Biogeographic history of the threatened species <Emphasis Type="Italic">Araucaria araucana</Emphasis> (Molina) K. Koch and implications for conservation: a case study with organelle DNA markers

Fragmentation of the habitat due to glaciations, fires and human activities affected the distribution range of Araucaria araucana in southern South America. On the borders of the Argentinean Patagonian steppe, the species is restricted to isolated patches without natural regeneration. Our objective is to test the hypothesis that these populations are relicts of pre-Pleistocene origin. A total of 224 individuals from 16 populations were sampled. Twenty chloroplast microsatellites, 19 non-coding chloroplast DNA regions and eight mitochondrial DNA fragments were screened for polymorphisms. A low transferability rate of universal primers from Pinaceae and also a low variation were detected for this ancient species. Only one non-coding region of the chloroplast DNA showed polymorphism allowing the identification of five haplotypes. A low genetic differentiation (G _ST  = 0.11; G′ _ST  = 0.267) and lack of geographic structure was found. Allelic richness was lower and genetic differentiation higher among the eastern isolated populations, suggesting a long lasting persistence. Conservation guidelines are given for these relictual populations, which are located outside the limits of the National Parks.     

Chloroplast DNA variation and postglacial recolonization of common ash (Fraxinus excelsior L.) in Europe

We used chloroplast polymerase chain reaction-restriction-fragment length polymorphism (PCR-RFLP) and chloroplast microsatellites to assess the structure of genetic variation and postglacial history across the entire natural range of the common ash (Fraxinus excelsior L.), a broad-leaved wind-pollinated and wind-dispersed European forest tree. A low level of polymorphism was observed, with only 12 haplotypes at four polymorphic microsatellites in 201 populations, and two PCR-RFLP haplotypes in a subset of 62 populations. The clear geographical pattern displayed by the five most common haplotypes was in agreement with glacial refugia for ash being located in Iberia, Italy, the eastern Alps and the Balkan Peninsula, as had been suggested from fossil pollen data. A low chloroplast DNA mutation rate, a low effective population size in glacial refugia related to ash's life history traits, as well as features of postglacial expansion were put forward to explain the low level of polymorphism. Differentiation among populations was high (GST= 0.89), reflecting poor mixing among recolonizing lineages. Therefore, the responsible factor for the highly homogeneous genetic pattern previously identified at nuclear microsatellites throughout western and central Europe (Heuertz et al. 2004) must have been efficient postglacial pollen flow. Further comparison of variation patterns at both marker systems revealed that nuclear microsatellites identified complex differentiation patterns in south-eastern Europe which remained undetected with chloroplast microsatellites. The results suggest that data from different markers should be combined in order to capture the most important genetic patterns in a species.     

Analyses of genetic population structure of two ecologically important mangrove tree species, Bruguiera gymnorrhiza and Kandelia obovata from different river basins of Iriomote Island of the Ryukyu Archipelago, Japan

We analyzed nuclear and chloroplast microsatellite makers to assess genetic diversity and examine genetic structure of two mangrove tree species, Bruguiera gymnorrhiza and Kandelia obovata recovered from nine major river basins of Iriomote Island of the Ryukyu Archipelago, Japan. The average number of alleles per nuclear locus per population was 2.6 in B. gymnorrhiza and 1.7 in K. obovata. Bayesian clustering analysis using InStruct identified two genetic clusters in B. gymnorrhiza and three clusters in K. obovata. Chloroplast microsatellites revealed two dominant haplotypes from B. gymnorrhiza and three haplotypes from K. obovata. The overall result suggests low genetic diversity for both species. AMOVA for nuclear microsatellites showed 9.3?% of population variation in B. gymnorrhiza. Although genetic differentiation between several populations was significant in this species, F _ST suggested low to moderate level of differentiations between most of the populations. Distribution of genetic clusters and chloroplast haplotypes also suggested weak differentiations among B. gymnorrhiza populations. In K. obovata, population variation was, however, relatively high (27.8?%). The high differentiation between K. obovata populations was also suggested from the F _ST and genetic clusters from nuclear microsatellites, and chloroplast haplotypes. A significant correlation between chloroplast genetic distances and coastline distances as well as haplotype distributions for both species suggest that propagules from each species mostly disperse to the neighboring river basins. While significant F _IS and higher percentage of admixed clusters from nuclear microsatellites suggested inbreeding, continual gene exchange by propagule dispersal among populations especially among neighboring populations was suggested for both species from maternally inherited chloroplast microsatellites analyses.     

