the haplotypes.(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.


Fucus radicans, endemic to the Baltic Sea, has separate sexes, but new attached thalli may also form asexually. We mapped the spatial distribution of clones  over macrogeographic (>500 km) and microgeographic (<100 m) scales in the Baltic Sea to assess the relationship between clonal spatial structure, sexual recruitment, and the potential for natural selection. Sexual recruitment was predominant in some areas, while in others asexual recruitment dominated. Where clones of both sexes were locally intermingled, sexual recruitment was nevertheless low. In some highly clonal populations, the sex ratio was strongly skewed due to dominance of one or a few clones of the same sex. The two largest clones  were distributed over 100–550 km of coast and accompanied by small and local MLGs formed by somatic mutations and differing by 1–2 mutations from the large clones. Rare sexual events, occasional long‐distance migration, and somatic mutations contribute new genotypic variation potentially available to natural selection. However, dominance of a few very large (and presumably old) clones over extensive spatial and temporal scales suggested that either these have superior traits or natural selection has only been marginally involved in the structuring of genotypes.In dioecious species with both sexual and asexual reproduction, the spatial distribution of individual clones affects the potential for sexual reproduction and local adaptation. The seaweed  Asexual reproduction results in genetically identical offspring, but single‐locus differences can be contributed by somatic mutations  are produced from the original parental individual and together they are part of the same genetic unit (the genet), making the primary outcome of clonal growth an increase in the size of the genet  the phalanx strategy, in which aggregated structures with genetically identical ramets are clumped together forming discrete clones in which ramets of other genets are more or less excluded; and (2) the guerrilla strategy, in which genets are intermingled through a more efficient spread of ramets, resulting in clones being spatially mixed and less discrete  origin from Baltic populations of F. vesiculosus  to almost full oceanic salinity outside the Danish straits. In the northern parts of the Baltic Sea, the two brown macroalgal species The mixed mode of reproduction will influence the spatial genetic structure, and at small spatial scales, this will affect the opportunities for sexual reproduction of individual plants and thus the species' potential to evolve local adaptation by forming new genotypes from recombination of existing ones   spatial distribution of clones identified from microsatellite genotypes in the dioecious seaweed s) Wulff . We usedWe furthermore mapped the macrogeographic distribution of clones over all 16 populations to find out whether clones were generally local or widespread. Using the microsatellite information, we also inferred which clones (MLGs) were likely of close ancestry (genotypes differing by 1–2 mutations only) and considered these to be members of the same multiclonal lineage (MLL)  from three populations  close to the northern distributional limit of the species along the Swedish coast of the Bothnian Sea , and distance along the tape for each sampled thallus. Samples were taken from between 0.5 and 6.0 m depth at three different sites; Swe N and Swe O were separated by 135 m, and Swe P was 20.1 km from the two others. For this microgeographic analysis, a total of 198 thalli were analyzed: 60 from Swe N, 79 from Swe O, and 59 from Swe P.For the microscale study, tissue from d July 200 from thIn the macrogeographic analysis, 13 additional populations were analyzed including samples from northern Finland (Bothnian Sea), eastern Sweden (Bothnian Sea), and Estonia   according to standard kit instructions. Samples were amplified and genotyped at nine microsatellite loci  were mechanically cleaned of epiphytes to reduce contamination and stored separately in plastic bags with silica gel to avoid DNA degradation. Dried tissue was pulverized in a milling instrument. DNA was extracted using the NucleoSpinHO and HE), deviations from Hardy–Weinberg equilibrium (HWE), and linkage disequilibrium (LD).The new sets of microsatellite data  or disperse  . Tests were also performed to check whether the number of sampled units (n) per area gave a good estimate of the clonal diversity of the area sampled. Alternative methods were used to approximate clonal diversity within the three localities such as genotypic richness, R = (G−1)/(N−1), where G is the number of multilocus genotypes and N is the sample size , or from sampling two thallus of the same genotype originating from two distinct sexual events. To separate these hypotheses, we determined the probability that thalli with identical genotypes derived from separate sexual reproductive events , and with previously published data from 10 localities across the known distributional range of FST) on genet level was obtained from GENEPOP. Using the web‐based program IDBWS . We used two different datasets: one including all genets from all 16 localities, and one in which we removed the four populations that differed the most (the two from Estonia and two southern Finnish populations) to increase the power of resolution among the remaining 12 populations. The optimal number of subpopulations was calculated for each analysis, where the K with the highest probability was indicated by the lowest Pr(X¦K) value . Multilocus lineages (MLLs) were manually created by grouping MLGs that differed by one or two mutations from a central (large) MLG, or differed by maximum one mutation from an MLG already connected to the central MLG. Also singletons  that differed by 1 or 2 mutations from a clone were considered when building MLLs.GENCLONE 2.0 was used to assign each thallus to a certain MLG and estimate the probability that each MLG was a true clone consisting of thalli originating from the same sexual event (HO and HE) as in populations surveyed earlier in this area of the species' distribution  to 1 (L94). Three loci  showed significant (P < 0.05) departures from Hardy–Weinberg equilibrium (HWE) in all six localities using GENEPOP 4.2. In Swe P, four additional loci  also deviated from HWE. Most loci showed a significant heterozygote excess, but in locus Fsp3 the deviation from Hardy–Weinberg was due to a heterozygote deficiency in Swe N and Swe O. Overall, results were similar when running separate analyses