Mitochondrial DNA Diversity of Orchid Bee Euglossa fimbriata (Hymenoptera: Apidae) Populations Assessed by PCR-RFLP

Euglossa fimbriata is a euglossine species widely distributed in Brazil and occurring primarily in Atlantic Forest remnants. In this study, the genetic mitochondrial structure of E. fimbriata from six Atlantic Forest fragments was studied by RFLP analysis of three PCR-amplified mtDNA gene segments (16S, COI-COII, and cyt b). Ten composite haplotypes were identified, six of which were exclusive and represented singleton mitotypes. Low haplotype diversity (0.085–0.289) and nucleotide diversity (0.000–0.002) were detected within samples. AMOVA partitioned 91.13% of the overall genetic variation within samples and 8.87% (?_st = 0.089; P < 0.05) among samples. Pairwise comparisons indicated high levels of differentiation among some pairs of samples (?_st = 0.161–0.218; P < 0.05). These high levels indicate that these populations of E. fimbriata, despite their highly fragmented landscape, apparently have not suffered loss of genetic variation, suggesting that this particular population is not currently endangered.     

Genetic Variation in Chinese Pine (Pinus tabulaeformis), a Woody Species Endemic to China

Random amplified polymorphic DNA markers were used to investigate genetic variation of the Chinese pine (Pinus tabulaeformis Carr.), a species endemic to China and the most widely distributed pine species in North China. The results revealed that P. tabulaeformis populations had a relatively high level of genetic diversity (H _t = 0.3268), distributed mainly within (79.2%) rather than among (20.8%) populations. The populations of Lingkong Mountain and Wuling Mountain had a higher level of diversity (0.2687) than the other four populations (0.2537). No statistically significant relationships were found between genetic diversity and climatic factors by correlation analysis and between genetic distance and geographic distance by the Mantel test. These results suggest that the partitioning of genetic diversity in each population might have been influenced not only by water and temperature conditions but also by other factors such as human activities and the Holocene postglacial history of these populations.     

Structure of genetic diversity in marginal populations of black poplar (Populus nigra L.)

It has been hypothesized that populations at the margins of the distributional range of a species show reduced genetic diversity and increased inter-population differentiation compared to central populations. Here, we test this hypothesis by examining the structure of genetic diversity in marginal populations of black poplar, Populus nigra L. (Salicaceae). This species occurs mainly in Europe but its range extends to central Asia. We collected 117 individuals from 10 populations at the edge of the distributional range of the species in central Asia to examine the structure of genetic diversity based on genetic polymorphisms at 20 microsatellite markers. As expected, the genetic diversity within these marginal populations is relatively low, with an average observed heterozygosity H_o of 0.337 and an average expected heterozygosity H_e of 0.466, compared to the genetic diversity of populations from central distributions. However, we recovered very low genetic differentiation between populations, with an average F_st of 0.0745, a value similar to those reported for central populations. AMOVA analyses confirmed this result, showing that only 9.2% of the total variation could be attributed to between-population variance (P < 0.001). Our findings do not fully support hypotheses about the structure of genetic diversity in marginal populations formed from observations on other species. We suggest that a high rate of outcrossing and possible postglacial colonization at the edge of the distributional range of this long-lived poplar may explain the observed structure of the genetic diversity.     

