Ne. We combined both approaches in evaluating genetic connectivity of a threatened ungulate, woodland caribou. We used least-cost paths to calculate matrices of resistance distance for landscape variables  and population-pairwise harmonic means of Ne, and correlated them with genetic distances, FST and Dc. Results showed that spatial configuration of preferred habitat and Ne were the two best predictors of genetic relationships. Additionally, controlling for the effect of Ne increased the strength of correlations of environmental variables with genetic distance, highlighting the significant underlying effect of Ne in modulating genetic drift and perceived spatial connectivity. We therefore have provided empirical support to emphasize preventing increased habitat loss and promoting population growth to ensure metapopulation viability.Landscape genetics provides a framework for pinpointing environmental features that determine the important exchange of migrants among populations. These studies usually test the significance of environmental variables on gene flow, yet ignore one fundamental driver of genetic variation in small populations, effective population size,  A lack et al. t, where Ht/H0 is the reduction in heterozygosity after t generations. Consequently, knowledge of Ne can be a powerful tool in conservation as a predictor of genetic diversity loss, inbreeding and, perhaps most important to landscape genetics studies, population differentiation.While landscape genetics can enhance conservation planning, it cannot ignore the fundamental evolutionary processes that underlie metapopulation dynamics and their spatial and temporal scales. For example, few studies have taken into account the potential impact of population size on patterns of genetic diversity and the potential for genetic drift to obstruct resistance models from identifying important landscape genetic relationships, particularly when dealing with endangered species. Genetic drift is an evolutionary process with important implications in conservation biology due to its sensitivity to population fluctuations and temporal and geographical isolation –13. The deration , and whiNe among the factors determining patterns of genetic diversity. Specifically, we analyzed the primary environmental and demographic variables that drive ungulate population substructure in endangered woodland caribou (Rangifer tarandus). Our a priori landscape models were chosen to test specific hypotheses regarding the factors thought to have the greatest conservation impact on threatened species, including caribou. We test four hypotheses that population genetic structure is influenced by: (i) preferred habitat availability, (ii) anthropogenic barriers, (iii) predation risk, or (iv) reduced Ne. If small populations are experiencing rapid genetic drift owing to small Ne, this may explain a large variance component in population-pairwise genetic distances and obscure the signal from landscape variables. Woodland caribou offer an ideal species to explore these hypotheses as their ecological and conservation challenges are well documented in the literature [In this study, we conducted a detailed landscape genetics analysis that incorporates the effect of terature –17 and gterature ,18.2.(a)2 region in west-central Alberta and eastern British Columbia, Canada  enveloping flat valley bottoms. The climate is characterized by long winters and short, dry summers and habitat types include montane, subalpine and alpine ecoregions that correspond to increasing elevation and decreasing annual productivity. Protected areas tend to be located in the more mountainous regions, in contrast to the areas of highest human impact occurring predominantly in the boreal foothill regions in the eastern portion of the study area. Roads, seismic lines, well pads and forestry cut blocks were more prevalent in the eastern portion of the study area.The study area encompassed an approximately 70 000 km, Canada  that lieNc; We studied caribou herds representing eight spatially distinct populations Nc;  were bas(b)et al. [doi:10.5061/dryad.gn22271h). DNA was derived from blood samples collected through agency monitoring efforts between 2001 and 2009.The 207 individuals analysed represent those herds previously analysed  for whicet al.  (DRYAD e(c)FST [Dc, [Dc emphasizes genetic drift over mutation, reflects decreases in populations better than other genetic distance metrics, and thus may be particularly suited for microsatellites and fine-scale landscape genetic analyses such as those used here [FST was calculated using 10 000 randomizations, correcting for type I errors using sequential Bonferroni adjustment. We also estimated deviations of observed heterozygosities from those expected under Hardy–Weinberg equilibrium (HWE), using population inbreeding coefficients (FIS) using FSTAT 2.9.3 [FIS > 0 indicates greater inbreeding than expected under HWE and FIS < 0 indicates greater heterozygosity than expected [For comparison with population-pairwise resistance distances (see below), we calculated standard pairwise estimates of FST  in MicroFST ). We alsFST [Dc, ). Dc empsed here ,22. SignAT 2.9.3 , where FNe within each studied population, we estimated Ne using the linkage disequilibrium (LD) method in LDNe [Ne was used to provide a quantitative, non-landscape factor that may affect patterns of genetic differentiation among populations. We constructed a population-pairwise matrix of the harmonic mean of Ne between each population pair.To calculate the  in LDNe . We repo in LDNe ) and wit in LDNe  demonstrFinally, herds were assessed for genetic effects of rapid reduction in population size using the program BOTTLENECK . A two-p(d)We considered four landscape variables to be potentially biologically important in determining gene flow between caribou populations. These include a combination of ecological and anthropogenic factors that are explained below.a, RSF; [et al. [figure 2a).Habitat loss and fragmentation has been cited as one key driver in caribou population declines . We usedfigure 2a, RSF; ) model ta, RSF; ,16. DeCe [et al.  estimateCanis lupus) as a mechanism to avoid predation [figure 2c; [figure 2b), each as potential sources of landscape resistance, including: forestry cut blocks, roads and other non-road linear features (seismic lines and maintained hiking trails).Caribou use a strategy of spatial separation from wolves . SimilaFinally, we evaluated the null hypothesis of a completely homogeneous landscape of resistance by relating population differentiation with geographical distance alone, in IBD analysis. It is important to note that while IBD is a common null model in landscape genetics studies that expresses the equilibrium between gene flow and genetic drift, it does not specifically account for the influence of genetic drift via demographic episodes of bottlenecking or founder events that are specifically related to population size.To determine whether landscape resistance has influenced past gene flow, and thus genetic differentiation among population pairs, we calculated cumulative cost distance of least-cost paths (LCP) between all pairwise population combinations for each hypothesized landscape resistance surface . LCPs ar(e)Ne. A review of the literature shows a recent emergence of diverse and computationally complex methodologies offered for landscape genetics analysis . Howevel et al. ,34 and al et al. . The star between genetic distances and landscape resistance distances after accounting for the effects of geographical distance (IBD) and the effects of Ne. This correlation provides a measurement of the strength of the environmental relationships after removing the influence of pure geographical distance and Ne.We first tested for a pattern of IBD where genetic differentiation is predicted to increase with geographical distance as expected under mutation/migration/drift equilibrium . We then3.(a)FST and Dc population-pairwise genetic distances ranged from 0.021 to 0.213 and 0.227 to 0.603, respectively . As would be predicted, FST and Dc were highly correlated . FIS results  as 95% confidence intervals included infinity. These results are likely due to the small sample sizes of these herds  and made the assumption that BNP and MAL had a similar ratio and from that calculated an Ne to use in the pairwise comparison matrix. For all other populations, the general trend showed that Ne was always lower than Nc, and usually Nc was not included within the 95% Ne confidence intervals  expected under bottleneck scenarios.After correction for multiple comparisons (strict Bonferroni), significant excess heterozygosity (one-tailed Wilcoxon test for H excess) at TPM of 20% was detected in RPC and BNP herds. As the TPM converged towards a purely stepwise mutation model (TPM of 10% and 5%), only BNP continued to show significant heterozygosity excess ((b)FST and Dc  in tests using Dc over FST. Although there was a significant pattern of IBD  gave different results . Again, Ne, partial Mantel r-values for all variables were significant. The r-values decreased for FST and increased for Dc, except for RSF, which decreased for both genetic metrics , that no other variables explained genetic variability after accounting for preferred habitat availability. RSF models have only recently begun to be applied in landscape genetics, but are already demonstrating superior results to traditional landscape models [Our study is one of the first to assess the impact of lability , RSF anden herds . This paariables , indicate models .Ne were significant in explaining the variability among genetic relationships in both FST (RSF was best followed by Ne) and Dc  was a significant explanatory variable. Interestingly, after controlling for Ne, the genetic distance correlations with the anthropogenic variables increased for Dc  in the threatened populations analysed here, modern anthropogenic features do not appear to yet have significant impacts on gene flow by themselves, but have been shown to reduce population size , thus le5.a, and the associated resistance surface of that habitat  lowers per patch immigration rate, thus inhibiting the rescue effect  and resuNe (Ne will tend to explain an increasingly larger proportion of the variability.The correlation that we found between genetic differentiation and low population numbers provides an empirical link between habitat loss and fragmentation ,6. The fNe , and if Ne, is an important variable that tends to be neglected in most landscape genetic studies [Ne component of genetic drift may lead to erroneous inferences of population structure strictly based on landscape features and, as a result, will fail to pinpoint crucial demographic processes important to conservation.Population size, and in particular  studies . Genetic


