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.     

Population genetic structure in European populations of Spiranthes romanzoffiana set in the context of other genetic studies on orchids

Spiranthes romanzoffiana Cham. is restricted in Europe to the British Isles, where it is recognised as a conservation priority species due to frequent extirpation of populations along with no evidence of seed set; vegetative reproduction has been invoked as the sole means of perpetuation and dispersal. To investigate the reproductive ecology of this species, 17 populations have been sampled for chloroplast microsatellites and amplified fragment length polymorphisms (AFLPs). These markers revealed a previously unsuspected genetic-geographic split in the species, which correlates with differences in patterns of within-population variation. Northern populations were fixed for one chloroplast haplotype but showed high levels of AFLP genotypic diversity consistent with sexual reproduction (proportion of genotypes distinguishable, PD = 0.98). More southerly populations showed fixed differences from the northern populations in their chloroplast haplotype and for 10 AFLP markers. They harboured only 12 unique multilocus genotypes among 113 individuals from six populations (PD = 0.11). These genotypes differed mostly by single bands, and none by more than 4/138 loci, with identical multilocus genotypes occurring in widely separated populations. This uniformity in southern populations is consistent with agamospermous or autogamous reproduction, and/or an extreme population bottleneck. Finally, the observed patterns of population differentiation in S. romanzoffiana are compared with other studies of orchids, revealing a wide range of values that belie recent contrasting published generalisations that claim that orchids show either higher, or lower, levels of population differentiation than other plant families.     

Phylogeography and host race differentiation in the European mistletoe (Viscum album L.)

Mistletoes are bird dispersed, hemi-parasitic shrubs infecting a large number of woody host plants. Chloroplast fragment length polymorphisms were used to study genetic differentiation among presumed host races, population genetic structure, and to elucidate the postglacial migration history of mistletoe ( Viscum album ) across the entire natural distribution range in Europe. The populations sampled belong to four closely related taxa, three of which are widely distributed and differ in their host trees, whereas a fourth taxon is rare and endemic to the Island of Crete. The molecular analysis of chloroplast DNA variation supported the distinction of these four taxa. We further found evidence for phylogeographical structure in each of the three widely distributed host races. Independent of host race, mistletoe haplotypes from Turkey were distinct and distant from those found elsewhere in Europe, suggesting that highly differentiated populations, and possibly new taxa, exist at the range limit of the species.     

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.     

Variation of colony types in the antFormica cinerea

Summary Formica cinerea is a rare ant species in northern Europe where it occurs in few isolated populations. Estimates of genetic relatedness among worker nestmates revealed very different colonial structures. Relatedness was g = 0.81 in one population, and g = –0.03 and = 0.01 in two others. These results indicate that some populations of the species have mainly monogynous colonies (perhaps with monandrous queens), whereas others consist of polygynous and possibly polydomous colonies. Genetic differentiation of closely located populations suggests restricted dispersal.     

Rangewide phylogeography of a bird-dispersed Eurasian shrub: contrasting Mediterranean and temperate glacial refugia

We studied the phylogeography of alder buckthorn (Frangula alnus), a bird-dispersed shrub or small tree distributed over most of Europe and West Asia and present in three of the four main refugia of West Palaearctic temperate woody plants: the Iberian Peninsula, the Balkans and Anatolia. A total of 78 populations from 21 countries were analysed for chloroplast DNA variation using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), and 21 different haplotypes were distinguished. We found a very strong overall population differentiation (GST = 0.81) and phylogeographical structure, and a sharp contrast between the haplotype-rich refugia and the almost completely uniform area of postglacial colonization. The haplotype network comprises three lineages made up of haplotypes from the Iberian Peninsula, Anatolia with the Caucasus, and temperate Europe. The Iberian and the Anatolian branches represent parts of a major lineage that spans over the whole northern Mediterranean Basin and some neighbouring areas and probably dates back to the Tertiary. Many haplotypes of this lineage are distributed locally and most populations are fixed for a single haplotype; these populations have apparently been very stable since their establishment, experiencing negligible gene flow and few mutations. The temperate European lineage consists of one very widespread and abundant plus six locally distributed haplotypes. Four of them are located in Southeast Europe, the putative refugium of all extant temperate European populations. Contrary to populations from Iberia and Anatolia, F. alnus populations from the southeastern European refugium have most genetic variation within populations. Bird-mediated seed dispersal has apparently allowed not only a very rapid postglacial expansion of F. alnus but also subsequent regular seed exchanges between populations of the largely continuous species range in temperate Europe. In contrast, the disjunct F. alnus populations persisting in Mediterranean mountain ranges seem to have experienced little gene flow and have therefore accumulated a high degree of differentiation, even at short distances. Populations from the southern parts of the glacial refugia have contributed little to the postglacial recolonization of Europe, but their long-term historical continuity has allowed them to maintain a unique store of genetic variation.     

