Differential cycles of range contraction and expansion in European high mountain plants during the Late Quaternary: insights from Pritzelago alpina (L.) O. Kuntze (Brassicaceae)

Nuclear DNA sequence variation of the internal transcribed spacer (ITS) and amplified fragment length polymorphisms (AFLPs) were used to illuminate the evolutionary history of Pritzelago alpina, a herbaceous perennial of (sub)alpine to nival habitats of the European high mountains. Maximum likelihood analysis of ITS sequences of P. alpina, Hornungia petraea and Hymenolobus procumbens (the 'Pritzelago alliance') resolved P. alpina and H. petraea as sister taxa. ITS divergence estimates support an origin for P. alpina in the Late Tertiary, while intraspecific diversification started in the Late Quaternary (0.4-0.9 million years ago). AFLP analysis of 76 individuals of P. alpina, representing 24 localities across its entire west-east distribution, identified four mountain lineages in Cantabria, the Pyrenees, (south-) western Alps, and northeastern Alps/Tatras/Carpathians. In an analysis of molecular variance (amova), 14.3% of the total variation derived from this separation. However, relationships among these lineages remained unresolved in neighbour-joining and principal co-ordinates analyses, suggesting a population history of near simultaneous vicariance. Comparison with our previous ITS/AFLP study of Anthyllis montana (Fabaceae) indicates that the two co-distributed but altitudinally differentiated plant species exhibit temporally concordant but spatially discordant patterns of genetic variation. Moreover, levels of AFLP divergence were significantly lower in P. alpina than in the submediterranean, lower-elevation A. montana. Together, these data are consistent with a 'displacement refugia model', which predicts that European mountain plant species associated with lower- and upper-elevation habitats had a different cycle of range contraction into (long-term) glacial and (short-term) interglacial refugia, respectively.     

Genetic diversity in an endangered alpine plant, Eryngium alpinum L. (Apiaceae), inferred from amplified fragment length polymorphism markers

Eryngium alpinum L. is an endangered species found across the European Alps. In order to obtain base-line data for the conservation of this species, we investigated levels of genetic diversity within and among 14 populations from the French Alps. We used the amplified fragment length polymorphism (AFLP) technique with three primer pairs and scored a total of 62 unambiguous, polymorphic markers in 327 individuals. Because AFLP markers are dominant, within-population genetic structure (e.g. FIS) could not be assessed. Analyses based either on the assumption of random-mating or on complete selfing lead to very similar results. Diversity levels within populations were relatively high (mean Nei's expected heterozygosity = 0.198; mean Shannon index = 0.283), and a positive correlation was detected between both genetic diversity measurements and population size (Spearman rank correlation: P = 0. 005 and P = 0.002, respectively). Moreover, FST values and exact tests of differentiation revealed high differentiation among populations (mean pairwise FST = 0.40), which appeared to be independent of geographical distance (nonsignificant Mantel test). Founder events during postglacial colonizations and/or bottlenecks are proposed to explain this high but random genetic differentiation. By contrast, we detected a pattern of isolation by distance within populations and valleys. Predominant local gene flow by pollen or seed is probably responsible for this pattern. Concerning the management of E. alpinum, the high genetic differentiation leads us to recommend the conservation of a maximum number of populations. This study demonstrates that AFLP markers enable a quick and reliable assessment of intraspecific genetic variability in conservation genetics.     

Geographic pattern of genetic variation in the European globeflower Trollius europaeus L. (Ranunculaceae) inferred from amplified fragment length polymorphism markers

The distribution of genetic variation and the phylogenetic relationships between 18 populations of the arctic-alpine plant Trollius europaeus were analysed in three main regions (Alps, Pyrenees and Fennoscandia) by using dominant AFLP markers. Analysis of molecular variance revealed that most of the genetic variability was found within populations (64%), although variation among regions (17%) and among populations within regions (19%) was highly significant (P < 0.001). Accordingly, the global fixation index FST averaged over loci was high (0.39). The among-population differentiation indicates restricted gene flow, congruent with limited dispersal of specific globeflower's pollinating flies (Chiastocheta spp.). Within-population diversity levels were significantly higher in the Alps (mean Nei's expected heterozygosity HE = 0.229) than in the Pyrenees (HE= 0.197) or in Fennoscandia (HE = 0.158). This finding is congruent with the species-richness of the associated flies, which is maximum in the Alps. We discuss the processes involved in shaping observed patterns of genetic diversity within and among T. europaeus populations. Genetic drift is the major factor acting on the small Pyrenean populations at the southern edge of T. europaeus distribution, while large Fennoscandian populations result probably from a founder effect followed by demographic expansion. The Alpine populations represent moderately fragmented relics of large southern ancestral populations. The patterns of genetic variability observed in the host plant support the hypothesis of sympatric speciation in associated flies, rather than recurrent allopatric speciations.     

