Allozyme Polymorphism of Swiss Stone Pine <Emphasis Type="Italic">Pinus cembra</Emphasis> L. in Mountain Populations of the Alps and the Eastern Carpathians

Swiss stone pine Pinus cembra L. is a species with fragmented range, occurring in the Alpine-East Carpathian mountain system. Seeds of P. cembra are dispersed by nutcrackers, which offers potential possibilities for gene exchange among populations. Using isozyme analysis, we have examined five samples from two parts of the Swiss stone pine range: the Alps (Switzerland and Austria) and the Carpathians (two samples from the northern macroslope of the Gorgany Ridge, Eastern Carpathians, Ivano-Frankivs'ka oblast and one sample from Zakarpats'ka oblast of Ukraine). The allele frequencies of 30 isozyme loci, coding for enzymes ADH, FDH, FEST, GDH, GOT, IDH, LAP, MNR, MDH, PEPCA, 6-PGD, PGI, PGM, SDH, SKDH, SOD, were analyzed using cluster analysis and Principal Component Analysis. Two clusters, corresponding to the isolated Alpine and Carpathian parts of the range, were found. The main contribution to these differences were made by loci Adh-1, Adh-2, Fest-2, Lap-3, Mdh-4, and Sod-4. The interpopulation differentiation proved to be somewhat higher than that typical for pines (F_ST = 7.4%), but within the limits characteristic for taxonomically close species. Thus, isolation of the populations did not lead to their marked differentiation, which may be explained by gene flow and balancing selection, which equalizes gene frequencies across the fragmented species area. Interlocus (F_ST heterogeneity (from 0.003 to 0.173) suggests adaptive significance of some of the allozyme polymorphisms or linkage of some loci with adaptive genes. The Carpathian populations were shown to have higher gene diversity than the Alpine ones (expected heterozygosities 0.095–0.114 and 0.060–0.080, respectively). A deficiency of heterozygotes (as compared to the Hardy-Weinberg proportions), observed in the embryo sample, was probably explained by inbreeding. The reduction in the area of Carpathian pine forests in Holocene, caused by the global climatic changes and the anthropogenic impact, is hazardous for the gene pool of the species. The maintenance of genetic uniqueness of both Carpathian populations of P. cembra in general, and individual stands in particular, requires special measures for protection of Swiss stone pine in the Eastern Carpathians.     

Genetic structure of Hypochaeris uniflora (Asteraceae) suggests vicariance in the Carpathians and rapid post-glacial colonization of the Alps from an eastern Alpine refugium

Aim The range of the subalpine species Hypochaeris uniflora covers the Alps, Carpathians and Sudetes Mountains. Whilst the genetic structure and post‐glacial history of many high‐mountain plant taxa of the Alps is relatively well documented, the Carpathian populations have often been neglected in phylogeographical studies. The aim of the present study is to compare the genetic variation of the species in two major European mountain systems – the Alps and the Carpathians. Location Alps and Carpathians. Methods The genetic variation of 77 populations, each consisting of three plants, was studied using amplified fragment length polymorphism (AFLP). Results Neighbour joining and principal coordinate analyses revealed three well‐supported phylogeographical groups of populations corresponding to three disjunct geographical regions – the Alps and the western and south‐eastern Carpathians. Moreover, two further clusters could be distinguished within the latter mountain range, one consisting of populations from the eastern Carpathians and the second consisting of populations from the southern Carpathians. Populations from the Apuseni Mountains had an intermediate position between the eastern and southern Carpathians. The genetic clustering of populations into four groups was also supported by an analysis of molecular variance, which showed that most genetic variation (almost 46%) was found among these four groups. By far the highest within‐population variation was found in the eastern Carpathians, followed by populations from the southern and western Carpathians. Generally, the populations from the Alps were considerably less variable and displayed substantially fewer region‐diagnostic markers than those from the south‐eastern Carpathians. Although no clear geographical structure was found within the Alps, based on neighbour joining or principal coordinate analyses, some trends were obvious: populations from the easternmost part were genetically more variable and, together with those from the south‐western part, exhibited a higher proportion of rare AFLP fragments than populations in other areas. Moreover, the total number of AFLP fragments per population, the percentage of polymorphic loci and the proportion of rare AFLP fragments significantly decreased from east to west. Main conclusions Deep infraspecific phylogeographical gaps between the populations from the Alps and the western and south‐eastern Carpathians suggest the survival of H. uniflora in three separate refugia during the last glaciation. Our AFLP data provide molecular evidence for a long‐term geographical disjunction between the eastern and western Carpathians, previously suggested from the floristic composition at the end of 19th century. It is likely that Alpine populations survived the Last Glacial in the eastern part of the Alps, from where they rapidly colonized the rest of the Alps after the ice sheet retreated. Multiple founder effects may explain a gradual loss of genetic variation during westward colonization of the Alps.     