Comparison between mitochondrial and chloroplast DNA variation in the native range of Silene vulgaris

A detailed survey of mitochondrial and chloroplast diversity in eight populations of Silene vulgaris from Central Europe was conducted for comparison with previously published data on diversity from S. vulgaris populations in the introduced range. Mitochondrial DNA (mtDNA) variation around the coxI gene was assessed with Southern blotting/restriction fragment length polymorphism methods. Chloroplast variation was assessed by sequencing the intergenic spacer separating the trnH and psbA genes. Thirty mtDNA haplotypes and 24 chloroplast DNA (cpDNA) haplotypes were found within 86 individuals. The overall genetic diversity h (0.941 for mitochondrial, and 0.893 for chloroplast markers) and within-population diversity were higher than reported in previous population studies of S. vulgaris in the USA and Europe. The frequency of private alleles was surprisingly high - more than 90% for both kinds of markers. Most of our populations were large and located in relatively undisturbed meadows, whereas surveys in Virginia consisted of smaller roadside populations. The slow rate of population turnover in European populations is discussed as a factor responsible for the relatively high diversity of S. vulgaris in undisturbed areas of its native range. Association between mtDNA and cpDNA haplotypes was also demonstrated. Finally, gender and mtDNA haplotype were associated in the Alps populations, where females were very rare.     

Population genetic analysis of European Prunus spinosa (Rosaceae) using chloroplast DNA markers

Chloroplast DNA diversity in Prunus spinosa, a common shrub of European deciduous forests, was assessed using the polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) technique. Thirty-two haplotypes were detected in 25 populations spread across the European continent. Ten haplotypes were shared by two or more populations, and 22 were private. The major proportion of the total cpDNA diversity (H(T) = 0.73) was located within the populations (H(S) = 0.49), and differentiation between populations was low (G(ST) = 0.33) compared with other forest species. Haplotype diversity was higher in southern Europe than in northern Europe, indicating probable localization of glacial refugia in southern Europe. The minimum-length spanning tree of haplotypes showed incongruency between the phylogeny of haplotypes and their geographic locations. This might be the result of intensive seed movements following recolonization, which thereby erased the phylogeographic structure in P. spinosa.     

Distribution of chloroplast DNA diversity within and among populations in gynodioecious Beta vulgaris ssp. maritima (Chenopodiaceae)

With the recent technical advances in molecular biology, chloroplast DNA (cpDNA) has become a marker used for the study of cytoplasmic differentiation of natural populations of plants. As chloroplasts are maternally inherited in most plant species, the seed component of gene flow is thus made accessible. We present here a study of cpDNA polymorphism within the maritima subspecies of the gynodioecious Beta vulgaris in which we try to assess the impact of such a reproductive system on seed flow. One hundred and eighty-eight wild beets were sampled from 20 hermaphroditic and 20 gynodioecious (i.e. containing both hermaphroditic and female plants) populations from the Atlantic coast of Europe. cpDNA variability in these populations was characterized with a rapid restriction fragment length polymorphism (RFLP) method. Eight cpDNA haplotypes were found. Strong differentiation among populations was observed ( F _ST = 0.43) and was consistent with isolation by distance, although most of the cpDNA haplotypes were ubiquitous. Gynodioecy seems to affect the distribution of cpDNA diversity: gynodioecious populations of Beta vulgaris ssp. maritima contained a greater number of cpDNA types but were less differentiated among themselves than hermaphroditic ones.     

Low genetic differentiation among seasonal cohorts in Senecio vulgaris as revealed by amplified fragment length polymorphism analysis

Common groundsel, Senecio vulgaris (Asteraceae), is a highly selfing semelparous ephemeral weed that belongs to the few plant species in central Europe capable of growing, flowering and fruiting all year round. In temperate climates, flowering S. vulgaris cohorts were found to appear up to three times per year. Using amplified fragment length polymorphism (AFLP) molecular markers we examined temporal genetic differentiation among spring, summer and autumn cohorts at each of seven sites located in two regions in Switzerland. Strong genetic differentiation among cohorts may indicate the existence of seasonal races of S. vulgaris, reproductively isolated by nonoverlapping flowering phenologies. Analysis of molecular variance (amova) revealed that < 2.5% of the AFLP variation resided among cohorts within sites, whereas there was significant genetic differentiation among plants from different sites (15.6%) and among individuals within cohorts (81.9%). Significant genetic differentiation was also observed between the two regions. Isolation-by-distance was found on a regional scale, but not on a local scale. Gene flow was estimated to be approximately 15-fold higher among cohorts within sites than among sites. We further found, on average, similar levels of genetic diversity within the three seasonal cohorts. The results of this study demonstrate that season of growth represents a weak barrier for genetic exchange among S. vulgaris populations and does not affect molecular variance. Therefore, there is no evidence for the existence of seasonally specialized races of S. vulgaris. We discuss some implications of the results for the biological control of S. vulgaris using a native rust fungus.     

