Population genetic structure of Titanotrichum oldhamii (Gesneriaceae), a subtropical bulbiliferous plant with mixed sexual and asexual reproduction

• Background and Aims Titanotrichum oldhamii is a monotypicgenus distributed in Taiwan, adjacent regions of China and theRyukyu Isands of Japan. Its conservation status is vulnerableas most populations are small and widely scattered. Titanotrichumhas a mixed system of reproduction with vegetative bulbils andseeds. The aim of this study was to understand the populationgenetic structure of Titanotrichum in relation to its specificreproductive behaviour and to determine possible implicationsfor conservation strategies. • Methods After an extensive inventory of most wild populationsof Titanotrichum in East Asia, samples from 25 populations withinits major distribution were carried out utilizing RAPD and inter-SSRmolecular fingerprinting analysis. • Key Results The findings support the conclusion thatmany populations reproduce predominantly asexually but thatsome genetic variation still exists within populations. However,significant amounts of variation exist between populations,perhaps reflecting population differentiation by drift. Thispartitioning of genetic diversity indicates that the level ofinter-population gene exchange is extremely low. These findingsare consistent with field observations of very limited seedproduction. The Chinese populations are similar to those ofNorthern Taiwan, while the Ryukyu populations fall within therange of variation of the north-central Taiwan populations.The Taiwanese populations are relatively variable and differentiationbetween north, east and south Taiwan is evident. • Conclusions The distribution of Titanotrichum seems tobe consistent with a former land connection between China, Taiwanand the Ryukyu Islands at a glacial maximum during the Quaternary,followed by progressive fragmentation of the populations. North-centralTaiwan is the centre of genetic diversity, possibly due to theproximity of the former land bridge between the regions, togetherwith the variety of suitable habitats in north Taiwan. The significanceof these findings for conservation is discussed.     

Fine-scale genetic structure among genetic individuals of the clone-forming monotypic genus Echinosophora koreensis (Fabaceae)

• Background and Aims For rare endemics or endangered plantspecies that reproduce both sexually and vegetatively it iscritical to understand the extent of clonality because assessmentof clonal extent and distribution has important ecological andevolutionary consequences with conservation implications. Asurvey was undertaken to understand clonal effects on fine-scalegenetic structure (FSGS) in two populations (one from a disturbedand the other from an undisturbed locality) of Echinosophorakoreensis, an endangered small shrub belonging to a monotypicgenus in central Korea that reproduces both sexually and vegetativelyvia rhizomes. • Methods Using inter-simple sequence repeats (ISSRs) asgenetic markers, the spatial distribution of individuals wasevaluated using Ripley's L(d)-statistics and quantified thespatial scale of clonal spread and spatial distribution of ISSRgenotypes using spatial autocorrelation analysis techniques(join-count statistics and kinship coefficient, F_ij) for totalsamples and samples excluding clones. • Key Results A high degree of differentiation betweenpopulations was observed (_ST(g) = 0·184, P < 0·001).Ripley's L(d)-statistics revealed a near random distributionof individuals in a disturbed population, whereas significantaggregation of individuals was found in an undisturbed site.The join-count statistics revealed that most clones significantlyaggregate at 6-m interplant distance. The Sp statistic reflectingpatterns of correlograms revealed a strong pattern of FSGS forall four data sets (Sp = 0·072–0·154), butthese patterns were not significantly different from each other.At small interplant distances (2 m), however, jackknifed 95% CIs revealed that the total samples exhibited significantlyhigher F_ij values than the same samples excluding clones. • Conclusion The strong FSGS from genets is consistentwith two biological and ecological traits of E. koreensis: bee-pollinationand limited seed dispersal. Furthermore, potential clone matesover repeated generations would contribute to the observed highF_ij values among genets at short distance. To ensure long-termex situ genetic variability of the endangered E. koreensis,individuals located at distances of 10–12 m should becollected across entire populations of E. koreensis.     