excluding different loci one by one.A total of 36 alleles were identified in the six new localities, and the average number of alleles per locus was 1.3–4.5, ranging from 8 (Ac) was generally low (0–0.2), although significant in two of the three populations (Table Ee), which may result in clonal diversity being somewhat overestimated . Two female clones were common in all three populations , and overall, there was an isolation‐by‐distance effect at the macrogeographic scale , although mostly driven by differences at the largest spatial scale (countries). The population structure analysis showed that a division into K = 6 clusters was most strongly supported (highest Pr(X¦K) value); in addition, some localities in close proximity differed substantially from each other , while the opposite was also sometimes true . When we reanalyzed the weakly differentiated populations separately (excluding the two southernmost Finnish populations and the two Estonian ones), K = 3 clusters were instead supported . This indicated that there were three genetic groups present in the north and west Bothnian Sea and that all 12 populations contained components of 2–3 of these groups . A majority of the study populations were genetically different in pairwise comparisons (see Psex < 0.05), and it was therefore concluded that all ramets with identical MLG shared a common origin and belonged to the same clone. Moreover, many of the small, local clones and individuals of unique MLGs (singletons) differed by 1–2 somatic mutations from a larger clone and were identified as members of large clonal lineages  will more likely include widespread individuals of the same clone  of the clones were geographically spread, and of these, the large female clone was outstanding. The many local mutant clones derived from this clone strongly suggest that this is a very old clone. It seems indeed likely that much of the species' distribution along the Swedish and northern Finnish coasts of the Bothnian Sea   and this showed an obvious change in the composition of genotypes. One MLL  not present at all in the first sampling had a frequency of 30% in the second sampling Fig. . Indeed,F. radicans gametes, zygotes, adventitious branches, and thalli all have negative buoyancy  and geographic distance (km). Scatter plot A shows pairwise FST versus geographic distances of 16 localities of F. radicans in the Baltic Sea showing significant correlation between geographic and genetic distance (P = 0.0001). Scatter plot B shows the pairwise FST versus geographic distances without the two Estonian populations (P = 0.001).Figure S2 A‐B. STRUCTURE analyses of nine microsatellite genotypes of genets from 16 sites  resp. 12 sites  of F. radicans in the Baltic Sea. Each vertical bar represents one individual. Fraction of color in an individual represents its estimated membership to a certain genetic group. Black lines with arrows separate the different localities. Sampling sites are labelled below the figure and region labelled above. K (number of groups) is 6 for S2 a, and 3 for S2 b, which was supported by values of Pr(X¦K).Figure S3 A‐C. The Pareto distribution analysis describes the frequency distribution of clonal membership for the micro‐geographic localities of F. radicans. A skewed distribution with very few, large clonal lineages containing the majority of ramets, will result in a shallow slope, as seen here.Table S1 Pairwise FST‐values of F. radicans.Click here for additional data file.


Araucaria are endemic to this territory. Their phylogenetic relationships remain largely unresolved. Using nuclear microsatellites and chloroplast DNA sequencing, we focused on five closely related Araucaria species to investigate among-species relationships and the distribution of within-species genetic diversity across New Caledonia.New Caledonia harbours a highly diverse and endemic flora, and 13 (out of the 19 worldwide) species of montana and A. laubenfelsii that were not differentiated and, at most, form a genetic cline. Given their apparent morphological and ecological similarity, we suggested that these two species may be considered as a single evolutionary unit. We observed cases of nuclear admixture and incongruence between nuclear and chloroplast data, probably explained by introgression and shared ancestral polymorphism. Ancient hybridization was evidenced between A. biramulata and A. laubenfelsii in Mt Do, and is strongly suspected between A. biramulata and A. rulei in Mt Tonta. In both cases, extensive asymmetrical backcrossing eliminated the influence of one parent in the nuclear DNA composition. Shared ancestral polymorphism was also observed for cpDNA, suggesting that species diverged recently, have large effective sizes and/or that cpDNA experienced slow rates of molecular evolution. Within-species genetic structure was pronounced, probably because of low gene flow and significant inbreeding, and appeared clearly influenced by geography. This may be due to survival in distinct refugia during Quaternary climatic oscillations.The species could be clearly distinguished here, except A. The study species probably diverged recently and/or are characterized by a slow rate of cpDNA sequence evolution, and introgression is strongly suspected. Within-species genetic structure is tightly linked with geography. We underline the conservation implications of our results, and highlight several perspectives.The online version of this article (doi:10.1186/s12862-014-0171-6) contains supplementary material, which is available to authorized users. Ther. Ther5])New Caledonia is a biodiversity hotspot  with morAraucaria Juss. (Araucariaceae) and were both interested in among-species relationships and the distribution of within-species genetic diversity across New Caledonia. The genus Araucaria comprises a total of 19 species worldwide, 13 of which are endemic to the archipelago. They usually occur as large populations, in a variety of ecological habitats . Import maquis; – except e season .Araucaria species form a monophyletic group and, using AFLP markers, Gaudeul et al. [A. montana, A. laubenfelsii Corbasson, A. rulei F.Muell., A. biramulata J. Buchholz, A. muelleri (Carrière) Brong. & Gris and A. humboldtensis J. Buchholz. In addition, the distinction of A. montana and A. laubenfelsii –described in 1871 and 1968, respectively [A. montana being found in the north while A. laubenfelsii is restricted to some localities in the south. As for many New Caledonian plant species, several Araucaria populations/species are threatened by habitat destruction [Araucaria trees are