High level of genetic variation within clonal orchid Goodyera repens

Type of reproduction has an important effect on the maintenance of particular populations and species persistence in time and space. This trait significantly influences the ecological and genetic structure of populations, and in consequence the evolution of species. The primary objectives of this study were: to estimate genetic diversity within and among populations of clonal species Goodyera repens from different populations in northeastern Poland, and to amount factors shaping the genetic structure of this orchid. Based on 451 rosettes of G. repens from 11 localities in northeastern Poland, we conducted a genetic population analysis using allozymes. We included information on population size, flowering, fruit set and seed dispersal to elucidate their influences on genetic diversity of this species. Populations differed according to demographic properties. The majority of seeds (86.4–94.8 %) were found at a distance of 0.2 m. We observed a high level of genetic (P _PL = 50 %, A = 1.68, H _O = 0.210, H _E = 0.204) and genotypic diversity (G = 163, G/N _S = 0.66, G _U = 30.2 %), and low but statistically significant genetic differentiation among populations (F _ST = 0.060; P < 0.001). We suggest that the genetic diversity of G. repens is mainly an effect of the abundance of pine and spruce forest communities suitable for this species in NE Poland and the high level of sexual reproduction.     

Genetic diversity and structure of Lilium pumilum DC. in southeast of Qinghai–Tibet plateau

Lilium pumilum DC. is a valuable species not only for its showy flowers but also for its edible and medicinal values. As one of the distribution areas of L. pumilum, Qinghai–Tibet plateau has unique environmental features which have high impact on the evolution of the species. No population genetic studies have been done for L. pumilum so far. To provide the first reference data for evolutionary study and understanding the influence of eco-geographic factors on the distribution of genetic variation in L. pumilum, interspecific simple sequence repeat markers were used to investigate genetic diversity and population structure of 28 populations sampled from southeast of Qinghai–Tibet plateau. Fifteen selected primers generated a total of 147 polymorphic bands. The genetic diversity was low within populations (average He = 0.173), but higher at the species level (He = 0.392). A clear population structure and high level of genetic differentiation (F _ST = 0.518) were detected by unweighted pair group method for arithmetic averages, principle coordinate analysis and Bayesian clustering. All clustering approaches supported a division of the 28 populations into 4 major groups for which analysis of molecular variance confirmed a significant variation among groups (34.3 %). These population genetic parameters suggest limited gene flow among populations and evidence for isolation by distance (r = 0.272, P < 0.0001) was found in this study. Altitude, AMT and AMP explained 9.5, 11.5 and 14.0 % of the total variance among populations indicating that eco-geographic factors have a significant effect. Considering the low within-population genetic diversity, high differentiation among populations and the increasing anthropogenic pressure on the species, in situ conservation measures were recommended to preserve L. pumilum in Qinghai–Tibet plateau.     

Genetic diversity and genetic structure in wild populations of Mexican oregano (Lippia graveolens H.B.K.) and its relationship with the chemical composition of the essential oil

Mexican oregano is an aromatic plant traditionally harvested from wild populations by rural communities; however, there is little information about population genetics aspects of this species. Moreover, considering that the variation in essential oil production of aromatic plants has been attributed to several environmental as well as genetic factors, in this study we estimated the genetic diversity and genetic structure from 14 wild populations of L. graveolens located in four different bioclimatic regions in southeastern Mexico using AFLP markers. The overall genetic diversity of L. graveolens described as the percentage of polymorphic loci (PPL = 60.9 %) and Nei’s gene diversity (H _j  = 0.17) was moderate, but not associated with the bioclimatic conditions. Genetic variation was analyzed at chemotype and population levels. Regarding chemotypes, thymol had the highest genetic diversity (PPL = 82.8 % and H _j  = 0.22). PCoA revealed that chemotypes exhibit a certain level of genetic differentiation. Maximum parsimony dendrogram showed a grouping of individuals with a predominant chemotype. Bayesian analyses revealed a low, but significant differentiation among chemotypes (θ _ΙΙ = 0.008). Regarding populations, gene diversity showed significant differences (F _13,1204 = 22.8, P < 0.001); populations dominated by individuals from the thymol chemotype showed the highest gene diversity (H _j  = 0.31–0.25), while populations with exclusively sesquiterpene chemotype showed the lowest value (H _j  = 0.058). Cluster and Bayesian analyses (θ _ΙΙ = 0.027) revealed a low level of genetic differentiation among populations. Correlation analysis showed a significant association between the distance matrices based on the genetic markers (AFLP) and chemical compounds of essential oil (r = 0.06, P < 0.001). Our results suggest an important genetic influence on the observed chemical profiles. Nevertheless, other biotic and abiotic environmental pressures also play an important role in determining the chemotype and structure found in this aromatic species.     

