The geographic distribution of genetic diversity and the population structure of tetraploid wheat landraces in the Mediterranean basin has received relatively little attention. This is complicated by the lack of consensus concerning the taxonomy of tetraploid wheats and by unresolved questions regarding the domestication and spread of naked wheats. These knowledge gaps hinder crop diversity conservation efforts and plant breeding programmes. We investigated genetic diversity and population structure in tetraploid wheats  using nuclear and chloroplast simple sequence repeats, functional variations and insertion site-based polymorphisms. Emmer and wild emmer constitute a genetically distinct population from durum and rivet, the latter seeming to share a common gene pool. Our population structure and genetic diversity data suggest a dynamic history of introduction and extinction of genotypes in the Mediterranean fields. Triticum aestivum L.). Although a ‘relic’ crop today, emmer wheat is used for bread making, animal feed and as a genetic resource for the improvement of durum and bread wheat varieties th century and its extinction was prevented only by inclusion of rivet accessions in germplasm bank collections Tetraploid wheats have played a critical role in human history. Durum is the primary wheat for pasta and semolina production and the second most cultivated wheat after bread wheat (th millennium BCE T. aestivum L.) has been demonstrated to have evolved from a domesticated tetraploid, most likely emmer, and the D-genome donor Aegilops tauschiiTetraploid wheats are genetically and morphologically diverse and their evolution under domestication has not been fully elucidated Triticum turgidum (for example in the Van Slageren system durum is T. turgidum subsp. durum). MacKey further classifies durum and rivet as convarieties of the subspecies T. turgidum subsp. turgidum (T. turgidum subsp. turgidum conv. durum). Conversely, Dorofeev and colleagues T. durum). Previous genetic studies based on expressed genes in the nuclear and chloroplast genomes have focused on the origin of the different genomes and the relationship between domesticated and wild wheats A rigorous taxonomical classification for wheat species is important for the conservation of wheat biodiversity We analysed four classes of genetic markers: nuclear simple-sequence repeats (nuSSRs), chloroplast SSRs (cpSSRs), insertion site-based polymorphisms (ISBPs) and functional markers in expressed genes to investigate within landrace and between landrace genetic diversity in the Mediterranean Basin. We addressed the following questions: 1) how genetically diverse are tetraploid wheat landraces?; 2) can the genetic diversity in neutral markers elucidate the taxonomy of tetraploid wheats?; 3) can the geographic distribution of genetic diversity reveal the evolution and history of tetraploid wheats in the Mediterranean basin?within landrace genetic diversity analysis, 75 individuals from seven tetraploid wheat landrace accessions from the Iberian Peninsula were analysed  preserved by cultivation in situ in Asturias, Spain. Population structure in the latter accessions was previously studied using chloroplast and nuclear SSRs For analysed . These abetween landrace genetic diversity analysis, 244 accessions were assayed  to ten (K = 10) clusters were tested using 1,000,000 MCMCs and 500,000 burn-in runs, with the admixture model. Twenty replicate runs were performed for each value of K. The best-fit model was determined by plotting the natural log probability of the data against KKQ-matrices from replicate runs in each value of K using the program CorrSieve 1.1 http://gmt.soest.hawaii.edu).We clustered landraces into populations based on the nuclear SSR data without any Genetic structure was further investigated on the 244 accessions by means of principal component analysis (PCA) based on microsatellite allele frequency data from the 29 nuSSR panel using the R packages ade4 CD genetic distance measure CD genetic distances were also calculated separately for the sets of microsatellites located in the A and B genomes. Finally, we grouped our landraces in eight a priori defined populations: emmer accessions, rivet accessions, and durum accessions grouped by regional geographical provenance i.e. Southwest Asia, Southeast Europe, Central Mediterranean, Southwest Europe, Northwest Africa and Northeast Africa  obtained by re-sampling the allelic frequency data. A majority-rule consensus tree was produced using the CONSENSUS routine in the PHYLIP package available in the Mobyle portal  and subsequently manipulated in Dendroscope version 2.5 Gene diversity, polymorphic information content (PIC) and allele richness for nuSSRs were calculated using PowerMarker t Africa . DC genewww.worldclim.org). These included altitude (r), mean annual temperature (s), maximum temperature of the warmest month (t), minimum temperature of the coldest month (u), mean annual precipitation (v), precipitation of the driest month (w) and precipitation of the wettest month (x). Environmental distances (ENVijD) between two locations i and j were calculated using equation 1  and the WorldClim database (Xgwm540) and 6 (Xgwm46) with a mean of 4 alleles per locus (Xgwm169) and 0.776 (Xgwm160 and Xgwm60) with a mean of 0.622  were detected in the 75 individual plants analysed with 15 nuSSRs. The number of alleles detected per locus ranged between 2  . The in ectively .T. aestivum outgroup). The number of alleles per locus ranged between 4 (WCt15) and 33 (Xgwm6) with a mean of 14.6 alleles per locus  were detected in the panel of tetraploid wheat accessions  followed by emmer (gene diversity of 0.708) and rivet (gene diversity of 0.682), while Northwest African durum was the least diverse (gene diversity of 0.546) , indicating that the phylogenetic information contained in the A and B genomes is different.Wild emmer was the most diverse of all ten f 0.546) . Among dr groups . Interesr groups . SoutheaT. aestivum landraces found well-supported population structure among wheats with a degree of admixture between clusters. In all models, most accessions were indicated to have received alleles from more than a single group. Analyses of ΔK, LnP(D) and Q-matrix correlations indicated that the solutions K = 2 and K = 6 were stable and well-supported , containing wild emmer, emmer and bread wheat, and Group II (red), including rivet and durum accessions  from Spain (Group Ib – yellow) from the remainder of Group I (Group Ia – dark green) (In the k green) . Group Ik green) . Group Ik green) . Group Ik green) . Group Ik green) . Group Ik green) . Table 4Q-matrices from the K = 6 model, we plotted clines of the six groups' allele frequencies on a map of the Mediterranean Basin (Using an Basin . Group IPCA corroborated the results obtained using STRUCTURE . The firT. aestivum) (T. aestivum var. Chinese Spring control), 2, 13 and 14 were the most frequent. Emmer and wild emmer had a high frequency of unique cp-haplotypes, indicating a higher diversity of maternal lineages in these forms. The frequencies of the four main cp-haplotypes in Southwest Europe, Northwest Africa and the Central Mediterranean were similar. Cp-haplotype 2 was very rare in emmer and rivet and its geographic distribution in durums was limited to accessions from Northwest Africa, Southwest Europe and Central Mediterranean (WCt22 (We identified 24 chloroplast haplotypes (cp-haplotypes) in our accession panel (excluding estivum) . Cp-haplerranean . 20 durun , Beladi Bouhi (Egypt) and Rubio o Rubial (Spain) with the ‘GS-105’ type deletion; Rubio (Spain) and Tri13912 (Libya) with the ‘GS-100’ type deletion; and Trigo Fuerte (Spain) where both deletions were detected. The presence of both mutations in this latter accession may be because bulks of 6 to 8 plants were used for DNA extractions and plants with the two types of mutations were included; at a phenotypic level all of the plants are expected to have the same photoperiod insensitivity response. However, these molecular results were not confirmed with the phenotypic analysis of their photoperiod response type. The accessions with the deletion were previously clustered in distinct groups by STRUCTURE.Almost all of the accessions had neither of the two VRN-A1 gene was obtained in 235 of the 244 accessions  in the intron 1 of the cessions . With onr = 0.211; p<0.001) was found between genetic and geographic distance  and in the Mediterranean European accessions . For the 53 Iberian durum accessions, there were no noteworthy correlations between CD and the logarithm of ENVijD or between CD and the individual environmental parameters, suggesting that none of them is having a substantial influence on microsatellite genetic diversity  was much higher than in emmer (3), durum (3) or rivet (2). Out of the 14 cp-haplotypes found in rivet and durum, 4 were also present in emmer accessions and 3 were present in wild emmer. Of the 7 cp-haplotypes found in emmer, 2 were also found in wild emmer. This suggests a scenario in which all three forms share a common maternal ancestral gene pool that would later become distinct between them due to different population histories. Zohary and Hopf Considering the strong genetic similarity between rivets and durums demonstrated in all our marker systems, it is likely that the two subspecies were originated from a common domesticated ancestor. Adaptation of plants to specific conditions as the species was introduced into Europe could have yielded landrace varieties with distinct morphological characteristics, such as the distinct head form in rivet or the latter's higher tolerance to frost and humidity in comparison with durum. These differences would be maintained by artificial selection giving rise to the agronomically distinct rivet and durum. These selective pressures were apparently not strong enough to create a distinct genetic pool between the two. Another possibility is that the distinction between rivet and durum is simply an artefact based on the criteria used by early botanists, focusing on differences rather than similarities between groups, whereas traditional farmers might have simply thought in terms of varieties with similar agronomic properties, like thresh-ability in situ and ex situ. In some of these Asturias accessions, Leigh and colleagues Xgwm169 could also be phylogenetically meaningful as they occurred only in Asturias accessions. Emmer landraces in Asturias can constitute relic varieties descending from the earliest cultivated forms that have been preserved from genetic erosion due to geographic isolation in mountainous terrain and small-scale farming Population structure was detected within emmer genotypes. Emmer accessions from Asturias were distinct from other emmers. These include accessions preserved both K = 6 model. It appears to be associated with cp-haplotype 2 which also shows an exclusive Western Mediterranean distribution  or natural selection . We investigated variation in our accession panel for two functional markers, one related to photoperiod response and another to vernalisation requirement. Only five accessions had a deletion in the Weak correlations between geographic and genetic distances suggest that a simple isolation by distance model does not apply to durum landraces in Iberia, North Africa or Mediterranean Europe. It is probable that durum wheat did not follow a linear and gradual route when it was introduced in Iberia, North Africa or Mediterranean Europe, or that subsequent historical processes have masked the primitive phylogeographic signal.The absence of correlations between genetic distances