Lachancea kluyveri (formerly Saccharomyces kluyveri), highlighting a broad genetic diversity (π = 17 × 10−3) compared to the yeast model organism, S. cerevisiae (π = 4 × 10−3). Here, we sought to generate a comprehensive view of the phenotypic diversity in this species. In total, 27 natural L. kluyveri isolates were subjected to trait profiling using the following independent approaches: (i) analyzing growth in 55 growth conditions and (ii) investigating 501 morphological changes at the cellular level. Despite higher genetic diversity, the fitness variance observed in L. kluyveri is lower than that in S. cerevisiae. However, morphological features show an opposite trend. In addition, there is no correlation between the origins  of the isolate and the phenotypic patterns, demonstrating that trait variation follows neither population history nor source environment in L. kluyveri. Finally, pairwise comparisons between growth rate correlation and genetic diversity show a clear decrease in phenotypic variability linked to genome variation increase, whereas no such a trend was identified for morphological changes. Overall, this study reveals for the first time the phenotypic diversity of a distantly related species to S. cerevisiae. Given its genetic properties, L. kluyveri might be useful in further linkage mapping analyses of complex traits, and could ultimately provide a better insight into the evolution of the genotype–phenotype relationship across yeast species.It is now clear that the exploration of the genetic and phenotypic diversity of nonmodel species greatly improves our knowledge in biology. In this context, we recently launched a population genomic analysis of the protoploid yeast  The budding yeast Saccharomyces cerevisiae and other closely related species, including S. paradoxus, are the most often used for both genetic and phenotypic variation surveys . This latter is a heterothallic yeast having the same life cycle as that described for S. cerevisiae. It can live at both haploid and diploid levels; the two different mating types (MATa and MATα) can be crossed to form a stable diploid that can undergo meiosis under unfavorable conditions  event, in contrast to post-WGD species  follow neither the population history nor the source environment. However, fitness profiles are more similar between closely related strains than distant isolates. Altogether, our study provides multiple insights into the phenotypic diversity of L. kluyveri, which will be useful to future studies of natural genetic variation in this organism.However, little is known about the phenotypic variation spectrum in Table S1). The media used to determine the phenotypes of each strain was the YPD medium  supplemented with various compounds (Table S2). In the “Carbon Utilization” class, dextrose was substituted by alternative carbon sources (Table S2). The minimal medium YNB  was also used during this analysis.The strains used in this study were stable haploids or obtained from random spore analysis of natural diploid isolates. These strains were isolated from different geographical origins and ecological niches  and cultivated for 48 hr in a microplate reader (Tecan Infinite F200) at 30°C unless otherwise specified. These plates were stirred for 8 min with orbital shaking (120 rpm) followed by 2 min with linear shaking (160 rpm) over the course of 48 hr. After each stirring cycle, absorbance was recorded using a narrow spectrum (595–605 nm). In each tested condition, we determined the specific growth rate (population doubling time), the lag time (population adaptation time), and the efficiency (biomass produced during the cultivation) using a “home-made” program. Briefly, after background correction, the specific growth rate was determined given the maximum linear regression line calculated over 15 consecutive absorbance records with a sliding window of 10 points. Lag time was estimated as the intersection between this regression line and the baseline absorbance level. The yield of biomass was determined as the maximal OD reached during the cultivation process. Each value was normalized using results obtained with standard YPD medium, representing 1485 data points for each of the three parameters.Strains were subjected to high-throughput phenotyping by a microcultivation strategy in biological duplicate (n = 2). Briefly, strains were inoculated in 150 μl of YPD medium and incubated overnight at 30°. Precultures were picked in 96-well microplates containing 150 μl of different media (http://www.r-project.org/). The Kruskal-Wallis test, a principal component analysis (PCA), and a hierarchical cluster analysis (HCA) were performed as described previously . Each PC was characterized in Table S3 using the morphological features by grouping the morphological traits as described previously . Phenotypic variance of the cell morphology among 27 strains was calculated from the Z values of 501 traits, which were estimated by the generalized linear model (GLM) as described previously . Phenotypic variance among growth rates was estimated as the average of the variance estimated in each environmental condition.All statistical analyses of the cell morphology were performed using the R programming language  are common parameters allowing for the characterization of the variation between and within species, particularly in yeasts. To obtain insight concerning intraspecific variation within the L. kluyveri species, we determined the specific growth rate, lag time, and the yield of biomass produced during the cultivation process of 27 L. kluyveri strains (listed in Table S1) grown in 55 environmental conditions, which were classified into three main groups: carbon utilization, toxins, and environments and metabolites (Table S2). With the exception of the strain 55-86.1, which was a strain prone to the flocculation phenotype, the same set of strains as those used for unraveling the genetic architecture within L. kluyveri were used  in most of the experimental conditions tested (with the exception of the substitution of glucose by mannitol or ethanol showing a null correlation). In contrast, the lag phase was independent from these fitness features   .As described in both S. cerevisiae strains are sensitive to   . This suS. cerevisiae yeast  and the elongated cell shape (mother axis ratio). The values of these three traits ranked strains in three different orders, indicating that there was no correlation among cell size, nuclear shape, and elongated cell shape. Thus, the variation of L. kluyveri cellular morphology represents a set of multiple independent traits with different sources of variability.One of the most striking variations in morphology concerned whole cell size . In FiguL. kluyveri species ranges up to 2.5% .The genetic divergence within the  origins . Except Table S3). We found that the strains were nearly evenly spaced with no particular subgroups apparent. Analysis of all spaces represented by the first five components  led to similar results , suggesting that L. kluyveri is characterized by a continuum of morphological features rather than discrete classes of distinct morphologies. In accordance with hierarchical clustering, strains from common ecological origins did not group together, indicating that differences in the morphological traits do not simply reflect adaptation to the simulated environments.Similarly, hierarchical clustering of the 501 morphological parameters revealed two clusters, each divided into two subgroups where no correlation with strain origin was distinguishable . We thenL. kluyveri species  was found when comparing fitness data among all the 27 strains, indicating that trait variation increases with the enhancement of genetic diversity   despite their high genetic conservation. However, a significant anticorrelation  . These rS. cerevisiae revealed a correlation with their genomic structure rather than to adaptation associated with their ecological niche. For example, S. cerevisiae strains isolated from Europe are more prone to be resistant to Na+ and Li+ ions given the pleiotropic roles of causal loci. In addition, clean lineages within the S. paradoxus species show similar trait profiles (L. kluyveri species: one corresponding to strains originated from North America (group 1), the second comprised of strains isolated from Europe (group 6) , demonstrating that growth profiles are more similar than that for S. cerevisiae species, where the variance was estimated to be approximately 0.75  is not defined by population history in L. kluyveri. Using PCA analysis and hierarchical clustering, we did not observe any correlation between the origin  and the phenotypic patterns, showing that trait variations are not linked to either the population structure or the source environment. However, the number of L. kluyveri strains used in this study is relatively low and their ecological origins are somehow similar. Increasing the number of strains would probably reveal more structured trait profiles.In contrast to fitness patterns in L. kluyveri were obtained through mapping against a reference genome (S. cerevisiae but are still not well known in L. kluyveri (Finally, we determined the correlation between pairwise trait variation and genetic diversity in order to have a better view of how genomes and phenotypes are related. Interestingly, there is strong evidence that genetic diversity is anticorrelated with fitness variation. However, this is not the case for differences among morphological parameters, showing a different global behavior of the two types of traits. We are here considering genetic diversity as SNPs but other genome variation can lead to trait profile differences. Genomes of the 27 strains of L. kluyveri and its trait variability, quantitative genetics is definitively applicable to this species and would lead to a better understanding of the genotype–phenotype relationship across diverse yeasts. Moreover, laboratory evolution experiments carried out for both S. cerevisiae and the pathogenic Candida albicans species have shown that the presence of an additional chromosome is linked to stress resistance acquisition (L. kluyveri would reveal if the acquisition of aneuploidy is a common mechanism across yeast species to improve fitness in stress conditions. Finally, further analysis will, without a doubt, provide deeper insight into functional diversity in this nonmodel organism.Given the genetic characteristics of