Population structure and genetic diversity of the only extant Baroninae swallowtail butterfly,Baronia brevicornis,revealed by ISSR markers

Due to its relict nature, the unique Baroninae swallowtail, Baronia brevicornis, is considered a “living fossil”. It is also one of the most enigmatic butterfly species with contentious origins and peculiar ecological characteristics. The aim of this study is to evaluate the genetic diversity and population structure of this endemic species of butterfly in Mexico. We sampled populations in two areas within its restricted geographical range in central Mexico and the isolated subspecies population in the state of Chiapas. Three ISSR primers produced 66 loci, indicating a high genetic diversity (P = 100 %, H _e  = 0.22) and variation range in these populations (62 % < P < 85 %, 0.18 < H _e  < 0.25). The Chiapas population presented the lowest values. The observed high values can be explained by the population dynamic of this species characterized by a very high density of individuals over very limited areas. Variation between populations appears to reflect both the age of colonization and locality perturbation level. Two methods of genetic structure analysis (Self-Organizing Map and Structure analysis) match to define three clusters. Natural and anthropogenic barriers may explain the separation between two clusters (cluster 1 and 2) of central Mexico but an unexpected result revealed that the Chiapas population is not genetically distinguishable from the central Mexico populations (cluster 3) leading us to hypothesize a possible “recent” separation or anthropogenic introduction. Habitat and host plant specificity probably limits the exchange of individuals between populations thus increasing fragmentation and leading to a complex genetic structure. We should put in place population monitoring schemes at different spatial scales, combining field occurrences and genetic tools, in order to reduce extinction susceptibility and keep track of recolonization events for this enigmatic species.     

Planchonella, first record of gynomonoecy for the family Sapotaceae

Inter-simple sequence repeat (ISSR) markers were used to investigate the levels and pattern of genetic variation within and among populations of Pteroceltis tatarinowii Maxim., an endangered plant endemic to China. Of the 76 primers screened, 11 produced highly reproducible ISSR bands. A total of 118 bands were presented from the 11 selected primers across all individuals of five natural populations, corresponding to an average of 10.73 bands per primer. The size of the ISSR bands ranged from 200 to 2,000 bp. The percentage of polymorphic loci at the population level ranged from 77.97 to 86.44%, with an average value of 82.54%. Genetic differentiation among populations was revealed based on Nei’s genetic diversity analysis (19.41%) and the nonparametric analysis of molecular variance (20.62%). The Mantel test showed a significant positive correlation between geographic distance and genetic distance (r = 0.7758, P < 0.05), indicating a role of geographic isolation in shaping the present population genetic structure of P. tatarinowii. The size of the natural populations of P. tatarinowii was noted in field observations to be very small, chiefly owing to habitat destruction and overexploitation in the past decades. Therefore, effective measures for preserving genetic diversity of this species at the population level are needed and should include protecting its natural habitats and increasing the numbers of individuals. To meet the commercial demand for this species, P. tatarinowii plantations and cultivation facilities should be established as an alternative source of raw materials.     

Genetic diversity of the endangered Chinese endemic herb Saruma henryi Oliv. (Aristolochiaceae) and its implications for conservation