New(t)s and views from hybridizing MHC genes: introgression rather than trans-species polymorphism may shape allelic repertoires

One of the key features of major histocompatibility complex (MHC) genes is the frequent occurrence of trans-species polymorphism, that is 'the passage of allelic lineages from ancestral to descendant species' (Klein et al. 2007). Selectively maintained ancestral polymorphism may, however, be hard to distinguish from introgression of MHC alleles between hybridizing species (Fig. 1). In this issue of Molecular Ecology, Nadachowska-Brzyska et al. (2012) present data that suggest that the latter can be observed in two closely related species of newts, Lissotriton vulgaris (Lv) and L. montandoni (Lm) from south-east Europe. Strikingly, allelic MHC variation displayed more structure between geographically separated populations of L. vulgaris than across species in the hybrid zone. This suggests that high MHC variation in L. montadoni may result from mainly unidirectional gene flow between species, while differentiation between northern and southern populations of L. vulgaris might reflect local adaptation.     

Phylogeography of two European newt species--discordance between mtDNA and morphology

The newts Triturus vulgaris and Triturus montandoni are sister species that exhibit contrasting levels of intraspecific morphological variation. Triturus vulgaris has a broad Eurasiatic distribution encompassing both formerly glaciated and unglaciated areas and shows substantial morphological differentiation in the southern part of its range, while T. montandoni, confined to the Carpathians, is morphologically uniform. We analysed sequence variation of two mtDNA fragments of the total length of c. 1850 bp in 285 individuals of both species collected from 103 localities. Phylogenetic analysis of 200 unique haplotypes defined 12 major clades, their age estimated at c. 4.5-1.0 million years (Myr). Most of the older clades were found in the southern part of the range, and also in central Europe, mainly in Romania. The distribution of mtDNA clades points to the existence of several glacial refugia, located in the Caucasus region, Anatolia, the Balkan Peninsula, Italy, and more to the north in central Europe. The concordance between mtDNA based phylogeny and the distribution of T. vulgaris subspecies was weak. Triturus montandoni haplotypes did not form a monophyletic group. Instead they were found in six clades, in five of them mixed with T. vulgaris haplotypes, most likely as a result of past or ongoing hybridization and multiple introgression of mtDNA from T. vulgaris to T. montandoni. Patterns of sequence variation within clades suggested long-term demographic stability in the southern groups, moderate and relatively old demographic growth in the populations inhabiting central Europe, and high growth in some of the groups that colonized northern parts of Europe after the last glacial maximum.     

Phylogeography of Scots pine in Europe and Asia based on mtDNA polymorphisms

We analyzed mitochondrial DNA polymorphisms to search for evidence of the genetic structure and patterns of admixture in 124 populations (N = 1407 trees) across the distribution of Scots pine in Europe and Asia. The markers revealed only a weak population structure in Central and Eastern Europe and suggested postglacial expansion to middle and northern latitudes from multiple sources. Major mitotype variants include the remnants of Scots pine at the north-western extreme of the distribution in the Scottish Highlands; two main variants (western and central European) that contributed to the contemporary populations in Norway and Sweden; the central-eastern European variant present in the Balkan region, Finland, and Russian Karelia; and a separate one common to most eastern European parts of Russia and western Siberia. We also observe signatures of a distinct refugium located in the northern parts of the Black Sea basin that contributed to the patterns of genetic variation observed in several populations in the Balkans, Ukraine, and western Russia. Some common haplotypes of putative ancient origin were shared among distant populations from Europe and Asia, including the most southern refugial stands that did not participate in postglacial recolonization of northern latitudes. The study indicates different genetic lineages of the species in Europe and provides a set of genetic markers for its finer-scale population history and divergence inference.     

