Adh) located within and near the In(2L)t inversion of Drosophila melanogaster at three different sites along a latitudinal cline on the east coast of Australia.Chromosomal inversions are increasingly being recognized as important in adaptive shifts and are expected to influence patterns of genetic variation, but few studies have examined genetic patterns in inversion polymorphisms across and within populations. Here, we examine genetic variation at 20 microsatellite loci and the alcohol dehydrogenase gene t chromosomes and colonization of Australia, although there was no consistent evidence of a recent bottleneck and simulations suggest that differences between arrangements would not persist unless rates of gene exchange between them were low. Genetic patterns therefore support the notion of selection and linkage disequilibrium contributing to inversion polymorphisms, although more work is needed to determine whether there are spatially varying targets of selection within this inversion. They also support the idea that the allelic content within an inversion can vary between geographic locations.The strong differentiation between arrangements and reduced variation in the inverted chromosomes are likely to reflect ongoing selection at multiple loci within the inverted region. They may also reflect lower effective population sizes of  Drosophila and other Diptera, inverted and noninverted (standard) forms of chromosomes often coexist within the same population. These inversion polymorphisms can be identified by examining the banding patterns of chromosomes in the larval salivary gland cells, and by the formation of loops during chromosomal pairing between inverted and standard arrangements, making them convenient genetic markers for studying evolution ; cn[1] bw[1] sp[1] strain (FlyBase ID: FBst0002057). The progeny from these crosses were preserved in 100% ethanol and stored at -20°C for genotyping.From each site between 118 and 189 isofemale lines were established from individual field collected females. These lines were maintained at 25°C under continuous light on a sugar (1.6% w/v), agar (3.2%), yeast (3.2%) and potato (1.6%) medium that was always treated with an antifungal agent (0.14% nipagin) and antibiotics (2% dihydrostreptomycin and 0.6% penicillin added to the medium surface). After two to 12 generations in the laboratory, virgin females from each isofemale line were crossed to males from the isogenic et al. ; cn[1] bw[1] sp[1] strain were also genotyped for each of the molecular markers.DNA extraction from individual flies, PCR protocols, and allele scoring followed methods outlined in Gockel et al. . Each fl)t Table . Primer a et al.  and Ando [et al.  respectiy[1]; cn[1] bw[1] sp[1] strain (the sire) from the genotype of each F1 individual at each genetic marker. The chromosome arrangement for each haplotype was also determined in the same way using the genotype of the In(2L)t genetic marker. These data were used for all subsequent analyses.A haploid, multilocus data set of known gametic phase was created by subtracting the allele present in the isogenic fstat software package [FST. Pairwise FST values, 95% confidence limits for these values and tests for differentiation among sites were calculated with the fstat software package [In(2L)t were quantified using the r2 coefficient. Estimates of r2, the significance level of the disequilibrium and AMOVA were calculated with the arlequin version 3 software package [For each site/chromosome arrangement combination, the level of genetic variation was quantified by calculating allelic richness  and gene diversity using the  package . Differe package  estimato package . We used package .FST outlier approach [lositan software package [FST and expected heterozygosity in an island model of migration with neutral markers. This distribution is used to identify excessively high or low FST values compared to neutral expectations. Such outlier loci are candidates for being subject to selection. Simulations were run using 10 000 replications, 95% confidence intervals and the neutral and forced mean options. An infinite allele mutation model was assumed. Analyses using the stepwise mutation model were also carried out, but they provided similar results, so only those with the infinite allele mutation model are presented.Tests for selection acting on marker loci were carried out using the approach ,41 and w package . The metstructure, version 2.1 [structure was run with the admixture model and correlated allele frequencies [To assess the extent of genetic mixing between chromosome arrangements at each site, a cluster analysis was performed using the program sion 2.1 . We assuquencies ,44. Fivebottleneck[Finally, tests for a severe reduction in effective population size (population bottleneck) were performed for each site/chromosome arrangement combination using the software package ottleneck. The metottleneck. In thisIn(2L)t only. Because sampling multiple chromosomes from within isofemale lines may influence LD, only a single chromosome from each isofemale line were used for the LD analysis. Corrections for multiple comparisons were applied to all tests.With the exception of the LD calculations and tests for loci under selection, analyses were performed using markers situated within the breakpoints of In(2L)t in Australian populations, simulated data sets were created using a modified version of the EASYPOP 2.0.1 computer program [In(2L)t each generation. Unfortunately there are no estimates of these processes for microsatellite markers, and two rates of gene exchange were used. The first was a rate of 9.4 × 10–4 per generation, which is equal to the probability of a crossover event occurring within a 5 cM region (1.2 – 3.4 Mb) inside each of the inversion breakpoints in heterokaryotypes (0.05 × 0.05 × 2q(1 – q), where q is the frequency of In(2L)t). The second was a rate of 1.0 × 10–4 per generation, which is an estimate of the rate of double crossovers between segregating inversions in Drosophila based on phenotypic markers located near the centre and inversion breakpoints [To determine whether there has been sufficient time for gene exchange to breakdown LD and genetic differentiation between chromosome arrangements in the middle part of  program . The simakpoints ,49.6 t chromosomes segregating in tropical populations (0.75 and 0.25 respectively). The mutation rate was set at 5.65 × 10–6, which is the weighted average of microsatellite mutation rates observed in D. melanogaster[The total number of chromosomes (i.e. both subpopulations combined) was set at 2.0 × 10e ~ 106, ). Howeveanogaster,52. BothD. melanogaster has been in Australia assuming 10 generations per year [FST ~ 0.3) allele frequency differences between subpopulations. In models with maximum genetic divergence, subpopulations were fixed for different alleles at each locus. Subpopulations with intermediate genetic divergences were set up by randomly selecting alleles from all possible allelic states for one subpopulation and randomly selecting alleles from a subset of possible allelic states for the other subpopulation. This also resulted in a difference in allelic richness between subpopulations, which is likely in natural populations given the smaller Ne and more recent origin of inverted chromosomes.The simulations ran for 1400 generations (the estimated number of generations per year ) and werarlequin to assess levels of LD.Each generation 44 chromosomes (the minimum sample size from mid and low latitude sites in our study) were sampled from each subpopulation and used to calculate the level of divergence between the subpopulations. Simulated data sets from generation 1400 were also analyzed with The authors declare that they have no competing interests.WJK and AAH conceived and designed the study. WJK collected and analysed the data. Both authors wrote the manuscript. Both authors read and approved the final manuscript.Repeat type and primer sequences for microsatellite loci.Click here for file


Solanum melongena L.) was domesticated in the Indo-Birmanian region, which is also the primary center of diversity for this crop. From there eggplant spread to other regions, and diversity accumulated in several secondary centers of diversity. We have assessed the diversity and relationships of 52 accessions of eggplant from three geographically distant secondary centers of diversity  using 28 morphological descriptors and 12 highly polymorphic genomic SSRs. A wide variation was found for most morphological traits, and significant differences among the three centers of diversity were detected for 22 of these traits. The PCA analysis showed that eggplants from the three origins were morphologically differentiated, and accessions from each of the three secondary centers of diversity presented a typical combination of morphological characteristics. In this respect, discriminant analysis showed that accessions could be correctly classified to their origin using only six traits. The SSR characterization identified 110 alleles and allowed obtaining a unique genetic fingerprint for each accession. Many alleles were found to be private to each origin, but no universal alleles were found for any of the origins. The PCA analysis showed that the genetic differentiation among origins was less clear than for morphological traits, although the analysis of the population structure shows that accessions mostly group according to the origin, but also provides evidence of migration among the three secondary centers of diversity. The genetic diversity (HT) within each origin was high, ranging between HT = 0.5400 (Sri Lanka) and HT = 0.4943 (China), while the standardized genetic differentiation (G’ST) among origins was moderate (G’ST = 0.2657). The correlation between morphological and SSR distances was non-significant (r = 0.044), indicating that both data are complementary for the conservation of germplasm and breeding of eggplant. These results are relevant for the management of genetic resources, breeding programmes, and evolutionary studies of eggplant.Eggplant ( Solanum melongena L.) is the most important Solanum crop native to the Old World, and it ranks as one of the most important vegetable crops in the world with an annual worldwide production of more than 41·106 t . This results in an average production of 6.1 kg/person/year or 16.7 g/person/day. Eggplant is one of the vegetables with highest antioxidant capacity Eggplant  Coffea arabica L.) Zea mays L.) Sorghum bicolor (L.)) Although a number of studies have been devoted to the study of the diversity of eggplant materials from specific countries or regions, like India The characterization of eggplant with both morphological descriptors and molecular markers has proved useful for the study of the diversity and relationships of different varietal groups of eggplant, as they sample different levels of diversity Here we assess the morphological and the molecular (SSR) diversity of a collection of eggplants from three geographically distant secondary centers of diversity: China, Spain, and Sri Lanka. Our objective is to obtain information on the diversity, relationships, and differentiation among accessions of these three different origins. These results will be of interest for the conservation of genetic resources, breeding, and study of the evolution of the cultivated eggplant.The collection of eggplants studied, as well as each of the three groups of accessions originating from China, Spain, or Sri Lanka, displayed a wide variation for most of the morphological traits studied . AlthougChinese accessions are less vigorous than those of Spain or Sri Lanka, so that the Chinese accessions have lower plant height (P-Height) and smaller leaves  than either the Spanish or Sri Lankan eggplants . RegardiFor flower traits, Chinese eggplants had, in general, a smaller number of flowers per inflorescence (Fl-Number) than either the Spanish or Sri Lankan eggplants . SpanishThe fruits of Sri Lankan accessions, in general, have higher fruit length (Fr-Length) and lower fruit breadth (Fr-Breadth) and, in consequence, are more elongated (Fr-Ratio) than those of China or Spain . SpanishAccording to the scree plot method, a total of five principal components were found to be relevant in the