and environmental parameters for the Iberian durum suggests that natural selection has not significantly affected SSR allele distribution in Iberia. This is not to say that some plants would not have been selected due to their better performance under different environmental conditions, but simply that the genetic signal of this selection was not imprinted in the SSR markers analysed. Gene flow (by human-driven migration of genotypes) seems thus to be the major evolutionary force affecting the phylogeographic pattern of this species as revealed by nuSSR markers.Our study revealed a distinct population structure in tetraploid wheat accessions using even a small number of SSR markers. It is likely that a larger panel of SSRs would reveal further substructure in these wheat types. It remains to be seen if high-throughput screening of thousands of genome-wide SNPs in these accessions would confirm or falsify the population structure here reported. Moreover, using next-generation-sequencing technologies a large number of homologous genes related to climatic or physiological adaptations could be sequenced in landraces and wild types, revealing selective pressures occurring during wheat adaptation to distinct environments. Although caution would be advised when comparing these different marker systems, namely due to different ascertainment bias levels in SNPs and in SSRs in situ preserved varieties. An individual wheat landrace accession cannot always be seen as a more-or-less homogeneous group of individual plants with most alleles being fixed, as landraces are not static entities and episodes of introduction of new genotypes may occur throughout their history.In this paper we have demonstrated that tetraploid wheat accessions in the Mediterranean show population structure that may reflect early agricultural movements. This structure is corroborated by different genetic marker systems and adds strength to the taxonomic classification proposed to tetraploid wheats by MacKey Details S1PCR details.(DOC)Click here for additional data file.Figure S1Determination of the best STRUCTURE model for the panel of tetraploid wheats. The most meaningful values of K were determined using the LnP(D) (blue line) and the ΔK (red line) methods.(TIF)Click here for additional data file.Figure S2STRUCTURE results for domesticated tetraploid wheats (top and middle) and naked wheats (bottom). For the domesticated tetraploid wheats, models K = 3 and K = 6 are presented. For the naked wheats, model K = 4 is presented Groups were identified with the same letter and colour as their equivalent groups in the STRUCTURE runs using the complete panel of accessions.(TIF)Click here for additional data file.Figure S3Geographical distribution of population structure in domesticated tetraploid accessions. Wild emmer is excluded. Pie charts indicate the proportional membership of each landrace to each one of the different groups as determined by STRUCTURE. A) K = 3 model. B) K = 6 model.(TIF)Click here for additional data file.Figure S4Plot of ΔK and LnP(D) for 20 STRUCTURE runs with a panel of 215 tetraploid wheat accessions.(TIF)Click here for additional data file.Table S1Accession sourcing and genotype data. NSG-USDA: National Small Grains Research Facility, Idaho, USA; IPK: Institute für Pflanzengenetik und Kulturpflanzenforschung, Gatersleben, Germany; CRF-INIA: Centro de Recursos Fitogenéticos, Madrid, Spain; ENMP-INIA: Estação Nacional de Melhoramento de Plantas, Elvas, Portugal.(XLS)Click here for additional data file.Table S2Markers used to characterise genetic diversity in 235 tetraploid wheat and 9 bread wheat accessions. For each marker chromosome location (Chr.), PCR annealing temperature (Tm), Dye used for labelling primers, number of alleles detected (Allele Richness), gene diversity and polymorphic information content (PIC) are provided. “Touchdown” PCRs are denoted by “t*” under annealing temperature.(XLS)Click here for additional data file.Table S3Genetic markers used to characterise the within landrace genetic diversity in seven tetraploid wheat accessions. For each marker measures of genetic diversity are provided. MAF: frequency of the most frequent allele; PIC: polymorphic information content.(XLS)Click here for additional data file.Table S4Genetic marker systems used in the characterisation of tetraploid wheat landraces. For each system the percentage of null alleles detected, average frequency of the most frequent allele (MAF), average number of alleles and average gene diversity detected and polymorphic information content (PIC) is presented.(XLS)Click here for additional data file.Table S5Chloroplast haplotypes identified in tetraploid wheat accessions combining the alleles of five chloroplast SSR markers.(XLS)Click here for additional data file.Table S6ISBP haplotypes detected in tetraploid wheat accessions. The haplotype found in the T. aestivum var. Chinese Spring control was labelled “I”. Numbers correspond to the number of accessions where haplotypes were detected.(XLS)Click here for additional data file.Table S7Haplotypes identified in tetraploid wheat accessions using four ISBP markers.(XLS)Click here for additional data file.


When introgression of undesired exogenous genetic material occurs in a population intended to remain pure, actions are necessary to recover the original background. It has been shown that genome-wide information can replace pedigree information for different objectives and is a valuable tool in the fields of genetic conservation and breeding. In this simulation study, molecular information provided by 50 000 SNP was used to minimise the molecular coancestry between individuals of an admixed population and the foreign individuals that originally introgressed a native population in order to remove the exogenous DNA.This management method, which detects the ‘purest’ individuals to be used as parents for the next generation, allowed recovery of the native genetic background to a great extent in all simulated scenarios. However, it also caused an increase in inbreeding larger than expected because of the lower number of individuals selected as parents and the higher coancestry between them. In scenarios involving several introgression events the method was more efficient than in those involving a single introgression event because part of the genetic information was mixed with the native genetic material for a shorter period.Genome-wide information can be used to identify the purest individuals via the minimisation of molecular coancestry between individuals of the admixed and exogenous populations. Removal of the undesired genetic material is more efficient with a molecular-based approach than with a pedigree-based approach. Depending on the situation, crossbreeding can be considered as a positive or negative process for the management of populations. Many studies have analysed the benefits of a new genetic input: gene flow between populations can offset the loss of genetic diversity and avoid the deleterious consequences of inbreeding,2. HowevLivestock breeds are important components of the world’s biodiversity. Local bIn a previous study, we used pedigree data as source of information to remove introgressed genetic material and recover the native genetic background. DiffereIn most realistic scenarios, reliable pedigree information is lacking and using molecular information is the only option. In the last decades, molecular markers have become a standard tool to characterize the genetic variation of populations . Analyses of this molecular variation help to set priorities in conservation programs, to improve traceability in livestock, to estimate genetic diversity among breeds, etc.-18. In aProgress in genotyping techniques has increased the number of available markers and made it possible to use marker information in a wide variety of analyses. The availability of dense molecular marker maps covering the whole genome can provide a more precise picture of the genetic background of a population than pedigree information. Some studies have investigated whether molecular marker information can be a substitute to genealogical information. Hayes et al. demonstrThe objective of this study was to analyse, through computer simulations, the consequences of substituting pedigree coancestry with molecular coancestry, calculated from genome-wide marker information, for the removal of exogenous genetic material from an introgressed population.Computer simulations comprised three stages: (1) generation of the two original populations ; (2) introgression of exogenous individuals into the native population followed by random mating of the resulting offspring for a variable number of generations; and (3) management of the admixed population to recover the genetic background of the native population.Native and exogenous populations included 100 individuals each  and the genome of each individual was made up of 20 chromosomes, each one Morgan long and carrying two types of biallelic loci: 2500 markers and 25 000 non-marker loci. All loci were equidistant and markers were evenly spaced between the non-marker loci.Initially, frequencies of alleles at each locus (marker and non-marker) were 0.5/0.5 in both populations. To create offspring, random mating was applied in both populations during 100 discrete generations maintaining a constant population size and sex ratio and crossovers were assumed to occur at random along the chromosomes (with no interference) and to follow a Poisson distribution (λ = 1). As a result, allelic frequencies (with a U-shaped distribution due to drift) were divergent between populations and loci were in linkage disequilibrium with a different pattern in each population.The 2500 markers per chromosome were used in the management of the removal of exogenous background. The non-marker loci were used only for evaluation (not for management), as alleles originating from the native population were distinguishable from those originating from the exogenous population. Therefore, the non-marker loci allowed evaluation of the percentage of genome of each individual that originated from native versus exogenous ancestors, and thus, evaluation of the efficiency of the de-introgression process.After generating the native and exogenous base populations, two introgression scenarios were simulated.To form the admixed population, 10, 20, 30, 40 or 50 exogenous individuals were added to the native population maintaining the total number of individuals at 100 over generations. Individuals were mated randomly during one to five discrete generations  over generations. In generation 1, the non-exogenous individuals originated from the native population and in subsequent generations from the already admixed population  without management. The population size was kept constant . A matrix X, composed of xij values is constructed for individuals in the current and exogenous populations and the matrix of genomic relationships between current (c) and exogenous (ex) individuals is calculated as:where Nmark is the total number of markers. To eliminate exogenous genetic material, at each generation of management, contributions of individuals to the next generation  were calculated by minimising an objective function which includes the relationship between current and exogenous individuals:where ci is the relative contribution of individual i to the next generation and ai,Ex is the sum of the genomic relationships between individual i and all the exogenous individuals obtained from equation (2). This genomic relationship is equivalent to the molecular coancestry based on identity by state, and