very long-lived  and hablumnaris ,41,42. TA. montana and A. laubenfelsii (which may form a single species), A. rulei, and A. scopulorum . Also, some species sometimes grow sympatrically and we suspected introgression, which may account for the difficulty to resolve the species phylogeny. Since A. biramulata may be involved in such events based on our field observations , it was also surveyed here. Ancient hybridization events and introgression may be detected through genetic admixture at nuclear loci  more broadly, to estimate the extent of among-species genetic variation and give some insight into their relationships; iii) to search for possible signs of introgression between species; iv) at the within-species level, to examine the spatial structure of genetic variability in order to infer the history of populations and major processes influencing their evolution .We examined the genetic structure of four species that are largely distributed in New Caledonia: see e.g. ), or thrsee e.g. ). The resee e.g. ,43-45 ansee e.g. ,46. HybrWe surveyed genetic variation on an extensive population sampling, using both nuclear (nDNA) microsatellite loci and chloroplast DNA (cpDNA) sequencing. Whereas the nuclear genome is diploid and biparentally inherited, the chloroplast genome is effectively haploid and uniparentally, paternally inherited in gymnosperms. Therefore, both genomes are dispersed through seeds and pollen but the chloroplast genome has a smaller effective population size, which makes it more sensitive to genetic drift and population/species differentiation . MoreoveA. montana, A. laubenfelsii, A. biramulata and A. scopulorum. Araucaria montana was sampled both on non-ultramafic (in Mt Panie and TonNon) and ultramafic soil substrates . The natural distribution of A. scopulorum is characterized by a disjunction between northwestern and eastern populations. In contrast, some (unsampled) A. rulei populations occur between the northernmost and more southern localities surveyed.Plant material was collected from 1999 to 2008 , whereas five samples were identified as A. montana; ii) in Mt Tonta, trees were identified and considered here as A. biramulata but they appeared morphologically somewhat intermediate between A. biramulata and A. rulei, although the ecological characteristics were more typical of A. biramulata.In two cases, the taxonomic identification was uncertain: i) in Pandop, close inspection of  herbarium vouchers identified the species as A. muelleri and A. humboldtensis, as well as of A. columnaris and A. nemorosa that belong to the third Araucaria clade , and 0.5 U Taq polymerase . The reaction profile was: 40 cycles of 30 s denaturation at 95°C, 30 s annealing and 1 min elongation at 72°C, followed by a final elongation step of 10 min at 72°C. Annealing temperature was 50°C for As167 and As190, and 52°C for As25 and As93. For Aru1, annealing was initiated at 62°C, reduced by 1°C for the next 10 cycles, and maintained at 52°C for the subsequent 29 cycles. PCR products were then mixed in a 2/3/6/6/5 ratio for As167/As190/As25/As93/Aru1, respectively and loaded on an automated sequencer . Microsatellite profiles were manually genotyped using GeneScan 3.7 and Genotyper 3.7. For all five loci, reproducibility was checked by performing the amplification and genotyping steps twice on 38 samples. The error rate was calculated as the proportion of mismatches between duplicated, single-locus genotypes.DNA was extracted using the DNeasy 96 Plant Kit . Seven nuclear microsatellite markers were tentatively amplified ,48 but thylogeny . PCRs wepsbA-trnH, a subregion of the intergenic spacer trnS-trnfM and the intergenic spacer atpH-atpI , and 0.5 U Taq polymerase (Taq CORE kit). The reaction profile was: 40 cycles of 30 s denaturation at 95°C, 30 s annealing at 50°C (except for atpH-atpI: 45°C) and elongation at 72°C, followed by a final elongation step of 10 min at 72°C. Elongation time was 30 s for psbA-trnH, 2 min 30 s for trnS-trnfM and 2 min for atpH-atpI. PCR products were then sequenced in both directions at the Centre National de Séquençage . The resulting sequences were manually inspected in Sequencher® 4.9 , and aligned in MEGA 4.0.1 [Three regions were sequenced: a subregion of the intergenic spacer pI Table . PCRs weGA 4.0.1 . Since cA. scopulorum were run on a dataset excluding locus Aru1 so that the data were comparable.Nuclear and chloroplast datasets were analyzed separately. For each dataset, some analyses were performed on the total sampling i.e. simultaneously considering all species, while others were computed within each species separately. For nDNA, among-species comparisons including A. montana and A. laubenfelsii were considered as a single species in the within-species analyses. The A. montana samples from Pandop and A. biramulata samples from Pic du Grand Kaori were removed from analyses at the population level because sample sizes were too low (see A. montana in Pandop). Last, we did not consider the geographic structure of genetic diversity in A. biramulata, because our sampling was too limited and two populations seemed to undergo genetic exchange with other species (see below), precluding reliable within-species inferences.Based on the results obtained on the multispecies dataset, K genetically distinct clusters. Structure was run ten times for each K-value from one to eight to check the consistency of the results across runs. Each run comprised a burn-in of 200000 iterations followed by 106 iterations. We adopted the admixture and correlated allele frequencies models. We used StructureHarvester v0.6.93 [K and (i) the probability of the data L(K) and (ii) the ad hoc statistic ΔK recommended by Evanno et al. [K) and/or ΔK. A similar analysis was run with a reduced dataset, only including A. montana and A. laubenfelsii populations.We used Structure version 2.3.3 ,53 to cl v0.6.93  to plot o et al. . We idenFST indices between all pairs of populations (see below), the relationships between populations were visualized using the network-building distance-based algorithm Neighbor-Net [Based on the matrix of hbor-Net ,57 implehbor-Net .