Xenopus gilli, is a frog distributed in only three disjunt areas within low-lying regions of the southwestern Cape. We determined the relationship between frogs from these three disjunct areas, by using a combination of morphometric analysis and mtDNA (ND2 and 16S fragments) sequences of 130 frogs from eight ponds. Coalescent analyses on molecular data dated the divergence in two major clades to around 4.6 Mya, a period during which major uplifting on the eastern side of the subcontinent caused climate changes throughout southern Africa. Principal components analysis showed significant morphometric differences between each clade on head and limb measurements. Consistent differences in ventral colouration and patterning were also observed. We report on increased levels of hybridisation with X. laevis throughout the range of X. gilli, which reaches at least 27% hybrids in some ponds. Urgent conservation actions are required to control habitat loss from alien invasive vegetation, and prevent introgression with the domestic-exotic, X. laevis.Low-lying areas of the Cape at Africa’s south-westernmost tip have undergone dramatic marine-remodelling, with regular changes in sea-level following glacial cycles. Species for which marine barriers are impenetrable underwent concomitant radical distribution changes which may account for current range disjunctions. The Cape platanna,  Today, southern Africa is primarily a semi-arid region with subtropical conditions along the east coast . The souThe high percentage of Cape floral endemics is associated with a wealth of faunal endemism in terrestrial, freshwater and marine ecosystems . CladogeXenopus gilli Rose & Hewitt, is one of several species with a disjunct distribution on either side of False Bay (X. gilli has three distinct areas of distribution (Amietophrynus pantherinus (Smith) (the western leopard toad) and Microbatrachella capensis (Boulenger) (the micro frog), share similar distributions and restricted dispersal ability , and dated their divergence at 8.5 Mya (4.8–13.4). However, X. gilli is a fynbos endemic, specialising in the acid-blackwater pools that are characteristic in this vegetation type, and the fynbos biome is only thought to have become established in the early Plio- Pleistocene (see below). The dating and genetic distance between these clades is larger than between other species in the genus Xenopus  but in the 1970s a population was discovered approximately 50 km to the east, on the opposite side of False Bay   see  which isdin) see . In SoutX. gilli .Xenopus species from the Cape of Good Hope Nature Reserve , all within an area of 2 km2. All other collection sites were on the east side of False Bay  and near Pearly Beach . We identified individuals as X. gilli from dorsal and ventral patterning, shape of head, and overall size  and South African National Parks . Ethics clearance was obtained from South African National Biodiversity Institute ethical clearance (number: 001/10).Xenopus gilli captured had a standard set of 14 measurements taken: snout-vent length (SVL), head length (HL), head width (HW), head height (HH), lower jaw length (LJL), ilial length (IL), ilial width (IW), femur (FM), tibia (TB), metatarsal (MT), longest toe (LT) on the hind limb, humerus (HM), radius (RD), and longest finger (LF) on the forelimb  for sequencing.Total genomic DNA was extracted from the tissue samples using a standard salt extraction procedure  and frag to vTrp  and 896  to vTrp . Polymer to 16Sb  of which to 16Sb . PCR annX. laevis  were considered to be hybrids and therefore removed from further analyses.Geneious Pro 5.0.4 was used to edit and align the DNA sequences from each sample using a global alignment  and the K = 2–9 putative populations to determine the maximum CTF value, the highest proportion of differences between populations due to genetic variation  was conducted , using Nariation . The resariation  was usedariation  was usedEGA v. 5 .SF was used to test for departures from mutation–drift equilibrium (t = τ/(2u), where t is time in generations, τ is the age of the expansion in mutational units (estimated in the model of demographic expansion) and u is the sum of the per nucleotide mutation rate for the region sequenced .To examine whether any population had experienced historical demographic changes, Tajima’s D and Fu’s ilibrium  using Arilibrium . For popilibrium . For popequenced . For theequenced  and u wat) between all the populations identified in SAMOVA (Cape Peninsula vs. eastern), using IMa2 (X. gilli lineages (8.5 Mya) in X. gilli: 4.8 and 13.4 Mya (http://cbsuapps.tc.cornell.edu/IMa.aspx).Coalescent analysis was used to investigate time of divergence  and from the eastern population (n = 59) were used as input variables for a principal components analysis (PCA) to reduce the 13 morphometric variables into linear combinations of correlated variables . The Keiser-Meyer-Olkin (KMO) measure of sampling adequacy was applied to determine whether the dataset was adequate to conduct a PCA. The analysis included a varimax rotation to minimise the number of variables with high loadings on each PC, which simplifies the interpretation of the PCs extracted. Variables with communalities greater than 0.5 were retained in the analysis and only PCs with eigenvalues greater than 1.0 were extracted. The principal component scores were saved, and ANOVA was carried out on the PCs extracted using R , data from both sexes were pooled for all further analyses. To examine population level differences in morphology, a multivariate approach was taken. Size corrected residuals from the Cape peninsula  unambiguously showed that the nine sampling sites could be grouped into two populations, corresponding to animals collected in the Cape peninsula (western) and combined eastern sites . For 16S, all four individuals sequenced from the Cape peninsula were of a single haplotype identical to X. gilli from Cape Point sequenced by X. gilli previously sequenced from Betty’s Bay by p-distance for 16S was found to be 1.7%.Sequences of 896 base pair fragment of ND2 were obtained from 128 individuals identified in the field as equenced . All anip-distance of 8.8% at the ND2 locus between animals from the Cape peninsula and the eastern population. The Cape peninsula had only three ND2 haplotypes amongst 38 individuals (p-distance within = 0.25%), while the 74 animals sequenced from the eastern population share 30 different haplotypes (p-distance within = 5.63%).The haplotype network for ND2 shows two clear groups separated by 74 base pair changes . SequencX. gilli populations had a higher ND2 nucleotide and haplotype diversity than animals from the Cape peninsula (h), but not nucleotide diversity (π). The low haplotype diversity on the Cape peninsula is also demonstrated by the haplotype network , but this was not detectable within the Cape peninsula. The model of demographic expansion fitted the observed distribution for the eastern population  suggesting the population has undergone a demographic shift. The value obtained for τ = 2.16 (95% CI: 1.29–2.78), provided an estimate for years since population expansion (t) of 75.7 thousand generations or ca. 151.4 Kya.The eastern eninsula . However network . Both TaThe coalescent analysis provided an estimated divergence time between the Cape peninsula and eastern population of ca. 4.6 Mya with a confidence interval from 3.2 to 6.4 Mya ; Fig. 3,n = 59) compared to those from the Cape peninsula (n = 50). The first test for sexual size dimorphism confirmed that in both populations females were significantly larger than males by around 25%: Cape Peninsula females had a mean SVL of 43.0 mm (SE 0.752), while males’ was 34.6 mm . For the eastern population, mean female SVL was 44.2 mm (SE 0.071), with males’ mean of 34.3 mm . No significant differences were found overall between sexes using size corrected measurements in a MANOVA . Additionally, there was no significant difference for the interactive term: sex and population , indicating that within populations, the sexes did not differ in their morphology. Thus, for all further tests sexes were pooled.Morphometric measures were made on a total of 109 individuals from all sites. Following the results of SAMOVA analysis we conducted morphometric tests on animals collected from all eastern populations  were extracted in the PCA, which accounted for 73.3% of the total variation in the dataset . The KMOXenopus gilli distributed on either side of False Bay. However, for the two disjunct clusters of sites in the East (Kleinmond and Pearly Beach), our analyses did not suggest that they represent separate populations. Despite the disturbance to population distributions caused by sea-level change, our results from coalescent analysis of the deep genetic division between the two populations, dates back to 4.63 Mya (3.17 to 6.38 Mya). This is considerably younger than the estimate provided by There is considerable genetic and morphological variation between the two identified populations of X. gilli by X. gilli from X. laevis. Relative to their size, the Cape peninsula animals had significantly broader and higher heads than those from the eastern population. In addition, animals from the eastern population had relatively shorter legs. These characters may relate to differences in performance between animals from the different habitat types  outcrop of sandstone which may have been able to support a population of X. gilli. It is not known whether X. gilli would have been capable of movement across the intermediary habitat, and there is a similar distance from this outcrop to the habitat extensions in the east. If dispersal was possible over these distances, there is the potential for movement of individuals across False Bay at each glacial maximum. Similarly, The Agulhas Bank was last exposed as recently as the last glacial