Genetic structure of island populations of <Emphasis Type="Italic">Prunus lannesiana</Emphasis> var. <Emphasis Type="Italic">speciosa</Emphasis> revealed by chloroplast DNA,AFLP and nuclear SSR loci analyses

The wild flowering cherry Prunus lannesiana var. speciosa is highly geographically restricted, being confined to the Izu Islands and neighboring peninsulas in Japan. In an attempt to elucidate how populations of this species have established we investigated the genetic diversity and differentiation in seven populations (sampling 408 individuals in total), using three kinds of genetic markers: chloroplast DNA (cpDNA), amplified fragment length polymorphisms (AFLPs), and 11 nuclear SSR polymorphic loci. Eight haplotypes were identified based on the cpDNA sequence variations, 64 polymorphic fragments were scored for the AFLP markers, and a total of 154 alleles were detected at the 11 nuclear SSR loci. Analysis of molecular variance showed that among-population variation accounted for 16.55, 15.04 and 7.45% of the total detected variation at the cpDNA, AFLPs, and SSR loci, respectively. Thus, variation within populations accounted for most of the genetic variance for all types of markers, although the genetic differentiation among populations was also highly significant. For cpDNA variation, no clear structure was found among the populations, except that of the most distant island, although an “isolation by distance” pattern was found for each marker. Both neighbor-joining trees and structure analysis indicate that the genetic relationships between populations reflect geological variations between the peninsula and the islands and among the islands. Furthermore, hybridization with related species may have affected the genetic structure, and some genetic introgression is likely to have occurred.     

Population genetics of Wolbachia‐infected,parthenogenetic and uninfected,sexual populations of Tetrastichus coeruleus (Hymenoptera: Eulophidae)

Wolbachia are endosymbiotic bacteria known to manipulate the reproduction of their hosts. These manipulations are expected to have consequences on the population genetics of the host, such as heterozygosity levels, genetic diversity and gene flow. The parasitoid wasp Tetrastichus coeruleus has populations that are infected with parthenogenesis‐inducing Wolbachia and populations that are not infected. We studied the population genetics of T. coeruleus between and within Wolbachia‐infected and uninfected populations, using nuclear microsatellites and mitochondrial DNA. We expected reduced genetic diversity in both DNA types in infected populations. However, migration and gene flow could introduce new DNA variants into populations. We therefore paid special attention to individuals with unexpected (genetic) characteristics. Based on nuclear and mitochondrial DNA, two genetic clusters were evident: a thelytokous cluster containing all Wolbachia‐infected, parthenogenetic populations and an arrhenotokous cluster containing all uninfected, sexual populations. Nuclear and mitochondrial DNA did not exhibit concordant patterns of variation, although there was reduced genetic diversity in infected populations for both DNA types. Within the thelytokous cluster, there was nuclear DNA variation, but no mitochondrial DNA variation. This nuclear DNA variation may be explained by occasional sex between infected females and males, by horizontal transmission of Wolbachia, and/or by novel mutations. Several females from thelytokous populations were uninfected and/or heterozygous for microsatellite loci. These unexpected characteristics may be explained by migration, by inefficient transmission of Wolbachia, by horizontal transmission of Wolbachia, and/or by novel mutations. However, migration has not prevented the build‐up of considerable genetic differentiation between thelytokous and arrhenotokous populations.     

Phylogeography of the red coral (<Emphasis Type="Italic">Corallium rubrum</Emphasis>): inferences on the evolutionary history of a temperate gorgonian

The red coral Corallium rubrum (Cnidaria, Octocorallia) is an exploited, long-lived sessile species from the Mediterranean Sea and the adjacent coastline in the Atlantic Ocean. Surveys of genetic variation using microsatellites have shown that populations of C. rubrum are characterized by strong differentiation at the local scale but a study of the phylogeography of this species was still lacking. Here, we used seven polymorphic microsatellite loci, together with sequence data from an intron of the elongation factor 1 (EF1) gene, to investigate the genetic structure of C. rubrum across its geographical range in the western Mediterranean Sea and in the Adriatic Sea. The EF1 sequences were also used to analyse the consequences of demographic fluctuations linked with past environmental change. Clustering analysis with microsatellite loci highlighted three to seven genetic groups with the distinction of North African and Adriatic populations; this distinction appeared significant with AMOVA and differentiation tests. Microsatellite and EF1 data extended the isolation by distance pattern previously observed for this species at the western Mediterranean scale. EF1 sequences confirmed the genetic differentiation observed between most samples with microsatellites. A statistical parsimony network of EF1 haplotypes provided no evidence of high sequence divergence among regions, suggesting no long-term isolation. Selective neutrality tests on microsatellites and EF1 were not significant but should be interpreted with caution in the case of EF1 because of the low sample sizes for this locus. Our results suggest that recent Quaternary environmental fluctuations had a limited impact on the genetic structure of C. rubrum.     