Tribulus terrestris L., commonly called puncture vine and gokhru, is an important member of Zygophyllaceae. The species is highly important in context to therapeutic uses and provides important active principles responsible for treatment of various diseases and also used as tonic. It is widely distributed in tropical regions of India and the world. However, status of its genetic diversity remained concealed due to lack of research work in this species. In present study, genetic diversity and structure of different populations of T. terrestris from north India was examined at molecular level using newly developed Simple Sequence Repeat (SSR) markers. In total, 20 primers produced 48 alleles in a size range of 100–500 bp with maximum (4) fragments amplified by TTMS-1, TTMS-25 and TTMS-33. Mean Polymorphism Information Content (PIC) and Marker Index (MI) were 0.368 and 1.01, respectively. Dendrogram showed three groups, one of which was purely containing accessions from Rajasthan while other two groups corresponded to Punjab and Haryana regions with intermixing of few other accessions. Analysis of molecular variance partitioned 76 % genetic variance within populations and 24 % among populations. Bayesian model based STRUCTURE analysis detected two genetic stocks for analyzed germplasm and also detected some admixed individuals. Different geographical populations of this species showed high level of genetic diversity. Results of present study can be useful in identifying diverse accessions and management of this plant resource. Moreover, the novel SSR markers developed can be utilized for various genetic analyses in this species in future.The online version of this article (doi:10.1007/s13205-016-0469-8) contains supplementary material, which is available to authorized users. Tribulus terrestris commonly known as puncture vine and Gokhru, belonging to family Zygophyllaceae, is an annual herbaceous plant. The plant is native to the South and East Europe and West Asia. It is widely distributed in the warm regions of Asia, Africa, Europe, America and Australia  for designing new SSR markers to evaluate the genetic diversity and population structure of this species in north India.To study genetic diversity and population structure at molecular level different types of DNA markers can be used. However, Simple Sequence Repeats  were analyzed. Of these, twelve samples were from Punjab, six from Haryana and eight from Rajasthan. A detatiled account of all the accessions is given in Table In present study twenty-six T. terrestris. A total of 637 nucleotide sequences were available as on 12th May 2015. These nucleotide sequences were retrieved and assembled using EGassembler .Sequence data at NCBI was searched for PCR reactions were performed in a 10 µL reaction volume as per Sharma et al. . PCR ampX|K) as per Pritchard et al. (K) and to get the best fit value of K for the data. Highest value was shown at K = 2. Therefore, STRUCTURE analysis was conducted for K = 2. However, we also performed STRUCTURE analysis at K = 3 to compare the different clusters in relation to the groupings of dendrogram. Genetic differentiation (Fst) estimates among inferred clusters were also measured by STRUCTURE software. Genetic relationships among the genotypes were also analyzed by factorial analysis using the software DARwin  was calculated using formula given by Roldán-Ruiz et al. , i.e., Pd et al. . STRUCTUd et al.  was usedd et al.  based prd et al.  was usedOnly unambiguous and reliable fragments amplified by 20 SSR primers were scored. In total, 20 primers amplified 48 fragments ranging from 1 to 4 with an average of 2.4 fragments. Size range of amplified fragments varied from 100 to 500 bp. Three primer namely, TTMS-19, TTMS-28 and TTMS-31 amplified minimum of 1 fragment while three primers namely, TTMS-1, TTMS-25 and TTMS-33 amplified maximum of 4 fragments. PIC value ranging from 0.142 for primer TTMS-8 to 0.499 for primer TTMS-7 with an average of 0.368  portion of genetic variation resided within different populations while 24 % genetic variance resided among populations.Dendrogram based on Jaccards similarity coefficient and UPGMA method showed three groups as shown in Fig. K at 2. However, when we analyzed data at K = 3, the clustering largely supported the grouping of dendrogram. At K = 2, all the accessions, namely, Pun-01, Pun-02, Pun-03, Pun-04, Pun-05, Pun-06, Pun-08, Pun-09, Pun-10, Pun-11, Pun-12, belonging to Punjab clustered in cluster-I except one accession namely, Pun-07 which was collected from Punjabi University campus. On the other hand, the maximum accessions belonging to Rajasthan  and Haryana  clustered in cluster-II. Mean values of Fst for cluster-I and cluster-II were 0.443 and 0.440, respectively. Percentages of pure accessions in cluster-I and cluster-II were 64.2 and 58.3 %, respectively  purely represented accessions from Rajasthan indicating conserved genetic nature of dry region germplasm. Furthermore, as this germplasm is cultivated repeated propagation of same parent germplasm may have lead to its conservative and more homozygous nature. Few exceptional groupings needs further research and analysis. Factorial analysis  rather than among populations (24 %). It points that allele sharing is more frequent within populations than in between populations. Baysean structure analysis indicated two genetic stocks for the entire germplasm included in present study. Cluster-I  belonging to Punjab showed that genetic background of accessions from Punjab was different than the accessions  from other two states which clustered in cluster-II. Mean values of Fst for cluster-I and cluster-II were 0.443 and 0.440, respectively, which were much higher than reported by Duran et al. (Larrea tridentata) populations and by Fuchs and Hamrick (Guaiacum sanctum (Zygophyllaceae). This showed that genetic differentiation was high in analyzed accessions of T. terrestris than reported in the other members of family. Relative high percentages of pure accessions in both clusters indicate low admixture and natural interbreeding of accession. The reason may be distantness of the accessions included in the study. At K = 3, some of the accessions from the Rajasthan clustered separately and formed a pure cluster representing T. terrestris germplasm from Rajasthan. These observations showed that although there present only two genetic stocks for entire germplasm analyzed in this study, geographical and environmental factors have a great effect in shaping the populations of T. terrestris.Various clustering methods give the estimate of genetic relatedness of analyzed accessions of a species. Among different clustering tools, the tools based on Baysean methods are considered more robust. Here we used both distance-based and simulation-based Baysean methods for inferring genetic relationships and structure of different sis Fig.  supporten et al.  in Creos Hamrick  in GuaiaT. terrestris is one such plant which is frequently uprooted to replace major crops thus shrinking in minor patches. Sarwat et al. Supplementary material 2 (XLSX 12 kb)Below is the link to the electronic supplementary material.


Maintenance of genetic variance in secondary sexual traits, including bizarre ornaments and elaborated courtship displays, is a central problem of sexual selection theory. Despite theoretical arguments predicting that strong sexual selection leads to a depletion of additive genetic variance, traits associated with mating success show relatively high heritability. Here we argue that because of trade-offs associated with the production of costly epigamic traits, sexual selection is likely to lead to an increase, rather than a depletion, of genetic variance in those traits. Such trade-offs can also be expected to contribute to the maintenance of genetic variation in ecologically relevant traits with important implications for evolutionary processes, e.g. adaptation to novel environments or ecological speciation. However, if trade-offs are an important source of genetic variation in sexual traits, the magnitude of genetic variation may have little relevance for the possible genetic benefits of mate choice. Darwin  developeMaintenance of genetic variation in sexually selected traits has attracted much attention from evolutionary biologists, since prominent hypotheses for the evolution of sexual preferences assume genetic benefits for choosy females. Such indirect benefits of mate choice can stem either from positive feedback arising because the choosy sex  will produce progeny inheriting not only sexual attractiveness but also genes for preferences Fisher , or becaA) in trait combinations associated with the direction of selection, even though there is ample VA in individual traits  is presumably usually lower than that of life-history traits, they are nevertheless costly to produce. Thus the elaboration of a sexual trait should depend on the resources available to an individual. Based on this, the “genic capture” hypothesis posits that genetic variation in the expression of sexual traits represents genetic variation in resource acquisition and utilisation efficiency, or “condition”  play in maintaining genetic variation either via balancing selection, or by affecting mutation-selection balance  in horn size. Horned males have higher reproductive success, but lower survival , resulting from sexually dimorphic effects of alleles on female and male phenotypes  in weaker selection against deleterious mutations. Consequently, sexually-antagonistic mutations are expected to segregate in populations for longer than mutations deleterious for both sexes, resulting in larger amounts of genetic variance segregating in populations  unlock a number of ways by which genetic variance can be maintained. In the next two sections we consider the consequences of the nature of that variation for sexual and natural selection.One of the leading hypotheses proposed to resolve the lek paradox is the genic capture mechanism . If benefits to females of mating with the most competitive males were assumed to be large, extreme values of male traits evolve. In case of small benefits, arguably a more likely situation in case of genetic benefits of mate choice, trade-offs caused cyclical dynamics of sexually selected traits, such that their elaboration can decrease at some points of the cycle, followed by directional selection leading again to extreme trait elaboration. Under this scenario, preferences for epigamic traits easily evolve and are maintained in populations  and species turnover rate  is likely to impose antagonistic selection on many genes involved in basic organismal processes. Consequently, in as much as such trade-offs contribute to the maintenance of genetic variation (Connallon and Clark Conversely, fluctuations in ecological conditions can have consequences for the evolution of sexually selected traits and mate choice that are also worth exploring more fully. In our opinion, the realisation of the consequences of sexual selection for the maintenance of genetic variance in sexually selected traits opens new research perspectives in several areas of evolutionary biology.