maximum, some 20 Kya with a sea-level fall of around 140 m . The chaoday see . East ofnd today . On the Xenopus lineage would have had a larger distribution. This may explain the star-shaped haplotype network , and this might explain why there was not a more recent genetic expansion during the last glacial maximum. Most recently however, anthropogenic factors have further reduced the ranges of these X. gilli populations, raising concern for the conservation of these species.Climatic change also followed this period of substantial uplifting on the eastern side of the subcontinent and is thought to have led to significant reductions of rainfall in the west and aridification of the interior of South Africa . The sta 3–5 Mya , on whos depends . This sa depends . Thus, tXenopus gilli over the past century. The type series was said to be collected from the Silvermine River and adjacent Cape Flats  and nucleotide (π) diversity between the two lineages is indicative of a bottleneck in the Cape peninsula, with a reduction in the number but not in the diversity of the remaining haplotypes. The distribution of pairwise differences between haplotypes  sampled in the eastern area, while none were found from 59 sampled from the CoGH reserve  but result from a X. laevis maternal lineage. From relatively low levels (2.5%) on the Cape peninsula, to 27% in ponds near Kleinmond where X. laevis and hybrids have come to dominate some historically known X. gilli sites. Ponds near Pearly Beach, which were previously thought to contain only pure X. gilli . Despite good and clear documentation on the threats to this species, all appear to continue unabated.Lastly, we suggest that the eastern lineage of reat see . The eas


The genetic differentiation among populations was estimated at a = 0.92. Estimates of genetic distances between the populations varied from 0.034 to 0.390. Genetic distance and the corresponding clustering analysis indicate the formation of two groups. The first consists of the Baía Formosa, Georgino Avelino, and São José do Mipibu populations and the second consists of the Japoatã, Pacatuba, and Praia do Forte populations. The correlation matrix between genetic and geographic distances was positive and significant at a 1% probability. Taken together, our results suggest a spatial structuring of the genetic variability among the populations. Geographically closer populations exhibited greater similarities. Coconut palms of the Tall group were introduced to Brazil from the Cape Verde Islands in 1553. The present study sought to evaluate the genetic diversity among and within Brazilian Tall coconut populations. Samples were collected of 195 trees from 10 populations. Genetic diversity was accessed by investigating 13 simple sequence repeats (SSR) loci. This provided a total of 68 alleles, ranging from 2 to 13 alleles per locus, with an average of 5.23. The mean values of gene diversity (H Cocos nucifera L.), the Tall (Typica) and the Dwarf (Nana) types, are known. Coconut is the most widely naturally distributed palm tree. It is extensively cultivated around the world and is considered to be one of the most important tropical species used by man , with the majority of the plants located on the Northeastern coast. These populations are considered to have adapted to distinct environmental conditions and have undergone genetic divergence , and leaf polyphenol investigations , 0.5 μL of forward primer (10 μM stock), 0.5 μL of reverse primer (10 μM stock), 0.5 μL of Taq DNA Polymerase (2 U/μL), 5 μL of genomic DNA (2.5 ng/μL), and 13.75 μL of sterile water. PCR reaction cycles consisted of an initial denaturation step at 94 °C for 5 min, followed by 36 cycles at 94 °C for 30 s for denaturation, one minute at 51 °C for primer annealing, and one minute at 72 °C for extension, plus an additional final extension step of 5 min at 72 °C.For the PCR reaction, a final volume of 25 μL was prepared. It contained a mixture of 2.5 μL of 10X PCR buffer, 2.0 μL of dNTP (2 mM of each dNTP), 0.25 μL of MgClThirteen fluorescence-labeled primer pairs that were designed and selected by F, θp, and f parameters  and gene diversity He  was obtained by the fixation index f for each population, assuming mating system equilibrium (a = (1-f)/(1+f).For each investigated locus, we did the adherence test to the Hardy-Weinberg proportions, according to ilibrium , so thatIn addition, the software STRUCTURE (e), and observed heterozygosity (Ho) for each SSR locus are shown in The total number of investigated alleles, gene diversity , according to the Mantel test. The smallest distances were found between the populations of Bahia Formosa and Georgino Avelino (0.034) and Georgino Avelino and São José do Mipibu (0.035). Relatively small distances were also found for the populations of Japoatã and Pacatuba (0.063), Baia Formosa and São José do Mipibu (0.071), Pacatuba and Praia do Forte (0.096), and Japoatã and Praia do Forte (0.099). The greatest genetic distances were observed between the populations of Santo Inácio and Georino Avelino (0.390), Santo Inácio and São José do Mipibu (0.381), Merepe and Praia do Forte (0.343), Merepe and Pacatuba (0.335), and Luis Correia and Pacatuba (0.335). The distance comparison results are summarized in f) for each population and the corresponding confidence intervals are given in a) are also shown in Estimates of the fixation index (Of the 130 tests for Hardy-Weinberg equilibrium (13 loci in 10 populations), only 16 were statistically significant. For most of the studied populations, statistical significance was found for only one or two loci. The exception was the population of Baia Formosa with four loci that exhibited a significant departure from the Hardy-Weinberg proportions among the 13 loci.The pattern of genetic divergence among the investigated populations of Brazilian Tall coconut that were obtained from the Nei genetic distance is shown as a dendrogram in K = 7  was similar to that found in other studies of coconut palm tree populations using SSR markers. e = 0.459) was lower than that found by ex situ collections of Tall coconut trees in Sri Lanka, with values ranging from 0.426 to 0.846 and an average of 0.682. The maximum possible value of gene diversity (He max) within a population, for a locus with A alleles, is He(max) = (A-1)/A. With A between 5 and 6, this value is 0.80 and 0.83, respectively, thus indicating that the investigated populations exhibited approximately 56% of the maximum, as a consequence of uneven values of allelic frequencies per locus within the populations.The gene diversity in the present study (Hf), despite being significantly different from zero, was small in magnitude (0.043), indicating a predominantly panmixia reproduction system among the populations. The low inbreeding rate detected may have resulted from intermating of related parents or from natural self-fertilization. Further insights on the predominant reproductive system of the investigated populations would require data from the offspring of maternal families. Considering the overall apparent outcrossing rate ta = 0.92 and, in the case of inbreeding by selfing, the apparent rate of self-fertilization is very small (The estimated intrapopulation fixation index (ST (0.086) was approximately one half of the corresponding θP value, although with a slightly overlapping confidence interval. These observations may indicate that the stepwise model is not the most appropriate means to explain the recent evolution of the populations. In fact, the allelic frequencies suggest an independent size distribution for the investigated loci. Therefore, estimate θP was considered to be more adequate to represent differentiation among populations in the present study. The P.The value of total fixation was relatively high   . For higThese results, along with historical records, suggest that the populations are undergoing a recent process of differentiation, meaning that a few generations have passed in the process of evolution from the ancestor populations. The first record of the introduction of coconut palms in Brazil dates back to 1553, as mentioned previously. However, individuals from the species can live from 80 to 100 years. This indicates that there have been relatively few generations in Brazil and that the present structuring is probably due to genetic drift, strongly influenced by a founder effect, and possibly due to indirect effect of artificial selection by humans.The correlation between the genetic and geographic distances matrix  may be considered to be intermediate to high, demonstrating that interpopulational genetic divergence is structured spatially and probably in a clinal variation pattern. These analyses indicate that a stochastic process is probably responsible for the differentiation, with genetic drift only partially counter-balanced by short-distance gene flow.The data suggest the possibility of clustering of the populations with genetic distances inferior to 0.1. Two population groups were formed: group 1 composed by the populations of Baia Formosa, Georgino Avelino, and São José do Mipibu; and group 2, consisting of the populations of Japoatã, Pacatuba, and Praia do Forte. Our data evidenced that the genetically most similar populations were also geographically closest. The differentiated pattern of the other populations did not allow consistent clustering .The results of the structure analysis were consistent with the dendrogram. The groups obtained resulted from similar clustering, namely: group 1, consisting of the population of Baia Formosa, Georgino, Avelino and São José do Mipibu and denominated Natal group; and group 2, consisting of the populations of Japoatã, Pacatuba, and Praia do Forte and called the Southern group .f), with an overall mean of 0.043 and values of the apparent outcrossing rates (ta) around the overall mean of 0.918, in addition to results of the test of goodness of fit to Hardy-Weinberg equilibrium, demonstrate that the majority of the populations studied reproduce predominantly by panmixia.Estimates of the fixation index (ex situ germplasm conservation, selection, and support of breeding programs in Brazil.The present study permitted the conclusion that microsatellite markers are effective in estimating genetic variation levels within and between the populations of Brazilian Tall coconut trees. Brazilian populations exhibited high genetic divergence detected by the employed markers. Diversity among the investigated populations is spatially structured, with a greater similarity among geographically close populations. The studied populations of Brazilian Tall coconut are preferentially allogamous, with a mean apparent outcrossing rate of 92%. Taken together, our results will provide important tools for