Genetic structure in Pinus cembra from the Carpathian Mountains inferred from nuclear and chloroplast microsatellites confirms post-glacial range contraction and identifies introduced individuals

Genetic differentiation of scattered populations at neutral loci is characterized by genetic drift counteracted by the remaining gene flow. Populations of Pinus cembra in the Carpathian Mountains are isolated and restricted to island-like stands at high-elevation mountain ranges. In contrast, paleobotanical data suggest an extended early Holocene distribution of P. cembra in the Carpathians and its surrounding areas, which has contracted to the currently disjunct occurrences. We analyzed the genetic variation of 11 Carpathian populations of P. cembra at chloroplast and, in part newly developed, nuclear microsatellites. Both marker types revealed low levels of genetic differentiation and a lack of isolation by distance, reflecting the post-glacial retraction of the species to its current distribution. Stronger effects of genetic drift were implied by the higher genetic differentiation found for haploid chloroplast than for diploid nuclear markers. Moreover, we found no association between the values of population genetic differentiation for the two marker types. Several populations indicated recent genetic bottlenecks and inbreeding as a consequence of decline in population sizes. Moreover, we found individuals in two populations from the Rodnei Mountains that strikingly differed in assignment probabilities from the remaining specimens, suggesting that they had been introduced from a provenance outside the studied populations. Comparison with Eastern Alpine P. cembra and individuals of the closely related Pinus sibirica suggests that these individuals presumably are P. sibirica. Our study highlights the importance of the maintenance of sufficiently large local population sizes for conservation due to low connectivity between local occurrences.     

Phylogeographical structure in the coastal species Senecio rodriguezii (Asteraceae), a narrowly distributed endemic Mediterranean plant

Aim Our goals were (1) to assess the levels of chloroplast DNA variation in a narrowly distributed plant restricted to continental islands, (2) to ascertain whether a phylogeographical structure is present in plants restricted to coastal linear systems, and (3) to interpret the results in the light of the known palaeogeography of these islands. Location The Eastern Balearic Islands (Majorca and Minorca) in the Western Mediterranean Basin. Methods Sampling included 134 individuals from 28 populations of Senecio rodriguezii covering the entire range of the species. Sequences of the chloroplast genome (trnT–trnL spacer) were obtained and parameters of population genetic diversity and substructure were determined (h_sh_t, G_st). The geographical structure of genetic variation was assessed by an analysis of molecular variance (AMOVA). Additionally, a spatial AMOVA (SAMOVA) was used to identify groups of populations that were geographically homogeneous and maximally differentiated from each other. Finally, a pattern of isolation by distance was assessed by testing the correlation between the matrix of pairwise Φ_ST values and the matrix of geographical distances between pairs of populations using a Mantel test. Results Seven haplotypes were detected in S. rodriguezii. Only two of them were shared between islands; all of the others were restricted to Majorca (two) or Minorca (three). Overall, we found high levels of genetic diversity and significant geographical structuring of cpDNA markers. Most of the variation detected can be attributed to differences among populations (84.6%), but there was also a significant differentiation between the islands. Main conclusions Our results support the view that the Balearic Islands constitute a reservoir of genetic diversity, not only for widespread Mediterranean taxa, but also for endemic ones. The intraspecific genetic structure found in S. rodriguezii suggests that its population history was dominated by both expansion and contraction events. This has resulted in a species that is highly structured genetically, showing very few shared haplotypes between islands, and a high number of haplotypes restricted to small geographical areas within the islands. Changes in habitat availability and dynamic processes of population fragmentation and connectivity due to repeated cycles of sea‐level changes during the Quaternary are the possible underlying factors that have shaped the cpDNA pool of this endemic species on a regional scale.     