Intercontinental Mediterranean disjunct mosses: morphological and molecular patterns

This study focused on three species that occur disjunctly between western North America and the Mediterranean region of southern Europe, northern Africa, and western Asia, forming the so-called Madrean-Tethyan distribution pattern. Quantitative morphological characters were measured in New and Old World plants to find any subtle phenotypic differentiation between the disjunct populations. Sequences from the nuclear ribosomal internal transcribed spacer region were obtained from the same populations to assess differentiation at the molecular level and to compare molecular diversity with patterns of morphological similarity among plants. Little or no morphological differentiation existed between New and Old World plants in any of the species, but internal transcribed spacer (ITS) sequences revealed some phylogeographic structure. Patterns of morphological similarity in all three species were incongruent with phylogeographic structure revealed by sequence data. New World populations were more variable than Old World populations at the molecular level in the three species. Despite some evidence for differentiation between disjunct plants, no plausible mutation rate would date the divergence at ≥20 million years ago (MYA), as implied by the Madrean-Tethyan hypothesis. Recent long-distance dispersal is a more likely explanation for intercontinental disjunctions in these species.     

Range-wide phylogeography of the European temperate-montane herbaceous plant Meum athamanticum Jacq.: evidence for periglacial persistence

Aim  The aim of this study is to analyse the genetic population structure of Meum athamanticum Jacq. in order to explore the alternative hypotheses (1) that the central and northern highland populations are the result of post-glacial recolonization from southern refugia, and the disjunct distribution of M. athamanticum can be explained by modern ecological conditions, or (2) that extant populations north of the Alps and Pyrenees persisted in situ during glacial periods.
Location  Europe.
Methods  Variation of amplified fragment length polymorphisms (AFLPs) was analysed for 23 populations from the entire range of the species. We used band-based approaches and methods based on allele frequencies to measure genetic diversity and to identify population structure.
Results  Our analyses reveal a north–south differentiation within M. athamanticum . High levels of genetic diversity, as well as private fragments, are found in populations both north and south of the Alps. Differentiation among populations is lower in the northern than in the southern population group, and significant isolation-by-distance is found only in the latter group.
Main conclusions  Our results indicate that M. athamanticum survived the last ice age in multiple refugia throughout its contemporary range and did not expand into areas north of the Alps from southern refugia. We found evidence that regional-scale migration in northern, formerly periglacial, parts of the species range has resulted in the intermingling of populations. In contrast, southern populations are characterized by long-term isolation. The south-west Alps represent an area where immigration and mixing of populations from northern and southern refugia appears to have taken place.     

Patterns of variation at a mitochondrial sequence-tagged-site locus provides new insights into the postglacial history of European Pinus sylvestris populations

Due to their maternal mode of inheritance, mitochondrial markers can be regarded as almost 'ideal' tools in evolutionary studies of conifer populations. In the present study, polymorphism was analysed at one mitochondrial intron (nad 1, exon B/C) in 23 native European Pinus sylvestris populations. In a preliminary screening for variation using a polymerase chain reaction-restriction fragment length polymorphism approach, two length variants were identified. By fully sequencing the 2.5 kb region, the observed length polymorphism was found to result from the insertion of a 31 bp sequence, with no other mutations observed within the intron. A set of primers was designed flanking the observed mutation, which identified a novel sequence-tagged-site mitochondrial marker for P. sylvestris. Analysis of 747 trees from the 23 populations using these primers revealed the occurrence of two distinct haplotypes in Europe. Within the Iberian Peninsula, the two haplotypes exhibited extensive population differentiation (PhiST = 0.59; P < or = 0.001) and a marked geographical structuring. In the populations of central and northern Europe, one haplotype largely predominated, with the second being found in only one individual of one population.     

Population genetic structure of Arabidopsis lyrata in Europe

Population genetic theory predicts that the self-incompatible and perennial herb, Arabidopsis lyrata, will have a genetic structure that differs from the self-fertilizing, annual Arabidopsis thaliana. We quantified the genetic structure for eight populations of A. lyrata ssp. petraea in historically nonglaciated regions of central Europe. Analysis of 20 microsatellite loci for 344 individuals demonstrated that, in accordance with predictions, diploid populations had high genome-wide heterozygosity (H(O) = 0.48; H(E) = 0.52), high within-population diversity (83% of total) compatible with mutation-drift equilibrium, and moderate differentiation among populations (F(ST) = 0.17). Within a single population, the vast majority of genetic variability (92%) was found at the smallest spatial scale (< 3 m). Although there was no evidence of biparental inbreeding or clonal propagation at this scale (F(IS) = 0.003), significant fine-scale spatial autocorrelation indicated localized gene flow presumably due to gravity dispersed seeds (Sp = 0.018). Limited gene flow between isolated population clusters (regions) separated by hundreds of kilometres has given rise to an isolation by distance pattern of diversification, with low, but significant, differentiation among regions (F(ST) = 0.05). The maintenance of geographically widespread polymorphisms and uniformly high diversity throughout central Europe is consistent with periglacial survival of A. lyrata ssp. petraea north of the Alps in steppe-tundra habitats during the last glacial maximum. As expected of northern and previously glaciated localities, A. lyrata in Iceland was genetically less diverse and highly differentiated from central Europe (H(E) = 0.37; F(ST) = 0.27).     