K = 13% and 5%, respectively) were delineated from the Quebec breeding program. Their genetic diversity and allele frequencies were compared with those of control populations of the same size and geographic origin to assess the effect of increasing the selection intensity. The two control populations were also compared to assess the effect of reducing the sampling size. On one hand, in all pairwise comparisons, genetic diversity parameters were comparable and no alleles were lost in the case populations compared with the control ones, except for few rare alleles in the large case population. Also, the distribution of allele frequencies did not change significantly (P ≤ 0.05) between the populations compared, but ten and nine SNPs (0.8%) exhibited significant differences in frequency (P ≤ 0.01) between case and control populations of large and small sizes, respectively. Results of association tests between breeding values for height at 15 years of age and these SNPs supported the hypothesis of a potential effect of selection on the genes harboring these SNPs. On the other hand, contrary to expectations, there was no evidence that selection induced an increase in linkage disequilibrium in genes potentially affected by selection. These results indicate that neither the reduction in the sampling size nor the increase in selection intensity was sufficient to induce a significant change in the genetic diversity of the selected populations. Apparently, no loci were under strong selection pressure, confirming that the genetic control of height growth in white spruce involves many genes with small effects. Hence, selection for height growth at the present intensities did not appear to compromise background genetic diversity but, as predicted by theory, effects were detected at a few gene SNPs harboring intermediate allele frequencies.A scan involving 1134 single-nucleotide polymorphisms (SNPs) from 709 expressed genes was used to assess the potential impact of artificial selection for height growth on the genetic diversity of white spruce. Two case populations of different sizes simulating different family selection intensities ( Commercial plantations have been established in numerous countries to respond to the increasing demand for forest products . ReforesWhen assembling their breeding populations and making selections for next generations, tree breeders must determine the optimum size of these populations and estimate the potential effect of their decisions on the level of genetic diversity maintained. Based on population genetics theory, this question can be addressed from two different perspectives: (i) the reduction in genetic diversity from sampling effects, which should affect all genes more or less equally, and (ii) the reduction in genetic diversity from selection intensity, which should affect only genes implicated in the selected phenotypic trait . By drawStudies were conducted in the past for a number of forest tree species with the aim to compare genetic diversity between natural and breeding populations . GloballPicea glauca (Moench) Voss.] is a boreal conifer species with transcontinental range in North America from Newfoundland to British Columbia, and it extends to the Lake States and New England in the United States  located in a large number of expressed genes distributed across the 12 linkage groups of the spruce genome . On the Picea glauca (Moench) Voss.] in Quebec can be found in the study of The complete details about the breeding strategy applied to develop genetically improved stock for white spruce [To study the effects of the selection intensity applied to delineate this improved population, a ‘large case population’ was assembled with 71 trees belonging to 38 different provenances , Table 1in silico from the redundancy of EST sequences in white spruce gene clusters following the methods outlined in the study of A total of 1506 SNPs and 30 indels  were chosen for the construction of a large SNP genotyping array. They were located on 822 different genes distributed over the 12 linkage groups of white spruce. This array, called PgLM1, was also used for white spruce gene mapping under the Arborea project . These chttp://www.illumina.com; Genotyping of the 142 sampled individuals was performed by constructing a 1536-SNP bead array (PgLM1) and using the Illumina GoldenGate SNP genotyping assay  at the 95% level, the average number of alleles per locus (A), observed heterozygosity (HO), expected heterozygosity or gene diversity (HE) corrected for small samples according to FIS), and allele frequencies for each SNP. Moreover, alleles were grouped into 10 classes based on their frequencies in each population, which made it possible to compare the distribution of allele frequency classes between populations. Alleles with frequencies lower than 5% were defined as rare. The heterogeneity of HO, HE, and FIS between populations was tested with paired t-tests using the statistics package of the software R version 2.6.1 (http://www.r-project.org). A Fisher’s exact test and a chi-square test (χ2-test) were used to check the heterogeneity of allele frequencies for each SNP and the distribution of allele frequency classes between populations, respectively. The same parameters were used to assess the effect of increasing the selection intensity by comparing same-size control and case populations: small case versus small control and large case versus large control populations.To determine the extent to which selection intensity affected genetic diversity, we compared a number of genetic diversity estimates between case and control populations of small size and between case and control populations of large size. To further determine the effect of sampling size, the genetic diversity estimates were compared between the small and large control populations. These genetic diversity estimates included the percentage of polymorphic SNPs (θRH). This parameter was proposed as an estimator of FST by FIS and FST (we use the term FST to indicate θRH) was tested with 10 000 permutations of alleles within populations and of samples between populations, respectively. All the genetic parameters were obtained and statistical tests conducted using Genetix version 4.05  were successfully genotyped with a GenTrain score higher than the conservative threshold of 0.25 set for this study  and withPO, A, HO and HE) were comparable between the two control populations of different sizes , as tested using 1000 bootstraps over loci. A total of 14 alleles were lost in the small control population as compared with the large control population , including rare alleles with MAF ≤ 0.05 . When considering each SNP individually, none showed significant differences in allele frequencies between the two control populations after correction for multiple testing using the false discovery rate (FDR)  between the two populations was not significantly different from zero.All parameters  , at a rePO, A, HO, HE and FIS) were similar between same-size case and control populations and did not show any significant statistical difference  , including the proportion of rare alleles with MAF ≤ 0.05 . When tested for each SNP individually, allele frequencies were not significantly different after correction with FDR even when we relaxed the confidence level to Q ≤ 0.10. Before correction, 36 and 38 SNPs were significantly different at P ≤ 0.05 between the two small (n = 28) and between the two large populations (n = 71), respectively. This sizeable number of significant SNPs before correction for multiple testing certainly contains false positives, but nine and ten SNPs maintained significant differences at a higher probability  was ten times higher between the two small than between the two large populations . While the differentiation between the two large populations was not significantly different from zero, that between the two small populations was significantly greater than zero, as tested using 1000 bootstraps over SNPs.All genetic diversity estimates ( > 0.05) . Also, tHO = 0.354 and 0.344, and HE = 0.347 and 0.343, for the populations of 158 and 71 trees, respectively), the difference being well within the standard errors of estimates. Only one of the alleles was lost in the large control population (71) as compared with the population of The decrease in population size between large and small control populations  induced a slight increase in the percentage of polymorphic loci and, at the same time, a slight decrease in the average number of alleles per locus and the total number of rare alleles in the small control population. However, all these changes were not statistically significant and not sufficient to induce a significant change in the frequencies of any SNP or in the overall distribution of allele frequency classes, even for rare alleles (MAF ≤ 0.05). Thus, genetic diversity was not reduced in the small control population, relative to the large control one. These findings could possibly be