Relative emissions intensity of dairy production systems: employing different functional units in life-cycle assessment.

This study aimed to assess the merit and suitability of individual functional units (FU) in expressing greenhouse gas emissions intensity in different dairy production systems. An FU provides a clearly defined and measurable reference to which input and output data are normalised. This enables the results from life-cycle assessment (LCA) of different systems to be treated as functionally equivalent. Although the methodological framework of LCA has been standardised, selection of an appropriate FU remains ultimately at the discretion of the individual study. The aim of the present analysis was to examine the effect of different FU on the emissions intensities of different dairy production systems. Analysis was based on 7 years of data (2004 to 2010) from four Holstein-Friesian dairy systems at Scotland's Rural College's long-term genetic and management systems project, the Langhill herd. Implementation of LCA accounted for the environmental impacts of the whole-farm systems and their production of milk from 'cradle to farm gate'. Emissions intensity was determined as kilograms of carbon dioxide equivalents referenced to six FU: UK livestock units, energy-corrected milk yield, total combined milk solids yield, on-farm land used for production, total combined on- and off-farm land used for production, and the proposed new FU-energy-corrected milk yield per hectare of total land used. Energy-corrected milk was the FU most effective for reflecting differences between the systems. Functional unit that incorporated a land-related aspect did not find difference between systems which were managed under the same forage regime, despite their comprising different genetic lines. Employing on-farm land as the FU favoured grazing systems. The proposed dual FU combining both productivity and land use did not differentiate between emissions intensity of systems as effectively as the productivity-based units. However, this dual unit displayed potential to quantify in a simple way the positive or negative outcome of trade-offs between land and production efficiencies, in which improvement in emissions intensity using one FU may be accompanied by deterioration using another FU. The perceived environmental efficiencies of different dairy production systems in terms of their emissions intensities were susceptible to change based upon the FU employed, and hence the FU used in any study needs to be taken into account in the interpretation of results.

Substantial heritable variation for susceptibility to Dothistroma septosporum within populations of native British Scots pine (Pinus sylvestris).

The threat from pests and pathogens to native and commercially planted forest trees is unprecedented and expected to increase under climate change. The degree to which forests respond to threats from pathogens depends on their adaptive capacity, which is determined largely by genetically controlled variation in susceptibility of the individual trees within them and the heritability and evolvability of this trait. The most significant current threat to the economically and ecologically important species Scots pine (Pinus sylvestris) is dothistroma needle blight (DNB), caused by the foliar pathogen Dothistroma septosporum. A progeny-population trial of 4-year-old Scots pine trees, comprising six populations from native Caledonian pinewoods each with three to five families in seven blocks, was artificially inoculated using a single isolate of D. septosporum. Susceptibility to D. septosporum, assessed as the percentage of non-green needles, was measured regularly over a period of 61 days following inoculation, during which plants were maintained in conditions ideal for DNB development (warm; high humidity; high leaf wetness). There were significant differences in susceptibility to D. septosporum among families indicating that variation in this trait is heritable, with high estimates of narrow-sense heritability (0.38-0.75) and evolvability (genetic coefficient of variation, 23.47). It is concluded that native Scots pine populations contain sufficient genetic diversity to evolve lower susceptibility to D. septosporum through natural selection in response to increased prevalence of this pathogen.

Development and use of microsatellite markers to study diversity, reproduction and population genetic structure of the cereal pathogen Ramularia collo-cygni.

Ramularia collo-cygni (Rcc) is a major pathogen of barley that causes economically serious yield losses. Disease epidemics during the growing season are mainly propagated by asexual air-borne spores of Rcc, but it is thought that Rcc undergoes sexual reproduction during its life cycle and may also disperse by means of sexual ascospores. To obtain population genetic information from which to infer the extent of sexual reproduction and local genotype dispersal in Rcc, and by implication the pathogen's ability to adapt to fungicides and resistant cultivars, we developed ten polymorphic microsatellite markers, for which primers are presented. We used these markers to analyse the population genetic structure of this cereal pathogen in two geographically distant populations from the Czech Republic (n=30) and the United Kingdom (n=60) that had been sampled in a spatially explicit manner. Genetic diversity at the microsatellite loci was substantial, Ht=0.392 and Ht=0.411 in the Czech and UK populations respectively, and the populations were moderately differentiated at these loci (Θ=0.111, P<0.01). In both populations the multilocus genotypic diversity was very high (one clonal pair per population, resulting in >96% unique genotypes in each of the populations) and there was a lack of linkage disequilibrium among loci, strongly suggesting that sexual reproduction is an important component of the life cycle of Rcc. In an analysis of spatial genetic structure, kinship coefficients in all distance classes were very low (-0.0533 to 0.0142 in the Czech and -0.0268 to 0.0042 in the Scottish population) and non-significant (P>0.05) indicating lack of subpopulation structuring at the field scale and implying extensive dissemination of spores. These results suggest that Rcc possesses a high evolutionary potential for developing resistance to fungicides and overcoming host resistance genes, and argue for the development of an integrated disease management system that does not rely solely on fungicide applications.