θ of FST [The overall population differentiation was quantified by the estimator θ of FST , calculaθ of FST , and hieθ of FST . AnalyseAR; mean number of alleles per locus based on the minimal sample size; [HE) using FSTAT. The correlation between population diversity  and latitude was tested through non-parametric Spearman rank correlations in Minitab 12.2 . To detect signs of recent bottlenecks, we examined deviations in heterozygosity from mutation–drift equilibrium in each population with the software Bottleneck [Within all populations, genetic diversity was estimated as allelic richness  and expttleneck . We assuFIS and among-population FST indices using FSTAT, and AMOVAs were used to partition the genetic variance into among-population and among-individual within population components. The statistical significance of FIS was assessed by permutations of alleles in each population at each locus, followed by a Bonferroni correction for multiple tests. FST indices were calculated both at the species level and among all pairs of populations. The overall divergence of each population was quantified as the mean pairwise FST between each population and all conspecifics. Exact tests of population differentiation were also performed among all pairs of populations using Genepop [Within each species, genetic structure was quantified by within-population  Genepop  and appl Genepop . In ordeAR, HE, FIS and overall within-species FST were assessed using permutation tests in FSTAT.Among pairs of species, differences in population We constructed both a haplotype and a population network, using the Neighbor-Net algorithm of Splitstree4 and based on the uncorrected p-distances (i.e. the number of changes between two haplotypes) and pairwise Nst indices (see below), respectively. Using Arlequin, we computed an AMOVA to estimate the proportion of the overall genetic variance found among species, among populations within species, and among individuals within population.HR; calculated in FSTAT by considering distinct haplotypes as distinct alleles of a single, always homozygous locus) and mean number of pairwise differences between pairs of individuals .Within all populations, genetic diversity was estimated as haplotype richness , and we performed 10000 permutations of rows and columns of the distance matrix between haplotypes to test whether NST > GST. Such a significant relationship suggests that distinct haplotypes are more related within populations than among them, and comparing NST and GST allows assessing the importance of mutation relative to other causes of genetic differentiation .HR was assessed using permutation tests in FSTAT, while t-tests were performed in Minitab 12.2 to check for differences in i) MNbPwDiff within populations, and ii) NST indices among all pairs of populations within species.Among pairs of species, differences in population psbA-trnH, partial trnS-trnfM and atpH-atpI) and comprised 25 variable sites  among which 16 nucleotide substitutions and 9 microsatellites displaying 2 to 5 alleles each. This allowed defining 71 haplotypes, each observed in one to 78 individuals.The five nuclear microsatellite loci displayed 23 to 35 alleles each, leading to 145 alleles observed in total. The overall reproducibility of genotyping was 92%. The concatenated, aligned cpDNA sequences were 1920 base pairs long . We therefore considered K = 7 as the biologically most plausible number of clusters. The major clusters were: 1) the three A. scopulorum populations from the northwestern coast; 2) the five A. scopulorum populations from the eastern coast; 3) all A. rulei populations; 4) the two northeastern A. montana populations (Mt Panie and TonNon); 5) the Koniambo A. montana population; 6) the remaining six A. montana populations (with the exception of Pandop and with quite strong admixture for the northernmost populations), three A. laubenfelsii populations and the A. biramulata Mt Do population; 7) the three A. biramulata populations Pandop, Kaala Gomen and Mt Tonta and two samples identified as A. montana in Pandop (which were admixed with the Koniambo A. montana cluster). At the sample level, we observed that some other individuals were admixed, with their genotype originating not only from a cluster characteristic of the species they belong to based on morphological grounds but also, to a large extent, to other clusters: some A. montana samples were admixed with the A. rulei and/or A. scopulorum clusters in Paeoua, Boulinda and Me Maoya; some A. scopulorum samples were largely assigned to the A. biramulata cluster in Bogota; and a few A. biramulata and A. rulei samples were largely assigned to an A. scopulorum cluster in various sites , two genetic clusters formed a rough genetic cline from north (Kopeto) to south (Ouinee) at K = 4 and, at K = 5, three clusters were retrieved that broadly consisted of the A. montana Kopeto-Paeoua-Boulinda-Me Maoya populations, A. montana Me Ori-Bwa Meyu populations, and A. laubenfelsii Mt Do-Mt Mou-Ouinee populations  and the A. biramulata Mt Tonta population appeared in an intermediate position between A. montana-A. laubenfelsii and A. scopulorum. As in the Structure analysis, we also observed the eastern vs. northwestern differentiation in A. scopulorum, and the strong divergence of the Panie, TonNon and to a lesser extent Koniambo populations compared to other A. montana-A. laubenfelsii populations. The A. montana Paeoua and Boulinda populations appeared genetically close to the A. rulei Paeoua and PinPin populations, in an intermediate position between the two species.The nDNA population network showed the same patterns: short branches suggested strong genetic relatedness among species but s Figure . HoweverFST was 0.143. The AMOVA showed a low proportion of the genetic variance among species (5.2%), most of it being observed among populations within species (11.5%) and within populations (83.3%). When restricting the analysis to A. montana and A. laubenfelsii, only 0.4% of the variance was observed between species, which was a much lower proportion than between A. montana and other species  displayed cpDNA haplotypes specific to A. biramulata (H05 to H08) although they clustered with A. montana and A. laubenfelsii based on nDNA (Table A. biramulata from Mt Tonta shared a haplotype that was otherwise only found in A. rulei (H65). In contrast, these samples clearly clustered with A. biramulata based on nDNA. Also, out of the five samples from Pandop and identified as A. montana, we observed that three harboured a cpDNA haplotype specific to A. biramulata (H07) whereas two displayed a cpDNA haplotype specific to A. montana (H58). All clustered with the A. biramulata nDNA cluster. Morphological identification was uncertain and we therefore revised it and considered the three samples with both nDNA and cpDNA related to A. biramulata as A. biramulata samples in population analyses. The two individuals with cpDNA haplotypes specific to A. montana were excluded from population analyses because of the low sample