Saruma henryi Oliv., the only representative of the monotypic genus Saruma Oliv. (Aristolochiaceae), is an endangered perennial herb endemic to China. It is a phylogenetically, ecologically, and medicinally important species. In the present study, inter-simple sequence repeat (ISSR) markers were employed to investigate the genetic diversity and differentiation of 14 populations. A total of 16 selected primers yielded 175 bright and discernible bands, with an average of 10.94 per primer. POPGENE analysis showed that the genetic diversity was quite low at the population level (h = 0.0447–0.1243; I = 0.0642–0.1853; PPB = 10.29–36.57%), but pretty high at the species level (h = 0.2603; I = 0.3857; PPB = 73.71%). The hierarchical analysis of molecular variance (AMOVA) revealed a high level of genetic differentiation among populations (67.18% of total variance components, P < 0.001), in line with the gene differentiation coefficient (G _ST = 0.6903) and the limited among-population gene flow (N _m = 0.2243). Both Principal Coordinates Analysis (PCoA) and UPGMA cluster analysis supported the grouping of all 14 populations into three geographic groups, among which there occurred a moderate level of genetic differentiation (33.18% of total variance components, P < 0.001) as shown by AMOVA analysis. In addition, Mantel test revealed a significant correlation between genetic and geographic distances among populations (r = 0.7792, P = 0.001), indicating the role of geographic isolation in shaping its present population genetic structure. The present levels and patterns of genetic diversity of S. henryi were assumed to result largely from its breeding system, geographic isolation, clonal growth, its unique biological traits and evolutionary history. The high genetic differentiation among populations implies that the conservation efforts should aim to preserve all the extant populations of this endangered herb.     

Genetic diversity of the African poplar (<Emphasis Type="Italic">Populus ilicifolia</Emphasis>) populations in Kenya

We evaluated the genetic diversity of the African poplar (Populus ilicifolia) populations found in Kenya compared with reference samples of five poplar species from North America and one species introduced in Kenya from India (KEFRI-Kenya). Amplified fragment length polymorphism (AFLP) was used with the objective of providing important information for breeding and in situ/ex situ conservation of this species. Samples collected from three locations along the species’ natural range (Athi, Ewaso Nyiro, and Tana rivers) were compared with four samples of locally planted Populus deltoides stand introduced from India and ten reference samples from North America. Six AFLP primer combinations produced 521 clear bands for analysis. The percentage polymorphic loci were lowest in Tana (20.4 %) and highest in Athi (40.6 %). The average heterozygosity across the studied populations was between 0.07 and 0.3. AMOVA revealed more genetic variation partitioning within population (87 %; P?<?0.01) than among populations (13 %; P?<?0.01) suggesting significant genetic variation between populations. Further, UPGMA delineation showed two clusters of the Tana, Athi, and Ewaso Nyiro populations clustered together compared to the North America and India/KEFRI reference samples. Moreover, the study showed that the Athi population is more diverse than those of Tana and Ewaso Nyiro and may be important for conservation, domestication, and improvement studies. The genetic differentiation (F _ST ?=?0.134) among Kenyan P. ilicifolia populations suggests limited possibility of gene flow between these populations.     

Comparative Analysis of Genetic Diversity and Population Structure of Sipunculus nudus as Revealed by Mitochondrial COI Sequences

Genetic diversity and population structure of Sipunculus nudus were evaluated using a 652 base pair fragment of the mitochondrial cytochrome oxidase I gene. The populations were collected from Beihai, Sanya, and Xiamen. A total of 71 polymorphic sites defined 16 distinct haplotypes. The mean haplotype diversity and nucleotide diversity of the three populations were 0.9354 ± 0.0168 and 0.0035 ± 0.0018, respectively. Analysis at the intrapopulation level showed that the Beihai population had the greatest haplotype and nucleotide diversity, followed by the Xiamen and Sanya populations. Analysis of molecular variance showed significant genetic differentiation among the three populations (Fst = 0.0796, P < 0.05). The present results revealed that S. nudus populations had a high level of genetic diversity and distinct population structures.     