Population genetic structure of Bombus terrestris in Europe: Isolation and genetic differentiation of Irish and British populations

The genetic structure of the earth bumblebee (Bombus terrestris L.) was examined across 22 wild populations and two commercially reared populations using eight microsatellite loci and two mitochondrial genes. Our study included wild bumblebee samples from six populations in Ireland, one from the Isle of Man, four from Britain and 11 from mainland Europe. A further sample was acquired from New Zealand. Observed levels of genetic variability and heterozygosity were low in Ireland and the Isle of Man, but relatively high in continental Europe and among commercial populations. Estimates of F_st revealed significant genetic differentiation among populations. Bayesian cluster analysis indicated that Irish populations were highly differentiated from British and continental populations, the latter two showing higher levels of admixture. The data suggest that the Irish Sea and prevailing south westerly winds act as a considerable geographical barrier to gene flow between populations in Ireland and Britain; however, some immigration from the Isle of Man to Ireland was detected. The results are discussed in the context of the recent commercialization of bumblebees for the European horticultural industry.     

Identification of conservation units in the European Mergus merganser based on nuclear and mitochondrial DNA markers

The conservation status of small breeding areas of the Goosander (Mergus merganser merganser) in Central Europe is unclear. Geographic isolation of these areas suggests restricted gene flow to and from large North-European populations. On the other hand, migrating Goosanders from northern Europe join the Central European breeding population for wintering. To evaluate the conservation status of the small breeding areas we assessed the genetic structure of M. merganser populations in Europe by examining two nuclear marker systems (microsatellites and Single Nucleotide Polymorphisms, SNP) and mitochondrial (mtDNA) control region sequence variation for Goosanders in 11 sampling areas representing three of five distinct breeding areas and two subspecies (M. m. merganser and M. m. americanus). Overall population differentiation estimates including both subspecies were high, both based on mtDNA () and nuclear markers (θ _ST = 0.219; 95% CI 0.088–0.398, SNP and microsatellites combined). Within Europe, mtDNA revealed a strong overall () and significant pairwise population differentiation between almost all comparisons. In contrast, both nuclear marker systems combined revealed only a small overall genetic differentiation (θ _ST = 0.022; 95% CI 0.003–0.041). The strong genetic differentiation based on female-inherited mtDNA but not on biparentally inherited nuclear markers can be explained by sex-biased dispersal and strong female philopatry. Therefore, small breeding areas in Europe are endangered despite large male-mediated gene-flow, because when these populations decline, only males—but due to strong philopatry not females—can be efficiently supplemented by migration from the large North European populations. We therefore propose to manage the small breeding areas independently and to strengthen conservation efforts for this species in Central Europe.     

PATTERNS OF GENETIC DIFFERENTIATION AND CONSERVATION OF THE SLABSIDE PEARLYMUSSEL, LEXINGTONIA DOLABELLOIDES (LEA, 1840) IN THE TENNESSEE RIVER DRAINAGE

The restoration and recovery of imperiled mussel species willrequire the re-establishment of populations into historicallyoccupied habitats. The possible existence of genetic differentiationamong populations should be considered before inter-basin transfersare made. Eighty individuals of the federal candidate speciesLexingtonia dolabelloides were sampled from populations in theNorth Fork Holston, Middle Fork Holston, Clinch, Paint Rockand Duck rivers of the Tennessee River basin in the southeasternUnited States. We sequenced 603 base-pairs of a mitochondrialDNA gene (ND-1) and 512 base-pairs of a nuclear DNA gene (ITS-1).Analyses of molecular variation (AMOVA) values for both genesindicated that the majority of variation in L. dolabelloidesresided within populations (82.9–88.3%), with 11.7–17.1%of variation among populations. Haplotype frequencies differedsignificantly among populations for both genes sequenced. Clusteringof haplotypes in minimum-spanning networks did not conform stringentlyto population boundaries, reflecting high within-populationand low between-population variability. Maximum parsimony analysisdid not identify any population as a monophyletic lineage. AMantel test showed no significant correlation between geographicalstream distance and genetic distance, thus not supporting apattern of isolation-by-distance. Overall, results providedsupport to manage fragmented populations of L. dolabelloidesin the Tennessee River drainage as two management units (MUs),but did not provide evidence for the existence of ESUs followingpublished molecular criteria. (Received 26 October 2004; accepted 29 April 2005)     