standardised. Since calculation of the genomic relationship ai,Ex is based on many markers, it becomes impossible to have two individuals with the same value. For this reason, minimising the expression in equation (3) selects just one male and one female, i.e., those with the minimum values of ai,Ex. To avoid this, the following restriction was applied: each possible parent in the population could only contribute 10 offspring (of any sex) resulting in 20 equally contributing parents  at each generation, which implies a theoretical rate of inbreeding (ΔF) of 0.0125 (assuming random selection and mating). After selection of the 20 parents, they were mated randomly to create the next generation.where ΔF = 0.006). These simulations were run only with the scenario involving a single introgression event.To further analyse the implications of the number of individuals contributing to the next generations, a new set of simulations was run, with a maximum number of offspring per individual at each generation of management equal to 5 . Then, molecular coancestry between individuals of the admixed population and the pure exogenous individuals of this foreign population was calculated and used to remove the exogenous genetic material. In all cases, the number of genotyped descendants and the number of exogenous individuals that originally entered the population were identical.For every generation, two variables were calculated to evaluate the efficiency of the strategies: native genetic representation and average inbreeding coefficient. Native genetic representation is the percentage of native genetic material recovered after one or ten generations of management estimated by the fraction of alleles originating from the native breed based on information from non-marker loci. Native genetic representation relates with the objective of the method itself. Average inbreeding coefficient based on pedigree information reflects the loss of genetic diversity due to the de-introgression process. The rate of inbreeding was calculated for all generations of native and exogenous populations and during admixture and management periods as:ΔF10:The increase in inbreeding accumulated over the ten generations of management is given by Twenty replicates per scenario were simulated and the results presented are averages across replicates.The percentages of native genetic material recovered after one or ten generations of management with the scenario involving a single introgression event  are shown in FigureIn cases involving just one generation of admixture or minimal introgression, removal of the exogenous genetic material was almost completely achieved in just one generation of management. With increasing percentages of introgression (and/or durations of admixture), more generations of management were required to reach the maximum value.Compared to the results obtained using pedigree data  is shown in FigureΔF10). The greater increase in F compared to expectations for unmanaged populations is a general consequence of the reduction in the number of contributing individuals with the removal method. Notwithstanding, the restriction imposed on the maximum contribution per breeding animal allowed the method to control this increase.The observed ΔF was higher than the theoretical value of 0.0125 in the first generations of management, during which the level of removal was greater. This is due to the fact that the 20 contributing individuals are probably more related to each other than average individuals. This also explains why ΔF is higher for scenarios in which recovery of native genetic material is less easily obtained. After a few generations, the maximum removal was almost achieved and the population had more homogeneous coancestry with the exogenous individuals. At this time, observed ΔF was close to the theoretical value (data not shown). The more generations the method required to recover the native genetic background, the higher was the total increase in inbreeding.Even when including a restriction on inbreeding, F values obtained with the pedigree-based management and the molecular-based management used here cannot be directly compared. In the pedigree-based method, the algorithm chose solutions with a larger number of contributing parents when several solutions with the same value of global coancestry existed. Therefore, there was an unspecific limitation of inbreeding, but no control over the individuals contributing as in the present study[F values) when introgression was between 10 and 20% and both management systems became similar for medium and high levels of introgression (30 to 50%).nt study. The perΔF10 for the scenarios in which the number of offspring per individual in each generation of management was set to 5. The increase in inbreeding was smaller than that obtained in the previous simulations, as expected, which is due to the larger number of contributing individuals. The recovery of native genetic background was lower in most of the scenarios, but exogenous material could still be completely removed if the number of generations of admixture was low.Table FigureAs observed in the scenario involving one introgression event, native genetic material was recovered to a large extent after one generation of management, especially in cases with a low level of introgression. With higher levels of introgression, more generations were necessary to achieve maximum recovery.Comparison of equivalent cases between scenarios including one or several introgression events, , the percentage of native genetic material recovered was higher than with the scenario including a single introgression event. This shows that the period of time between when introgression takes place and when removal begins is an important factor. In the scenario involving several introgression events, part of the introgression occurs later than in the scenario with a single introgression event and, thus, the exogenous material is easier to remove.Other approaches based on marker genotypes have also been shown to be useful to recover an introgressed population. Using iThe availability of genome-wide information for natural populations is not yet as widespread as for farmed animals, thus our method is expected to be more easily applied in the latter. However, recent progress in next-generation sequencing approaches makes it possible to identify SNP in species without a reference genome,28 thus Knowledge on the original frequencies of the alleles in the populations is required to correct the genotypes as shown in equation (1). These frequencies should be estimated in the pure population (when still available). If the reference populations are not large, the sampling variance can be corrected as done in other methods to calculate the genomic relationship matrix, e.g., Yang et al..Besides genealogies and molecular markers, phenotypic characteristics can be used in livestock to characterise a breed. Phenotypes can also be applied to de-introgression as in Fernández et al. and willGenome-wide information can be used to remove introgressed genetic material and to completely recover the native genetic background when the contribution of the exogenous population is limited to 30-40% and the number of generations of admixture is not too high (one to three generations). Molecular coancestry proved to be an effective tool to recover native genetic material and even more efficient than pedigree information or inforEach situation is different and needs to be carefully studied, but even with relatively few SNP, molecular coancestry can be calculated and used in the absence of proper pedigree information for de-introgression. Regardless of the possibilities for recovering a native genetic background, when dealing with introgression it is essential to minimise undesirable exogenous inputs of genetic material as much as possible, because the de-introgression process involves an increased rate of inbreeding that can represent a significant cost for an endangered breed.The authors declare that they have no competing interests.All authors designed the experiment. CA developed the simulations and methods, and drafted the manuscript. THEM and JFM helped in drafting the manuscript. All authors read and approved the final manuscript.


Nematocarcinus lanceopes Bate, 1888 is found in the deep sea around Antarctica and sub-Antarctic islands. Previous studies on mitochondrial data and species distribution models provided evidence for a homogenous circum-Antarctic population of N. lanceopes. However, to analyze the fine-scale population genetic structure and to examine influences of abiotic environmental conditions on population composition and genetic diversity, a set of fast evolving nuclear microsatellite markers is required.The shrimp Nematocarcinus lanceopes (Crustacea: Decapoda: Caridea). Microsatellite markers were screened in 55 individuals from different locations around the Antarctic continent. All markers were polymorphic with 9 to 25 alleles per locus. The observed heterozygosity ranged from 0.545 to 0.927 and the expected heterozygosity from 0.549 to 0.934.We report the isolation and characterization of nine polymorphic microsatellite markers from the Antarctic deep-sea shrimp species Nematocarcinus lanceopes populations in the Southern Ocean and monitor effects of ongoing climate change in the region on the populations inhabiting these.The reported markers provide a novel tool to study genetic structure and diversity in  Nematocarcinus lanceopes Bate, 1888 [N. lanceopes population [Convention on the Conservation of Antarctic Marine Living Resources). The reported microsatellite markers allow monitoring the genetic composition of populations, and identifying declines in genetic diversity. Further they enable the identification of genotypes with selective advantages in regions with increased warming or higher rates of primary production [The shrimp te, 1888 . It has te, 1888 . Previoupulation . These fpulation . Currentpulation . Howeverpulation ,7. The ipulation . The conoduction .© Blood and Tissue Kit, Qiagen GmbH, Hilden, Germany), following the manufacturer´s protocol. Two different approaches of microsatellite isolation were performed: First we created a small microsatellite enriched library following the Reporter Genome Protocol (RGP) [Nematocarcinus lanceopes (see [http://www.ruhr-uni-bochum.de/ecoevo/cm/cm_phobos.htm). Suitable oligonucleotide primers were designed using Primer3 [We extracted DNA from pleon muscle tissue using a commercial extraction kit (QIAmpol (RGP) . As a repes  ranged from 0.545 to 0.927 and the expected heterozygosity (HE) from 0.529 to 0.934. Null allele frequencies ranged between 0 and 0.077. The software GENEPOP v4.1.4 [http://datadryad.org/) and accessible via the link http://dx.doi.org/10.5061/dryad.gg3rn.Amplification success and polymorphism of the reported microsatellite loci were assessed using 55 individuals of P v4.1.4  was usedNematocarcinus lanceopes will help to analyze the fine-scale genetic structure around the Antarctic continent. These results will help us to test, whether there is regional differentiation that we have not yet detected relying on a single mitochondrial marker only [The reported set of polymorphic microsatellite loci from ker only , whetherAll authors declare that they have no competing interest .JD, FL and MJR did the study design and drafted the manuscript. JD, JS and FL carried out the laboratory work. CM, JD and JS carried out the computational work for loci identification and the statistical analysis. All authors wrote the manuscript and all authors read and approved the final manuscript.


