Mimulus guttatus, in Copperopolis, California, which recently evolved tolerance to soil contaminants from copper mines and simultaneously, as an incidental by-product, hybrid lethality in crosses with some off-mine populations. However, in new work, Wright and colleagues show that hybrid lethality is not a pleiotropic consequence of copper tolerance. Rather, the genetic factor causing hybrid lethality is tightly linked to copper tolerance and spread to fixation in Copperopolis by genetic hitchhiking.The modern evolutionary synthesis codified the idea that species exist as distinct entities because intrinsic reproductive barriers prevent them from merging together. Understanding the origin of species therefore requires understanding the evolution and genetics of reproductive barriers between species. In most cases, speciation is an accident that happens as different populations adapt to different environments and, incidentally, come to differ in ways that render them reproductively incompatible. As with other reproductive barriers, the evolution and genetics of interspecific hybrid sterility and lethality were once also thought to evolve as pleiotripic side effects of adaptation. Recent work on the molecular genetics of speciation has raised an altogether different possibility—the genes that cause hybrid sterility and lethality often come to differ between species not because of adaptation to the external ecological environment but because of internal evolutionary arms races between selfish genetic elements and the genes of the host genome. Arguably one of the best examples supporting a role of ecological adaptation comes from a population of yellow monkey flowers,  New species arise when populations gradually evolve intrinsic reproductive barriers to interbreeding with other populations Origin of Species to the first problem: as the sterility or lethality of hybrids provides no advantage to parents, how could the genetic factors involved possibly evolve by natural selection? The second problem was recognized much later AA and aa) produce, say, sterile hybrids (Aa) due to an incompatibility between the A and a alleles, then how could, e.g., the AA genotype have evolved from an aa ancestor in the first place without passing through a sterile intermediate genotype (Aa)? Not only does natural selection not directly favor the evolution of hybrid sterility or lethality, but there is reason to believe natural selection positively prevents its evolution.Most reproductive barriers arise as incidental by-products of selection—either ecological adaptation or sexual selection. For these cases, the genetic basis of speciation is, effectively, the genetics of adaptation. But hybrid sterility and lethality have historically posed two special problems. Darwin aabb), with the A allele becoming established in one population (AAbb) and the B allele in the other (aaBB); while A and B alleles must function on their respective genetic backgrounds, there is no guarantee that the A and B alleles will be functionally compatible with one another. Hybrid sterility and lethality most likely result from incompatible complementary genetic factors that disrupt development when brought together in a common hybrid genome. Dobzhansky Together these problems stymied evolutionists and geneticists for decades. T.H. Huxley Today, there is no doubt that the Dobzhansky-Muller model is correct, as the data for incompatible complementary genetic factors is now overwhelming Mimulus guttatus, from Copperopolis . In the last ∼150 years, the Copperopolis population has evolved tolerance to the tailings of local copper mines , Wright et al. Tol1 and Nec1, respectively. Hybrid lethality is not a pleiotropic consequence of copper tolerance. Instead, the tolerant Tol1 allele spread to fixation in Copperopolis, and the tightly linked incompatible Nec1 allele spread with it by genetic hitchhiking. In a turn of bad luck, the loci happen to fall in a heterochromatic pericentric region, where genome assemblies are often problematic, putting identification of the Tol1 and Nec1 genes out of immediate reach. Wright et al. Tol1 and place Nec1 within a 10-kb genomic interval that contains a Gypsy3 retrotransposon, raising two possibilities. First, the Gypsy3 element is unlikely to cause hybrid lethality directly; instead, as transposable elements are often epigenetically silenced in plants, it seems possible that the Nec1-associated Gypsy3 is silenced with incidental consequences for gene expression on a gene (or genes) in the vicinity Nec1 interval is 10-kb in the reference genome of M. guttatus, it could be larger in the (not-yet-sequenced) Copperopolis population, perhaps harboring additional genes.In this issue, Wright and colleagues Tol1 and Nec1 mapped near and to particular genomic scaffolds, respectively, Wright et al. were able to investigate the evolutionary history of the genomic region. Given the clear adaptive significance of copper tolerance in Copperopolis plants, we might expect to see the signatures of a strong selective sweep in the Tol1 region—a single Tol1 haplotype may have spread to fixation so quickly that all Copperopolis descendant plants bear the identical haplotype and thus show strongly reduced population genetic variability in the Tol1-Nec1 region relative to the rest of the genome Tol1 reflects an adaptation to mine tailings established just ∼150 generations ago, there would have been little time for such a recovery. And yet, while Wright et al. find evidence of moderately reduced genetic variability in the Tol1-Nec1 genomic region, the magnitude of the reduction is hardly dramatic relative to the genome average.With Tol1-Nec1 region swept to fixation in Copperopolis in fewer than ∼150 generations and yet left no strong footprint of a hitchhiking event? One possibility is that rather than a single, unique Tol1-Nec1 haplotype contributing to fixation, causing a “hard sweep," multiple Tol1-Nec1 haplotypes sampled from previously standing genetic variation contributed to fixation, causing a “soft sweep" Tol1 and Nec1 both segregate in the local off-mine ancestral population and if the two were, coincidentally, found on the same chromosome more often than expected by chance . Then, after the copper mines were established, multiple plants with multiple Tol1 haplotypes  could have colonized the newly contaminated soils of the mine tailings. Tol1 segregates at ∼9% in surrounding populations, suggesting that standing genetic variation for copper tolerance may well have been present in the ancestral populations.How, then, is it possible that the Tol1-Nec1 story, and both would be readily advanced by identification of Tol1 and Nec1. The first question concerns the history of Tol1 haplotypes in Copperopolis and surrounding off-mine populations. As Nec1-mediated hybrid lethality is incomplete, the ∼9% Tol1 frequency in surrounding populations could reflect its export via gene flow from the Copperopolis populations. Conversely, if there was a soft sweep from standing Tol1 variation in surrounding off-mine populations, then Tol1 and Nec1 may still be in linkage disequilibrium in those populations (assuming ∼150 years of recombination has not broken up the association). Resolving these alternative possibilities is a matter of establishing the history of movement of Tol1 haplotypes into or out of the Copperopolis population. The soft sweep scenario, if correct, presents a population genetics puzzle: during the historical time that mutations accumulated among the multiple tolerant but incompatible Tol1-Nec1 haplotypes in the ancestral off-mine populations, why did recombination fail to degrade the association, giving rise to tolerant but compatible haplotypes?Two big questions remain for the Nec1 . The answer bears on one of the new emerging generalizations about genetic incompatibilities in plants ArabidopsisNec1-mediated lethality provides yet another instance.The second question concerns the identity of


Nacella concinna) sampled from Adelaide and Galindez Islands near the base of the Antarctic Peninsula. Three sites within Ryder Bay, Adelaide Island  were each sub-sampled three times, yielding a total of 405 samples that were genotyped at 155 informative Amplified Fragment Length Polymorphisms (AFLPs). Contrary to our initial expectations, limpets from Anchorage Island were found to be subtly, but significantly distinct from those sampled from the other sites. This suggests that local processes may play an important role in generating fine-scale population structure even in species with excellent dispersal capabilities, and highlights the importance of sampling at multiple spatial scales in population genetic surveys.Several recent empirical studies have challenged the prevailing dogma that broadcast-spawning species exhibit little or no population genetic structure by documenting genetic discontinuities associated with large-scale oceanographic features. However, relatively few studies have explored patterns of genetic differentiation over fine spatial scales. Consequently, we used a hierarchical sampling design to investigate the basis of a weak but significant genetic difference previously reported between Antarctic limpets ( A classical paradigm in marine population biology is that broadcast-spawning species exhibit little or no genetic structure relative to otherwise ecologically equivalent brooders Nacella concinna, one of the most abundant and widespread of all shallow-water Antarctic marine macro-invertebrates Fst = 0.003–0.007, P<0.05) from the other Peninsula sites.An interesting case in point is provided by the Antarctic limpet, The genetic distinctness of Adelaide Island poses a conundrum due to the absence of any obvious oceanographic barriers to gene flow in this region. One possible explanation could be that larval transport between Galindez and Adelaide Islands is restricted by some form of previously overlooked, large-scale hydrological barrier such as a gyre or eddy system. This is plausible given that the Antarctic Circumpolar Current runs northwards along the Peninsula, the Antarctic Coastal Current runs southwards closer to the shore and there are indications of a series of semi-isolated gyres between the two N.concinna, we designed a hierarchical sampling strategy embracing three separate regions within Ryder Bay, each sub-sampled three times to facilitate the detection of any potential fine-scale differences  approach of Benjamini and Hochberg The N.concinna within Ryder Bay, Adelaide Island, a total of 405 individuals from nine sites were genotyped at seven selective AFLP primer combinations . Fst values obtained in pairwise comparisons among the sites were also low (P<0.01 and one at P<0.05) and a further seven approached significance (0.08<P<0.05). Following table-wide FDR correction for multiple tests Fst values remained significant at P<0.05. No relationship was observed between the shortest geographic distance by sea and genetic distance . However, significant P-values were only encountered among pairwise comparisons involving sites from Anchorage Island, an imbalance that is itself statistically significant .Population genetic differentiation was weak but statistically significant overall  near the base of the Antarctic Peninsula. Limpets from Anchorage Island were found to be subtly, but significantly distinct from those sampled from the other sites, with implications for understanding the population genetic connectivity of marine species and more generally for the design of population genetic surveys.In this study, we combined a highly informative panel of AFLPs with a hierarchical sampling design within Ryder Bay to explore the fine-scale population structure of Antarctic limpets  and hence that statistical significance may only have been reached due to a combination of large sample sizes of individuals (average n = 45) and polymorphic loci (n = 155). The demographic consequences of such subtle genetic differences can be difficult to assess Fst, although this should decrease with increasing numbers of markers. Nevertheless, there is good reason to believe that the differences we report are genuine. First, efforts were made to drive the genotyping error rate downwards by scoring only bands that could be clearly distinguished as either present or absent in all individuals, resulting in an overall error rate of only 0.011 per reaction. Second, not only was overall Fst significant in a global permutation test, but also all but one of the significant values obtained in pairwise population comparisons were robust to table-wide FDR correction. Third, if most or all of the significant tests obtained were attributable to Type I error, they should be distributed randomly, which was clearly not the case in this study. Individually significant Fst values were only found in pairwise comparisons involving Anchorage Island  and this pattern became even stronger when marginally significant P-values (0.08<P<0.05) were taken into account .Antarctic limpets are prolific broadcast-spawners with