morphological PCA study. These five principal components account for 63.4% of the total variation. However, given that the first and second components account, respectively, for 22.9% and 16.2% of the total variation , and thaThe projections of the accessions on the PCA graph show that with the exception of two accessions (C02 and L11) the accessions of China, Spain and Sri Lanka plot in different parts of the PCA graph . The ChiThe discriminant analysis of the 28 morphological traits revealed that 100% of the accessions were correctly classified to their country of origin. The forward stepwise discriminant analysis showed that a correct classification of all the accessions could be achieved using a minimum set of six traits: L-Length, Fr-L*, Fr-UnderC, Fr-b*, P-Prickles, and Fr-Weight. The two discriminating functions for this model were highly significant (P<0.001). For all these traits, with the exception of Fr-b*, important and highly significant (P<0.001) differences were found among origins .The twelve SSR loci evaluated were polymorphic in the materials evaluated and amplified 110 alleles  . The numWhen considering the number of alleles present in accessions from different origins, the Sri Lanka materials presented the highest number of alleles (63), followed by Chinese (52), and Spanish (50) accessions, which corresponds, respectively, to 57.3%, 47.3%, and 45.5% of the total number of alleles detected . HoweverThe first and second components of the PCA analysis performed with SSR data account for 22.0% and 19.1% of the total variation, respectively. Although in this analysis accessions of the different origins are intermingled , accessiK) was 3. The inferred population structure for K = 3 obtained with the STRUCTURE software showed that most of the accessions of each origin are assumed to belong to the same population (iq) is used to assign one accession to a population, for the Chinese accessions, 14 out of the 20 accessions are assigned to the CH population; for the rest of Chinese accessions, C14, C16, C17, and C18 are assigned to the SP population, and C01 and C11 to the SL population. In the case of the Spanish accessions, 10 out of the 14 accessions are assigned to the SP population; three of the rest of Spanish accessions  to the CH population, while the remaining one (S10) to the SL population. Finally, the 15 out of the 18 Sri Lankan accessions are assigned to the SL population; two out of three of the rest of accessions (L05 and L11) to the CH population, and the last one (L02) to the SP population  could be considered as admixed (iq≤0.8). Seven of the admixed accessions were from China , five from Spain , and four from Sri Lanka  (The Evanno’s test indicated that the most informative number of populations (pulation . Therefopulation . For mosand L10) .T), which ranges from HT = 0.4943 for Chinese accessions to HT = 0.5400 for Sri Lankan accessions (÷). The partition of the total diversity shows that most of the genetic diversity is found within each of the groups (DST = 0.0481 and HS = 0.4909), which results in a moderate value of the relative magnitude of genetic differentiation (GST = 0.0892). However, the standardized GST (G’ST) reached a much higher value (G’ST = 0.2657)  . The valThe lowest genetic distance (and consequently highest genetic identity) values are found between the Chinese and Sri Lankan origins, with a value of 0.1204 . The genMorphological and molecular data are not only complementary for the study of the diversity and relationships of eggplants from these distant centers of diversity, but also synergistic, as information of interest becomes conspicuous when comparing both type of data. Several studies are available on the relationships between morphological and molecular data in eggplant, but these studies have been restricted to specific cultivar groups The morphological data show that within each of the origins, a considerable diversity exists, confirming what had already been observed within Spanish eggplants The wide range found for most morphological traits within each of the origins suggests that genetic drift may not have been an important force in the morphological differentiation of eggplant within each of the three secondary centers of diversity The important differences among origins for morphological traits may be relevant for eggplant breeding, and indicates that sources of variation of interest for several relevant agronomic traits of materials from a center of diversity may be found in exotic materials from other centers of diversity. For example, when considering some traits of interest for breeding modern cultivars The high morphological variation in the materials studied is matched by high levels of molecular diversity. In this sense, we have found that the 12 genomic SSR loci were polymorphic and allowed the detection of a total of 110 SSR alleles . These 12 SSR loci were developed by Vilanova et al. iq to one of the populations higher than 0.8 The multivariate analysis of SSR data shows a less clear picture of the differentiation among centers of diversity than the analysis of morphological data, with a higher degree of admixture among accessions of different origins in the SSR PCA plots than in the morphological PCA graph. In any case, the combination of the first and second components separate most of the Sri Lankan accessions from most of the Chinese accessions, while the third component differentiates mostly the Spanish and Sri Lankan accessions. Therefore, despite the fact that some of the accessions are intermingled, differentiation is observed among the three origins in the PCA analysis. The Bayesian-based analysis without a priori assignment of accessions to populations resulted in the creation of three populations, each of which was formed mostly by accessions from a single origin, which suggests an important differentiation among origins T) parameter The analysis of the genetic diversity with the total diversity (HST) is considerably lower than the genetic diversity within groups (HS), the standardized genetic differentiation (G’ST), which is more adequate than GST for SSR data Despite the fact that the genetic diversity among groups  found by Muñoz-Falcón et al. The lack of correlations in our collection suggests that both types of data sample different levels of diversity and, therefore, both of them should be considered for the management and conservation of germplasm The results show that eggplant accessions from the three geographically distant secondary centers of diversity studied  present a wide morphological and molecular diversity. At the morphological level, a clear differentiation exists among the different origins, indicating that different selection criteria have been applied in each secondary center of diversity, leading to a typical syndrome of traits for each origin. The molecular data also show that considerable differentiation exists at the molecular level among the three origins, although in this case there is evidence of migration between the different origins, as revealed by the study of the population structure. The lack of correlation between morphological and molecular diversity shows that both types of data provide complementary information and that both of them should be taken into account in the management of germplasm and formation on core collections No specific permits were required for the described field studies, which took place in an experimental field plot at the Universitat Politècnica de Valencia. This field plot is used by the authors of this paper affiliated to the aforementioned institution  for field trials for characterization of germplasm of cultivated species.S. melongena, of which 20 originated in China (C codes), 14 in Spain (S codes), and 18 in Sri Lanka (L codes) were used for the present study  Fifty-two accessions of nt study . The matFor each accession five plants raised from seed were grown in an open-air field plot  in Valencia (Spain) following a completely randomized experimental design. Plants were spaced 1 m between the rows and 0.8 m apart in the row. The standard horticultural practices for eggplant production in the area of Valencia were followed.Individual plants were characterized using 23 primary descriptors, most of which were developed by EGGNET Genomic DNA was extracted from a mixture of young leaf from the five plants morphologically evaluated according to the CTAB method procedure 2, 0.05 µM of forward primer, 0.25 µM of reverse primer, 0.2 µM fluorescent-labelled M-13 primer, 0.2 mM dNTPs and 1 unit of Taq polymerase in 1X PCR buffer. Conditions of the PCR amplification were as follows: 1 cycle for 2 minutes at 94°C, 35 cycles of 15 s at 94°C, 30 s at appropriated annealing temperature (For the present study we used twelve simple sequence repeat (SSR) markers  that properature , 45 s atThe range and mean values for the morphological traits for each of the groups of Chinese, Spanish, and Sri Lankan eggplants were calculated from the means of each accession. Analyses of variance were used to detect differences for the traits studied among the three groups of accessions. Kolmogorov-Smirnov and Bartlett tests were performed to test, respectively, the normality of data and homogeneity of variances among different origins. Principal components analysis (PCA) were performed for standardised morphological and agronomic data using pairwise Euclidean distances among accessions. Eigenvalues were calculated for each of the principal components, and relevant components were identified using the spree plot method K, from 1 to 10, in 10 independent runs K was identified using the statistic ΔKK = 3 and using 50000 iterations. Genetic diversity was estimated with the total diversity (HT) ST), and within origins (HS). The relative magnitude of genetic differentiation among origins (GST) was calculated as the ratio DST/HTST (G’ST), which standardizes the observed GST value with the maximum possible value that GST could obtain given the amount of observed diversity, was calculated as (GST(1+HT))/(1−HS) i2), where pi is the frequency of the ith allele For the SSR data, pairwise genetic similarities were estimated using the Dice (Sorensen) similarity coefficient. Principal components analyses (PCA) were performed using the pairwise genetic similarities. Possible population structure associated to origin and likelihood of assignment of each accession to population was estimated using the Bayesian-based model procedure implemented in the software STRUCTURE v2.3.3