Caretta caretta) nesting in Cape Verde as a model system to investigate the link between adaptive potential and philopatry. We produced a dataset of three complementary genomic regions to investigate female philopatric behaviour , male-mediated gene flow (microsatellites) and adaptive potential . Results revealed genetically distinct nesting colonies, indicating remarkably small-scale philopatric behaviour of females. Furthermore, these colonies also harboured local pools of MHC alleles, especially at the margins of the population's distribution, which are therefore important reserves of additional diversity for the population. Meanwhile, directional male-mediated gene flow from the margins of distribution sustains the adaptive potential for the entire rookery. We therefore present the first evidence for a positive association between philopatry and locally adapted genomic regions. Contrary to expectation, we propose that philopatry conserves a high adaptive potential at the margins of a distribution, while asymmetric gene flow maintains genetic connectivity with the rest of the population.Without genetic variation, species cannot cope with changing environments, and evolution does not proceed. In endangered species, adaptive potential may be eroded by decreased population sizes and processes that further reduce gene flow such as philopatry and local adaptations. Here, we focused on the philopatric and endangered loggerhead sea turtle ( The evoeratures . Thus, peratures . The hypCaretta caretta) nesting in Cape Verde as a model system. Sea turtles are important models for understanding a wide variety of biological phenomena such as animal migrations [To tackle these questions, we used the endangered and philopatric loggerhead sea turtle  control region allowed us to characterize female philopatric behaviour ,23, wherOur study system in the archipelago of Cape Verde is the second largest nesting aggregation of loggerhead turtles in the Atlantic Ocean ,32. The 2.(a)n = 19) found dead (killed by poachers) were also sampled. In order to avoid duplicates in sample collection, nesting turtles were tagged with external metal Inconel tags  on the front flippers and all carapaces of dead turtles were marked with paint. Samples were individually preserved in 96 per cent ethanol for later DNA analysis.Tissue samples from 142 female loggerhead turtles were collected during the 2010 nesting season on four different islands of the Cape Verde Archipelago (i)n = 26).All tissue pieces were washed in distilled water for 1 min and then air-dried for 15 min. DNA extraction was performed using the DNeasy 96 blood and tissue kit . DNA extraction failures mainly occurred in samples taken from turtles found dead on the beach ((ii)All samples were amplified for an approximately 720 base pair (bp) fragment in the mtDNA control region . ResultEight polymorphic microsatellite loci were genotyped on an ABI 3130 Genetic Analyzer: Cc-10, Cc-17, Cc-22, Cc-16, Cc2 , 7C04, 2et al. [−1. Then, for each individual, two independent PCRs were carried out using MHC-class-I-specific primers extended with 6 bp MID individual-based barcodes [The MHC class I exon 2 was sequenced on a 454 platform for the 142 sampled individuals, following Stiebens et al. . Brieflybarcodes ,41. For (c)(i)odonCode Aligner v. 3.5 (CodonCode Corporation) and then classified following the nomenclature of the Archie Carr Centre for Sea Turtle Research (ACCSTR). All new sequences were submitted to both ACCSTR and GenBank (accession numbers: KF021625 (CCA1.9) and KF021626 (CCA69.1)). A haplotype data file was created with the software dnasp v. 5.10.01 [etwork v. 4.6.1.0 [MtDNA control region sequences were aligned in C 5.10.01 , and hap 5.10.01 . To eluc 4.6.1.0 .n = 128 individuals). For the islands where the datasets overlapped , we computed φST pairwise tests (50 000 permutations) and exact tests of population differentiation (Markov chain length was 500 000 with 10 000 dememorization steps) based on an expanded test analogous to the Fisher exact test [rlequin v. 3.1.5.2 [φST values rely on Wright's island, model and the list of assumptions in this model are rarely met , the two datasets were pooled. For the combined dataset, we then used φST and the exact test to assess population structure across the entire sampled nesting range. Multiple testing was accounted for by applying the modified false discovery rate (FDR) threshold [To better understand female philopatric behaviour in Cape Verde and further increase statistical power, we combined our mtDNA dataset with one previously generated . BecausφST values to geographical distances, a Mantel test was conducted using the vegan package of R v. 2.15.0 (R core Development Team). Geographical distances were estimated as the shortest possible swimming distance between islands using Google Earth (v. 5.2.1.1588). The relationship was tested using 10 000 permutations (Pearson's correlation), φST/(1−φST) and the log geographical distance between islands as suggested for an isolation by distance event in two dimensions, when using FSTs [With the purpose of relating ing FSTs .(ii)eneMarker v. 1.91 , and the data were imported into Arlequin v. 3.1.5.2 to estimate departure from Hardy–Weinberg equilibrium, observed and expected heterozygosity  and the mean number of alleles over all loci.Microsatellite alleles were called in GFST and exact tests (same parameters as stated above) were also computed in Arlequin, and multiple testing was accounted for with the modified FDR method. Isolation by distance was assessed with Mantel tests in the same manner as previously mentioned, replacing φST with FST. Estimates of FST assume symmetric migration rates and equal population sizes, however, these assumptions are rarely met in natural populations [igrate v. 3.2.17. ENI was obtained by multiplying asymmetrical mutation-scaled immigration rates  with the mutation-scaled effective population sizes (Θreceiving pop.) for each possible migration across nesting islands [M and Θ were performed using the settings modified after Bowen et al. [http://www.r-project.org).Pairwise ulations . Therefoulations  was impl islands . Computan et al. . Five re(iii)FST. Even though alleles may originate from different duplicated loci, for the sake of simplicity, we named the different sequence variants ‘alleles’ (All MHC sequences are deposited on GenBank with accession numbers: KF021627 to KF021666).Individuals harboured up to four different alleles suggesting the presence of up to four different loci , preventW = 0.868, p-value = 0), thus variation among nesting colonies was tested with a Kruskal–Wallis rank sum test.The mean number of alleles per individual were not normally distributed . To further control for a possible effect of geographical distance, we conducted a partial Mantel test with the R statistic correlated against the log , whereas controlling for the neutral divergence represented through 3.(a)p = 0.001). In particular, exact tests revealed that the population structure was more pronounced in turtles nesting in the west: the further west the turtles nested, the more pairwise comparisons were significant (S. Vicente (four significant tests), Sta. Lucia (three), S. Nicolau (two), Sal and Boavista (one); table 1a). Although slightly weaker, this gradient remains even after correction for multiple testing (table 1a). Pairwise φST tests revealed similar structure arising from turtles nesting at the most northwestern island of S. Vicente (table 1a). It is noteworthy that the observed structure did not arise from isolation by distance .In 133 turtles, eight distinct mtDNA control region haplotypes (717 or 723 bp) were found . Pairwis(b)We genotyped eight microsatellite loci for 111 nesting turtles from four different islands . Diversity indices showed identical levels of variability across nesting colonies . However, levels of observed heterozygosity increased in an eastward gradient .p < 0.001). Pairwise exact tests suggested significant differences between the most geographically distant islands (Boavista and S. Vicente); however, none of the exact tests was significant after FDR correction for multiple testing (b). On the contrary, pairwise FST revealed clear structure with low but significant FST values (ranging from 0.009 to 0.025) between the most distant sampled nesting colonies, even after correction for multiple testing (b). Surprisingly, for such a large migratory species, this pattern suggests significant reproductive isolation by distance .The global exact test also revealed significant genetic structure across nesting colonies  was correlated to the direction of the migration, with a higher rate of migrations towards the east χ²3,112 = 6.959, p = 0.073).We sequenced a 216 bp long fragment of the MHC class I region in 112 individuals using 454 next-generation sequencing technology . We detected 44 different variants ) of which 16 (36.36%) were found to be unique to one specific island . Geneticc and MHC class I allele frequency distributions were different between the most distantly separated islands, which remained significant even after correction for multiple testing , suggesting an adaptive pattern of MHC divergence. Additionally, a partial Mantel test of the MHC divergence against geographical distance while controlling for neutral genetic structure represented through FST indicated no significant correlation , further confirming that the observed genetic structure was not solely a result of neutral drift.Then, we tested whether the observed differences among allele pools were simply due to the distance between islands: the Mantel test correlating MHC divergence with geographical distance revealed no statistically significant association , consistent with a high accuracy of female philopatry as reported for other rookeries [φST and exact tests for S. Vicente turtles. The slightly weaker genetic structure in the east was consistent with the much higher nesting densities there [Even though distances between islands of this oceanic rookery only ranged from 80 to 260 km, we found a clear pattern of genetic structure of mtDNA haplotypes among nesting islands a, consisookeries ,57. Thises there . Non-phies there . Thus, aes there . Nonethees there . The difAnother striking discovery was that genetic structure based on the bi-parentally inherited microsatellites, unlike that of the maternally inherited mtDNA, followed a pattern of isolation by distance. This pattern of isolation by distance revealed the possible existence of male philopatry to specific mating grounds. Our data support the existence of male philopatry at least at a regional scale (e.g. east versus west). Nevertheless, the observed pairwise comparisons at nuclear markers appeared lower than for that of the mtDNA control region. Although this pattern may arise from slower allelic fixation of microsatellites  and a fn = 112); Namibian cheetah: 10 MHC class I alleles (n = 108) [n = 387) [n = 99) [n = 14) [Our original hypothesis stated that philopatry in endangered species would deplete the overall adaptive potential of the rookery because of reduced gene flow and smaller nesting colony sizes. Contrary to that expectation, the genetic diversity at MHC loci was not low: each nesting colony displayed more than 20 MHC class I alleles, which, compared with other endangered species seems to be high  ; Tasmanin = 387) ; Europea[n = 99) , but see[n = 14) ). It is [n = 14) . To conf[n = 14) ,35. ThisThese results allow us to conclude that despite high gene flow, selection has contributed to sort different MHC alleles among nesting colonies. To the best of our knowledge, this represents the first evidence for an association between philopatry and locally adapted genomic regions. We therefore propose the alternative hypothesis that philopatry may be acting to maintain a high adaptive potential in sea turtles by facilitating the retention of locally adapted genetic polymorphism. Although we cannot clearly point out how selection occurs, it may be possible that there are differences in the incubation environment, which include factors such as parasites .Our results also emphasize the synergistic interaction of asymmetric gene flow and the maintenance of genetic diversity in a philopatric species. One remarkable discovery was that the smallest nesting colony at the extreme westward margin held the most differentiated set of MHC alleles c. IncreaIn the scope of conservation biology, the Cape Verde rookery should not be considered as a single population but rather various nesting colonies that harbour important genetic variation necessary for future adaptations, especially in the face of climate change and the maintenance of a healthy metapopulation. We uphold the suggestion that marginal colonies should not be neglected as inconsequential components of a population  and in sIn summary, we showed that local immunogenetic adaptation may be a driver for the evolution of philopatry and that philopatry in the endangered loggerhead turtles maintains the adaptive potential of the species. Furthermore, we showed that the edges of populations should be considered as an important reservoir of genetic diversity, particularly in the face of current rapid global changes.