Comparative allozyme and microsatellite population structure in a narrow endemic plant species, Centaurea corymbosa Pourret (Asteraceae)

Centaurea corymbosa Pourret (Asteraceae) is a narrow endemic species known only from six populations located in a 3-km2 area in the south of France. Earlier field experiments have suggested that pollen and seed dispersal were highly restricted within and among populations. Consistent with the field results, populations were highly differentiated for five allozyme loci and among-population variation fitted an isolation-by-distance model. In the present study, we investigated the genetic structure of C. corymbosa using six microsatellite loci. As with allozymes, microsatellites revealed no within-population structure and a large differentiation among populations. However, allozyme loci were less powerful than microsatellites in detecting the extent of gene flow assessed by assignment tests. The patterns of structuration greatly varied among loci for both types of marker; we suggest that differences in single-locus pattern could mainly be an effect of stochastic variation for allozymes and an effect of variation in mutation rate for microsatellites. In contrast to the multilocus results, the two most polymorphic microsatellite loci did not show any isolation-by-distance pattern. Our results suggest that highly variable loci might not always be the best suited markers to quantify levels of gene flow among populations.     

Genetic variation at MHC, mitochondrial and microsatellite loci in isolated populations of Brown trout (Salmo trutta)

We have studied levels and distribution of genetic variation in nine isolated populations of Brown trout in NW Spain. In the present study, we have tried to test the importance of preservation of genetic variability for the survival of a set of isolated Brown trout (Salmo trutta) populations from the same river drainage. We screened genetic variation in three different markers, mitochondrial, microsatellites and Major Histocompatibility Complex (MHC), presumed to be under different selective pressures. Overall, genetic diversity varied considerably across populations and the distribution of genetic variation was similar at MHC and microsatellites; highly polymorphic populations at the microsatellite loci were also highly polymorphic at the MHC. We also observed high levels of differentiation among populations. Although we found evidence suggesting that balancing selection has influenced the long term evolution of the MHC, genetic drift seems to have eroded the effect of selection, becoming the predominant evolutionary force shaping genetic variation in some of the smaller populations. Despite current lack of variation at the MHC, these small populations seem to have remained viable for a long time.     

Near panmixia at the distribution‐wide scale but evidence of genetic differentiation in a geographically isolated population of the Terek Sandpiper Xenus cinereus

Populations from different parts of a species range may vary in their genetic structure, variation and dynamics. Geographically isolated populations or those located at the periphery of the range may differ from those located in the core of the range. Such peripheral populations may harbour genetic variation important for the adaptive potential of the species. We studied the distribution‐wide population genetic structure of the Terek Sandpiper Xenus cinereus using 13 microsatellite loci and the mitochondrial DNA (mtDNA) control region. In addition, we estimated whether genetic variation changes from the core towards the edge of the breeding range. We used the results to evaluate the management needs of the sampled populations. Distribution‐wide genetic structure was negligible; the only population that showed significant genetic differentiation was the geographically isolated Dnieper River basin population in Eastern Europe. The genetic variation of microsatellites decreased towards the edge of the distribution, supporting the abundant‐centre hypotheses in which the core area of the distribution preserves the most genetic variation; however, no such trend could be seen with mtDNA. Overall genetic variation was low and there were signs of past population contractions followed by expansion; this pattern is found in most northern waders. The current effective population size (N_e) is large, and therefore global conservation measures are not necessary. However, the marginal Dnieper River population needs to be considered its own management unit. In addition, the Finnish population warrants conservation actions due to its extremely small size and degree of isolation from the main range, which makes it vulnerable to genetic depletion.     