Genetic diversity and geographic differentiation in endangered Ammopiptanthus (Leguminosae) populations in desert regions of northwest China as revealed by ISSR analysis

• Background and Aims The desert legume genus Ammopiptanthuscomprises two currently endangered species, A. mongolicus andA. nanus. Genetic variability and genetic differentiation betweenthe two species and within each species were examined. • Methods Inter-simple sequence repeat (ISSR) marker datawere obtained and analysed with respect to genetic diversity,structure and gene flow. • Key Results Despite the morphological similarity betweenA. mongolicus and A. nanus, the two species are geneticallydistinct from each other, indicated by 63 % species-specificbands. Low genetic variability was detected for both populationlevel (Shannon indices of diversity H_pop = 0·106, percentageof polymorphic loci P = 18·55 % for A. mongolicus; H_pop= 0·070, P = 12·24 % for A. nanus) and specieslevel (H_sp = 0·1832, P = 39·39 % for A. mongolicus;H_sp = 0·1026, P = 25·89 % for A. nanus). Moderategenetic differentiation was found based on different measures(AMOVA _ST and Hickory ^B) in both A. mongolicus (0·3743–0·3744)and A. nanus (0·2162–0·2369). • Conclusions The significant genetic difference betweenthe two species might be due to a possible vicariant evolutionaryevent from a single common ancestor through the fragmentationof their common ancestor's range. Conservation strategies forthese two endangered species are proposed.     

Genetic variation and island biogreography: Microsatellite and mitochondrial DNA variation in island populations of the Australian bush rat, Rattus fuscipes greyii

To understand the impact of various factors on the maintenance of genetic variation in natural populations, we need to focus on situations where at least some of these factors are removed or controlled. In this study, we used highly variable, presumably neutral, microsatellite and mtDNA markers to assess the nature of genetic variation in 14 island and two mainland populations of the Australian bush rat, where there is no migration between islands. Thus we are controlling for selection and gene flow. Both marker sets revealed low levels of diversity within the small island populations and extreme differentiation between populations. For six microsatellite loci, all of the small island populations had less genetic variation than the mainland populations; reduction in allelic diversity was more pronounced than loss of heterozygosity. Kangaroo Island, the large island population, had similar levels of diversity to the mainland populations. A 442 base pair (bp) section of the mtDNA control region was screened for variation by outgroup heteroduplex analysis/temperature gradient gel electrophoresis (OHA/TGGE). Only three of the 13 small island populations showed haplotypic diversity: Gambier (2), Waldegrave (2), and Eyere (3). The level of haplotypic diversity in the small island populations was similar to that on the mainland, most likely reflecting a recent population bottleneck on the mainland. In contrast, Kangaroo Island had 9 mtDNA haplotypes. The dominant factor influencing genetic diversity on the islands was island size. No correlation was detected between genetic diversity and the time since isolation or distance form the mainland. The combination of genetic drift within and complete isolation among the small island populations has resulted in rapid and extreme population divergence. Population pair-wise comparisons of allele frequency distributions showed significant differences for all populations for all loci (F _st = 0.11–0.84, R _st = 0.07–0.99). For the mtDNA control region, 92.6% of variation was apportioned between populations; only the Pearson islands shared a haplotype. Mantel tests of pair-wise genetic distance with pair-wise geographic distance showed no significant geographical clustering of haplotypes. However, population substructuring was detected within populations where sampling was conducted over a broader geographical range, as indicated by departures from Hardy-Weinberg equilibrium. Thus substructuring in the ancestral population cannot be ruled out. The dominant evolutionary forces on the islands, after the initial founder event, are stochastic population processes such as genetic drift and mutation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Absence of founder effect and evidence for adaptive divergence in a recently introduced insular population of white‐tailed deer (Odocoileus virginianus)