explained in two interconnected ways. The first explanation can be linked to the way the small control population was set up. In the present study, the individuals in the small control population were not selected randomly among the provenances of the large control population, as expected under genetic drift. They were rather selected within provenances that also included families with the highest breeding values for height, because the objective was to set up a control population for the small case population with the same geographic background, so as to neutralize the possible confounding effect of different geographic backgrounds between small case and large case populations. Consequently, the sampling scheme used should not be considered as strictly equivalent to a simulation of genetic drift. One could also argue that, because the size of both control populations was much lower than the effective size of natural populations they originated from, a bottleneck effect might be already associated with the large control population. To address this potential issue, we compared the level of genetic diversity of the large control population (size of 71) with that of a set of 158 trees representative of natural populations in the same geographic area, assembled by FIS) compared with what was expected from Hardy–Weinberg equilibrium was also observed in the small control population  for the 43 trees of the large control population that were not selected to be part of the small control population, whereas that of the 28 trees making up the small control population was 0.319 (SE = 0.028). As a consequence, a significant excess of heterozygotes (pulation .PO), the number of alleles per locus (A), heterozygosity (HO and HE), FIS, and the frequency of rare alleles. It is likely that biallelic markers such as those used in the present study do not offer much sensitivity for identifying differences in some of these genetic diversity parameters on a per locus basis, although a large number of loci was sampled, which should have led to high power in detecting differences in heterozygosity. However, we noticed that when retaining a smaller proportion of families under the tested scenario of high selection intensity (down to the best 5%), a small but proportionally large tenfold increase in genetic differentiation (FST) was induced between the small case and control populations, compared with the scenario where the best 13% of the families were retained (large case versus control populations). This increase in overall genetic differentiation is significant and indicates that even in species highly diversified genetically such as spruces and even at an early stage of domestication, a sample of much reduced size resulting from high selection intensity can lead to increased genetic differentiation.The comparison of same-size case and control populations did not result in notable differences in standard genetic diversity estimates, namely the proportion of polymorphic loci , which will generally reduce the additive genetic variance usable for gains in the future . Such a r2) as a measure of LD. It was possible to estimate r2-values for 205 pairs of SNPs. The average r2 values were similar for each of the four populations and not significantly different , ranging from 0.319 to 0.333. For three of the SNPs potentially affected by artificial selection in the large populations . For the set of small populations, LD decreased for the two SNPs potentially affected by selection for which we had r2 estimates: for 08pg02761g, r2 decreased from 0.3749 to 0.1532 for control and case populations, and for 09121m, r2 respectively, decreased from 0.3819 to 0.2396. Thus, evidence for any increase in LD after artificial selection was very weak in the present study. However, the present study design was not optimal to obtain accurate estimates of LD. Only a large series of candidate genes potentially affected by selection with a good coverage of SNPs evenly distributed along the gene sequences would make it possible to obtain sound estimates of LD and test the hypothesis of increase in LD after selection. At the same time, larger population sizes would be required while maintaining selection intensities to increase statistical power in detecting significant shifts in LD.To test whether LD might have been induced in the selected populations, we estimated unphased LD between each pair of SNPs within genes, i.e., for genes for which more than one SNP had been mined, using the squared allelic correlation coefficient (ulations  and for FIS) it harbored compared with expectations from Hardy–Weinberg equilibrium. At a first glance, this excess combined with the increase in genetic differentiation between the small case and the small control populations may support the hypothesis of a positive relationship between heterozygosity and growth, survival or fitness. This trend has been observed for knobcone pine  in all population comparisons after correction for multiple analyses were not surprising. The FDR method provides an increased power to detect differentiation between paired samples but remains conservative, although less conservative than the Bonferroni correction (P ≤ 0.05 between the two large populations (38), and the two small populations (36), cannot be explained biologically and may contain a fair proportion of false positives. Discarding all these SNPs would nonetheless likely eliminate valuable information about some true-positive SNPs. Therefore, we increased the threshold level to P ≤ 0.01, which allowed us to reduce the possible number of false-positive SNPs while resulting in the identification of a small number of significantly differentiated SNPs that could be potentially affected by selection.The statistically non-significant differences between SNP allele frequencies  and detected only one or a few significantly different loci e.g.,  or one oP ≤ 0.01 could be related to growth and reproductive processes and, to a lesser extent, to plant response to biotic or abiotic stress (08Pg02761) significantly different between the small case and the small control population belongs to a gene of the pectate lyase family, which is often linked to plant growth, development, and response to chemical stimuli (08pgsb08a and 08pgsb08b) belong to a gene from the proteasome maturation factor UMP1 family protein, which is involved in the proteasomal degradation pathway. It is essential for many cellular processes, including the cell cycle, the regulation of gene expression, and responses to oxidative stress (10614t2), which pertains to an arabino-galactan protein gene reported to be involved in wood formation in Pinus taeda L.  but about 100 cM apart. The two other QTLs are on linkage groups 2 and 5. Hence, these SNPs are well dispersed throughout the genome, and their corresponding QTLs are all distinct.Among the seven SNPs found significant in the present study and whose corresponding genes were also reported to be associated with QTLs , four weP ≤ 0.05, and two more at 0.05 > P ≤ 0.10. Each of them could explain between 3% and 9% of the total variation in breeding values. Similarly, for the set of the two small populations, seven of the nine detected SNPs were found significantly associated with breeding values for height at P ≤ 0.10 and six at P ≤ 0.05, and up to 27% of the observed variation in breeding values could be explained by the most significant SNPs (f < 0.50). This trend provides further support to the idea that they are indeed affected by selection, given that the SNPs expected to be the most influenced by selection have been predicted to be those harboring intermediate frequencies  also strengthen our belief that these SNPs are throughly affected by selection. Indeed, among the 10 detected SNPs in the pair of large control and large case populations, five were significant at ant SNPs . While sant SNPs . One addant SNPs  have intquencies .FST) in neutral genetic markers has already been observed between the second generation of Douglas-fir orchards and their wild progenitors compared with the corresponding first generation , most studies did not provide clear figures about the selection intensity that their breeding populations experienced. Authors also based their conclusions upon the comparisons of populations of different sizes e.g., , groups The main contribution of this study consisted in surveying a much expanded sample of the expressed plant genome in assessing the effects of artificial selection on natural genetic diversity. While no significant loss in genetic diversity was noted after selecting for one generation, subtle effects were nevertheless observed, implicating differentiation of allele frequencies at certain gene loci and significant associations with phenotypic selection criteria. Whether these SNPs may harbor good predictive value of breeding values in marker-aided selection schemes remains to be verified in large association populations. As for the issue of gene conservation, previous studies suggested that 30 to 70 individuals should be sufficient to ensure that the genes most influenced by selection (i.e., allele frequencies in the intermediate range,