Genetic differentiation and species cohesion in two widespread Central American Begonia species.

Begonia is one of the ten largest plant genera, with over 1500 species. This high species richness may in part be explained by weak species cohesion, which has allowed speciation by divergence in allopatry. In this study, we investigate species cohesion in the widespread Central American Begonia heracleifolia and Begonia nelumbiifolia, by genotyping populations at microsatellite loci. We then test for post-zygotic reproductive barriers using experimental crosses, and assess whether sterility barriers are related to intraspecific changes in genome size, indicating major genome restructuring between isolated populations. Strong population substructure was found for B. heracleifolia (FST=0.364, F'ST=0.506) and B. nelumbiifolia (FST=0.277, F'ST=0.439), and Bayesian admixture analysis supports the division of most populations into discrete genetic clusters. Moderate levels of inferred selfing (B. heracleifolia s=0.40, B. nelumbiifolia s=0.62) and dispersal limitation are likely to have contributed to significant genetic differentiation (B. heracleifolia Jost's D=0.274; B. nelumbiifolia D=0.294). Interpopulation crosses involving a divergent B. heracleifolia population with a genome size ∼10% larger than the species mean had a ∼20% reduction in pollen viability compared with other outcrosses, supporting reproductive isolation being polymorphic within the species. The population genetic data suggest that Begonia populations are only weakly connected by gene flow, allowing reproductive barriers to accumulate between the most isolated populations. This supports allopatric divergence in situ being the precursor of speciation in Begonia, and may also be a common speciation mechanism in other tropical herbaceous plant groups.

Next-generation hybridization and introgression.

Hybridization has a major role in evolution-from the introgression of important phenotypic traits between species, to the creation of new species through hybrid speciation. Molecular studies of hybridization aim to understand the class of hybrids and the frequency of introgression, detect the signature of ancient hybridization, and understand the behaviour of introgressed loci in their new genomic background. This often involves a large investment in the design and application of molecular markers, leading to a compromise between the depth and breadth of genomic data. New techniques designed to assay a large sub-section of the genome, in association with next-generation sequencing (NGS) technologies, will allow genome-wide hybridization and introgression studies in organisms with no prior sequence data. These detailed genotypic data will unite the breadth of sampling of loci characteristic of population genetics with the depth of sequence information associated with molecular phylogenetics. In this review, we assess the theoretical and methodological constraints that limit our understanding of natural hybridization, and promote the use of NGS for detecting hybridization and introgression between non-model organisms. We also make recommendations for the ways in which emerging techniques, such as pooled barcoded amplicon sequencing and restriction site-associated DNA tags, should be used to overcome current limitations, and enhance our understanding of this evolutionary significant process.

Early evolution in a hybrid swarm between outcrossing and selfing lineages in Geum.

Although often considered as evolutionary dead ends, selfing taxa may make an important contribution to plant evolution through hybridization with related outcrossing lineages. However, there is a shortage of studies examining the evolutionary dynamics of hybridization between outcrossing and selfing taxa. On the basis of differential pollinator attractiveness, production and competitive ability of pollen, as well as levels of inbreeding depression, we predict that the early products of hybridization between outcrossing and selfing lineages will be F1s and first-generation backcrosses sired mainly by the outcrossing lineage, together with selfed F2s containing a limited genetic contribution from the outcrosser. These predictions were tested using amplified fragment length polymorphism and chloroplast markers to analyze the composition of a recent hybrid swarm between predominantly outcrossing Geum rivale and predominantly selfing Geum urbanum. In line with predictions, the hybrid swarm comprised both parental species together with F1s and first-generation backcrosses to G. rivale alone. Chloroplast data suggested that G. rivale was the pollen parent for both observed hybrid classes. However, there was no evidence for F2 individuals, despite the fact that the F1 was fully self-compatible and able to auto-pollinate. The pollen fertility of F1s was only 30% lower than that of the parental taxa, and was fully restored in backcross hybrids. Predicting future evolution in the hybrid swarm will require an understanding of the mating patterns within and among the mix of parental, F1 and backcross genotypes that are currently present. However, these results support the hypothesis that introgression is likely to be asymmetrical from selfing to outcrossing lineages.