Siderastrea siderea and Montastraea cavernosa, were able to maintain gene flow among populations separated by as much as 1,200 km along the coast of Brazil. In contrast, brooding species, such as Favia gravida and Siderastrea radians, had more restricted gene flow along the Brazilian coast.Among Atlantic scleractinian corals, species diversity is highest in the Caribbean, but low diversity and high endemism are observed in various peripheral populations in central and eastern Atlantic islands and along the coasts of Brazil and West Africa. The degree of connectivity between these distantly separated populations is of interest because it provides insight into processes at both evolutionary and ecological time scales, such as speciation, recruitment dynamics and the persistence of coral populations. To assess connectivity in broadly distributed coral species of the Atlantic, DNA sequence data from two nuclear markers were obtained for six coral species spanning their distributional ranges. At basin-wide scales, significant differentiation was generally observed among populations in the Caribbean, Brazil and West Africa. Concordance of patterns in connectivity among co-distributed taxa indicates that extrinsic barriers, such as the Amazon freshwater plume or long stretches of open ocean, restrict dispersal of coral larvae from region to region. Within regions, dispersal ability appears to be influenced by aspects of reproduction and life history. Two broadcasting species,  The ability to disperse over long distances and maintain connectivity over large geographical areas has important repercussions for the population dynamics of a species. Over ecological time scales, connectivity contributes to the persistence of populations Many marine organisms have broad geographical distributions, but it is unclear whether these distributions reflect the ability to maintain frequent long-distance dispersal or if populations are fragmented across their range. In the Atlantic, the most extensive and diverse tropical reef ecosystems are found in the Caribbean Sea, but reefs with low overall species richness yet high endemism are also found along the coast of Brazil, on mid-Atlantic islands and in West Africa Dispersal in benthic marine organisms can be achieved in various ways. Long-lived pelagic larvae are capable of traversing hundreds of kilometers Corals support biodiversity in the oceans by providing the substrate and architecture for a host of other marine species. Understanding the processes that limit or promote dispersal in coral species can provide insight into how and why populations persist and evolve. Although connectivity and dispersal have been explored for a number of co-distributed Pacific corals Corals exhibit a variety of reproductive strategies, making them an interesting group for evaluating the role of reproductive traits in determining dispersal potential. Corals reproduce sexually either by internal fertilization and brooding of larvae or by external fertilization of broadcast gametes followed by development in the water column The reproductive output of a coral species may increase its ability for long distance dispersal, simply because the chances of successful dispersal events increase as a greater number of propagules are generated. The number of larvae produced by a colony varies according to colony size, the number of eggs produced per polyp, number of reproductive cycles per year, and fertilization success Madracis asperula, M. decactis, M. pharensis, Montastraea cavernosa, Porites astreoides, Siderastrea radians, S. siderea, S. stellata and the invasive Tubastraea coccinea. In addition, Favia fragum (Caribbean and West Africa) and F. gravida (Brazil and West Africa) were previously considered synonymous Meandrina braziliensis, Siderastrea stellata, or Scolymia cubensis, is debated among taxonomists and biogeographers  in 2002 and 2007. Genomic DNA was preserved and extracted following a protocol described previously β-tubulin and the intron of the Pax-C gene, which have previously been shown to contain sufficient intraspecific variation in corals for population studies P. astreoides, for which only β-tubulin could be amplified. For P. astreoides, the mitochondrial control region  was amplified and sequenced for 32 individuals from Panamá and Brazil, but because all individuals contained identical nucleotide sequences at this locus, this marker was not used for further analysis.Coral tissue samples were collected from colonies of six coral species: β-tubulinPax-Cβ-tubulin or 50–54°C/45 sec for Pax-C, 72°C/90 sec, with a final extension step at 72°C for 5 min. An additional forward primer was designed for the amplification and sequencing of Pax-C for F. fragum (PaxFsh1: 5′- GGA GGA GCT TGC GAA TAA GA -3′). Multiple bands were amplified in P. astreoides and the Siderastrea species in β-tubulin, and for S. siderea in Pax-C. Bands at ∼750 bp and ∼600 bp were extracted and purified for products of β-tubulin and Pax-C respectively, using the Qiaquick Gel Extraction kit (Qiagen). The remaining PCR products were purified either by gel extraction or by elution through a silica-membrane column using the appropriate Qiaquick purification system (Qiagen).Amplification of the two nuclear loci was performed by polymerase chain reaction using published primer sequences for β-tubulin for S. radians (site 549) and S. siderea (site 549), and in sequences of Pax-C for S. radians (site 134), F. fragum (site 205) and F. gravida (site 205). Because only a small number of individuals were heterozygous for indels within any species and indel positions were straightforward to identify, indel sites were kept and considered in the analysis.Sequencing of all loci was performed directly on purified PCR products for both forward and reverse directions on an ABI 3130xl genetic analyzer with the BigDye Terminator v3.1 chemistry (Applied Biosystems). Sequence chromatographs were viewed and edited using the Sequencher v4.5 software (Gene Codes Corp). Heterozygous alleles were identified by double peaks observed in sequences from both directions. Indels were observed in the introns of Haplotypes for heterozygous individuals were reconstructed using PHASE v 2.1.1 ST ALL). In this AMOVA, populations of F. fragum (n = 1) and F. gravida (n = 3) were pooled to assess levels of genetic differentiation between these two closely related species. A second AMOVA tested for differentiation among populations within Brazil, the only region for which multiple populations were sampled (ΦST BR). Estimates of pairwise φst used distances between haplotypes. Parsimony haplotype networks were constructed with TCS Haplotype frequencies, molecular diversity indices, tests of neutrality, analysis of molecular variance (AMOVA) and estimates of population differentiation were calculated using Arlequin v.3.11 β-tubulin and 191 for Pax-C. Haplotype counts for each species can be found in A total of 238 individuals across all species were sequenced for S. stellata in Brazil contained alleles that were either (1) identical or had 1–2 mutations difference to alleles of S. siderea from Caribbean or West Africa, or (2) had a combination of haplotypes identified in S. siderea and S. radians at one or both loci. The presence of S. siderea in Brazil has been debated S. siderea and S. radians haplotypes indicates that S. stellata could be the result of hybridization between these two species. Although most individuals with hybrid genotypes were found in Brazil (n = 20), one hybrid was found in Panamá and four were found in São Tomé. Further investigation is required to confirm the hybrid status of S. stellata. For the purposes of this study, data collected from hybrid individuals were kept in the analysis, but only populations of S. radians and S. siderea were considered. Therefore, haplotypes obtained from S. stellata were included as part of either a S. radians or S. siderea population, depending on similarity/identity with haplotypes from those species.Interestingly, individuals identified in the field as h), which estimates the probability that two randomly sampled haplotypes in a population are different, ranged from 0–0.863 in Pax-C and 0.235–0.986 in β-tubulin, indicating that while some populations are dominated by a common haplotype, others have a large diversity of haplotypes. Nucleotide diversity (π) ranged from 0.0009–0.0090 in Pax-C and 0.0003–0.0158 in β-tubulin, and the average number of differences between haplotypes of a population (k) ranged from 0.324–3.293 in Pax-C and 0.235–10.505 in β-tubulin. The mean and standard deviation for each molecular diversity index in each population is shown in S. siderea in São Tomé (West Africa) had lower average gene diversity than populations in the Caribbean and Brazil. Reduced gene diversity was also observed for F. gravida in West Africa in the Pax-C locus; however, this trend was not observed in the β-tubulin locus. S. radians and P. astreoides showed overlap in mean values of gene diversity at both loci across Caribbean, Brazilian and West African populations (π) and average number of differences (k) overlap among populations for nearly all species in both loci  had significantly negative values of Tajima's D , except for P. astreoides . The strongest levels of differentiation were observed among the pooled populations of F. fragum and F. gravida , but not among populations of the broadcasters S. siderea or M. cavernosa within Brazil   no significant differentiation was observed between populations of P. astreoides in Brazil and the Caribbean and (3) populations of Abrolhos and Fortaleza (both in Brazil) and São Tomé in S. radians were not significantly different at Pax-C, most likely because similar allele frequencies were observed for two common haplotypes (SRP1 and SRP2). Haplotype frequency pie charts were plotted for each locus and each species on a map to illustrate differences across populations  so that a comparison of haplotype frequencies could be made between these two species  . Signifi p>0.05) , with onulations . Since i species .F. fragum and F. gravida were plotted together to show levels of divergence among haplotypes of the two species. Haplotype networks were typically composed of a small number of haplotypes (H<15), although the network for β-tubulin in S. siderea had 60 haplotypes. No loops were observed in the haplotype networks of F. fragum and F gravida for both loci, or in the network of P. astreoides for β-tubulin. One loop was observed in the networks of S. radians at each locus. For S. siderea, no loops were observed in Pax-C; however, numerous loops were observed in β-tubulin, suggesting that in this locus, several recombination events have occurred. The four gamete test indicates that only a few recombination events have occurred at each locus for most species (Rm = 0–4), except for β-tubulin of S. siderea, where a minimum of 12 recombination events are inferred. Recombination has also been observed in β-tubulin1 and β-tubulin2 of M. cavernosaβ-tubulin in S.siderea, where 72% of haplotypes were observed only once.Statistical parsimony haplotype networks for each species and locus