Many coastal species occupying the temperate rainforests of the Pacific Northwest in North America comprise endemic populations genetically and ecologically distinct from interior continental conspecifics. Morphological variation previously identified among wolf populations resulted in recognition of multiple subspecies of wolves in the Pacific Northwest. Recently, separate genetic studies have identified diverged populations of wolves in coastal British Columbia and coastal Southeast Alaska, providing support for hypotheses of distinct coastal subspecies. These two regions are geographically and ecologically contiguous, however, there is no comprehensive analysis across all wolf populations in this coastal rainforest.By combining mitochondrial DNA datasets from throughout the Pacific Northwest, we examined the genetic relationship between coastal British Columbia and Southeast Alaska wolf populations and compared them with adjacent continental populations. Phylogenetic analysis indicates complete overlap in the genetic diversity of coastal British Columbia and Southeast Alaska wolves, but these populations are distinct from interior continental wolves. Analyses of molecular variation support the separation of all coastal wolves in a group divergent from continental populations, as predicted based on hypothesized subspecies designations. Two novel haplotypes also were uncovered in a newly assayed continental population of interior Alaska wolves.We found evidence that coastal wolves endemic to these temperate rainforests are diverged from neighbouring, interior continental wolves; a finding that necessitates new international strategies associated with the management of this species. Evaluating phylogeographic patterns across multiple species can aid in deciphering the historical processes that drive community assemblage and species diversification throughout regions of interest Canis lupus) in western North America Recent molecular studies of wolves supported both phylogeographic and ecological mechanisms for diversification of wolves .The sampling regime emphasized localities within continental and coastal Southeast Alaska and British Columbia , as descST statistics ST estimates.Phylogenetic relationships among haplotypes were examined by constructing a network using the median-joining method available in Network v.4.6 a priori subspecies groups, we used analyses of molecular variance . Thus, if there is concordance between the distribution of genetic subdivisions, and presumed subspecies delineations, values of ΦCT should be significant, and larger than alternative groupings.To test significant geographic divisions of hypothesized ce AMOVA , in Arleses i.e.  and the ses i.e. ;, we alsF-statistics based spatial analysis of molecular variance (SAMOVA) was performed using SAMOVA 1.0 FCT while minimizing FSC were assumed to be the most probable geographical subdivisions.To investigate the distribution of genetic diversity over the contemporary samples, an A and M, and C and L) from Weckworth et al. A and C were included in this manuscript. Two previously undocumented haplotypes, S and U, were discovered in the Yukon-Charley population. This population also shares additional haplotypes with interior Alaska wolf populations (represented in this study by Fairbanks (FAI) area samples). Three haplotypes  are restricted to coastal British Columbia and Southeast Alaska. Haplotypes G and H are shared between coastal wolves in Southeast Alaska and coastal British Columbia. Haplotype I, however, is found only in Southeast Alaska wolves. The most common haplotype (F) found in the coastal region is also shared with wolves in interior British Columbia.The genetic diversity described here is similar to the results of previously published studies e.g. . Based oA) that was ubiquitous across northern Alaska and western Canada populations. Contemporary haplotypes in Phylogroup 1 (haplotypes A and S) have a distribution corresponding to C. l. occidentalis. However, the addition of the lu52 haplotype from a historical specimen collected in Oklahoma C. l. nubilus, and is not consistent with Nowak's The haplotype network  includesF, present in 184 individuals from Southeast Alaska, and coastal and interior BC. This phylogroup also included haplotype H which is restricted to coastal BC and the southern mainland coastal area in Southeast Alaska. Haplotype G is shared between the North Pacific Coast and the Yukon, corroborating the southern refugial origin of some individuals C. l. nubilus . The presence of haplotypes from extirpated southern populations  in Phylogroup 2 (I is in Phylogroup 3) as found by Weckworth et al. Coastal populations were predominately found in Phylogroup 2, which included haplotype ogroup 2  indicateD, K, and C). However, the displaced haplotype I, which is only found in one population in Southeast Alaska, is found within this phylogroup, which would suggest mixed refugial origins of coastal wolves, or contemporary gene flow between interior and coastal wolves. Individuals with haplotypes in phylogroup 3 (except for haplotype I) are within the defined range of C. l. occidentalis. Phylogroup 4 represents a distinct southern refugial group identified by Leonard et al. C. l. baileyi), and is diverged from other North American wolves (Phylogroup 3 included haplotypes found within interior Alaska and the Yukon (n wolves .P = 0.002), 34.1% of variation was apportioned within populations (P<0.0001), and 13.0% was relegated among populations within groups (P<0.0001). Model A partitions the populations into two groups that correspond to either the distribution of C. l. nubilus or C. l. occidentalisC. l. fuscus) results in a reduction of ΦCT, although the value is still significant . Overall, the results of the AMOVA analysis suggest that C. l. ligoni is genetically distinct from C. l. occidentalis when coastal British Columbia is included.Population information for the haplotypes from extirpated populations is not available and consequently these sequences were not included in AMOVA analyses. When populations were divided into coastal and continental groups nificant , Model Bnificant . FinallyFCT = 67.06) while differentiation between populations within groups dropped below zero (FSC = −0.15). All of the continental populations and MCN were each in a group by themselves. The sixth group was composed of all the coastal populations (except for MCN).Population subdivision based on mtDNA was calculated using SAMOVA. The results indicated that genetic differentiation among groups was maximized at six groups  that was unique to five individuals from the Juneau region of the northern mainland coastal area (MCN) of Southeast Alaska. Populations of island wolves in coastal BC generally possess multiple haplotypes, whereas most island wolves in Southeast Alaska were monotypic for the common coastal haplotype (F), suggesting that either gene flow between mainland coastal and island wolves is higher in BC than Southeast Alaska, or that island wolves in Southeast Alaska have been subjected to extreme genetic drift, perhaps due to small founding populations or subsequent bottlenecks. Weckworth et al. e]) occurred more than 100 generations (ca. 4Ne) ago Although both coastal British Columbia and Southeast Alaska wolves share similar evolutionary histories, genetic diversity varies among the regions. Haplotype diversity was similar across the combined regions, with the exception of a fourth haplotype (F and G) were found in continental populations. Haplotype F was found in 87% of all coastal individuals, and was identified in some continental populations , and C. l. crassodon on Vancouver Island C. l. nubilus, which extended to wolf populations across most of the conterminous US and into eastern Canada. Subsequent molecular perspectives C. l. ligoni, C. l. fuscus and C. l. crasodon.Weckworth et al. Coastal wolves have been described as distinct Management Units Canis lupus is listed as vulnerable across its global range  C. l. ligoni) restricted to temperate rainforests of coastal Southeast Alaska and British Columbia. In the U.S., 80% of the terrestrial biome is managed by the U.S. Forest Service . These coastal wolves are considered a species The high dispersal capabilities cited by the FWS presumably suggest that recruitment of wolves from outside of Southeast Alaska would mitigate declining populations and loss of genetic diversity. This study and previous work Previous studies focusing on wolves in the continental United States found minimal variation among populations e.g. . In highHabitat of wolves of the Yukon-Charley National Preserve is protected by the National Park Service, which is explicitly mandated to assess and maintain variability of wildlife populations The coastal wolves in Southeast Alaska and coastal British Columbia represent a distinct portion of the genetic diversity for all wolves in North America. Moreover, increased sampling across continental populations will reveal additional variation as exhibited in Yukon-Charley wolves. Given the intensity of current efforts to control wolves in many areas, our assessment of phylogeographic structure across the North Pacific region suggests that a much more refined understanding of genetic variation is needed to ensure the persistence of this high profile carnivore throughout the region.th century. The extension of such international collaborations to include Alaska, British Columbia and Yukon Territory will encourage recognition of the importance of wolf populations in regions outside the extirpation zone, and support the establishment of trans-boundary management plans that maintain the important ecological and genetic diversity of these shared northern populations.Wolves are a trans-boundary species and, as demonstrated here, exhibit metapopulation dynamics that encompass habitats in both Canada and the US. This necessitates increased international cooperation for wolf management and conservation. The success of such geographic integration of management programs has been demonstrated for other taxonomic groups, particularly migratory birds Text S1Sample accession numbers. Description of Canis lupus GenBank mitochondrial DNA sequences and the location of original tissue sample if known. Listed by original publication are populations (abbreviations as given in text), GenBank accession numbers and when available, in corresponding order, the voucher number from either University of Alaska Museum of the North (UAM) or Museum of Southwestern Biology (MSB).(DOCX)Click here for additional data file.