relatively long-lived planktotrophic veliger larvae and high larval densities Fst = 0.028 and 0.114 respectively), consistent with their being separated by deep ocean channels that may act as a barrier to larval transport. In contrast, five sites sampled from Antarctic Peninsula  were genetically indistinguishable from one another, yielding Fst values of zero in all ten pairwise comparisons. This further reinforces the assertion that significant positive values obtained in comparisons involving Anchorage Island are genuine.It is also helpful to consider our results in the context of a previous study spanning most of the Antarctic Peninsula and the islands of Signy and South Georgia N. concinna populations at Potter Cove, King George Island, on the South Shetland Islands, although these primarily involved comparisons between the intertidal and subtidal morphs, which differ markedly from one another in shell size and physiology Fst values almost an order of magnitude higher than those found in this study, despite sample sizes being far smaller . However, two subsequent studies conducted independently at other localities found no genetic differences between intertidal and subtidal N. concinna individuals using larger panels of AFLPs A previous study by de Aranzamendi et al. Although relatively few studies have explored the potential for fine-scale genetic structure in Antarctic marine organisms, the limited evidence available suggests that straits of deep water as narrow as 30km can significantly impact population connectivity Haliotis rubra, tidal currents may be sufficient to impart fine-scale population structure due to an unusual behavioural pattern in which the larvae synchronise swimming with periods of low or no water flow N. concinna and, unless tidal flows differ markedly across Ryder Bay, this would in any case be expected to generate uniform fine-scale structuring. A second possibility is that coastal eddies around Anchorage Island could be advecting larvae back towards the shore, a mechanism invoked to explain local ‘hot spots’ of larval retention encountered in computer simulations N. concinna spawns in the early austral summer, with larvae present in the water column throughout late summer and early winter The second possible mechanism based on local currents is more difficult to evaluate, but could potentially involve different types of water flow. The first of these, tidal currents, move particles relatively short distances, in the order of hundreds of metres along the shore and back again. In the abalone A third possibility relates to the fact that permanent ice cover and ice scour can profoundly impact communities of relatively sessile benthic Antarctic organisms. Coastal glaciers and ice shelves have been retreating for some time along the Antarctic Peninsula Finally, it is important to note that temporal stochasticity has also been implicated as a factor that could drive fine-scale population structure in planktonically dispersing marine invertebrates Using a large panel of AFLP markers, we detected weak fine-scale population genetic structure among Antarctic limpets sampled from Ryder Bay. Although the underlying mechanisms as yet remain unclear, our study emphasises the importance of sampling over multiple spatial scales. It also contributes to a growing list of factors including differential microhabitat use


Efforts have been made to eliminate wild poliovirus transmission since 1988 when the World Health Organization began its global eradication campaign. Since then, the incidence of polio has decreased significantly. However, serotype 1 and serotype 3 still circulate endemically in Pakistan and Afghanistan. Both countries constitute a single epidemiologic block representing one of the three remaining major global reservoirs of poliovirus transmission. In this study we used genetic sequence data to investigate transmission links among viruses from diverse locations during 2005-2007.In order to find the origins and routes of wild type 1 poliovirus circulation, polioviruses were isolated from faecal samples of Acute Flaccid Paralysis (AFP) patients. We used viral cultures, two intratypic differentiation methods PCR, ELISA to characterize as vaccine or wild type 1 and nucleic acid sequencing of entire VP1 region of poliovirus genome to determine the genetic relatedness.One hundred eleven wild type 1 poliovirus isolates were subjected to nucleotide sequencing for genetic variation study. Considering the 15% divergence of the sequences from Sabin 1, Phylogenetic analysis by MEGA software revealed that active inter and intra country transmission of many genetically distinct strains of wild poliovirus type 1 belonged to genotype SOAS which is indigenous in this region. By grouping wild type 1 polioviruses according to nucleotide sequence homology, three distinct clusters A, B and C were obtained with multiple chains of transmission together with some silent circulations represented by orphan lineages.Our results emphasize that there was a persistent transmission of wild type1 polioviruses in Pakistan and Afghanistan during 2005-2007. The epidemiologic information provided by the sequence data can contribute to the formulation of better strategies for poliomyelitis control to those critical areas, associated with high risk population groups which include migrants, internally displaced people, and refugees. The implication of this study is to maintain high quality mass immunization with oral polio vaccine (OPV) in order to interrupt chains of virus transmission in both countries to endorse substantial progress in Eastern-Mediterranean region. Since 1988 the world has come very close to eradicate polio through global polio eradication initiative . HoweverThe Polio Eradication Initiative was launched in Pakistan in 1994 with remarkable success, the number of confirmed cases declined from 1015 in 1997 to 32 wild polio cases including 19 WPV1 and 13 WPV 3 in only 18 of 120 districts of Pakistan ,7. In AfDespite intensified mass immunization activities, the two countries failed to interrupt poliovirus transmission. Persistent pockets of polio transmission along the border between Afghanistan and Pakistan are key epidemiological challenges due to insecurity and continued conflict ,11.PV1, PV2, and PV3; each with a slightly different outer capsid protein which define cellular receptor specificity and virus antigenicity. Therefore infection with one serotype does not prevent infection with another serotype [PV1 is the most common type encountered in nature, however all three types are extremely infectious and can circulate independently [-2 substitutions/site/year [Poliovirus, the causative agent of poliomyelitis, is a human enterovirus and member of family Picornaviridae . It is cserotype ,15. PV1 endently ,17. Longendently -20. The ite/year ,22 whichite/year ,24. Geneite/year ,26.In current study 111 wild type1 poliovirus isolates collected during 2005-2007 from Pakistan and Afghanistan were sequenced for complete VP1 gene to study their genetic diversity and to reveal the indigenous genotype in this region that would be helpful to track the transmission patterns in Pakistan and Afghanistan being a single epidemiological block.Stool samples of children with AFP  and from apparently healthy contacts were collected as per WHO guidelines within 14 days of paralysis onset and were sent to WHO Regional Reference Laboratory for Polio Eradication Initiative, Department of Virology, National Institute of Health, Islamabad, Pakistan.These samples were processed for isolation and identification of poliovirus by standard techniques as described in World Health Organization manual . BrieflyTo differentiate Sabin (SL1) and Wild type (NSL1) Polioviruses, PCR and ELISA were used according to WHO recommendations ,31.®, Applied Biosystems) with the overlapping primers  and subsequently stored in aliquots at-70°C. Reverse transcription Polymerase chain reaction (RT-PCR) was performed in single step of 50 μl reaction volume. RT-PCR was used to amplify the entire VP1 capsid protein coding region of 906 nucleotides. The primers used in these reactions listed in  which provides different methods to edit and align sequence fragments into larger structure (contigs). Phylogenetic analysis of wild type 1 polioviruses was carried out by using Molecular Evolutionary Genetic Analysis (MEGA v.4) . EvolutiA total of 25,965 and 4,485 AFP cases including contact cases from Pakistan and Afghanistan respectively were processed during 3 years time period and 111 wild type1 polioviruses were detected as shown in  corresponding lineages  different lineages  isolates, represents a hypothetical genetic founder.As a result all 111 isolates were grouped into three major clusters A, B, & C respectively. Within these clusters, a definite pattern of virus distribution was observed representing lineages i.e., cluster s Figure  and cluss Figure . While ta, b, c, d, e & f  of district Rodat, one isolate AFG/01/06/562 from (Baghlan) district Puli Khumri, three isolates AFG/08/06/508,541,543 from (Helmand) district Bust and two isolates from this lineage AFG/08/06/461,532 from kandhar with districts SpinBoldak and Maywand. Similarly in Pakistan during same period, polio wild type 1 virus circulation was seen in Killa Abdullah and Quetta districts , Bajour district (KP) and Karachi Korangi district (Sindh) while in 2007, Kambar & Thatta (Sindh); Lasbela ; Nowshera, South Waziristan & Khyber (KP) were infected. However, no strain of this lineage was observed during 2005-2007 in Punjab.b'  which has sequence similarity with the 2004 wild type 1 polioviruses Figure . The corThe covert circulations of 11 wild orphan polioviruses (isolates with ≥ 2% sequence diversity with their closely related isolate) have been observed in several districts/provinces of both countries Table . During et al., has been detected multiple genotypes of wild type 1 polioviruses in Karachi district of Sindh province in Pakistan [The data presented here illustrates molecular characterization of wild type 1 polioviruses endemic in Pakistan and Afghanistan during last 3 years, 2005-2007. The sequence of VP1 gene was employed to determine the epidemiological links among the wild type 1 polioviruses circulating across the both countries. The VP1 gene comprised of 906 nucleotide bases with sufficient heterogeneous motifs that clearly discriminate between various genotypes. In 1993 Huovilianen Pakistan . HoweverPakistan ,41.Despite this monophyletic character, distinctive reservoirs of wild type 1 polioviruses have been observed that confined to specific geographic regions in both countries. Based on sequence diversity of ≤ 5% among the VP1 nucleotide sequence, the wild type 1 polioviruses have been classified into certain groups called 'clusters'. A phylogenetic tree was reconstructed on the genetic relationship of VP1 gene sequence for the wild polioviruses detected during the 3 year period which clearly displayed the distribution of isolates among three clusters  Figure . Within The molecular data obtained from this study explicitly reveals the transmission pattern of wild type 1 polioviruses circulation in Pakistan and Afghanistan as a successful tool to monitor the AFP surveillance as well as to formulate the mop-up immunization activities established as a reference standard policy to interrupt the wild poliovirus multiplicity within population. The underlying factors posing a incessant threat to achieve the polio-free country status are the areas with poor security and hard to reach children communities leaving a number of children unimmunized despite a number of supplementary immunization campaigns in the country. Furthermore, parallel circulation of some lineages/multiple chains among both countries highlights the ongoing continuous presence of wild polioviruses.et al. [The factors behind this worrisome situation are attributed to the security concerns and also to the unchecked cross-border movement as well. The similar concern has already been shown by Kew., et al. . Howeveret al. -44. But Current molecular data also explores the evolutionary pattern of wild type 1 poliovirus isolates in relation to their ancestral descent. Based on the 1% (in vivo) nucleotide evolutionary rate of VP1 gene, any wild poliovirus isolate indicating equal to or more than 2% nucleotide sequence divergence from its ancestor is recognised as an "orphan virus" . FindingMoreover, epidemiological records and results of this study clearly highlights that transmission of wild type 1 polioviruses is still uncontrolled. However, significant progress has been made to localize the wild poliovirus. The number of infected districts has been significantly decreased from 2005 to 2007 ; MMA contributed in write-up, overall coordination; SaS contributed in data collection; AK participated in laboratory work (Virology testing); SZ participated in the study conception and design and all study was performed under his supervision. All authors read and approved the final manuscript.