Evolution in Aeschynanthus (Gesneriaceae) inferred from ITS sequences

 Aeschynanthus Jack, an epiphytic genus with c.160 species, is widespread in SE Asia. We selected 50 species for ITS nrDNA sequencing, to include all biogeographic areas and all infrageneric groupings, which are currently based on seed morphology. Some species were sequenced directly from PCR product; others cloned because of ITS length polymorphisms. The clone sequences were analysed individually and combined in an elision matrix. Results extend earlier findings that Aeschynanthus is divided into two clades, one occurring primarily in mainland SE Asia and the other in Malesia. This pattern is interpreted as indicating an ancient vicariance event followed by dispersal and plate fusion. Clade I has straight or clockwise spiral orientation of the testa cells and clade II anticlockwise spiral orientation. In clade I some species of section Microtrichium form a basal group with other sections being polyphyletic or paraphyletic. In clade II the monophyletic section Aeschynanthus is nested within the paraphyletic basal Microtrichium. Received February 8, 2001 Accepted June 8, 2001     

Genetic variation of Calycophyllum spruceanum in the Peruvian Amazon Basin, revealed by amplified fragment length polymorphism (AFLP) analysis

An understanding of the level, structure and origin of genetic variation within and among populations of tropical trees is essential for devising optimum management strategies for their sustainable utilization and conservation. Here, amplified fragment length polymorphism (AFLP) analysis was used to partition genetic variation within and among nine populations of the predominantly riverine tree, Calycophyllum spruceanum , sampled across a wide geographical range along river tributaries of the Peruvian Amazon Basin. Analysis of molecular variance ( AMOVA ) employed 65 AFLP markers and revealed most variation among individuals within populations (91%), although variation among populations was highly significant ( P < 0.001). Calculation of genetic distances and nested AMOVA indicated a degree of structuring among populations based on geographical proximity, although clustering did not depend on geographical distance alone. No firm evidence was obtained for unidirectional seed dispersal by water playing an important role in determining genetic structure over the geographical range sampled. Implications of data for optimising genetic management of the species are discussed and areas for further study identified.     

Complete exclusion of nonsires in an analysis of paternity in a natural plant population using amplified fragment length polymorphism (AFLP)

The utility of the new polymerase chain reaction (PCR)-based multilocus DNA fingerprinting technique amplified fragment length polymorphism (AFLP) for paternity analysis in natural plant populations was assessed. In a natural population of 25 plants of Persoonia mollis (Proteaceae), three AFLP primer pairs generated 147 dominant loci. Of these, 125 (85%) were polymorphic, with a mean recessive allele frequency of 0.735. The theoretical expected percentage of offspring for which all males except the true father can be excluded ( P _ET) was 99.9% for this population. The estimates of P _ET drop marginally to 99.6% and 97.6% for larger populations of 100 and 1000 individuals, respectively. A preliminary investigation confirmed the power of AFLP for paternity analysis by assigning paternity, or excluding all known potential sires, for 242 of 252 (96.0%) naturally pollinated seeds. Ambiguous paternity for the remaining 10 seeds was quickly resolved by utilizing two further AFLP primer pairs, ultimately generating over 200 polymorphic loci and resulting in the exclusion of all nonsires for all 252 (100%) seeds. This study highlights the utility of AFLP for paternity analysis because: (i) it generates sufficiently large numbers of highly reproducible polymorphic loci, that are (ii) quickly and accurately scored using an automated DNA sequencer and dedicated software, and (iii) unlike microsatellites, requires no sequence knowledge so it is more easily applied to new study species.     