Comparing RADseq and microsatellites for estimating genetic diversity and relatedness — Implications for brown trout conservation

The conservation and management of endangered species requires information on their genetic diversity, relatedness and population structure. The main genetic markers applied for these questions are microsatellites and single nucleotide polymorphisms (SNPs), the latter of which remain the more resource demanding approach in most cases. Here, we compare the performance of two approaches, SNPs obtained by restriction‐site‐associated DNA sequencing (RADseq) and 16 DNA microsatellite loci, for estimating genetic diversity, relatedness and genetic differentiation of three, small, geographically close wild brown trout (Salmo trutta) populations and a regionally used hatchery strain. The genetic differentiation, quantified as F_ST, was similar when measured using 16 microsatellites and 4,876 SNPs. Based on both marker types, each brown trout population represented a distinct gene pool with a low level of interbreeding. Analysis of SNPs identified half‐ and full‐siblings with a higher probability than the analysis based on microsatellites, and SNPs outperformed microsatellites in estimating individual‐level multilocus heterozygosity. Overall, the results indicated that moderately polymorphic microsatellites and SNPs from RADseq agreed on estimates of population genetic structure in moderately diverged, small populations, but RADseq outperformed microsatellites for applications that required individual‐level genotype information, such as quantifying relatedness and individual‐level heterozygosity. The results can be applied to other small populations with low or moderate levels of genetic diversity.     

Genetic diversity and gene flow in a Caribbean tree <Emphasis Type="Italic">Pterocarpus officinalis</Emphasis> Jacq.: a study based on chloroplast and nuclear microsatellites

We analysed the molecular diversity of Pterocarpus officinalis, a tree species distributed in Caribbean islands, South and Central America to quantify the genetic variation within island, to assess the pattern of differentiation and infer levels of gene flow; with the overall goal of defining a strategy of conservation. Two hundred two individuals of 9 populations were analysed using three chloroplast and six nuclear microsatellite markers. The observed heterozygosity varied markedly among the populations for nuclear (H _Onuc = 0.20–0.50) and chloroplast microsatellites (H _cp = 0.22–0.68). The continental population from French Guyana showed a higher value of H _Onuc than island populations, and the differences were significant in some cases. The fixation index F _IS ranged from −0.043 to 0.368; a significant heterozygote deficit was detected in 7 populations. The heterozygosity excess method suggested that two populations in Guadeloupe have undergone a recent bottleneck. Global and pairwise F _ST were high for both nuclear (F _STnuc = 0.29) and chloroplast microsatellites (F _STcp = 0.58). The neighbour-joining tree based on both markers, presented a differentiation pattern that can be explained by the seed dispersal by flotation and marine stream. The comparison of Bayesian approach and the method based on allelic frequency demonstrate a very limited number of migrants between populations.     

Genetic consequences of past climate and human impact on eastern Mediterranean <Emphasis Type="Italic">Cedrus libani</Emphasis> forests. Implications for their conservation

Summary This study demonstrates the impact of natural factors and human activities on biodiversity at gene level on a keystone Mediterranean forest ecosystem species. We monitored the within and among population gene diversity of Cedrus libani, a forest tree species of the Eastern Mediterranean mountains. We used paternally inherited chloroplast microsatellites (57 haplotypes) and bi-parentally inherited isozymes (12 loci) to estimate allelic richness, heterozygosity, and differentiation in 18 natural and 1 planted populations from Turkey and Lebanon. We showed that there is a phylogeographic structure in C. libani, and that forests from Lebanon and Turkey constitute two genetically isolated groups which probably arose from distinct refugia after the last Quaternary glacial cycle. We found extensive gene flow and relatively low differentiation in Turkey, as well as little evidence of genetic drift within populations. However, one population we analyzed, which was planted more than 20 centuries ago, and is isolated from core populations in Turkey, demonstrated extremely low genetic diversity and deserves high conservation priority. In contrast, we found low gene flow, high differentiation and severe cases of genetic drift in Lebanon. As forests there are the remnants of millennia-long extensive deforestation, all deserve high conservation priority.     

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.     

Chloroplast DNA markers (cpSSRs,SNPs) for <Emphasis Type="Italic">Miscanthus,Saccharum</Emphasis> and related grasses (Panicoideae,Poaceae)

Miscanthus and Saccharum are closely related perennial C₄ grasses. Miscanthus has recently attracted interest as a non-food crop for energy and fibre production. However, molecular genetic tools for the selection of new Miscanthus genotypes and study of its genetic resources are limited. We have identified six chloroplast (plastid) marker loci,containing both microsatellites (cpSSRs) and single nucleotide polymorphisms (SNPs) and developed primers to amplify and sequence these regions. The primers were designed using the complete chloroplast genome sequence of sugarcane and were tested on a collection of 164 Miscanthus genotypes and 14 related species of the subfamily Panicoideae. The cpSSR markers were highly polymorphic, with the number of alleles ranging from 10 to 16 per locus. Within the six cpSSR marker loci, the hybrid M. ×giganteus exhibits virtually no cpDNA variation compared with its putative parents M. sinensis and M. sacchariflorus. These SNP markers enable the differentiation of most Miscanthus species and detect infraspecific variation suitable for defining cytoplasmic genepools of Miscanthus for breeding purposes.     

