Akan and Gaa-Adangbe from Ghana, Yoruba and Igbo from Nigeria) using data from 372 autosomal microsatellite loci typed in 493 unrelated persons (986 chromosomes).Africa contains the most genetically divergent group of continental populations and several studies have reported that African populations show a high degree of population stratification. In this regard, it is important to investigate the potential for population genetic structure or stratification in genetic epidemiology studies involving multiple African populations. The presences of genetic sub-structure, if not properly accounted for, have been reported to lead to spurious association between a putative risk allele and a disease. Within the context of the Africa America Diabetes Mellitus (AADM) Study (a genetic epidemiologic study of type 2 diabetes mellitus in West Africa), we have investigated population structure or stratification in four ethnic groups in two countries  showed that the between-population component of genetic variance is less than 0.1% in contrast to 99.91% for the within population component. Pair-wise genetic distances between the four ethnic groups were also very similar. Nonetheless, the small between-population genetic variance was sufficient to distinguish the two Ghanaian groups from the two Nigerian groups.There was little evidence for significant population substructure in the four major West African ethnic groups represented in the AADM study sample. Ethnicity apparently did not introduce differential allele frequencies that may affect analysis and interpretation of linkage and association studies. These findings, although not entirely surprising given the geographical proximity of these groups, provide important insights into the genetic relationships between the ethnic groups studied and confirm previous results that showed close genetic relationship between most studied West African groups. ST (the classic measure of population subdivision) from mitochondrial DNA are much higher in Africa than other populations, as summarized by Tishkoff et al [ST values for African populations [Africa is inhabited by populations that show high levels of genetic diversity compared to most other continental populations today and it is thought to be the ancestral home of modern humans. African populations have the largest number of population specific autosomal, X-chromosomal and mitochondrial DNA haplotypes with non-African populations having only a subset of the genetic diversity present in Africa . Estimatff et al . In addiulations -4. Recenulations ,6. Howevulations . AvailabSeveral genetic epidemiologic studies mapping complex disease-causing genes have been designed to take advantage of the population genetic characteristics of contemporary African populations for fine mapping of informative genomic regions. These characteristics include lower linkage disequilibrium values -9 and smAkan and Gaa-Adangbe from Ghana, Yoruba and Igbo from Nigeria) using data from 372 autosomal microsatellite loci [see We investigated population structure or stratification in four ethnic groups in two countries in West Africa  and between-ethnic group variation is only 0.09%  shows that most of the variance in the sample is attributable to 9% Table . Locus-bST values were low suggests little evidence for genetic differentiation between the ethnic groups. The fixation index for the entire sample as estimated by FST is 0.00093. Allele-sharing distances are also similar between the groups , they reported that inferred clusters for some of the African populations did not correspond to predefined groups, unlike groups from America, Oceania and Eurasia [Using data from 372 microsatellite loci typed in 493 unrelated persons from four major ethnic groups in Nigeria and Ghana, we sought for evidence of population structure using several methods. Our results did not show any significant population substructure and no ethnic group corresponded to inferred clusters. This finding has been reported by others . Althoug Eurasia .The within-population component of genetic variation accounts for most of the diversity in the sample. This is consistent with previous findings  showing It is important to point out that despite the small amount of genetic differentiation in the sample as a whole, it was possible to distinguish between the groups from each country using a hierarchical AMOVA model and a dendrogram algorithm. Thus, the absence of significant population structure between the four groups did not mean that the groups could not be distinguished from each other. Rather, the data in Table From the disease-mapping point of view, population stratification is important in the analysis of association genetic data, especially when that data is being used to infer the contribution of genetics to a disease. The presence of undetected population structure can mimic association  or mimic lack of association  . While tth percentile upper bound on the level of stratification to within 10% of the true value [Typically, efforts are made to minimize the effect of stratification during study design and data analysis, including a careful selection of cases and controls  and by conducting family-based association tests. However, for the size of study needed to detect typical genetic effects in common diseases, even modest levels of population structure within population groups cannot be safely ignored . Given tue value .In summary, there was little evidence for significant population substructure in the four major West African ethnic groups represented in the AADM study sample. Classification of individuals into clusters showed symmetry, with roughly the same proportion of each ethnic group assigned to each cluster(s). Ethnicity apparently did not introduce differential allele frequencies that may affect analysis and interpretation of linkage and association studies. These findings, although not entirely surprising given the geographical proximity of these groups, provide important insights into the genetic relationships between the ethnic groups studied and confirm previous results that showed close genetic relationship between most studied West African groups.Igbos (28%) and Yorubas (28%); the two centers in Ghana (Accra and Kumasi – see figure Akan (25%) and Gaa-Adangbe (11%). For this analysis, 493 unrelated persons were studied, comprising 147 Akan, 61 Gaa-Adangbe, 129 Yoruba and 156 Igbo participants.The AADM study is an affected sibling pair (ASP) design with enrolment of available spouses as controls. Recruitment strategies and eligibility criteria for the families enrolled in this report have been described in a previous publication . The thrGenotyping was done at the Center for Inherited Disease Research (CIDR). The CIDR marker set is composed primarily of trinucleotide and tetranucleotide repeats and consists of 392 primer pairs with average spacing of 8.9 cM throughout the genome. There are no gaps in the map larger than 18 cM. The average marker heterozygosity is 0.76. Approximately 10% of the marker loci are different between the current CIDR marker set and the Marshfield Genetics screening set version 8. Almost all reverse primer sequences have been modified from the version 8 sequences in order to reduce '+A' artifacts. The resulting PCR products are sized using a capillary sequencing platform. Data for the markers are generated with 218 PCR reactions . Each primer pair has undergone extensive optimization to improve performance and reliability. Error rate was 0.1% per genotype. Inconsistency rate was 0.11%. Extensive quality checks were carried out to verify consistency of marker genotyping as previously described .aadeyemo@howard.edu or crotimi@howard.edu.)For this analysis, all 372 typed autosomal microsatellite markers were included. The markers comprised 272 (73%) tetranucleotide, 46 (12%) trinucleotide and 54 (15%) dinucleotide microsatellites. The markers and their characteristics are provided see . The rawstructure program version 2.1 [K populations (where K may be unknown), each of which is characterized by a set of allele frequencies at each locus. Individuals in the sample are assigned probabilistically to populations, or jointly to two or more populations if their genotypes indicate they are admixed. It is assumed that within populations, the loci are at HWE and linkage equilibrium. This method has the advantage that it does not assume any particular mutation model and it can be applied to microsatellite, SNP and RFLP data. The data was analyzed under an admixture model, assuming correlated allele frequencies between populations as previously described [6 estimation iterations (admixture model) or 2 × 106 estimation iterations (non-admixture model). Each run was carried out several times to ensure consistency of the results. Posterior probabilities for each K were computed for each set of runs.We used a model-based clustering method for inferring population using genotype data consisting of unlinked markers as implemented in the sion 2.1 . The modescribed ; these aescribed . The anaArlequin 2000 [within populations and variation between populations. In addition, AMOVA can be used for a hierarchical analysis of three genetic-variance components – those due to genetic differences (i) between individuals within groups, (ii) between populations within groups, and (iii) between groups. We conducted AMOVA analyses on the study sample using two models (a) a model in that partitioned the genetic variance into that within each ethnic group and that between ethnic groups, (b) a hierarchical model with the country as the first level and the ethnic group within each country as the second level. Additional locus-by-locus AMOVA analysis was done  to 1 (complete isolation or extreme division), and FST values of up to 0.05 represents negligible genetic differentiation. Allele-sharing genetic distances [Analysis of molecular variance (AMOVA) was done using data from all 372 loci as implemented in uin 2000 . AMOVA edone see . Signififficient , was alsistances  were alsAA and CR conceived and designed the study; AA did the statistical genetic analyses; AA and CR drafted the manuscript. GC and YC contributed to the interpretation of the results and development of the manuscript.The 372 microsatellite markers on the 22 autosomes studiedClick here for fileSingle locus AMOVA for all 372 loci studiedClick here for file