are shown in Montastraea cavernosaThe data collected for six coral species plus an existing dataset for The most likely biogeographic barrier separating Caribbean and Brazilian coral populations are the deltas and low salinity waters of the Amazon, Orinoco and numerous rivers along the coast of northern South America. The Amazon is the largest among them, and accounts for 16% of the annual freshwater discharge into the world's oceans F. fragum and South Atlantic F. gravida were the most pronounced among all of the studied species , P. astreoides has one common haplotype that is observed both in Brazil and Panamá at similar frequencies, with all other haplotypes being unique to each respective population  and P. astreoides, for which specimens were only taken in João Pessoa  and can suffer loss of diversity via genetic drift. In addition, peripheral populations colonized by a small group of founders may only contain a subset of the full diversity of alleles observed in the main population. However, loss of genetic diversity in Brazil and West Africa does not appear to be a consistent trend among the coral species studied here, or if differences in genetic diversity exist among regions, they have not been captured with the markers used here.This finding is at odds with patterns observed in populations of the coral F. fragum, F. gravida, P. astreoides and S. radians were much lower than for the broadcasters S. siderea and M. cavernosa. For example, >40 haplotypes were observed for M. cavernosa and S. radians, while the number of haplotypes observed for the four brooding species ranged from 6–12. High levels of polymorphism in M. cavernosa may have made the patterns of reduced variation in the peripheral populations more pronounced and readily detected. In S. siderea, decreasing trends in gene diversity (h) are observed, but there is overlap in the mean values of other diversity indices among the three biogeographic regions . Differences in eN between the species may result in different rates in the loss of genetic diversity. Founder effects may also be less pronounced if peripheral populations have received multiple founding events. Some of the brooding species studied have multiple mating cycles per year and reproductive strategies that may allow them to establish larger populations over shorter time periods, thereby increasing eN and buffering the loss of genetic diversity. Another possible scenario is that historically some of the other species were able to maintain gene flow up until more recently, while gene flow for M. cavernosa ended a longer time in the past – subjecting the species to a longer period of time for drift to act upon.Regional differences in genetic diversity may have gone undetected, but another reasonable alternative is that loss of variation has occurred in peripheral populations of Patterns of connectivity across broad regions of the Atlantic are congruent across multiple species of corals, suggesting that barriers to dispersal such as the Amazon freshwater plume and the long distances that separate the east and west South Atlantic are effective for most coral species. At shorter, regional scales, it appears that some aspects of reproduction such as mode of larval development can influence gene flow, although this pattern cannot be generalized to all scenarios. Broadcasters have greater dispersal ability in the Atlantic, but several exceptions exist in the Indo-Pacific, suggesting that complex interactions between biotic and abiotic factors can limit connectivity rather than single traits of a species. Loss of genetic diversity in peripheral populations of corals is also not a general trend observed among all amphi-Atlantic corals, likely due to differences in population size and timing of isolation across species. Regional isolation may mean that the persistence of peripheral populations relies primarily on local to regional recruitment since migration from other regions must occur rarely. These populations may be more vulnerable to disturbance on ecological time scales as a result of their isolation. On the other hand, isolated peripheral populations with small effective population size may become the sites for local adaptation and allopatric speciation. In both scenarios, these regions require special attention for conservation as a result of their potential vulnerability to environmental change, but also their importance in generating diversity.Table S1Coral species list for the Atlantic. Species occurrence in each biogeographic region is indicated by an X. Total species count for each region is found at the bottom of each column.(PDF)Click here for additional data file.Table S2Haplotype count for each species, locus and population.(PDF)Click here for additional data file.Table S3Two-locus genotypes for sampled individuals.(PDF)Click here for additional data file.Table S4Genbank accession number for all sequenced haplotypes.(PDF)Click here for additional data file.


Araucaria angustifolia, Copaifera langsdorffii, Myracrodruon urundeuva and Hymenaea stigonocarpa, using microsatellite markers and parentage analysis. More specifically, in these studies we tried to answer the following questions: Can spatial isolation of tree populations following forest fragmentation block seed and pollen migration (gene flow)? What is the distance and patterns of seed and pollen dispersal in populations of tree species living within fragmented stands? Is there intra-population spatial genetic structure (SGS) in the adults and regeneration, and where is it higher, in adults or regeneration after forest fragmentation? Can forest fragmentation really reduce the genetic diversity and effective population size and increase the inbreeding and relatedness within populations? What is the necessary number of seed trees to collect seeds aiming at ex situ conservation and environmental reforestations plans with a reference effective population size that provides a minimum evolutionary potential?Tree species are key organisms of forest ecosystems, due their size and extended life, providing the environmental setting for many other living organisms. The levels of genetic diversity and the effective population sizes are essential issues for the maintenance and survival of tree species populations, because they direct adaptation to the current environment and future environmental changes. Forest fragmentation around the world is a fact, affecting many animal and plant populations in all continents. In Brazil, forest fragmentation and selective logging are the two main problems affecting populations of tropical tree species. For example, the Atlantic Forest was intensively destroyed and fragmented during the last century. Today, only between 11 and 17% of the original areas remain, generally split into small forest fragments or isolated trees in pastures and agricultural lands. In the Atlantic Forest, only 3% of the original area of the Araucaria Forest biome in southern Brazil, remains. Forest fragmentation is especially drastic in tropical forest biomes, due to the very high species diversity, associated to low population density (<1 tree/ha). Thus, after forest fragmentation takes place, the remaining fragmented forest may contain the same species diversity, but the populations are strongly reduced, and, in some cases, less than a dozen reproductive individuals remain. The bottleneck (size reduction of the reproductive population) caused by forest fragmentation associated to spatial isolation of the remaining stands may cause the loss of genetic diversity, an increase in inbreeding and relatedness, reducing the effective population size and blocking seeds and pollen migration inside the stands. These hypotheses have been tested in some tropical tree species of the Araucaria Forest and semicidual Atlantic Forest, including in situ and ex situ conservations, tree breeding and environmental restoration plans. As many tropical trees are long lived individuals, established before forest fragmentation had occurred (<100 years), seeds, seedlings and juveniles, established after forest fragmentation have been included in the samples. Thus, these studies have been based on the sampling of all reproductive trees regenerates , as well as open-pollinated seed . All adult trees and regenerates were also mapped (x and y coordinates), the diameter at breast height (dbh) and/or total height measured and in the case of dioecious species, all reproductive trees were also sexed. We used a set of microsatellite loci to arrive to a high exclusion power, to guarantee that each candidate parent displays a unique multilocus genotype for parentage analysis. Our results showed an essentially perfect isolation of seed gene flow, with total absence of seed immigration, probably due to the large distance separating the remaining forest fragments, associated to an intensive use of surrounding land . Within populations, although long distance seed dispersal events were detected, seeds generally disperse intensively near the mother trees following a classic isolation by distance model. These hypotheses have also been supported by studies of SGS in both adults and juveniles and mating system, revealing significant rates of mating among relatives. In contrast, substantial realized and effective pollen immigrations have been detected in the studied species (ranging from 4 to 10%), evidencing longer-distance pollen dispersal than seed dispersal. Within populations, although the estimates are downward biased due to a substantial rate of pollen immigration in the stands, pollen generally disperses with high intensity at short distances, in a near neighbour pollen dispersal pattern, determined by the individual flowering patterns and behaviour of pollination vectors. Matings are not random, due to selfing, mating among relatives and correlated mating, resulting in higher inbreeding relatedness and lower effective population size in open-pollinated seeds than expected under a panmitic model. High inbreeding and relatedness and lower effective population size were also seen in the regeneration. Lower genetic diversity has been found in seeds, seedlings and juveniles when compared to the adult population, suggesting the occurrence of genetic drift. Taken together our results in the targeted species has indicated a requirement to maintain the genetic connectivity among the forest fragments using strategies of reforestation in riparian areas and forest corridors. Our results also suggest the need to collect seeds from a large number of trees for ex situ conservation, tree breeding and environmental reforestation, due to a typically limited effective population size contained in open-pollinated individuals. This small number of species studied so far does not represent all the genetic, demographic, reproductive and ecological characteristics of the tree diversity in the Atlantic Forest. To provide a stronger support to our hypotheses and results to date, new studies of gene flow, mating system, SGS, genetic diversity and effective population size are underway with other species. Finally, it is never enough to reiterate the importance of conserving tropical tree diversity for future generations so that they can benefit from them as we currently do. Although, in situ conservation is the indicated method for this purpose, conserving not only the target species, but also other organism in the same biome using ex situ strategies is also necessary due the continued fragmentation and degradation of the Atlantic Forest.Knowledge of these issues is crucial to delineate strategies for