Cataglyphis emmae at both, local and regional scales. We find that C. emmae is monogynous and polyandrous. Lack of detection of any population viscosity and population structure with nuclear markers at the local scale suggests efficient dispersal, in agreement with a lack of inbreeding. Contrasting demographic differences before and during the mating seasons suggest that C. emmae workers raise sexuals in peripheric nest chambers to reduce intracolonial conflicts. The high genetic differentiation recovered from the mtDNA haplotypes, together with the significant correlation of such to geographic distance, and presence of new nuclear alleles between areas  suggest long-term historical isolation between these regions, indicative of limited dispersal at the regional scale. Our findings on the ecological, social and population structure of this species increases our understanding of the patterns and processes involved under independent colony foundation.Dispersal has consequences not only for individual fitness, but also for population dynamics, population genetics and species distribution. Social Hymenoptera show two contrasting colony reproductive strategies, dependent and independent colony foundation modes, and these are often associated to the population structures derived from inter and intra-population gene flow processes conditioned by alternative dispersal strategies. Here we employ microsatellite and mitochondrial markers to investigate the population and social genetic structure and dispersal patterns in the ant  Dispersal is a pivotal process with important ecological and evolutionary consequences Ants represent interesting model systems to study population genetic consequences of various dispersal strategies at different spatial scales. Many ant species are considered sessile organisms that can occupy the same nests during years while dispersal is mainly determined by the ability of sexuals to fly, often associated to distinctive mating systems Rhytidoponera, which exhibits both reproductive strategies, suggests that the shift from ICF to DCF is gradual and ICF is mostly attributed to advantages of aerial dispersal in open habitats DCF is expected to lead to an increase in population structure and promoting an isolation by distance pattern at a local scale Although many studies have addressed ant sociogenetics in details, there is limited information on the effect of colony foundation mode on population genetic structure at different spatial scales. The mode of colony founding, dispersal and population genetic structure are generally associated, but they are conceptually different phenomena, so that one should not be deduced from the other. Moreover, other factors such as evolutionary history, past environmental and geographical changes, habitat richness and homogeneity, flight efficiency and other behavioral aspects could have important consequences in the population structure. For example, it was suggested that frequent colony relocations in DCF species could compensate for short dispersal distances leading to population mixing Cataglyphis is composed of species with DCF  and ICF . Populations of the former are genetically structured while those of the latter show no pattern of isolation by distance C. cursor has shown that its genetic structure at different spatial scales was largely dependent on the sampling distance considered, habitat patchiness and the sex specific dispersal capability. As expected, males contribute more to gene flow at local and regional scales, leading to a pattern of isolation by distance. The limited on foot dispersal of queens (a few meters) results in population viscosity where male gene flow is insufficient to homogenize the female genetic background C. mauritanica (DCF) and C. bicolor (ICF). Both species show contrasting genetic mtDNA backgrounds with structured populations in the former but not in the latter Cataglyphis is interesting for the great variability of social systems, exhibiting monogyny, polygyny and/or monandry, polyandry, social hybridogenesis and thelythokous parthenogenesis The desert ant genus Cataglyphis emmae is a common species distributed along presaharian wadis  of Northern Africa ? Are males responsible for most gene flow at the local scale and is there a correlation of isolation by distance at the local scale?n Africa . ColonieP<0.001). Because nests were excavated at the beginning of their diurnal activity we did not expect any demographic differences by not collecting the few foraging ants. Furthermore, this small sampling error, difficult to avoid, should not produce any biased results. Moreover, only 25 out of 33 nests excavated during the 2009 and 2010 mating periods . Numeral sex ratio was not significantly different between queenless (QL) and queenright (QR) nests .We found contrasting demographic differences between nests collected one month before and during the mating period. Nests collected in early April 2011 were composed of 702.5±78.5 workers . All had a queen, small larvae and a few colonies had pupae that were not counted. We found a few large pupae that most likely were sexual offspring but no adult sexuals were found. By contrast, nests excavated in late April-early May 2010 contained significantly less workers . Given that the two tests could be significant by chance alone, we considered the seven microsatellites to be independent. Between four and 13 alleles were detected per locus with a mean observed heterozygosity Ho = 0.47 (range: 0.033–0.664) and a mean expected heterozygosity He = 0.36 (range: 0.021–0.503). Genetic descriptive statistics are given in −1; Clark & Evans R = 1.060, P = 0.25) (P>0.0007 after Bonferroni correction). The fixation index ISF was significantly different from zero  suggesting strong outbreeding. STF estimate  indicated high genetic divergence between nests within the plot. Yet it was not significantly different from the overall STF calculated over 74 colonies genotyped in the Ait Ibourk area .None of the microsatellite loci showed significant deviations from HWE in 140 tests (20 re-sampled datasets × 7 loci). Of the 300 tests of linkage disequilibrium, only 2 were significant (0.004<  = 0.25) . Neighboq-wr = 0.5±0.04 (mean ± SEjacknife N = 24) was not significantly different from 0.5 expected under strict monogyny . Genetic relatedness between nestmate workers w-wr = 0.43±0.03 (mean ± SEjacknife N = 620) was significantly lower than 0.75 expected between full sibs . However, all typed workers were assigned to the queen present in their nest. When no queen was found in a nest, worker genotypes were compatible with a single inferred queen. Field sample pedigree analyses from workers and queens of 21 nests showed that queens mated with up to 5 males. Absolute and effective patriline numbers were Mp = 3.04±1.04 and Me,p = 2.74±1.09, respectively (non-detectP  = 7.70−14). Moreover, queens’ male mates were unrelated to each other  not significantly different from 0 ).Genetic relatedness between workers and the queen ectively . Ten worWe investigated the parentage of 41 males collected from five queenless nests. Thirty males collected from four of these nests carried only queen alleles at all loci and were therefore queens' sons. However eleven males from one queenless nest carried at least one non-queen allele at one locus and seven of them carried two non-queen alleles, which were present in the workers. A total of four distinct non-queen genotypes were found. Thus, the males of this nest derived from worker arrhenotokous parthenogenesis.q-fr = 0.62±0.08 did not differ significantly from that between queens and workers q-wr = 0.50±0.04 .All 26 alate females encountered in four nests bore a non-queen allele at one locus, which indicated that they had been produced sexually and not through thelytokous parthenogenesis. Consistent with this view, the relatedness between the queens and alate females th 2010. They were all genotypically different and no other genotyped colony showed the exact same alleles combinations except for one queen showing the same alleles at all loci than two ants sampled in a nest 60 m away from the plot, suggesting a possible origin at that colony.Ten dealated queens were found walking throughout the plot on April, 28ad hoc statistic, ΔK, which provides a better predictor of the (K) clusters at the uppermost hierarchical level Between-nest nuclear genetic differentiation did not correlate significantly with geographic distance at a scale of a few meters (nests sampled within the mapped plot in 2010) or tens of meters (nests sampled in 2009 near Ait Ibourk), indicating no isolation by distance within this population . At a laR = 0.47, P<0.001, −lnL = 1095.4026, BIC = 2306.5980). The Bayesian Inference phylogram was congruent with the MJ network and recovered two monophyletic clades . Overall, both methods tended to show an East-West genetic subdivision along the wadis, with exception of H7 grouped with the Eastern haplotypes  showed marked differences between nuclear and mtDNA data. Hierarchical analyses of molecular variance  results in a significant reduction of the genetic relatedness among nestmate workers (w-wr = 0.43) compared to strict monandry/monogyny. Although monogamy is the ancestral sociogenetic organization of social Hymenopterans, promoting the evolution of eusociality Cataglyphis species. So far, the combination of strict monogyny and monoandry has been reported only in C. hispanicaC. emmae have been reported in C. cursor  and adult sexuals are generally found in peripheral queenless nests C. emmae, the high Fst values encountered at the local scale, the fact that all nests were different genetic entities and the absence of observation of social transport during our field-work may discard the possibility of polydomy. An alternative explanation is that, though in winter all the workers gather in the royal chamber, in spring sexuals are transported to peripheral chambers connected to the royal chamber through complex and ramified galleries. Enhanced nest complexity may therefore have led us to excavate only a fraction of the