New Caledonian crows exhibit considerable variation in tool making between populations. Here, we present the first study of the species’ genetic structure over its geographical distribution. We collected feathers from crows on mainland Grande Terre, the inshore island of Toupéti, and the nearby island of Maré where it is believed birds were introduced after European colonisation. We used nine microsatellite markers to establish the genotypes of 136 crows from these islands and classical population genetic tools as well as Approximate Bayesian Computations to explore the distribution of genetic diversity. We found that New Caledonian crows most likely separate into three main distinct clusters: Grande Terre, Toupéti and Maré. Furthermore, Toupéti and Maré crows represent a subset of the genetic diversity observed on Grande Terre, confirming their mainland origin. The genetic data are compatible with a colonisation of Maré taking place after European colonisation around 1900. Importantly, we observed (1) moderate, but significant, genetic differentiation across Grande Terre, and (2) that the degree of differentiation between populations on the mainland increases with geographic distance. These data indicate that despite individual crows’ potential ability to disperse over large distances, most gene flow occurs over short distances. The temporal and spatial patterns described provide a basis for further hypothesis testing and investigation of the geographical variation observed in the tool skills of these crows. Corvus moneduloides) are omnivorous forest birds endemic to the French Territory of New Caledonia. They live on only two of the larger islands of New Caledonia: Grande Terre and Maré Pseudophyllanax imperialis) that were destroying coconut trees (pers. comms. to GRH).New Caledonian crows  across nine sites, which were spread over the island (2 in area) on the south east side of Grande Terre  in inland forest where Pandanus spp. trees were absent. The 14 crows at Bourail (site 6) were also caught close to the sea, at the foot of hills where candlenut trees (Aleurites moluccana) were common. Crows in candlenut tree areas use tools year round to extract large wood-boring grubs from dead candlenut wood On Grande Terre we sampled crows at sites that varied with respect to habitat and associated tool-making behaviour. For calculations based on local frequencies the sample size at sites had to be at least five  . To maximise the recovery of DNA, the tubes containing the feathers were centrifuged at 3000 g for 5 mins. Ethanol was then removed and the tubes were inverted on a bench to dry. The pelleted material was then re-suspended in 50 µl of water and added to the buffer/proteinase K mix, along with the feather tips cut into pieces. DNA was finally re-suspended in a 50 µl volume of T.E.C. kubaryiC. brachyrhynchosWe used nine microsatellite markers that we had previously optimised to reliably genotype New Caledonian crows We used neutral genetic markers to investigate genetic structure in the New Caledonian crow in order to see how genetic variation is structured geographically and to what degree gene flow exists between populations .a priori assumptions on population delimitation. The most likely number of population units (K) in the complete dataset  was inferred using a fully Bayesian clustering method as implemented in STRUCTURE 2.3.2 K varying from 1 to 6. We used a classical model with admixture without a priori information on population membership with correlated frequencies. Although these models make it easier to distinguish closely related populations, there is a risk of overestimating KK was estimated using Evanno’s ΔK method The presence of null alleles was tested following Chapuis & Estoup Based on the results of this grouping method, standard genetic parameters such as allele frequencies, mean number of alleles per locus and heterozygosity were calculated to estimate genetic diversity. These parameters were computed using the software GENALEX 6 D. We used Jost’s D rather than the classical Fst because it is more rigorous at taking into account differences in heterozygosity between sites D values was tested using bootstrapping methods with resampling (n = 1000). These calculations were carried out using the R package DEMEtics We estimated the level of genetic variation between sample sites with at least five samples using Jost’s D as a measure of genetic divergence  and conducted all combinations of normal and log-transformed distances. The significance of the Z statistics was estimated through a randomization procedure (1000 randomizations). Moreover, the strength of the correlation (r2) was estimated using a Reduced Major Axis regression. These calculations were performed using IBDWS To test for isolation by distance on Grande Terre, we used a Mantel test with p-value was obtained using permutation methods with 1000 replicates.We also used the Mantel test to see if there was any geographical pattern in the shapes of stepped pandanus tools that might mirror variation in genetic structure . Specifically, we tested if distance between sites where stepped tools were made on Grande Terre was correlated with variation in the mean length of these tools between the sites. We used 18 of the stepped tool collection sites with adequate samples sizes described in Hunt & Gray We also conducted analyses to determine the genetic relationship between the crow populations on Grande Terre, Toupéti and Maré. We compared the number and identity of alleles for each locus between the two islands to see if Grande Terre was a likely source for the crow population on Maré. We then tested different colonisation scenarios involving the three islands with Approximate Bayesian Computation methods (ABC) using software DIYABC v1.0 K = 3, approximately representing the islands of Grande Terre, Maré and Toupéti . For K = 3, crows from Toupéti are separated from those on Grande Terre (K = 3 as the hypothesis for the main population groupings for three reasons: (1) Evanno’s method is known for underestimating KK from 2 to 3, and (3) the mean likelihood gives an optimal value for K = 3.Clustering analysis conducted on the whole dataset indicates that the most likely number of distinct genetic entities is  Toupéti . The anacreasing . A highenno’s ΔK . For K =de Terre . From th−3 for as few as five loci. Genotyping errors were low and approximate zero. We found no discrepancies in the 10% of the data that were genotyped twice to test for errors. Global departures from Hardy-Weinberg (n = 40) rather than on Toupéti (n = 8), the difference in genetic diversity increases . Ten private alleles were found on Grande Terre, three on Maré and none on Toupéti.No significant null alleles were detected in the three populations . Moreover, the probability of identity dropped under 10Weinberg  were detD between each pair of the five sites that had at least five samples  were not significantly different. Excluding Toupéti, values of differentiation on Grande Terre ranged from 0.085 to 0.199. Comparisons between Toupéti and the five Grande Terre sites showed higher values ranging from 0.316 to 0.504. These values are even higher than that obtained for the comparison between Maré and the Grande Terre sites, which ranged from 0.13 to 0.358. The highest value of genetic divergence was between Maré and Toupéti (0.519).To quantify the level of genetic differentiation in the New Caledonian crows that we sampled, we computed Jost’s  samples . Only twr2 values ranged from 0.321 to 0.499). When Toupéti is included, isolation by distance is only significant when both distances are log-transformed, resulting in a lower r2 (0.246). Similar results are obtained when using Fst as a measure of genetic distance rather than Jost’s D (data not shown). These results indicate two important findings: (1) a correlation between geographical separation and genetic similarity exists on Grande Terre, and (2) the crow population on Toupéti does not fit well into this correlation, possibly because the narrow sea channel between Toupéti and Grande Terre creates a much more restricted gene flow than occurs on Grande Terre.We also tested for isolation by distance using the five Grande Terre sites with larger sample sizes and Toupéti . When Top = 0.879, r2 = −0.09).We found no significant correlation across the 18 stepped-tool making sites between geographical distance and variation in mean tool length , we tested four scenarios using DIYABC . Scenario 2 provides the same colonisation pattern as scenario one, but with a larger time constraint (0 to 1000 generations instead of 30 to 50). We next estimated the posterior distribution of the parameters used in our model  still stays close to teuro with a mean of 67 generations even though it could potentially have had a value from 1 to 1000. The estimation of NM1  is relatively large at more than 200 individuals.We compared scenarios by computing their posterior probabilities using a logistic regression approach based on the best 40,000 datasets (1%). The logistic regression favoured scenario 1 . Finally, we evaluated the confidence in scenario choice by estimating type I and type II errors. We first computed 100 datasets under each competing scenario . Then we calculated (1) the number of times scenario 1 did not have the highest posterior probability when it was the true scenario (Type I error), and (2) the number of times scenario 1 had the highest posterior probability when the true scenario was either scenarios 2, 3 or 4 (type II error). The results suggested that our methodology to discriminate between the four competing scenarios was rigorous (type I error rate was 0.08 and the mean type II error rate was 0.032).Using nine neutral markers, we conducted the first study of the population genetic structure of the tool-using New Caledonian crow over its geographical range. Our analyses produced four main findings: (1) significant genetic differentiation between sites on Grande Terre, (2) a general signal of isolation by distance on Grande Terre, (3) populations on the islands of Grande Terre, Toupéti and Maré are distinct genetic entities, and (4) confirmation that both Toupéti and Maré populations were founded from Grande Terre.Our finding that crows on Maré are significantly genetically differentiated from those on Grande Terre was not unexpected given the 110 km of open water between the two islands that should have prevented the birds mixing. The genetic composition of the Maré population is almost a strict sub-sample of the genetic diversity found on Grande Terre, however. Our colonisation scenario analysis is also in agreement with the anecdotal evidence that birds were introduced to Maré from the mainland after European colonization began ca. 1850 The genetic distinctness of the small crow population on the small inshore island of Toupéti is also a robust finding. Despite crows’ ability to fly, the genetic data suggests that the birds on Toupéti rarely mix with those on Grande Terre. New Caledonian crows are tropical forest birds and as such may be extremely reluctant to cross even a narrow expanse of open water et al.et al.A recent study by Rutz Nonetheless, both studies suggest that genetic structure may exist at fine-scales in New Caledonian crows. Rutz et al. suggest that the genetic structure they found demonstrates the potential for genetic and/or cultural isolation to cause variation in tool skills between crow populations. However, caution is needed in extrapolating the genetic structure between their three sites to the Grande Terre as a whole. This is because only one of their three sites was in forest habitat; the other two sites (farmland and holiday settlement) were highly human modified habitat. Dispersal dynamics of crows between human modified and forest habitat is likely to be different from the dynamics within forest. Most of our sampling sites with five or more crows were in forest habitat. Small sample sizes, though, means that we cannot conclude that fine-scale genetic structure is absent in forest crows on Grande Terre. Importantly, in spite of small sample sizes we found weak isolation by distance in forest where the majority of tool manufacture by crows occurs.The isolation by distance effect that we found enables us to make inferences about the social system of tool making crows in forest habitat on Grande Terre. It suggests that the species’ social system and dispersal behaviour even more than environmental boundaries  are responsible for patterns of gene flow. A field study on Maré The patterns of gene flow that we found on Grande Terre do not allow us to make inferences about the reasons for the geographical variation in crows’ tool skills. However, the gene flow and the likely closely associated dispersal dynamics of crows on Grande Terre raise the possibility of a genetic effect on the observed local specialization of tool skills, along with both ecological and social effects and genetic data to untangle the processes and mechanisms responsible for the variation in crows’ tool skills. Recent work on chimpanzees clearly shows that such data needs to investigate patterns of genetic and behavioural dissimilarity associated with individual behaviours Parus major) Our work emphasizes the need for detailed behavioural, ecological Figure S1Four different colonisation scenarios of New Caledonian islands by crows. NGT: current effective size of Grande Terre; NM0: current effective size of Maré; NT: current effective size of Toupéti; NM1: effective size of Maré during the post-colonisation bottleneck; teuro: constrained colonisation time on Maré around 1900 in scenario 1, corresponding to 30 to 50 generations; db: duration of the post-colonisation bottleneck; t1: time before present of colonisation of Maré in scenario 2, 3 and 4; t2: time before present of colonisation of Toupéti; M: time of second colonisation on Maré in scenario 3 after the first one at t1; r: admixture rate during the second colonisation event on Maré in scenario 3.(TIF)Click here for additional data file.