Phelipanche ramosa is a parasitic plant that infects numerous crops worldwide. In Western Europe it recently expanded to a new host crop, oilseed rape, in which it can cause severe yield losses. We developed 13 microsatellite markers for P. ramosa using next-generation 454 sequencing data. The polymorphism at each locus was assessed in a sample of 96 individuals collected in France within 6 fields cultivated with tobacco, hemp or oilseed rape. Two loci were monomorphic. At the other 11 loci, the number of alleles and the expected heterozygosity ranged from 3 to 6 and from 0.31 to 0.60, respectively. Genetic diversity within each cultivated field was very low. The host crop from which individuals were collected was the key factor structuring genetic variation. Individuals collected on oilseed rape were strongly differentiated from individuals collected on hemp or tobacco, which suggests that P. ramosa infecting oilseed rape forms a genetically diverged race. The microsatellites we developed will be useful for population genetics studies and for elucidating host-associated genetic divergence in P. ramosa. Orobanche ramosa L. Pomel), is the most widespread species. It occurs throughout the South-East of Europe, West Asia and North Africa and was accidentally introduced in several other parts of the worlds . It is a noxious weed that infects numerous crops, among which tobacco, tomato and hemp, particularly in countries surrounding the Mediterranean basin and in Central Europe [P. ramosa to oilseed rape fields has been observed since the beginning of the 1990s [Among flowering plants, approximately 3000 species 1%) are parasitic. These parasitic plants form a close connection with the vascular system of their host plant through a specialized organ, the haustorium, through which they remove water, mineral salts, and carbon elements % are par. Broomral Europe . From thl Europe . In westhe 1990s ,7, causihe 1990s ,8.P. ramosa have been fairly limited up to now. All previous studies used dominant markers, either RAPD [P. ramosa, which might be associated with host specificity. Among the molecular markers available to study genetic variation in natural plant populations, microsatellites or SSRs (simple sequence repeats) are especially valuable because of their high reproducibility, high degree of polymorphism and codominant inheritance. Despite the economic importance of broomrapes, up to now SSRs have been developed for only one species, the sunflower broomrape Orobanche cumana [P. ramosa and investigate whether these markers reveal some genetic divergence in relation with the host crop, and especially with the ability to infect oilseed rape.Investigations of the extent of genetic variation in her RAPD ,9, ISSR her RAPD  or AFLP her RAPD . Their re cumana . Here, w2.2.1.The 454-sequencing data retrieved from the Sequence Read Archive (SRA) contained a total of 1,516,538 reads, with an average read length of 563 bp. After quality filtering, we retained a reduced set of 172,364 reads (11%), with a mean length of 395 bp (range: 250–483 bp). After discarding sequences without microsatellite motifs using the QDD software, the remaining sequences were filtered for redundancy and multiple copies in the sequence data set. In total, 2091 microsatellite loci were identified, and automated primer design was successful for 425 loci.We selected 63 primers pairs with highest quality based on a low self-end complementarity and high end-stability of the primers. PCR amplification was assayed using 6 DNA samples. After these initial PCR assays, 13 microsatellites markers reliably yielding a single PCR product were retained. Based on Sanger sequencing, these markers were all confirmed to contain the expected microsatellite motif.2.2.P. ramosa individuals from six populations. Among the 13 loci, 2 were found to be monomorphic  ranged from 0.000 to 0.025 and the expected heterozygosity (He) ranged from 0.308 to 0.601 , while one single genotype was predominant in hemp (frequency 0.79), and one single genotype was also predominant in oilseed rape (frequency 0.82). Finally, a multivariate analysis of the genetic variation at the eleven polymorphic microsatellites perfectly distinguished among st crops . The firGeographic effects might have contributed to the genetic structure observed. However, this seemed unlikely here. Population PO (hemp) was localized in the north-east of France, more than 400 km away from the other five populations, which were all localized in the same area (West of France), at less than 50 km from one another . Thus ge3.3.1.P. ramosa that had severely infected hemp, oilseed rape and tobacco fields in 2001 and 2002. The plants sampled were distributed throughout each cultivated field. Geographic origins of the populations are shown in Parasite seeds were collected from French natural populations of 3.2.P. ramosa genome was retrieved from the NCBI Sequence Read Archive (http://www.ncbi.nlm.nih.gov/sra). The experiment was reported in [Raw sequence data for the orted in . It conshttp://edwards.sdsu.edu/prinseq) [After converting raw sequence data to the fastq format, quality control and filtering was performed using the PRINSEQ web (prinseq) . BrieflyMicrosatellites were identified by running the QDD pipe-line  using th3.3.2, 17 mM (NH4)2SO4, 10 mM β-mercaptoethanol, 0.05% (w/v) polyoxyethylene-ether W1, 0.2 mg/mL bovine serum albumin, 200 mM of each dNTP, 10 ng genomic DNA, 0.5 units of Taq DNA polymerase, and 0.2 μM each of reverse and forward primers. The PCR program used consisted of 5 min at 95 °C, followed by 37 cycles of 5 s at 95 °C, 10 s at 60 °C and 30 s at 72 °C. Amplicons were visualised under UV light by electrophoresis on a 3% (w/v) agarose gel stained with ethidium bromide. When a single, intense amplicon was obtained from all 6 plants, it was sequenced on both strands to confirm the presence of the expected microsatellite motif.PCR amplification was first assayed on a subsample of 6 individuals. PCR amplifications were performed using a Mastercycler  thermocycler, in a 20 μL reaction mix containing 70 mM Tris-HCL, 2 mM MgClFor genotyping, the DNA extracts of all plants were diluted 50-fold prior to genotyping with fluorescent dye-labelled markers . PCR products were assayed on an ABI 3730XL sequencer  using GeneScan 500 LIZ dye size standard (Applied Biosystems). Amplicon sizes were analyzed with the software Peak Scanner 1.0 (Applied Biosystems).3.4.Na) per locus, observed heterozygosity (Ho), expected heterozygosity (He), and inbreeding coefficient (Fis) were estimated using GenAlex 6.5 [The number of alleles (Alex 6.5 . OverallAlex 6.5 .4.Phelipanche ramosa. Eleven out of the 13 markers were polymorphic on a set of 96 individuals of P. ramosa sampled in France. The level of polymorphism was low, especially within populations. This low level of variation may be explained by a high selfing rate in this species and possibly by bottlenecks associated with founding events for the studied populations. Genetic variation was strongly structured by the three host crops on which individuals were collected: tobacco, hemp and oilseed rape. Our results suggest that P. ramosa infecting oilseed rape in France belong to a recently evolved but highly genetically divergent host race. Analyzing more populations collected from different hosts will be necessary to confirm this. The microsatellites developed here will be useful for future population genetics studies in P. ramosa and for elucidating the presence of genetically diverged host races.We report the development and characterization of 13 microsatellites markers in the branched broomrape,


Recent data support the beneficial role of gesturing during mental practice. The present study examined whether coupling motor imagery (MI) with some movement sequences (dynamic imagery condition) impacted motor performance to a greater extent than performing MI while remaining motionless.A group of active high jumpers imagined their jump both with and without associated arm movement. Three outcome variables were measured: the number of successful attempts, the temporal congruence between MI and actual jump performance, and the technical quality of the jump.Data revealed that dynamic imagery enhanced both MI quality and temporal congruence between MI and motor performance, and further improved the technical efficacy of the jump. Athletes also reported more vivid representation while coupling MI with actual movement.These data support the hypothesis that performing dynamic imagery might contribute to enhance MI quality and efficacy, and sketch potentially fruitful new directions for MI practice. Motor imagery (MI) is one of the remarkable capacities of the mind enabling everyone to mentally simulate an action without engaging in actual physical execution . MI and Among others, motor execution issues during MI should be close to those related to actual execution, that is, the spatial and temporal characteristics should be preserved during MI . For insduring imagined actions. Practically, enhancing imagery effectiveness by incorporating movement during MI has been promoted early on in applied studies [With reference to the definition we gave in the above paragraphs, the participants are usually requested to perform MI in the absence of movement during mental rehearsal sequences . A large studies ,28, and  studies . For ins studies  stated t studies  investig studies  later pr