High genetic diversity and distinct origin of recently fragmented Scots pine (<Emphasis Type="Italic">Pinus sylvestris</Emphasis> L.) populations along the Carpathians and the Pannonian Basin

Historical evolutionary events highly affect the modern-day genetic structure of natural populations. Scots pine (Pinus sylvestris L.), as a dominant tree species of the Eurasian taiga communities following the glacial cycles of the Pleistocene, has survived in small, scattered populations at the range limits of its south-eastern European distribution. In this study, we examined genetic relationships, genetic divergence and demographic history of peripheral populations from central-eastern Europe, the Carpathian Mountains and the Pannonian Basin. Four hundred twenty-one individuals from 20 populations were sampled and characterized with both nuclear and chloroplast simple sequence repeat (SSR) markers. Standard population genetic indices, the degree of genetic differentiation and spatial genetic structure were analysed. Our results revealed that peripheral Scots pine populations retained high genetic diversity despite the recently ongoing fragmentation and isolation of the persisting relict populations. Analysis of molecular variance (AMOVA) showed 7% among-population genetic differentiation, and there was no isolation by distance among the island-like occurrences. Genetic discontinuities with strong barriers (99–100% bootstrap support) were identified in the Carpathians. Based on both marker types, populations of the Western Carpathians were delimited from those inhabiting the Eastern Carpathians, and two main genetic lineages were traced that most probably originate from two main refugia. One refugium presumably existed in the region of the Eastern Alps with the Hungarian Plain, while the other was probably found in the Eastern Carpathians. These findings are supported by recent palynological records. The strongest genetic structure was revealed within the Romanian Carpathians on the basis of both marker types. With only some exceptions, no signs of recent bottlenecks or inbreeding were detected. However, Carpathian natural populations of Scots pine are highly fragmented and have a small census size, though they have not yet been affected by genetic erosion induced by isolation.     

Phylogeography and genetic structure of the orchid Himantoglossum hircinum (L.) Spreng. across its European central–marginal gradient

Aim This study aims to link demographic traits and post‐glacial recolonization processes with genetic traits in Himantoglossum hircinum (L.) Spreng (Orchidaceae), and to test the implications of the central–marginal concept (CMC) in Europe. Location Twenty sites covering the entire European distribution range of this species. Methods We employed amplified fragment length polymorphism (AFLP) markers and performed a plastid microsatellite survey to assess genetic variation in 20 populations of H. hircinum located along central–marginal gradients. We measured demographic traits to assess population fitness along geographical gradients and to test for correlations between demographic traits and genetic diversity. We used genetic diversity indices and analyses of molecular variance (AMOVA) to test hypotheses of reduced genetic diversity and increased genetic differentiation and isolation from central to peripheral sites. We used Bayesian simulations to analyse genetic relationships among populations. Results Genetic diversity decreased significantly with increasing latitudinal and longitudinal distance from the distribution centre when excluding outlying populations. The AMOVA revealed significant genetic differentiation among populations (F_ST = 0.146) and an increase in genetic differentiation from the centre of the geographical range to the margins (except for the Atlantic group). Population fitness, expressed as the ratio N_R/N, decreased significantly with increasing latitudinal distance from the distribution centre. Flower production was lower in most eastern peripheral sites. The geographical distribution of microsatellite haplotypes suggests post‐glacial range expansion along three major migratory pathways, as also supported by individual membership fractions in six ancestral genetic clusters (C1–C6). No correlations between genetic diversity (e.g. diversity indices, haplotype frequency) and population demographic traits were detected. Main conclusions Reduced genetic diversity and haplotype frequency in H. hircinum at marginal sites reflect historical range expansions. Spatial variation in demographic traits could not explain genetic diversity patterns. For those sites that did not fit into the CMC, the genetic pattern is probably masked by other factors directly affecting either demography or population genetic structure. These include post‐glacial recolonization patterns and changes in habitat suitability due to climate change at the northern periphery. Our findings emphasize the importance of distinguishing historical effects from those caused by geographical variation in population demography of species when studying evolutionary and ecological processes at the range margins under global change.     