Pocillopora damicornis  has recently been found to comprise at least five distinct genetic lineages in Eastern Australia, some of which likely represent cryptic species. Due to similar and plastic gross morphology of these lineages, field identification is often difficult. Here we present a quick, cost effective genetic assay as well as three novel microsatellite markers that distinguish the two most common lineages found on the Great Barrier Reef. The assay is based on PCR amplification of two regions within the mitochondrial putative control region, which show consistent and easily identifiable fragment size differences for the two genetic lineages after Alu1 restriction enzyme digestion of the amplicons. Many scleractinian coral species show high morphological variability that has been related to differing environmental conditions. Reciprocal transplant experiments have demonstrated that the same genet may exhibit different growth forms in response to light and water movement intensities Pocillopora damicornis  is a key model scleractinian coral species displaying a wide spectrum of morphotypes throughout its range P. damicornis: Flot et al. P. damicornis sensuP. damicornis should be re-visited, using the newly identified genetic lineages as the units of observation. Although the genetic lineages seem to be associated with phenotypic characteristics, phenotypic plasticity and cryptic appearance complicate confident identification, especially for the untrained eye. Currently, the only practical technique to differentiate among these genetic lineages is sequencing mitochondrial regions characteristic for each lineage P. damicornis from the Great Barrier Reef (GBR), Types α and β P. damicornis sensuThis paper presents a quick and relatively low-cost genetic assay to reliably identify the two most common, and best resolved genetic lineages of All necessary permits were obtained for the described field studies. Specimens for this study were collected under permit numbers G08/28215.1 and G09/30237.1, issued by the Australian Government’s Great Barrier Reef Marine Park Authority. The locations of sample collection are not privately-owned, and no endangered or protected species were collected.atp8 and cox1 genes P. damicornis Type α and a six-bp deletion is unique for Type β, sensu Schmidt-Roach et al. Pocillopora spp. and genetic lineages, hereafter “other Pocillopora”). Primer sequences are Pdam-F 5′-AAG AAG ATT CGG GCT CGT TT-3′ and Pdam-R 5′-CGC CTC CTC TAC CAA GAC AG-3′. These primer sequences do not occur in the mtDNA genome of the pocilloporid genera Seriatopora and Stylophora. The detection of such small amplicon size differences is challenging. Therefore, to facilitate rapid and reliable identification, i.e. to enable the use of simple agarose gels with reasonable electrophoresis time without compromising identification accuracy, we included a digestion step with Alu1. This restriction enzyme digest generates a unique banding pattern for Type α , Type β  and “other Pocillopora”  protocol, using 0.5 pmol of the M13-tailed F primers, and 2 pmols of all other primers (All 329  primers . ThermalThe Bayesian clustering method implemented in STRUCTURE v2.3.3 Pocillopora DNA sample (Pocillopora”, and classified the Type α LHI samples as “other Pocillopora” (Pocillopora”. Since the GBR samples originate from 10 different reefs, spanning more than 12° of latitude, these results provide high confidence of the appropriateness of the assay as an identification tool for the whole of the GBR. Testing the assay on populations outside the East coast of Australia was beyond the scope of this study, therefore we recommend sequencing a representative number of specimens in any non-GBR population to determine whether the type-specific deletions are present, prior to adopting the presented assay as an identification tool.Consistent with our expectations, three easily distinguishable banding patterns were seen on the agarose gels after running the assay on any A sample . The assllopora” . The taxPocillopora” specimens among the 329 samples from Lizard Island. The Bayesian model clustered specimens according to their mtDNA lineage identity (The PCR RFLP assay identified 205 Type α, 93 Type β, and 31 “other identity . Similaridentity . The conP. damicornis. Furthermore, while the recruits of pocilloporid species are readily distinguished from the recruits of other scleractinian families by their characteristic skeletal structures Pocillopora spp. DNA, and not the DNA from other pocilloporid genera. The assay is therefore also suitable to reliably identify Type α and Type β P. damicornis from among pocilloporid recruits, which will open doors to genetic lineage-specific recruitment studies.The genetic assay presented here provides a simple means to ensure that future studies avoid sample misidentification in


The timing of motor imagery has recently received attention from a number of researchers, culminating in a comprehensive review by Guillot and colleagues. This paper aims to further explore this issue, building upon the said review to suggest a number of other important timing-related issues. Specifically, we consider the possible role of bio-informational theory Lang, , 1985 an Imagery is one of the most popular psychological techniques used in sports skill learning. However, despite growing knowledge of how skills are best learned, there is still some lack of agreement regarding the most effective ways to implement imagery interventions. One issue that has received a great deal of recent research scrutiny is the speed at which the imagery should be conducted to have the greatest performance benefits. Imagery can be performed in real time, or there can be a divergence between the time taken to perform a movement and to mentally simulate it. This may be deliberate or because an individual is not capable of producing a vivid image in real time. For example, individuals may perform slow motion imagery deliberately when developing a skill, to enable them to focus more on key aspects of that skill than would be possible when performing real-time imagery . Preliminary results have generally revealed a positive impact on performance regardless of imagery speed. However, the real time and slow motion groups have shown the largest performance increases. Therefore, this evidence does not unequivocally support the idea that real time imagery should generally be used to facilitate the behavioral matching process. Indeed, depending on the stage of learning of the performer or their particular performance goals, slow motion may be equally effective, as slow motion imagery has been shown to have advantages for athletes trying to correct a bad habit . However, an increased speed of imagery could well have a detrimental effect on the quality of the imagery, though this is an issue that remains to be investigated. It is therefore important to fully understand the benefits and drawbacks of the varying timings of imagery, in order that the correct intervention can be matched to the age, performance level and sport of the individual. As such, we recommend future research should focus on the potential motivational effects of imagery timing, the link to meaning and the potential overlap with producing rhythmical action.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Since 1997, the Victorian Colorectal Cancer Family Study (VCCFS) has been studying a large number of individuals who have had colorectal cancer and their relatives. When a genetic mutation is identified by the VCCFS, family members are offered the chance to have genetic testing through a family cancer clinic to learn of their result. Between 17% (before August 2003) and 49% (after August 2003) of individuals offered genetic testing decline the offer [A sample of participants in the VCCFS who had been offered genetic testing between 2003 and 2008 were invited to take part in a qualitative interview about genetic testing decision-making in the area of colorectal cancer. The sample included both those who declined genetic testing (decliners) and those who accepted genetic testing (acceptors). Thematic analysis was performed to determine the main reason individuals gave for accepting or declining the offer of genetic testing.A total of 15 interviews have been conducted, six with decliners, and nine with acceptors. All participants recalled the offer of genetic information, and knew of the hereditary genetic mutation in their family. Each participant was able to clearly describe their decision. There were four types of decliners and four types of acceptors. Individuals declined because 1) Lynch Syndrome had been confirmed by other means; 2) fear about insurance implications; 3) genetic testing ‘wouldn’t change anything’ or 4) a positive result would cause too much worry. The main reason individuals accepted the offer of genetic testing was for the sake of 1) their children; 2) their own health; 3) curiosity; or 4) research. We consider how this decision was related to both risk perception and screening behaviour.The key perception informing the genetic testing decision was the consequence of receiving results. In the case of decliners, receiving genetic results was perceived to have either no consequences or negative consequences . For acceptors, genetic testing was perceived to have positive consequences either for their children, themselves, or society. This research highlights the need to explore risk perceptions, genetic testing and screening decision making in more detail in families eligible for genetic testing, in order to increase the uptake of genetic testing in this group.