size in this site (n = 2). Other cases of cpDNA vs. nDNA incongruence are summarized in Table In the haplotype network, most haplotypes clustered according to taxonomy Figure . In partA. montana and A. laubenfelsii, whose populations were grouped although the A. laubenfelsii populations appeared at the extremity of the A. montana-A. laubenfelsii portion of the network. Also, the A. biramulata Mt Tonta population was retrieved with A. rulei, which was expected given haplotype sharing. Similarly to what was found for nDNA, A. scopulorum populations separated into two geographic groups.In the population network based on cpDNA data, the distinction among species was clear and even more pronounced than based on nDNA Figure . As withNST was 0.646. Consistent with the clear discrimination of species on the population network, the AMOVA showed that 59.8% of the variation was found among species, 10.0% within species among populations, and 30.2% within populations. When considering pairs of species, the among-species component of the variance represented 17.5% of the total variance between A. montana and A. laubenfelsii, which was much lower than between other pairs of species . The among-population differentiation index Nst was statistically higher than Gst in A. biramulata  and in A. scopulorum . The relationship was however not significant anymore when we restricted the analysis to the northwestern and eastern parts of the A. scopulorum distribution area, respectively. In A. montana-A. laubenfelsii and A. rulei, GST estimates were 0.185 and 0.144.There was no significant difference across species in population differentiation at nuclear loci, with P = 0.026; r = −0.64), with the northernmost populations Mt Panie and TonNon displaying very low diversity and central/southern populations harbouring relatively high numbers of (often private) haplotypes  and split in two distinct clusters at K = 6. Other genetic clusters were less clear-cut  increased both towards the north and the south of the island, leading to a significant Mantel test (P < 0.001).CpDNA haplotypic richness was significantly correlated with latitude (es Table . No suchFIS = 0.131). Based on nDNA, the most adequate number of clusters was K = 5, but Structure actually suggested the existence of four genetic groups .There was no correlation between diversity indices and latitude. Three populations showed significant deficits of heterozygotes: Poum, Cap Bocage and Thio  at nuclear loci, they segregated in somewhat different Structure clusters only after some other, conspecific populations were separated; ii) they shared several cpDNA haplotypes; iii) all A. montana and A. laubenfelsii populations were grouped in nDNA and cpDNA distance-based networks; and iv) the distinction between the two species accounted for only 0.4 and 17.5% of the nuclear and chloroplast total genetic variance, respectively, whereas the distinction between other pairs of species explained three to more than ten times more variance. Using the same five nuclear markers as in the present study, Kettle et al. [A. columnaris and A. nemorosa, although the two species belong to the same AFLP clade [Araucaria montana and A. laubenfelsii therefore seem to belong to a single gene pool. The Structure analysis and cpDNA network suggested that A. montana and A. laubenfelsii rather form a genetic cline, which may be due to spatial separation given the more southern distribution of A. laubenfelsii. The reproductive compatibility between A. montana and A. laubenfelsii is not known but their morphological distinction appears difficult. Therefore, we suggest that they may be considered as a single species. The subspecies status may nevertheless be justified by their geographic separation and slight genetic differentiation.In contrast to AFLP markers , nucleare et al.  observedLP clade . AraucarInterestingly, our results highlighted cases of incongruence between nDNA and cpDNA markers: some individuals possess nDNA predominantly from one species, and a cpDNA haplotype generally associated with another species. Such incongruent patterns could be explained either by gene flow among species (introgression) or by incomplete sorting of ancestral polymorphisms . TheA. rulei may have been present in Mt Tonta when among-species gene flow occurred, giving rise to the adult trees that we sampled and that are probably > 100 years old. Alternatively, introgression in Mt Tonta may result from occasional long-distance transport of pollen grains since A. rulei grows in sites that are only a few kilometres distant . However, obs. and ). Such i America .A. biramulata, possessed A. biramulata cpDNA haplotypes but were predominantly assigned to A. laubenfelsii for nDNA. In contrast, in Mt Tonta, trees were morphologically identified as A. biramulata  and predominantly assigned to A. biramulata for nDNA, but possessed cpDNA otherwise specific to A. rulei. Since cpDNA is paternally inherited in conifers, this indicates that A. biramulata pollen grains fertilized A. laubenfelsii ovules in Mt Do, whereas A. rulei pollen grains fertilized A. biramulata ovules in Mt Tonta. At nuclear loci, that are biparentally inherited, the genetic composition of the introgressed trees was predominantly influenced by one parental species, as opposed to a mixed influence of the two parents: on average, in Mt Do, the A. montana-A. laubenfelsii clusters contributed 76% to the nuclear genotype of the introgressed individuals, whereas the A. biramulata cluster contribution was only 15% . In Mt Tonta, the A. biramulata cluster was largely predominant in the genotype of all introgressed trees, and contributed as much as 88% to their genotype on average. This suggests, first, that most introgressed trees that we sampled do not belong to the F1 generation, which would show equal contributions from the two parental species, and that hybridization is more ancient. Second, it implies that the initial hybrids backcrossed much more extensively with one parent than with the other one: hybrids would have mostly backcrossed with A. laubenfelsii in Mt Do, and with A. biramulata in Mt Tonta. This is congruent with the relative abundance of the parental species in the two sites, since A. laubenfelsii is dominant in Mt Do (based on our field observations) and A. rulei was not recently observed in Mt Tonta. At each generation, the proportion of the other parent genome in the nucleus of the introgressed tree was halved, ultimately leading to a very low contribution. It is intriguing that such extensive backcrossing with A. laubenfelsii, in Mt Do, gave