Morus boninensis, is an endemic plant of the Bonin (Ogasawara) Islands of Japan and is categorized as "critically endangered" in the Japanese red data book. However, little information is available about its ecological, evolutionary and genetic status, despite the urgent need for guidelines for the conservation of the species. Therefore, we adopted Moritz's MU concept, based on the species' current genetic structure, to define management units and to select mother tree candidates for seed orchards.FST and RST analysis. Although multiple clusters were detected in the putative man-made populations, the pattern of genetic diversity was monotonous in comparison to the natural populations.Nearly all individuals of the species were genotyped on the basis of seven microsatellite markers. Genetic diversity levels in putative natural populations were higher than in putative man-made populations with the exception of those on Otouto-jima Island. This is because a limited number of maternal trees are likely to have been used for seed collection to establish the man-made populations. A model-based clustering analysis clearly distinguished individuals into nine clusters, with a large difference in genetic composition between the population on Otouto-jima Island, the putative natural populations and the putative man-made populations. The Otouto-jima population appeared to be genetically differentiated from the others; a finding that was also supported by pairwise The genotyping by microsatellite markers revealed strong genetic structures. Typically, artificial propagation of this species has ignored the genetic structure, relying only on seeds from Otouto-jima for replanting on other islands, because of a problem with inter-specific hybridization on Chichi-jima and Haha-jima Islands. However, this study demonstrates that we should be taking into consideration the genetic structure of the species when designing a propagation program for the conservation of this species. Morus boninensis, a plant native to the Bonin Islands , is only endemic to Otouto-jima, Chichi-jima and Haha-jima Islands; it is categorized as "critically endangered" in the Japanese Red Data Book [Morus boninensis used to be one of the main species constituting the canopy in the moist tall forest on the Bonin Islands, some invasive trees, mainly Bischofia javanica, have replaced it in recent years [M. acidosa, has been observed and has been confirmed by molecular marker analysis [ex situ conservation and promoting the propagation of individuals for the next generation are put in place. One proposal by Moritz [ata Book ). Althount years -5. In ouanalysis . To promanalysis ). To achanalysis . Therefoy Moritz  was thaty Moritz  identifiy Moritz -11. BecaMos0008 and Mos0050 loci, respectively. Although alleles with the highest frequency were common between the operational populations at three loci, the other four loci did not share the highest frequency alleles between the operational populations , observed and expected heterozygosity (Ho and He), are responsible for the highest values in the KWK and SKM populations . Based on their geographic distribution, these individuals were assigned to one of the six operational populations . All individuals growing in the OTJ operational population were assigned to a single cluster  remembered that the species had been grown in plantations on this Island. He recollected that part of KWK and LPS were planted by humans. Although the origin of the IGM population was not clear, the small, relatively uniform trees in IGM are distributed in quite a small area. This might indicate that, whilst these trees were not planted, they are the result of simultaneous regeneration from a few maternal trees, such as those of an original plantation. The amounts of within population genetic diversity of plantation trees (LPS) and IGM were lower than those of SKM and KWK. According to the model-based clustering, we identified a similar pattern of genetic composition between the planted populations (part of KWK and LPS) and IGM – the clusters represented by mostly green and pink colours Fig. . This miM. boninensis is known to be an extremely long-lived woody species [The individuals in the SKM population exhibited many components of different clusters Fig. . As a re species . AlthougWhen we estimated the pairwise kinships between individuals within a cluster and compared their average between clusters, the difference in average pairwise kinships between cluster 6 and all individuals was significant Fig. . This meEx situ conservation would seem to be necessary to conserve M. boninensis, because it is difficult to obtain seeds free of hybridization with M. accidosa. The exception is the OTJ population [Bischofia javanica [M. boninensis seedlings, controlled crossing and ex situ conservation are all necessary to ensure propagation of the species. To achieve this, we must consider Moritz's management units in order to select appropriate mother trees and we must undertake controlled crossing to obtain seed sources for propagation. In terms of genetic diversity, there was a large difference between the putative natural populations, with the exception of OTJ and IGM  and putative plantation trees. According to the model-based clustering analysis, planted individuals are classified into two main clusters, green and pink. This genetic differentiation, which is supported by the model-based clustering, should be used in selecting the MUs. We were able to identify at least seven main MUs for M. boninensis, which were OTJ, natural trees in CCJ, natural trees in KWK, SKM, man-made trees in CCJ and KWK and two major clusters including individuals in IGM and LPS (Green and Pink). Natural populations retained many elements of the different clusters. On the other hand, most of the plantation trees were simply classified into a single cluster. Hence, natural individuals were prime candidates for use as maternal trees in controlled crossing and ex situ conservation by grafting. According to analyses of pairwise FST and RST and the model-based clustering, we should avoid controlled crossing and instead should establish new seed orchards by open pollination of individuals from different clusters. To date, the OTJ population has been the only one to produce pure M. boninensis seeds. These seeds, therefore, have been the only ones used for propagation. There is a hybridization problem with the introduced species in the KWK and SKM populations. Because the MU of OTJ is definitely different from the other natural populations, we need to independently conduct controlled crosses or ex situ conservation (and subsequently develop seed orchards) within each cluster. Only then will propagated seedlings be available to use for propagation of the species in the forest according to the MUs identified in this study.pulation . In addijavanica . TherefoGenotyping using microsatellite markers revealed that the pattern of genetic variation was different between the OTJ population, the north CCJ putative man-made population, the south CCJ putative natural population, the natural population in Haha-jima (most of SKM and a part of KWK), IGM and the man-made population in Haha-jima (a part of KWK and LPS). These differences should guide the selection of the MUs. The putative natural populations exhibited higher genetic diversity than those of the man-made populations, IGM and OTJ. These are, therefore, important genetic resources for the propagation of a new generation. To date, propagation of the species has ignored its genetic structure by relying only on seeds from the OTJ population, even for planting on the other islands. This has been because of the inter-specific hybridization problem on Chichi-jima and Haha-jima Islands. However, this study has demonstrated the high level of genetic differentiation within the species. This needs to be considered with respect to any propagation program aimed at conserving the evolutionary range of the species.M. boninensis. Summary of field survey concerning with background of each population, location, tree size and vegetation was shown in Table M. boninensis or hybrids between M. boninensis and M. acidosa was conducted, following sampling, using the three SCAR markers [Between 1998 and 2002, we conducted a field survey to find the unknown remnant trees of M. boninensis is a putative autotetraploid species, we determined the allele copy number of partial heterozygotes based on dosage of electrophoretogram peaks.DNA was extracted from 100 mg of the petioles of each of the sampled remnant trees using a DNeasy plant-mini Kit  and following the manufacturer's instructions. The genomic DNA concentration was adjusted to 5 ng/μL by dilution after measuring the concentration of extracted DNA using a spectrophotometer . For all trees, the genotypes of seven microsatellite markers were assigned according to the procedure described by Tani et al. . BecauseA); the average number of alleles per individual (Ai); the average number of four allele genotypes (G); and observed heterozygosity (Ho). These were obtained for each locus using the program AUTOTET [He) and fixation indices (Fis) were calculated under two assumptions: 1) random mating and random chromosome segregation (RceS); and 2) random mating and some level of chromatid segregation (RcdS), for which we selected the maximum theoretical double reduction rate, α = 1/7 [F-statistics and R-statistics, based on the infinite allele model and the stepwise mutation model, respectively, were estimated in order to evaluate genetic diversity between the operational populations [FST and RST, with the aid of two software packages, Mega ver. 2.1 and SPAGeDi [We based the composition of the six operational populations on geographical distribution and individual tree aggregations. For these we calculated population genetic statistics as follows: the number of alleles  of clusters into which the sample data (X) were fitted with posterior probability Pr(X|K), using a model with admixture and uncorrelated allele frequency [6 iterations following a burn-in period of at least 30,000 iterations. The genotypes of the Mos0157-2 locus were omitted from the data set because Mos0157-2 was constructed from same sequence as Mos0157-1, and hence these loci were closely linked to each other. K provides only a rough guide for determining which models are consistent with the data. Therefore, we examined various K values, from 1 to 20, within the simulation, and searched for a suitable K value to maximise the posterior probability Pr(X|K). The Structure software estimates the proportion of ancestry  from each of the K clusters for each individual and the 90% probability intervals. Assignment of individuals into inferred populations was conducted using the Q values and their probability intervals. Only individuals supported by 90% probability intervals were assigned to each cluster. A kinship coefficient between individuals within a cluster was estimated using SPAGeDi software [To identify any unknown population structure, we used a model-based clustering method implemented by the program Structure ver. 2.0. This estimated the number (requency . We condsoftware ,20. The NT performed the sampling, molecular analyses, data interpretation, wrote the manuscript and was responsible for this study of M. boninensis. HY performed the sampling, constructed the database for the remnant trees and interpreted the data. TK performed the sampling and data interpretation. YH, FN and TY performed the survey of the remnant trees and the sampling. All authors read and approved the final manuscript.