colonies. In addition, important forager mortality in spring not replaced by new-born ants may contribute to explain the reduction of worker numbers.Although genetic data were compatible with monogyny in all the sampled colonies, no fertile queen was found in 8 out of 33 nests collected during the mating season. Interestingly, adult sexuals were present in all the queenless nests. By contrast, before the mating season, just at the end of the hibernation, all nests contained a queen, more workers and no sexual adults. The fact that the presence of a fertile queen tends to inhibit or delay the development of sexual larvae is relatively common in ants C. emmae, MJJ pers. obs.) lay haploid eggs by arrhenotokous parthenogenesis. However, the reality of this phenomenon has rarely been reported in nature. Our data are therefore interesting in showing the males encountered in one out of 5 queenless nests were workers' sons. Worker egg laying may result from a selfish strategy or from a hopeless situation after the death of the mother queen C. cursor, C. hispanica, C. mauritanica and C. veloxC. emmae queens or workers.In laboratory conditions, queenless workers of many ant species  shared between the two wadis near the Ouarzazate lake. The low mtDNA genetic diversity observed at a local scale suggests recent and rare maternal lineage colonization events as well as efficient female dispersal within localities. Evidence of this can be seen in the last 30 km of irection .C. emmae is exclusively distributed along the wadis. The high level of mtDNA differentiation between Tamnougalt and the other localities is likely due to a lack of gene flow provoked by arid mountains where we were unable to locate C. emmae while other Cataglyphis species were present  during our census , there is growing evidence that the physical or mechanical ability to fly is not the primary cause limiting female dispersal and that ethological factors may be more important for this purpose r census . Howeverr census  suggestir census . SimilarCataglyphis ICF species  where queen dispersal occurs after mating. Males fly in swarms on relatively long distances to copulate with queens near their mother nest entrance Cataglyphis, male-biased dispersal was also shown in other ICF species , in which queens bear long wings but contribute less than males to within population gene flow The contrasting genetic differentiation between the mtDNA and nuclear markers within wadis could be an indication of higher within-population gene flow by males than by queens. This is also evident from the many nuclear alleles unique to regions and/or localities Cataglyphis emmae population structure is similar to those of other monogynous and polyandrous ICF Cataglyphis species like C. sabulosa, C. livida, C. bombycina, C. theryiC. emmae same mtDNA haplotypes clusters  many kilometers distant have also been observed in C. bicolor (ICF) but not in DCF species such as C. mauritanica and C. cursorC. emmae population structure maybe associated to their habitat requirement and wind current migration through the wadis, following a directional dispersal pattern. Future comparisons of C. emmae with C. floricola-tartessica species complex is likely to give better insight into the evolutionary and the ecological consequences of divers mating systems and how and what factors may influence the transition from ICF (C. emmae) to DCF (C. floricola-tartessica).No ethics guidelines are required or established by the Spanish or Moroccan authorities to work on ants. The project licence to work in Morocco was granted by the AECID  by the Secretario General Juan Pablo de Laiglesia (Project Number A4774/06). Work was carried out on Moroccan public and non-protected land and the model ant was not a protected species.C. emmae sociogenetics and population genetic structures, we first excavated 16 nests in late April-early May 2009 (during the mating period) in a zone about 1.8 km South of the locality of Ait Ibourk (All sampling was conducted in the South of the Atlas Mountains (Morocco). In order to investigate t Ibourk . These n2) plot at about 50 m from the 2009 site. All nests within the plot were mapped to estimate nest density and spatial distribution. Active nests were located by offering a piece of biscuit to workers and following them to the nest. The map was considered complete when no new nest was found and all foragers reached previously marked nests. Sinclair's (1985) correction of Clark & Evans (1954) statistics nSR) was calculated for each nest containing at leat one adult sexual as the proportion of female sexuals over the total number of sexauls. nSR was compared between queenless (QL) and queenright (QR) nests (2009–2010) with a Kruskal-Wallis test (hereafter KW).A finer scale sampling was conducted at the same site in late April 2010. To that end, we delimited a 34×34 m (1166 mC. emmae (one worker per colony) colonies from 14 sites along a 150 km transect , before the Atlas Mountains and the most Eastern nests (31 km from Ouarzazate) were just 3 km East from Skoura. Two colonies were sampled from an additional site located 54 km Southeast from Ouarzazate, 3 km before the locality of Tamnougalt, possibly the last locality of C. emmae before the dune formations of the Saharan desert. At all localities samples were taken from two nests less than 50 m apart, with the only exception of one single nest found at locality number 19 . DNA was extracted from the brain and surrounding musculature. This soft tissue proved more suitable for efficient PCR amplification than other body parts containing quitine, such as legs and thorax, which inhibited the PCR reactions. DNA extraction followed the HotShot method C. cursor  were carried out in pairs (duplex reaction) or individually. Each 20 µl PCR volume contained approx. 50 ng DNA, 200 µM of each dNTP, 0.15 µM of each primer, 2 µl Buffer 10X, µl Mgcl2 and 0.1 unit of taq polymerase (QIAGEN). The thermal cycle profile was as follows: an initial denaturation step of 2 min at 94°C; 35 cycles of denaturation at 30 s at 94°C, annealing for 30 s at 52°C and extension for 45 s at 72°C; and a final extension for 5 min at 72°C. Following the PCR reactions, excess primers and dNTPs were removed using enzymatic reaction of E. coli Exonuclease I, Antartic phosphatase and Antartic phosphatase buffer . Sequencing was carried out in both directions using the BigDye® Terminator v1.1 cycle sequencing kit (Applied Biosystems) according to the manufacturer’s instructions. Labelled fragments were resolved on an automated A3130xl genetic analyzer (Applied Biosystems). Incomplete terminal sequences and PCR primers were removed. Control for genotyping errors due to null alleles and allele drop-outs was performed with Micro-checker Seven microsatellite markers developed for p-uncorrected distances (%) analyses were performed with PAUP*4.b.10 Mitochondrial DNA variation was assessed from all colonies sampled from the regional scale transect . The priC. bombycina, from the C. bombycina group, and basal to the C. emmae group The most appropriate substitution model for the Bayesian Inference (BI) analyses was determined by the Bayesian Information Criterion (BIC) in jModeltest v.0.1.1 i.e., the number of alleles, allele frequencies, observed heterozygosity and expected heterozygosity) and Wright’s F-statistics  were computed with FSTAT Descriptive genetic statistics (r were estimated in Relatedness (v 5.0.8) according to C. emmae being strictly monogynous (see Results), when no queen was found during excavation, the genotype of the presumed queen was reconstructed from worker genotypes (N = 9 queens).Relatedness coefficients Individuals were assigned as belonging to different matrilines if they did not share an allele with the (presumed) queen at least at one locus. Assignment of individuals to matrilines was confirmed with the maximum-likelihood methods implemented in the program COLONY 1.2 using of 21 colonies with a minimum of 10 workers (average  = 15.8) were run in COLONY p, the minimum number of males inferred from worker genotypes) was estimated on the basis of mother-offspring allele combinations. Because males may contribute unequally to the offspring, we estimated the effective mating frequency  following The absolute number of matings per queen . Queen sons must carry a queen derived allele at a loci, and as a group, they should not display more than two alleles at a single loci. Workers' sons can carry with the same probability a mother or father derived allele. A male with non-queen alleles is a workeŕs son. Nevertheless, worker sons may carry queen alleles at all loci by random chance. To account for this probability of non-detection, we estimated it following C. emmae asexual reproduction for queen production, as seen in C. cursorExamination of ST/ (1-FST)) coefficients between pairs of colonies against the logarithm (ln) of geographical distances The pattern of isolation by distance was tested by plotting (FHaplotype frequencies and reduction were estimated using a Median joining (MJ) network constructed with Network 4.5 The Bayesian clustering software Structure 2.1 C. emmae from all sampled Wadis  was evaluated by Fst and Φst statistics using the analysis of molecular variance (AMOVA) in ARLEQUIN F-statistics were assessed by permutations  of the data sets.The extent of geographical structuring of genetic variation between Figure S1Plots showing the ants nests sampled starting from a larger (2009 and 2010 nest sampling) to a smaller scale (only 2010 sampling). The vertices indicate the measured plot area from where nests were sampled. Lines delimit different independent approximate transects to assess genetic divergence by distance (mantel tests) and the circle delimits a mantel test performed for all nests in such circle. Line 1; (5 nests), line 2; (6 nests), line 3; (6 nests); lower section of genotyped nests from 2009 (12 nests in circled area); all nests in mapped plot (31 nests), all nests combined (49 nests). See (JPG)Click here for additional data file.Figure S2ST/(1-FST).Relationship between geographical distance and mitochondrial genetic differentiation between nests for the transect, estimated as F The correlation is highly significant .(JPG)Click here for additional data file.Table S1Mantel tests analysed and statistics for genotyped nests from nuclear data (microsatellites). All results were non significant. See (DOC)Click here for additional data file.