Islands are generally colonized by few individuals which could lead to a founder effect causing loss of genetic diversity and rapid divergence by strong genetic drift. Insular conditions can also induce new selective pressures on populations. Here, we investigated the extent of genetic differentiation within a white‐tailed deer (Odocoileus virginianus) population introduced on an island and its differentiation with its source mainland population. In response to their novel environmental conditions, introduced deer changed phenotypically from mainland individuals, therefore we investigated the genetic bases of the morphological differentiation. The study was conducted on Anticosti Island (Québec, Canada) where 220 individuals were introduced 120 years ago, resulting in a population size over 160,000 individuals. We used genotyping‐by‐sequencing (GBS) to generate 8,518 filtered high‐quality SNPs and compared patterns of genetic diversity and differentiation between the continental and Anticosti Island populations. Clustering analyses indicated a single panmictic island population and no sign of isolation by distance. Our results revealed a weak, albeit highly significant, genetic differentiation between the Anticosti Island population and its source population (mean F_ST = 0.005), which allowed a population assignment success of 93%. Also, the high genetic diversity maintained in the introduced population supports the absence of a strong founder effect due to the large number of founders followed by rapid population growth. We further used a polygenic approach to assess the genetic bases of the divergent phenotypical traits between insular and continental populations. We found loci related to muscular function and lipid metabolism, which suggested that these could be involved in local adaptation on Anticosti Island. We discuss these results in a harvest management context.     

Population Genetic Structure of the Endangered <Emphasis Type="Italic">Brasenia schreberi</Emphasis> in South Korea Based on Nuclear Ribosomal Spacer and Chloroplast DNA Sequences

Genetic variation of nuclear ribosomal ITS (nrITS) and chloroplast DNA (cpDNA) regions was investigated in Brasenia schreberi (Cabombaceae) to assess the population structure and to infer the evolutionary relationship among 12 populations distributed in South Korea. The combined sequence of the two regions was aligned to 2,069 bp, of which 28 sites were variable. In total, 20 genotypes were identified from 240 individuals of B. schreberi. Genotype diversity (Gd) and nucleotide diversity (Pi) on Jeju Island (Gd = 0.2511, Pi = 0.00012) were higher than those of the mainland of South Korea (Gd = 0.1358, Pi = 0.00005). The relatively low level of genetic variation of the mainland populations is associated with its higher genetic differentiation (G _ST = 0.095 on mainland and 0.039 on Jeju Island) and human activities. Minimum spanning network analysis demonstrated that the investigated populations of B. schreberi were subdivided into two geographical groups: the mainland of South Korea and Jeju Island. In addition, analysis of molecular variation showed that a large proportion (73.55%) of genetic differentiation existed between the two regions. These results strongly suggest the presence of significant barriers to gene flow between regions. Thus, the management unit for B. schreberi should be carefully designed to avoid the potential risk that can results from the admixture of individuals from the mainland and Jeju Island regions.     

A holistic approach to taxonomic evaluation of two closely related endangered freshwater mussel species, the oyster mussel Epioblasma capsaeformis and tan riffleshell Epioblasma florentina walkeri (Bivalvia: Unionidae)

Species in the genus Epioblasma have specialized life historyrequirements and represent the most endangered genus of freshwatermussels (Unionidae) in the world. A genetic characterizationof extant populations of the oyster mussel E. capsaeformis andtan riffleshell E. florentina walkeri sensu late was conductedto assess taxonomic validity and to resolve conservation issuesfor recovery planning. These mussel species exhibit pronouncedphenotypic variation, but were difficult to characterize phylogeneticallyusing DNA sequences. Monophyletic lineages, congruent with phenotypicvariation among species, were obtained only after extensiveanalysis of combined mitochondrial (1396 bp of 16S, cytochrome-b,and ND1) and nuclear (515 bp of ITS-1) DNA sequences. Incontrast, analysis of variation at 10 hypervariable DNA microsatelliteloci showed moderately to highly diverged populations basedon F_ST and R_ST values, which ranged from 0.12 to 0.39 and 0.15to 0.71, respectively. Quantitative variation between specieswas observed in fish-host specificity, with transformation successof glochidia of E. capsaeformis significantly greater (P<0.05)on greenside darter Etheostoma blennioides, and that of E. f.walkeri significantly greater (P<0.05) on fantail darterEtheostoma flabellare. Lengths of glochidia differed significantly(P<0.001) among species and populations, with mean sizesranging from 241 to 272 µm. The texture and colourof the mantle-pad of E. capsaeformis sensu stricto is smoothand bluish-white, whereas that of E. f. walkeri is pustuledand brown, with tan mottling. Based on extensive molecular,morphological and life history data, the population of E. capsaeformisfrom the Duck River, Tennessee, USA is proposed as a separatespecies, and the population of E. f. walkeri from Indian Creek,upper Clinch River, Virginia, USA is proposed as a distinctsubspecies. (Received 23 February 2005; accepted 16 January 2006)     