Fenneropenaeus chinensis were investigated using sequence data of the complete mitochondrial control region (CR). Fragments of 993 bp of the CR were sequenced for 93 individuals from five localities over most of the species' range in the Yellow Sea and the Bohai Sea. There were 84 variable sites defining 68 haplotypes. Haplotype diversity levels were very high (0.95 ± 0.03-0.99 ± 0.02) in F. chinensis populations, whereas those of nucleotide diversity were moderate to low (0.66 ± 0.36%-0.84 ± 0.46%). Analysis of molecular variance and conventional population statistics (FST ) revealed no significant genetic structure throughout the range of F. chinensis. Mismatch distribution, estimates of population parameters and neutrality tests revealed that the significant fluctuations and shallow coalescence of mtDNA genealogies observed were coincident with estimated demographic parameters and neutrality tests, in implying important past-population size fluctuations or range expansion. Isolation with Migration (IM) coalescence results suggest that F. chinensis, distributed along the coasts of northern China and the Korean Peninsula (about 1000 km apart), diverged recently, the estimated time-split being 12,800  years ago. Genetic variation and evolutionary demography of the shrimp  Fenneropenaeus chinensis, commonly called the Chinese shrimp or fleshy prawn, is a commercially important species in northern China and Korea  coast population, more numerous and larger in individual size, and the other from the western Korean peninsula (KW) coast population, with relatively smaller numbers and size , where they remain in the so called `nursery grounds' for over two months. By late August to early September, juveniles return to deeper waters, where they mate in October. In mid-November, the shrimps migrate southward to the Yellow Sea overwintering grounds, arriving there in late January. It is worthy to note that overwintering grounds of the two stocks are slightly different,with those from the China Seas being located more eastward, and those from the Korean Peninsula more westward  have been applied to the study of genetic variability at the population level  (H = 0.24) was insufficient for detecting significant genetic structure in this species.It has been shown in a Fenneropenaeus merguiensis, Farfantepenaeus aztecus, Litopenaeussetiferus, Farfantepenaeus duorarum, Marsupenaeusjaponicus,  to examine sequence variability and geographic structure of this species, particularly whether the two populations of F. chinensis distributed along the coasts of north China and the Korean Peninsula (about 1000 km apart) have diverged; (2) to infer historical population processes , or present-day processes  that might have affected the current distribution of this species.Thus, we analyzed samples of F. chinensis were collected from five localities over most of its range during 1997 to 2001 , 0.1 mM primers, 2.0 mM MgCl2, 0.1 mM dNTPs and 2.5 μL 10x PCR buffer, with approximately 30 ng of DNA as template. Cycling conditions were as follows: an initial 3 min at 95 °C, followed by 36 cycles of 50 s at 95 °C, 1 min at 50 °C, and 90 s at 72 °C, with a final 5 min at 72 °C. The PCR products were visualized on 1% agarose gels, and purified with a Takara Agarose Gel DNA Purification Kit . The purified products were used as template DNA for cycle sequencing reactions performed using a BigDye Terminator Cycle Sequencing Kit , whereas sequencing itself was undertaken on an ABI Prism 3730 (Applied Biosystems) automatic sequencer with both forward and reverse primers. The primers used for sequencing were the same as those for PCR amplification. Control region sequences were deposited in the GenBank database under Accession numbers GQ283010-GQ283077.Genomic DNA was isolated from muscle tissue using the standard phenol-chloroform method and subsequently re-suspended in 50 μL TE buffer. Mitochondrial control region (CR) sequences were amplified using the primers DLA: 5'-AAGAACCAGCTAGGATAAAACTTT -3' . PCR ampSequences from both directions in each specimen were aligned with CLUSTAL X1.81 , hierarcF. chinensis was by way of molecular variation analysis  was tested by 1,000 permutations for each pairwise comparison.The examination of significant population structure in s AMOVA,  model, wHaplotype phylogenetic trees were constructed using PAUP  and MrbaD test , based on three parameters, θ0, θ1 (before and after population growth) and τ   . Mismatcal time) . Arlequiapproach . τ valuet, and that descendant populations may exchange migrants in both directions at unequal rates , genetic diversity in the ancestral population (θA = Neμ), divergence time (T = tμ) and migration rates (m12 = m/μ) between the two diverging populations . In addition, the program can register an estimate for the most recent common ancestor (TMRCA) for each locus. The IM method can be used for closely related populations or species; here it was applied to closely related populations defined by the tagging study. This should shed light on the geographical distribution of genetic diversity, divergence time of the two populations and their mutual gene flow.The Hey and Nielsen (2004) IM program was also used to estimate genetic diversity, migration rates and divergence time. The IM-model (isolation with migration) assumes that an ancestral population splits into two populations at a time M, and T were calculated and the values with the highest posterior probability accepted as the best estimates. Values for Nef, t, and TMRCA were calculated using a generation time of 1 year and mutation rates estimated at 1.9 x 10-7 substitutions/site/year  of this sequence was consistent with that of counterpart sequences from other Penaeus species . When regions of ambiguous alignment were removed, 84 segregating sites were detected, defining 68 haplotypes, which included 72 transitions, 12 transversions and 3 indels.A 993-bp CR fragment was bidirectionally sequenced for 93 individuals of i.e., observed in only one location. Two were found in more than one individual, but only in one specific sample locaton, whereas each of the remainder was found in only one individual, respectively. Twelve haplotypes were shared among locations, five  being coincident on both the coasts of China and the Korean peninsula (.The number of detected haplotypes within samples ranged from 15 (KS) to 19 (LD) , Table 3eninsula .h), nucleotide diversity (π), and other sample-specific diversity indices are presented in F. chinensis populations, whereas those of nucleotide diversity were at a moderate to low level (0.66 ± 0.36%-0.84 ± 0.46%). In addition, on comparing genetic variation with that of several other penaeid species  and none statistically significant (p > 0.05). In the 10 possible comparisons, four of the FST estimates were negative  revealed a smooth unimodal pattern, characteristic of population expansion  to a very large size, with a 95% confidence interval (CI) in all cases  years ago, based on a mutation rate of 19%/MY for CR gene  and the Korean Peninsula (KX/KS). Results strongly indicated unimodal posterior distribution at four parameter estimates . Smoothed θ1 posterior distribution peaked at 197.7 (95% HPD: 54.7-1892.8), and that of θA at 23.5 (95% HPD: 7.8-70.4). However, posterior distributions of θ2 were mostly across a broad range of values  suggest that F. chinensis has undergone population expansion. The densities of migration rate parameters are fairly flat (Data not shown), thereby indicating that the data contain little information on migration within the framework of an IM model. Since the model assumes a constant rate of gene flow after population separation, it is expected that migration rates between populations that have only recently split, as appears to be the case, would be difficult to estimate. The posterior distribution of t  peaked at 2.4 (95% HPD: 1.4-3.5), which, when converted to time in years, implies that the two populations began diverging about 12,800 bp . Our estimate of TMRCA substantially predates these estimated times of divergence, and therefore some of the extant diversity was most likely present prior to population divergence. Posterior probabilities of TMRCA peaked at 5.5, indicating that all the sampled haplotypes coalesced at approximately 29,300 bp.IM coalescence was used to further examine whether the two populations of f values , the taiFST population statistics were insignificant , thereby indicating that no significant population structure exists throughout the F. chinensis range, which is consistent with findings from previous research , has only recently diverged. The estimated split-time was 12,800  years ago, suggesting that the two populations were recently derived from a single population. Consequently, these mutual and shallow genetic relationships among F. chinensis populations are consistent with recent divergences (after the Pleistocene), thereby playing an important role in shaping the current genetic structure of Chinese shrimps.Two mutually different, but not exclusive, factors might be responsible for this low level of divergence, ocations . Howeverocations . Moreoveh and moderate π values were clearly observed in the samples of F. chinensis , but diploid loci are expected to be less sensitive in this respect, as a four-fold decrease in population size would be needed to show a reduction comparable with that observed in haploid mtDNA. MtDNA diversity gradients most likely emanated from extinction and recolonization in marginal areas. MtDNA diversities in the newly abundant stocks are expected to be reduced, compared with those in neighboring regions.F. chinensis showed lack of genetic differentiation in this species. However, an RAPD analysis (F. chinensis geographic populations in the Yellow Sea and the Bohai Sea. In their study, shrimp samples were collected from only three localities, two from the Korean Peninsula and one from the coast of China. The subsequent study by the same group using microsatellites detected little genetic differentiation between the populations (F. chinensis, which is extensively distributed in the Yellow and Bohai Seas, consists of two independent populations. Our data, as presented here, are consistent with this result.Most previous studies of the population structure of F. chinensis from the coasts of China and Korea should be treated as separate management units, and fishery management should be conducted separately by regional authorities. Efforts should be made to sustain population stability, since genetic diversity is a prerequisite for evolutionary adaptation to a changing environment (Generally, a single panmictic population is capable of recovering through increased recruitment by propagation . Neverthironment .Our work also shows the potential of coalescence-based models in inferring evolutionary processes, and illustrates the value of a historical perspective in developing a full understanding of contemporary genetic patterns and in designing adequate conservation strategies. On considering that historical population expansion masks low migration, with the subsequent tendency to upgrade gene flow estimates, the low level of differentiation between localities as detected could be explained accordingly.