Knowing the extent and structure of genetic variation in germplasm collections is essential for the conservation and utilization of biodiversity in cultivated plants. Cucumber is the fourth most important vegetable crop worldwide and is a model system for other Cucurbitaceae, a family that also includes melon, watermelon, pumpkin and squash. Previous isozyme studies revealed a low genetic diversity in cucumber, but detailed insights into the crop's genetic structure and diversity are largely missing. We have fingerprinted 3,342 accessions from the Chinese, Dutch and U.S. cucumber collections with 23 highly polymorphic Simple Sequence Repeat (SSR) markers evenly distributed in the genome. The data reveal three distinct populations, largely corresponding to three geographic regions. Population 1 corresponds to germplasm from China, except for the unique semi-wild landraces found in Xishuangbanna in Southwest China and East Asia; population 2 to Europe, America, and Central and West Asia; and population 3 to India and Xishuangbanna. Admixtures were also detected, reflecting hybridization and migration events between the populations. The genetic background of the Indian germplasm is heterogeneous, indicating that the Indian cucumbers maintain a large proportion of the genetic diversity and that only a small fraction was introduced to other parts of the world. Subsequently, we defined a core collection consisting of 115 accessions and capturing over 77% of the SSR alleles. Insight into the genetic structure of cucumber will help developing appropriate conservation strategies and provides a basis for population-level genome sequencing in cucumber. Cucumis sativus L.) is the fourth most important vegetable worldwide Cucumis and afforded novel insights into several important biological processes such as biosynthesis of cucurbitacin and “fresh green” odor. Cucumber is being developed as a new model species in plant biology due to its small number of genes, rich diversity of sex expression, suitability for vascular biology studies, short life cycle (three months from seed to seed), and accumulating resources in genetics Commonly known as cucurbits or gourds, the botanical family Cucurbitaceae includes a number of cultivated species of global or local economical importance The rapid advance of Next Generation Sequencing (NGS) technologies makes it affordable to re-sequence multiple genotypes of a given species to generate a haplotype map that displays the genome-wide patterns of genetic variation at a single base resolution C.s. var. sativus and the wild cucumber C.s. var. hardwickii (Royle) Alef. The cultivation of cucumber seems to have spread rapidly from India to Western Asia, and then to Southern Europe. It has been reported that cucumber was introduced into China through the Silk Route by the diplomat Zhang Qian (164 B.C.–114 B.C.) during the Han Dynasty and subsequently spread to East Asia Cucumber is indigenous to India Genetic diversity in the U.S. cucumber collection  maintained by the National Plant Germplasm System (NPGS) was investigated in a series of studies by analysis of variation at 21 isozyme loci In this study we assembled a mega-collection consisting of all available cucumber accessions from the national germplasm centres of China, the Netherlands and U.S. We fingerprinted a total of 3,342 accessions with 23 highly polymorphic Simple Sequence Repeat (SSR) markers. The SSR data were used to assess overall genetic diversity and to reveal a clear population structure in cucumber. In addition, the data allowed the construction of a core set of 115 accessions capturing over 77% of the total allelic diversity observed.http://www.ars-grin.gov/npgs/), 759 accessions from the Centre for Genetic Resources, the Netherlands , 2 accessions from the IPK (http://www.ipk-gatersleben.de/), and 6 accessions from the East-West Seed International Ltd. The mega-collection included 235 accessions originating from India, 1,361 from China (excluding Xishuangbanna), 208 from Xishuangbanna, 209 from Southeast Asia, 141 from Western Asia, 802 from Europe, 176 from the Americas, 50 from Africa and 8 from Oceania. Additionally, 152 accessions of unknown origin were included in the study. Passport information about the mega-collection is listed in A mega-collection of 3,342 cucumber accessions was used in this study. A total of 1,692 accessions were collected from the China National Vegetable Germplasm Bank located at the Institute of Vegetables and Flowers at the Chinese Academy of Agricultural Sciences (IVF-CAAS), 883 accessions from the U.S. National Plant Germplasm System , carboxytetramethylrhodamine (TAMRA), hexachloro-6-carboxyfluorescein (HEX) and ROX (carboxy-X-rhodamine). Multiplex PCRs were performed in a 11 µl volume containing approximately 20–50 ng template DNA, 0.4 pmol of each primer, and 5 µl Multiplex PCR Master Mix . Reactions were performed in an ABI 9700 thermocycler with an initial denaturation step of 15 min at 95°C, followed by 35 cycles of 95°C for 30 s, 57°C for 90 s and 72°C for 70 s, and a final extension at 60°C for 30 min. For PCR fragment size determinations, 0.25 µl of an internal size standard  was mixed with 1 µl of diluted PCR product (1/100) and 9 µl formamide. The mixture was heated at 94°C for 3 minutes and then cooled within icy water. Electrophoresis was carried out on an ABI 3130×l genetic analyzer (Applied Biosystems). Analyses were performed using the Genemapper 4.0 software.The model-based program STRUCTURE STF statistics with the AMOVA means in Arlequin 3.11 For the entire collection as well as the subpopulations, allele richness, gene diversity and the number of unique alleles were calculated with PowerMarker version 3.25 For sampling core collections, we used the Maximization (M) algorithm implemented in MSTRAT software version 4.1 We fingerprinted 3,342 cucumber accessions using 23 SSR markers. In total, 94.9% of PCR reactions yielded clear peaks in electrophoresis and generated 72,960 data points. The remaining 5.1% resulted in multiple bands with low quality and consequently were treated as missing values in subsequent analyses. Out of the 72,960 data points, 61,976 (84.9%) corresponded to single peaks that were regarded as homozygous for the given SSR locus, while 8,929 (12.2%) corresponded to double peaks that were regarded as heterozygous. Only 2,055 (2.8%) of the data points corresponded to more than two clear peaks, of which only the two highest peaks were recorded that were regarded as heterozygous. This simplified scoring may cause under-estimation of rare alleles.The 23 SSR markers generated a total of 316 alleles in the mega-collection . Out of The population structure of the cucumber mega-collection was firstly inferred using STRUCTURE 2.3.1 The unrooted neighbour-joining tree  is consiWe also performed Principal Component Analysis (PCA) on 1,026 accessions without any missing SSR data . This anIn summary, the model-based ancestry analysis, the phylogenetic tree and the PCA strongly supported that cucumber has three well-differentiated genetic populations and admixtures.The classification of populations appeared highly correlated with the geographical distribution of the cucumber accessions . P1 inclIn PCA analysis , P3 can STF using AMOVA indicated a high degree of differentiation between the three model-based populations with values ranging from 0.30 to 0.33 .AMOVA analysis of the populations P1, P2 and P3 indicated that 68.1% of the variation was due to differences within populations, while 31.9% was due to differences among populations . PairwisGenetic parameters for all accessions and the three model based populations are given in Among the three model based populations, P3 possessed the highest diversity (He = 0.52), though there was no significant differences in Na and Ne. Even with the least number of accessions involved, P3 displayed the largest number of population specific alleles (n = 40). This supports the notion that cucumber was originated from India For all populations, the observed heterozygosity was lower than expected heterozygosity, which may be caused by inbreeding during seed propagation by germplasm banks.The Maximization or M strategy algorithm was used to select the core set of the mega-population. The M curve reached the plateau when approximately 115 accessions were selected . Taking In this study, we carefully selected 23 highly polymorphic SSR markers and used them to fingerprint a large cucumber collection. The dataset allowed us to visualize the population structure of cucumber and to assemble a core collection of 115 accessions that cover over 77% of the observed SSR alleles.Out of the 316 SSR alleles scored, 59% and 80% are present in less than 1% and 5% of the mega-collection, respectively. As the level of gene flow is negatively related to the number of rare alleles The results also supports that independent migrations of cucumber have occurred from India to other parts of the world. The migration route to Europe and then to the Americas and Africa are clearly through Central and West Asian countries. Cucumber accessions from China and other East Asian countries are genetically differentiated from those in Central and West Asia. It is well documented in Chinese history that cucumber was introduced from there through the Silk Route by the diplomat Zhang Qian (164 B.C.–114 B.C.) Cucumber plants in India and Xishuangbanna bear short, ovary to round fruits with sparse spines . The occThe Xishuangbanna cucumbers are affiliated to the Indian population. As semi-wild landraces, they apparently lost the unfavourable fruit bitterness but still possess the dependence of short daylight for flowering and fruit setting. The latter trait was discarded in most cucumber cultivars. Accessions from Xishuangbanna are genetically close to each other, suggesting that a single dispersal occurred from India. It is worth mentioning that among the African accessions, six lines are present from Zambia and Zimbabwe classified as P3 and admixture P2P3 Click here for additional data file.Table S2Cucumber core collection.(XLSX)Click here for additional data file.Figure S1Pictures of typical cucumber accessions from the three model based populations. CG1149, “Chinese Long”; CG4357, Southern China type; CG5786, Dutch greenhouse; CG6600, Pickling; CG9191, Xishuangbanna; CG8039, Indian(PDF)Click here for additional data file.