Genetic diversity and integrity of German wildcat (Felis silvestris) populations as revealed by microsatellites,allozymes, and mitochondrial DNA sequences

As a consequence of persecution and habitat fragmentation, wildcats (Felis silvestris silvestris) in Western Europe have experienced a severe reduction in population numbers and sizes. The remaining wildcat populations are considered to be endangered by losses of genetic variability and by hybridisation with free-ranging domestic cats. To investigate genetic diversity within and among wild and domestic cat populations in Germany and to estimate the extent of gene flow between both forms, we analysed a total of 266 individuals. PCR-amplification and sequencing of 322 base pairs of a highly variable part of the mitochondrial control region (HV1) of 244 specimens resulted in 41 haplotypes with 31 polymorphic sites. Additionally, eight microsatellite loci were examined for those 244 cats. Moreover, a total of 46 wildcats and 22 domestic cats could be genotyped for 13 polymorphic out of 31 enzyme loci. Genetic variability in both groups was generally high. Variability in domestic cat populations was higher than in wildcat populations. Almost no differentiation between domestic cat populations could be found (F_ST for microsatellites=3%). In contrast, wildcat populations differed significantly from one another (F_ST for microsatellites=9.55%) Within the smaller wildcat populations, a reduction of genetic diversity was detectable with regard to the nuclear DNA. Wildcat and domestic cat mitochondrial haplotypes were separated, suggesting a very low level of maternal gene flow between both forms. In microsatellites and to a somewhat lesser extent in allozymes, wildcats and domestic cats showed distinct differentiation, suggesting an only low extent of past hybridisation in certain populations. The microsatellite data set indicated a significantly reduced effective population size (bottleneck) in the recent past for one German wildcat population.     

Low genetic diversity in small peripheral populations of a rare European tree <Emphasis Type="Italic">(Sorbus torminalis)</Emphasis> dominated by clonal reproduction

Analysis of nuclear microsatellites in six small populations of Sorbus torminalis on its northern distribution limit was carried out to evaluate the dominating mode of reproduction as well as genetic diversity within and among populations. Within populations there was a low genetic diversity with heterozygote excess in five populations. Genetic differentiation was high among populations and the amount of clonally arisen recruits was 94–100%. This suggests that clonal growth, small population size and a possible self-incompability system maintain heterozygosity, although some genetic drift would also be expected. The results are discussed with respect to potential conservation strategies for S. torminalis in the study region.     

Leading-edge populations do not show low genetic diversity or high differentiation in a wind-pollinated tree

Climate changes can shift species’ ranges. Knowledge on genetic variation of the leading-edge populations provides critical information to understand responses and adaptation of plants to projected climate warming. To date, the research into genetic variation of leading-edge populations has been limited, particularly in the role of wind-mediated pollen flow in maintaining high genetic variation. Castanopsis sclerophylla (Fagaceae) is a wind-pollinated and gravity-dispersed tree. In the present study, we used seven polymorphic microsatellites to genotype 482 samples from five leading-edge and 12 non-edge populations. Significant effects of recent population bottleneck events were found in three of the five leading-edge populations, indicating that the leading-edge populations might have been recolonized after the Last Glacial Maximum. Genetic diversity was higher, though not significantly, in leading-edge than in non-edge populations. Relationship between genetic diversity and latitude indicated an increasing trend of genetic diversity towards leading-edge populations. No significant difference in genetic differentiation was found between leading-edge and non-edge populations. The inconsistence with the general predictions by leading-edge colonization model could be explained by high gene flow via pollen grains. Pollen-mediated gene flow could maintain high genetic diversity within and low differentiation among leading-edge populations. In response to climate warming, high genetic variation may provide leading-edge populations raw materials for evolutionary adaptation to future environmental conditions.     