Strix occidentalis in North America and the mountain pygmy-possum Burramys parvus in Australia. Using previously published mitochondrial and microsatellite data, we construct phylogenetic networks and score each population by its relative genetic distinctiveness. In both cases, our phylogenetic networks capture the geographic structure of each species: geographically peripheral populations harbor less-redundant genetic information, increasing their conservation rankings. We note that our approach can be used with all conservation-relevant distances  and suggest it be added to the assortment of tools available to wildlife managers for allocating effort among threatened populations.In the face of inevitable future losses to biodiversity, ranking species by conservation priority seems more than prudent. Setting conservation priorities within species  may be critical as species ranges become fragmented and connectivity declines. However, existing approaches to prioritization  are based on phylogenetic trees, which may be poor representations of differentiation below the species level. In this paper we extend evolutionary isolation indices used in conservation planning from phylogenetic trees to phylogenetic networks. Such networks better represent population differentiation, and our extension allows populations to be ranked in order of their expected contribution to the set. We illustrate the approach using data from two imperiled species: the spotted owl  Extinctions due to human impacts are now unavoidable: even optimistic scenarios predict significant changes in biodiversity by the year 2100 e.g., www.edgeofexistence.org). In the United States, taxonomic distinctiveness is one of several explicit criteria for prioritizing conservation attention One prime conservation goal is to preserve genetic variation evolutionarily significant units (ESUs) to identify populations with genetic variation in need of long-term conservation; this was expanded by Moritz management units (MUs) and distinct population segments (DPS), respectively, for species that had undergone more recent range fragmentation. All these population-based approaches have enjoyed wide usage in population genetic studies  and the mountain pygmy-possum (Burramys parvus Broom 1896). The new approach to assessing population differentiation might be of immediate practical use to those tasked with managing discrete populations of a threatened species, and may allow for new policy associated with conservation triage Previous authors have shown that the logic of measuring and maximizing phylogenetic diversity We present our approach for prioritizing populations in three steps. First, we briefly review the various approaches for measuring diversity and evolutionary isolation on bifurcating trees of taxa. We then review the properties of NeighborNet networks as a representation of pairwise evolutionary distances and describe how to prioritize taxa by their expected contribution to biodiversity. In expected diversity. Rather than score taxa individually, this approach seeks to identify the set of taxa that will retain the most diversity on a future tree, given some measure of diversity and a probability of persistence for each potential combination of taxa. Although Weitzman's original diversity metric was rather general, he did consider an example of biological character-state differences that could be represented on a phylogenetic tree.The concept of evolutionary isolation can be understood in terms of a species' biological distinctiveness, which we might measure by comparing its adaptive or non-adaptive traits to those of related species. More generally, our goal is to measure a taxon's contribution to the current and/or future “diversity” in a set of taxa. Several different approaches for quantifying such diversity have been proposed. One of the earliest, described by Weitzman edge) linking it to all other taxa  or in raw or inferred genetic distances. Looking specifically at biological systems, Witting and Loeschcke phylogenetic diversity (PD), the latter which specifically calculates the sum of all branch lengths on a tree (see next section). Like Weitzman expected PD approach can be used to identify a set of taxa that maximizes the amount of total tree length retained, given a set of extinction probabilities for the tips.On such a tree, every taxon contributes an amount of unique evolutionary information denoted by the length of the branch  on a tree of size PD complementarity of a given taxon.The related i.e., the average length of the edge linking the taxon to all possible future trees), one can rank taxa in order of their relative impact on future diversity. This Shapley metric (SH) is almost identical to the ad-hoc evolutionary distinctness (ED) metric used by the Zoological Society of London in their Edge of Existence programme (www.edgeofexistence.org). The major difference between the two is that the ED metric is explicitly measured on a rooted tree, as opposed to the more general undirected graph that SH takes as input An independently-derived approach based on Game Theory . HED is the expected contribution of a given taxon to future subsets of taxa where the subsets are weighted by their probability of persistence. In this case, the focal taxon is assumed to persist . On trees, HED is formally equivalent to a form of PD complementarity where the contribution of a taxon is measured with respect to all possible subsets, each weighted by their probability of persistence expected PD complementarity.The Shapley metric was further refined by Steel et al. k of n problem to maximize diversity for a given subset size. They referred to this metric as split diversity (SD).As a final antecedent, Minh In this context it should be possible to measure the PD contribution of individual taxa on a phylogenetic network. Critically for our purposes, the two metrics we use here (SH and HED) do not require a rooted phylogenetic tree, and so can be adapted to networks in the same way that PD indices can FST) may yield many alternative trees, none of which accurately reflect the actual relationships among these populations  the resulting NeighborNet output will resemble a phylogenetic tree. Where there is no tree-like history, a network representation should be more informative. Indeed, for many distance matrices , the assumptions necessary to produce a tree are not met, and a neighbor-joining tree, for example, produces negative edge lengths. Here, a network representation would definitely be preferred NeighborNet ns e.g., . The Neisplit  separating any two taxa. Thus, just as a phylogenetic tree represents a collection of weighted splits  represents the observed distance in the distance matrix.An example of a very simple matrix of pairwise distances and the resulting network is depicted in phylogenetic diversity (PD) Whether represented on a tree or a network, every split system contains information on the overall diversity of its constituent taxa where PD complementarity (PD_c) metric can be expressed asA very simple approach for measuring an individual taxon's PD contribution, illustrated in where e.g., e.g., et al. We can also extend the metrics SH and HED from trees to NeighborNet networks using similar ideas for extending PD calculations from trees e.g.,  to netwoet al. where is 0.870 .i.e., those connected to the rest of the network by long edges, like taxon As with a phylogenetic tree, the sum of Shapley values will always equal the sum of all parallel split weights in the network. Because the shape of a network reflects the relative distances among its taxa, we should expect outlying taxa , the size of each population (and hence the probability of extinction for each population) is not known. In the second example (B), population sizes are known, allowing us to estimate separate probabilities of extinction for each population.We present SH and HED ranking for two datasets based on putatively neutral genetic markers. In the first example , (2) its distribution is fragmented , (3) sampling efforts have covered its entire range, and (4) genetic analyses have been published or the raw sequence data made publicly available.We selected our two examples based on the following criteria: (1) The species as a whole is of conservation interest  are distributed throughout late-succession conifer forests in western North America S. o. caurina from southern British Columbia to northwest California, S. o. occidentalis in California and Nevada, S. o. lucida in Utah, Colorado, Arizona, New Mexico, and northern Mexico, and S. o. juanaphillipsae in central Mexico S. varia Barton 1799) S. o. caurina (the northern spotted owl) and S. o. lucida (the Mexican spotted owl) are threatened subspecies under the United States' Endangered Species Act, and S. o. occidentalis  is a subspecies of special concern in the state of California S. o. lucida) are particularly fragmented and perhaps most suitable for population-level conservation Spotted owls (ST). This procedure generated a pairwise differentiation matrix for the 32 populations  Spotted owl mitochondrial sequences were obtained from Genbank  and aligned in Mega v. 5 ulations . A Neighulations  [26] wasBurramys parvus) are alpine specialists restricted to three small regions of the Australian Alps  based on data from 8 microsatellite loci We used a published matrix of genetic differentiation  or close to zero, indicating higher levels of genetic differentiation within these populations than among them As expected for a set of lineages with a recent history of gene flow, the network for spotted owls is quite non tree-like . HoweverT values . ConversS. o. lucida exhibit a more star-like phylogenetic network that may reflect historical isolation in the “sky islands” of the American Southwest S. o. caurina and northern S. o. occidentalisWe observe strong geographic structure across the United States consistent with current subspecific designations . PopulatThe results of our SH and HED ranking are shown in As with spotted owls, the most genetically differentiated populations of mountain pygmy-possums, namely those in the northern and southern areas of their range, occupy nodes that are separated from most other populations by long edges . Overalla priori group assignments based on sampling location, the 13 populations still partition into northern, central, and southern regions. Again, outlying populations on the network tend to receive higher SH and HED scores. Unsurprisingly, the small and isolated Mount Buller population consistently ranks highest. For HED, no bias towards small or large populations is apparent; populations with high extinction probabilities do not necessarily receive high scores The ranking results are shown in all other taxa. Thus, although the three northern populations are closely related to each other, they still receive high SH and HED scores because of the long branches separating them from the central and southern populations  Salmo trutta Linnaeus 1758) Thunnus albacares Bonnaterre 1788) e.g., pikas (Ochotona princeps Richardson 1828) The premise of conservation below the species level is not novel. Faith's original e.g., i.e., subject to change if extinction alters the shape of the network. Ours is the first ranking scheme to consider a taxon's contribution to all possible future networks  can be used.We acknowledge that the mathematical shortcomings of Φe.g., niche use differences among populations).Several properties of the networks described here invite further investigation. In both our heuristic datasets, geographically peripheral populations are more genetically isolated, meaning they would rank highly on SH and HED. However, this was based on only very few putatively neutral markers. Two related questions concern how processes such as demographic history and current patterns of gene flow map onto genetic isolation as we measure it here, and also how phylogenetic networks map onto networks produced from ecological data  might also be considered. We note that in our examples, low-ranking populations tend to be geographically close to one another, meaning that their haplotypes are often shared. Important differences in ecology and adaptability We do not advocate relying solely on genetic isolation when deciding where and how resources should be allocated at the population level. Total population genetic diversity Click here for additional data file.Table S1ST) for spotted owl  populations based on data from Barrowclough et al. Pairwise genetic distances (Φ(PDF)Click here for additional data file.