Dendroctonus frontalis Zimmerman (Coleoptera: Curculionidae) is an economically important pest species throughout the southeastern United States, Arizona, Mexico, and Central America. Previous research identified population structure among widely distant locations, yet failed to detect population structure among national forests in the state of Mississippi. This study uses microsatellite variation throughout the southeastern United States to compare the southern pine beetle's pattern of population structure to phylogeographic patterns in the region, and to provide information about dispersal. Bayesian clustering identified east and west genetic groups spanning multiple states. The east group had lower heterozygosity, possibly indicating greater habitat fragmentation or a more recent colonization. Significant genetic differentiation  followed an isolation-by-distance pattern  among samples, and a hierarchical AMOVA indicated slightly more differentiation occurred between multi-state groups. The observed population structure matches a previously identified phylogeographic pattern, division of groups along the Appalachian Mountain/Apalachicola River axis. Our results indicate that the species likely occurs as a large, stable metapopulation with considerable gene flow among subpopulations. Also, the relatively low magnitude of genetic differentiation among samples suggests that southern pine beetles may respond similarly to management across their range.The southern pine beetle,  Dendroctonus frontalis Zimmerman (Coleoptera: Circling) is an economically important pest species that occurs throughout the southeastern United States, and in Arizona, Mexico, and Central America  and turpentine. Traps were placed in 19 locations over eight states and 26 to 100 individuals were screened from each site , Mississippi Holly Springs National Forest (32 km apart), Mississippi Tombigbee National Forest (3 km apart), Mississippi Beinville National Forest (7 km apart), Mississippi Homochitto National Forest (36 km apart), and Mississippi De Soto National Forest (53 km apart). These samples were combined to represent a single location given the lack of genetic differentiation observed at these sites and distances in Schrey et al. . Southerwww.qiagen.com). Collected individuals (n = 1198) were screened at eight microsatellite loci following the methods detailed in Schrey et al. (www.appliedbiosystems.com), and genotypes were determined using GENESCAN 3.2.1 and GENOTYPER v 2.5 (Applied Biosystems). Allele size data were binned after visualization on scatter plots. FSTAT version 2.9.3 ) to pairwise geographic distance (as log10 Euclidean distance in meters) with POPTOOLS  than among samples within Bayesian clustering defined groups . Within the Bayesian clustering defined east group, 5 of 55 comparisons were significant (p < 0.001) and indicated a positive correlation (r = 0.42) between geographic distance and genetic distance . Pairwise θT values  ranged fnificant . Within nificant . The Newdistance .o t-test p = 0.002, He t-test p < 0.001; Observed heterozygosity and expected heterozygosity were significantly higher in the west group than in the east group . However, two of the three methods used found congruent results among our samples. These methods may have lacked sufficient power to distinguish the New Jersey samples from the remaining samples with Bayesian clustering. If BAPS is forced to form three genetic groups, New Jersey forms an independent group, with the remaining samples assigning identically as with two groups. It is possible that additional samples spanning the range from New Jersey to North Carolina/Kentucky would provide additional information as to the placement of the New Jersey samples.The southern pine beetle exhibited genetic differentiation among regions within the southeastern United States, which was weakly compartmentalized into at least two large, multistate groups. Bayesian clustering identified two widespread groups: east samples , and west samples . The two groups were not geographically congruent. The easternmost samples in New Jersey were assigned to the west group. However, estimates of FObserved and expected heterozygosity were greater in the west group than in the east group. Also, allelic richness was slightly higher and private allelic richness was slightly lower in the west group. The greater diversity and fewer private alleles in the west group suggest that this area may have larger, more connected populations that have been longer established. The genetic diversity of the east group would be consistent with smaller, more fragmented populations.Pinus echinata, and the loblolly pine, Pinus taeda (Southern pine beetle genetic differentiation conformed to the previously identified major phylogeographic pattern, which divides the southeast into east and west groups at the Appalachian Mountains/Apalachicola River axis (reviewed by us taeda . The twoIps confusus (Dendroctonus ponderosae (Dendroctonus mexicanus (Tomicus destruens (Ips typographus (Significant isolation-by-distance, albeit at low magnitudes, was found among southern pine beetles in the eastern United States. Isolationby-distance has been observed in other pine forest beetle species: confusus , Dendrocnderosae , Dendrocexicanus , and Tomestruens . While Iographus  lacked gographus  indicateOur results expand the previous genetic studies of the southern pine beetle. The allozyme studies  showed gManaging the southern pine beetle as a pest has proven difficult because the species is wide-ranging and may exist in large metapopulations. Our results are consistent with southern pine beetle outbreaks originating from geographically proximate individuals. The relatively low estimates of genetic differentiation observed could be caused by gene flow among regions and/or by extremely large populations experiencing low magnitude genetic drift. Evidence for rapid changes in local allele frequencies or widespread significant differences in allele frequencies were not found over short distances. Thus, there does not appear to be large immigrations of beetles from other areas. The lack of genetic differentiation over large geographic areas suggests that successful management practices in one location would be expected to be successful in other locations.


Hevea genotypes, consisting of Asiatic, Amazonian, African and IAC clones, and pertaining to the genetic breeding program of the Agronomic Institute (IAC), Brazil, was estimated. Analyses were based on phenotypic multivariate parameters and microsatellites. Five agronomic descriptors were employed in multivariate procedures, such as Standard Euclidian Distance, Tocher clustering and principal component analysis. Genetic variability among the genotypes was estimated with 68 selected polymorphic SSRs, by way of Modified Rogers Genetic Distance and UPGMA clustering. Structure software in a Bayesian approach was used in discriminating among groups. Genetic diversity was estimated through Nei's statistics. The genotypes were clustered into 12 groups according to the Tocher method, while the molecular analysis identified six groups. In the phenotypic and microsatellite analyses, the Amazonian and IAC genotypes were distributed in several groups, whereas the Asiatic were in only a few. Observed heterozygosity ranged from 0.05 to 0.96. Both high total diversity (HT' = 0.58) and high gene differentiation (G st' = 0.61) were observed, and indicated high genetic variation among the 60 genotypes, which may be useful for breeding programs. The analyzed agronomic parameters and SSRs markers were effective in assessing genetic diversity among Hevea genotypes, besides proving to be useful for characterizing genetic variability. Genetic diversity of 60  Hevea genus belongs to the Euphorbiaceae family and comprises 11 species native to the Amazon region (Hevea brasiliensis (Willd. ex Adr. de Juss.) Muell-Arg. is the only cultivated species and the main source of natural rubber.The Hevea brasiliensis is cultivated in several tropical countries, most of which have active plant-breeding programs .Average values of five agronomical descriptors, each based on three replicates, were subjected to multivariate analysis. They comprised average of seven years of girth growth increment at juvenile immature phase before tapping, average of three years of girth growth increment in adult trees on tapping, average of three years of dry rubber yielding, virgin bark thickness in opened panel tapping; and the total number of latex vessel rings. These data were collected over a period of ten years. Measurements were taken as described by Gonçalves Multivariate procedures consisted of Standard Euclidian Distance (SED), Tocher Clustering and Principal Component Analysis (PCA). The contribution of each variable to genetic divergence was calculated by the criteria of Hevea microsatellites. Redundancies were identified using BLASTN software search utilities in GenBank  at a difference of up to 10 °C. After an initial denaturing step of 1 min at 94 °C, the PCR amplification was performed in 30 cycles of 1 min at 94 °C, 1 min at the specific Ta and 1 min at 72 °C, followed by a final extension at 72 °C for 5 min and then kept at 15 °C. Alternatively, some SSRs could only be amplified by Touchdown. Amplification products were resolved on 6% (w/v) denaturing polyacrylamide gels and silver stained, according to PCR amplifications were carried out in a 25 μL volume containing 100 ng of DNA, 1 U n is the number of alleles; and fi and fj are the frequencies of the ith and jth alleles, respectively (Data on the presence (1) or absence (0) of SSR bands were transformed into genotypic data in order to identify loci and alleles. The Polymorphism Information Content (PIC) value for each locus was calculated using the PIC formula = Genetic distances were calculated by using Modified Rogers Genetic Distance (MRD) according to The SED and MRD dissimilarity matrices were correlated using the Genes software . Both thK = 3 to K = 20, and ten runs of each K were conducted using the admixture model and correlated allele frequencies, a 200,000 burn-in period and 500,000 MCMC. Ad hoc statistics was related to rate changes in the log probability of data according to the number of Ks proposed by K being used as a predictor of the ideal number of clusters. In addition, the ideal number according to Pritchard and Wen (2004) was used as the criterion for defining the number of groups (k). The most trustworthy value was estimated based on the lowest negative number of Ln (the log-likelihood of the data) and the lowest standard deviation found during statistical analysis.The Bayesian approach of Hevea benthamiana was a common ancestor for seven of the 15. The most divergent genotype pair was IAC 318 - PB 235.The 15 most divergent genotype pairs identified by the SED matrix are listed in Through Tocher analysis, the 60 rubber-tree genotypes were clustered into 12 groups . Among tPCA for phenotypic data accounted for 80.88% of the total variation in the first three principal components. The average dry rubber yield was the variable that contributed the most in the estimation of the genetic divergence among the 60 genotypes. The number of latex vessel rings was the least important variable, and so could be discarded.Of the 80 characterized SSRs (Table S1) 68 were polymorphic and informative. In the SSR IAC-Hv34 genotype, two distinct bands were amplified and considered as two distinct loci . Thus, 69 polymorphic and informative SSR loci were identified and characterized. The electrophoretic profile obtained with SSR IAC-Hv72 can be observed in Figure S1. Polymorphic information content varied from 0.11 to 0.87, with an average of 0.57. The mean allele number per locus was 5.88, ranging from 2 to 13, the extremes being attributed to SSR IAC-Hv36 and SSR IAC-Hv20, respectively.Hevea pauciflora, and showed transferability in relation to Hevea brasiliensis, using the same PCR amplification procedure.Six SSRs  were efficient at amplifying o) based on Nei's estimates varied from 0.05 to 0.96, with an average of 0.45  and high gene differentiation (Gst' = 0.61) were observed among all the 60 genotypes. SSR PCO accounted for 19.66% of the total variation in the first three axes. When selecting the most divergent pairwise distance in the Rogers modified genetic distance matrix  were distributed into only two groups . This distribution pattern is in agreement with the data obtained through Tocher analysis of phenotypic data and the UPGMA dendrogram based on SSRs.Matrix correlation between both kinds of genetic distances was significant by t-test and Mantel test . Genetic distances estimated from phenotypic and molecular traits were correlated. Pair-wise distances within and among groups were separated in the dendrogram according to the respective group association pattern . SignifiH. benthamiana x H. brasiliensis  detected higher divergence between the clone IAC 318 and the Asiatic clone PB 235, the latter having been derived from a crossing between two Malayan clones. This dissimilarity possibly occurred through IAC 318 has the clone Fx 3899 as male parental, which is an interspecific hybrid of iliensis . Among tiliensis , H. bentiversity .Total variance (80.8%), as explained by the three principal components of the phenotypic data, was expressive. However, it was less than that described by Polymorphic information content was high for SSR loci, and indicated a substantial genetic information content in the clones analyzed with microsatellites. Microsatellite marker analysis is very efficient when examining genetic diversity (ST' = 0.61) was high, indicating 61% of total variation to be exploited among the available genotypes. A total of 52 SSRs presented high coefficients of genetic differentiation (GST' > 0.50). This value was higher than that observed for other open pollinated species belonging to the same family as rubber trees, such as cassava  and cassava (Manihot utilissima).Genomic transferability of the SSR K = 6, the arrangement was such as to correspond to dendrogram clustering. The coherence in genotype clusters indicated a non-random distribution of alleles and their frequencies. In fact, clones in most groups were gathered according to ascendancy as previously described. The lack of consistency in various dendrogram clusters could be associated to low bootstrap node support  in some of the major groups. Low bootstrap nodes could be associated with the lack of genetic structure, and the incapacity of a clearing clustering tendency supported by high cophenetic values.What makes the Bayesian approach interesting in the study of population genetic structures is the facility in detection without the need for prior information on individual origin  reported that Amazonian wild genotypes do not always contribute desirable traits to rubber tree genetic breeding. Even though, Amazonian wild genotypes were introduced into rubber tree breeding by crossing, so as to widen the genetic basis of Asiatic clones. The Amazonian genotypes evaluated in this study arose from prior breeding and selection procedures, thereby possibly constituting an interesting genetic background to be exploited in rubber tree breeding programs. Indeed, these clones showed the highest genetic diversity when compared to the other improved clones analyzed, and appear to be attractive for rubber breeding.In phenotypic and microsatellite analyses, both the Amazonian and IAC genotypes were clustered into several groups, thereby indicating high genetic diversity among these genotypes. On the other hand, Asiatic genotypes were distributed in only a few groups, thus indicating low diversity, with, most certainly, a narrow genetic base . Taking into account the molecular dendrogram clustering and correlated genotypes, significant associations were detected between intergroup genetic distances (phenotypic and SSR-based GDs) in Groups 1x3; 1x5 and 1x6. Currently, associations between molecular and phenotypic data tend to be stronger in crosses between genotypes of similar pedigrees (Despite being significant, matrix correlation for all genetic distances of different marker systems exhibited poor association, which was also observed by Roldán-Ruiz edigrees .The genotypes analyzed in the present work had been previously selected and evaluated in breeding programs, and had demonstrated good performance. Thus, prior screening of the most divergent genotype pairs identified through both methods is suggested for evaluation of the relative agronomic performance of their hybrids. In a traditional breeding program, thousands of crosses are normally performed and evaluated in experimental designs. According to the data described in this work, SSR-based genetic distances could be useful in selecting superior crosses between rubber tree clones derived from a population with a broad genetic base. Hence, the application of SSR markers in rubber-tree breeding could be instrumental in reducing the number of single-cross hybrids to be evaluated. SSRs are easily assayed by the Polymerase Chain Reaction, and have proved to constitute a potent tool for characterizing genetic diversity. Furthermore, in perennial plant species, such as rubber trees, this molecular marker technology has the additional advantage of shortening breeding time by allowing for the screening of seedlings and juvenile plants.The Following online material is available for this article:Sequence of primers and gene annotations.SSR IAC-Hv72 electrophoretic profile of rubber tree genotypes on 6% polyacrylamide gel.This material is available as part of the online article from http://www.scielo.br/gmb.


Genetic testing remains low among racial/ethnic minority populations in the United States. We aimed to determine the prevalence and correlates of awareness of direct-to-consumer (DTC) genetic tests and the prevalence of genetic test use in a population-based sample of adults in Puerto Rico.We analyzed data from adults aged 18 years or older who completed information on genetic test awareness  from the Health Information National Trends Survey conducted in Puerto Rico in 2009. Odds ratios with 95% confidence intervals were estimated by using logistic regression models to identify factors associated with awareness of DTC genetic tests.The majority of respondents (56%) were aware of direct-to-consumer genetic tests, and approximately 4% had ever undergone any genetic test. Respondents who had never been married were less likely to be aware of DTC tests, as were current smokers. Respondents who ever sought cancer information were more likely to be aware of these tests.We provide the first published data on the awareness of DTC genetic tests and on use of genetic testing in Puerto Rico. Forty-four percent of our sample of Puerto Rican adults were unaware of direct-to-consumer genetic tests. Given the lack of clear benefits of DTC genetic tests to the general population, educational interventions should be developed to increase awareness and specific knowledge regarding the appropriate use of DTC genetic tests among people who are already aware of their existence. With the completion of the Human Genome Project , geneticDespite increasing availability and public interest, studies on genetic testing are few, and most have focused on specific cancer types, as disease-specific tests have become more available in recent years. Previous research suggests that not all sociodemographic groups have benefited equally from genetic counseling and testing for cancer susceptibility ; their uGenetic tests are increasingly being offered as direct-to-consumer (DTC) services through the Internet and other venues, providing people access to these tests without the involvement of a health care professional ,15. For Demographic differences between people who are aware of and use genetic tests have been observed for both DTC tests and for tests administered in clinical settings ,14,19; sWe performed a secondary data analysis from a sample of adults aged 18 years or older who participated in the cross-sectional 2009 HINTS survey conducted in Puerto Rico (HINTS-PR). Data collection procedures for the 2009 HINTS-PR survey have been described in detail elsewhere  was fielThe main outcome variables included in our analysis were awareness of DTC genetic tests and use of genetic tests, assessed with the following questions: "Genetic tests that analyze your DNA, diet, and lifestyle for potential health risks are currently being marketed by companies directly to consumers. Have you heard or read about these genetic tests?" and "Have you ever had a genetic test?" Study covariates included demographic characteristics such as age, sex, education, marital status, and employment status. Information on health insurance coverage and personal and family history of cancer was also assessed. Other covariates of interest included body mass index, daily consumption of 5 or more servings of fruit or vegetables, and having a regular health care provider. Current smokers were defined as people who had smoked at least 100 cigarettes in their lifetime and continued to smoke at the time of the interview.We also assessed information about health, cancer-specific information seeking, and sources of information. Respondents were asked, "Have you ever looked for information about health or medical topics from any source?" and "Have you ever looked for cancer information from any source?" Respondents who sought health or cancer information were asked to identify the source: "The most recent time you looked for information about health or medical topics, where did you go first?" Responses were categorized into 4 groups: Internet, printed materials , health care professionals, and other .2 test. On the basis of the results of the bivariate analysis, we constructed a multivariate logistic regression model to estimate odds ratios and their 95% confidence intervals for the association between covariates of interest and genetic test awareness. Interactions and goodness of fit of the model were tested with the likelihood ratio test and the Hosmer-Lemeshow test, respectively.All analyses were weighted to estimate standard errors of point estimates for the complex survey data by using jackknife repetition as an estimator. Data were weighted according to total estimates of sex, age, education, and marital status obtained from the American Community Survey for Puerto Rico  to proviThe majority of respondents (56%) were aware of DTC genetic tests, and approximately 4% had ever used any genetic test . Lower a2 = 2.50; P = .96). Awareness of DTC genetic tests was independently associated with marital status: people who were married or living together and those who were divorced, separated, or widowed were more likely to be aware of genetic tests than people who were never married. Current smokers were less likely than nonsmokers to be aware of genetic tests, and respondents who had ever looked for cancer information were more likely to have heard or read about DTC genetic tests than those who had not (We found no significant interactions in the multivariate model and a good model fit (Hosmer-Lemeshow test: χ had not .Although our result for awareness of DTC genetic tests (56%) was higher than that reported in the United States in the 2006 HealthStyles Survey 14%) and for Hispanic (25%) and non-Hispanic (30%) respondents in the 2008 US HINTS  using the same survey item % and for,23, the Similar to what has been seen in the US mainland , insteadEven though this lack of professional information could be because limited data are available to support the products currently on the market , the rapConsistent with the results of our study, previous research from US HINTS has identified a positive relationship between health information seeking and genetic test awareness . WhereasHealth disparities exist in cancer occurrence by race/ethnicity . Some ofOur study is subject to limitations. First, because HINTS-PR was a telephone-based survey, it includes data only from residents who have a working landline telephone. Consequently, our data may not be generalizable to the entire adult Puerto Rican population. Nonetheless, the weighted extended interview response rate in the 2009 HINTS-PR (76.4%) was higher than that reported for the 2007 US HINTS (60.4%) . Second,To our knowledge, this study provides the first published data on the awareness of DTC genetic tests and use of genetic tests in Puerto Rico. These baseline data are valuable for tracking trends in awareness of DTC tests and use of genetic tests in Puerto Rico and can inform the development of policy and educational efforts regarding the appropriate use of these tests. We expect that the proportion of people who are aware of and use these tests will continue to increase, as more of these tests become available, as their marketing increases, and as the Internet and other sources of information become more widely available to the general population. Given the lack of clear benefits of DTC genetic tests to the general population, educational interventions should be developed to increase awareness and specific knowledge regarding the appropriate use of DTC genetic tests among people who are already aware of their existence (primary audience). As a clear message about these tests is in circulation, people who are unaware (secondary audience) may start to become more adequately aware of them. Future studies should try to elucidate remaining questions in this area of genetic testing, such as the prevalence of disease-specific genetic testing in clinical settings, and other psychosocial correlates of genetic testing awareness, including attitudes, beliefs, and cultural or religious norms regarding their use.