studies . A more  studies . Specifi studies , we are The present study was designed to determine whether coupling MI with actual movement (dynamic imagery) impacts motor performance to a greater extent than performing MI without any overt body movement, and further contributes to achieve the temporal congruence between MI and motor performance. Based on the assumption that MI and overt movement are intimately related one to another both at the behavioral ,26 and nWe used a within-subjects design allowing reduction in error variance associated with individual differences. However, to avoid that participation in one imagery condition affects performance in the other imagery condition, all MI trials were scheduled in a random order.A sample of high jumpers was recruited for the experiment as this motor skill is appropriate for imagery training , and eliA first session was scheduled one week before the experiment to select the athletes and determine their knowledge about MI. In particular, they were questioned about the frequency and the nature of their imagery use, in order to exclude athletes who regularly performed imagery routines including movements, and/or who would have been unfamiliar with motionless imagery. Participants were also given descriptions of internal visual imagery, external visual imagery, and kinaesthetic imagery, and were advised that they will be able using kinaesthetic imagery in combination with whichever visual support they found beneficial. All athletes self-reported using more frequently and easily internal visual imagery along with kinaesthetic imagery. This combination was therefore considered in the imagery scripts (see below).very hard to see” to 7 = “very easy to see”), the ability to perceive the sensations usually elicited by the movement during kinaesthetic imagery with the second (from 1 = “very hard to feel” to 7 = “very easy to feel”). The MIQ-R has demonstrated adequate reliability and validity with alpha coefficients of .79 for both subscales.The individual imagery ability of the participants was evaluated to ensure that the sample did not include athletes with extremely high or low imagery ability, which could have influenced the capacity to imagine in real time. The revised version of the Movement Imagery Questionnaire  was commotionless imagery), the other five trials being coupled with actual movement (dynamic imagery), i.e. athletes were asked to freely mimic the actual jump phases with their arms. All participants performed MI in a position which was compatible with the motor skill, i.e. in a standing position. Practically, we define dynamic imagery as a specific sequence of MI which is accompanied by external movements miming in part those which are mentally represented, in particular specific movement features related to temporal or spatial invariance. Athletes were thus asked to mimic the actual movement using simple upper-limb movements, but without engaging in the actual motor act and while keeping the lower limbs motionless. The main functional aim of associating movement to MI was to provide sensory feedback including somatosensory inputs likely to facilitate the mental representation of the jump, and further help participants to formalize the spatio-temporal configuration of the movement. Specifically, movements performed during MI brought temporal boundaries, and thus had a temporal function to facilitate calibration of time. Finally, lower-limb movements were excluded to limit the intensity of the motor act during the mental representation . They were also asked to report the experimental condition in which they formed more easily mental images of the movement.Finally, individual debriefings were scheduled to control that MI instructions were respected, and to determine whether participants encountered difficulty in forming mental images. Accordingly, participants were required to rate the vividness of the mental images they attempted to form during each condition, using a Likert-type scale (from 1 = “2) for ANOVA results are also provided. The results are presented as a mean (standard deviation), and a level of p < 0.05 was considered critical for assigning statistical significance.There was no significant gender difference on MI and actual times or on any rating provided by the two experts, and thus there was no need to subdivide the group by gender. One-way repeated measures analyses of variance (ANOVA) with two conditions  were performed to compare chronometric data. Repeated measures ANOVAs were also computed to compare the number of successful jumps (“hit/miss”), the overall technique score, and expert ratings in each experimental condition. Data were then split into hits and misses and an additional 2 (Dynamic vs. Motionless imagery) x 2 (Hit vs. Miss) repeated measure ANOVA was performed to compare actual and MI times. Effect sizes, including partial eta-squared  = 20.02, p < 0.001, η2 = .64). There was no participant with extremely high or low MIQ-R score (two SD above or below the mean), and current MIQ-R mean scores were comparable to those observed in previous MI studies [Mean MIQ-R scores (SD) were 39.50 (3.90). Mean score was 21.58 (1.98) for the visual subscale, and 17.92 (2.78) for the kinaesthetic subscale. Visual scores were higher than kinaesthetic scores  = 24.72, p < 0.001, η2 = 0.69, Figure F = 0.02, p = 0.91, η2 = 0.001, Figure When MI was performed while remaining motionless, mean MI time was 5.12 s (0.88), and mean corresponding actual time was 4.24 s (0.78). The ANOVA revealed that this difference reached significance  = 8.98, p = 0.003), but no effect of performance  = 0.97, p = 0.33) nor significant imagery condition x performance interaction  = 0.10, p = 0.75).An additional 2 (Dynamic vs. Motionless imagery) x 2 (Hit vs. Miss) repeated measure ANOVA was performed to compare actual and MI times after splitting jump performance into hits and misses. Data revealed a main effect of the imagery condition  = 4.97, p < 0.05, η2 = 0.31). Mean ratings for the approach item were 8.48 from 10 (0.49) during dynamic imagery and 8.33 (0.53) during motionless imagery  = 9.58, p < 0.005, η2 = 0.47). Likewise, greater scores were obtained during dynamic imagery than during motionless imagery for the curve item  = 9.73, p < 0.005, η2 = 0.47), mean scores being 8.11 (0.65) and 7.89 (0.68), respectively. Greater scores were also found during dynamic imagery than during motionless imagery for the impulsion item  = 3.91, p = 0.04, η2 = 0.26), mean scores being 7.75 (0.70) and 7.58 (0.72), respectively. Finally, greater scores were obtained during dynamic imagery than during motionless imagery for the bar clearance item  = 3.50, p < 0.05, η2 = 0.24), respective mean scores being 7.93 (0.83) and 7.76 (0.78).Evaluating motor performance in terms of hit and miss revealed a higher percentage of success during dynamic imagery (45%) than during motionless imagery 35%). Comparing expert ratings measuring the quality of the jump then revealed systematic greater performance during dynamic imagery compared to motionless imagery  = 14.19, p < 0.003, η2 = 0.56).During the debriefings following MI sessions, all participants reported using motor imagery as outlined in the scripts. All participants combined internal visual, auditory, and kinaesthetic imagery without switching to external visual imagery, nor changing the imagery script to suit individual needs. Accordingly, they were able to report the series of movements with an explicit knowledge of each key-component of the physical execution. Finally, respective mean ratings given by athletes when evaluating the vividness of MI were 4.67 (0.98) during dynamic imagery and 3.58 (1.24) during motionless imagery. The ANOVA revealed that the difference reached significance , as well as time lack which prevented us from spanning the study over a longer period during the new competition period, we were not able including a control condition. Including a condition with performance of actual movement without any MI may thus be particularly helpful in future studies.The present within-subjects design showed that performing dynamic imagery might be of interest in a sample of athletes. Firstly, integrating some movements during MI may contribute to improve the ability to achieve temporal congruence between MI and actual performance, which has been shown to positively influence the efficacy of MI. Secondly, data tend to suggest that the technical efficacy of the jump also improved, along with an increased number of successful trials, therefore opening a way for fruitful imagery applications in such populations of athletes.Written informed consent was obtained from the participant for publication of this report and any accompanying images.aA copy of the imagery script is available from the corresponding author upon request.ANOVA: Analysis of variance; MI: Motor imagery; MIIMS: Motor imagery integrated model in sport; MIQ-R: Movement Imagery Questionnaire – Revised version; PETTLEP: Physical, Environment, Task, Timing, Learning, Emotion and Perspective.The authors declare that they have no competing interests.AG conception and design, data analysis and interpretation, writing of the manuscript, final approval of the submitted version. KM Conception and design, data acquisition and collection. CC Conception and design, data analysis and interpretation, revision of the manuscript. All authors read and approved the final manuscript.