Heliconius butterfly species in the northern portion of Brazil's Atlantic Forest to estimate the potential impact of habitat fragmentation on population connectivity in butterflies with home-range behavior.Brazil's Atlantic Forest is a biodiversity hotspot endangered by severe habitat degradation and fragmentation. Habitat fragmentation is expected to reduce dispersal among habitat patches resulting in increased genetic differentiation among populations. Here we examined genetic diversity and differentiation among populations of two Heliconius erato specimens from eight collecting locations and 146 H. melpomene specimens from seven locations. Population genetic analyses of the data revealed high levels of genetic diversity in H. erato relative to H. melpomene, widespread genetic differentiation among populations of both species, and no evidence for isolation-by-distance.We generated microsatellite, AFLP and mtDNA sequence data for 136 Heliconius butterflies among neighboring habitat patches. The results also lend support to the observation that fine-scale population genetic structure may be common in Heliconius. If such population structure also exists independent of human activity, and has been common over the evolutionary history of Heliconius butterflies, it may have contributed to the evolution of wing pattern diversity in the genus.These results are consistent with the hypothesis that the extensive habitat fragmentation along Brazil's Atlantic Forest has reduced dispersal of  Landscape structure has a fundamental influence on the distribution of populations, affecting their demography and genetics . Some poGene flow determines the potential for genetic differentiation among populations and for local adaptation and the spread of novel adaptations ,4. In buExtensive studies using molecular markers on butterflies have shown how fragmentation leads to the reduction of gene flow among populations in different habitat patches and increases genetic differentiation among populations ,13-15. IHowever, even though information on the relationship between fragmentation and genetic diversity in butterflies is available, many of these studies have been conducted with temperate species ,14,17,18Heliconius butterflies are a well-studied group of tropical organisms [Heliconius have shown that populations remain fairly stable over time, usually at low densities [Heliconius adults establish themselves in areas where they remain for the rest of their lives [Heliconius are organized in moderately sedentary units, with little movement of individuals, apparently as a result of home range behavior [rganisms -30, commrganisms . Mark-reensities . After air lives . Thus, pbehavior . These bHeliconius populations and the effects of fragmentation on population connectivity. Most studies have employed mark-recapture methods to infer population structure [Heliconius charithonia using allozymes and found low genetic diversity and no evidence of genetic subdivision in south Florida. Additional studies with allozymes have found little evidence for genetic differentiation in other Heliconius species [Heliconius species in Costa Rica using AFLP markers.Yet, little is known about geographical structure of tructure -39, but tructure  analyzed species -42. In c species  found evHeliconius also has important implications for the evolutionary dynamics of mimicry. Heliconius erato and H. melpomene are distantly-related species belonging to different clades within the genus [H. erato and H. melpomene were thought to have occurred in parallel across time and space, possibly facilitated by Pleistocene rainforest refugia [H. erato radiating first and establishing the diversity of wing patterns which H. melpomene later evolved to mimic. A remaining issue then is how H. erato originally diversified, given the strong stabilizing selection expected on warning coloration. One hypothesis is that stochastic events in local populations have occasionally allowed novel phenotypes to drift over the frequency threshold necessary to become a learned, and hence protected, warning pattern [Heliconius; allozymes have generally shown little structure while AFLPs have revealed more.Population genetic structure in he genus ,44. Thes refugia . However refugia -47, supp refugia , with H. pattern ,50 and t pattern -53. ThisHeliconius was hence motivated by two factors. First, we were interested in determining the potential impact of the highly fragmented habitat along Brazil's Atlantic Forest on population connectivity in butterfly species with home range behavior. Second, we were interested in whether replicate analyses of population genetic structure in different geographic regions, and with a diversity of molecular markers, provide evidence for or against substantial population genetic structure in Heliconius. The generality of this phenomenon has important implications for the potential role of drift in color pattern diversification, as long as it has also occurred throughout the evolutionary history of Heliconius butterflies and independent of human activity. To address these questions, we analyzed the population genetic structure of co-mimics H. erato and H. melpomene throughout a portion of Brazil's Atlantic Forest using three types of molecular markers; microsatellites, AFLPs, and mitochondrial DNA sequences. Our results reveal substantial genetic differentiation among populations and intriguing differences between species and among molecular markers, reflecting the unique population biology of our study system and its geographic context.Our current analysis of population genetic structure in Heliconius butterfly species, H. erato and H. melpomene. Our analysis included multiple populations of both species and three distinct classes of molecular markers, allowing us to compare and contrast patterns across species, populations, and types of molecular data.Brazil's Atlantic Forest is a biodiversity hotspot endangered by extreme deforestation. To examine the potential impact of this habitat fragmentation on butterfly dispersal, we measured genetic diversity and differentiation among remnant forest patches for two co-mimetic H. erato displayed greater genetic diversity than H. melpomene  described by Flanagan et al. , four , observed heterozygosity (HO), and deviations from Hardy-Weinberg equilibrium using Arlequin 3.0 [ST) and to perform Mantel tests, comparing population pairwise FST values to straight line geographic distances, to test isolation-by-distance (IBD).For each locus we calculated standard population genetic statistics such as allele frequencies, expected heterozygosity  and separated fragments with an ABI3730 Genetic Analyzer. Three selective primer combinations were used to generate fragments: EcoRI-ACT/MseI-CAT, EcoRI-ACT/MseI-CTG and EcoRI-ACA/MseI-CTG. We separated fragments with an ABI3730 Genetic Analyzer and scored fragments between 50 and 500 bp using ABI GeneMapper software v. 3.7. For each species, we calculated AFLP gene diversity using the Bayesian method of Zhivotovsky . We calcet al. [ST values as well as perform Mantel tests. DNA sequences were submitted to GenBank under accession numbers GU330064 - GU330070, GU330108 - GU330114, and HQ701917 - HQ702184.We PCR amplified and sequenced a 1600 bp region of mitochondrial DNA from each individual using the primers and methods described by Béltran et al. . This reST: Wright's fixation index; msat: microsatellite; mtDNA: mitochondrial DNA; PCR: polymerase chain reaction; SNP: single nucleotide polymorphism.AFLP: amplified fragment length polymorphism; AMOVA: analysis of molecular variance; FPAM, MZC and MRK planned the project; PAM and MZC collected specimens; PAM and SPQ generated molecular data; PAM and MRK analyzed the data; PAM, MZC and MRK wrote the paper; all authors read and approved the final manuscript.We thank Katia Scortecci (Genome Lab-UFRN) for support during the early stages of this project, Luis Vicente Burle Maciel for assistance with the map, and Durrell Kapan and reviewers for comments on the manuscript. Butterflies were collected under MMA (Brazil) permit #10894-1 to MZC. This project was funded by grants from CAPES and the Graduate Program in Ecology at UFRN to PAM, FAPERN/CNPq (PPP/2007) to MZC, and NIH NIGMS grant GM068763 to MRK.