Triatoma infestans, a common vector of this disease, has recently expanded its range into rapidly developing cities of Latin America. We aim to identify the environmental features that affect the colonization and dispersal of T. infestans in an urban environment. We amplified 13 commonly used microsatellites from 180 T. infestans samples collected from a sampled transect in the city of Arequipa, Peru, in 2007 and 2011. We assessed the clustering of subpopulations and the effect of distance, sampling year, and city block location on genetic distance among pairs of insects. Despite evidence of genetic similarity, the majority of city blocks are characterized by one dominant insect genotype, suggesting the existence of barriers to dispersal. Our analyses show that streets represent an important barrier to the colonization and dispersion of T. infestans in Arequipa. The genetic data describe a T. infestans infestation history characterized by persistent local dispersal and occasional long-distance migration events that partially parallels the history of urban development.Chagas disease is a vector-borne disease endemic in Latin America.  T. infestans, an important vector of the etiologic agent of Chagas disease, in an expanding urban environment. Samples were obtained along a transect in Arequipa, Peru, that includes old, well-developed communities and new communities characterized by rudimentarily constructed houses. We assessed the clustering of subpopulations and the effect of distance, sampling year, and city block on genetic distance among pairs of insects. Our analyses show that streets represent an important barrier to the colonization and dispersion of T. infestans in Arequipa. The genetic data describe a T. infestans infestation history characterized by persistent local dispersal and occasional long-distance migration events that partially parallels the history of urban development.The colonization and dispersal of disease vectors in new and expanding urban areas pose important health risks. The population and demographic dynamics of these vectors are often unclear, and their temporal and spatial associations with urbanization are unknown. Here, we use molecular markers to describe the genetic structure of populations of  Triatoma infestans, a true bug that commonly harbors the etiologic agent of Chagas disease, Trypanosoma cruzi, has historically occurred throughout southern South America [Chagas disease is a vector-borne disease endemic in Latin America that poses important public health risks . Triatom America ,3. Its r America –6. Rapid America  and with America .T. infestans along a west-to-east transect within the city of Arequipa, Peru.Urban landscapes are a patchwork of habitats composed of a heterogeneous mosaic of city blocks separated by inhospitable streets . FeatureT. infestans [The study transect is located in the Mariano Melgar district of Arequipa, Peru . The tranfestans .T. infestans samples used in this study were collected in 2008 and 2011 from homes in the transect area; Tetramethrin  was sprayed into the cracks in stone walls or other small structures that provide suitable T. infestans habitats to flush out the insects. More than 3,000 individuals were collected, placed into individual tubes, and stored at -20°C. From this total, 180 were chosen for genetic analyses (90 from each sampled year). To choose specific samples for analysis, 30 houses were randomly chosen from each third of the transect  and one fifth instar or adult individual was randomly selected from each chosen home.The Genomic DNA was extracted from two legs of each sample following the insect tissue protocol provided with the Qiagen DNEasy Blood and Tissue kit . The 13 microsatellites used in this study are well characterized, commonly used for both macrogeographical and microgeographical studies e.g., ,12–14, aK, ranging from 2 to 12) assuming correlated allele frequencies, admixture, and no location data as a prior, with 500,000 Markov Chain Monte Carlo (MCMC) iterations and a 20% burn-in. The output of the STRUCTURE analyses was extracted in STRUCTURE HARVESTER [ΔK method implemented by Structure Harvester was used to determine the optimal number of clusters [Potential differences between collection years were assessed with the exact test of sample differentiation in Arlequin version 3.5.1.2  using 10ARVESTER  and the ARVESTER . The ΔK clusters . The cluclusters  as a pieWe used a permutation-based linear regression to quantify the effect of Euclidean distance, sampling year, and city block  on the genetic distance between pairs of insects using the Brat-Curtis dissimilarity index . Null diFIS), and the allelic richness around each individual sample in the defined radius considering all samples within that radius. Data points with fewer than 10 individuals within the radius were excluded from the analyses. The optimal distance was defined as the distance at which the number of valid samples levels off. Radii of 100, 125, 150, 175, 200, 225, and 250 m were considered. The indexes obtained with the selected radius were plotted using ArcMap 10. To assess the presence of trends across the transect, the calculated indexes , and allelic richness) were regressed against the horizontal position in the transect using JMP 10.Point estimates of population genetic diversity across the transect were calculated using sGD . The anahttp://doi.org/10.5061/dryad.5tt50.Microsatellite data generated in this study have been deposited to DRYAD and are available from the Dryad Digital Repository: T. infestans populations are very finely spatially structured within the sampled transect. The majority of city blocks can be characterized by a single, dominant subpopulation as defined by the STRUCTURE algorithm, which incorporates information from all analyzed loci  in genotype frequencies between the samples collected in 2008 and those collected in 2011. The genetic analyses showed that zed loci . Sampleszed loci . The optzed loci . by the K method ; howeverThe fine-scale spatial patterns are also visible for each of the 13 analyzed loci .. In nea2 = 0.01, p < 0.001) was found in the complete dataset.The location of samples within or between city blocks has an important effect on the genetic dissimilarity among samples, which is visually evident in the violin plots . The genFIS; Fig. The optimal radius distance for the neighborhood genetic diversity analyses was 225 m .. The agT. infestans and, consequently, that particular genotypes tend to be specific to a given city block.The expansion of cities alters the environment and creates new habitats for urban species. In an expanding city, the genetic signature of an invading population is expected to be closely related to the history of urban development. However, urban environments contain many migratory barriers that may interfere with such expansion . The datT. infestans characterized by constant local dispersal and occasional long-distance migration events. Local dispersal events mainly involve households on the same city block, resulting in city blocks colonized by closely-related individuals . These results confirm that T. infestans has been present for much longer in the older parts of the transect than in the newer parts. However, the analysis of the allelic richness adds a layer of complexity, as high levels of allelic richness are scattered throughout the transect . This mixed pattern can be explained by the connectivity of the transect to surrounding areas. These sections of great allelic richness could represent the location of the initial colonization or the location of subsequent contact with other T. infestans invasions.The genetic signatures typically associated with the recent introduction of a population, including limited heterozygosity and high inbreeding coefficients, were mostly associated with the newest section of the transect Fig. , 5. ThesT. infestans; however, these effects are very difficult to rigorously quantify. The genetic signatures detected in this study confirm that the demographic dynamics of T. infestans infestations are significantly affected by urbanization.The transition from new, recently urbanized communities to older, better established communities coincides with physical changes in housing structures; more specifically, land tenure tends to result in the acquisition of fully mortared walls and more domestic animals. Further changes can occur with increased capital, including the presence of fewer food animals and more companion animals . These cT. infestans infestation or reinfestation are typically controlled by the application of insecticide around infested households in a ring-like fashion. Our results suggest that the initial application of insecticide should be focused on the city block where the infestation was first detected. Moreover, even though streets represent barriers for these insects, our results and those of previously published works [Our conclusions may guide the research and development of strategies to control the emergence and re-emergence of vector populations in urban environments. Isolated instances of ed works  indicateed works ,34,39–42S1 TableThis list includes the name of the primer, the source, the fluorescent dye used, forward and reverse sequences, and PCR cycling conditions.(DOCX)Click here for additional data file.S2 TableFIS), and the allelic richness. N/A indicates that the samples were not used because the neighborhood size was smaller than 10. Note that the geographical position has not been included to protect resident’s privacy.The table details the ID of the sample, the size of the genetic neighborhood , the observed heterozygosity, the inbreeding coefficient ((DOCX)Click here for additional data file.S1 FigK is four groups.This method is implemented by Structure Harvester. The selected number for (DOCX)Click here for additional data file.S2 FigIndividuals are represented by circles where each colored half circle represents one allele. Single color circles are homozygotes and two-color circles are heterozygotes. Additional circles over individuals highlight the presence of uncommon alleles in distant parts of the transect . Alleles are presented in consecutive panels following the order reported in (DOCX)Click here for additional data file.S3 Figd.f. = 132, p = 0.0014).The analysis is restricted to pairs of samples with matching distances (up to 1 m). The differences are significant Click here for additional data file.S4 Fig2 = 0.0048; n = 133; p = 0.4277) or different city block , Euclidean distances have no effect on genetic distances. In the complete dataset, Euclidean distances have small but significant effects on genetic distances .In the restricted dataset, which only considers equally distanced pairs of individuals in the same Click here for additional data file.S5 FigThe selected distance (225 m) is indicated with an arrow. The optimal distance was considered to be the distance at which the increase in the number of valid samples levels off.(DOCX)Click here for additional data file.