Causes of Size Homoplasy Among Chloroplast Microsatellites in Closely Related <Emphasis Type="Italic">Clusia</Emphasis> Species

Chloroplast DNA sequences and microsatellites are useful tools for phylogenetic as well as population genetic analyses of plants. Chloroplast microsatellites tend to be less variable than nuclear microsatellites and therefore they may not be as powerful as nuclear microsatellites for within-species population analysis. However, chloroplast microsatellites may be useful for phylogenetic analysis between closely related taxa when more conventional loci, such as ITS or chloroplast sequence data, are not variable enough to resolve phylogenetic relationships in all clades. To determine the limits of chloroplast microsatellites as tools in phylogenetic analyses, we need to understand their evolution. Thus, we examined and compared phylogenetic relationships of species within the genus Clusia, using both chloroplast sequence data and variation at seven chloroplast microsatellite loci. Neither ITS nor chloroplast sequences were variable enough to resolve relationships within some sections of the genus, yet chloroplast microsatellite loci were too variable to provide any useful phylogenetic information. Size homoplasy was apparent, caused by base substitutions within the microsatellite, base substitutions in the flanking regions, indels in the flanking regions, multiple microsatellites within a fragment, and forward/reverse mutations of repeat length resulting in microsatellites of identical base composition that were not identical by descent.     

Mitochondrial DNA and nuclear microsatellites reveal high diversity and genetic structure in an avian top predator,the white‐tailed sea eagle,in central Europe

We analysed 123 white‐tailed sea eagles (Haliaeetus albicilla) from (primarily central) Europe with respect to variability and differentiation based on 499 bp of the mitochondrial control region and genotypes at seven unlinked nuclear microsatellites. Variability was high (overall expected heterozygosity, haplotype and nucleotide diversity being 0.70, 0.764 and 0.00698, respectively) and both marker systems showed a subdivision into two main genetic clusters (microsatellites) or haplogroups (mtDNA). In line with earlier analyses focusing on populations from northern and eastern Europe, as well as from Asia, we found a high level of admixture in Europe and no signs of a bottleneck – despite a severe decline of white‐tailed sea eagle populations during the 20^th century. Europe is thus a global stronghold for this species not only with respect to the number of breeding pairs but also regarding the proportion of species‐wide genetic diversity. Our dense sampling revealed a possibly clinal variation within central Europe from north‐west to south‐east that was reflected by the distribution of mtDNA haplotypes as well as the two microsatellite‐based clusters. This population differentiation in central Europe probably originated from a geographically structured postglacial colonization and was later enhanced by recent demographic fluctuations. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99 , 727–737.     

Imprint of post-glacial history in a narrowly distributed endemic spruce, Picea alcoquiana, in central Japan observed in nuclear microsatellites and organelle DNA markers

Aim We aim to infer the post‐glacial history of the narrowly distributed endemic spruce Picea alcoquiana in Japan, and to assess the hypothesis that the existing narrow natural range of the species has resulted from the diminution of the past natural range since the last glacial period. Location Mountainous forests in central Honshu Island in Japan. Methods We assessed the geographical patterns of genetic variation across nine populations of P. alcoquiana across its natural range using five nuclear microsatellites and polymerase chain reaction–restriction fragment length polymorphism markers for chloroplast (cp) and mitochondrial (mt) DNA. This study focuses on the genetic differentiation between northern peripherally isolated and southern core populations within the natural range of the species in central Honshu. In addition, based on diagnostic morphological traits of the cone, we used reliable macrofossil evidence to gain further insight into the past natural range of the species. Results Picea alcoquiana shows a level of genetic differentiation among populations (Φ_ST = 0.082) that was higher than the levels observed in nuclear microsatellites for other anemophilous and widespread tree species in Japan. A notable finding for the nuclear microsatellites was the loss of rare alleles and strong evidence of a recent bottleneck in the peripherally isolated populations. Analysis of molecular variance in cpDNA and mtDNA markers showed an absence of genetic differentiation between the peripherally isolated and core populations. Macrofossil evidence indicated that the past natural range of the species extended to a lower elevation and c. 135 km north of the existing peripherally isolated populations during the last glacial period (c. 28,000 yr bp ). Main conclusions During the last glacial period, P. alcoquiana had a wider natural range in which the peripherally isolated and core populations could have been geographically continuous with each other, and effective gene flow by means of pollen and seeds might have occurred. The recent bottleneck and consequent loss of rare alleles in the peripherally isolated populations might have evolved during the diminution and retreat of the northern part of the past natural range southwards to the core area and during the subsequent separation of the peripherally isolated populations from the core area since the last glacial period.