High genetic diversity at the extreme range edge: nucleotide variation at nuclear loci in Scots pine (Pinus sylvestris L.) in Scotland.

Nucleotide polymorphism at 12 nuclear loci was studied in Scots pine populations across an environmental gradient in Scotland, to evaluate the impacts of demographic history and selection on genetic diversity. At eight loci, diversity patterns were compared between Scottish and continental European populations. At these loci, a similar level of diversity (θ(sil)= ~0.01) was found in Scottish vs mainland European populations, contrary to expectations for recent colonization, however, less rapid decay of linkage disequilibrium was observed in the former (ρ=0.0086±0.0009, ρ=0.0245±0.0022, respectively). Scottish populations also showed a deficit of rare nucleotide variants (multi-locus Tajima's D=0.316 vs D=-0.379) and differed significantly from mainland populations in allelic frequency and/or haplotype structure at several loci. Within Scotland, western populations showed slightly reduced nucleotide diversity (π(tot)=0.0068) compared with those from the south and east (0.0079 and 0.0083, respectively) and about three times higher recombination to diversity ratio (ρ/θ=0.71 vs 0.15 and 0.18, respectively). By comparison with results from coalescent simulations, the observed allelic frequency spectrum in the western populations was compatible with a relatively recent bottleneck (0.00175 × 4N(e) generations) that reduced the population to about 2% of the present size. However, heterogeneity in the allelic frequency distribution among geographical regions in Scotland suggests that subsequent admixture of populations with different demographic histories may also have played a role.

Paternity analysis of pollen-mediated gene flow for Fraxinus excelsior L. in a chronically fragmented landscape.

Paternity analysis based on microsatellite marker genotyping was used to infer contemporary genetic connectivity by pollen of three population remnants of the wind-pollinated, wind-dispersed tree Fraxinus excelsior, in a deforested Scottish landscape. By deterministically accounting for genotyping error and comparing a range of assignment methods, individual-based paternity assignments were used to derive population-level estimates of gene flow. Pollen immigration into a 300 ha landscape represents between 43 and 68% of effective pollination, mostly depending on assignment method. Individual male reproductive success is unequal, with 31 of 48 trees fertilizing one seed or more, but only three trees fertilizing more than ten seeds. Spatial analysis suggests a fat-tailed pollen dispersal curve with 85% of detected pollination occurring within 100 m, and 15% spreading between 300 and 1900 m from the source. Identification of immigrating pollen sourced from two neighbouring remnants indicates further effective dispersal at 2900 m. Pollen exchange among remnants is driven by population size rather than geographic distance, with larger remnants acting predominantly as pollen donors, and smaller remnants as pollen recipients. Enhanced wind dispersal of pollen in a barren landscape ensures that the seed produced within the catchment includes genetic material from a wide geographic area. However, gene flow estimates based on analysis of non-dispersed seeds were shown to underestimate realized gene immigration into the remnants by a factor of two suggesting that predictive landscape conservation requires integrated estimates of post-recruitment gene flow occurring via both pollen and seed.

Identifying the early genetic consequences of habitat degradation in a highly threatened tropical conifer, Araucaria nemorosa Laubenfels.

The early genetic effects of habitat degradation were investigated in the critically endangered conifer Araucaria nemorosa. This species occurs in New Caledonia, a global biodiversity hotspot where the world's greatest concentration of endemic conifer species coincides with an extremely high level of habitat destruction due to fire and mining. Using seven microsatellite loci, estimates were made of genetic marker variation, inbreeding coefficients and population differentiation of adult and seedling cohorts of A. nemorosa. These were contrasted with equivalent estimates, made over similar spatial scales and with the same marker loci, in the locally common and more widespread sister species Araucaria columnaris. There were no significant differences in population genetic parameters between adult populations of the two species, despite their different abundances. However, in A. nemorosa, the juvenile cohort showed a loss of rare alleles and elevated levels of inbreeding when compared to the adult cohort. These genetic differences between the cohorts were not observed in the locally common A. columnaris. This suggests that recent environmental degradation is influencing the genetic structure of A. nemorosa populations. Although this is not detectable among predisturbance adult populations, an early warning of these impacts is evident in more recently established seedling cohorts. The conservation implications of these results are discussed.