Solanum quitoense Lam.) and the tree tomato or tamarillo (Solanum betaceum Cav. Sendt.) are both Andean tropical fruit species with high nutritional value and the potential for becoming premium products in local and export markets. Herein, we present a report on the genetic characterization of 62 accessions of lulos (n = 32) and tree tomatoes (n = 30) through the use of PCR-based markers developed from single-copy conserved orthologous genes (COSII) in other Solanaceae (Asterid) species. We successfully PCR amplified a set of these markers for lulos  and tree tomatoes (26 out of 41) for molecular studies. Six polymorphic COSII markers were found in lulo with a total of 47 alleles and five polymorphic markers in tree tomato with a total of 39 alleles in the two populations. Further genetic analyses indicated a high population structure (with FST > 0.90), which may be a result of low migration between populations, adaptation to various niches and the number of markers evaluated. We propose COSII markers as sound tools for molecular studies, conservation and the breeding of these two fruit species. The Lulo or naranjilla ( Solanum quitoense Lam.) and the tree tomato or tamarillo (Solanum betaceum Cav. Sendt.) are edible fruits belonging to the Solanaceae family. The primary center of diversity for lulo comprises the Andean region of Colombia, Ecuador and Peru  includes Universal Primers for Asterid species (UPA) from COS genes designed to amplify intronic (iUPA) and/or exonic (eUPA) regions, thereby providing greater flexibility in the identification of polymorphisms  agarose gels.Thirty accessions of lulos, two entries of the related species artwegii , with fiin silico from more than 400 markers, based on the presence of Single Nucleotide Polymorphisms (SNPs) or Insertions/Deletions (InDels) in the lulos and tree tomatoes themselves  were considered different alleles of the same COSII locus. The number of alleles per COSII locus was counted, whereupon each allele was assigned a consecutive number. A matrix of alleles per locus vs. accessions was generated.Stained PCR products were visualized with GeneSnap software for Windows XP, using the 1 kb plus DNA ladder as standard . Products showing different sizes (pb) IS: inbreeding coefficient, FIT: measure of the genetic differentiation over populations, FST: variance of allele frequencies among populations) (Ho (observed heterozygosity) and He (expected heterozygosity) were calculated as described by lations) .Principal component (PC), genetic diversity, and population structure analysis were performed using Genetix 4.05 , as thisPolymorphism assessed in lulos showed that among 46 COSII markers initially selected, 34 were successfully transferred and PCR amplified, six being polymorphic by InDel as observed by agarose gel electrophoresis. The polymorphic COSII markers were previously classified as iUPAs due to amplification of intronic regions and also mapped to four different tomato chromosomes . The sixIn tree tomatoes, among the 41 initially selected COSII markers, 26 were successfully transferred, five of these being polymorphic by InDel as observed by agarose gel electrophoresis. The polymorphic COSII markers were mapped to five different tomato chromosomes and classified as iUPAs, with the exception of locus C2_At3g15430, which was identified as eUPA, due to amplification of the exonic region .To our knowledge, this is the first report describing the use of Asterid COSII polymorphisms for studies on diversity, based on the size of PCR products. However, it is possible that genetic diversity is thereby underestimated, due to the low resolution provided by 2% agarose gels, since differences in a few base pairs (less than 20) are not detected on analysis. Other reports are based on COS sequence information, probably more informative and reliable than that founded on PCR product size  in Asterids. We found that the number of COSII polymorphic markers identified herein  and are in accordance with the low Ho and He values per accession and locus  explaining 36.82% of the total genetic variation in lulos and 35.27% in tree tomatoes, each factor contributing a similar percentage of variance (8.04-10.57% for lulos and 7.65-10.69% for tree tomatoes). The distribution of populations in the three dimensions revealed by the first three PCs showed defined groups Figures  and 2. IS2 and S3), displayed clusters with low to high statistical support, with bootstrapping values ranging from 0.02%-100% for lulos and 0.004%-100% for tree tomatoes. In both cases, accessions 05T1688028 for lulo and 06TA042 for tree tomatoes were chosen as outgroups, with 100% bootstrapping values clustering out of the other accessions . In lulos, distances were the highest and clusters independent in accessions 06L082, 06L098 and 06L085, although bootstrapping values were low . In tree tomatoes, high distances and independent clusters with low bootstrapping support prevailed in accessions 06TA031, 06TA24, 06TA045 and 06TA41 . These results are consistent with PCA output. Moreover, the low bootstrapping values observed might be related to high genetic differentiation possibly arising from high homozygosity within accessions and the low number of mutually shared alleles, propitious for the formation of several cluster possibilities, each with low statistical support.To estimate genetic distances, we used the Cavalli-Sforza distance measure . The conS. hirtum 05T1688028 (a wild relative to S. quitoense) and S. corymbiflora 06TA042 (a wild relative to S. betaceum) were chosen as outgroups, since PCA output showed these accessions to be consistently and tri-dimensionally separate from the others. This is due to their both coming from different countries as compared to the majority of the accessions tested (05T1688028 was from Venezuela and 06TA042 from Brazil) . S. hirtum and S. corymbilora were under-represented in the tested sample, with only two accessions in the former and one in the latter ( Brazil) . This sebetaceum . S. hirte latter .S. hirtum accession from Santander, Colombia (06L063) grouped distantly from S. hirtum from Venezuela (05T1688028) might be consistent with their different geographic origins. Nevertheless, S. hirtum 06L063 did group together with other S. quitoense accessions, thus possibly indicating allelic homoplasy (the close relationship between species), which may have occurred through the same-sized alleles on agarose not necessarily being homologs, consequently with different and recent common ancestors. Thus, differences in sequence, if any, could be masked as the alleles were of the same size. The same principle might also apply to the close relationships between S. betaceum, S. hartwegii var racemosa, and S. diversifolia, species which grouped together. We also observed a lack of clustering associated with geographic origin in Colombian regions, as well as any other attribute. This might reflect the lack of specific sampling for different geographic and distribution regions. Similar results were obtained by The fact that the other S. quitoense, where genetic variation is low, based on the morphology of entries from Ecuador  origin, whereas the name “naranjilla”, used in Ecuador, is of Spanish origin. Furthermore, these authors, as well as Heiser (1969), mentioned a possible founder effect associated with human dispersion from a common center, namely Colombia.The pronounced variability observed among lulo accessions is in agreement with previous studies based on morphologic and agronomic traits, thereby indicating the high variation among accessions, most of which represented Colombian entries  or multi-allelic (InDels) nature, we may infer that COSIIs are adequate for diversity analysis. Due to the novel nature of using COSIIs based on the size of PCR products for diversity, we resorted to a small marker sample, which might thus bias analysis. Consequently, care should be taken in interpretation, with confirmation by means of a larger sample of polymorphic markers. Notwithstanding, this study represents an important guiding line for future research.COSII markers are being used for studies on diversity based on sequence data in tomatoes (S. hirtum and S. quitoense (Heiser, 1972; S. betaceum and S. unilobum accessions (. Finally, there should be a high probability of success for gene-map construction and QTL analysis among accessions. Hence, we have observed high levels of COSII polymorphism distributed among the 12 tomato chromosomes in different lulo and tree tomato parental combinations (Based on the high genetic differentiation among lulo and tree tomato accessions, we might venture upon some inferences. Firstly, Colombia could be the primary center of diversity as has already been suggested (Heiser, 1969; Finally, we illustrate the potential of the SOL genomics initiative, wherefore the use of COSII markers is just one example of the transferability of genomics tools to less developed Solanaceae species such as lulos and tree tomatoes for their future conservation and breeding.The following online material is available for this article:Consensus tree using bootstrapping of 10,000 replicates for lulo.Consensus tree using bootstrapping of 10,000 replicates for tree tomato.COSII Markers selected for screening.Genetic distances for tree tomato and relatives.This material is available as part of the online article from http://www.scielo.br/gmb


Mya arenaria Linnaeus, 1758) is native to the northwestern region of the Atlantic Ocean. This species has been introduced in the northeast Pacific and along the European coasts, due to intense naval transports and aquaculture, and it is now present in all the European seas. In this paper we describe seven new microsatellite loci for Mya arenaria. The isolated loci are polymorphic with a number of alleles per locus between 6 and 14. The observed and expected heterozygosities ranged from 0.417 to 0.951, and from 0.643 to 0.895, with an average of 0.716 and 0.775, respectively. These microsatellite markers should be useful in analyzing this species’ genetic diversity, which could explain various processes of its invasion history.The invasive softshell clam ( Mya arenaria is one of the most important commercial clams in the USA, being used as a food source [M. arenaria has reinvaded the eastern regions of the Atlantic, most likely as a result of human activity, and it is thought that the re-invasion of the European coasts has begun somewhere in the thirteenth century as a result of Viking expeditions [d source . Its curd source . After teditions . The sofeditions .M. arenaria were conducted using only allozymes [Mya arenaria.Genetic population studies on invasive species are useful in obtaining information about the geographic origins of invasive populations, the route(s) of invasion, number of introductions, and levels of genetic diversity and gene flow. Lately, DNA microsatellite markers have become the most utilized tool in studying such processes. However, the microsatellite loci available for molluscs species are still limited  and untillozymes  and COI llozymes . Recentlllozymes . Computellozymes  thereforAll of the sequenced clones contained repetitive motifs, but only 25 of them were suitable for primer design. Out of the 25 primer pairs, only 7 gave consistent amplification and were subsequently used for polymorphism screening .p < 0.05). The results of the Micro-Checker testing showed that three loci  exhibited an excess of homozygotes, most likely due to the presence of null alleles. While some authors reported these phenomena  as common for bivalve species [The number of alleles per locus ranged from 6 to 14, with an average of 8.9. The observed and expected heterozygosities ranged from 0.417 to 0.951, and from 0.643 to 0.895, with an average of 0.716 and 0.775, respectively . Signifi species , we also species , we did Mya arenaria specimen using a phenol-chloroform protocol optimized for molluscs [Genomic DNA was extracted from the adductor muscle of a single molluscs . We isolmolluscs ,14. The M. arenaria were genotyped from two populations sampled in 2011: 35 individuals from Modern Beach, Constanţa, Romania  and 41 individuals from Marină Beach, Mangalia, Romania . All individuals were preserved in 96% ethanol. Total genomic DNA was extracted from muscle tissue using a Macherey-Nagel NucleoSpin® Tissue kit, according to the producer’s specifications.A total of 76 individuals of 2, each dNTP at 0.1 mM, each primer at 0.05 μM, 0.02 μM of IRD700 or IRD800 labeled M13 primer and 0.5 units of Taq DNA polymerase . The temperature profile of the PCR reaction consisted of an initial denaturation step at 95 °C for 2 min followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at a specific temperature for each locus  of this species in the Black Sea, explaining if the invasion was a step-by-step process (from previously established European populations), or the Black Sea populations of Mya arenaria were established directly from a North American source.In addition to the previously described markers, these new seven highly polymorphic microsatellite markers are a useful resource for further studying the genetic diversity of native and invasive populations of