Chloroplast DNA phylogeography of the shrub Cistus ladanifer L. (Cistaceae) in the highly diverse Western Mediterranean region

This study investigated the phylogeographic structure of Cistus ladanifer, in order to locate its Quaternary refugia, reconstruct its recolonisation patterns and assess the role of geographical features (mountain ranges, rivers and the Strait of Gibraltar) as barriers to its seed flow and expansion through the Western Mediterranean. Thirty-eight populations were screened for length variation of polymorphic chloroplast simple sequence repeats (cpSSRs). Statistical analyses included estimation of haplotypic diversity, hierarchical analysis of molecular variation (amova) and fixation indices. Mantel tests, SAMOVA and BARRIER analyses were applied to evaluate the geographical partitioning of genetic diversity across the entire species range. Pollen data from bibliography were used to complement molecular inferences. Chlorotype diversity within populations was similar throughout the natural range of C. ladanifer (mean haplotypic diversity=0.32). High differentiation among populations was estimated (G(ST)=0.60). Our data suggest that the barriers of the Strait of Gibraltar and the Betic ranges may have favoured the divergence during glacial periods of four different lineages of populations inferred with SAMOVA. The main northward colonisation of in the Iberian Peninsula occurred from refugia in southwest Iberia. This process may have been influenced by human activities (forest clearance, livestock grazing and even commerce) in the Iberian Peninsula. In contrast, populations in the Betic area have conserved a specific haplotype.     

Haplotype variation in a mitochondrial tandem repeat of Norway spruce (Picea abies) populations suggests a serious founder effect during postglacial re-colonization of the western Alps

Populations from 13 elevational transects of Norway spruce [Picea abies (L.) Karst] across the Alpine range were sampled to elucidate the geographical pattern of genetic variation in relation to postglacial re-colonization and to study elevational effects on haplotypic diversity. We assessed fragment length variation in a tandem repeat region of the mitochondrial (mt) nad1 intron 2. This maternally inherited genetic marker is suited to infer migration as it is dispersed by seed only. A total of 10 haplotypes was found, most of which were due to repeat copy number variation. An analysis of molecular variance (amova) showed that overall population differentiation was high (F(ST)=0.41), and it revealed a significant differentiation between monomorphic western and moderately to highly variable eastern Alpine populations. This phylogeographic pattern may be explained by a founder effect during postglacial re-colonization. An early arriving haplotype, assumed to originate from a western Carpathian refugium, could expand into suitable habitats, reducing the chances for establishment of subsequently arriving haplotypes. On the other hand, the high variation in populations within an Italian transect of the south-eastern Alps may be the consequence of merging migration pathways from and close distance to putative glacial refugia, most likely those assumed in the Carpathian mountains and on the Balkan peninsula or possibly in the central plains of Italy. An effect of elevation on haplotypic diversity was not evident, though a low, but significant, partition of total genetic variation was attributed to among-population variation in one Italian transect. Various factors, such as vertical seed dispersal and forest management, may account for blurring an otherwise established pattern of genetic variation on a small geographical scale.     

Genetic diversity and differentiation of <Emphasis Type="Italic">Fagus orientalis</Emphasis> Lipsky in Hyrcanian forests revealed by nuclear and chloroplast microsatellite markers

Oriental beech (Fagus orientalis Lipsky) is a widespread monoecious and wind-pollinated tree species. It is one of the major components of the Hyrcanian forests of Iran and it is of both ecological and economical importance. Twelve beech stands were surveyed at 9 chloroplast (cp) and 6 nuclear (n) polymorphic microsatellite loci (simple sequence repeats, SSR) to provide information on distribution of genetic diversity within and among populations and on gene conservation and silvicultural management of this species. High levels of genetic differentiation were detected for the chloroplast genome (F _ST = 0.80 and R _ST = 0.95), in sharp contrast to the nuclear genome (F _ST = 0.06, R _ST = 0.05). The analysis of molecular variance (AMOVA) showed that 48% of the total cpSSR variation was attributable to differences among regions and 30% to differences among populations within regions, suggesting multiple origins of beech populations in Hyrcanian forests. Nuclear SSRs confirmed the presence of significant differentiation among populations and among geographic regions, even if, as expected, this was less pronounced than that found with cpSSRs (based on AMOVA, differences among regions and among populations within regions each contribute 5% to total nSSR variance). A highly significant correlation between genetic (nSSRs) and geographic distances (R ² = 0.522) was estimated, thus showing an isolation by distance effect. The application of spatial analysis of molecular variance (SAMOVA) using both marker data allowed identification of genetically homogeneous groups of populations. Possible applications of these results for the certification of provenances and/or seed lots and for designing conservation programs are presented and discussed.     