nevertheless rise to trees that were morphologically identified as A. biramulata. However, only a very small number of loci may be crucial in bringing about the morphological differences that distinguish the species, and the five loci used here may not be involved in such differences [Introgression in Mt Do and Mt Tonta also differ in the genetic signature left in the chloroplast and nuclear genomes relative to the morphological identification of the introgressed trees: in Mt Do, trees were morphologically identified as ferences .A. montana-A. laubenfelsii and A. biramulata in Pandop (as suggested by the nDNA vs. morphology and cpDNA incongruence of two A. montana samples) and between A. rulei and A. montana-A. laubenfelsii in Paeoua and Ouinee (as suggested by the cpDNA vs. morphology and nDNA incongruence in one A. rulei sample in each site). The involved species co-occur in these geographic locations and, in Pandop, the larger assignment of the putative introgressed samples to A. biramulata compared to A. montana-A. laubenfelsii based on nuclear data is in agreement with the higher abundance of A. biramulata in this site. A lower degree of confidence is associated with the possible cases of introgression in Paeoua and Ouinee, because the putative introgressed samples do not display full genetic (cpDNA haplotype) identity with their potential paternal species A. montana-A. laubenfelsii, but only close genetic relatedness. However, it is interesting to note that the population network based on nDNA data indicated close genetic relatedness between the A. montana-A. laubenfelsii and A. rulei populations in Paeoua, and that the Structure analysis showed nuclear admixture between the two species at this site. This would be congruent with the occurrence of hybridization and introgression. Introgression may also have happened in Boulinda, where both species also co-occur and where A. montana-A. laubenfelsii samples appeared genetically close to A. rulei based on the nDNA population network, and admixed with A. rulei at nuclear loci based on the Structure analysis.Hybridization and introgression may also have happened between Introgression may also explain cases of nuclear admixture observed at other geographic locations, although the involved species do not always co-occur. This would suggest a high potential for pollen dispersal across sites, and deserves further investigations.A. montana, A. biramulata, A. rulei, A. muelleri and A. humboldtensis, between A. columnaris and A. nemorosa, and even between species that do not belong to the same AFLP clade . Some other haplotypes that were previously shared may have gone extinct in some species while not in others. This may be the case e.g. for the A. montana haplotypes H11 in Paeoua and H64 in Mt Panie. Shared ancestral polymorphism suggests that species diverged relatively recently, have large effective sizes (delaying the fixation of polymorphisms) and/or experience slow rate of cpDNA sequence evolution [Another possible explanation for incongruence between chloroplast and nuclear data is the incomplete sorting of shared ancestral polymorphism in one of the two genomes. It is more likely at loci exhibiting a lower degree of variation, and therefore much more plausible in DNA sequences of cpDNA intergenic regions than at nDNA microsatellite loci. Shared cpDNA haplotypes were observed between volution .FST indices ranged from 0.109 to 0.156 and 10.1 to 12.1% of the total nDNA variance was observed among populations, depending on the species, whereas Hamrick et al. [FST of 0.073 for various gymnosperms based on allozymes and Petit et al. [FST of 0.116 at various biparental markers in conifers. This is most likely explained by the fact that New Caledonian Araucaria species do not conform to the general model of conifers growing as large, wide-ranging populations with no physical barriers over long distances, which usually leads to high gene flow and low differentiation in temperate, northern Hemisphere species . In agres  and the non-significance of most Mantel tests offer limited support to this hypothesis. Second, different genetic clusters could correspond to groups of populations originating from distinct refugia, where the species would have survived periods of adverse environmental conditions and from which they would have colonized their current geographic ranges when conditions became suitable. A retreat of (at least some) Araucaria species towards refuge areas probably happened during the Quaternary climatic oscillations and genetic divergence could therefore result from prolonged isolation. More genetic, paleontologic and paleoclimatic data are needed to propose hypotheses on the timing and location of potential refugia [NST and GST in A. montana-A. laubenfelsii and A. rulei . Subsequent fragmentation of the ancestral population could have occurred in response to e.g. competition, erosion of ultramafic soils , or morA. montana populations of Mt Panie and TonNon was particularly striking. The divergence of the Mt Panie population was previously reported [Some populations displayed especially strong divergence, and the case of the reported . The TonA. nemorosa [A. araucana and A. angustifolia [At the population level, we observed significant deficit of heterozygotes in eight populations out of 31. This could be due to the occurrence of null alleles, but we did not observe PCR failure at just one particular locus, as expected in individuals that are homozygous for a null allele. Therefore, the deficit of heterozygotes may rather be explained by the existence of geographic substructure within populations (Wahlund effect) due to selfing or biparental inbreeding following reproduction among related individuals. Such biparental inbreeding was previously shown in nemorosa  and the stifolia ,82.A. montana and A. laubenfelsii may be considered as a single evolutionary unit in conservation plans. In contrast, we observed a clear differentiation of the northernmost A. montana populations of Mt Panie and TonNon. This may be related to different soil adaptations, and suggests the need to protect these populations as they could represent a distinct Evolutionary Significant Unit .A. biramulata in particular. Such ancient hybridization events and introgression can be seen as a motor of evolution, potentially given rise to evolutionary novelties [A. biramulata grows in only a few sites and the risk of genetic assimilation by more widespread species should be carefully monitored and minimized in order to maintain the species [Our data showed strong signs of