Piper , may provide an exception. Species in this genus often have aggregated, high-density populations with self-compatible breeding systems; characteristics which the BFH would predict lead to high local genetic differentiation. We test this prediction for five Piper species on Barro Colorado Island, Panama, using Amplified Fragment Length Polymorphism (AFLP) markers. All species showed strong genetic structure at both fine- and large-spatial scales. Over short distances (200–750 m) populations showed significant genetic differentiation , with values of spatial genetic structure that exceed those reported for other tropical tree species (Sp = 0.03–0.136). This genetic structure probably results from the combined effects of limited seed and pollen dispersal, clonal spread, and selfing. These processes are likely to have facilitated the diversification of populations in response to local natural selection or genetic drift and may explain the remarkable diversity of this rich genus.Fifty years ago, Baker and Fedorov proposed that the high species diversity of tropical forests could arise from the combined effects of inbreeding and genetic drift leading to population differentiation and eventually to sympatric speciation. Decades of research, however have failed to support the Baker–Fedorov hypothesis (BFH), and it has now been discarded in favor of a paradigm where most trees are self-incompatible or strongly outcrossing, and where long-distance pollen dispersal prevents population drift. Here, we propose that several hyper-diverse genera of tropical herbs and shrubs, including  Evolutionary biologists have long sought an explanation for the high diversity of closely related species that coexist in tropical forests. One of the first micro-evolutionary explanations for the origin of this diversity, the Baker–Fedorov hypothesis (BFH), was based on the expectation of restricted gene flow in tropical trees. Trees differ from shrubs and herbs in several respects. First, trees tend to occur at lower population densities , which rPiper , Psychotria , Miconia , and Inga (>300 species) are composed mostly of shrubs and small trees that often grow sympatrically , and two in clearings . Piper species share pollinators , Panama  that is described in detail by Piper is an important component of tropical forests worldwide and is represented by 22 species on BCI  located 200 m apart in the laboratory clearing. In plot 3, we collected leaves from 33 plants of P. dilatatum and 43 of P. marginatum. In plot 4, we collected leaves from 43 plants of P. dilatatum and 41 of P. marginatum. The location of each plant in each plot is available in To include both species variation in life history characteristics, and to assess population genetic differentiation, we analyzed five Leaves were collected and kept on ice until they were processed in the laboratory. On the day of collection, leaves were surface cleaned with 95% alcohol and left to dry in silica gel for 1 week. Twenty milligrams of dry tissue was ground using the FastPrep FP120 . DNA was extracted using a DNeasy 96 plant extraction kit  following the manufacturer's protocol. DNA concentrations were established by running DNA samples with Low DNA Mass™ Ladder  of known concentration on agarose gels.xl capillary electrophoresis machine, and the presence or absence of fragments was scored using Genescan and Genotyper software . Clones and genetically distinct individuals were identified using the Genotype software  for dominant markers developed by I = 0 for P. marginatum, which we found to be self-incompatible, and an inbreeding coefficient of FI = 0.5 for all remaining species because they had a mixed breeding system . We also tested other inbreeding coefficients and found only a slight effect on patterns of spatial genetic structure (SGS). Given that this estimate has been proven to be fairly robust to moderate errors made on the assumed inbreeding coefficient ), was compared with those obtained after 1,000 random permutations of individuals among positions. This procedure has the advantage that all the information is contained in one single-test statistic, and the results are independent of arbitrarily set distance intervals , and 2, 5, 10, 20, and 40 m (for light-demanding species). To test the hypothesis that there was significant SGS, the observed regression slope of ntervals .Sp’ statistic as Sp = –b/(1 – F1), where b is the slope of the regression of ijF on ln(distance), and F1 is the mean ijF between individuals in the first distance class . The Sp statistic is a synthetic way of calculating the strength of spatial genetic structure and enables simple comparisons to be made among species and to other published studies because it is less sensitive to sampling schemes than other measures of spatial genetic structure intensity . First, we calculated Wright's Fst statistic from estimates of allelic frequencies under different inbreeding scenarios  to observe the range of possible outcomes for each species. An inbreeding coefficient of Fis = 0 is typical for random mating, and, a coefficient of Fis = 1 indicates complete selfing. The significance of the genetic differentiation among populations was tested by comparison of the observed Fst with a distribution of Fst under the hypothesis of no genetic structure, obtained by means of 10,000 random permutations of individuals among populations. We also calculated Фpt, a measure of population genetic differentiation that is analogous to Fst and is used specifically for AFLP and other binary data, using GenAlEx 6 . This method uses a Markov chain Monte Carlo (MCMC) algorithm to account appropriately for the genotypic ambiguity inherent in dominant markers like AFLP (K) that are most appropriate for interpreting the data (see below). Then, we examined the clustering of individuals in the K populations to identify the admixed individuals. On each run, the program places individuals into K panmictic groups, by minimizing deviations from Hardy–Weinberg equilibrium and linkage equilibrium. The program calculates an estimate of the posterior probability of the data for a given K, Pr(X|K)  .K and obtain insight into how the genetic variation was organized based on the clustering of individuals, we ran the program Structure (ver. 2.2) without prior information on the population of origin using values from K = 1 to K = 5. We generated a series of 10 independent runs for each value of K and we used the ad hoc statistic ΔK . Because the results were similar, we present only the results from the analysis of the entire dataset.All The spatial autocorrelation analysis revealed strong and significant SGS in all species and all sites. The slope of the regression line (b) between the kinship coefficient and the natural logarithm of the distance between individuals was highly significant in all instances, even after removing clones from the analysis . However(1) ranging between 0.02 and 0.43. Values of 0.25 and 0.125 are expected for full and half-siblings, respectively; therefore some clonal spread seems to contribute to population growth. However, some F(1) values remained above 0.25 even when ramets are excluded, suggesting that some biparental inbreeding is taking place. While clonality is not the only factor responsible for the strong SGS observed, slopes were two or three times higher when clones were included in the analysis , probably as a result of its populations having aggregations dominated by related neighboring plants. These values of Sp for Piper are higher than those reported for 45 species of trees and six species of shrubs  were significant and were in the range of 0.11–0.46. Values of Фpt diverged significantly from zero, based on the permutation test and were in the range of 0.14–0.49  for all species with little admixture for four of the five species studied  derived from the other population  were found to be immigrants or to have had an immigrant ancestor. Most of them (9/10) were immigrants coming from plot 2 to plot 1  were immigrants or descendents of immigrants, with most migration (10/14) from plot 2 to 1. For P. cordulatum, only two of 132 individuals (1.5%) were immigrants or descendents of immigrants  were found to be immigrants or had an immigrant ancestor. Most (4/5) moved from plot 4 to plot 3. On the other hand, P. dilatatum had the largest number of immigrants; 21 of 76 (27%). For this species, the majority of movement was in the opposite direction of the movement observed for P. marginatum  than the vast majority of trees , from seeds sired by self-pollen or pollen of relatives (limited pollen flow), or from both. The observation that Piper populations as it does in other clonal plant species  ied here . Both sePiper seeds in most parts of the Neotropics are Carollia perspicillata and Carollia castanea , and ants (26%)   both of ts (26%) . FurtherPiper flowers consist of Hymenoptera and Diptera, particularly bees of the genus Megachile and Trigona, and Syrphid flies , or understory herbaceous taxa . Likewise, families that include both shrubs and trees have much higher diversity in shrub than canopy genera  and Chamaedorea (107 species).Ecologists have long recognized that there is no single ultimate mechanism that can account for the latitudinal richness gradient; instead they suggest that several contributory mechanisms may be in action . Some coTrees differ from shrubs in many characteristics  that may lead to greater seed and pollen movement in trees relative to shrubs, which in turn should slow population differentiation and, possibly, sympatric speciation. Moreover, differences in intrageneric diversity between trees and shrubs may reflect different speciation rates among groups. More evidence has recently emerged that suggests that rates of mutation and molecular evolution changes with life history in flowering plants , betweenEugenia , Miconia , Piper , Inga (∼300 species), and Psychotria  contribute disproportionally to total diversity in the tropics. Many congeners in these groups also occur sympatrically and