Genetic and epigenetic variation in mass selection populations of Pacific oyster Crassostrea gigas

Selective breeding often produces an improvement in phenotype. Much of the phenotypic change within a species is a consequence of genetic variation. However, there is growing evidence for phenotypic change even in the absence of DNA sequence polymorphisms, termed epigenetic variation. This study’s goal was to investigate the genetic and epigenetic variation in the mass selection populations of the Pacific oyster (Crassostrea gigas), determine if any correlation exists between the genetic and epigenetic variations. This can serve as a first step in investigating the potential role epigenetic variations have in selective breeding. Amplified fragment length polymorphism analysis and methylation-sensitive amplified polymorphism methodology were used to monitor genetic and epigenetic variation in two populations (the base stock and the third selected generation) from a mass selection line in the Pacific oyster. The correlation between genetic and epigenetic variation was evaluated by Co-Inertia Analysis. The genetic difference was mainly found in the gene frequency shift revealed by the F _ST value (0.0151, P < 0.01) and no significant reduction in genetic diversity was detected. The percentage of methylation in C. gigas was 26.4 %. No significant difference was observed on the average state of methylation, but a few bands showed different frequencies between the two populations. Co-Inertia Analysis revealed a significant association between the genetic and epigenetic profiles (P < 0.01).     

Chloroplast DNA-based genetic diversity and phylogeography of Pyrus betulaefolia (Rosaceae) in Northern China

Pyrus betulaefolia Bunge, considered as an intermediate between oriental and occidental pear groups, is one of the most important wild pear species. The number of its populations is decreasing because of habitat destruction, fragmentation, and continuous exploitation, so protection and conservation measures are urgently needed. Assessment of its genetic diversity and phylogeography are imperative for its efficient conservation. Two chloroplast DNA intergenic fragments were used to detect genetic diversity and phylogeography of 320 individuals from 18 wild P. betulaefolia populations. Haplotype variation, genetic differentiation, and historical events of the populations were estimated. The results showed that P. betulaefolia populations sampled in northern China contained a high level of genetic diversity (H _T?=?0.826). A significant isolation-by-distance value (r?=?0.587, P?<?0.001, 1,000 permutations) among all 18 populations indicated a correlation between genetic divergence and geographic distance. Four population groups were identified in a neighbor-joining tree based on the genetic distance. Analyses of molecular variation showed that the genetic variation mainly existed among population groups, representing 64.61 % of the total variation. Phylogeographic analyses indicated that the populations of P. betulaefolia experienced a scenario of rapid range expansion, which probably occurred between 608,000 and 204,580 years ago. Meanwhile, both the restricted gene flow with isolation by distance and allopatric fragmentation were crucial processes responsible for shaping the genetic patterns of P. betulaefolia. The occurrence of specific haplotypes might be ascribed to an ancestral introgression or joint retention of an ancestral polymorphism with other Pyrus species at the northern edge of the distribution of P. betulaefolia. Three populations displaying a high level of haplotype diversity and unique haplotypes were assumed to be relict populations of Quaternary glaciation and should have conservation priority. Three additional large populations should also be preferentially protected by building natural preservation zones.     

Genetic Divergence and Signatures of Natural Selection in Marginal Populations of a Keystone,Long-Lived Conifer,Eastern White Pine (Pinus strobus) from Northern Ontario

Marginal populations are expected to provide the frontiers for adaptation, evolution and range shifts of plant species under the anticipated climate change conditions. Marginal populations are predicted to show genetic divergence from central populations due to their isolation, and divergent natural selection and genetic drift operating therein. Marginal populations are also expected to have lower genetic diversity and effective population size (N _e) and higher genetic differentiation than central populations. We tested these hypotheses using eastern white pine (Pinus strobus) as a model for keystone, long-lived widely-distributed plants. All 614 eastern white pine trees, in a complete census of two populations each of marginal old-growth, central old-growth, and central second-growth, were genotyped at 11 microsatellite loci. The central populations had significantly higher allelic and genotypic diversity, latent genetic potential (LGP) and N _e than the marginal populations. However, heterozygosity and fixation index were similar between them. The marginal populations were genetically diverged from the central populations. Model testing suggested predominant north to south gene flow in the study area with curtailed gene flow to northern marginal populations. Signatures of natural selection were detected at three loci in the marginal populations; two showing divergent selection with directional change in allele frequencies, and one balancing selection. Contrary to the general belief, no significant differences were observed in genetic diversity, differentiation, LGP, and N _e between old-growth and second-growth populations. Our study provides information on the dynamics of migration, genetic drift and selection in central versus marginal populations of a keystone long-lived plant species and has broad evolutionary, conservation and adaptation significance.     