Characterization and physical mapping of ribosomal RNA gene families in Plantago

• Background and Aims The organization of rRNA genes incultivated Plantago ovata Forsk. and several of its wild allieswas analysed to gain insight into the phylogenetic relationshipsof these species in the genus which includes some 200 species. • Methods Specific primers were designed to amplify theinternal transcribed spacer (ITS1 and ITS2) regions from sevenPlantago species and the resulting fragments were cloned andsequenced. Similarly, using specific primers, the 5S rRNA genesfrom these species were amplified and subsequently cloned. Fluorescencein-situ hybridization (FISH) was used for physical mapping of5S and 45S ribosomal RNA genes. • Results The ITS1 region is 19–29 bp longer thanthe ITS2 in different Plantago species. The 5S rRNA gene-repeatingunit varies in length from 289 to 581 bp. Coding regions arehighly conserved across species, but the non-transcribed spacers(NTS) do not match any database sequences. The clone from thecultivated species P. ovata was used for physical mapping ofthese genes by FISH. Four species have one FISH site while threehave two FISH sites. In P. lanceolata and P. rhodosperma, the5S and 45S (18S-5·8S-25S) sites are coupled. • Conclusions Characterization of 5S and 45S rRNA geneshas indicated a possible origin of P. ovata, the only cultivatedspecies of the genus and also the only species with x = 4, froma species belonging to subgenus Psyllium. Based on the studiesreported here, P. ovata is closest to P. arenaria, althoughon the basis of other data the two species have been placedin different subgenera. FISH mapping can be used as an efficienttool to help determine phylogenetic relationships in the genusPlantago and show the interrelationship between P. lanceolataand P. lagopus.     

The Temporal and Spatial Invasion Genetics of the Western Corn Rootworm (Coleoptera: Chrysomelidae) in Southern Europe

This study describes the genetics of the western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte in southern Europe during the introduction (1996–2001) and establishment/spread (2002–2011) phases of its invasion. The Diabrotica microsatellite core-set was used to perform traditional population genetics analyses. Our results indicated that during the introduction phase genetic diversity and population genetic structure were lower overall as compared to the establishment/spread phase. Unusually high genetic differentiation was found between the Italy and southern Europe comparisons, including high differentiation between Italian populations separated by a short distance during the establishment/spread phase. STRUCTURE analysis revealed two genetic clusters during the introduction phase and two genetic clusters during the establishment/spread phase. However, bottlenecked populations were only detected during the invasion phase. A small but significant isolation by distance effect was noted in both phases. Serbia was the geographic source of WCR to Croatia and Hungary in the introduction phase, while the United States of America was the possible source of WCR to Italy in 2001. These introductory populations were the subsequent source of individuals sampled during the establishment/spread phase. Repeated introductions and admixture events in southern Europe may have resulted in genetically diverse WCR populations that have attained 83% of all known alleles worldwide.     