Pomacea canaliculata, a pest registered in the list of “100 of the world’s worst invasive alien species”. The fast isolation by AFLP (Amplified Fragment Length Polymorphism) of sequences containing repeats (FIASCO) method was used to isolate microsatellite loci, and polymorphism was explored with 29 individuals collected in an invasive region from China. These primers showed a number of alleles per locus ranging from three to 13. The ranges of observed and expected heterozygosity were 0.310–0.966 and 0.523–0.898, respectively. These microsatellite markers described here will be useful for population genetic studies of P. canaliculata.We report the characterization of 16 polymorphic microsatellite markers in the golden apple snail,  Pomacea canaliculata (Lamarck 1822) , commonly known as the golden apple snail, is native to freshwater wetlands of South America [P. canaliculata is gonochorism. Females are homogametic (XX), while males are heterogametic (XY) [P. canaliculata is the only aquatic snail listed as one of the 100 of the world’s worst invasive alien species by the IUCN Species Survival Commission Invasive Species Specialist Group [The invasive species  Lamarck 822 (Caentic (XY) . The spetic (XY) , becomin Lamarck 822 (Caentic (XY) . P. canast Group . Since ist Group . It has P. canaliculata [P. canaliculata in China have focused on its distributions, local pernicious effects and issues related to chemical control. In the case of invasive species, the study of their genetic diversity in the invaded area in comparison to the native area could help to infer important aspects of the invasion process, like the route(s) of invasion, the time of invasion, etc. Microsatellite markers are useful tools to investigate the genetic diversity in wild populations because they are highly polymorphic and codominant. However, reports on the development, characterization and use of microsatellite loci in P. canaliculata are still scarce. The present study provides 16 polymorphic microsatellites isolated from P. canaliculata, which will allow investigating genetic diversity and population genetic structure of P. canaliculata in its native and invasive range, as well as tracing its invasion history.However, only a few genetic studies have been conducted to decipher the evolutionary processes associated with the invasion of liculata ,7. Most A total of 80 recombinant clones were sequenced. From the 30 successfully amplified primer pairs, 16 loci showed polymorphism in the 29 individuals. The number of alleles per locus ranged from three to 13, whereas the observed and expected heterozygosities ranged from 0.310 to 0.966 and from 0.523 to 0.898, respectively . Nine lose I and ligated to double-stranded MseI linker (MseI F: 5′-TACTCAGGACTCAT-3′ and MseI R: 5′-GACGATGAGTCCTGAG-3′) [seI-N primer (5′-GATGAGTCCTGAGTAAN-3′) [15 probe for 1 h at 65 °C. Hybridized probe-DNA was captured using streptavidin-coated magnetic beads . Nonspecific binding and unbound DNA were removed by several nonstringent and stringent washes. These microsatellite-enriched DNA fragments were PCR-amplified again and then ligated into pGEM-T Easy vectors (Promega) and transformed into JM109 competent cells (Takara). Recombinant clones were identified using blue/white screening on Luria–Bertani agar plates containing ampicillin, X-gal and IPTG. Insert positive bacterial clones were amplified using one primer for the vector and a second repeat-containing oligonucleotide. Positive clones were sequenced using an ABI PRISM 3730 automated sequencer (Applied Biosystems). Primer sets were designed for microsatellite sequences using PRIMER 3 [Genomic DNA was extracted from foot tissue of one individual collected from Zhaoqing City of Guangdong Province in China using the DNeasy Tissue Kit (QIAGEN). About 250 ng DNA was digested using MTGAG-3′)  for 3 h TAAN-3′) . After dPRIMER 3 .P. canaliculata, sampled from two sites of Guangdong Province (Guangzhou City and Zhaoqing City), in the southern part of China. PCR amplifications were conducted using the following conditions: an initial denaturation step at 95 °C for 5 min, followed by 30 cycles of 30 s at 95 °C, 30 s at the locus specific optimal annealing temperature (see Levels of locus polymorphism were assessed in 29 assumed unrelated individuals of ture see , and 30 Levels of expected and observed heterozygosities were calculated using CERVUS 3.0 software . Tests fP. canaliculata, which should help improve management strategies for this species.The microsatellites described here will be useful for investigating the genetic diversity, in particular the genetic structure within populations of


Hordeum vulgare ssp. spontaneum) in geographic and environmental space in the context of current, past and potential future climates is important for conservation and for breeding the domesticated crop (Hordeum vulgare ssp. vulgare). Spatial genetic diversity in wild barley was revealed by both nuclear-  and chloroplast-derived (5 cpSSR) markers in 256 widely-sampled geo-referenced accessions. Results were compared with MaxEnt-modelled geographic distributions under current, past  and mid-term future  climates. Comparisons suggest large-scale post-LGM range expansion in Central Asia and relatively small, but statistically significant, reductions in range-wide genetic diversity under future climate. Our analyses support the utility of ecological niche modelling for locating genetic diversity hotspots and determine priority geographic areas for wild barley conservation under anthropogenic climate change. Similar research on other cereal crop progenitors could play an important role in tailoring conservation and crop improvement strategies to support future human food security.Describing genetic diversity in wild barley ( Ecological niche modelling of the distributions of crop wild relatives in present, past and future climates can provide important insights into the past evolution and future trajectories of crop progenitors and domesticates Hordeum vulgare ssp. spontaneum), the progenitor of the agriculturally important domesticated H. vulgare ssp. vulgareWild barley  is the most comprehensive geo-referenced collection of de novo at 24 of the barley nSSR loci described by Ramsay et al. de novo at five of the cpSSR loci designed for Hordeum by Provan et al. Three molecular marker data sets were analysed in the current study. First, SNP data derived from two Illumina barley oligonucleotide pool assay platforms were used . After trial runs, K was set at five because log Pr(X/K) values in STRUCTURE had started to plateau at this point K.) The ‘no admixture’ model in STRUCTURE was used to assign single states to individuals. Other analysis options were kept at default settings. STRUCTURE was run five times for each data set and the most common group assignments used as the basis for the interpretation of results (most accessions placed in the same groups in separate runs).STRUCTURE analysis was not designed for predominantly inbreeding species such as barley, but it has been widely applied to cultivated and wild barley populations to reveal interesting genetic features (see discussion in www.diva-gis.org) and ArcGIS 10 www.esri.com/software/arcgis/). Two approaches were used, the first based on allelic (or haplotype) richness and the second based on K groupings. In the first, allelic (BOPA SNP and nSSR) and haplotype (cpSSR) richness estimates were calculated for groups of accessions. Groups were circumscribed using a circular neighbourhood diameter of four degrees and a grid size of 30 minutes (method described in K groupings (K = 5) revealed by STRUCTURE for BOPA SNPs and nSSRs were used instead of allelic/haplotype richness estimates, in order to reveal genetic differentiation at a local geographic scale. Apart from this, the method of analysis was the same as applied in the first approach.To overlay genetic diversity onto geographic maps we employed DIVA-GIS 7.3 Circular neighbourhood analysis with rarefaction, as conducted in both the above approaches, has the advantage of allowing unbiased comparisons of genetic diversity across geographic space. However, it necessarily excludes accessions from analysis where sampling intensity is low. In the current study, a total of 38 accessions were thereby excluded .www.worldclim.org/) , modelling was based on CCSM and MIROC models . The decrease in similarity observed with geographic distance is, however, not a simple trend for any of our three data sets. Differences in profiles are also observed between marker types. For both BOPA SNPs and nSSRs, an increase in similarity at a distance class of around 1,000 km is observed, after which similarity declines again. For cpSSR haplotypes, an obvious increase in similarity is also observed at a distance class of around 500 km. Spatial autocorrelation analysis therefore indicates that a simple isolation-by-distance model does not fully explain genetic structure across the geographic range of wild barley tested  in the Eastern Mediterranean than in Central Asia  comparing wild barley from the Eastern Mediterranean region and environs with Central Asia. For example, Volis et al. Hordeum. The results of our ecological niche modelling are presented in Our intention in this study is to compare patterns of genetic variation in wild barley with the modelled distributions of the taxon under all three conditions of current, past and future climates, something which to our knowledge has not been undertaken before for any member of the genus Considering first the modelled present-day geographic distribution of wild barley, visual assessment confirms that the accessions included in our study for genetic analysis provide good coverage of most of the taxon's range in the Fertile Crescent and Central Asia compare . The mosThe apparent relatively recent range expansion of wild barley in Central Asia as revealed by ecological niche modelling is consistent with our findings of lower levels of genetic diversity in the region compared to the Eastern Mediterranean, in which latter region it can be postulated that a continuous presence of the taxon has led to the accumulation of genetic diversity  for each chromosome, and then averaged across chromosomes, in EXCEL. Results were then expressed in graphical form comparing LD estimates across sub-samples   .Our comparison of LD estimates indicates that at shorter chromosome distance intervals values of LD are relatively higher in Central Asia than in the Eastern Mediterranean, but that with increased distance along chromosomes values become more equal. Linkage disequilibrium values are difficult to interpret because of the many influencing factors, including population structure, selection pressures, mating patterns and changes in population size and range in situ conservation measures A comparison of present-day plant distributions with predictions for the 2080s is useful for devising responses to anthropogenic climate change N = 155, i.e., excluding accessions in ‘lost’ habitat, as indicated in N = 243). We chose nSSRs as the estimator for this analysis because of their high allelic variability and hence sensitivity in describing diversity differences. Our analysis indicated a relatively small reduction in allelic richness under climate change . Further assessment, based on BOPA SNPs and cpSSRs, revealed a relatively modest 108 SNP alleles (4.6% of SNPs in the comparison) and four chloroplast haplotypes unique to 2080s ‘lost’ habitat. Our analysis therefore suggests that, overall, mid-term future losses in genetic diversity due to climate change are expected to be relatively low. A comparison of modelled current, past and future distributions  populations included in the same study and sampled at the same dates. This suggests different responses to climate change by different cereals in the Eastern Mediterranean region. Third and finally, our current assessment was based on genetic markers that are presumably (mostly) neutral with regard to phenotype, so we are not able to determine whether or not there will be important losses in functional genetic diversity under anthropogenic climate change.It is important to consider a number of provisos when interpreting our findings. First, we have not considered in our analysis that existing wild barley populations in habitat that will be lost under climate change could migrate to (newly) environmentally-matched sites. Such migration will presumably be easier in areas with greater micro-geographic environmental heterogeneity, as the distances to be moved are then smaller. This would suggest migration is more feasible in the Eastern Mediterranean region see . WhetherOur analyses are consistent with the view that climate change has played a role in determining the levels of present-day genetic variation observed in wild barley in different portions of its natural range. Our data support the utility of ecological niche modelling of current and past plant distributions for predicting geographic areas of high genetic diversity, and suggest limited future losses of genetic diversity in wild barley under mid-term future climate change. We have explored the use of chromosome-position-mapped SNPs for discriminating between different hypotheses to explain diversity patterns in wild barley, but more research is required on this topic, ideally using SNPs that have been physically positioned in the genome. With the promise soon of a complete genome sequence for barley Click here for additional data file.