Maculinea butterflies have been particularly affected because of their specialized lifecycles, requiring both specific food-plants and host-ants. However, the interaction between dispersal, effective population size, and long-term genetic erosion of these endangered butterflies remains unknown. Using non-destructive sampling, we investigated the genetic diversity of the last extant population of M. arion in Denmark, which experienced critically low numbers in the 1980s.Fragmentation of terrestrial ecosystems has had detrimental effects on metapopulations of habitat specialists. Using nine microsatellite markers, we show that the population is genetically impoverished compared to nearby populations in Sweden, but less so than monitoring programs suggested. Ten additional short repeat microsatellites were used to reconstruct changes in genetic diversity and population structure over the last 77 years from museum specimens. We also tested amplification efficiency in such historical samples as a function of repeat length and sample age. Low population numbers in the 1980s did not affect genetic diversity, but considerable turnover of alleles has characterized this population throughout the time-span of our analysis.M. arion is less sensitive to genetic erosion via population bottlenecks than previously thought, and that managing clusters of high quality habitat may be key for long-term conservation.Our results suggest that  While gene flow decreases the differentiation among populations, it may increase genetic diversity within them. Population connectivity is therefore important to maintain overall genetic diversity across small local populations that would otherwise "erode" because of drift . The effEndangered species often occur in small isolated populations where demographic and environmental stochasticity impose additional risks of local extinction. This has made some researchers question the role that genetic factors play in driving population extinction , becauseResearchers have traditionally been forced to evaluate present day diversity of endangered populations against other contemporary populations of the same or closely related species. Such studies are valuable, but since populations rarely have identical demographic and environmental histories, precise identification of the factors that caused extant genetic differences remains impossible. Recent technical advances in the extraction and amplification of old DNA have made the large resources of natural history collections (NHC) available for population genetic studies, providing direct and highly relevant reference points for studies of genetic diversity in endangered populations. Particularly taxa with long histories of collection by entomologists, such as beetles, butterflies and hoverflies, have thus become very useful for long term population studies of genetic change over time.The number of studies utilizing NHC material for evolutionary genetic studies is increasing, and many focus on past and present genetic diversity in endangered populations . DespiteMaculinea arion, is one of many butterfly species that have declined in Europe during the last century, both in terms of population numbers and population connectivity  but the duration of these 'natural' population oscillations are normally shorter than the ones observed on Møn . At preca. 8 ha (breeding area). The site, a grassland habitat, had been left almost untouched in the period 1915-1991, allowing for trees, scrub and larger grasses to invade the area with negative consequences for the food-plant distribution. Since 1991 the population has been managed and monitored every year, and now has a breeding area of ca. 10 ha. The number of imagos was determined using transect walks, a standard method for assessing year to year changes in butterfly abundance [c N≈ 3.5 × the number of imagos counted on the best day. Recent evaluations of butterfly monitoring methods conclude that caution is needed when estimating population census sizes from transect counts [In 1991 a critically low number of imagos was observed flying at the single remaining Høvblege site, which at this point was bundance . Populatbundance , assumint counts -43. The In 1991-1996 the population size was consistently around 50-85 individuals, but increased to much larger numbers in 1997-2005 , to subsequently drop to 70-105 individuals in 2006-2008 Figure . The lacM. arion populations in south and central Sweden  and 2007 (n = 17) using a non-invasive sampling technique, collecting 2 × 2 mmM. arion specimens exist in the collections, reflecting the rarity of the butterfly, which was finally declared protected in Denmark in 1992. Forceps used for the collection of legs were cleaned in bleach (1% sodium hypochlorite) between each sampling to prevent cross contamination.The historical samples were kindly provided by the Danish Natural History Museums in Copenhagen and Aarhus, and included specimens collected at Høvblege and the two nearby, now extinct, sites Jydelejet and Møns Klint. Unless there is a need to distinguish them, we will collectively refer to all these sites as Møn. One middle leg per museum specimen was sampled from years in which a reasonable number of specimens were collected, which gave a time series with 4-12 years between samples, covering 77 years (Table DNA from the contemporary samples was extracted by homogenizing the wing fragment in a solution of 100 μl 5% chelex-TRIS (10 mM) and 5 μl proteinase K (0.75 units). The samples were then incubated at 56°C for 90 min, boiled at 99°C for 15 min, and centrifuged at 13000 rpm for 3 min. The supernatant was stored at -20°C.et al. [2, 2% sodium dodecyl sulphate (SDS), 40 mM dithiothreitol (DTT) and 10% proteinase K . The samples were incubated for 24 h at 56°C with gentle agitation. The extracted DNA was purified using a Qiagen PCR purification kit (QIAquick), re-suspended in 30 μl elution buffer and stored at -20°C. Extraction and PCR-setup was performed in dedicated ancient DNA clean-laboratories at the Centre for GeoGenetics at the Natural History Museum in Copenhagen, where only pre-PCR work occurs. According to standard protocols for work with low quality/quantity DNA [DNA from the historical samples was extracted using a buffer slightly modified from Gilbert et al. , which ctity DNA , contamiENEMAPPER 4.0 (Applied Biosystems).Two sets of nuclear microsatellite markers were employed corresponding to the two research questions . Instead, two independent amplifications were performed for each sample at each locus. If the same genotype was obtained, this was recorded as the consensus genotype. Conversely, if two different genotypes were found (e.g. one homozygote and one heterozygote) a third PCR was conducted. Genotypes were only scored when every allele was observed at least twice, and in cases where a consensus genotype was not found after three PCRs, it was recorded as missing. Genotype error rates were calculated as recommended by Pompanon et al. [We applied a multiple tube approach when genotyping the historical samples, as they were expected to be prone to genotype errors such as allelic dropout and false allele amplification . The amon et al. , i.e. thPCR amplification success was analysed by fitting a general linear model (GLM) with binomial errors and logit link, correcting for over-dispersion, and using the number of samples successfully amplifying at each microsatellite locus as the response variable and the total number of samples as the binominal denominator. The maximum allele length amplified at each locus (in base pairs), the age of the samples (in years), and their interaction were used as explanatory variables. The analysis was carried out in JMP 7.02 (SAS Institute Inc.).STAT 2.9.3[ENALEX 6.3[ICRO-CHECKER 2.2.3 [REENA[ST Fvalues among the historical samples were re-calculated after applying the ENA correction for null alleles as implemented in FREENA and then correlated with the temporal difference between samples using Mantel tests (Mantel 1967) in FSTAT, using 2000 permutations. The contemporary populations did not show signs of null alleles, and pairwise ST Fvalues among the seven Scandinavian populations were calculated in FSTAT, using 1000 permutations. A second measure of genetic diversity, allelic richness, was computed in FSTAT for both contemporary and historical samples and differences in allelic richness and expected heterozygosity among samples were tested using repeated-measures ANOVA in JMP.Linkage disequilibrium among pairs of microsatellite loci was tested using FTAT 2.9.3. The proNALEX 6.3 was usedER 2.2.3  was used.3 [REENA. High nu.3 [REENA,51. Pairk) than the range of allele sizes (r), leading to a smaller M-ratio (= k/r) in size-reduced populations compared to equilibrium populations. In order to evaluate the empirical M-ratio, an equilibrium population was simulated based on parameters describing the evolution of the analysed microsatellite loci  and the effective population size of pre-bottlenecked populations (eN). These parameters are difficult to estimate in empirical samples and each sample estimate of M-ratio was thus tested under different evolutionary scenarios as suggested by Guinand and Scribner [g Δvarying between 2 and 4  equal to 0.02, 0.04, 0.1 and 0.2. For each sample, an equilibrium population was simulated 10000 times using these parameter settings. The empirical M-ratio averaged across loci was compared to the distribution of simulated M-ratios, in order to evaluate the likelihood of a bottleneck event having taken place (95% criterion).Evidence of recent genetic bottlenecks in the temporal samples was tested using the software developed by Garza and Williamson . The proScribner . The scelliamson  suggest LVU carried out the experimental work, and analysed the data together with DRN. PSN contributed with the survey data. The study was designed by LVU, JJB and DRN, who also wrote the manuscript. All authors have approved of the final version of the manuscript.Table S1 - Microsatellite loci used in the study. Name, GenBank accession numbers, repeat motif and primer sequences  are given for newly developed microsatellite loci (for previously developed loci see references below). Product size in base pairs and the optimal annealing temperature in degrees Celsius for M. arion are also provided. N = number of study populations; n = number of genotyped individuals; k = observed number of alleles; Ho = observed heterozygosity. The genotype error rate calculated per locus and the fraction of positive PCRs per locus is given separately for historic samples (1930-1975) and contemporary samples (2005-2007).Click here for fileM. arion populationFigure S1 - Presence of 'Ghost' alleles in the isolated Danish . Allele frequencies per microsatellite locus found in each sampling year. Twelve alleles are only present in the historical samples (black arrows), whereas two alleles are unique to the contemporary (and 1975) samples (white arrows). Microsatellite loci name abbreviations: Ma = Macari, Mu = Macu.Click here for fileTable S2 - Detection of recent genetic bottlenecks. Empirical M-ratio values averaged over the ten microsatellite loci for each historic (1930-1957) and contemporary  sampling year. An equilibrium population was simulated 1000 times for the parameter combination of Θ, Δg and ps, and P-values for the occurrence of a genetic bottleneck computed. Grey cells show evidence of a bottleneck (P< 0.05), whereas black cells show no evidence of a bottleneck.Click here for file