Phylogeography contributes to our knowledge of regional biotas by integrating spatial and genetic information. In New Zealand, comprising two main islands and hundreds of smaller ones, phylogeography has transformed the way we view our biology and allowed comparison with other parts of the world. Here we review studies on New Zealand terrestrial and freshwater invertebrates. We find little evidence of congruence among studies of different taxa; instead there are signatures of partitioning in many different regions and expansion in different directions. A number of studies have revealed unusually high genetic distances within putative species, and in those where other data confirm this taxonomy, the revealed phylogeographic structure contrasts with northern hemisphere continental systems. Some taxa show a signature indicative of Pliocene tectonic events encompassing land extension and mountain building, whereas others are consistent with range expansion following the last glacial maximum (LGM) of the Pleistocene. There is some indication that montane taxa are more partitioned than lowland ones, but this observation is obscured by a broad range of patterns within the sample of lowland/forest taxa. We note that several geophysical processes make similar phylogeographic predictions for the same landscape, rendering confirmation of the drivers of partitioning difficult. Future multi-gene analyses where applied to testable alternative hypotheses may help resolve further the rich evolutionary history of New Zealand's invertebrates. As originally circumscribed, phylogeography deals primarily with the structuring of populations within species . This foIn its simplest form, two types of information can be gleaned from phylogeographic studies: first, the spatial distribution of genetic variation  and second, the extent of differentiation between genetic variants . Approaches founded in population genetic theory and latterly statistical phylogeography using coalescent modeling ,8 allow Phylogeographic analysis uses sampling in current time, but has the goal of making inferences about past populations. As with all phylogenetically-based methods, what results is a hypothesis, and the fit of the hypothesis to some other (prior) information is usually used as the basis for “testing” between alternative historic scenarios. An obvious limitation is that the method may not be able to discriminate among alternative historic processes that predict the same or similar distribution of genetic diversity. Even with the advantage of molecular clock calibration, there remains uncertainty about which extrinsic factors have influenced gene flow, because: (a) drivers may be contiguous or even coincide in time; and (b) divergence time estimates are imprecise for many reasons at the scale that is relevant in phylogeography –11.1.1.2 situated in the south-western Pacific Ocean [New Zealand is an archipelago of nearly 270,000 kmic Ocean  , and thAs in most parts of the world, the New Zealand fauna is dominated by invertebrates and in particular insects . This s1.2.Spatial variation in climate, topography and vegetation generates fairly steep north-south, and in some places east-west gradients . Spatial1.3.Identifying the pattern of genetic structuring among populations (or other samples) is just a first step in phylogeography, as the main motivation of such work is to inform on the nature and timing of extrinsic processes that have driven population structuring. Despite a traditional focus in New Zealand biogeography on ancient processes , most if not all species-level diversity is much younger and coincides with the geophysically active period during the Plio-Pleistocene . As phylA general expectation in biogeography and phylogeography is that organisms occupying the same spatial range are likely to have been subject to the same evolutionary drivers and this should be evident in their similar phylogeographic signatures ,99–101. 1.4.The scope of studies of New Zealand invertebrates that involve some spatial and genetic information is enormous, ranging from multispecies to single species, widely sampled to narrowly sampled, with shallow genetic diversity to variation at the informative limits of cytochrome oxidase I (COI) sequencing .Paracalliope fluviatilis [Peripatoides novaezealandiae [Hemideina thoracica confirmed that cryptic species were not present [Prodontria [Pristoderus bakewelli and Epistranus lawsoni [Paranephrops zealandicus [More than 50 studies deal with species-level variation, usually with relatively small samples of individuals from several species , and havviatilis  and morpalandiae . In cont present ,109 and odontria . High mt lawsoni , and koulandicus  await fuTaxon selection in phylogeographic studies tends to be biased toward widespread species, because these are perceived as having the potential to provide data about large-scale phenomena. Widespread species are more likely to be chosen because their ranges will more often encompass geophysical features of interest. These species by their very nature, however, are more likely to have high gene flow and connectedness , and may2.Variation in the level of mtDNA diversity may by used to infer population expansion from an area of high diversity towards one of low diversity . Half ofWe note from an inventory of regional New Zealand insect endemicity  that divAcanthoxyla; 0.022 uncorrected) and large .With only 37 independent intraspecific phylogeographic studies of invertebrates in New Zealand we find few statistically supported patterns although trends are revealing. Forest (n = 17) and aquatic (n = 11) species show all manner of phylogeographic patterns . In contKikihia subalpina, Potamopyrgus antipodarum, Orthopsyche fimbriata, Acanthophlebia cruentata, Clitarchus hookeri, Argosarchus horridus) might have expanded their ranges southward in a similar and relatively recent time-frame as maximum mtDNA distances within each are similar and relatively small  and tree weta (Hemideina crassidens and H. thoracica) were expanding southwards at the same time, as they show quite different intraspecific pairwise mtDNA distances . A refugium (at some undefined time in the past) in the Nelson region (northern South Island) is possible for centipedes (mtDNA genetic distances up to 32%), koura   . For exaotamius) . There iotamius) , aquaticotamius) . Speciesotamius) . These tdisation , and theThe most obvious gap in terrestrial habitat in mainland New Zealand is Cook Strait, a narrow seaway minimum 25 km) between North Island and South Island . This fe km betwe4.Zelandoperla fenestrata, found all three to be quite highly structured, consistent with the field observation that the winged form is rarely, if ever, seen in flight [Z. decorata showed much less structure. In a cicada, lineages are finely subdivided, despite the cicada being widespread and apparently a good flier [With their flighted and flightless forms, insects display a wide range of dispersal capability and behaviour. Flightlessness evolves quickly and repeatedly, even showing polymorphism within species , presentn flight . In contod flier . Studiesod flier . Successod flier  include od flier , isopod od flier , cicada od flier , winglesod flier , beetlesod flier  and wormod flier .5.A hypothesized effect of glaciation in South Island is vicariance (division) of widespread species into isolated populations . Glaciat6.The current data indicate that all manner of phylogeographic patterns are identifiable among New Zealand invertebrate taxa , sustainversus Pleistocene effects of habitat availability.Even with the best multigene data to infer phylogeographic patterns, it will remain difficult to distinguish among some events that might be drivers of population partitioning and speciation in New Zealand. The geophysical events that have shaped New Zealand often overlap in their temporal influence, so future phylogeographic studies should concentrate on alternative hypotheses that can be distinguished, such as Pliocene In the meantime, we have to remember that inferences about history are susceptible to the general problems of biogeographic testing and the difficulty of assigning confidence . It is e


Bombus species  and one declining species (B. ruderatus) in an agricultural landscape in Southern England, UK. The study landscape contained sown flower patches representing agri-environment options for pollinators. We found that, as expected, the B. ruderatus population was characterized by relatively low heterozygosity, number of alleles and colony density. Across all species, inbreeding was absent or present but weak (FIS = 0.01–0.02). Using queen genotypes reconstructed from worker sibships and colony locations estimated from the positions of workers within these sibships, we found that significant isolation by distance was absent in B. lapidarius, B. hortorum and B. ruderatus. In B. terrestris and B. pascuorum, it was present but weak; for example, in these two species, expected relatedness of queens founding colonies 1 m apart was 0.02. These results show that bumble bee populations exhibit low levels of spatial genetic structure at fine spatial scales, most likely because of ongoing gene flow via widespread queen dispersal. In addition, the results demonstrate the potential for agri-environment scheme conservation measures to facilitate fine-scale gene flow by creating a more even distribution of suitable habitats across landscapes.Land-use changes have threatened populations of many insect pollinators, including bumble bees. Patterns of dispersal and gene flow are key determinants of species' ability to respond to land-use change, but have been little investigated at a fine scale (<10 km) in bumble bees. Using microsatellite markers, we determined the fine-scale spatial genetic structure of populations of four common  Land-use change and the consequent loss and degradation of habitats have fragmented the ranges of many insect pollinator species, leading to significant declines in population size and an increased risk of extinction (Potts Bombus spp.) are important pollinators of a range of native plant species and commercial crops and thus contribute significantly to global crop yields and the persistence of plant communities  across an agricultural landscape in Southern England, UK. Four of these species are nationally common and widespread, whereas B. ruderatus has suffered significant declines in the UK in recent decades and is now restricted to a few sites in southern and central England  shows reduced genetic diversity and higher levels of inbreeding than the common species; and (ii) if bumble bee populations are well mixed on a fine scale, then related queens, for example sisters or cousins, do not tend to nest in proximity to one another, leading to an absence of isolation by distance at fine scales. We also assess the implications of our results for the ability of agri-environment schemes to support foraging and nesting behaviours, and promote gene flow, in common and declining bumble bees.We addressed these issues by conducting a genetic study of five social species of bumble bee  and have longer-tongued workers that specialize in foraging at flowers with long corolla tubes , on biodiversity at the farm scale  , combined with a digital canopy height model derived from LiDAR, produced a high-resolution (0.5 × 0.5 m pixels) map containing 18 land use or land cover (LULC) types including arable crops, intensive grassland, trees and buildings . For further details on the collection and processing of the LiDAR and hyperspectral data, see Redhead et al.  and hyperspectral [Specim AISA Eagle (400–970 nm)] data. All data sets were geo-referenced and preprocessed. Supervised classification of the hyperspectral data set .The study area was divided into 250 × 250 m grid cells. Within every cell, bumble bee workers were sampled across all potential habitat patches with sampling intensity (i.e. search effort) being broadly proportional to the relative cover of suitable nesting and foraging habitats present. Hence, searches were focussed mainly on field boundaries and other noncrop habitat parcels (defined areas of continuous land use) but also included field centres. Within linear parcels, the full width of the boundary or hedgerow was searched. Within nonlinear parcels, searches focussed within a 6-m-wide transect following a zigzag pattern across the field or woodland. Flowering crops were searched by walking along tramlines and sampling workers within 2 m to either side. Within the few villages or hamlets present, gardens were visited where possible or searches conducted along public rights of way. All workers of the five study et al. Worker sampling was conducted on 4–5 days per week between 20 June and 5 August 2011. Habitats within each grid cell were searched at least once during this period. Workers were caught and held in a cooled container until a given habitat parcel or grid cell had been searched . The tarsal tip was nonlethally removed from the right mid-leg of