Haplotype richness in refugial areas: phylogeographical structure of <Emphasis Type="Italic">Saxifraga callosa</Emphasis>

This paper illustrates the phylogeographical structure of Saxifraga callosa in order to describe its genetic richness in refugial areas and to reconstruct its glacial history. S. callosa is a species spread throughout south-east France and Italy with a high distribution in the Maritime Alps. Four chloroplast microsatellite and AFLP markers were analyzed in populations of S. callosa. The size variants of all tested loci amount to 11 different haplotypes. Intrapopulational haplotype variation was found in two of the populations analyzed: on the Mt. Toraggio in the Maritime Alps, and in the Apuan Alps. On the other hand, no intrapopulational variation was found in 25 populations, most of which were sampled from isolated areas. Analysis of the haplotype distribution showed that population subdivision across all populations was high (G _ST = 0.899). Moreover, its genetic structure was studied using AMOVA and STRUCTURE analysis. The study legitimated inferred conclusions about the phylogeographical structure of the species and identified centers of diversity. Considerations concerning genetic structure and divergence among three major clades (Maritime Alps, Apuan Alps and Apennines), the patchy distribution of haplotypes, and the high number of private haplotypes support the proposal that S. callosa survived in some refugia within the Italian Peninsula refugium, and that mainly northern populations of refugia were involved in postglacial recolonization.     

Refugia within refugia: the case study of a canopy tree (Eurycorymbus cavaleriei) in subtropical China

Aim Eurycorymbus cavaleriei (Lévl.) Rehd. et Hand.‐Mazz. (Sapindaceae) is a Tertiary relict tree endemic to subtropical China. This area is a centre for speciation and evolution within the East Asia biome and one of its most important refugial locations. In this study we aim to elucidate the phylogeographical patterning in E. cavaleriei, in order to identify the locations of the species’ main refugia and the predominant patterns of migration that have led to the contemporary spatial genetic structure of chloroplast variation. Location Subtropical China. Methods We sampled 18 populations of E. cavaleriei throughout its geographical range. Chloroplast DNA (cpDNA) sequence data from two non‐coding regions ((trnS/trnG and pl20/5′‐rps12) were obtained from 170 individuals for phylogeographical analyses. Relationships among cpDNA haplotypes were determined using median‐joining networks. Genetic structure was examined by spatial analysis of molecular variance (SAMOVA). Population differentiation was estimated by G_ST and N_ST statistics. Results Ten distinct haplotypes were identified. The level of differentiation among populations was relatively high (G_ST = 0.817), and N_ST was significantly higher than G_ST (P < 0.05), indicating that strong phylogeographical structure is exhibited by this species. The SAMOVA revealed five diverging groups of related haplotypes, which coincide with major landscape features in this region. Main conclusions The high differentiation among populations of E. cavaleriei may be a combined effect of historical and contemporary processes, such as the low effective population size for the chloroplast genome of a dioecious species, long‐term range fragmentation and limited seed dispersal for the species. Clear‐cut geographical distributions of ancestral haplotypes of the species suggest multiple potential refugia across subtropical China. The identified refugial regions have long been recognized as centres of plant diversity and endemism for China and have also been suggested as glacial refugia for many other plant species. The combination of these factors means that these locations should be considered as the highest priority for inclusion in conservation policies and sustainable forest management strategies for subtropical China.     