Genetic differentiation in Scottish populations of the pine beauty moth, Panolis flammea (Lepidoptera: Noctuidae).

Pine beauty moth, Panolis flammea (Denis & Schiffermüller), is a recent but persistent pest of lodgepole pine plantations in Scotland, but exists naturally at low levels within remnants and plantations of Scots pine. To test whether separate host races occur in lodgepole and Scots pine stands and to examine colonization dynamics, allozyme, randomly amplified polymorphic DNA (RAPD) and mitochondrial variation were screened within a range of Scottish samples. RAPD analysis indicated limited long distance dispersal (FST=0.099), and significant isolation by distance (P<0.05); but that colonization between more proximate populations was often variable, from extensive to limited exchange. When compared with material from Germany, Scottish samples were found to be more diverse and significantly differentiated for all markers. For mtDNA, two highly divergent groups of haplotypes were evident, one group contained both German and Scottish samples and the other was predominantly Scottish. No genetic differentiation was evident between P. flammea populations sampled from different hosts, and no diversity bottleneck was observed in the lodgepole group. Indeed, lodgepole stands appear to have been colonized on multiple occasions from Scots pine sources and neighbouring populations on different hosts are close to panmixia.

The relationship between flower size, inbreeding coefficient and inferred selfing rate in British Euphrasia species.

The genus Euphrasia in Britain comprises a taxonomically complex group of self-compatible, morphologically similar, hemi-parasitic, annual plant species of high conservation importance. The 19 diploid and tetraploid taxa currently recognised show striking variation in flower size. The objective of this paper is to determine whether a relationship exists between flower size and breeding system within Euphrasia. Following a survey of flower size variation among the 19 taxa, seven diploid populations, encompassing a broad range of flower sizes, were selected for detailed study. Four nuclear microsatellite loci were used to estimate the inbreeding coefficient Fis within each population. Fis values varied from to 0.17-0.77 and showed a significant, negative correlation with flower size. These results are best explained as the consequence of variation in selfing rate among the Euphrasia populations, with selfing rate increasing as flower size decreases. The potential factors influencing breeding system evolution in Euphrasia are discussed, together with the role of autogamy in generating taxonomic complexity and facilitating lineage differentiation within the genus.

Breeding systems and continuing evolution in the endemic Sorbus taxa on Arran.

The Arran whitebeams Sorbus arranensis and S. pseudofennica are two endemic woody plant taxa that have evolved on Arran through hybridisation. S. arranensis is a triploid hybrid between the widespread diploid S. aucuparia and the rare tetraploid S. rupicola. S. pseudofennica is a tetraploid formed by crossing between S. arranensis and S. aucuparia. In order to determine the mating systems of the two endemic species six maternal trees of each taxon together with 10-12 of their seed offspring were scored for their phenotype at three microsatellite loci and one nuclear intron locus. All seeds of S. arranensis were identical in phenotype to their maternal parents. In S. pseudofennica, 17.5% of all seeds differed in marker phenotype from their maternal parent. The proportion of seed with nonmaternal phenotypes varied significantly among maternal trees of S. pseudofennica. The results suggest that the triploid S. arranensis is an obligate apomict, whereas the tetraploid S. pseudofennica is a facultative apomict. Molecular marker analysis of three trees from Arran with an unusual leaf morphology indicates that they are the product of sexual reproduction by S. pseudofennica, and may originate from hybridisation with S. aucuparia. This research demonstrates that the Sorbus taxa on Arran are participants in an active evolutionary process generating novel biodiversity. Conservation programmes for these taxa should aim to preserve this evolutionary process rather than the individual taxonomic entities that it produces.

Neighbour joining trees, dominant markers and population genetic structure.