each bee  using a LIZ 500 size standard. Genotypes were resolved using genemapper software v. 4.1.6. All species were genotyped at loci BL03, BL11, BT10, BT26 and BTMS0125. Bombus terrestris was additionally typed at BTERN01, BTMS0045, BT18, B96, BTMS0033, BL06, B10, B124 and B126; B. lapidarius at BL02, BL06, B10, B11, B131, BTERN02, BTMS0057 and BTMS0136; B. pascuorum at BL02, B96, BL06, B10, B124, B126, B131, B132 and BTMS00125; B. hortorum at BTERN01, B96, BTMS0045, BT18 and BTMS0136; and B. ruderatus at BTERN01, BT18, BTERN02, B131, BTMS0045 and BTMS0136. In total, 2577 workers were successf-ully sampled and genotyped across the five study species.DNA was isolated using the HotSHOT protocol  and expected heterozygosity (HE) were calculated in arlequin v. 3.5  averaged over all loci. Tests for deviation from Hardy–Weinberg equilibrium and linkage disequilibrium were also conducted in arlequin. Hardy–Weinberg P-values were obtained using a Markov chain of 100 000 steps. P-values for linkage disequilibrium were obtained using Fisher's exact test with 10 000 permutations. Significance levels were adjusted for multiple testing with P < 0.05 as appropriate.To remove any possible confounding effects of family structure, the sampled workers were reduced by eliminating all but one sibling per inferred family (colony) before the basic population genetic analyses were conducted. Number of alleles (A), observed heterozygosity  the mean centre locations to the nearest LULC class that might have formed suitable nesting habitat . In this case, ‘nearest’ was taken as the closest point on the boundary of the relevant habitat parcel orthogonal to the initially estimated colony location. Alternative approaches tested in preliminary analyses were heavily influenced by either outlying worker locations  or clusters of workers (e.g. median centre). In addition, because the mean centre method involved a purely statistical single-point output requiring no additional parameters or analysis, no prior assumptions regarding likely foraging distances were required. However, the method still yielded similar estimated colony locations to the kernel density estimation method used previously in the same landscape  exhibited significantly positive FIS values, whereas B. ruderatus exhibited a marginally significantly positive FIS value  and, given the lack of deviation of the populations from Hardy–Weinberg equilibrium, it appears that inbreeding was either absent from the study populations or, if present, very weak.Across all the study species, the number of alleles per locus (A) varied from 10.55 to 19.50, and the effective number of alleles (AE) from 4.39 to 9.55  exhibited significant isolation by distance, with pairwise relatedness between colony queens decreasing as intercolony geographic distance increased . In addition, estimated relatedness between close neighbours was very low and declined to zero over a relatively short distance. For example, in B. terrestris, if two queens had founded colonies 1 m apart, their expected pairwise relatedness would have been 0.02 . In B. terrestris, the expected pairwise relatedness falls to the sample mean at a colony separation distance of only 493 m. These findings suggest that isolation by distance in B. terrestris and B. pascuorum, if present, is very weak.Within the study landscape, two species (B. ruderatus) shows reduced genetic diversity and higher levels of inbreeding than the common species; and (ii) related queens do not tend to nest in proximity to one another at fine spatial scales. We found that, in all species, inbreeding was either absent or, if present, extremely weak. In terms of genetic diversity, we found that, in contrast to the closely related B. hortorum, the declining species B. ruderatus had the second lowest observed heterozygosity, the lowest allelic diversity and the second lowest effective number of alleles among the study species. The results therefore support our first hypothesis with respect to genetic diversity but fail to demonstrate greater inbreeding levels in the declining species relative to those shown by the common species at the study site. The results also support the second hypothesis, because we found that isolation by distance was either absent or only very weakly present. Together, these results suggest that gene flow in these populations of common and declining bumble bees is unconstrained at a fine spatial scale. In particular, they show that, in agricultural landscapes at this scale, queen dispersal and settlement patterns are such that bumble bee colonies nesting near one another are essentially unrelated and hence that populations are well mixed.We conducted population genetic analyses of the fine-scale spatial structure of four widespread and one declining bumble bee species occurring sympatrically within an agricultural landscape. Specifically, we tested the hypotheses that (i) the declining species  is consistent with previous results showing an association in bumble bees between population decline and a reduction in levels of genetic variation  was likely to have stemmed from the absence of obvious physical barriers to queen and male premating dispersal within the study landscape. In other populations of B. terrestris and B. pascuorum within agricultural and urban habitats, no evidence of inbreeding has been found . Such targeted agri-environment conservation measures have been shown to increase bumble bee abundance and potentially reduce worker foraging distances (Carvell et al. Finally, our results potentially inform conservation management for bumble bees. Jha & Kremen's  finding


Anastrepha fraterculus Wiedemann is a horticultural pest which causes significant economic losses in the fruit-producing areas of the American continent and limits the access of products to international markets. The use of environmentally friendly control strategies against this pest is constrained due to the limited knowledge of its population structure.A. fraterculus from four genomic libraries, which were enriched in CA, CAA, GA and CAT microsatellite motifs. Fifty microsatellite regions were evaluated and 14 loci were selected for population genetics studies. Genotypes of 122 individuals sampled from four A. fraterculus populations were analyzed. The level of polymorphism ranged from three to 13 alleles per locus and the mean expected heterozygosity ranged from 0.60 to 0.64. Comparison between allelic and genotypic frequencies showed significant differences among all pairs of populations.We developed microsatellite markers for A. fraterculus. This information will be used to identify and characterize candidate strains suitable to implement effective pest control strategies and might represent a first step towards having a more comprehensive knowledge about the genetics of this pest.This novel set of microsatellite markers provides valuable information for the description of genetic variability and population structure of wild populations and laboratory strains of  Anastrepha fraterculus Wiedemann (Diptera: Tephritidae) is an important pest of commercial fruit in the American continent. In subtropical and temperate regions of South America, this pest shares its habitat with the Mediterranean fruit fly Ceratitis capitata Wiedemann (Diptera: Tephritidae), and both species cause significant economic losses in fruit-producing areas. The presence of these species limits access to international markets due to quarantine restrictions imposed by fruit-fly-free countries. In Argentina, the National Control and Eradication Program (PROCEM) acts to control pest fruit fly species by using the only currently available control measures for A. fraterculus which are traps and pesticides.The South American fruit fly A. fraterculus is still poorly understood, so that the development of suitable molecular tools might greatly help in providing a more complete scenario for the effective implementation of control strategies against this pest.Biological studies on the reproductive behavior  .In order to select suitable polymorphic regions, 144 nucleotide sequences from the four libraries (75-83% enriched), containing di- or tri-nucleotide repeats, were analyzed. The percentage of enrichment was calculated based on the proportion of microsatellite sequences obtained from all the clones sequenced. To detect  2.2.22+ . To excl 2.2.22+ . All thehttp://simgene.com/Primer3)  based on the protocol described by Baruffi et al. . These molecular tools will be useful to be applied in a comprehensive investigation of the population diversity of an invasive pest of economic importance in the American continent and in the development and implementation of improved control strategies taking into account the genetic context of this species.Microsatellite markers may also be helpful to elucidate the species status within the  species ,12. In ty et al.  and Brazy et al. ,40,65 haA. fraterculus sp. 1 microsatellite sequences were generated and analyzed. Selecting 14 microsatellite loci was useful to analyze four populations of A. fraterculus. The study of the genetic variability both in lab strains and wild populations represents a first step to explore the genetic forces modulating the levels of genetic variability during artificial rearing in this species. The microsatellite markers developed here will provide valuable insights into the population genetics, colonization patterns and phylogenetic relationships of this species and into ecological strategies in the field. In addition, these tools characterize a source of polymorphic molecular markers for species identification in the fraterculus complex of cryptic species and could represent a valuable tool for the analysis of the genetic variability of other congeneric species.A total of 144 A. fraterculus, and particularly to identify and characterize A. fraterculus candidate strains to be used in environmentally friendly control strategies against fruit fly pests, such as SIT, which allow diminishing the use of chemical control and toxic baits.The results provided here are of paramount importance for the integral genetic knowledge of The authors declare that they have no competing interests.SBL analyzed the SSR nucleotide sequences, characterized the microsatellite markers, performed most of the analyses described in the manuscript and drafted the largest part of the manuscript. MJ participated in the characterization of the microsatellite markers and carried out molecular genetic studies. AB participated in the characterization of SSR markers. LG participated in sequence analysis and helped to draft the manuscript. ACS participated in the drafting of the manuscript. DFS helped in the design of the study and to draft the manuscript. JLC participated in the design of the study and helped to draft the manuscript. AM and GG hosted a part of the research performed in their laboratory, participated in the design of the study and helped to draft the manuscript. All authors read and approved the final manuscript.A. fraterculus microsatellite loci. Detailed legend: N: number of individuals analyzed; Dye: fluorescent label incorporated in forward primers; T: optimized annealing temperature; [MgCl2]: optimized magnesium chloride concentration; Size: size of PCR product predicted by Primer 3 software. A: Number of alleles detected. NA: no PCR amplification observed; ND: number of alleles not determined; MP: multiple peaks or bands observed in agarose gels or capillary electrophoresis analysis; CP: complex peaks: complex pattern of peaks observed in capillary electrophoresis analysis, difficult allele scoring. * Primers designed by GIS Company using Designer PCR software v 1.03 .Basic characteristics and PCR amplification conditions of 50 Click here for fileA. fraterculus. Detailed legend: aN: number of alleles per locus; OH: observed heterozygosity; EH: expected heterozygosity; Null: estimated null allele frequency [P < 0.05). A total of 122 individuals  were analyzed.Microsatellite analysis of wild populations and laboratory strains of requency ; *value Click here for fileTable S1 Allelic frequencies obtained from GENEPOP for IGEAF and IPCL laboratory strains and Concordia and Puerto Yeruá wild populations  for each locus (14 loci).Click here for fileTable S2 Observed and expected genotypic frequencies obtained from GENEPOP for each population  and locus (14 loci).Click here for file