appear to have similar morphology, reproductive syndromes, and habitat preferences, raising yet again the enigmatic issue of coexistence and speciation . For these old genera, part of their tremendous species diversity can probably be ascribed to their age and pantropical distribution, yet the existence of many Piper and Psychotria sympatric species of small geographic range and relatively recent origin (3–12 Ma; Inga (The tropical rain forest contains strikingly large numbers of plant species, many of which are closely related sympatric species and many might be accounted for by explosive speciation . Hyperdieciation . Some of2–10 Ma; , while o ∼40 Ma; , or even3–12 Ma;  suggestsMa; Inga . The patPiper, and perhaps to other species-rich genera. This mechanism represents a modified version of BFH that adds extended clonal growth to the limited gene flow and genetic drift proposed by them to favor the establishment and subsequent survival of small reproductively isolated populations—the conditions under which much of the speciation in tropical woody plants has probably occurred (Piper, are commonly found in aggregations and are also animal-dispersed. Some, however, like Inga and Psychotria differ from Piper in their mating systems and include many species that are self-incompatible (Here, we propose yet another mechanism that that may contribute to diversification in occurred . Membersoccurred  and, likmpatible  or exhibmpatible .Psychotria species using allozymes, a marker less variable than AFLP, and found low but significant values of genetic differentiation among subpopulations separated by only 120 m, and detected that neighboring plants were either siblings or parent and offspring; again evidence of limited dispersal. Evidence from another six tropical shrubs species, together with these five Piper species indicates that shrubs have more restricted gene flow than the majority of trees studied so far (Whether restricted gene flow also occurs in those speciose tropical shrub genera remains an open question. d so far . Whether


Arvicola sapidus), a habitat specialist whose habitat patches are distributed through less than 2% of the study area  and whose populations form a dynamic metapopulational network. We predict that individuals will require a high ability to move through the inhospitable matrix in order to avoid genetic and demographic isolations. Genotypes (N = 142) for 10 microsatellites and sequences of the whole mitochondrial Control Region (N = 47) from seven localities revealed a weak but significant genetic structure partially explained by geographic distance. None of the landscape models had a significant effect on genetic structure over that of the Euclidean distance alone and no evidence for efficient barriers to dispersal was found. Contemporary gene flow was not severely limited for A. sapidus as shown by high migration rates estimates (>10%) between non-neighbouring areas. Sex-biased dispersal tests did not support differences in dispersal rates, as shown by similar average axial parent-offspring distances, in close agreement with capture-mark-recapture estimates. As predicted, our results do not support any preferences of the species for specific landscape attributes on their dispersal pathways. Here, we combine field and molecular data to illustrate how a habitat specialist mammal might disperse like a habitat generalist, acquiring specific long-distance dispersal strategies as an adaptation to patchy, naturally fragmented, heterogeneous and unstable habitats.Habitat specialists inhabiting scarce and scattered habitat patches pose interesting questions related to dispersal such as how specialized terrestrial mammals do to colonize distant patches crossing hostile matrices. We assess dispersal patterns of the southern water vole ( Individuals disperse as an effective strategy for the avoidance of inbreeding, resource competition, and kin competition glue that connects different scales and disciplines Animal dispersal is commonly defined as the movement of individuals away from their home ranges with no subsequent return  effective dispersal (i.e. when it is followed by breeding success) and is expected to homogenize the genetic variation among populations and counteract the structuring effects of drift. Therefore, species might show strong genetic structure when gene flow among populations is reduced, either because the geographic distance exceeds average dispersal distance or because effective barriers (or filters) to dispersal separate the populations. Genetic structure will thus be greater for low mobility than for highly mobile species at a particular geographical scale. Classical analyses of patterns of gene flow have usually addressed their extent and distance components, often revealing a monotonic decrease of gene flow with distance (isolation-by-distance), where geographic distance is calculated as the Euclidean distance separating individuals or populations. This approach implicitly assumes that dispersing individuals travel in a straight line across a homogeneous or irrelevant landscape matrix. A more recent approach has highlighted the relative importance of the landscape matrix heterogeneity on the dispersal behavior of species, by showing a better correlation of gene flow with landscape-modified distances than with purely Euclidean distances Gene flow is one of the important consequences of Species are often classified into habitat generalist or specialists based on habitat requirements: while the former can exploit multiple habitat types or food sources, the latter are restricted to only one or few habitats. Like habitat generalists, specialists in large and continuous habitats can move rather freely across space, rendering populations with reduced spatial and genetic structure. Quite often, however, habitat specialists are restricted to more or less scarce and scattered patches of suitable habitat embedded in an unsuitable habitat matrix. Given that small and isolated populations have increased risks of extinction, highly specialized species inhabiting patchy habitats require a high ability to move through the matrix in order to avoid genetic and demographic isolation Arvicola sapidus) as a case study of a species tightly associated to naturally fragmented habitats embedded in heterogeneous but largely hostile habitat matrices (see below). We first estimate gene flow among the populations of this rodent in the study area through indirect and direct approaches based on neutral autosomal microsatellite genotypes and mitochondrial control region sequences. We will then evaluate the relative role of the landscape matrix in shaping gene flow patterns through several landscape genetic approaches. According to our prediction, a high ability of southern water voles to disperse must be reflected on a weak genetic structure and minor effects of landscape on gene flow patterns (i.e. isolation-by-distance pattern more pronounced than any other landscape-based model). Finally, we will try to elucidate whether the observed genetic structure is a result of sex-biased dispersal, as found for many mammals. Patterns of dispersal inferred from genetic markers will be compared to those obtained in previous field studies We set out to test this prediction using southern water vole (ISF = 0.297) reveals a deficit of heterozygotes potentially caused by null alleles. Overall ISF, which was moderate and significant when AV14-2 was included , became low and non significant when this locus was excluded from the dataset . Consequently, this locus was excluded in global tests of HWE for each population, but all loci were considered for the rest of analyses. Significant deviations from Hardy-Weinberg Equilibrium across loci , while significant heterozygote excess was found at RES1  and ROC .A total of 142 individuals distributed in seven sampling areas throughout the region were genotyped using 10 polymorphic loci. All loci were highly variable, the number of alleles per locus ranging from 8 to 29. Allelic richness averaged over loci, and adjusted for the minimum sample size of 8 individuals, was between 2.889 and 10.713. Average expected population heterozygosities varied from 0.6860 to 0.7808 . The onloci HWE,  were detSTF = 0.072, 95% confidence interval [0.058–0.089]. Exact Pairwise STF values ranged from 0.028 to 0.116 . Nucleotide diversity (π) varied throughout Doñana from π = 0 (MAR) to π = 0.006 (RES3) (H) were slightly higher in Northern localities . The distribution of haplotypes throughout Doñana did not show any obvious geographical pattern, although haplotypes 7 (n = 1) and 10 (n = 1) were only found in RES1 and ABA2, respectively, and four haplotypes were distributed only in the Northern region  . Pairwise STF ranged from −0.0177 to 0.4717 with no significant pattern of isolation-by-distance  . Haplotyand ROC) . We founGeographic (Euclidean) distance between sampled areas explained 34% of the microsatellite genetic variance  . At the POND = 0.096±0.004; CVDRE = 0.171±0.007). LCDs based on vegetation, drainages and ponds were all positively correlated with genetic distances in Doñana and explained a slightly higher proportion of the observed genetic variance than Euclidean distances . However, none of these landscape effects remained significant once the effects of Euclidean distance were discounted . Moreover, delta differences of AICc between the model with lowest AICc (Euclidean distance) and landscape modified line models were always less than two.Least Cost Distances calculated from different landscape attributes were robust to variations in the arbitrarily assigned maximum cost values , although none of sex-biased dispersal tests rejected the null hypothesis of equal dispersal between sexes . The same result was obtained when only adults trapped in ABA1 and ABA2 after dispersal but before breeding were considered . Finally, very similar average axial parent-offspring distances were estimated for males and females  from the slopes of the individual-based IBD analyses  and considering an effective density of 5–10 ind./km2.Observed differentiation indices were generally higher for males than for females . This pattern suggests a moderate metapopulation dynamics within the study area with either low or relatively high immigration rates proceeding from nearby populations.Most sampling locations in Doñana showed low recent immigration rates , with thOur results provide the first insight into the genetic structure and patterns of gene flow of Southern water voles in a Mediterranean patchy habitat. This, together with the existing knowledge of its natural history and habitat preferences, contributes to the understanding of the patterns and significance of dispersal in the species and illustrates the potential contribution of molecular markers to the study of dispersal in small mammals.E = 0.74), slightly lower than that previously reported in metapopulations of European water voles (Arvicola terrestris) A. terrestris in Scotland MicrotusWe found a moderate level of population genetic diversity . Low levels of haplotype diversity might be reflecting recent founder or bottleneck effects affecting females and absence of recent gene flow by females (but see below). Besides, we found a moderate mitochondrial genetic structure reflecting significant differences in haplotype frequencies, but without an obvious geographic pattern . Furthera priori a putative barrier for dispersal of SWV . This pattern may result from a single or few correlated events of migration and illustrates the inherent stochasticity associated to demographic events under a metapopulational dynamic