Plasmodium falciparum is a key issue for better underÂ¬standing of mortality, morbidity and effective control of malaria. However, little is known about the genetic makeup of P. falciparum populations.Genetic diversity of P. falciparum is a parasite of man, its demographic history is expected to be intimately associated with that of modern humans. To assess the relative importance of human demographic history, we characterised spatial patterns of genetic diversity of P. falciparum populations by analyzing a worldwide sample of >500 isolates sequenced for two housekeeping genes (serca and adsl: 63 SNPs from 5.0 kb per isolate). We observed a strong negative correlation between within-population genetic diversity and geographic distance from Africa over Africa, Asia and Oceania, presenting evidence for isolation by distance pattern in P. falciparum. This negative correlation was also noted for two surface antigen genes (msp1 and csp: 165 SNPs from 5.5 kb per isolate).It is generally assumed that the parasite population structure has been shaped by a variety of factors including transmission intensity, past intervention efforts and evolutionary history. Since P. falciparum populations. The striking geographic patterns of isolation by distance mirror the ones previously documented in modern humans. Age estimates of P. falciparum populations, based on genetic distance between P. falciparum and P. reichenowi, a chimpanzee malaria parasite, strongly suggest that modern humans were infected prior to their exit out-of-Africa and carried the parasite along during their colonization of the world.In contrast, regional variation in transmission intensity and recent control initiatives seem to have had a negligible impact on the distribution of genetic diversity, indicating that geography is the primary predictor of the genetic diversity of


The domestic goat is one of the important livestock species of India. In the present study we assess genetic diversity of Indian goats using 17 microsatellite markers. Breeds were sampled from their natural habitat, covering different agroclimatic zones.A measure of genetic distance between pairs of breeds indicated that the lowest distance was between Marwari and Sirohi (0.135). The highest distance was between Pashmina and Black Bengal. An analysis of molecular variance indicated that 6.59% of variance exists among the Indian goat breeds. Both a phylogenetic tree and Principal Component Analysis showed the distribution of breeds in two major clusters with respect to their geographic distribution.The mean number of alleles per locus (NA) ranged from 8.1 in Barbari to 9.7 in Jakhrana goats. The mean expected heterozygosity (He) ranged from 0.739 in Barbari to 0.783 in Jakhrana goats. Deviations from Hardy-Weinberg Equilibrium (HWE) were statistically significant (P < 0.05) for 5 loci breed combinations. The DOur study concludes that Indian goat populations can be classified into distinct genetic groups or breeds based on the microsatellites as well as mtDNA information. Goats, like other livestock species, are recognised as important components of world diversity and may play a useful role in sustainable agriculture in future. The goats in India are distributed in all climatic regions and evolved in isolation for a long period due to varying selective pressures and genetic drift. Goat breeds are mainly defined by their geographical position, morphological characteristics and production performance. Goats in India can also be divided into large, medium and small breeds, which are mainly distributed into four major geographical regions: temperate Himalayan region, northwestern region, southern peninsular region and eastern region . Goat brAn assessment of genetic variability in domestic goats is a first step towards conservation of genetic resources for maintaining breeding options. In the changing phase of agricultural practices, a few breeds have been used on a large scale for immediate economic gain. Therefore, locally adapted native breeds have been neglected or displaced without knowing their genetic importance. DNA markers have been used to study the genetic variation in livestock, human and other populations -5. GenetOut of 23 STR markers analysed, 4 failed to amplify in any of the samples, 2 showed monomorphic patterns and the remaining 17 were polymorphic. The total number of alleles and allele size range for each locus are presented in Table The most polymorphism was detected at the SRCRSP9 locus (0.85) and the least polymorphism at the ILST S005 locus (0.597). Breed specific alleles were observed at different loci for different breeds with low frequency. Measures of genetic variability are shown in Table The mean number of alleles per locus (NA) varied from 8.1 in Barbari to 9.7 in Jakhrana goats. The mean number of alleles per locus (NA) corrected for sample size  is presented in Table ST values for each pair of populations varied from 0.036 to 0.088. The average GST values over all loci was 0.080, indicating that a 8.0% of total genetic variation corresponded to differences among populations, whereas 92.0% was explained by difference among individuals. The average RST value (based on the stepwise mutation model) over the loci was 0.177. Mean pairwise comparisons between breeds showed that RST values were 2–4 folds higher than GST values. An exact test for population differentiation for all pairs of breeds across all loci showed that all breeds were significantly (P < 0.001) different from each other.FFurther, an AMOVA analysis was carried out to analyze the variation within and between breeds. The AMOVA revealed that percentage of variation among populations was 6.59% and within populations was 93.41%. Variance components among population were highly significant for all the studied loci , indicating the existence of a multivariate compromise structure.PCA was performed for each of the 17 markers. Corresponding scree plots are presented in Figure PCA was performed using the frequencies of the 242 alleles of the 17 markers. The first three principal components explained 65% of the total variation. The global principal component analysis for the first three principal components is presented in Figure The contributions of markers to the construction of axes are plotted in Figure On the other hand, BM4621 and ILST005 participated in the construction of only one or two axes. ILSTS005 participated in the construction of the first axis. The corresponding PCA plot indicates that Pashmina and Barbari breeds were isolated from some other populations, as in the fist axis of the global analysis, but the clusters Jakhrana, Jamunapari, Black Bengal and Sirohi, Marwari were not exhibited by this marker. On the other hand, BM4621, which contributes to the construction of the second and third axes, exhibited these clusters, but did not isolate the Barbari and Pashmina breeds. BM4621 revealed three clusters  in contrast to the four clusters exhibited by the global analysis .Our genetic analysis of seven Indian goat breeds with 17 microsatellite markers showed higher gene diversity as compared to European and Asian goat breeds. Barker, 1994  and TakeST values shows the fraction of total variance of allele size between populations, the estimated RST was more than twice the size of GST and FST, suggesting that goat breeds differ in both allele frequency and allele size. In addition, AMOVA indicated that 6.59% of the total genetic variation is between breeds of goats, confirming higher within population diversity in the Indian subcontinent. Mitochondrial DNA analysis in Indian goats showed 83% of variation within breeds and 17% among breeds [As no systematic effort has been made to create distinct goat breeds in India, founder effects and genetic drift may have played major role in differentiation of Indian goat breeds. Population subdivision in Indian goats is also supported by the average proportion of genetic differentiation among breeds 8.03%). As Rg breeds . The bet.03%. As g breeds .A and DC are the most efficient means of obtaining a correct tree topology on the basis of microsatellite analysis when within population variation is high and distances between each pair of populations are used to build a NJ tree.Takezaki and Nei, 1996  have shoThe genetic data revealed that the smallest distance is between Marwari and Sirohi and the largest distances are between Pashmina and Black Bengal, Barbari and Black Bengal, and Barbari and Jakhrana. The highest geographical distance between Black Bengal and Pashmina corresponds to the highest genetic distance. The phylogenetic analysis indicated that breeds were grouped according to their geographic locations except Barbari goats. A similar observation of population clustering according to their geographic origin has been reported in cattle . This shBreeds cluster according to their geographic location. Similar population clustering according to geographic location was previously observed in microsatellite analyses of humans , cattle In conclusion, this analysis showed that microsatellites as well as mtDNA analysis can be used to classify Indian goat populations into distinct genetic groups or breeds. Phylogenetic and principal component analysis showed the clustering of goats according to their geographical origin. Although the breeding tracts of goats are overlapping and they are spread over all the parts of the country, they still maintain genetic distinctness in their natural habitat.A total of 302 goats representing 7 major breeds of India were sampled from their natural habitat. The breeds studied have been grouped into 5 different types based on their utility and size Table . SummariAbout 10 ml of blood samples were collected from each animal's jugular vein using EDTA vacutainers and stored at 4°C until DNA isolation. An effort was made to collect samples from unrelated individuals based on information provided by farmers. The geographical distribution of the breeds from different regions sampled for the study is described in Table 2, 1 U of AmpliTaq Gold (Perkin Elmer) and 10 pM of each primer. Amplification conditions for these markers were as follows: Initial denaturation at 95°C for 10 min. followed by 95°C for 1 min, specific annealing temperature for each marker as given in Table The markers were chosen from the existing bovine, ovine and caprine genetic maps -22 with One micro liter of PCR amplicons and 0.5 μl of size standard (GS-ROX500) were mixed with 2.5 μl of loading dye , denatured (94°C for 2 mins) and electrophoresed in 5% Long Ranger (FMC) gel, using ABI 377 automated DNA sequencer (Perkin Elmer). GeneScan (Perkin-Elmer) software was used to analyse the gel image and Genotyping software (Perkin-Elmer) was used to get the allele size of each amplicons.Exact tests for deviations from Hardy-Weinberg equilibrium (HWE) were performed by the GENEPOP Package . The proST, was calculated which accounts for variance in allele size and was defined for genetic markers undergoing a stepwise mutation model. The DA genetic distance [Analysis of molecular variance (AMOVA), Fst and pairwise difference were computed using ARLEQUIN ver 3.11 ,27. A sedistance  was compdistance  to estabdistance . The sigAs admixture in the breeding tract of goats is very common and some populations are known to be admixed, we used a multivariate procedure to represent population relationships. Multivariate procedures are recommended in this situation because the admixed populations are not original evolutionary units and may be misrepresented by phylogeny-based tree-building techniques . Prior tPKR, LS and KT conceived and designed the experiments. MBJ and AM performed the experiments. PKR, MBJ, AM and DL performed the data analysis. PKR, MBJ and KT wrote the manuscript. All authors read, revised and approved the final manuscript.