Distribution of genetic diversity in Pinus pinaster Ait. as revealed by chloroplast microsatellites

 Using nine chloroplast simple sequence.repeats (cpSSRs) markers, we evaluated haplotypic variation within and among natural populations of Maritime pine (Pinus pinaster Ait.) in order to shed light on the history of this species. Seven out of the nine cpSSRs analysed were polymorphic, giving a total of 24 different variants. The 24 variants combined in 34 different haplotypes. The populations which generally showed the lowest level of haplotypic diversity are those located in Portugal. The Landes (France) and Pantelleria (Italy) populations represent the two main reservoirs of haplotypic diversity. The proportion of genetic differentiation among populations, estimated using R_st, which is a measure based upon a strict stepwise mutation model, was 0.235. The high level of differentiation was also confirmed by the AMOVA analysis (Φ_ST=0.254, P<0.001). Four main groups of populations were identified on the basis of Principal Component Analysis, with the differences being statistically significant (Φ_CT=0.299, P<0.001). Based on our results the presence of refugia located in the South of Portugal, previously proposed for this species, may be excluded, and a different possible recolonization process of Maritime pine in the post-glacial period has been proposed. Populations from North Africa and France might have represented a starting point of the recolonization process of Portugal and of the Italian part of the natural range, respectively. This hypothesis seems to be confirmed by the analysis of the distribution of the pairwise differences among individuals within populations: Landes and Pantelleria populations showed a bimodal distribution, as would be expected for ancient gene pools. Received: 5 November 1997 / Accepted: 5 January 1998     

Genetic diversity and population differentiation in the yellow drum Nibea albiflora along the coast of the China Sea

Nibea albiflora (yellow drum) is an important seafood fish species in East Asia. We explored the population genetic variation of N. albiflora along the coastal waters of the China Sea using microsatellite markers to facilitate a selective breeding programme that is undertaken in China. A total of 256 alleles were detected at 12 loci in four wild populations. A high level of genetic diversity was observed with the mean number of alleles and the observed and expected heterozygosity in each population ranging from 7.917 to 14.083, 0.701 to 0.764 and 0.765 to 0.841, respectively. Pairwise fixation index (F_ST) analysis indicated significant but weak genetic differentiation among populations from four localities (F_ST?=?0.030, P?<?0.01), which was also confirmed by analysis of molecular variance (AMOVA). Significant genetic differentiation was detected between Ningde and the other populations (F_ST?=?0.047–0.056, P?<?0.01). Structure analysis suggested that N. albiflora within the examined range might be composed of two stocks. The data of the present study revealed high genetic diversity and low genetic differentiation among the N. albiflora populations along the coast of the China Sea. This baseline information could be valuable for future selective breeding programmes of N. albiflora.     

Disentangling the effects of geographic peripherality and habitat suitability on neutral and adaptive genetic variation in Swiss stone pine

It is generally accepted that the spatial distribution of neutral genetic diversity within a species’ native range mostly depends on effective population size, demographic history, and geographic position. However, it is unclear how genetic diversity at adaptive loci correlates with geographic peripherality or with habitat suitability within the ecological niche. Using exome‐wide genomic data and distribution maps of the Alpine range, we first tested whether geographic peripherality correlates with four measures of population genetic diversity at > 17,000 SNP loci in 24 Alpine populations (480 individuals) of Swiss stone pine (Pinus cembra) from Switzerland. To distinguish between neutral and adaptive SNP sets, we used four approaches (two gene diversity estimates, F_ST outlier test, and environmental association analysis) that search for signatures of selection. Second, we established ecological niche models for P. cembra in the study range and investigated how habitat suitability correlates with genetic diversity at neutral and adaptive loci. All estimates of neutral genetic diversity decreased with geographic peripherality, but were uncorrelated with habitat suitability. However, heterozygosity (H_e) at adaptive loci based on Tajima's D declined significantly with increasingly suitable conditions. No other diversity estimates at adaptive loci were correlated with habitat suitability. Our findings suggest that populations at the edge of a species' geographic distribution harbour limited neutral genetic diversity due to demographic properties. Moreover, we argue that populations from suitable habitats went through strong selection processes, are thus well adapted to local conditions, and therefore exhibit reduced genetic diversity at adaptive loci compared to populations at niche margins.     