Bulinus (Planorbidae) serve as intermediate host for flukes belonging to the genus Schistosoma . Despite its importance in the transmission of these parasites, the evolutionary history of this genus is still obscure. In the present study, we used the partial mitochondrial cytochrome oxidase subunit I (cox1) gene, and the nuclear ribosomal ITS, 18S and 28S genes to investigate the haplotype diversity and phylogeny of seven Bulinus species originating from three endemic countries in Africa .Snails species belonging to the genus Bulinus sequences. High levels of genetic diversity were detected at all loci in the seven studied species, with clear segregation between individuals and appearance of different haplotypes, even within same species from the same locality. Sequences clustered into two lineages; (A) groups Bulinus truncatus, B. tropicus, B. globosus and B. umbilicatus; while (B) groups B. forskalii, B. senegalensis and B. camerunensis. Interesting patterns emerge regarding schistosome susceptibility: Bulinus species with lower genetic diversity are predicted to have higher infection prevalence than those with greater diversity in host susceptibility.The cox1 region showed much more variation than the ribosomal markers within Bulinus is essential to understand the epidemiology of many schistosome parasites.The results reported in this study are very important since a detailed understanding of the population genetic structure of  Bulinus  serve as intermediate hosts for larval development of the parasite species belonging to the Schistosoma haematobium species group in Africa, the Eastern Mediterranean and Madagascar [S. haematobium blood flukes are responsible for 112 million infections in Africa with an incidence exceeding 50% in some communities [Bulinus genus have an extensive distribution throughout Africa, some areas of the Middle East and the Mediterranean countries [B. africanus, B. forskalii, B. reticulatus and the B. truncatus/tropicus complex [Schistosoma parasites. However, the role of each species in the transmission of the disease varies considerably between and within countries [B. truncatus is more implicated than B. globosus in the transmission of urinary schistosomiasis [B. globosus, B. senegalensis and B. umbilicatus are known as the main intermediate hosts for S. haematobium [Snails of the genus  Müller, 781 servemunities . Indeed,ountries . It is dountries . For exasomiasis , while tBulinus species. Furthermore, such information is essential for assessing the geographical distribution of variation in genes associated with resistance in bulinid snails. Previous studies showed that the above four Bulinus species groups were stable throughout the majority of phylogenetic analyses and demonstrated the monophyletic status of these groups with both variable and highly conserved genes [The interpretation of these specific snail-parasite relationships requires a deeper understanding of the past events that have affected the population diversity and distribution of ed genes -10. HoweBulinus species collected from endemic areas in three African countries , using one mitochondrial gene (the protein coding cytochrome oxidase subunit I (cox1) gene and three nuclear genes ). We tried to explain their present diversity in relation with schistosomiasis transmission.In this investigation, we used a suite of population genetic and phylogenetic analyses to characterize populations of seven B. umbilicatus to 0.99 for B. senegalensis. Furthermore, our estimate of nucleotide diversity ranged from 0.016 in B. camerunensis population to 0.052 for B. senegalensis . All mtDNA sequences were found to be highly variable. Within species, the global haplotype diversity was very high in our samples ranging from 0.85 in B. truncatus unique haplotypes found in the Northern region of Africa (Egypt) is lower than that found in the Sub-Saharan Africa . There is also higher nucleotide diversity found within B. truncatus populations sampled from Cameroon and Senegal compared to Egypt.Within countries, as expected haplotype diversity is lower but exceeds 0.800 in the three countries Table . The numB. senegalensis species (Hd = 0.86), while the lowest one was observed within B. truncatus (Hd = 0.157), confirming the results obtained from the cox1 data .A data matrix of 855 bp was used in this analysis, the sequences were found to be more variable than 18S, and therefore higher values of haplotype and nucleotide diversity were observed  between the seven studied species was calculated using the combined data matrix , whereas those of B. truncatus and B. tropicus are the most closely related (Fst = 0.206), as expected since these two species are part of the same group.The diversification index , maximum parsimony (MP) and neighbor-joining (NJ)). All analyses confirm the monophyletic status of the Bulinus specimens, as this improved resolution and resulted in higher bootstrap values for internal nodes compared to the single gene trees. The three different tree-building algorithms were largely congruent , the first subclade C contains sequences that are restricted to either B. forskalii and B. camerunensis, while subclade F contains sequences that are restricted to B. senegalensis, which is itself split into two groups: the first with Cameroonian haplotypes and the second with Senegalese haplotypes  confirm the major clades described by both the combined data matrix and the cox1 region Figure . Howeverr Figure .Figure 3B. truncatus and B. forskalii (from Cameroon) where one snail out of six to seven individuals had different mtDNA haplotypes using the same fragment [The mtDNA sequences showed high haplotype and nucleotide diversity, with a clear segregation according to species and locality. Many different haplotypes were found, with approximately one out of three to four snail specimens having a different haplotype within the same locality. This high level of genetic diversity was higher than that found by Nalugwa et al.  in B. trfragment .Bulinus taxa representing the four species groups currently recognized in this genus from different African countries [Of the nuclear fragments, 18S was the least variable, while 28S showed an intermediate level of nucleotide variation compared to the ITS fragment which was the most variable. These results are comparable to that found in the work of Jørgensen et al., [i)Bulinus populations investigated in the present study originated from geographically distant countries, limiting gene flow between them. This reproductive isolation might have allowed independent evolution and divergence of these populations leading to distinct lineages. Indeed, our results showed that B. truncatus and B. senegalensis populations from the same country clustered together and formed different lineages Bulinus species are hermaphrodite and can reproduce both by cross-fertilization and/or self-fertilization [B. globosus and B. senegalensis are mainly outcrossing and therefore they showed high levels of genetic variability.Secondly, lization , and eaclization . B. globiii)B. truncatus is preferentially self-fertilizing, it is a tetraploid species [Thirdly, despite the fact that  species , and stu species , which mThis high genetic diversity detected at all loci, both within and between species is probably influenced by several factors:Schistosoma and Bulinus species is very specific and varies in according to geographic location [The relationship between location ,16. For location . This inlocation .B. truncatus than for B. globosus and B. senegalensis at all loci. In fact, according to Schmid-Hempel [B. truncatus populations. Indeed, experimental infections of B. globosus and B. truncatus from Cameroon with different isolates of local miracidia were carried out to study snail-parasite compatibility [B. truncatus might be more implicated than B. globosus in S. haematobium transmission.In Cameroon, our results showed lower levels of genetic diversity for d-Hempel , and Cold-Hempel , a loss d-Hempel ,19, basitibility . Their fB. forskalii is ubiquitous in Cameroon and it is often present in association with B. senegalensis. It acts as intermediate host for S. guineensis [. The results reported in the present study revealed very high levels of genetic diversity in B. forskalii at all loci, which is in accordance with the work of Gow et al. [B. forskalii populations in Cameroon. Furthermore, according to Tchuem Tchuenté et al. [S. guineensis transmission in some major foci in Cameroon, indicating a progressive extinction for this parasite species in this country [S. guineensis and S. haematobium, but also is maybe influenced by the high levels of genetic diversity within Cameroonian B. forskalii populations since a gain of genetic variation can decrease susceptibility to parasitism [On the other hand, ineensis . The resw et al. , who havé et al. , there i country . This parasitism .B. truncatus acts as intermediate host for urinary schistosomiasis. B. truncatus snails are recorded in water bodies throughout Egypt. It is most abundant in large canals, and decreases in density as the water approaches and flows into drains [B. truncatus snails are year-round infected with schistosomes [B. truncatus populations. In parallel, according to El-Khoby 2000, there is a decrease in the prevalence of S. haematobium in Egypt during the last years, and S. mansoni has almost totally replaced S. haematobium in Lower Egypt and is spreading into Upper Egypt [S. haematobium in Upper Egypt sites was higher than that found in the Nile Delta [B. truncatus in Nile Delta.In Egypt, only o drains . In thisstosomes . In the er Egypt . Furtherle Delta . This miB. truncatus populations than those in Egypt and Cameroon. This might be linked to the fact that Senegalese B. truncatus populations are implicated in the transmission of S. bovis and S. haematobium x S. bovis hybrids, but not of pure S. haematobium [In Senegal, our results showed higher diversity in matobium , suggestB. truncatus populations originating from North Africa (Egypt) compared to those originating from Sub-Saharan Africa  at all loci. Many factors can explain this lower diversity in Egypt such as selection, drift or probably a relatively recent introduction since specimens from Cameroon and Senegal are basal to them in lineage C . Snails were collected from their habitats and conserved in 70% ethanol until genetic analyses.Snails of the Genomic DNA was extracted from snails using the QiAamp DNA Mini Kit  following manufacturer’s instructions. A Nanodrop ND-1000 spectrophotometer was used to quantify and check DNA purity.Four markers were selected for sequencing: the protein coding cytochrome c oxidase subunit I (cox1), and three nuclear ribosomal genes . Partial sequences from the four genes were obtained after PCR amplification. Various combinations of the used primers are presented in Table For cox1 and ITS, 25 μL PCR mixtures were set up with 0.5 U Taq polymerase (Qiagen), 5 μL 10x buffer, 2–4 μL MgCl2 (depending on template and primers), 0.8 μL dNTPs, 0.5 μL from each primer (20 ng/μL). For 18S and 28S rRNA; amplification was performed with High Fidelity Taq DNA polymerase (Roche) in a 50 μL total reaction volume with standard reaction conditions since this enzyme is designed to increase yield and fidelity when amplifying longer fragments. A standard concentration of 500 ng/μL was used as template for all PCR reactions.The PCR conditions varied for each primer combination. Cycling conditions for both cox1 and ITS were as follows: preheat step at 95°C for 5 min followed by 35 cycles of denaturation at 94°C for 10 sec, annealing at 40°C for 30 sec and extension at 72°C for 1 min; final extension step at 72°C for 10 min. Amplification for both 18S and 28S involved i) a preheat step at 94°C for 2 min, ii) 10 cycles, each including denaturation at 94°C for 15 sec, annealing at 43°C for 30 sec and extension at 72°C for 45 sec, iii) 15 cycles including the same conditions except the extension step which was expanded to 5 sec per cycle, iv) a final extension step at 72°C for 10 min.PCR products were purified using QIAquick PCR Purification Kit , then sequenced using an ABI Prism BigDye® Terminator v1.1 Cycle Sequencing Ready Reaction Kit  following manufacturer’s instructions, and run on an ABI 377 automated sequencer.The obtained sequences were used to perform BLAST searches  via the (Fst) between each pair of species.DnaSP v5.10.1  was usedMEGA v5  was usedIndoplanorbis exustus  (Planorbidae: Bulininae) was selected as outgroup in analyses of cox1 (Folmer and Asmit regions), 18S and 28S, as Morgan et al., [Bulinus [. Trees using ITS data matrix were constructed with Biomphalaria helophila as outgroup since no ITS sequence of I. exustus was available. Parsimony inference was performed via a heuristic search using 1000 replicates of random sequence entry, tree-bisection-reconnection (TBR) branch swapping. Maximum likelihood and NJ trees were performed with the inferred substitution models as mentioned above.In the case of interpopulation diversity, data matrices were constructed (with unique haplotypes) for individual genes separately, and then for all combined genes. All phylogenetic analyses were carried out using MEGA. Evolutionary relationships between the haplotypes were inferred using maximum parsimony (MP), maximum likelihood (ML), and Neighbor-joining (NJ) methods. The different types of analyses were subjected to 1000 bootstrap replicates as a means for testing the reliability of individual branches within the generated tree. The data sets supporting the results of this article are available in the Dryad.org repository, doi:10.5061/dryad.hf63h.