Population genetic theory for 'traditional' codominant loci showing low levels of allelic diversity (eg allozymes) has been well characterised and evaluated. In contrast, appropriate methods for the analysis of data from more recently developed marker systems are still being refined. For multilocus dominant markers such as amplified fragment length polymorphisms (AFLPs) and randomly amplified polymorphic DNA (RAPDs), the methods of data analysis can be split into two main categories. In population-based approaches, population allele frequencies are compared to obtain some measure of the partitioning of genetic diversity into within- and between-population components. In contrast, individual-based approaches use individual multilocus genotypes as the unit of analysis. Inferences on population processes such as gene flow are based on inter-relationships among individual samples as visualised on phenetic diagrams such as neighbour joining trees. Using a simulation approach coupled with neighbour joining analyses, we show that while the underlying population genetic structure is an important determinant of tree shape in the analysis of dominant data, the number of loci examined also affects the topology. At low levels of population differentiation (eg FST=0.07), mutually exclusive clustering of individuals into their respective populations can occur when sufficiently large numbers of loci are scored (eg 250 loci, typical of many AFLP studies). In contrast, unresolved star-shaped topologies can be recovered at higher levels of population differentiation (FST= >0.15) when lower numbers of loci are employed (eg 50 loci, typical of many RAPD studies). Thus, the relationship between tree topology and the extent of genetic structuring of populations is contingent upon the number of dominant loci scored. The consequences of these findings for the biological interpretation of individual-based analysis of dominant data sets are discussed.

Genetic effects of chronic habitat fragmentation on tree species: the case of Sorbus aucuparia in a deforested Scottish landscape.

Sustainable forest restoration and management practices require a thorough understanding of the influence that habitat fragmentation has on the processes shaping genetic variation and its distribution in tree populations. We quantified genetic variation at isozyme markers and chloroplast DNA (cpDNA), analysed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in severely fragmented populations of Sorbus aucuparia (Rosaceae) in a single catchment (Moffat) in southern Scotland. Remnants maintain surprisingly high levels of gene diversity (HE) for isozymes (HE = 0.195) and cpDNA markers (HE = 0.490). Estimates are very similar to those from non-fragmented populations in continental Europe, even though the latter were sampled over a much larger spatial scale. Overall, no genetic bottleneck or departures from random mating were detected in the Moffat fragments. However, genetic differentiation among remnants was detected for both types of marker (isozymes Theta n = 0.043, cpDNA Theta c = 0.131; G-test, P-value < 0.001). In this self-incompatible, insect-pollinated, bird-dispersed tree species, the estimated ratio of pollen flow to seed flow between fragments is close to 1 (r = 1.36). Reduced pollen-mediated gene flow is a likely consequence of habitat fragmentation, but effective seed dispersal by birds is probably helping to maintain high levels of genetic diversity within remnants and reduce genetic differentiation between them.

Multiple hybrid origins, genetic diversity and population genetic structure of two endemic Sorbus taxa on the Isle of Arran, Scotland.

Understanding the processes that have given rise to polyploid hybrid taxa is central to our understanding of plant evolution. In this study, we use an array of genetic markers in a population analysis to elucidate the hybrid origins of the Arran whitebeams Sorbus arranensis and S. pseudofennica, two woody plant taxa endemic to the Isle of Arran, Scotland. It has been proposed that S. arranensis was derived by hybridization between S. aucuparia and S. rupicola, and that subsequent hybridization between S. arranensis and S. aucuparia gave rise to S. pseudofennica. Analyses of species-specific isozyme, nuclear intron and chloroplast DNA markers confirm the proposed origin of S. arranensis, and indicate that S. aucuparia was the female parent in the hybridization. Analysis of microsatellite markers suggests that there have been at least three origins of S. arranensis on Arran. Microsatellite markers also support the proposed hypothesis for the origin of S. pseudofennica, and indicate at least five hybrid origins of this taxon. In total, three multilocus genotypes of S. arranensis and eight of S. pseudofennica were detected on Arran and multilocus genotypic diversity levels Hg were 0.09 and 0.63, respectively. Genetic differentiation (thetaST) values based on multilocus genotypes are substantial (0.344 and 0.470 for S. arranensis and S. pseudofennica, respectively) implying limited seed flow among populations. These results indicate that the endemic Sorbus taxa on Arran are the products of multiple and ongoing evolutionary events. This information must be incorporated into management policies for their future conservation.

Chloroplast DNA diversity of the dioecious European tree Ilex aquifolium L. (English holly).