Patterns of Genetic Variation in Swertia przewalskii, an Endangered Endemic Species of the Qinghai-Tibet Plateau

Swertia przewalskii Pissjauk. (Gentianaceae) is a critically endangered and endemic plant of the Qinghai-Tibet Plateau in China. RAPD and ISSR analyses were carried out on a total of 63 individuals to assess the extent of genetic variation in the remaining three populations. Percentage of polymorphic bands was 94% (156 bands) for RAPD and 96% (222 bands) for ISSR. A pairwise distance measure calculated from the RAPD and ISSR data was used as input for analysis of molecular variance (AMOVA). AMOVA indicated that a high proportion of the total genetic variation (52% for RAPD and 56% for ISSR) was found among populations; pairwise Φ _ST comparisons showed that the three populations examined were significantly different (p < 0.001). Significant genetic differentiation was found based on different measures (AMOVA and Hickory θ_B) in S. przewalskii (0.52 on RAPD and 0.56 on ISSR; 0.46 on RAPD and 0.45 on ISSR). The differentiation of the populations corresponded to low average gene flow (0.28 based on RAPD and 0.31 based on ISSR), whereas genetic distance-based clustering and coalescent-based assignment analyses revealed significant genetic isolation among populations. Our results indicate that genetic diversity is independent of population size. We conclude that although sexual reproduction and gene flow between populations of S. przewalskii are very limited, they have preserved high levels of genetic diversity. The main factors responsible for the high level of difference among populations are the isolation and recent fragmentation under human disturbance.     

Genetic diversity and phylogeographic analysis of Pinus leiophylla: a post-glacial range expansion

Aim Mexico is a centre of diversity for species of the genus Pinus, most of which have restricted geographical distributions. An exception is Pinus leiophylla Schiede and Deppe, which is widely distributed throughout most of Mexico’s mountainous regions. We attempt to reconstruct the phylogeographic history of this species, in order to determine if its current broad distribution is associated with major events of environmental change that occurred during the Quaternary. Location Coniferous forests in Sierra Madre Occidental, Eje Volcánico Transversal and Sierra Montañosa del Norte de Oaxaca, Mexico. Methods A total of 323 individuals of both P. leiophylla var. leiophylla and P. leiophylla var. chihuahuana sampled from 22 populations were screened for variation at six paternally inherited chloroplast DNA microsatellite markers (cpSSR). In addition to haplotypic diversity estimates and neutrality tests, the following clustering methods were employed: principal components analysis (PCA), analysis of molecular variance (AMOVA), spatial analysis of molecular variance (SAMOVA), haplotype network and a technique similar to Croizat’s panbiogeographical method of individual and generalized tracks. Results The combination of mutations at the six microsatellites yielded a total of 92 different haplotypes. The percentage of shared haplotypes between varieties (P. leiophylla var. leiophylla and P. leiophylla var. chihuahuana) was only 2.2%. The average haplotypic diversity for the species was H = 0.760. PCA and SAMOVA indicate the presence of four main genetic clusters. The estimated divergence time between the two most frequent haplotypes was between 75,000 and 110,000 years. Significantly large negative F_s values suggest that most of the sampled populations are currently expanding. Individual and generalized tracks identified three potential zones that may have harboured ancestral populations of P. leiophylla and from which the expansion of this species started, as well as two secondary contact zones between the two varieties. Main conclusions The results indicate that one of the three potential areas hypothesized to have harboured ancestral populations of P. leiophylla may be related to the origin of P. leiophylla var. chihuahuana, while the other two may be related to the origin of P. leiophylla var. leiophylla. The current broad distribution of P. leiophylla is probably associated with its strong colonization ability.     

Increased genetic structuring of isolated Salamandra salamandra populations (Caudata: Salamandridae) at the margins of the Carpathian Mountains

The traditional southern Pleistocene refugia hypothesis in Europe has lately been challenged for several animal and plant species. The Carpathian Basin, especially at the marginal regions, is one of the recently recognized biodiversity hotspots in Europe. Marginal populations are prone to have lower genetic diversity and higher genetic differentiation than central populations. Here, we examined one mitochondrial DNA fragment (D‐loop) and nine nuclear (microsatellite) loci to describe the genetic diversity and phylogeographical pattern of fire salamander (Salamandra salamandra) populations in the Carpathian Basin with focusing on the southern margins of the Western Carpathians, where isolated populations of this species are present. Analyses of microsatellites indicated reduced genetic diversity for most of the isolated populations. Based on the mitochondrial DNA, only two haplotypes were found, whereas the analyses with the nuclear markers revealed a more recent genetic split between Western (Alpine) and Eastern (Carpathian) populations, and separated the Apuseni Mountains population (part of the Western Carpathians). Using approximate Bayesian computation analyses, we identified the most probable colonization scenario for the isolated North Hungarian Carpathian Basin populations. The split between isolated salamander populations from the central populations in the Carpathian Mountains dates back to the beginning of the Late Pleistocene, while the split between most of the Hungarian populations can be associated with the Last Glacial Maximum. We found evidence for long‐time isolation between the marginal Carpathian Basin and central populations. Our results also show that S. salamandra survived glacial periods in the temperate forests of north‐east Pannonia (North Hungarian Mountains), confirming that the Carpathian Basin served as important northerly refugia during the Pleistocene climatic oscillations.     