Pan-European phylogeography of the aquatic snail Theodoxus fluviatilis (Gastropoda: Neritidae)

Investigating the geographical distribution of genetic lineages within species is critical to our understanding of how species evolve. As many species inhabit large and complex ranges, it is important that phylogeographical research take into account the entire range of widespread species to clarify how myriad extrinsic variables have affected their evolutionary history. Using phylogenetic, nested clade, and mismatch distribution analyses on a portion of the mitochondrial COI gene, I demonstrate that the wide-ranging freshwater snail Theodoxus fluviatilis possesses in parallel many of the phylogeographical patterns seen in less widespread freshwater species of Europe. Fragmentary forces play a major part in structuring the range of this species, with 12 of 14 geographically structured nested clades displaying a distribution consistent with fragmentation or restricted dispersal. Certain regions of southern Europe harbour the majority of genetic diversity (total haplotype diversity, H = 0.87), particularly Italy (H = 0.87) and areas surrounding the Black Sea (H = 0.81). Post-Pleistocene range expansion is pronounced, with the majority of northern European populations (95% of sample sites) having arisen from northern Italian individuals that initially colonized northern Germany. Additionally, two highly divergent haplotype lineages present in northern Germany imply that there were at least two postglacial recolonization routes. Estuaries may also provide a means of dispersal given that no genetic differentiation was found between estuarine populations and neighbouring freshwater populations. Taken together, these data reveal a species with a complex genetic history resulting from the fragmentary effects of European geology as well as continuous and discrete range expansion related to their aquatic biology.     

Glacial survival may matter after all: nunatak signatures in the rare European populations of two west-arctic species

Biogeographers claimed for more than a century that arctic plants survived glaciations in ice-free refugia within the limits of the North European ice sheets. Molecular studies have, however, provided overwhelming support for postglacial immigration into northern Europe, even from the west across the Atlantic. For the first time we can here present molecular evidence strongly favouring in situ glacial persistence of two species, the rare arctic-alpine pioneer species Sagina caespitosa and Arenaria humifusa. Both belong to the 'west-arctic element' of amphi-Atlantic disjuncts, having their few and only European occurrences well within the limits of the last glaciation. Sequencing of non-coding regions of chloroplast DNA revealed only limited variation. However, two very distinct and partly diverse genetic groups, one East and one West Atlantic, were detected in each species based on amplified fragment length polymorphisms (AFLPs), excluding postglacial dispersal from North America as explanation for their European occurrences. Patterns of genetic diversity and distinctiveness indicate that glacial populations existed in East Greenland and/or Svalbard (A. humifusa) and in southern Scandinavia (S. caespitosa). Despite their presumed lack of long-distance dispersal adaptations, intermixed populations in several regions indicate postglacial contact zones. Both species are declining in Nordic countries, probably due to climate change-induced habitat loss. Little or no current connectivity between their highly fragmented and partly distinct populations call for conservation of several populations in each geographic region.     

Geographical partitioning of goat diversity in Europe and the Middle East

Thirty microsatellite markers were analysed in 1426 goats from 45 traditional or rare breeds in 15 European and Middle Eastern countries. In all populations inbreeding was indicated by heterozygosity deficiency (mean FIS = 0.10). Genetic differentiation between breeds was moderate with a mean FST value of 0.07, but for most (c. 71%) northern and central European breeds, individuals could be assigned to their breeds with a success rate of more than 80%. Bayesian-based clustering analysis of allele frequencies and multivariate analysis revealed at least four discrete clusters: eastern Mediterranean (Middle East), central Mediterranean, western Mediterranean and central/northern Europe. About 41% of the genetic variability among the breeds could be explained by their geographical origin. A decrease in genetic diversity from the south-east to the north-west was accompanied by an increase in the level of differentiation at the breed level. These observations support the hypothesis that domestic livestock migrated from the Middle East towards western and northern Europe and indicate that breed formation was more systematic in north-central Europe than in the Middle East. We propose that breed differentiation and molecular diversity are independent criteria for conservation.