introgression, involving ovelties . However species .Last, the negative effects of recent population fragmentation were not detected in the genetic structure of contemporary adult populations, but they may be observed in the future ,86. TherAraucaria species form a complex of closely related species, which probably diverged recently and/or are characterized by a slow rate of molecular evolution, and among which ancient hybridization and introgression is strongly suspected. Future studies on the evolutionary dynamics of this group should adopt an integrative approach –combining e.g. genetics, ecology and morphology– and simultaneously consider multiple species given their tight relationships and probable gene flow [A. montana and A. laubenfelsii should also be assessed based on morphological and ecological characters, in order to confirm or reject our hypothesis that they form a single species. Within A. montana, the potential adaptation to different soil substrates (acidic vs. ultramafic) and its influence on genetic differentiation would deserve attention. Last, the hybridization and introgression processes offer many exciting questions to tackle, e.g. how are the introgressed individuals related to their parental species in terms of morphology and ecology? When did hybridization occur, how common were such events and do they still occur nowadays? Could they lead to the emergence of new, self-sustainable and reproductively isolated hybrid species?New Caledonian ene flow . Mitochoene flow ,88. In o


Littorina saxatilis exhibits extreme morphological variation between and within geographical regions and represents an excellent model for assessing local adaptation. Previous studies support the hypothesis of parallel evolution in sympatry of two morphologically different ecotypes (named as RB and SU) that co-inhabit different habitats from Galician rocky shores (NW Spain), and which are interrupted by sheltered areas inhabited by a different morph never studied before (named as SRB). Here, we use morphological and mitochondrial DNA (mtDNA) sequence data to test hypotheses on the origin and diversification of SRB snails and to assess their evolutionary relationships with RB and SU ecotypes. Our results show that the SRB morph displays the largest size and shell elongation and the smallest relative shell aperture, representing an extreme type of the RB vs. SU polymorphism, which has been linked to adaptation to sheltered ecological factors. Phylogenetic analysis shows that the SRB morph shares ancestry with RB and SU ecotypes, rejecting the hypothesis that the SRB morph marks relict populations from which these ecotypes evolved in Galician coasts. Our data support that genetic differentiation among SRB, RB and SU morphs results from a general pattern of restricted gene flow and isolation by distance linked to the colonization of Galician coasts by two independent mtDNA lineages, rather than from a random fragmentation of the initial distributional range. Therefore, the confinement of distinct lineages to specific geographical areas denote evident limits to the distances these snails can disperse. Morphological analysis indicates no association between mtDNA lineage and a specific morphotype, and suggests the independent gain of convergent morphological patterns within each mtDNA lineage in populations occupying contrasting habitats following the colonization of Galician coasts.The marine snail  Littorina saxatilis is an example of incomplete ecological speciation that can be observed along the wave exposed-rocky shores in Galicia (NW Spain) . Galiciiewed in . In the iewed in –6. A simiewed in , 7.Two hypotheses have been proposed to explain the origin of the above ecotypes: I) divergence in allopatry followed by secondary contact, and II) parallel non-allopatric evolution of the ecotypes at both regional and local scales , 8, 9. RAlthough RB and SU ecotypes have been extensively studied (reviewed in ), questiL. saxatilis, previous work [Numerous studies have successfully used mitochondrial DNA (mtDNA) for assessing phylogenetic relationships in presence of gene flow to elucidate the relative importance of historical and ecological factors on intraspecific geographic variation e.g. –18). The. The18])ous work , 9, 10 hous work –12. A glous work . These lous work .L. saxatilis snails. Under this hypothesis, haplotypes from each morph and lineage would cluster essentially randomly within the phylogeny.Here, we analyze mtDNA sequences as well as phenotypic data (shell morphometrics) of samples from both exposed shores (inhabited by RB and SU ecotypes) and sheltered shores  from Galicia covering the Spanish distribution range of the species. We aimed to distinguish between three main scenarios. In scenario A, the SRB morph is the relict form from which RB and SU ecotypes evolved in Galician coasts. Under this scenario, haplotypes from all the sheltered sites should cluster together in phylogenetic trees into a separated and highly diverged clade owing to their ancestral condition. In scenario B, genetic differentiation among SRB, RB and SU morphs results from a general pattern of restricted gene flow and isolation by distance linked to the colonization of Galician coasts by at least two independent mtDNA lineages (I and II). Under this hypothesis, haplotypes should cluster in phylogenetic trees by regional geographical origin, with a positive relationship between geographical and genetic distance within each major mtDNA lineage. In scenario C, an initial population bearing at least two divergent mtDNA lineages (I and II) and lacking population structure experienced vicariance events that had a location and timing independent of the geographical distribution of A total of 48 adult snails inhabiting the most sheltered areas (the sheltered SRB morph) from four Galician estuaries were sampled covering the distribution range of the species in Spain . We alsoEach snail was photographed with a Leica MZ12 stereoscopic microscope and Leica digital ICA video camera. Adult shell images were analyzed using 11 landmarks positioned on the digitized shell image following Conde-Padín et al. . For eacmorph  and locality , following Underwood  and log-transformed geographical distances between samples.Contigs were assembled using Seqman  and alignments were performed with ClustalX . Alignmer  and the tatistic  were comtatistic . This program zt  was used6 generations using default values and trees saved every 1000 generations. The initial 2 x 106 generations were discarded for burn-in. The analysis was repeated twice and checked for