Myodes glareolus.Since Darwin's pioneering work, evolutionary changes in isolated island populations of vertebrates have continued to provide the strongest evidence for the theory of natural selection. Besides macro-evolutionary changes, micro-evolutionary changes and the relative importance of natural selection vs. genetic drift are under intense investigation. Our study focuses on the genetic differentiation in morphological and life-history traits in insular populations of a small mammal the bank vole VA) for that trait. Furthermore, our analyses of differentiation in neutral marker loci (Fst) indicate that VA is less than expected on the basis of genetic drift alone, and thus, a lower VA in insular populations could be caused by natural selection.Our results do not support the earlier findings for larger adult size or lower reproductive effort in insular populations of small mammals. However, the individuals living on islands produced larger offspring than individuals living on the mainland. Genetic differentiation in offspring size was further confirmed by the analyses of quantitative genetics in lab. In insular populations, genetic differentiation in offspring size simultaneously decreases the additive genetic variation (We believe that different selection pressures (e.g. higher intraspecific competition) in an insular environment might favour larger offspring size in small mammals. Island selection for larger offspring could be the preliminary mechanism in a process which could eventually lead to a smaller litter size and lower reproductive effort frequently found in insular vertebrates. Population genetics models  emphasisDifferences between mainland and island populations of mammals have often been referred to as the Island rule or Island Syndrome -11. The Myodes glareolus. Our previous studies have indicated a large additive genetic variation in reproductive traits (e.g size and number of offspring) in the mainland population of our study species [Here we focused on the possible genetic differentiation in morphological and life-history traits between insular and mainland populations of a small mammal, the bank vole  species . Further species . In the  species .We observed neither morphological differentiation in the breeding females, nor a significant difference in their reproductive effort between insular and mainland populations of the bank vole. By contrast, females that originated from the islands produced significantly larger offspring than those from the mainland Fig , Table 1VA ± S.E. = 0.047 ± 0.020 and h2 ± S.E = 0.96 ± 0.41), whereas genetic variance was zero or very low among island fathers (Table P < 0.002)  Table .Fst) [Fst ± S.E. = 0.177 ± 0.021; P < 0.001) but not according to the additive genetic variance (VA ≈ 0)  did not differ according to neutral markers . Fst values differed significantly between mainland and island populations (P = 0.001).The relative importance of natural selection and random drift on offspring size divergence can be tested by comparing among-island differences based on additive genetic variation and measures based on neutral marker genes (Fst) . Here th0) Table . This meThe present results are in agreement with earlier suggestions that offspring size might be the first life-history characteristic to evolve in insular populations of vertebrates ,23. LargThe breeding density of territorial bank vole females seems to be lower in open populations   comparedst) indicate that VA is less than expected on the basis of genetic drift alone [The phenomenon to produce large offspring can also be linked to the genetic dispersal tactics of individuals ,31. The ft alone . Here, wft alone .In contrast to the island populations, additive genetic variation in offspring body mass has been observed in mainland populations  and here-34. In aWe supposed that if density fluctuations and other ecological parameters related to them are more stable in insular environments, selection for large offspring size could also be stable long term. Theoretically, strong selection might decrease additive genetic variation found in our islands ,38,39. MThe bank vole is a common mammal in coniferous forests of northern Europe . The breThe study was carried out in central Finland . The data are based on 898 individuals caught from 37 islands (0.12–70 ha) in lake Konnevesi and from 20 mainland localities within 5 km of the lake during the summer 1999. Pregnant females were caught from 20 islands and all mainland areas, and thus only these populations were included in the present study. The shortest distance between neighbouring study islands varied between 50 to 500 m and the mean distance from islands to the mainland was 631 m (S.E. = 75 m). Dispersal between islands during summer is very low, indicated by a separate study during the autumn where we recaptured 106 island individuals (three to five months from the first capture); none of these individuals left their home islands. The mainland trapping areas were sufficiently far apart  to decrease dispersal between different mainland localities. We were not able to estimate dispersal rate between mainland localities, but according to earlier studies , it shoun = 241) and mainland .All trapped voles were taken into the laboratory where each individual was sexed, weighed to the nearest 0.1 g, and measured for maximum head width to the nearest 0.1 mm with a digital calliper . Males aRE(1) = L × Mo0.75/Mm0.75, where L is litter size; Mo is pup mass at birth, and Mm is weight of the female after delivery [RE(2) = L × Mo/Mm, where litter mass was divided by mother body mass.The reproductive effort of females was estimated using two formulas. First, we used the formula:delivery ,48. In tdelivery . The motdelivery . Since tdelivery ,53), we The genetic basis of offspring characteristics was analyzed in the laboratory. In the analyses, we compared the effect of male origin (island/mainland) on the characteristic of their offspring. Bank vole males do not rear their offspring and male quality does not affect the amount of maternal care , hence wt = 0.255, df = 73, P = 0.800; body mass: t = 1.651, df = 73, P = 0.103). Body mass and head width of the males that originated from islands did not differ from the males that originated from the mainland ;origin fixed and population random factor; head width: F1,15.7 = 1.21, P = 0.287; body mass: F1,10.9 = 1.13, P = 0.311). To obtain an estimate of heritability (h2) and additive genetic variance (VA) for the body mass of offspring in the mainland and island populations, we performed standard half-sib analyses  for the effects of island size and island distance to mainland on the breeding characters of insular females. Size of island and distance to mainland were used as fixed factors (covariates) and population as a random factor in the analyses. ndf = numerator degrees of freedom, ddf = denominator degrees of freedom.Click here for file


SNP data of goats of three Mediterranean countries were used for population studies and reconstruction of geographical patterning. 496 individuals belonging to Italian, Albanian and Greek breeds were genotyped to assess the basic population parameters.A total of 26 SNPs were used, for a total of 12,896 genotypes assayed. Statistical analysis revealed that breeds are not so similar in terms of genetic variability, as reported in studies performed using different markers. The Mantel test showed a strongly significant correlation between genetic and geographic distance. Also, PCA analysis revealed that breeds are grouped according to geographical origin, with the exception of the Greek Skopelos breed.Our data point out that the use of SNP markers to analyze a wider breed sample could help in understanding the recent evolutionary history of domestic goats. We found correlation between genetic diversity and geographic distance. Also PCA analysis shows that the breeds are well differentiated, with good correspondence to geographical locations, thus confirming the correlation between geographical and genetic distances. This suggests that migration history of the species played a pivotal role in the present-day structure of the breeds and a scenario in which coastal routes were easier for migrating in comparison with inland routes. A westward coastal route to Italy through Greece could have led to gene flow along the Northern Mediterranean. Capra hircus) has been an important livestock resource since its domestication [The domestic goat  to 0.197 (Capore) (Table Expected heterozygosity of the loci ranged from 0.016 (FABP4) to 0.495 (GHR), with a mean of 0.300. e) Table  ranged fThe frequencies of the major alleles are reported in table Significant deviations from Hardy-Weinberg equilibrium over all populations  were observed in three loci Table . SignifiBos taurus chromosome 23 and 7, respectively; the two SNPs on PRP are 458 bp apart on B. taurus chromosome 13 . The log-likelihood based test of genotypic differentiation for all populations showed statistically significant values  except for GHR, IL2_1 and FABP4 loci which have very little informativeness.Significant deviations from linkage equilibrium  were observed for two of the analysed locus pairs: HLA-DQA_2 and GDF9 loci, PRP_1 and PRP_2. Loci have not yet been mapped in the goat; HLA-DQA_2 and GDF9 are located on FST of 0.063. That is, 6.3% of allelic variation was accounted across breeds and 93.7% within breeds. Weir and Cockerham's [FST per locus ranged from 0.008 (IL2_1) to 0.144 (IL4), with a mean of 0.059 , Orobica (0.19), Camosciata (0.22), Girgentana (0.18) e Argentata dell'Etna (0.15) populations, were higher than all other pairwise comparisons suggesting the Skopelos as the most diverse population.The analysis revealed an overall kerham's  estimate 0.008 IL_1 to 0.1The Mantel test showed a strongly significant correlation between genetic and geographic distances .Genetic relationships were also explored by means of principal component analysis. The coefficients of the linear combinations reveal which SNPs most affect the component value. As for the first component, SNP IL4 presents extreme positive and SNP LGB extreme negative values, respectively. Likewise, the second component is mostly affected by the SNPs ACVR2B and MTNR1A, with positive sign, and by the SNPs HLA-DQA_2, IL4 and LGB with negative sign. To examine the overall pattern of population differentiation, PCA was conducted with the first two axes, which cumulatively explained 52% of the total inertia contained in the data set . In post-Neolithic times, some colonists may have improved local livestock as well as importing stock from overseas. The transport of animals made by sea has been already proposed for cattle ,17 and gFIS = 0.122, not the highest value in our breeds (max FIS = 0.197 in Capore), but to the lack of admixture with other populations since