Conservation genetics of the annual hemiparasitic plant <Emphasis Type="Italic">Melampyrum sylvaticum</Emphasis> (Orobanchaceae) in the UK and Scandinavia

Melampyrum sylvaticum is an endangered annual hemiparasitic plant that is found in only 19 small and isolated populations in the United Kingdom (UK). To evaluate the genetic consequences of this patchy distribution we compared levels of diversity, inbreeding and differentiation from ten populations from the UK with eight relatively large populations from Sweden and Norway where the species is more continuously distributed. We demonstrate that in both the UK and Scandinavia, the species is highly inbreeding (global F _IS = 0.899). Levels of population differentiation were high (F’_ST = 0.892) and significantly higher amongst UK populations (F’_ST = 0.949) than Scandinavian populations (F’_ST = 0.762; P < 0.01). The isolated populations in the UK have, on average, lower genetic diversity (allelic richness, proportion of loci that are polymorphic, gene diversity) than Scandinavian populations, and this diversity difference is associated with the smaller census size and population area of UK populations. From a conservation perspective, the naturally inbreeding nature of the species may buffer the species against immediate effects of inbreeding depression, but the markedly lower levels of genetic diversity in UK populations may represent a genetic constraint to evolutionary change. In addition, the high levels of population differentiation suggest that gene flow among populations will not be effective at replenishing lost variation. We thus recommend supporting in situ conservation management with ex situ populations and human-mediated seed dispersal among selected populations in the UK.     

Genetic structure of the Korean black scraper Thamnaconus modestus inferred from microsatellite marker analysis

The Korean black scraper, Thamnaconus modestus, is one of the most economically important maricultural fish species in Korea. However, the annual catch of this fish has been continuously declining over the past several decades. In this study, the genetic diversity and relationships among four wild populations and two hatchery stocks of Korean black scraper were assessed based on 16 microsatellite (MS) markers. A total of 319 different alleles were detected over all loci with an average of 19.94 alleles per locus. The hatchery stocks [mean number of alleles (N _A) = 12, allelic richness (A _R) = 12, expected heterozygosity (He) = 0.834] showed a slight reduction (P > 0.05) in genetic variability in comparison with wild populations (mean N _A = 13.86, A _R = 12.35, He = 0.844), suggesting a sufficient level of genetic variation in the hatchery populations. Similarly low levels of inbreeding and significant Hardy–Weinberg equilibrium deviations were detected in both wild and hatchery populations. The genetic subdivision among all six populations was low but significant (overall F _ST = 0.008, P < 0.01). Pairwise F _ST, a phylogenetic tree, and multidimensional scaling analysis suggested the existence of three geographically structured populations based on different sea basin origins, although the isolation-by-distance model was rejected. This result was corroborated by an analysis of molecular variance. This genetic differentiation may result from the co-effects of various factors, such as historical dispersal, local environment and ocean currents. These three geographical groups can be considered as independent management units. Our results show that MS markers may be suitable not only for the genetic monitoring of hatchery stocks but also for revealing the population structure of Korean black scraper populations. These results will provide critical information for breeding programs, the management of cultured stocks and the conservation of this species.     