Momordica charantia are widely cultivated vegetables throughout the tropics and subtropics, but not in Taiwan, a continental island in Southeast Asia, until a few hundred years ago. In contrast, the related wild populations with smaller fruit sizes are native to Taiwan. Because of the introduction of cultivars for agricultural purposes, these two accessions currently exhibit a sympatric or parapatric distribution in Taiwan. In this study, the cultivars and wild samples from Taiwan, India, and Korea were collected for testing of their hybridization and evolutionary patterns. The cpDNA marker showed a clear distinction between accessions of cultivars and wild populations of Taiwan and a long divergence time. In contrast, an analysis of eight selectively neutral nuclear microsatellite loci did not reveal a difference between the genetic structures of these two accessions. A relatively short divergence time and frequent but asymmetric gene flows were estimated based on the isolation-with-migration model. Historical and current introgression from cultivars to wild populations of Taiwan was also inferred using MIGRATE-n and BayesAss analyses. Our results showed that these two accessions shared abundant common ancestral polymorphisms, and the timing of the divergence and colonization of the Taiwanese wild populations is consistent with the geohistory of the Taiwan Strait land bridge of the Last Glacial Maximum (LGM). Long-term and recurrent introgression between accessions indicated the asymmetric capacity to receive foreign genes from other accessions. The modern introduction of cultivars of M. charantia during the colonization of Taiwan by the Han Chinese ethnic group enhanced the rate of gene replacement in the native populations and resulted in the loss of native genes.The landrace strains of  Momordica charantia, has been widely cultivated and domesticated for a long time  substitution model). The divergence time (T) between these two accessions is 0.519 Mya (±0.354 Mya) using the formula T = DA/2μ, where μ is the mutation rate, while a longer time to the most recent common ancestor  between the two accessions was estimated by using the BEAST. The large difference between the divergence time and the coalescent time between the accessions might be due to the high variance of the single-locus estimation or to the long time allowed for gene flow at the beginning of speciation [The cpDNA cp id lines . The twoeciation .atpB-rbcL sequences were obtained from 29 lines of cultivars, hybrids, and wild populations of M. charantia. The aligned cpDNA atpB-rbcL data matrix was 954 bp in length. The sequences from this study can be found under the GenBank accession numbers: HE585487-HE585491. Four segregating sites were examined, and low genetic diversity was estimated. The nucleotide diversity (π) and genetic diversity (θW) of total samples (including hybrids) is 0.00108 and 0.00100, respectively. The index of Tajima’s D of the total sample is 0.1977 and is not significant so the null hypothesis of neutral evolution cannot be rejected. Genetic diversity indices of two accessions are listed in In total, five haplotypes of 164 cp et al. [FST-outliers by criteria of 99% confidence intervals (CI). CMBR114-1 and CMBR31, which are thought to have evolved under positive selection (extremely high FST) and balancing selection (extremely low FST), respectively (FST-outlier loci. The locus CMBR114-1 is monomorphic in wild population of Taiwan samples but polymorphic in cultivars. The fixation of CMBR114-1 in Taiwan accession is probably reasoning for the extremely high FST among accessions. We estimated the genetic diversity by expected heterozygosity (Hexp) and genetic diversity index (θ) of the remaining eight microsatellite loci for each accession and obtained a wide range of diversity for each locus: for the cultivar accession Hexp ranges from 0.097 to 0.738, and θ ranges from 1.506 to 5.186; and for the Taiwan accession Hexp ranges from 0.264 to 0.791, and θ ranges from 1.506 to 2.527 . From the review of genetic differentiation by Storz et al. (2005), genetic differentiation will be significant if the genetic variation of one of two populations  is significantly lower than the other. In our study, the high percentage of genetic variation within accessions combined with insignificant genetic differentiation between accessions seems to imply that the degree of genetic variation of the Taiwan accession, the wild bitter gourd, is not very different from the genetic variation among multiple cultivar lines of M. charantia. However, the accession-specific RST values of the two accessions are 0.00139 and 0.01758 in the cultivar accession and the Taiwan accession, respectively. The difference in RST between the two accessions is larger than 10-fold, indicating a larger genetic differentiation of the wild population, which seems unreasonable because different sources (lines) of the cultivars have been suggested to have higher differentiation than the local populations. Therefore, clustering patterns of these samples were rechecked with PCoA. We also used the PCoA to reassign the clustering of collected samples without any assumption of prior classification. The result of the PCoA revealed mixture patterns of individuals between the two accessions in two axes with 52.81% explanation  , which indicates that the genetic differentiation between the two accessions is not significant (fixation index ctively) . This rectively) . To undet = 0.135 (95% CI: 0.065–0.545) has the highest posterior probability  (95% CI: 8.70 kya~72.96 kya), which is much smaller than the 0.519 Mya estimated from cpDNA atpB-rbcL spacer. The difference in divergence time inferences might be due to the intrinsic factor of rapid evolutionary rate of microsatellites compared with cpDNA, resulting in faster coalescence of the wide ranges of evolutionary rate of microsatellites, and extrinsic factors such as the pollen flow. In addition, we also noticed that the posterior probability is zero when t = 0, indicating that these two accessions are truly diverged. This result does not support the inference of the admixture pattern of genetic structure analyses .The isolation-with-migration (IM) model  that allbability  estimatem = 9.95 while the opposite direction of migration has a maximum posterior probability of m = 0.01 , indicating a unidirectional gene flow (or introgression) from the cultivar accession to the Taiwan accession (θA = 3.3572) is considerably larger than both current populations of the cultivar accession (θC = 0.3314) and the Taiwan accession (θT = 0.1767) (θC = θT) cannot be rejected , which suggests that these two accessions preserved equal weighted genetic diversity  . The fouiversity . Both thio tests .MC→T = 507.5, 97.5% CI: 475.0~535.0, where the suffixes C and T indicate the cultivar accession and the Taiwan accession, respectively) are larger than the opposite direction   between two accessions. This result is consistent with the inference of unidirectional gene flow by IMa analysis. Such asymmetric gene flow was also detected in the recent migration events by the assignment test of BayesAss [7 simulations, and (2) the rate of gene flow from the cultivar accession to the Taiwan accession is 0.0707 (95% CI: 0.0026~0.1963), and the opposite direction is 0.0151 (95% CI: 0.0005~0.0522)  . The migIGRATE-n  was scal~0.0522) . The ratM. charantia spans thousands of years [The first aim of this study was to test whether the cultivars of bitter gourds are genetically differentiated from the wild populations of Taiwan or whether these two accessions are a genetic mixture. Although the cultivars and the wild populations of Taiwan are taxonomically defined as the same taxon, which is also supported by AMOVA , PCoA F, and Bayof years , and thet) estimated by using the IMa analysis is zero at minimum t  in Taiwan. After the LGM , the geographic barrier of the Taiwan Strait isolated the island populations from mainland China. The perfect match of the divergence time of the two accessions and the age of the LGM suggests a relation between the evolutionary history of M. charantia and the geohistory of Southeast Asia. In addition, the shorter divergence time estimated by microsatellites than cpDNA implied the continuous gene transport by pollen flow between accessions after the divergence of maternally inherited materials since the middle Pleistocene, i.e., a long-term process of speciation (divergence).There are two explanations for the admixture pattern of the genetic composition of the two accessions: (1) incomplete divergence by sharing abundant common ancestral polymorphisms and (2) gene flow after divergence. Although the posterior probability of the splitting time  of the two accessions  is in contrast to the relatively small population sizes of the derived accessions, reflecting a prominent stochastic effect of lineage sorting among multiple loci [ca. 18 kya) inferred by microsatellites and that the cultivar accession is not native to Taiwan Island, the native population of the Taiwan accession most likely colonized Taiwan during the late Pleistocene glaciations . Certain examples, such as lizards [Furthermore, the large ancestral population size  than the wild population