long time and possibly a natural selection versus local environment. The Skopelos breed is largely differentiated from the other goat populations in Greece, both morphologically and in terms of performance. According to the inhabitants of the Skopelos island, the goat used to live in an uninhabited small island of Northern Sporades, and it was recently domesticated. The breed is also said to have some relationship with the wild goat of the Gioura island, originated from the homonymous island [FIS = 0.030), but to isolation of this breed in a very secluded valley of Italian Alps.The Greek Skopelos breed results the most distant one, reflecting the fact that it has been raised only in a island and on the mainland of Magnisia. The distance is not due to inbreeding as s island . BreederAmong the Italian southern breeds, it is interesting that the lowest distance is seen between Argentata dell'Etna, from Sicily, and Sarda, original from Sardinia. The two islands, although quite far apart, were important trade posts of Phoenician, Punic and Roman traders.The analysis carried so far excluded SNPs that were proven under selection . If we iOur data point out that the use of SNP markers to analyze a wider breed sample, until now scarcely employed for genetic population studies in livestock, could help in understanding the recent evolutionary history of domestic goats.We found correlation between genetic diversity and geographic distance. Also PCA analysis shows that the breeds are well differentiated, with good correspondence to geographical locations, thus confirming the correlation between geographical and genetic distances. This suggests that migration history of the species played a pivotal role in the present-day structure of the breeds. Instead, the limited genetic similarity within main geographical areas suggests that breed differentiation could have occurred in more recent times, after the main migrations.On the basis of the observed gradient of genetic diversity decreasing from south-east to north-west, and of the signals of the northward dispersal of populations from the domestication centre, we hypothesize that coastal routes from the domestication centre to Italy through Greece could be a likely explanation for the observed gene flow along the Northern Mediterranean.Blood samples of a total of 496 goats, about one third male, were collected in farms spread over the traditional rearing area of each breed Figure . No more. Generally, accuracy greater than 99% was achieved. Quality control criteria were adopted . All the SNPs described in [SNPs characterization has been described elsewhere . SNP ascribed in  were genFSTAT 2.93[HO and HE, respectively), Weir and Cockerham's [FIS per population, of FST per locus and population pairs were calculated for each locus using GENEPOP 4.0 [Allele frequencies were calculated using STAT 2.93. Observekerham's  estimateEPOP 4.0 . The samR 2.6.0 open source software (publicly available at ) to estimate the correlation between pairwise FST values and pairwise geographic distance using 1000 replicates to test significance. The matrix of the geographic distance was computed using the coordinates obtained with a global positioning system (GPS).Isolation by distance  has beeR 2.6.0 open source software.A principal component analysis (PCA) was performed on the covariance matrix of SNP frequency data to investigate spatial patterns of genetic variation using the POWERMARKER[Nei  genetic WERMARKER.LP: designed the study, carried out SNP data analysis and drafted the manuscript. AC: completed most of the statistical analysis. CL: provided Greek samples and formulated parts of the paper. PAM: leader of the Econogene project, provided DNA samples and guidance for the project. AV: participated in developing ideas, in supervision and revision of the manuscript. All authors read and approved the final manuscript.Table S1. Frequency of the Major allele per SNP and per population.Click here for file


Rousettus leschenaulti) and the greater short-nosed fruit bat (Cynopterus sphinx) are two abundant and widely co-distributed Old World fruit bats in Southeast and East Asia. The former species forms large colonies in caves while the latter roots in small groups in trees. To test whether these differences in social organization and roosting ecology are associated with contrasting patterns of gene flow, we used mtDNA and nuclear loci to characterize population genetic subdivision and phylogeographic histories in both species sampled from China, Vietnam and India. Our analyses from R. leschenaulti using both types of marker revealed little evidence of genetic structure across the study region. On the other hand, C. sphinx showed significant genetic mtDNA differentiation between the samples from India compared with China and Vietnam, as well as greater structuring of microsatellite genotypes within China. Demographic analyses indicated signatures of past rapid population expansion in both taxa, with more recent demographic growth in C. sphinx. Therefore, the relative genetic homogeneity in R. leschenaulti is unlikely to reflect past events. Instead we suggest that the absence of substructure in R. leschenaulti is a consequence of higher levels of gene flow among colonies, and that greater vagility in this species is an adaptation associated with cave roosting.The fulvous fruit bat ( Comparative studies taxa offer a powerful approach to elucidate how population processes and historical events have acted in shaping organisms' current distribution and genetic structure. Spatially matched taxa are often expected to show similar signatures of structure based on their shared histories Dispersal ability is a key demographic force shaping natural populations Rousettus leschenaulti) and the greater short-nosed fruit bat (Cynopterus sphinx) are co-distributed throughout much of their range in Southern Asia R. leschenaulti predominantly roosting in caves consisting of mixed sex colonies that can consist of up to 10,000 individuals C. sphinx typically roosts in palm trees during the breeding season, and is characterized by a single male with a harem of females The fulvous fruit bat  and microsatellite markers. We predict that R. leschenaulti will have a high dispersal ability that will be characterized by the identification of low population genetic structure across its range, while the opposite will be found for C. sphinx due to its predicted limited dispersal ability.By occupying a similar range but having different roosting preferences, Sampling was approved by the Administrative Panel on Laboratory Animal Care  of the Guangdong Entomological Institute in China, which also incorporates the South China Institute of Endangered Animals.Rousettus leschenaulti was sampled at four localities in China  and one in India (Cheranmahadevi). Cynopterus sphinx was sampled at six localities in China , one in India (Mandiyoor) and one in Vietnam (Pu Huong)  rather than by hand-netting. Tissue samples were taken from the wing-membrane using a 3-mm diameter biopsy punch and stored in 80% ethanol until processing u Huong) . To avoiR. leschenaulti was genotyped at eight microsatellite loci while C. sphinx was genotyped at six . Forward primers were 5′-fluoro-labelled (MedProbe). PCRs were performed using a PTC-200 thermal cycler (MJ Research) with the following profile: denaturation at 95°C for 15 min, 35 amplification cycles  and an extension step at 72°C for 20 min. PCR products were genotyped using an ABI 3100 DNA sequencer (Applied Biosystems) and alleles were sized using the programs GENESCAN version 2.1 and GENOTYPER version 2.5 (Applied Biosystems).DNA was extracted using DNeasy Tissue kits (Qiagen).  six see . To amplb gene (∼1100 bp) using the published primers cy1 (5′-AAA TCA CCG TTG TAC TTC AAC-3′) and cy2 (5′-TAG AAT ATC AGC TTT GGG TG-3′) Five to eight individuals from each population  were amplified at the cytochrome S) per locus per population, which accounts for unequal sample sizes, in FSTAT. To estimate the power of the markers, we calculated the total probability of identity’ (PID) for each locus and for all loci combined in GENALEX We tested for deviations from linkage equilibrium between loci in FSTAT version 2.9.3.2 ST values using the software GENETIX version 4.02 ST values, which accounts for the stepwise mutation model of microsatellite alleles ST values in which allele sizes were permuted (1000 times) among alleles (pRST) ST exceeds pRST is thus a measure of the extent to which phylogenetic distance among ordered alleles explains the observed pattern of genetic differentiation. We then assessed the relative contribution of phylogeography between the two species by using a paired sample t-test on equivalent pairwise values of RST - pRST.To quantify genetic structure, we calculated pairwise FST and RST against log-transformed geographic distances (km) to test for isolation by distance (IBD), using a Mantel test with permutations (1000 times) to assess significance in GENEPOP. An analysis of molecular variance (AMOVA) was used to examine genetic variation within and among the different populations, based on all samples, with and without the Indian colony . We assessed whether the derived indices were significantly different from zero using a permutation procedure  in the software ARLEQUIN version 3.1 We also plotted linearized pairwise values of both FPopulation structure was further investigated using Bayesian clustering analysis. We estimated the likelihood of different numbers of clusters (K) in the data using the method in STRUCTURE version 2.0 b sequences were aligned in BioEdit version 7.0.5 Cytochrome ST values and tested for significance by permutation (10 000). AMOVAs were used to examine genetic variation following the methods described above. Finally, to test whether any detected differences in population structure between the two species could be explained by contrasting demographic histories, we performed sequence mismatch distribution analyses in ARLEQUIN. Mismatch distributions are typically ragged or multimodal for populations at stationary demographic equilibrium, but smooth or unimodal for populations that have undergone a demographic expansion u, where Τ (tau) is a parameter of the time to expansion in units of mutations, and u is the mutation rate per generation for the DNA sequence. We used a mutation rate of 2% per Myr Tests for genetic differentiation among samples (n≥5), calculated using pairwise ΦRousettus leschenaulti and Cynopterus sphinx showed no evidence of linkage disequilibrium between loci, and there was no significant deviation from Hardy-Weinberg equilibrium detected in either species following Bonferroni correction for multiple tests.Samples of both R. leschenaulti, the mean number of alleles per locus ranged from 12.1 (Cheranmahadevi) to 15.5 (Haikou) while the