at the spatial and temporal scales considered. Furthermore, the observed patterns might be justified by recent experimental studies of dispersal in Arvicola terrestris in Scotland, where it was shown that juveniles disperse to distant patches making temporary stops at suitable sites between dispersal movements (“stepping-stone”) In our study, we have investigated dispersal and nuclear gene flow using two complementary approaches that provide estimates at two temporal scales and whose comparison allows us to address the existence of migration-drift equilibrium and the temporal dynamics of gene flow. Direct estimates of recent gene flow are clearly bimodal, with most pairwise estimates being low (mean 0.06%) and significant migration rates from ABA1 to ABA2, ABA1 to ROC, MAR to RES2 and RES3 to RES2, suggesting directional and simultaneous or recurrent events of dispersal between the same two populations. All four cases of recent migration occur between nearby  populations and are clearly asymmetrical, identifying a source and a recipient population. For example, ABA1 appeared as a recent source of immigrants into ABA2 and ROC, RES3 into RES2 and MAR into RES2 . While tet al. (2005) In contrast, population structure  seems to follow an isolation-by-distance pattern across the region, as expected under migration-drift equilibrium in a stepping-stone model. Both inter-individual and inter-population genetic distances increase linearly with geographic distance see  and the A. terrestris in an area of grassland and open forests in France  STF: 0.09) STF values reported above were estimated using different but overlapping sets of loci, sharing five and eight loci with those used in this study . Genetic estimates of average dispersal distance  do not differ from the average distances estimated for each sex using capture-mark-recapture analyses  lower dispersal rates in females may be compensated by their higher rates of survival and reproduction, resulting in similar effective dispersal distances for both sexes.Quantitatively, the level of genetic structure observed for microsatellite markers is moderate and intermediate between those found for the fossorial A. terrestris dispersal in more fragmented and unstable settings seems to support the prediction that female dispersal may be an adaptation to patchy unstable habitats, as females can improve their success by prospecting for suitable habitat when turnover rates in local patches are high Long-distance dispersal in both sexes et al. (2001) A. terrestris might show overland instead of waterway dispersal and Telfer et al. (2003) overland mean? A plentiful literature distinguishes habitat (used patches) from non-habitat (matrix) when a classification of land uses for species is intended. This binary division assumes the permeability of patches and the reluctance of individuals to cross inhospitable habitat matrices (barriers). There are, however, many species that apparently differentiate at least three kinds of habitat types: breeding habitat, dispersal habitat and the intervening landscape matrix Habitat specialists inhabiting scarce and scattered habitat patches must develop dispersal abilities to move through hostile matrices and colonize distant and/or empty patches, in order to avoid very small population sizes, reduced genetic variability and high extinction risks. Although occasionally small mammals may disperse by passive strategies After a few failed attempts to monitor radio-collared individuals, our genetic approach has improved our knowledge of dispersal habits in SWV. Ponds, drainages and vegetation cover apparently explained the genetic structure in SWV better than Euclidean distances (see Mantel tests) but none of the landscape models were significantly better than a model based on Euclidean distance alone. Accordingly, our results do not support any preferences of SWV for specific landscape attributes on their dispersal pathways for the establishment of breeding territories.A. terrestris, using a similar approach, although in this case the landscape model explained less of the genetic variance than the null Euclidean model It must be noted, though, than even when our sampling was widespread throughout Doñana, both the spatial distribution of landscape variables and the limited resolution of the landscape tessellation resulted in least cost distances that were highly correlated with Euclidean distances, what may have limited our power to detect any landscape effects. However, although landscape tessellation may not be appropriate for the study of dispersal in SWV at local scales (e.g. within geographic –sampling- populations in this study), high gene flow between non-neighbouring areas  may justify its suitability for the study of dispersal through the overall region of Doñana. Therefore, considering this limitation and the fact that models incorporating landscape attributes performed slightly better in explaining genetic variance between populations, the influence of landscape in SWVs movement and dispersal cannot be completely discarded. No preference for grassland over non-grassland could be demonstrated for where and when individuals choose to stop dispersing and settle their new breeding territory  . Field studies in the area have suggested a sex-biased dispersal  and rather long average dispersal distances , although the limitations of ecological methods and the limited scale of the study area may have impeded an accurate estimate of dispersal movements et al. (2002) The patterns of ponds occupancy by SWV in Doñana described by Fedriani A. terrestrisMicrotus oeconomusA. sapidus. Two more markers (AV10 and AV14) were used after primer redesign based on A. sapidus sequences obtained from products amplified with the original primers. These new primers are AV10-2 reverse (5′-CAAGGCTTGGAGCTTGGATA-3′), AV14 -2 forward (5′-TCCTCCCTCCCCAGCAAT-3’) and AV14-2 reverse (5′-GCAGAAGGGGGCAGATAAT-3′).We tested 24 pairs of microsatellite primers developed for Taq buffer, 2 mM MgCl2 (0.9 mM for AV1), 0.25 mM dNTPs, 0.25 mM sequence-specific reverse and fluorescently-labelled M13 primers, 0.017 µM sequence-specific forward primer with M13 extension, 0.1 mg/ml BSA (Roche) and 0.5 U/ml Taq DNA polymerase . AV1, AV10-2 and AV14-2 (those with 5′- labelled forward primers) required 0.25 mM forward and reverse primers. PCRs for all loci but AV1 involved temperature steps of 94°C for 5 min, 17 touchdown cycles 92°C for 30 s, 66°C for 30 s and 72°C for 30 s, decreasing one Celsius degree per cycle, and followed by 27 cycles of 92°C for 30 s, 50°C for 30 s and 72°C for 30 s. PCRs for AV1 were as follows: 94°C for 5 min, 34 cycles of 92°C for 30 s, 60°C for 30 s and 72°C for 30 s. All PCR reactions finished with 72°C for 5 min. PCR products were checked on a 2% agarose gel, run on an Applied Biosystems 3130×l Genetic Analyzer and scored with GeneMapper software v3.0 (Applied Biosystems).PCR products were indirectly labeled using a M13 5′ extension 5′-TCCCCACCATCAGCACCCAAAGC-3′ designed by 5′-ATGTTTTATCGTCCATACGTTCC-3′; F15872, 5′-AATCAGCCCATGCCTAACAT-3′; R15946, 5′-TAGCCGTCAAGGCATGAAG-3′; RCRasa 5′-AAAAACAACTCAAAATTCCAAAA-3′) The complete mitochondrial control region (1024 bp) was sequenced from a subset (n = 47) of the samples using primers F15708 and R92 O) and unbiased expected heterozygosities STF and linearized pairwise F-statistics [STF/(1- STF)] were estimated with GENETIX 4.05.2 ra) k groups in a way that the proportion of genetic variance among groups (ΦCT) is maximized. We ran SAMOVA with 1000 simulated annealing processes for k values ranging from 2 to 10. If our sampling scale was adequate to dispersal patterns of SWV, we would expect k = 7 (i.e. the number of geographic populations used in this study), where each group (k) must include those ponds embraced by the 3 km diameter. On the other hand, k<7 would suggest higher gene flow than expected between separated geographic populations.GENEPOP v3.4 STFF-statistics derived from mitochondrial and nuclear data assume equilibrium under different effective populations sizes, they need to be calibrated before comparison. For this purpose, we used the expression STFn = STFmt/(4–3 STFmt) derived from STFn and STFmt are F-statistics inferred using nuclear and mitochondrial markers, respectively. Because extensive homopolymer sequences hamper the sequencing reaction of the entire mitochondrial control region, we only sequenced a subset of 47 samples Global and per population nucleotide (π) and haplotype (H) diversities, number of segregating sites (S) and average number of pairwise differences (k) were estimated with DnaSP 4.5 STF values were plotted against log-transformed Euclidean distances To calculate both Euclidean and Least Cost Distances (LCD) (see below) we used the weighted mean spatial coordinate of all individuals trapped as the geographic coordinate of each sampling area. Linearized pairwise k is the number of landscape features in the model, n is the number of populations and RSS is the residual sum of squares. Because of the non-independency of the data, no proper method to estimate AIC for pairwise data has been developed. Nevertheless, we used the number of populations as degrees of freedom instead of the number of pairwise comparisons as a better fit of Mantel's test to p-values of ordinary least squares regression has been shown We evaluated the effects of ponds surface, drainages length and vegetation cover on the genetic structure of SWV, habitat features previously described as determinants for the settlement of the colonies in the study area Hence, two models whose delta difference on their AICc was lower than two could be considered as equally likely STF and an assignment index (AI) per sex and population pair; these two indices have been shown to be most powerful and least sensitive to changes in the magnitude of sex biased dispersal STF values, negative mean assignment indexes (mAIC) or larger variances of the AI (vAIC) are expected for the dispersing sex. The method proposed by Goudet et al. (2002) females = 14, Nmales = 16) in order to avoid the effect caused by including predispersing individuals in the dataset.Sex-biased dispersal promotes differences in genetic structure between sexes. We tested differences in dispersal rates between sexes using two approaches. First, we regressed pairwise genetic distances (see above) on geographic distances using an independent datasets for males (n = 68) and females (n = 74) and compared both slopes. Second, we used FSTAT v2.9.3 to calculate 2σ), which can be interpreted as an average dispersal distance, using the slope (b) of the regression of interindividual pairwise genetic distances  on the geographical distances 2 as effective density (D)  we can estimate a sex-specific σ2 using their respective slope obtained in the IBD analyses b using the whole  and sex-filtered datasets. Lower values of b would result from higher gene flow and higher average dispersal distance.Assuming IBD in a two-dimensional space, we can estimate the average squared axial parent-offspring distance  and geographic distance .Linearized pairwise (DOC)Click here for additional data file.Table S2(a). Control Region nucleotide diversity by sampling area and overall study system (b). Sample size (N), number of segregating sites (S), nucleotide diversity (π), number of haplotypes (h), haplotype diversity (H) and mean number of nucleotide differences (k) are reported. Standard Deviations are given in parenthesis (SD).Haplotypes of Control Region and their geographical distribution all over the Natural Region of Doñana (DOC)Click here for additional data file.