华东地区中华蜜蜂六地理种群的遗传多样性及遗传分化

目的】利用23对微卫星标记对来自于南昌、黄山、桐庐、费县、宜兴、武夷山6个华东地区的中华蜜蜂Apis cerana cerana种群进行遗传多样性及遗传分化分析。【方法】通过计算多态信息含量、平均杂合度、等位基因数、遗传距离、基因流、F 统计量等参数, 评估各中蜂种群遗传多样性和各种群间遗传分化。【结果】各座位的等位基因数为5（A014）至30（AP043）。所有种群均显示较高水平的期望杂合度, 其中, 武夷山中蜂最低, 为0.4280; 南昌中蜂最高, 为0.6329。各中蜂种群间存在极显著的遗传分化, 平均分化系数(Fst)为0.344。基于Nei氏遗传距离运用NJ聚类法将6个中蜂种群划分为3类。【结论】华东6个中蜂种群的遗传多样性较高, 遗传分化显著; 分析遗传分化与地理距离的关系发现, 华东6个中蜂种群间的遗传分化与地理距离不存在显著相关。     

Allozyme Variation and Phylogenetic Relationships in Picea jezoensis (Pinaceae) Populations of the Russian Far East

Genetic variation and differentiation of 12 populations of Picea jezoensis from the Russian Far East were studied using 20 allozyme loci. The mean number of alleles per locus was 2.63, the percent of polymorphic loci was 88.1%, the observed heterozygosity was 0.181, and the mean value of expected heterozygosity amounted to 0.189. The values of expected heterozygosity of the northern and central mainland populations were higher than in the southern part of the natural range. A significant bias of Hardy–Weinberg heterozygosity to equilibrium heterozygosity (H_eq) suggests that most of the mainland populations have recently experienced a severe expansion in population size while populations from Kamchatka Peninsula have undergone a reduction in population size. Unbiased Nei’s genetic distance values were low within and between the mainland and Sakhalin Island populations (D_N=0.008). The largest values (D_N=0.063) were found between the mainland/Sakhalin and Kamchatka Peninsula populations. Based on genetic distance, P. jezoensis and P. kamtschatkensis could be considered as distinct taxa, but P. ajanensis, P. microsperma, and P. komarovii do not warrant taxonomic recognition.     

Flow cytometric and microscopic analysis of the effect of tannic acid on plant nuclei and estimation of DNA content

• Background and Aims Flow cytometry (FCM) is extensivelyused to estimate DNA ploidy and genome size in plants. In orderto determine nuclear DNA content, nuclei in suspension are stainedby a DNA-specific fluorochrome and fluorescence emission isquantified. Recent studies have shown that cytosolic compoundsmay interfere with binding of fluorochromes to DNA, leadingto flawed data. Tannic acid, a common phenolic compound, maybe responsible for some of the stoichiometric errors, especiallyin woody plants. In this study, the effect of tannic acid onestimation of nuclear DNA content was evaluated in Pisum sativumand Zea mays, which were chosen as model species. • Methods Nuclear suspensions were prepared from P. sativumleaf tissue using four different lysis buffers (Galbraith's,LB01, Otto's and Tris.MgCl₂). The suspensions were treated withtannic acid (TA) at 13 different initial concentrations rangingfrom 0·25 to 3·50 mg mL^–1. After propidiumiodide (PI) staining, samples were analysed using FCM. In additionto the measurement of nuclei fluorescence, light scatter propertieswere assessed. Subsequently, a single TA concentration was chosenfor each buffer and the effect of incubation time was assessed.Similar analyses were performed on liquid suspensions of P.sativum and Z. mays nuclei that were isolated, treated and analysedsimultaneously. FCM analyses were accompanied by microscopicobservations of nuclei suspensions. • Key Results TA affected PI fluorescence and light scatterproperties of plant nuclei, regardless of the isolation bufferused. The least pronounced effects of TA were observed in Tris.MgCl₂buffer. Samples obtained using Galbraith's and LB01 bufferswere the most affected by this compound. A newly described ‘tannicacid effect’ occurred immediately after the addition ofthe compound. With the exception of Otto's buffer, nuclei ofP. sativum and Z. mays were affected differently, with pea nucleiexhibiting a greater decrease in fluorescence intensity. • Conclusions A negative effect of a secondary metabolite,TA, on estimation of nuclear DNA content is described and recommendationsfor minimizing the effect of cytosolic compounds are presented.Alteration in light scattering properties of isolated nucleican be used as an indicator of the presence of TA, which maycause stoichiometric errors in nuclei staining using a DNA intercalator,PI.     