observed heterozygosity ranged from 0.85 (Cheranmahadevi) to 0.95 (Maoming). Allelic richness (RS) ranged from 11.0 (Wuming) to 12.2 (Menglun)  to 12.6 (Xishuangbanna) while the observed heterozygosity (Ho) ranged from 0.73 (Beihai) to 0.84 (Zhongshan). The allelic richness (AR) ranged from 6.5  to 8.9 (Xishuangbanna)  . For Cynngbanna) . Total panalyses .ST for C. sphinx was considerablly greater  than that recorded for R. leschenaulti . Pairwise FST values among C. sphinx samples ranged from 0.0090 (non-significant) for Pu Huong vs. Jiangmen to 0.0801 (P<0.001) for Jiangmen vs. Mandiyoor with 22 out of 28 pairwise values found to be significant   . AlthougST was not significantly different from global pRST (P<0.05) in either species indicating that stepwise mutation had not contributed to observed differentiation and that FST is a more suitable estimator in this study. Hierarchical AMOVAs based on FST revealed that more genetic variance was partitioned among individuals within populations than among populations in C. sphinx (96.62% versus 3.38% respectively) and R. leschenaulti (99.48% versus 0.52% respectively). Removing the populations from India did not significantly alter the result. C. sphinx was also found to exhibit a significant pattern of isolation by distance based on pairwise FST . This analysis was not undertaken for R. leschenaulti due to the smaller number of pairwise comparisons.Global RC. sphinx, K = 4 showed highest probability, however, forcing values of K from 2 to 8 recovered hierarchical population structure. At K = 2, Mandiyoor in India and Xishuangbana in SW China formed a single cluster with all other populations forming a second cluster, though partial admixture was observed. At K = 3, Mandiyoor and Xishuangbana remained as a single cluster, while the five populations from China showed partial membership to the remaining two clusters, one of which being dominated by the colony from Haikou (Hainan Island). Pu Huong appears to share mixed ancestry from all the sampled populations. At K = 4 the Mandiyoor and Xishuangbana populations split with Xishuangbana largely forming a new cluster, although some admixture is seen, while the populations from China and Pu Huong show very little structure and show partial membership to all the clusters apart from the one for Mandiyoor. Clustering analyses revealed no substructure in the R. leschenaulti samples, where the most probable number of clusters was one.In agreement with F-statistics, Bayesian clustering analyses also revealed differences between the two taxa . For C. b in 29 individuals of R. leschenaulti and recorded 27 haplotypes and 52 polymorphic sites (GenBank accessions FJ489931 - FJ489957). For C. sphinx we sequenced 1075 bp of cytochrome b in 59 individuals and found 34 haplotypes with 71 polymorphic sites (GenBank accessions FJ489958 - FJ489992). No insertions or deletions were observed in either species. For each sample, the number of haplotypes, haplotype diversity, the number of polymorphic sites, average number of differences and nucleotide diversity are given in We sequenced 1100 bp of cytochrome R. leschenaulti, haplotypes formed a single network at a 95% parsimony threshold, and were scattered with respect to locality. No evidence of any structure was present. The most common haplotype was geographically widespread, recorded in both Maoming and Cheranmahadevi. In C. sphinx, a parsimony threshold of 95% yielded two subnetworks, one comprising only haplotypes from Mandiyoor and the other comprising the remaining haplotypes. The most common haplotype was found mainly in Guangzhou, Zhongshan and Jiangmen but was also recorded in Pu Huong. Based on its interior position and high level of connectedness, it is likely to be ancestral. The lack of monophyly seen in haplotypes from Haikou, Beihai and Xishuangbanna points to past mixing among these localities.Parsimony-based haplotype networks revealed differences between the two taxa . For R. R. leschenaulti populations. In C. sphinx, significant genetic differentiation was detected  with pairwise values indicating that this is caused by the population sampled at Mandiyoor which was consistently differentiated from all other colonies sampled in China and Vietnam (P<0.05). Apart from the tests including Mandiyoor, no significant differentiation was detected between any other pairwise values.No genetic differentiation was detected based on mtDNA data  for R. leschenaulti showed no significant genetic variance among populations, with or without Cheranmahadevi, with 3.90% of the variance explained by among-population variation, and 96.10% by within-population variation . When all eight populations were included in the analysis (AMOVA I), 55.11% of the total genetic variance was explained by among population differences, and 44.89% by within population differences, however, when Mandiyoor was excluded (AMOVA II), these values were reduced to 3.04% and 96.96%, respectively (AMOVAs carried out for ariation . AMOVAs ectively .R. leschenaulti failed to reject the model of population expansion (PSSD>0.05 and raggedness index PR>0.05) with an estimated timing of expansion occurring around 200 000 years BP (95% CI: 130 000–250 000 years BP). In C. sphinx, separate mismatch distribution analyses undertaken for all localities also failed to reject the expansion model (PSSD>0.05 and raggedness index PR>0.05), with an estimated timing of expansion of around 107 000 years BP (95% CI: 16 000–560 000 years BP).Mismatch distribution analysis for Rousettus leschenaulti and Cynopterus sphinx. We show that in spite of their similar ranges, these two fruit bat species exhibit highly contrasting patterns of genetic structure, and that these differences are more likely to reflect differences in vagility rather than demographic history.In this study we applied mtDNA and microsatellite analyses to characterize the phylogeographic histories of co-distributed populations of R. leschenaulti were characterized by a lack of genetic structure at both classes of molecular marker. Our MSN network showed that haplotypes were not geographically structured while tests of AMOVA indicated that 96% of the genetic variance was due to within-population variation. Microsatellite data also revealed complete mixing of localities based on clustering analyses of genotypes which was further supported by limited evidence of genetic differentiation from FST statistics.Populations of C. sphinx showed a lack of genetic structure among all populations sampled across China and Vietnam but significant genetic differentiation between each of these populations and that from Mandiyoor (India). These results were also reflected in the distribution of haplotypes in the MSN network, in which the Indian haplotypes formed a monophyletic sub-network. Greater substructure was detected from microsatellite data, with significant genetic differentiation seen among most pairs of populations, including 11 pairwise comparisons within China. Bayesian clustering of genotypes also revealed clear subdivision. Specifically, we found that samples from Yunnan in Southwest China showed greater similarities with samples from India than those from elsewhere in China and Vietnam. Sampling of additional populations in the adjoining regions would help to verify these results and delimit the geographical extent of these haplotype affiliations.In contrast, haplotype data for R. leschenaulti occurred around 200,000 years BP, which was earlier than the date of inferred growth for C. sphinx . Our results therefore indicate that the lack of genetic structure in R. leschenaulti is unlikely to reflect a recent expansion but instead results from a high level of gene flow among localities, both contemporary and in the past. In C. sphinx, however, the comparatively higher level of structure appears to have resulted from restricted gene flow since a more recent expansion.A lack of genetic structure can reflect either gene flow or a recent expansion. We tested whether broad differences between our two focal taxa could be explained by differences in their demographic history. Although we detected expansions in both taxa, we estimated that the timing of population growth in the more genetically homogenous R. leschenaulti and C. sphinx are consistent with our predictions based on their different roosting ecology. Studies in India and China have shown that R. leschenaulti migrates seasonally, with maternity colonies reaching numbers of up to 20,000 individuals during the summer and decreasing to only a few individuals each winter C. sphinx forms smaller colonies, often in palm trees, and typically comprising a single male with multiple females C. sphinx at foraging sites rather than at roosts was undertaken to reduce the potential over-representation of relatives in our samples; however, we cannot rule the possibility that this has contributed to observed subdivision.Although our results are based on few and unevenly distributed populations, and so should be treated cautiously, the observed differences in genetic structure observed between C. sphinx from Yunnan also revealed a marked discontinuity in allele frequencies within China and Vietnam, pointing to a phylogeographic signature. In particular, we found that bats sampled from Yunnan clustered with more geographically distant individuals from India rather than with other bats in China and Vietnam. In recent years, deep genetic subdivision in Southwest China, with associated suggestions of multiple glacial refugia and contact zones in this region, have been reported for several taxa, including the greater horseshoe bat b sequence data based on a parsimony threshold of 95% confirmed the monophyly of the Indian samples previously described C. sphinx in India and China probably belong to different subspecies In addition to the overall trend of restricted gene flow, our results of Comparative studies of co-distributed taxa provide a powerful means of disentangling the relative impact of biotic and abiotic factors on population genetic structure e.g. . A recenTable S1Rousettus leschenaulti and b) Cynopterus sphinx.PCR conditions and details for loci used in this study for a) (0.06 MB DOC)Click here for additional data file.Table S2Cynopterus sphinx populations. Bold  =  significant differentiation at P<0.05.Pairwise ΦST  and FST estimates  for eight (0.04 MB DOC)Click here for additional data file.Table S3Rousettus leschenaulti populations. Bold  =  significant differentiation at P<0.05.Pairwise ΦST  and FST estimates  for five (0.03 MB DOC)Click here for additional data file.Table S4Cynopterus sphinx and b) Rousettus leschenaulti populations. AMOVA I includes all populations, AMOVA II excludes the populations from India.Results of AMOVA based on microsatellite and mtDNA data for a) (0.03 MB DOC)Click here for additional data file.