Variation in the chloroplast genome of Ilex aquifolium (English holly), a dioecious evergreen tree native to south, west and central Europe, was analysed using polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLPs) and microsatellites. Differentiation between populations was high (GST = 0.595) and evidence for phylogeographical structure was detected (NST = 0.697, significantly higher than GST). Two chloroplast lineages were inferred originating from putative glacial refugia in southern Europe (Iberia, Italy and possibly the Balkans). The GST value was higher than reported for endozoochorous hermaphrodite species and for anemochorous dioecious species investigated over a similar geographical scale. It appears that dioecy has contributed to strong differentiation between refugia and that this has been maintained following postglacial recolonization as a result of limited seed flow. Palynological records for I. aquifolium are poor, thus these results give an important insight into patterns of glacial isolation and postglacial recolonization of this species.

Variation in host resistance and pathogen selective value in the interaction between Pinus sylvestris and the fungus Crumenulopsis sororia.

There have been many studies of plant pathogen evolution in systems showing gene-for-gene control of host resistance. However little is known about situations, exemplified by Scots pine, Pinus sylvestris, and its fungal pathogen Crumenulopsis sororia, where variation in host resistance is quantitative. In a field experiment genetically marked isolates of C. sororia from three natural populations were reciprocally inoculated on 1- and 2-year-old branch tissue of P. sylvestris in the three sites from which they had been collected. Quantitative variation in host resistance was measured by comparing the performance of the same inocula on different host populations, individuals and tissues. The selective value of isolates derived from different populations was estimated by comparing the frequency of genotypes in lesion re-isolations with those in the initial inoculum mixtures. Host resistance varied significantly among populations, individuals within populations and between 1- and 2-year-old branch tissue of P. sylvestris. Large differences in the relative selective values of C. sororia isolates from different populations were detected. The selective value of pathogens was independent of the host population on which they were inoculated. However, their selective value did depend on the age of the tissue on which they grew. The implications of these results for modelling evolution in pathogen-host interactions that lack gene-for-gene determination of host resistance are discussed.

Patterns of genetic variation in Pinus chiapensis, a threatened Mexican pine, detected by RAPD and mitochondrial DNA RFLP markers.

Pinus chiapensis (Pinaceae) is a large conifer, endemic to central and southern Mexico and north-western Guatemala. In order to assess the extent of genetic variation within and between populations of this species, samples were obtained from throughout the natural range and analysed using random amplified polymorphic DNA (RAPD) and mtDNA RFLPs markers. Probes for the CoxI mitochondrial gene enabled two mitotypes to be observed. Populations from the eastern and western limit of the range of the species were fixed for one mitotype ('A'), whereas two populations distributed near the centre of the range were fixed for another ('B'). When the samples were screened with eight 10-mer RAPD primers, a total of 12 polymorphic bands were detected. The proportion of polymorphic bands was unusually low (24.5%) compared with other tree species. AMOVA analysis indicated that a significant proportion of the variation (P < 0.002) was distributed between populations; the extent of population differentiation detected (Phi(st) = 0.226; G(ST ) = 0.194) was exceptionally high for a pine species. Pair-wise comparison of Phi(st) values derived from AMOVA indicated that populations were significantly (P < 0.05) different from each other in virtually every case. These results are interpreted in the context of the evolutionary history of the species, and the implications for its in- and ex situ conservation are discussed.

Chloroplast DNA diversity in Calluna vulgaris (heather) populations in Europe.

Variation in the chloroplast genome of Calluna vulgaris (heather), the dominant species of northwest European heath communities, was analysed using polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLPs) and microsatellites. No length polymorphisms were detected in the 100-200 base pair (bp) fragments amplified by the conserved microsatellite primers, and sequencing revealed that the repeat regions were interrupted relative to the corresponding sequence in Nicotiana tabacum. In contrast, PCR-RFLP analysis revealed high levels of haplotype diversity within populations (hS = 0.443, hT = 0.842), as well as substantial differentiation between populations (GST = 0.473). Diversity and differentiation were higher in southern Europe than in northern Europe. Interpreted in the light of data from allozyme studies and pollen core records, the results suggest that the main glacial refugia for C. vulgaris were located in southwest Europe, including northern Spain, the Pyrenees and the Massif Central region of France. There is also evidence for diffuse survival of the species at more northerly latitudes.