Invaded range of the blackberry pathogen Phragmidium violaceum in the Pacific Northwest of the USA and the search for its provenance

Field surveys in 2006 confirmed that the exotic rust fungus Phragmidium violaceum was widespread on Rubus armeniacus and Rubus laciniatus in the Pacific Northwest of the USA. The origin and dispersal pattern of this obligate biotrophic pathogen in the USA were investigated by comparing the genetic diversity and structure of 27 isolates each from the USA and Europe, and 20 isolates from Australia where an invasion occurred in 1984. Analysis of 11 microsatellite loci revealed 74 unique genotypes, with the European population having a significantly higher level of allelic diversity and number of private alleles compared to populations from the USA and Australia. Principal coordinate analysis (PCA), analysis of molecular variance and pairwise comparisons of Φ confirmed a strong level of differentiation among continental populations, with little divergence between isolates from the USA and Europe, but a high level of differentiation between these isolates and those from Australia. These results were broadly supported by the Bayesian cluster analysis, which indicated that at K = 3 the clustering of the isolates corresponds to their geographic origin. Bayesian clustering, PCA as well as insignificant migration estimates from Europe to the USA suggest that the USA population is not a direct descendant from the European P. violaceum population. There was a weak association between genetic and geographic distance among the USA isolates, suggesting invasion was initially localized prior to dispersal or that the population may have been present for some time prior to first detection in 2005.     

Low genetic variability and strong differentiation among isolated populations of the rare steppe grass Stipa capillata L. in Central Europe

Stipa capillata L. (Poaceae) is a rare grassland species in Central Europe that is thought to have once been widespread in post‐glacial times. Such relict species are expected to show low genetic diversity within populations and high genetic differentiation between populations due to bottlenecks, long‐term isolation and ongoing habitat fragmentation. These patterns should be particularly pronounced in selfing species. We analysed patterns of random amplified polymorphic DNA (RAPD) variation in the facultatively cleistogamous S. capillata to examine whether genetic diversity is associated with population size, and to draw initial conclusions on the migration history of this species in Central Europe. We analysed 31 S. capillata populations distributed in northeastern, central and western Germany, Switzerland and Slovakia. Estimates of genetic diversity at the population level were low and not related to population size. Among all populations, extraordinarily high levels of genetic differentiation (amova : φ_ST = 0.86; Bayesian analysis: θ^B = 0.758) and isolation‐by‐distance were detected. Hierarchical amova indicated that most of the variability was partitioned among geographic regions (59%), or among populations between regions when the genetically distinct Slovakian populations were excluded. These findings are supported by results of a multivariate ordination analysis. We also found two different groups in an UPGMA cluster analysis: one that contained the populations from Slovakia, and the other that combined the populations from Germany and Switzerland. Our findings imply that S. capillata is indeed a relict species that experienced strong bottlenecks in Central Europe, enhanced by isolation and selfing. Most likely, populations in Slovakia were not the main genetic source for the post‐glacial colonization of Central Europe.     

Population differentiation of sessile oak at the altitudinal front of migration in the French Pyrenees

To assess the effects of altitude on the level and structure of genetic diversity, a genetic survey was conducted in 12 populations of sessile oak (Quercus petraea) located between 130 and 1660 m in two parallel valleys on the northern side of the Pyrenees Mountains. Genetic diversity was monitored at 16 nuclear microsatellite loci and 5 chloroplast DNA (cpDNA) markers. The cpDNA survey suggested that extant populations in both valleys shared the same source populations from the plain. There was no visible trend of nuclear genetic diversity along altitude, even if indirect estimates of effective population sizes revealed a consistent reduction at higher altitudes. Population differentiation, although low, was mostly present among populations of the same valleys and reached similar levels than differentiation across the range of distribution of sessile oak. Contribution to the overall differentiation in the valleys was mostly due to the genetic divergence of the highest populations and the altitudinal variation of allelic frequencies at a few loci. Bayesian inference of migration between groups of populations showed that gene flow is preferentially unidirectional from lower altitudes in one valley to other groups of populations. Finally, we found evidence of clonal reproduction in high altitude populations. The introgression of Quercus robur and Quercus pubescens was also more frequent at the altitudinal margin suggesting that this mechanism may have contributed to the present migration and adaptation of Q. petraea and may also facilitate its future upslope shift in the context of climate change.     