Platypus quercivorus, is the vector of oak wilt, one of the most serious forest diseases in Japan. Population genetics approaches have made great progress toward studying the population dynamics of pests, especially for estimating dispersal. Knowledge of the genetic structuring of the beetle populations should reveal their population history. Using five highly polymorphic microsatellite loci, 605 individuals from 14 sampling sites were assessed to infer the ongoing gene flow among populations as well as the processes of expansion of damaged areas.The ambrosia beetle, FST = 0.047, G'ST = 0.167) was moderate and two major clusters were detected by several methods, dividing the samples into north-eastern and south-western populations, a similar genetic divergence was reported in host oak trees. Within the north-eastern populations, the subgroups mostly corresponded to differences in the collection period. The genetic characteristics of the population might have changed after 2 years due to the mixing of individuals between populations with enhanced migration related to population outbreaks. Because isolation by distance was detected for whole populations and also within the north-eastern populations, migration was considered to be limited between neighbouring populations, and most populations were suggested to be in genetic equilibrium of genetic drift and gene flow. Recent bottlenecks were found in some populations with no geographical bias; however, they were all from newly emerged oak wilt forests. The emergence of oak wilt should have induced intense fluctuations in the beetle population size.Population differentiation (Because the genetic boundaries coincide, we suggest that the geographical structuring of the beetle was formed by co-evolution with the host species. Our findings indicate the oak wilt expansion process. Platypus quercivorus , and infer the ongoing gene flow among populations.Understanding the processes that drive the spread of plant diseases by vector beetles is critical for developing plans to protect against such damage. Population genetic approaches have made great progress toward studying the population dynamics of pests, especially for estimating dispersal e.g., ,2). Here. Here2])Quercus crispula Blume, due to wilt, at a rate of approximately 2,000 ha/year  Standard (Applied Biosystems) were added to 12 μl of deionised formamide. The products were separated using capillary electrophoresis  and assigned scores using 310 GeneScan software (Applied Biosystems) and by eye.In total, 20-154 individuals were examined per site (Table HO) and expected heterozygosities (HE) were calculated to quantify the genetic diversity of each population. Allelic richness , total gene diversity (HT) and the GST values calculated by SMOGD  showed significant linkage disequilibrium after correcting for multiple tests.We were able to successfully genotype 605 individuals from 14 sampling sites using all five loci. No null alleles or scoring errors were detected by Micro-Checker. The number of alleles varied from 8 (Pq3422) to 16 (Pq3423), with an average of 12.4 alleles per locus. Nine of 150 tests  exhibited similar genetic components supported by an 83% bootstrapping value in cluster 4.Moderate population differentiations were found , as illustrated in Fig r = 0.714, P < 0.001; 2007, r = 0.495, P = 0.001). In addition, the IBD within north-eastern populations in the 2005 collection  was tested to eliminate any effects of differentiation between the north-eastern and south-western populations and the collection year. IBD was also detected in the north-eastern populations for the 2005 collection .The Mantel test showed that the between-population differentiation increased significantly with geographical distance , increased from K = 1 to 7 . This partition corresponded to the subgrouping shown in the SAMOVA and NJ analyses. Populations in Tenryu  were divided from the south-western cluster when K = 4. Population 12 (NGY) and populations in the Kyoto prefecture [13 (ODN) and 14 (MMD)] were discriminated when K = 5. These partitionings were also consistent with those of the SAMOVA and NJ tree analyses. Higher F values were exhibited in the north-eastern clusters when K = 2-5  were compared for their genetic diversity and components, the collection method (trapping flying adults or direct collection from trunks) had little effect on the overall results. Because no null alleles were detected, all of the genotyped data were considered useful for the analysis.A bottleneck was detected in some populations from recently infested forests. Since there was no damage, these individuals may have originated as a small number of colonisers, which would have entailed a founder effect. If they were not colonisers, they may have been residents, in which case sudden changes in the availability of resources might have resulted in fluctuations in the population size of the beetle in damaged oak forests.Linkage disequilibrium was also detected in recently infested forest area populations. The lack of linkage disequilibrium in most populations suggests that the loci used may not be in close proximity to one another on the chromosomes. Population bottlenecks and/or admixturing of differentiated populations (mentioned below) might be related to linkage disequilibrium in populations from recently infested areas.FST = 0.047, G'ST = 0.167) detected in P. quercivorus suggests that gene flow among populations is balanced with genetic drift for the entire study area. Genetic analysis using microsatellite markers revealed two main genetic groups of P. quercivorus in Japan. These are divided into north-eastern and south-western populations. Using chloroplast DNA, Okaura et al. [Q. crispula and three related species, which are the main hosts of P. quercivorus. Quang et al. [Q. crispula and inferred its history of colonisation into Japan. These studies showed that a genetic boundary for Q. crispula was found between the north-eastern and south-western populations and coincides with the Itoigawa-Shizuoka Tectonic Line, a major tectonic depression that runs through Nagano Prefecture and separates sites 1 (NKH) through 9 (SNN) from 10 (TRL) through 14 (MMD). Given that the beetles exhibited the same pattern of genetic differentiation between the eastern and western populations, it is possible that they share a population history with Quercus species.The moderate level of population differentiation , and there was no oak wilt. Site 11 (TRT) is near to site 10 (TRL). Beetle populations from sites 11 (TRT) and 10 (TRL) showed similar genetic compositions with high genetic diversity. Therefore, they may not represent descendants of long-distance dispersers from severely damaged areas, and may instead be endemic populations. The beetle populations within sites 10 (TRL) and 11 (TRT) must have persisted without a break.There was no visible damage near site 10 (TRL), and it was not clear whether the beetles therein that attacked The NJ, SAMOVA and clustering by STRUCTURE analyses indicated that within the north-eastern populations, genetic components were substituted between 2005 and 2007. Because IBD was detected among north-eastern populations in 2005, most of the migration should be limited to neighbourhoods. This substitution may have been attained by gradual expansion of population distributions and mixing of population structures prior to the emergence of oak wilt damage. The migration of the beetles among populations might be enhanced by increased oak wilt damage. Therefore, samples collected in 2007 in north-eastern regions may contain individuals from broader areas. Although the beetles may seldom disperse long distances beyond a regional scale, migration within a regional scale may be frequent.Locusta migratoria. They showed that population outbreaks reduce population differentiation by enhancing migration and/or effective population sizes. Thus, an increased P. quercivorus population size might alter the population structure in a similar manner to that seen with L. migratoria. Our STRUCTURE analysis produced an F value that indicated a high value for clustering of individuals in north-eastern populations mostly collected in 2005 when K = 3. However, the F value decreased in the 2007 collection cluster. This result indicates that the north-eastern population collected in 2005 was influenced by strong genetic drift after divergence from the common ancestral population, and that the effective population size is small. By incorporating independent demes, outbreak populations from 2005 to 2007 might have made the effective population sizes larger.Chapuis et al.  examinedThe genetic structure of the beetles supported Kobayashi and Ueda's prediction  that chaTo prevent the expansion of the disease to undamaged areas, it is important to manage beetle populations to maintain low densities and thus stopping the sequence of activation. Therefore, we need to trap beetles and/or monitor oak wilt damage to provide an indication of the population density of the beetles in continuous oak forests. Once damage is confirmed, the health of the oak forest can be maintained if immediate action is taken before Allee effects  become P. quercivorus, which corresponds to the genetic structuring of the host oak trees. Based on a combination of methods of statistical analysis, the genetic structure is presented with a high degree of confidence. The geographical structuring of the beetle was thought to be formed by co-evolution with its host species.We found genetic differentiation in the ambrosia beetle ESK participated in the design of the study, marker selection, genotyping and analyses, and drafted the manuscript. SS, MO, AN and KN collaborated on the study design, sample collection and the interpretation of the results. YT contributed to the improvement of the statistical analyses. All authors read and approved the final manuscript.