Apodemia mormo) species complex occurs as isolated and phenotypically variable colonies in dryland areas across western North America. Lange’s Metalmark, A. m. langei, one of the 17 subspecies taxonomically recognized in the complex, is federally listed under the U.S. Endangered Species Act of 1973. Metalmark taxa have traditionally been described based on phenotypic and ecological characteristics, and it is unknown how well this nomenclature reflects their genetic and evolutionary distinctiveness. Genetic variation in six microsatellite loci and mitochondrial cytochrome oxidase subunit I sequence was used to assess the population structure of the A. mormo species complex across 69 localities, and to evaluate A. m. langei’s qualifications as an Evolutionarily Significant Unit.The Mormon Metalmark  contains supplementary material, which is available to authorized users. Global loss of biodiversity, fueled by unprecedented anthropogenic influences, has elevated the importance of conservation biology in mainstream public consciousness. Despite this increased attention, one of the most fundamental challenges of conservation remains unsolved and contentious: how do we accurately identify and delimit manageable units of biodiversity -9? Sinceetc.) that exclusively cluster individuals or populations using the phylogenetic species concept )A. mormo . IsolatiA. mormo ,51,52). cificity ,50 restrcificity . This scA. m. mormo, exemplifies this point at the largest geographic scale considered in this study. From California to British Columbia and from New Mexico to Saskatchewan these butterflies can be more-or-less phenotypically identical, but exhibit deep genetic divergences in both their mitochondrial and nuclear genomes. Regionally, geographic proximity is a better predictor of genetic similarity than phenotypic appearance. However, within the three broad regions considered here, some areas exhibited stronger taxonomic/genetic/geographic correspondence than others. Northern Californian populations, for example, display higher correspondence between genetic variation and geography than in southern California , individuals from Antioch Dunes form a monophyletic mtDNA subclade . Several individuals from this population shared some of A. m. langei’s distinct phenotypic characters, but exhibited divergent mtDNA (clade W2) and high levels of admixture with microsatellite loci. The habitats of these populations are not particularly similar, and the use of different Eriogonum hosts , not with individual-based Bayesian clustering methods (STRUCTURE). Ecologically, A. m. langei is potentially distinctive, as its host Eriogonum nudum var. psychicola Reveal 2007 is also an endemic of the Antioch Dunes    and geog(1-FST)] , respect(1-FST)]  implemen(1-FST)] . A Bonfea priori populations. The program Poptree2 [a priori populations. 1000 bootstrap replicates were performed.In order to provide a more direct comparison between microsatellites and mtDNA, a phylogenetic tree was generated using 154 unique mtDNA haplotypes from samples for which we also had microsatellite genotypes. We generated a maximum-likelihood tree in Garli 1.0  under thPoptree2  was usedPoptree2  of populA. mormo species complex recognized by [A. mormo complex (as documented by [Eleven wing characters were selected to objectively relate the genetic data produced in this study to the existing taxonomic nomenclature of the 17 subspecies in the nized by  (Additioented by ) were inented by , examinaented by , and geoThe sequence dataset supporting the results of this article are available in the GenBank repository (Additional file


Genetics and genomic medicine in Mali: challenges and future perspectives. Mali is a landlocked country located in the center of West Africa and is surrounded by seven other countries Fig. . With a Genetic and genomic studies in Mali have been limited, leading to an underestimation and neglect of genetic diseases and the genetic contribution in common diseases. This may be due to social factors surrounding these diseases, limited resources preventing patients from seeking care, and a lack of infrastructure and health professionals. In fact, the low literacy rate in Mali, as in many developing countries, may foster a low understanding of basic genetic concepts by the general population. For instance, Malians often consider genetic diseases to be a result of bad fate, which leads to their stigmatization. In addition, limited resources have led authorities to place less emphasis on education in genetics, often mistakenly considered by medical students as a difficult science and not within their reach.Genetic diseases often cause premature death, severe disability and loss of productivity, resulting not only in high health care costs, but also in high loss of disability‐adjusted life years (DALYs). While these numbers are estimated in more developed countries such as in the United States and Europe , the majority of funds go to subsidize primary education. Until now, only a subset of medical doctors is sponsored by the government to get their specialty degree. Almost all specialists are concentrated in the capital city Bamako or in regional centers, and the recent unrest in the northern 2/3 of the country has left the populations of this region with almost no highly skilled health care practitioners. Despite the recent expansion of residency programs in almost all medical specialties, there still is no such program in genetics. This is due to the lack of qualified trainers, but also to health policies focusing primarily on common and communicable diseases.Despite the progress made in recent years regarding healthcare services, Mali is still lagging behind in terms of genetic healthcare delivery. Until recent years, there were few medical residencies in certain specialties in the country. Most medical doctors went to neighboring countries to get further training at their own expense. With an overall education expenditure of 4.8% of the GDP . Although it is one of the most common inherited diseases in the world, SCD was recognized by WHO as public health concern only in 2006. Mali has a prevalence of 3% with 5–6000 newborns with SCD each year . Interestingly, the same mutation in C19ORF12 was found in a Brazilian family with some clinical overlap, and haplotype reconstruction suggested that the Malian and Brazilian families may be related  started screening patients with hereditary neurological disorders. Genetic testing and counseling were performed under a joint protocol approved by the IRBs of both institutions. As the Malian population had never been exposed to genetic testing, the knowledge, attitudes, and beliefs of Malian families with hereditary neurological disorders regarding genetic testing were first assessed in pre‐and posttest questionnaires. The results showed that in general, the majority favored genetic testing and some gained knowledge from genetic counseling . While the overall pace of government intervention in research has been slow in Africa, leaders such as Mauritian President Ameenah Gurib‐Fakim are increasingly advocating for more investment in research . Current advances in genomic medicine propose the delivery of individualized treatment according to an individual's genetic background. This initiative, entitled Precision Medicine and supported by the US government, will lead to the sequencing of millions of human genomes and will substantiate treatment of several diseases. To date, no known Malian genome is published. If no effort is made by African governments, the disparities in genetic health will be similar to those seen with other new technologies. Few research projects are funded in Mali, and except for SCD, no genetic disease has been funded by the Malian government.Although the African Union has suggested African countries invest 1% of their GDP into research during their 2006 summit, this has not been fully translated into action yet , and nongovernmental organizations such as The Bill and Melinda Gates Foundation  and thousands of phenotypes showing Mendelian inheritance, many more disease genes remain to be identified, especially in unexplored populations. Although several thousand genes have now been associated with diseases, it remains important to find new genes to further our understanding of their function and interactions and to increase our knowledge of common disease mechanisms. Because of the population diversity in Africa and the limited studies done on the continent, genetic and genomic research in Africa will certainly answer many health questions that could not be solved by studying other populations.With only about 20% of human genes found to have disease‐associated mutations to date (Although medical genetics and research studies of genetic diseases are in their infancy in Mali, tremendous progress has been made during this decade to improve genetic research. A strong partnership between Malian scientists and their international collaborators has been the reason for this improvement. To ensure sustainability, the Malian government should be fully engaged to empower genetic training and research funding. With this support, Malian scientists and clinicians would be ready to meet emerging medical genetics and genomic challenges.None declared.