Genetic diversity and population differentiation of Capparis spinosa (Capparaceae) in Northwestern China

To explore the influence of vicariance on differentiation patterns of taxa in arid regions, we systematically assessed the genetic diversity and variability of Capparis spinosa, a typical xerophyte that is widely distributed in the Tianshan Mountains and adjacent areas. In total, 300 individuals from 25 populations were sampled, and 14 haplotypes were identified using two cpDNA sequences (rpS12-rpL20 and ndhF). A high level of total genetic diversity (H_T = 0.859) was detected, and this was attributed to the extensive distribution range, which included numerous large populations. The SAMOVA results suggested that the 25 populations were clustered into 4 major geographical groups; a similar divergence trend was found by constructing a BEAST phylogenetic tree and a network diagram. The AMOVA results revealed that significant genetic differentiation occurred among groups. Our results indicated a considerable correlation between genetic divergence and geographical distribution. Isolation due to complex mountain and desert geography might limit gene exchange between disjunct populations, resulting in high differentiation between geographical groups.     

Genetic Diversity and Geographical Differentiation of <Emphasis Type="Italic">Desmodium triflorum</Emphasis> (L.) DC. in South China Revealed by AFLP Markers

High levels of genetic variation enable species to adapt to changing environments and provide plant breeders with the raw materials necessary for artificial selection. In the present study, six AFLP primer pairs were used to assess the genetic diversity of Desmodium triflorum (L.) DC. from 12 populations in South China. A high percentage of polymorphic loci (P = 76.16%) and high total gene diversity (H _T = 0.310) were found, indicating that the genetic diversity of D. triflorum is high at the species level. Genetic diversity was also relatively high at the population level (P = 55.85%, H _e = 0.230). The coefficient of gene differentiation among populations (G _ST) was 0.255, indicating that while most genetic diversity resided within populations, there was also considerable differentiation among populations. AMOVA also indicated 24.29% of the total variation to be partitioned among populations (Φ_ST = 0.243). UPGMA clustering analysis based on genetic distances showed that the 12 populations could be separated into three subgroups: an eastern, a western, and a central-southern subgroup. However, a Mantel test revealed no significant correlation (r = 0.286, p = 0.983) between the geographical distances and genetic distances separating these populations; mountain barriers to gene flow and human disturbance may have confounded these correlations. The present study has provided some fundamental genetic data that will be of use in the exploitation of D. triflorum.     

Low genetic differentiation among widely separated populations of the pearl oyster Pinctada fucata as revealed by AFLP

Genetic variation within and among five populations of the pearl oyster Pinctada fucata, from China (Daya Bay, Sanya Bay and Beibu Bay), Japan (Mie Prefecture) and Australia (Port Stephens) was studied using AFLP. Three primer pairs generated 184 loci among which 91.8-97.3% is polymorphic. An overall genetic diversity of 0.38 among populations and an average of 0.37 within populations (ranging from 0.35 in Japanese population to 0.39 in Beibu Bay population) were observed. Genetic differentiation among the five populations is low but significant as indicated by pairwise G_ST (0.0079-0.0404). AMOVA further shows that differentiation is significant among the five populations but is not significant at a broader geographical scale, among the three groups of Chinese, Japanese and Australian populations or among the two groups of Australian and north Pacific populations. The low level of genetic differentiation indicated that P. fucata populations in the west Pacific are genetically linked. Among the five populations, the Australian one is more differentiated from the others, based on both pairwise AMOVA and G_ST analyses, and is genetically isolated by distance as indicated by Mantel test. However, genetic differences among the three Chinese populations are not correlated with the geographic distances, suggesting that Hainan Island and Leizhou Peninsula may act as barriers blocking gene flow.