Carychium minimum O.F. Müller, 1774 and Carychium tridentatum   – by using COI sequence data and species potential distribution models analyzed in a statistical phylogeographical framework. Additionally, we examined disjunct transatlantic populations of those taxa from the Azores and North America. In general, both Carychium taxa demonstrate a genetic structure composed of several differentiated haplotype lineages most likely resulting from allopatric diversification in isolated refugial areas during the Pleistocene glacial periods. However, the genetic structure of Carychium minimum is more pronounced, which can be attributed to ecological constraints relating to habitat proximity to permanent bodies of water. For most of the Carychium lineages, the broader Alpine Region was identified as the likely origin of genetic diversity. Several lineages are endemic to the broader Alpine Region whereas a single lineage per species underwent a postglacial expansion to (re)colonize previously unsuitable habitats, e.g. in Northern Europe. The source populations of those expanding lineages can be traced back to the Eastern and Western Alps. Consequently, we identify the Alpine Region as a significant ‘hot-spot’ for the formation of genetic diversity within European Carychium lineages. Passive dispersal via anthropogenic means best explains the presence of transatlantic European Carychium populations on the Azores and in North America. We conclude that passive (anthropogenic) transport could mislead the interpretation of observed phylogeographical patterns in general.The Alpine Region, constituting the Alps and the Dinaric Alps, has played a major role in the formation of current patterns of biodiversity either as a contact zone of postglacial expanding lineages or as the origin of genetic diversity. In our study, we tested these hypotheses for two widespread, sympatric microgastropod taxa –  Alternations of glacial and interglacial periods as results of climatic fluctuations of the Pliocene and Pleistocene periods most likely triggered the formation of current patterns of biodiversity. Species reacted to the changing environmental conditions in diverse and individualistic ways Terrestrial gastropods are particularly suitable for the testing of phylogeographical hypotheses Carychium , which comprises a group of Holarctic microgastropods (shell height 1.5–2.5 mm) inhabiting superficial subterranean habitats Carychium minimum Müller, 1774 Carychium tridentatum  In this study, we focused on the taxon Carychium minimum and C. tridentatum. Our aim was to investigate the individual responses to past climatic fluctuations and to analyze whether the broader Alpine Region (Alps+Dinaric Alps) constitutes a ‘hot-spot’ (refugium) or ‘melting-pot’ (suture zone) of genetic diversity. We tested our two competing hypotheses by the application of multiple lines of evidence using independent methods based on genetic and bioclimatic data, respectively, and by the comparative investigation of co-distributed gastropod taxa. We further benefit from the hermaphroditic nature of Carychium gastropods Carychium taxa and one would expect high genetic diversity, with endemic lineages and/or haplotypes to be found in this region. Furthermore, the Alpine Region could have served as an important source for postglacial population expansions We performed a comparative phylogeographic approach including data for the two closely related and mostly sympatric microgastropod species Carychium minimum  and C. tridentatum  were sampled during the years 2008–2011 throughout their native European range . Specimens were immediately preserved in 70–99% ethanol after collection. All individuals were identified by an integrative taxonomic approach using the combined investigation of conventional conchological characters for Carychiidae In total, 742 specimens from 92 sampling localities , Table 1an range  and froman range . No specCarychium’ (PHYCA) and ‘Barcoding Carychiidae microsnails’ (BARCA) stored at the Barcode of Life Data System (BOLD) 2, 0.3 µL (20 mM) dNTPs, 0.3 µL Taq-polymerase, 0.25 µL (0.5 M) tetramethylammonium chloride, 1.5 µL (10 mg/mL) bovine serum albumin, 11.15 µL ddH2O and 5 µL template DNA. PCR cycles were run at the following conditions: 1 min at 95°C, followed by 30 cycles of 30 s at 95°C, 30 s at 52°C and 30 s at 72°C, and finally, 3 min at 72°C. Single PCR products were visualized on a 1.4% agarose gel and cleaned with the GeneJET PCR Purification Kit . In cases where multiple PCR products were detected the QIAquick Gel Extraction protocol (Qiagen) was used. PCR products were bidirectionally sequenced using the PCR primer pair and the BigDye® Terminator v.3.1 Cycle Sequencing Kit  on an ABI 3730 xl capillary sequencer following the manufacturer's instructions.DNA of freshly conserved specimens was extracted using the DNeasy Blood and Tissue Kit  and the respective protocol. Prior to DNA extraction, visceral and shell material were removed to minimize contamination risk. E-voucher data  can be obtained from the projects ‘Phylogeography of Sequences were edited and aligned using Geneious 5.4 (Biomatters Ltd.) and BioEdit 7.0.9 Analyses of genetic data can provide insights into population structure and events shaping their demographic history, e.g. the impact of glacial periods with the formation of isolated allopatric lineages. A spatial-genetic correlation can further point to regions of high genetic diversity as potential refugial areas or zones of secondary contact.The software DnaSP v5 rd level nesting for the less genetically structured CT resulted in only two units, and would thus provide few insights into the spatial distribution of genetic diversity.Haplotype networks were constructed by using Network 4.6 (Fluxus Technology Ltd.) for Median-Joining th generation. Final samples were taken from the stationary phase of the runs. The convergence of relevant parameters was assessed using Tracer v1.5 Tests for population expansion events were conducted using the software BEAST 1.6.1 The McDonald-Kreitman test for a combined dataset of CM+CT was conducted in DnaSP v5. This test is based on the comparison of synonymous and non-synonymous differences within and between species SS measures are significant below the 0.05 and 0.02 p-level, respectively, which were calculated in DnaSP v5 . Deviations of the observed data from a simulated model of population expansion For selected MOTUs additional Tajima's D The comparison of species potential distribution models under recent and past bioclimatic conditions can help to identify (re-)colonization events and to locate permanently suitable regions (stable habitat) that with high probability, could have served as refugial area.a priori selection, we screened for important environmental variables to yield an even better goodness-of-fit than a mere model containing all variables. Consequently, the spatial and temporal transferability will be enhanced if such variables are omitted from the model runs Recent and past species potential distribution models were estimated using presence-only maximum entropy modeling implemented in Maxent 3.3.3 In distinguishing between ‘hot-spot’ (refugia) and ‘melting-pot’ (secondary contact) regions, it is important to detect past population expansion events and to localize their potential origins in congruence with spatial-genetic patterns and bioclimatic data.The refugium localization reconstruction approach (RLR) combines methods used in phylogenetic ancestral character reconstruction and population genetics for inferring the ancestral locality of the most recent common ancestor (MRCA) of a given lineage We used 250 randomly-sampled COI coalescent trees of the stationary phase of a BEAST-run applying the same parameters as mentioned for the demographic history analyses. BEAST-runs under constant size and exponential growth scenarios were compared using Bayes Factor tests . Runs unCarychium minimum, CM) and 39 . We detected the same number of haplotypes (n = 39) for both taxa  than in CT . The number of haplotypes per locality extends from 1–5 (The COI datasets have a length of 590 homologous base pairs (bp). Total numbers of variable sites are 52 (oth taxa . All diffrom 1–5 . Geneticfrom 1–5 . Samplinfrom 1–5  are reprMOTU1 – CMMOTU5) following the results of a 3rd level nesting. CMMOTU1 demonstrates a star-like topology comprising the most frequent haplotype H1 , northern Spain (CMMOTU3) and the Alpine Region (CMMOTU3+CMMOTU5). Haplotypes of CMMOTU5 are also present at two localities on the Black Sea coast . The application of a 3rd level nesting separates CTMOTU1-4 from CTMOTU5-8 (MOTU1-4 and CTMOTU5-8 occurring more likely northwards and southwards (respectively) of this virtual barrier .Resolved Median-Joining and Statistical Parsimony networks provide the same results . As a co barrier . Main exMOTU4  demonstrating haplotype H2 and individuals from Vancouver, Canada haplotype H1. Thus, six out of seven transatlantic sampling localities possess identical  or closely related haplotypes (CT: H2) as found on the European mainland.The seven transatlantic localities of CM  and CT  are characterized by only one haplotype per locality . The samMOTU4 . HaplotyModel runs for CM  and CT  ‘present-Europe’ and ‘LGM-Europe’ potential distribution models performed well. The average values for training AUC of both datasets are close to 1 . Thus, these models possess a high predictive performance and transferability.The ‘present-Europe’ models for CM and CT demonstrate a wide area of potential distribution for each taxon . Except Potential distribution in the ‘LGM-Europe’ models is restricted to several isolated regions . For CM,At this point, it should be highlighted that the potential distribution does not necessarily reflect the realized distribution. Hence, regions with positive model results should be regarded only as potential distribution areas including (micro-) habitats in which the taxa are able to survive.CM = 2.099; BFCT = 6.482). The complete COI-dataset of CM provides no indication for an event of past population expansion , 4 polymorphic non-synonymous (Pn), 6 fixed synonymous (Ms) and no fixed non-synonymous differences (Mn). Due to the lack of Mn, the McDonald-Kreitman test does not indicate that differences have been subject to positive selection. However, the selective pressure could have operated upon linked genes within the (single-molecule) mitochondrial genome. Moreover, even a significant result for positive selection does not necessarily exclude population expansion, as these differences could have enabled population expansion in the first place.Bayes factor (BF) tests of both datasets favor a scenario of constant population size over a