Begomovirus, in the Family Geminiviridae. The begomoviruses all share the same whitefly vector, are highly recombinogenic and are distributed throughout tropical and subtropical regions where they seriously threaten the food security of the world's poorest people.The rapidly growing availability of diverse full genome sequences from across the world is increasing the feasibility of studying the large-scale population processes that underly observable pattern of virus diversity. In particular, characterizing the genetic structure of virus populations could potentially reveal much about how factors such as geographical distributions, host ranges and gene flow between populations combine to produce the discontinuous patterns of genetic diversity that we perceive as distinct virus species. Among the richest and most diverse full genome datasets that are available is that for the dicotyledonous plant infecting genus, We focus here on using a model-based population genetic approach to identify the genetically distinct sub-populations within the global begomovirus meta-population. We demonstrate the existence of at least seven major sub-populations that can further be sub-divided into as many as thirty four significantly differentiated and genetically cohesive minor sub-populations. Using the population structure framework revealed in the present study, we further explored the extent of gene flow and recombination between genetic populations.Begomovirus population structure revealed here could facilitate population genetics studies into how central parameters of population genetics namely selection, recombination, mutation, gene flow, and genetic drift shape the global begomovirus diversity.Although geographical barriers are apparently the most significant underlying cause of the seven major population sub-divisions, within the framework of these sub-divisions, we explore patterns of gene flow to reveal that both host range differences and genetic barriers to recombination have probably been major contributors to the minor population sub-divisions that we have identified. We believe that the global  The study of genome-wide patterns of sequence variation within and between closely related virus species can be used to efficiently infer the fine-scale genetic structures of virus populations. Information on population structures - particularly that pertaining to stratification and admixture (i.e. gene flow) - is valuable in a variety of situations. These include the establishment of sensible species/subspecies/strain classification criteria, the detection of geographical or biological barriers to gene flow, and the identification of demographic, epidemiological or evolutionary processes responsible for virus differentiation -3. More Begomovirus that are responsible for economically devastating diseases of many leguminaceous, solanaceous, curcurbitaceous and malvaceous crop species throughout the tropical and subtropical regions of the world [Bemasia tabaci, and have circular single stranded one (i.e. monopartite) or two (i.e. bipartite) component genomes ranging in size from ~2.7 Kb (for monopartite species) to ~5.4 Kb (for bipartite species) [It is therefore surprising that there have been no studies specifically aimed at identifying global-scale population genetic structures within agriculturally significant groups of plant pathogenic viruses such as the geminiviruses, potyviruses, tospoviruses, cucumoviruses and sobemoviruses. For example, virtually nothing is known about population stratification amongst the various geminivirus species within the genus he world -15. Begospecies) .Relationships amongst DNA-A and DNA-A-like sequences are widely used in formalized begomovirus species, strain and variant demarcation schemes -19. BaseBegomovirus taxonomic classification system is based almost entirely on traditional phylogenetic reconstruction and pairwise genetic distance estimators (such as Hamming or p-distances) [The current stances) -20,22. THowever, frequent inter-species genetic recombination is a prominent feature of begomovirus evolution -27 that Here we use such a population-genetics model-based clustering approach both to verify the existence of defined sub-populations within the global begomovirus meta-population and to track the movement of genetic material between these populations. Besides identifying hitherto unappreciated genetically discreet begomovirus sub-populations, our study provides interesting insights into how constraints on genetic recombination imposed by geographical distance and/or host range differences may contribute to taxonomically relevant patterns of begomovirus diversity.SA). Monte Carlo simulations indicated that although pairs of sites within the begomovirus genome did indeed display evidence of significant linkage disequilibrium , the corresponding ISA was 0.0367 - a low value providing evidence that many of the polymorphic loci considered are effectively in linkage equilibrium. ISA is expected to be zero when there is no linkage among pairs of polymorphisms. The estimated ISA value for our global begomovirus dataset was, for example considerably lower than that approximated for Helicobacter pylori (0.0607) [The admixture model implemented in STRUCTURE assigns individual genomes to populations under the assumption that all polymorphic sites within the genomes are in linkage equilibrium. We therefore tested the degree of linkage equilibrium that is evident within begomovirus genomes using LIAN 3.4 to calculate a standardized index of association between genome sites (I(0.0607)  and slig(0.0607) . In both2, against the physical distance separating pairs of sites  against physical distance. This analysis indicated that there was no systematic LD bias in our begomovirus dataset that might seriously impact its use in the inference of gross population structure.To investigate further the degrees of LD displayed by pairs of polymorphic sites we plotted two standard measures of LD, |D'| and rtes Fig. . There wOur initial analysis of population structure within the full begomovirus dataset aimed at discriminating between two to twelve sub-populations i.e. K = 2 to12) failed to yield an estimate of the true optimal sub-population number in that the value of Ln P(D) increased consistently with increasing K. However, the second-order rate of change of the likelihood function (ΔK) showed a clear peak at K = 8, reflecting the existence of at least eight genetically cohesive begomovirus sub-populations each displaying distinctive nucleotide distribution patterns. Although according to ΔK, the optimal number of sub-populations for the complete begomovirus dataset was eight, we chose the more conservative K = 7 for further analysis because this number of sub-populations yielded reasonably consistent clustering in repeated analysis runs. With K = 8, either the sweet potato-infecting viruses within the larger swepovirus-Asian legumovirus sub-population , the New Delhi tomato-Asian Cucurbit-infecting viruses (NDT-ACU), the Indo-Pak cotton-South Indian tomato viruses (IPC-SIT), and China-Japan-Southeast Asia viruses (Ch-J-SEA).For the sake of clarity, we named the seven sub-populations identified in the K = 7 analysis based on both the geographical location and hosts of the viruses assigned with the sub-populations. Schematic representations of the population structures revealed by our analysis are summarised in Fig The sequential increase in population stratification noted in the analysis series with K values ranging from two through seven Fig.  providesSince inconsistent sub-population splits were obtained with K > 7, we attempted to identify further population structures within the seven consistently defined sub-populations obtained with K = 7. Each one of these sub-populations was treated as a main population and each was analysed separately under the admixture model with uncorrelated allele frequencies.A second layer of population structure analysis was performed on each of the seven major sub-populations in isolation Fig . STRUCTUThe major IPC-SIT and Af-Med sub-populations apparently each contained four genetically cohesive minor sub-populations . The hierarchical AMOVA of the seven major sub-populations and the 34 minor sub-populations indicated that most of the observable genetic diversity is collectively attributable to fixed genetic differences between the 34 minor sub-populations (40.96% of the diversity) and seven major sub-populations  were applied to the various population partitions. The null hypothesis of no population structure was rejected with p-values < 0.001 by the Z-test [ST statistics (pairwise measures of population differentiation), were calculated for each of the 34 minor sub-populations [see additional file ST scores ranged from 0.09 to 0.92. Whereas an FST value of 0 between two populations would indicates that they were completely undifferentiated, a score of 1 would indicate that every observable genetic difference between individual members of the two populations could be used to distinguish between the populations. Overall, a very high degree of differentiation was noted between the Tomato chino La paz virus group and the African cassava mosaic virus minor sub-population. (FST = 0.92). The lowest degree of differentiation (FST = 0.09) was observed between the New World Tomato rugose and chloratic mottle and Tomato golden mottle virus groups. With the exceptions highlighted (the numbers in bold) in Table S1 [see additional file ST values.To further test whether the 34 identified minor sub-populations would be considered genetically distinct using alternative methodologies, two other statistical tests of population differentiation  consist of viruses that have collectively been sampled from six or more different host species, others contain viruses that have only ever been sampled from one species. Interestingly, the "broad host range" minor sub-populations are also apparently more admixed than the "narrow host range" minor sub-populations. Unfortunately we cannot tell from our analysis either whether recombination has facilitated the increased host-ranges that are apparent within these sub-populations or whether increased host ranges drive increased inter-sub-population recombination frequencies.It must, however, be stressed firstly that very little is known about the natural host ranges of any of these virus groups and, secondly, that there exist blatant sampling biases in favor of begomovirus species/strains that cause crop diseases. The fact remains however, that whereas certain of the minor sub-populations  of pairwise distances between groups of closely related sequences that apparently share a recent common ancestry , is computed and compared to the variance expected when all loci are in linkage equilibrium (VE). Only polymorphic sites were included for the analysis and a 5% critical value was obtained as described [2 [Testing for the presence and degree of linkage disequilibrium (LD) evident in a group of sequences is a significant aspect of population genetics. Moreover, the model-based approach we used to investigate the structure of begomovirus populations assumes that different polymorphic sites along the genomes being investigated display only limited degrees of LD. From the perspective of global begomovirus diversity it is very probable that, because of the extent of inter-species genetic exchange amongst begomoviruses, many sites will be effectively in linkage equilibrium. However it was essential that we test the degree of linkage equilibrium evident within our worldwide begomovirus population sample. A null hypothesis of linkage equilibrium was tested by Monte Carlo simulations using the program LIAN (version 3.4) . LIAN pe dataset . This prescribed . In addiescribed  and r2 [ribed [2 , were esribed [2 .Global begomovirus population structure was investigated using the program STRUCTURE (Version 2.0) . This prad hoc STRUCTURE runs were performed to determine the optimum number of iterations for the initial burn-in and estimation phases of the analysis so as to ensure the reliability of posterior probability estimates. Burn-in and parameter estimation iterations ranging from 20,000 to 40,000 did not yield significantly different results. From these preliminary analyses we determined that an initial burn-in of 40,000 iterations followed by 40,000 iterations for parameter estimation was sufficient. To estimate the number of populations (the K parameter), the begomovirus dataset was analyzed allowing the value of K to vary from 1 to 12. Five independent runs were carried out for each K value . As advised in the STRUCTURE user's manual, we set most of the parameters to their default values [opt) was identified as previously described [In the beginning, t values . Specifit values . This mot values . We co-et values . The lamescribed .opt, each individual was then assigned to one of the sub-populations, according to their respective estimated membership scores  for each of the different sub-populations. Individuals that could be assigned to two or more different sub-populations each with membership scores of 0.15 or higher were considered to be admixed. It is important to note that despite our expecting the admixture model to identify the correct number of sub-populations we also expected it to generally overestimate the proportion of admixed individuals by ignoring linkage between polymorphic nucleotide sites that were physically very close to one another within the begomovirus genomes. We also applied the linkage model for Kopt in order to account for potential physical linkage between loci when refining the sub-population assignment of difficult to assign individuals. For this model the burn-in and MCMC run lengths were set at 20000 and 40000 respectively, with a 10000 iteration admixture burn-in length.For Kad hoc parameter as described in [We used the first hierarchical sub-population cluster classification inferred by STRUCTURE to study finer-scale clustering within major begomovirus sub-populations. Each of the seven established major begomovirus sub-populations was considered as a major sub- population and analysed separately under the admixture model with uncorrelated allele frequencies . We used ΔK, an ribed in  to deterST statistics). The fixation indices estimated from the begomovirus sequence analyses were tested using a non-parametric permutation approach as described in [ST based estimates of population structure was also tested in ARLEQUIN using a permutation test (with 1000 randomised iterations) as in [The population stratifications inferred by STRUCTURE were tested by analysis of molecular variance (AMOVA) as implemented in ARLEQUIN (ver. 3.0) . AMOVA mribed in . Furthers) as in . Also, Ds) as in  was useds) as in . PermutaThe authors declare that they have no competing interests.HCP conceived, designed the study. HCP, DPS, AV, MS, BS and MR performed sequence alignments and population structure analysis. HCP and DPM interpreted data and wrote the manuscript. All authors have read and approved the final manuscript.Table S1. Differentiation between the 34 minor begomovirus sub-populations identified in this study. Data provided represent pairwise measures of population differentiation (FST). Non significant FST values based on permutation test are highlighted.Click here for file