Genetic diversity and structure of natural Pinus tabulaeformis populations in North China using amplified fragment length polymorphism (AFLP)

Chinese pine (Pinus tabulaeformis carr.), endemic to China, is a conifer species with extensive and fragmented distribution in North China. In this study, the genetic diversity and structure of 20 natural populations of this species were investigated using amplified fragment length polymorphism (AFLP) markers. A total of 445 fragments were revealed with 8 pairs of primers, 379 (85.17%) of which were polymorphic. A moderate level of genetic diversity was detected at the species level (Shannon's information index I = 0.356, Nei's gene diversity H_E = 0.271) and at the population level (I = 0.219, H_E = 0.206). Most of genetic variation was within populations while a considerable level of genetic differentiation was detected (G_ST = 0.352, Ф_ST = 0.304). The high differentiation could be attributed to the complex and fragmented habitats, and a limited gene flow among populations (N_m = 0.572). The Mantel test indicated that there was significant correlation (r = 0.455, P < 0.001) between Nei's genetic distance and geographical distance among all the populations. The results suggested that proper countermeasures should be taken to prevent the habitat further deterioration and maintain the genetic diversity of this species.     

Population genetic differentiation of Schisandra chinensis and Schisandra sphenanthera as revealed by ISSR analysis

Schisandra chinensis (Turcz.) Baill. and Schisandra sphenanthera Rehd. et Wils. are well-known Chinese medicinal plants. The population genetic variation of the two species was studied using inter simple sequence repeat (ISSR) molecular markers. High levels of genetic diversity are revealed in both S. chinensis (P = 88.36%, h = 0.2894, I = 0.4396) and S. sphenanthera (P = 84.09%, H = 0.2782, I = 0.4280). However, the population genetic differentiation is significantly different between the two species. The S. sphenanthera harbors as high as 27% of the genetic variation among populations but 73% within populations, whereas in S. chinensis 17% of the genetic variation occurs among populations and 83% within populations. Both significant (P < 0.05) heterozygosity excess and shifted mode of allele frequency distribution are detected in four out of six populations of S. chinensis and one out of five populations of S. sphenanthera, suggesting the occurrence of recent population bottlenecks in the two species. The different patterns of genetic variation in S. chinensis and S. sphenanthera are discussed in relation to their differences in pollination mechanism, geographic distribution and historical events, and the level of gene flow and genetic drift.     

Genetic structure in the paleoendemic and endangered Petagnaea gussonei (Spreng.) Rauschert (Saniculoideae, Apiaceae) and implications for its conservation

Our investigation aims to understand the genetic structure and evolutionary history of Petagnaea gussonei, an ancient and endangered species belonging to the Saniculoideae subfamily (Apiaceae). It is paleoendemic to Sicily, with a small number of populations in the Nebrodi Mountains. A total of seven chloroplast microsatellite repeat loci and 12 AFLP primer combinations were used to screen 115 individuals corresponding to 17 populations. The ratio of seed to pollen flow was also calculated using the modified Ennos equation. A relatively high level of genetic diversity was detected with AFLPs (e.g., 0.045 < H < 0.278), and a moderate variation was also found using cpSSRs (0 < Hk < 0.667). Two different haplotypes (B and W) were identified, with five populations being monomorphic for haplotype B. There was no genetic differentiation on the basis of haplotypic frequency (G _ST) and similarity (R _ST), and no phylogeographic structure was detected among the populations. AFLP values also confirmed that the populations are not very genetically differentiated. The principal component analysis based on pairwise genetic differences showed three groupings without a geographical correlation. The AMOVA analysis indicates that the amount of variation is higher within populations (82 %) than among populations (18 %). Results of the pollen flow/seed flow ratio indicated positive values for each population, indicating that gene flow by seed is not more efficient than by pollen. Instead, the total pollen/seed flow for all population presents a negative value, suggesting that pollen dispersal does not appear to be more effective over the long range for gene flow than seed dispersal. This differentiation level supports the hypothesis that the fragmentation and isolation of the residual populations is in progress. This phenomenon is due not only to post-ice age climate changes, but also to direct and indirect anthropic actions.