Disjunct distribution of Hypericum nummularium L. (Hypericaceae): molecular data suggest bidirectional colonization from a single refugium rather than survival in distinct refugia

Hypericum nummularium has a strongly disjunct, bi‐areal distribution in Europe: it is abundant in the Pyrenees and grows in a very restricted part of the Alps, more than 1000 km away. My aim was to estimate the genetic divergence between these areas and to identify the factors responsible for the disjunction: glacial relicts, bidirectional colonization from a common refugium, long‐distance dispersal and/or human introduction? Internal transcribed spacers (ITS) sequencing (680 bp) and amplified fragment length polymorphism (AFLP) fingerprinting (104 polymorphic markers) showed very low differentiation between populations in the Alps and the Pyrenees, indicating that H. nummularium probably survived in a single refugium. Moreover, levels of genetic diversity were similar in the two areas, making human introduction and long‐distance dispersal unlikely. Thus, the species probably survived in one refugium, subsequently colonizing both areas more or less simultaneously. The comparison of genetic and geographical distances suggested a step by step migration in the Alps (isolation by distance), whereas random dispersal events were more likely in the Pyrenees. Finally, I discuss possible causes for the restricted distribution area of H. nummularium in the Alps (e.g. unsuitable habitat, low dispersal capacities) and conclude that strong human disturbance is probably the major limit to the expansion of the species in this region. © 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 87 , 437–447.     

Pleistocene refugia and recolonization routes in the southern Andes: insights from Hypochaeris palustris (Asteraceae, Lactuceae)

Hypochaeris palustris (Phil.) De Wild. is a species growing in the southern Andean chain. To elucidate potential Pleistocene refugia and recolonization routes in the southern Andes, we analysed amplified fragment length polymorphisms (AFLPs) in 206 individuals in 21 populations of H. palustris from the coastal Cordillera, the central, northern, and eastern ranges of the southern Andes, and Patagonia. Populations from the coastal Cordillera harboured more private AFLP fragments, and exhibited a higher frequency of polymorphic fragments as well as higher Shannon diversity than all other areas investigated. The comparison among pooled AFLP profiles of each region revealed that the central Andean ranges shared most fragments with populations from the margins of the distributional area in the Andes, in the N, E, and S (Patagonia). Phenetic analysis indicated close relationships among populations of the central ranges. Populations of the coastal Cordillera were shown to be highly differentiated from the Andean populations. It is very likely therefore that (1) H. palustris recolonized the central ranges of the southern Andes from nearby refugia, possibly unglaciated areas N, E, and/or S of its present distributional area; (2) the postglacial spread of H. palustris in the central ranges of the southern Andes occurred rapidly; and (3) the coastal Cordillera served as a refugium for H. palustris, but these populations did not contribute to the recolonization of the central Andean ranges.     

Genetic structure of coastal and inland populations of the annual halophyte Suaeda maritima (L.) dumort. in Central Europe, inferred from amplified fragment length polymorphism markers

Naturally occurring inland salt habitats are highly threatened due to increasing fragmentation and area reduction, while the surroundings of former potash mining dumps have experienced a massive invasion by halophytes over the last 20 years. We reconstructed colonisation patterns of these purely anthropogenic inland salt sites using molecular markers in the obligate halophyte Suaeda maritima (L.) dumort. (Chenopodiaceae), a typical plant in such areas. In the present study, 120 individual plants from 40 coastal and inland populations in Central Europe were subjected to AFLP analysis with nine primer combinations. A total of 243 AFLP band positions were scored as presence/absence characters. Genetic diversity values were not significantly different in populations from natural and anthropogenic inland salt sites as compared to coastal habitats. Results from principal coordinate analysis, neighbour-joining analysis and analysis of molecular variance ( amova ) all indicated that most of the genetic variation is preserved within populations, while genetic differentiation among populations is comparatively low. We conclude that S. maritima has repeatedly and independently colonised the surroundings of former potash mining dumps in Central Germany. However, the absence of founder effects and the lack of phylogeographic structure prevented us from identifying putative donor populations.     

Intraspecific and interspecific variation in the second internal transcribed spacer sequence for Metastrongylus (Nematoda: Metastrongyloidea) detected by high resolution PCR-linked restriction fragment length polymorphism

Metastrongylus species are important parasites of free-range pigs and wild boar, but little is known about the genetic make-up of natural populations. This study was undertaken to examine sequence variation in internal transcribed spacer 2 of ribosomal DNA within and among three species of Metastrongylus using PCR-linked restriction fragment length polymorphism analysis. In contrast to many other species of bursate nematodes, significant intraspecific variation was detected in restriction fragment length polymorphism profiles among individual worms. In spite of this, it was possible to identify the three species by their distinctive restriction profiles. The findings suggest that the internal transcribed spacer 2 region should be useful for analysing population variation within Metastrongylus species.