R. brelichi exhibits the lowest mitochondrial diversity of all so far studied Rhinopithecus species. In contrast, the genetic diversity of nuclear DNA is high and comparable to other Rhinopithecus species, i.e. the examined microsatellite loci are similarly highly polymorphic as in other species of the genus. An explanation for these differences in mitochondrial and nuclear genetic diversity could be a male biased dispersal. Females most likely stay within their natal band and males migrate between bands, thus mitochondrial DNA will not be exchanged between bands but nuclear DNA via males. A Bayesian Skyline Plot based on mitochondrial DNA sequences shows a strong decrease of the female effective population size (Nef) starting about 3,500 to 4,000 years ago, which concurs with the increasing human population in the area and respective expansion of agriculture. Given that we found no indication for a loss of nuclear DNA diversity in R. brelichi it seems that this factor does not represent the most prominent conservation threat for the long-term survival of the species. Conservation efforts should therefore focus more on immediate threats such as development of tourism and habitat destruction.To evaluate the conservation status of a species or population it is necessary to gain insight into its ecological requirements, reproduction, genetic population structure, and overall genetic diversity. In our study we examined the genetic diversity of Rhinopithecus brelichi by analyzing microsatellite data and compared them with already existing data derived from mitochondrial DNA, which revealed that  Trachypithecus delacouri] in Vietnam Canis lupus] in the northern Rocky Mountains, United States Perisoreus infaustus] in Finland Many animal species are threatened by extinction and for a number of species long-term survival is questionable without a proper conservation and population management Rhinopithecus brelichi). This endangered species is endemic to China and it occurs only in a single population of less than 900 individuals in the Fanjingshan National Nature Reserve (FNNR), Guizhou Province of southern China R. roxellana and R. bieti, R. brelichi shows a low diversity of mitochondrial DNA (mtDNA) ef) sometime in the past and does not give feasible information about the total genetic variation of the species. To get a better understanding of the genetic diversity of a population or species, a combined analysis of nuclear DNA (nDNA) and mtDNA diversity is more powerful. Since for R. brelichi only data on mtDNA diversity is available R. brelichi samples at eight microsatellite loci to gain information on nDNA diversity. Since the population size of R. brelichi is relatively small, we expect to find a similar impoverished nuclear genetic diversity as for mtDNA, which then would make a population genetic management plan more important Nearly 50% of the World's primate species are threatened , 3 µl 10x buffer , 4 µl BT buffer , 1 µl of each primer (10 µM), 0.2 µl dNTPs (25 mM), 0.2 µl Taq DNA polymerase  and 1–2 µl (50–100 ng) DNA. The PCR profile was 2 min at 94°C; then 40 cycles of 30 seconds at 94°C, 30 seconds at varying annealing temperatures (® v3.7 software (Applied Biosystems) was applied to determine allele sizes and genotypes. To avoid false inferences, the genotyping was repeated multiple times following the approach proposed by Morin et al. DNA extraction followed methods outlined in Yang et al. eratures , 30 secoK (assumed number of populations) from 1–3. GENPOP v4.0.10 was applied to test for linkage disequilibrium, using default settings  o) and expected (He) heterozygosities, and fixation index (Fis) to measure deviation from Hardy-Weinberg Equilibrium (HWE) based on heterozygosity excess, with significance of deviations from zero assessed through bootstrapping with 1,000 replications across loci to generate 95% confidence intervals First, we tested if there is any population structuring in our data set using a Bayesian approach in STRUCTURE v2.3.4 is value indicates heterozygous excess, contrarily, a significantly positive Fis value indicates heterozygous deficit (homozygote excess) in the population. Hamilton A significant negative FR. brelichi, we used Bottleneck v1.2.02 e > Heq: He based on allele frequencies; Heq: heterozygosity expected at mutation equilibrium, based on the number of alleles) was tested with the Two-Phase Model (TPM) e > Heq) indicates a recent bottleneck event To detect indication for a recent bottleneck or a reduction of population size in R. brelichi Nef, we performed a Bayesian Skyline Plot (BSP) in BEAST v1.7.4 R. brelichi population at FNNR is not sub-structured. Hence, false inferences from the BSP due to population structure are unlikely To infer timing and magnitude of past changes in For the BSP, we applied the HKY model a), effective number of alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He), and the fixation index (Fis) are shown in a per locus ranges from 3 to 13 with a mean of 6.63±3.34. The mean Ne resulted in 3.84±1.43 ranging between 2.23 and 5.77. The mean He across eight polymorphic loci (0.70±0.12) is similar to the mean Ho (0.71±0.12). All values indicate that all eight microsatellite loci are highly polymorphic in the R. brelichi population . Thus, no deviation from HWE is indicated. Similarly, the overall Fis value for the eight microsatellite loci is not significantly different from zero (p = 0.72) also suggesting that the R. brelichi population does not depart from HWE. However, D1S533 (Fis = 0.14) and D17S1290 (Fis = 0.22) show significant positive Fis values (p<0.05) indicating heterozygosity deficits. A test for linkage disequilibrium revealed no significant linkage pattern.Of the 146 collected samples, genotyping showed that they derived from 141 different individuals. We found no evidence for null alleles, large allelic dropout, scoring errors due to stuttering or sub-structuring in our samples. The observed number of alleles  revealed no significant differences between both species . Additionally the allele frequency distribution does not indicate any distortion form a normal L-shape distribution  exhibits a low diversity in mtDNA as compared to its congeners R. roxellana  and R. bieti . In contrast to the low diversity in mtDNA, diversity in nDNA does not differ from other Rhinopithecus species. All eight microsatellite loci were highly polymorphic as suggested by the high allelic richness (6.63±3.34) and heterozygosity . Similar allelic richness (7.5) and heterozygosity  were found in R. bietiR. roxellana  As shown by Yang et al. R. brelichi remained, we could not compare the genetic diversity among (sub-)populations to detect a loss of the overall genetic diversity, but we matched our results with respective data from R. bietiR. roxellana is highly polymorphic at microsatellite loci. Thus, with similar heterozygosity values, a similar high level of genetic diversity as in R. roxellana can be found in R. brelichi implying no indication for a reduction in terms of nDNA diversity in this species.Since only one panmixing population of is = 0.14) and D17S1290 (Fis = 0.22). The deviation from HWE might be caused by null alleles or consanguineous mating taking place in this population R. brelichi and reduces their fitness (inbreeding depression) R. brelichi could have a strong natural avoidance for inbreeding, which could lead to a decline of progeny and so to a decrease of population size A heterozygosity deficit was detected at two of the eight loci: D1S533  in R. brelichi over at least 25,000 to 35,000 years, a strong decline is indicated 3,500 to 4,000 years ago , which was close to extinction in the 1930s R. brelichi. In historical times, when the species had a much larger distribution with several demes High nDNA diversity combined with low mtDNA diversity is uncommon and not often observed in mammals. One example is the Scandinavian brown bear  and try to remove or limit them. An enlargement of the reserve would be advantageous, but it seems even more important to establish at least one additional, but geographically isolated population Our results suggest that the overall genetic diversity of the endangered R. brelichi is not reduced to a degree that it might become a direct threat for the long-term survival of the species. In contrast, due to the very limited distribution and population size of