Genetic diversity assessment plays an important role in plant improvement. It becomes more significant when evaluation is done at different ploidy and geographical origin levels. The present study provides a better understanding of the genetic association of Indian and Turkish hexaploid and tetraploid wheat. The Turkish hexaploid population demonstrated its close association with Indian hexaploid and tetraploid varieties. This confirmed their relatedness within the diverse gene pool. The results revealed in this study can be effectively used by breeders and evolutionary biologists for the development of genetically diverse, promising and healthier wheat varieties. Triticum species have not been assessed to date. In this study, genetic association of 95 tetraploid and hexaploid wheat genotypes originating from India and Turkey was determined for the first time. Combined analysis of random amplified polymorphic DNA and inter-simple sequence repeat markers disclosed 177 polymorphic bands, and both the dendrogram and two-dimensional scatterplot showed similar groupings of the wheat genotypes. Turkish hexaploid varieties were basically divided into two clusters, one group showed its close association with Indian hexaploid varieties and the other with Indian tetraploid varieties. Analysis of molecular variance revealed high (77 %) genetic variation within Indian and Turkish populations. Population structure analysis elucidated distinct clustering of wheat genotypes on the basis of both geographical origin and ploidy. The results revealed in this study will support worldwide wheat breeding programmes and assist in achieving the target of sustainable wheat production.Genetic diversity among plant species offers prospects for improving the plant characteristics. Its assessment is necessary to help tackle the threats of environmental fluctuations and for the effective exploitation of genetic resources in breeding programmes. Although wheat is one of the most thoroughly studied crops in terms of genetic polymorphism studies, phylogenetic affinities of Indian and Turkish  Genetic variability in natural plant populations holds the potential to deal with multiple biotic and abiotic stresses. The potential to select a superior line increases with genetic diversity, the discovery of which becomes an important tool in plant breeding. On depletion of genetic variability, plants are unable to cope with unfavourable environmental conditions or pathogens and pests. Diversity studies also facilitate the conservation and management aims of a particular plant species. For the effective use of genetic diversity in plant breeding, knowledge of its extent and distribution plays a crucial role. Considering its significance, a large number of studies have been performed to estimate genetic diversity employing various methodologies in multiple plant species. Assessment of genetic variability employing molecular markers has proved to be a keystone to understanding the genomic constitution, categorizing the genes responsible for important traits, the classification and conservation of genetic variation in plant germplasm and developing selective proliferation approaches for plant propagation.Wheat production in developing countries moved from defective to surplus  during tDuring the course of evolution, wheat gained sufficient genetic diversity along the road from einkorn to bread wheat. Today, however, its diversity is weakening due to repeated cultivation of landraces for specific characters, narrow adaptation, farmers' varietal selection and the requirement of uniform varieties in industrial seed grain processing  and hexaploid (Triticum aestivum L.) wheat cultivars (Table The object of the present diversity study was a collection of 95 Indian and Turkish wheat genotypes including tetraploid (rs Table . Well-kn−1 as templates for polymerase chain reactions (PCRs).Two to three weeks grown seedlings were utilized for total wheat DNA extraction following the cetyltrimethylammonium bromide (CTAB) method  with somTaq buffer containing ammonium sulfate (except ISSR F3 where KCl was used), 3 μL of 25 mM MgCl2, 0.4 μL of 25 mM dNTP, 0.5 μL of 10 μM primer, 1.5 units of Taq DNA Polymerase and 100 ng of template DNA. The two-step ISSR–PCR reactions were performed in a Eppendorf Master Cycler. The physical reaction conditions and the number of initial and final PCR cycles were optimized for each individual ISSR primer.Twenty-seven ISSR primers (Metabion) were examined for distinguishing the polymorphism patterns, and among those 10 primers showed positive outcomes Table  against Taq buffer with ammonium sulfate, 2.5 μL of 25 mM MgCl2, 3 μL of 1 mM dNTP, 3 units Taq DNA polymerase (Thermoscientific), 1.5 μL of 5 μM RAPD primer and 50 ng of template DNA in a total volume of 15 μL. Polymerase chain reaction amplifications were carried out utilizing a Eppendorf Master Cycler with initial denaturation at 94 °C for 3 min, followed by repeated cycles of denaturation at 94 °C for 45 s, annealing as per the primer's melting temperature for 1 min and primer extension at 72 °C for 1 min. On completion of the repeated number of cycles, final extension was performed at 72 °C for 10 min.For RAPD reactions, a total of 43 primers (MWG Biotech-AC) were screened for polymorphism using selected genotypes, and 10 primers were selected for the final reactions  coefficient by Numerical Taxonomy and Multivariate Analysis System (NTSYS-PC) version 2.02e software . This siOn the basis of SM coefficients, the similarity matrix was double centred using the DCENTER module of NTSYS-PC. Then eigen analyses were performed using the EIGEN module of NTSYS-PC to construct two-dimensional scatterplots by the R package. Scatterplots were drawn for the substantiation of the dendrograms and verification of genotypes clustering according to both ploidy and geographical origin.To explain the population structure of Indian and Turkish wheat genotypes, analysis of molecular variance (AMOVA) was performed using GenAlEx 6.5 software , 2012 wiK populations was employed for genetically homogenous group estimation in Indian and Turkish wheat germplasm. A parameter of 50 000 burn-in period and 100 000 Markov Chain Monte Carlo replication, along with the admixture model and correlated allele frequencies, was used in STRUCTURE, version 2.3.4 (K (from 1 to 4 assumed)  and cRAPD2 was the most prolific RAPD primer (11). In total, 94.8 and 81.7 % bands were found to be polymorphic among ISSR and RAPD markers. The average number of polymorphic bands per primer was 11.0 and 6.7 for ISSR and RAPD primers, respectively . On the basis of ploidy, AMOVA detected higher genetic variation within tetraploid and hexaploid populations (92 %); however, the genetic variation between ploidies was only 8 %   Table .Table 4Principal coordinate analysis (PCoA) serves as a platform to provide a spatial illustration of the comparative genetic distances between the individuals. It also assesses the robustness of the differentiation among the groups classified by the dendrogram A. SimilaK clusters, an Evano test was performed and yielded K = 3 as the highest log-likelihood. This means that 3 was the optimum number of subpopulations, indicating that the two major population groups actually represent three distinct clusters.For population genetic structure analysis, Bayesian clustering modelling was executed in the STRUCTURE software using genotyping data generated by 177 RAPD and ISSR loci. As the clustering model presumes the underlying existence of K) from 1 to 4. Indian and Turkish populations involved in this procedure showed separation from each other in accordance with clusters obtained in PCoA. At K = 3, wheat genotypes were divided into three clusters with two main populations, 1 and 2  in comparison to landraces (44.8 %)  was found to be higher than that in a previous study by The dendrogram obtained in this study clearly clustered the genotypes according to their ploidy level, consistently with the evolution of wheat .P = 0.010)  Table . The resSimilar separation of Indian and Turkish wheat varieties was observed by PCoA on the basis of ploidy and geographical region. The outcomes of the two methods  were comparable. Both of them classified 95 wheat genotypes mainly into three clusters and offered similar alignment of the genotypes with a few negligible discrepancies. The groups attained were in agreement with the recognized geographical origin as well.K = 3. In PCoA, some of the Indian and Turkish varieties showed close association with each other, and similar varieties demonstrated admixture in Structure analysis confirming their relatedness within the diverse gene pool. In dendrogram also, these varieties showed distinct clustering with the main population groups. Closeness of some of the Turkish hexaploid genotypes with Indian hexaploid genotypes in PCoA was also supported by the population structure as well as the dendrogram. Similar and mutually supportive results from all the statistical analyses demonstrated the capability of RAPD and ISSR markers to distinguish Indian and Turkish wheat varieties efficiently.Population structure analyses indicated that wheat accessions can be efficiently categorized on the basis of both geographical origin and ploidy. Using the maximum membership probability in STRUCTURE, Indian and Turkish populations showed similar grouping to the UPGMA and PCoA clustering. The PCoA clustering divided Indian and Turkish populations basically into similar clusters to those produced by the Structure bar plot at Genetic diversity evaluation serves as a crucial platform in plant improvement. The present study provides a detailed understanding of the genetic association of Indian and Turkish hexaploid and tetraploid wheat. The Turkish hexaploid populations showed their closeness to Indian genotypes, confirming their alliance within the diverse gene pool. The present genetic diversity study of wheat material obtained from diverse regions will support breeders in expanding the genetic variation of breeding accessions and utilizing the studied wheat resources more effectively.The Scientific and Technological Research Council of Turkey) for performing the present research work. Also, a part of the research was funded by BAP (Turkish Scientific Research Project agency) under Project No. 14401106.M.K.K. has been granted ‘2216 Research Fellowship for Foreign Citizens’ by TÜBİTAK (M.K.K. initiated and obtained the funding for the research work. M.K.K. and A.P. contributed in performing the research work and preparation of the manuscript. M.K.K., A.P., S.A.K. and Y.O. carried out all statistical analyses. All other authors have provided suggestions and guidance for the successful completion of the work. All authors read and approved the final manuscript.None declared.