Comparison of four nuclear isolation buffers for plant DNA flow cytometry

• Background and Aims DNA flow cytometry requires preparationof suspensions of intact nuclei, which are stained using a DNA-specificfluorochrome prior to analysis. Various buffer formulas weredeveloped to preserve nuclear integrity, protect DNA from degradationand facilitate its stoichiometric staining. Although nuclearisolation buffers differ considerably in chemical composition,no systematic comparison of their performance has been madeuntil now. This knowledge is required to select the appropriatebuffer for a given species and tissue. • Methods Four common lysis buffers (Galbraith's, LB01,Otto's and Tris.MgCl₂) were used to prepare samples from leaftissues of seven plant species (Sedum burrito, Oxalis pes-caprae,Lycopersicon esculentum, Celtis australis, Pisum sativum, Festucarothmaleri and Vicia faba). The species were selected to covera wide range of genome sizes (1·30–26·90pg per 2C DNA) and a variety of leaf tissue types. The followingparameters were assessed: forward (FS) and side (SS) light scatters,fluorescence of propidium iodide-stained nuclei, coefficientof variation of DNA peaks, presence of debris background andthe number of nuclei released from sample tissue. The experimentswere performed independently by two operators and repeated onthree different days. • Key Results Clear differences among buffers were observed.With the exception of O. pes-caprae, any buffer provided acceptableresults for all species. LB01 and Otto's were generally thebest buffers, with Otto's buffer providing better results inspecies with low DNA content. Galbraith's buffer led to satisfactoryresults and Tris.MgCl₂ was generally the worst, although ityielded the best histograms in C. australis. A combined analysisof FS and SS provided a ‘fingerprint’ for each buffer.The variation between days was more significant than the variationbetween operators. • Conclusions Each lysis buffer tested responded to a specificproblem differently and none of the buffers worked best withall species. These results expand our knowledge on nuclear isolationbuffers and will facilitate selection of the most appropriatebuffer depending on species, tissue type and the presence ofcytosolic compounds interfering with DNA staining.     

Genetic variation among endangered Irish red grouse (<Emphasis Type="Italic">Lagopus lagopus hibernicus</Emphasis>) populations: implications for conservation and management

Extant populations of Irish red grouse (Lagopus lagopus hibernicus) are both small and fragmented, and as such may have an increased risk of extinction through the effects of inbreeding depression and compromised adaptive potential. Here we used 19 microsatellite markers to assay genetic diversity across 89 georeferenced samples from putatively semi-isolated areas throughout the Republic of Ireland and we also genotyped 27 red grouse from Scotland using the same markers. The genetic variation within Ireland was low in comparison to previously published data from Britain and the sample of Scottish red grouse, and comparable to threatened European grouse populations of related species. Irish and Scottish grouse were significantly genetically differentiated (F_ST = 0.07, 95% CI = 0.04–0.10). There was evidence for weak population structure within Ireland with indications of four distinct genetic clusters. These correspond approximately to grouse populations inhabiting suitable habitat patches in the North West, Wicklow Mountains, Munster and Cork, respectively, although some admixture was detected. Pair-wise F_ST values among these populations ranged from 0.02 to 0.04 and the overall mean allelic richness was 5.5. Effective population size in the Munster area was estimated to be 62 individuals (95% CI = 33.6–248.8). Wicklow was the most variable population with an AR value of 5.4 alleles/locus. Local (Munster) neighbourhood size was estimated to 31 individuals corresponding to an average dispersal distance of 31 km. In order to manage and preserve Irish grouse we recommend that further fragmentation and destruction of habitats need to be prevented in conjunction with population management, including protection of the integrity of the existing population by refraining from augmenting it with individuals from mainland Britain to maximise population size.