of Taiwan, which implied multiple sources of different cultivated strains  followed by artificial hybridization for breeding purposes. Multiple independent domestication events from local strains might have resulted in the rapid fixation of domestication-related genes while most unlinked loci of the genome retained the degree of diversity that was present before the initial domestication. This mechanism might account for the high genetic diversity of the nuclear microsatellites of the cultivars of M. charantia. Another possibility is that crops restored diversity through recent gene flow from wild populations after domestication [M. charantia. However, the assignment tests by Bayesian clustering analysis (mT→C = 0.01) by the IM model, and the hypothesis of no migration (mT→C = 0) cannot be rejected (mC→T = 9.95), suggesting that the genetic admixture is a result of strong introgression from cultivars to wild populations of Taiwan. This inference is also supported by the analyses of historical and current gene flow by using the MIGRATE-n [In addition, the relatively smaller effective population size of the wild population of Taiwan (approximately a quarter of the cultivars) is truly a reflection of the locally restricted distribution in Taiwan. Reduced genetic diversity is a common feature of domesticated genomes because of a “genetic bottleneck” caused by the domestication process  or a seltication . We are analysis , which irejected . On the IGRATE-n  and BayeIGRATE-n , respectIGRATE-n ,46.M. charantia cultivars and wild populations were collected from the field by our investigators or by exchange with cooperating laboratories. Most of the cultivars were provided by the National Plant Genetic Resources Center of the Taiwan Agricultural Research Institute, Council of Agriculture of Taiwan. The lines of varieties are listed in M. charantia were grow or collected freshly matured leaves, purified genomic DNA, sequenced the atpB-rbcL intergenic spacer of cpDNA, and genotyped SSR, independently, to test the homogenization. In total, 164 individuals were tested on this study and listed in Momordica lines were incubated for four days in 30 °C to allow them germinate. Subsequently plants were cultivated in thermostatic chamber in 25 °C for two weeks and then transplanted into greenhouse conditions for the follow-up study. Freshly matured leaves were collected for molecular experiments.Seeds or adult leaves of both Cucumis melo L. [M. charantia  and the primers atpB-107 and rbcL-188 were derived from Chiang et al. [2, 0.1% Triton X-100, 100 mM Tris-HCl (pH 8.3), 2 mM dNTPs, 2 μM primers (forward and reverse), 20 ng template DNA, 1 μg RNase, and 0.5 U Taq polymerase . The program for amplifying atpB-rbcL was as follows: initial denaturation at 94 °C for 5 min followed by 35 cycles of 40 sec at 94 °C, 1 min annealing at 55 °C, 1 min at 72 °C and a subsequent 10 min final extension at 72 °C. The purified PCR products were sequenced in both directions using a BigDye® Terminator v 3.1 Cycle Sequencing Kit , the ABI 377XL automated sequencer  and the ABI PRISM® 3700 DNA Sequencer  at the Genomics BioSci & Tech. Co., Ltd., Taipei, Taiwan.Total DNA was extracted from leaves according to the protocol of the Plant Genomic DNA Extraction Miniprep System . Following the commercial protocol, 100 mg fresh leaves from each individual were used for purifying genomic DNA. Totally, extracted genomic DNA from 164 individuals was dissolved in 200 μL of TE buffer (pH 8.0) and stored at −20 °C for further experiments. Seventy-two primer sets developed from  melo L.  were tesharantia . The theharantia , 1 min eg et al. . The PCRπ) and the index of genetic diversity estimated by segregating sites (θW) were calculated using DnaSP ver. 4.0 [D neutrality tests [π and θW, were performed using DnaSP ver. 4.0 [The amplified cpDNA sequences from 164 individuals were aligned directly by ClustalX  and editver. 4.0 . The Tajty tests , estimatver. 4.0 .M. charantia, the phylogenetic Bayesian Markov chain Monte Carlo simulations were performed using BEAST v. 1.6.1 [−9 per site per year [Neighbor-joining phylogeny and Bayesian phylogenetic relationships of chlorotypes were constructed using MEGA v. 5.05 . The setv. 1.6.1 . The Marv. 1.6.1 . The Yulper year .i.e., the loci with any repeat-number variation) to ensure the exclusion of the influence of natural selection. We used the method of Beaumont and Nichols [FST outlier for each locus individually. The idea of outlier loci is based on the hypothesis of extremely high FST between populations for positively selected loci (the positive outliers) than for the neutral loci and a reduced FST between populations for loci under balancing selection (the negative outliers). The FST outlier approach is performed by LOSITAN [FST by empirical data followed by forcing the simulation according to the mean FST; (2) we ran LOSITAN as in strategy 1 but simulated by removing the loci outside the 99% CI as recommended by Antao et al. [Before extending our analysis to genetic structure and speciation model tests, it was necessary to perform a neutrality test on the polymorphic microsatellite loci  of the “neutral” microsatellite loci were estimated using the stepwise mutation model (SMM) by Arlequin v. 3.0.1 [RST) from random variation (no genetic differentiation) was tested using 1000 permutations. PCoA was performed using GenAlEx 6.3 [K = 1~10) was estimated by using the MCMC method with 10 independent runs to evaluate the consistency of the results using 3 million steps with a 500,000-step burn-in for each run. Better grouping numbers were evaluated by log estimates of the posterior probability of K.The expected heterozygosity and index of genetic diversity , and population sizes: (1) Are the cultivar accession and the Taiwan accession really divergent? (2) Is there a change in population size after their divergence? (3) Has any migration occurred since the divergence of two accessions? (4) How do the degrees of migration (gene flow) differ between the two accessions? The IMa uses the MCMC sampling strategy to simulate parameters  of population size , migration rate , and divergence time . The model of molecular evolution employed was the SMM with an inheritance scalar of 1 for the nuclear microsatellite loci. MCMC runs were carried out with 100,000-step burn-in followed by 10 million iterations of the Markov chain. Because the mutation rate for the microsatellite loci might vary and cannot be determined, the default mutation rate (μ = 1) was set. Most effective sample sizes (ESS) for the microsatellite loci are never higher than 50; thus, the lack of trends in the parameter plot for t was used to determine whether convergence had been reached. Log-likelihood ratio (2LLR) tests were performed to test the significance of various nested models (null models) implemented in the IMa. We used null models that included various combinations of equal population sizes of the ancestral (θA) and the current populations (θC for the cultivar accession and θT for the Taiwan accession), equal bidirectional migrations (mC→T = mT→C), and a fixed value of zero for migrations . The FST estimates were used as starting values for the initial analysis. For all other analyses, the ending parameters of the previous run were used as the starting values for the next run until results equilibrated at approximately the same values. We used the settings of three long chains with 15 million sampled, 15,000 recorded, and 25% discarded at the beginning of the chain. Adaptive heating was applied with temperature specifications of 1.00, 3.71, 9.14 and 20.00 to assure convergence, and the last three replicates were combined for estimation.The historical and recent gene flows were estimated with the assistance of the MIGRATE-n v. 3.0  and the 7 iterations with every 2000 iteration of sampling were performed, and the first 10% iterations were discarded as burn-in. Three delta parameters of allele frequency, migration, and inbreeding were set as 0.15, 0.15, and 0.20, respectively, based on the preliminary runs.The pattern of recent gene flow between accessions was inferred by using the Bayesian inference with the assistance of BayesAss v. 1.3 . This apM. charantia suggest incomplete divergence and a high capacity for gene exchange. In fact, artificial hybridization between the cultivars (as the pollen donor) and the wild populations of Taiwan has been carried out, named “Hua Lien Bitter Gourd No. 2” and “No. 15”, by the Hualien District Agricultural Research and Extension Station (http://www.hdais.gov.tw/bred). The successful artificial hybridization suggested the possibility of natural hybridization, especially for the crop-to-wild introgression by pollen flow. Different estimates of divergence time implied that the long-term duration of the speciation process might be caused by recurrent asymmetric gene flow, an idea that is supported by inferences of both historical and current rates of migrations. In fact, the crop-to-wild introgression is much more substantial than we had previously thought [The unresolved genetic structure and the high migration rates between cultivars and the wild populations of Taiwan of  thought . A serio thought . In our  thought . Therefo