scenario with exponential growth (BFxpansion . Howeverxpansion . GoodnesMOTU1) and CT (MOTU1 and b) the second most widespread lineage of each taxon providing sufficient occurrence data . Results of the RLR approach reveal significant relative probability scores (RPS) for three lineages  and a pooled locality in the region of the Sudetes (RPSBO+RE+SL: 35.9) possess maximal scores. The pooled sample of PL+PO in the Dinaric Alps demonstrates the highest RPS (34.0) for the ancestral locality of CMMOTU4. A West-Alpine pooled locality (LB+LC+LM+LT) demonstrates the maximal RPS (34.3) for CTMOTU1. In the case of CTMOTU5, no clear signal is revealed with four localities showing comparable relative probability scores .The inference of the ancestral locality was conducted for a) the most widely distributed and potentially expanding haplotype lineages of CM ; CMMOTU1lineages . For theCarychium. Our objective was to infer the presence of glacial refugia, events of postglacial recolonization and whether the Alpine Region (Alps+Dinaric Alps) constitutes an origin (‘hot-spot’) or crossroads (‘melting-pot’) of genetic diversity The generalness of hypotheses dealing with the location of glacial refugia, migration routes and the origin of suture zones is challenged by increased recognition of the individuality of species responses to past climatic changes Carychium taxa (MOTU3 (Alps), CMMOTU5 (Southern Alps and Dinaric Alps), CTMOTU3 , CTMOTU7 (Dinaric Alps) and CTMOTU8 (Eastern Alps and Dinaric Alps). Many of these areas were likewise modeled as potential distribution for both taxa at the time of the Last Glacial Maximum (LGM)  and the independent identification of this region in former phylogeographical studies dealing with other species  with limited geographical distribution can be identified for both ium taxa , 4. We eum (LGM) . This waum (LGM) , again fum (LGM)  BA, ER. refugium , the preies e.g.  allows tLimousin .Despite overlapping potential distribution models for both taxa and time periods , the genMOTU1, most likely stretched from the Western Alps, and was able to expand and occupy previously uninhabitable areas in Northern, Western and Southwestern Europe  that are composed of more than one MOTU per species , 4, i.e.r system . A high s region , 7A.Carychium taxa were able to expand into surrounding areas to occupy previously unsuitable regions. The recolonization of northern Europe was only achieved by a single lineage in both taxa.To sum up, our results provide consistent, independent evidence for the Alpine Region (Alps+Dinaric Alps) serving as an origin of genetic diversity (Alpine Region ‘hot-spot’ hypothesis). This condition was most likely achieved through high habitat heterogeneity and varying habitat suitability in this mountainous area Carychium microgastropods. Originally endemic to Europe and Western Asia, populations of CM and CT have been recorded from the Azores and from North America Carychium microgastropods are prone to phenotypic plasticity and taxonomic identifications based on conchology alone are oftentimes inadequate for this taxonomic group Since humanity has moved around the planet, ongoing globalization via migration, urbanization, and air and ship traffic has fostered homogenization of communities while simultaneously obliterating primal phylogeographical patterns Carychium populations. Moreover, these microgastropods are well-known entities of European greenhouses MOTU4), which otherwise is restricted to the broader peri-Alpine region in Europe   potentially indicates a multitude of human-mediated passive dispersal events.The high similarity or identity of haplotypes found on the Azores and in North America to European MOTUs of CM and CT suggest a recent origin , Fig. 3.n Europe , 4A. Altaplotype . Thus, a (MOTU1) , Fig. 4BCarychium populations on the European mainland reached and expanded into their postglacial distributions by either actively migrating or passively dispersing via wind and water Carychium microgastropods actively migrated and were passively dispersed by wind , water , birds and large mammals including humans .In conclusion, passive (anthropogenic) dispersal must be equally considered as a likely factor contributing to the formation of apparent phylogeographical patterns in Europe. However, we can not conclusively verify whether the Table S1th generation. After Bayes Factor (BF) tests for refugium localization reconstruction (RLR) approach. All values had an effective sample size (ESS) greater than 100. At least 10,000,000 generations were run. Tree sampling was conducted each 1,000(DOCX)Click here for additional data file.Table S2Carychium minimum (CM) and Carychium tridentatum (CT). Locality abbreviations correspond to Refugium localization reconstruction for expanding lineages of (DOCX)Click here for additional data file.


Bufo calamita) is distributed across a wide geographic range that qualifies the species as interesting for a geographic analysis of its genetic variability. Five populations of B. calamita in the Sierra de Gredos (Spain) were studied in an altitudinal gradient ranging from 750 to 2270 m using microsatellite markers. In addition, we analyzed the latitudinal genetic variation in B. calamita within a global European distribution using genetic diversity parameters  obtained from our results and those published in the literature. The low level of genetic differentiation found between populations of B. calamita (Fst ranging from 0.0115 to 0.1018) and the decreases in genetic diversity with altitude  can be interpreted by the combined effects of discontinuous habitat, produced mainly by the high slopes barriers and geographic distance. In the latitudinal gradient, genetic diversity decreases from south to north as a consequence of the colonization of the species from the Pleistocene refugium. We conclude that the genetic variability in B. calamita along its wide altitudinal and latitudinal geographic distribution mainly reflects the colonization history of the species after the last glacial period.Across altitudinal and latitudinal gradients, the proportion of suitable habitats varies, influencing the individual dispersal that ultimately can produce differentiation among populations. The natterjack toad ( Genetic variation is required for the evolution of populations in response to environmental changes  is distributed across a wide geographic range  of the southern Iberian Peninsula found no genetic differentiation between populations separated by more than 100 km  evaluate the consequences of mountains as barriers across European distribution, studying its latitudinal genetic variation using genetic diversity parameters obtained from published studies.This study analyses the impact of both altitude and geographic distance, in an effort to expand what is known about B. calamita)   were stualamita)  Fig. 2)B. calamialamita) . Precipialamita) . The breToads were captured at the local breeding ponds during the spring reproduction period. They were released in situ after sex determination, snout-vent length (SVL) measurement, and toe-clipping (third toe of the right hind limb). The toes were stored in 70% ethanol at room temperate. The DNA was extracted from the first phalange of a toe clipped using the Chelex100 protocol described in  and Rowe et al. . Following the methodology used by  in 20 μL of PCR buffer 1×. The PCR amplification was performed by first denaturing at 94°C for 5 min, followed by 40 cycles of 1 min at 94°C, 1 min at 62°C, and 1 min at 72°C. A final extension step was carried out by incubating samples during 10 min at 72°C. Amplified products were resolved by capillary gel electrophoresis on a Genetic Analyser 3130  using POP-7 polymer. Allele sizes were determined using GeneScan-500 LIZ standard marker . Genotype calls were obtained using GeneMapper Software v. 4.0.Genetic analysis was based on the study of eight microsatellite loci previously described by Rowe et al.  , and expected (HE) and observed (Ho) heterozygosity were estimated using the Microsatellite Toolkit (Park AR) was obtained for each population using FSTAT v. 2.9.3  and geographic and altitudinal distance matrices using the PASSAGE v.2. software  for the accurate estimation of Mantel test statistics.The analysis of genetic structure was made with an initial comparison within and among the three sampling regions  less than 1000 m: Nav750 and Deh920, (ii) between 1000 and 2000 m: LaC1470, and (iii) more than 2000 m: Cav2100 and Nas2300). The data were analyzed with a molecular analysis of variance (nested AMOVA) using ARLEQUIN v. 3.1  and the expected heterozygosity  were used as estimators of genetic diversity. The influence of latitude on the Ma and on the HE was estimated by fitting data in two-simple regression models: the lineal model and the exponential model, which represent two predefined hypothesis. The lineal model is expected when genetic diversity decrease constantly along the latitudinal gradient following an isolation by distance pattern. However, the exponential model is expected in the presence of barriers bursting the continuous decrease in genetic diversity from the glacial refuge across the latitudinal gradient. We also analyzed the data separately between the populations from Spain  and those past the Pyrenees  in order to evaluate the expansion of the species. The analysis was performed using the statistical package STATGRAPHICS Plus 5.0.In addition to data obtained in this study, Bcalμ7 to 36 for Bcalμ4. We identified a total of 174 different alleles; 32 were present in all populations  and 72 were observed in unique populations . HE ranged from 0.44 at Cav2100 to 0.93 at Nav750 . The low deviation from linkage equilibrium observed could be interpreted as a relaxed population structure.The eight microsatellite markers used were polymorphic in all populations and the number of alleles per locus varied from 14 for HE  with geographic distance according to the hypothesis of isolation by distance. In addition, Fst  correlated inversely with altitude. In contrast, the results from AMOVA indicated a lack of population structure, with a 94.95% of overall variation within populations . The landscape features seem to be adequate for gene flow, influencing genetic variations within and between populations  genetic variability decreases in the latitudinal gradient. These results are consistent with the hypothesis that the Iberian Peninsula was a refuge for B. calamita during the Pleistocene, where all populations expanded from the south to the north during the postglacial period (Beebee and Rowe HE correlated strongly with the geographic distance measured from south Spain. Our new data show that this correlation is not significant in the Iberian Peninsula that present a high genetic diversity; although it is strongly notable from the barrier of the Pyrenees to the north.As we noted, the B. calamita showed throughout its geographic distribution area mainly reflects the colonization history of the species after the last glacial period. This was suggested by previous studies in the latitudinal gradient (Beebee and Rowe In conclusion, the pattern of genetic diversity of