convergence. In addition, a statistical parsimony network [The optimal nucleotide substitution model was selected based on the Akaike Information Criterion in Modeltest 3.7 . The sel network  was buil network  using thΨ) obtained from this randomization process was used as an indicator of phylogeographic association [Ψ obtained when the relationship between the genealogy and sampling localities was not randomized.The program PhyloMapper v.1b1  was usedociation , and comWe used a constrained tree analysis as implemented in RAxML  to bettelocality was also significant for CS and RW1, but the percentage of variance explained was lower than 16% (data not shown), thus suggesting that although the differences between morphs were not identical across localities, these differences might be minor. Morphological differences between pairs of morphs were always statistically significant using a post-hoc Student–Newman–Keuls (SNK) tests for U1, RW1 and CS. The magnitude, however, was not always identical. For example, the difference in relative aperture size (RW1) was somewhat larger between RB and SU than between RB and SRB  and shape  among morphs. According to the results derived from the nested ANOVA, the 91% (CS), 39% (U1) and 67% (RW1) of the morphological variation observed for each variable was due to differences among morphs. The means (and standard deviations) of these variables for each of the morphs and localities in addition to the results of the ANOVAS are summarized in  SRB see . In summ SRB see .r2 = 0.104; P = 0.435). No evidence of population expansion was found at any site, as the raggedness r index and Fu´s Fs were never statistically significant.DNA sequence data revealed a substantial level of variation in the assayed mtDNA fragment among the 47 sheltered SRB specimens examined. There were 20 different haplotypes in the sheltered SRB populations, with a total of 59 variable sites. No evidence was found of genetic recombination for the entire data set (N = 95) including RB, SU and the 47 newly generated SRB mtDNA sequences. We observed no significant differences in overall nucleotide diversity between SRB (π = 0.0067 ± 0.0006), RB (π = 0.0078 ± 0.0009) and SU (π = 0.0079 ± 0.0010) morphotypes see . Levels P < 0.001) genetic differentiation, with FST values ranging from 0.971  to 0.191 (Cíes SU vs Ons SU). Geographical distance was positively correlated with the level of genetic differentiation  but not with morphological divergence . A hierarchical analysis of molecular variance revealed no significant (P = 0.858) differences in the level of geographic differentiation of SRB, SU and RB ecotypes, whereas most of molecular variance (89%) was explained by differences between localities within each ecotype (P < 0.001).Genetic variation was highly geographically structured among localities. All pairwise comparisons among morphotypes from different localities resulted in a highly significant . The geographically proximate Ons, Cíes and Mogor populations shared two different haplotypes and formed a separate and well defined monophyletic clade. Interestingly, two haplotypes found at low-frequency in Tapia most likely represent a recent event of gene flow from the nearby location Arealonga. The Shimodaira-Hasegawa (SH) test assuming monophyly of individuals from the same location (ln L = -858.63) was not significantly different from the unconstrained tree . In contrast, a constrained tree topology assuming that all SRB populations cluster in a single monophyletic clade was significantly different from the unconstrained tree .The phylogenetic analysis clearly indicated a global pattern in which haplotypes clustered according to their regional geographical origin with a high node support . BayesiaL. saxatilis snails, we added data from a previous study by Doellman et al. [L. saxatilis is composed of two major and broadly distributed mtDNA lineages (I and II) [Ψ = 23.15; P < 0.001) and a significant correlation between geographical distance and genetic differentiation . This analysis was not feasible for lineage I due to its small clade size.The haplotype network  containen et al. , which i and II) . GaliciaThe results above allow us to reject the scenario A, which predicts a major and ancestral monophyletic clade including all sheltered SRB individuals and scenario C, which predicts that haplotypes from each morph and lineage would cluster essentially randomly within the phylogeny. Instead, our results agree with scenario B, because this hypothesis predicts the monophyly of haplotypes from the same regional geographical origin, and a general pattern of restricted gene flow and isolation by distance linked to the colonization of Galician coasts by two independent mtDNA lineages.Littorina saxatilis inhabiting the Galician coast (NW Spain). Our results indicate a clear morphological distinctness of the SRB morph  sequence data to test hypotheses on the origin and diversification of the intertidal snail RB morph . PhylogeRB morph  shows thL. saxatilis mtDNA is in fact polyphyletic, including derived haplotypes from two ancient lineages (I and II) originated around the British Isles about 0.64 Ma [Our data confirm the previous claim that Galician  0.64 Ma . The spl 0.64 Ma  belonged 0.64 Ma , since tThe observation of two major ancient mtDNA lineages complicates the former identification of scenarios of repeated evolution , becauseL. saxatilis populations have been historically isolated from those living in northern areas, heavily affected by population size fluctuations and dramatic changes in distributional ranges [L. saxatilis . For exiewed in  reported inhabit –47.Nucella lapillus and Melarraphe neritoides) showed a similar parallel trend in shell shape (RW1) across the same environmental cline [Our results demonstrate for the first time the morphological distinctness of SRB snails. This is particularly interesting because the shell size and shape differences existing between the RB and SU morphs have been previously interpreted in adaptive terms  using a much smaller mtDNA fragment. Grey indicates L. arcana; black indicates L. compressa. Haplogroup designations (A to J) and colours as in Doellman et al. [L. saxatilis. In Galicia, only two haplogroups were found: haplogroup D from lineage I (in green), and haplogroup H from lineage II (in red).(a) Statistical parsimony network for North Atlantic populations of n et al. . Galicia(PDF)Click here for additional data file.S1 TableL. saxatilisEstimates of mtDNA nucleotide variation for each ecotype and locality of Galician (PDF)Click here for additional data file.