Antheraea assama), popularly known as muga silkmoth, is a semi-domesticated silk producing insect confined to a narrow habitat range of the northeastern region of India. Owing to the prevailing socio-political problems, the muga silkworm habitats in the northeastern region have not been accessible hampering the phylogeography studies of this rare silkmoth. Recently, we have been successful in our attempt to collect muga cocoon samples, although to a limited extent, from their natural habitats. Out of 87 microsatellite markers developed previously for A. assama, 13 informative markers were employed to genotype 97 individuals from six populations and analyzed their population structure and genetic variation.The Indian golden saturniid silkmoth (ST = 0.301) was due to high genetic diversity contributed by WWS-1 population. Analysis of the remaining five populations (excluding WWS-1) showed a marked reduction in the number of alleles at all the employed loci. Structure analysis showed the presence of only two clusters: one formed by WWS-1 population and the other included the remaining five populations, inferring that there is no significant genetic diversity within and between these five populations, and suggesting that these five populations are probably derived from a single population. Patterns of recent population bottlenecks were not evident in any of the six populations studied.We observed highly significant genetic diversity in one of the populations . Further analysis with and without WWS-1 population revealed that dramatic genetic differentiation (global FA. assama inhabiting the WWS-1 region revealed very high genetic diversity, and was genetically divergent from the five populations studied. The efforts should be continued to identify and study such populations from this region as well as other muga silkworm habitats. The information generated will be very useful in conservation of dwindling muga culture in Northeast India. Antheraea assama, Lepidoptera: Saturniidae), popularly known as muga silkmoth, is a semi-domesticated, golden colored silk producing insect, endemic to Northeast India Comparison of genetic structure among populations of a species, which have carved their own niche in a narrow ecological range, can illuminate our understanding of environmental factors affecting their adaptation and selection forces operating on them to retain such species for a long time Ecological research on insect species provide invaluable information on population structure, speciation, gene flow and genetic diversity, and offers an explanation on insect diversity based on their interaction with environmental factors, either biotic (including other species) or abiotic. Molecular marker data help to distinguish populations of a species as well as taxonomic relationships of a species in question A. assama collected from different locations of Northeast India. We have also attempted to analyze the genetic structure and test for signs of a bottleneck in these populations.Recently, we reported 12 informative SSR markers, in a muga silkworm population, out of 87 derived from Expressed Sequence Tags (EST-SSRs) and a repeat enriched genomic library (genomic-SSRs) A. assama with 13 informative microsatellite markers In the present study, we genotyped 97 individuals from 6 populations  of A. asAmong 13 polymorphic microsatellite markers used on 6 populations, five loci showed within population polymorphism in all the populations . Number Information on percentage polymorphism, number of private alleles, number of alleles per locus etc., of six populations under study is presented in IS (inbreeding coefficient) is the proportion of variance in the subpopulation contained in an individual. In the present analysis we observed high FIS values when all the six populations were considered (FIS = 0.502) (IS = 0.447)   and also= 0.447) . High FIThe degree of correspondence between the geographic distance matrix and genetic distance matrix was analyzed using Mantel test The observed and expected heterozygosities varied depending on the microsatellite loci. Eighteen of the 50 population-loci combinations showed no significant departures from HWE . Three oad hoc statistical analysis based on the second-order rate of change of the likelihood function with respect to K (ΔK), as described previously Population structure was investigated using the STRUCTURE program to estimate the number of genetically distinct populations (K). To calculate the most appropriate K value formed by the 97 individuals, we utilized an A. assama moths can fly for up to 20 Km distance, there seems to be migration between populations. This is evident from the structure analysis that showed shared genetic clusters between populations. Though there was no big physical barrier that restricts the migration of A. assama from WWS-1 region, we speculate that the presence of bamboo thicket and tall trees (such as Shorea and Tectona species) around this region may act as a barrier, thus limiting the migration of A. assama from the WWS-1 region.All the populations except WWS-1 formed one cluster and WWS-1 formed a separate cluster. As all the six populations are less than 100 km apart from each other and as the Since WWS-1 was found to form a separate cluster in STRUCTURE analysis, we carried a of population structure analysis excluding WWS-1. The five populations did not show any distinct structure to suggest that they are individual populations. The analysis revealed the presence of two sub-clusters in five populations, indicating that there is a mix of two populations .A neighbour joining (NJ) tree was constructed using Populations program with Nei’s minimum genetic distance matrix values . PopulatAMOVA was calculated for all the six populations and also separately for five population, excluding WWS-1, to examine the effect of highly diverse population WWS-1 on overall variability. AMOVA revealed that variation among populations  was almost same as that observed among individuals within populations  when all the six populations were considered for analysis. Interestingly, when only five populations were considered (excluding WWS-1), among populations variation was found to be only 9% (variance component 0.11), which is far less than that observed among individuals within populations . Variation within individuals was similar when WWS-1 was included  and when it was excluded (37%) from the populations. These results indicate that high among population diversity was due to genetically diverse population, WWS-1.P = 0.009), all the other populations did not show any significant heterozygote deficiency or excess. This heterozygote deficiency may be due to presence of null alleles, as our analysis revealed possible occurrence of null alleles in three of the loci tested.Populations that have experienced a recent reduction in their effective population size exhibit a correlated reduction in allele numbers and heterozygosities at polymorphic loci. It is expected that the allelic diversity is reduced faster than the heterozygosity, i.e. the observed heterozygosity is larger than the heterozygosity expected from the observed allele number where the locus is at mutation-drift equilibrium Low between population genetic diversity and high within population diversity, and lack of apparent population structure and higher number of shared alleles between 5 populations (excluding WWS-1) indicate that the five populations excluding WWS-1 may indeed constitute a single population.A. assama. Our analysis showed no real evidence of a significant correlation between genetic and spatial distance of different populations. Among the six populations examined in this study, WWS-1 population showed very high genetic diversity and was genetically divergent from the remaining five populations. On the other hand, the five populations, which we had collected from different regions were quite similar and are likely to have been founded from a single population. The A. assama populations like WWS-1, can be utilized in the conservation efforts of the muga silkworm. Though present analysis gave a reasonable insight into population genetics of A. assama, the analysis with a bigger sample size and including many more forest-based population, would give a much larger picture. However, difficulty in collecting more samples owing to hostile conditions particularly inaccessible forest terrains, politically sensitive border areas and prevailing insurgency conditions is hampering further studies on population genetics of this silkmoth.This is the first known report describing population genetics of A. assama in whole of Northeast India.Since the sampling is only from a part of the Northeast Indian region inhabited by this silkmoth, it may not account for the genetic variability existing in the populations in other regions. Efforts are being made to collect insect samples from other regions. These efforts, if successful, would give a clear picture of population structure and other details of population variability of A. assama were collected from six different geographical locations of Northeast India . The reactions were carried out in a Vapo-protect Gradient thermal cycler . The PCR conditions were; 94°C for 3 minutes as initial denaturation, and 35 cycles of: 94°C denaturing for 20 seconds, the appropriate annealing temperature for 10 seconds and 72°C extension for 45 seconds and 72°C for 10 minutes as final extension. All the amplicons were run on an ABI PRISM 3730 DNA analyzer, with Pop-7 as sieving matrix, HiDi Formamide as single-stranded DNA stabilizer and GeneScan 500 ROX standard as a size marker. ABI PRISM GeneMapper software version 3.0 was used to size the alleles.The primers were labelled at 5′ end using two different fluorescent dyes, namely TAM (yellow) and FAM (blue) . All polA), percentage of polymorphic loci (P), mean unbiased estimates of genetic diversity (HE) O) were estimated using GenAlex 6 software http://bioinformatics.org/~tryphon/populations/). A NJ tree was also constructed on per individual basis to test whether individuals from WWS-1 form different cluster, using Populations software and considering Nei minimum distance method (http://bioinformatics.org/~tryphon/populations/).The level of polymorphism was tested with 13 microsatellite markers, which were evaluated earlier for their informativeness, by genotyping all the individuals from six populations. For the EST-SSRs and genomic SSRs, mean number of alleles Click here for additional data file.Table S1Details of microsatellite loci used in this study . Fluorescent labelling was done for only forward primers of all the loci.(DOCX)Click here for additional data file.Table S2Summary of private alleles by population.(DOCX)Click here for additional data file.Table S3Summary of Chi-square tests for Hardy-Weinberg equilibrium.(DOCX)Click here for additional data file.