Isolation and characterization of microsatellite DNA markers in the grass Poa alpina L.

The important fodder grass Poa alpina L. occurs at several ploidy levels with common aneuploidy. We isolated and characterized five polymorphic microsatellite markers for the study of molecular genetic variation of this species. As first examples of the value of the developed markers for population genetic analyses, we show that plants with more chromosomes have more microsatellite bands and that isolation by distance plays a small role in shaping microsatellite diversity of P. alpina in the Swiss Alps.     

Polyploidisation and Geographic Differentiation Drive Diversification in a European High Mountain Plant Group (Doronicum clusii Aggregate,Asteraceae)

Range shifts (especially during the Pleistocene), polyploidisation and hybridization are major factors affecting high-mountain biodiversity. A good system to study their role in the European high mountains is the Doronicum clusii aggregate (Asteraceae), whose four taxa (D. clusii s.s., D. stiriacum, D. glaciale subsp. glaciale and D. glaciale subsp. calcareum) are differentiated geographically, ecologically (basiphilous versus silicicolous) and/or via their ploidy levels (diploid versus tetraploid). Here, we use DNA sequences (three plastid and one nuclear spacer) and AFLP fingerprinting data generated for 58 populations to infer phylogenetic relationships, origin of polyploids—whose ploidy level was confirmed by chromosomally calibrated DNA ploidy level estimates—and phylogeographic history. Taxonomic conclusions were informed, among others, by a Gaussian clustering method for species delimitation using dominant multilocus data. Based on molecular data we identified three lineages: (i) silicicolous diploid D. clusii s.s. in the Alps, (ii) silicicolous tetraploid D. stiriacum in the eastern Alps (outside the range of D. clusii s.s.) and the Carpathians and (iii) the basiphilous diploids D. glaciale subsp. glaciale (eastern Alps) and D. glaciale subsp. calcareum (northeastern Alps); each taxon was identified as distinct by the Gaussian clustering, but the separation of D. glaciale subsp. calcareum and D. glaciale subsp. glaciale was not stable, supporting their taxonomic treatment as subspecies. Carpathian and Alpine populations of D. stiriacum were genetically differentiated suggesting phases of vicariance, probably during the Pleistocene. The origin (autopolyploid versus allopolyploid) of D. stiriacum remained unclear. Doronicum glaciale subsp. calcareum was genetically and morphologically weakly separated from D. glaciale subsp. glaciale but exhibited significantly higher genetic diversity and rarity. This suggests that the more widespread D. glaciale subsp. glaciale originated from D. glaciale subsp. calcareum, which is restricted to a prominent Pleistocene refugium previously identified in other alpine plant species.     

Past,present and future of mountain species of the French Massif Central – the case of Soldanella alpina L. subsp. alpina (Primulaceae) and a review of other plant and animal studies

Aim Our goals were: (1) to investigate patterns of genetic variation in the French Massif Central (MC) of Soldanella alpina (Primulaceae), an alpine plant species that has only one known population in the region; (2) to analyse these patterns in order to deduce the Quaternary history of the population and to predict how current climatic warming may affect it; and (3) to review molecular analyses from the MC to evaluate the importance of the region for the conservation of genetic diversity. Location Europe, with a special focus on the French Massif Central and adjacent regions. Methods Amplified fragment length polymorphisms (AFLPs) were analysed for 192 individuals (nine populations) of S. alpina (subsp. alpina) representing the MC, Pyrenees and south‐western Alps. Population genetic diversity was assessed by various parameters (e.g. H_E, Shannon’s I). Neighbor‐Net and Bayesian approaches, and analysis of molecular variance (AMOVA) were used to infer population genetic relationships and structure. Results Individuals generally clustered according to populations within mountain regions. Hierarchical AMOVA indicated significant variation among mountain ranges (33.2% of the total variance), but there was also strong differentiation between populations (26.3%). The single population of S. alpina from the MC was identified as a distinct lineage of high genetic diversity. Our literature survey indicated that taxa with low and with high genetic diversity exist in the MC, and that genetic relationships to surrounding regions are diverse. Main conclusions The high genetic diversity and distinctiveness of S. alpina in the MC suggests the long‐term persistence of the single population in this region, which might have been favoured through elevational range shifts in response to past climatic change. This interpretation partly accords with other studies indicating that several plant and animal populations in the MC contain comparatively high genetic diversity, represent genetically independent lineages, and/or are likely descendants of populations that persisted in the MC throughout the Quaternary. These data underline the conservation importance of the MC as a key area for the long‐term persistence of species with often high levels of intraspecific genetic diversity.     

The origin of the serpentine endemic Minuartia laricifolia subsp. ophiolitica by vicariance and competitive exclusion

Serpentine soils harbour a unique flora that is rich in endemics. We examined the evolution of serpentine endemism in Minuartia laricifolia, which has two ecologically distinct subspecies with disjunct distributions: subsp. laricifolia on siliceous rocks in the western Alps and eastern Pyrenees and subsp. ophiolitica on serpentine in the northern Apennines. We analysed AFLPs and chloroplast sequences from 30 populations to examine their relationships and how their current distributions and ecologies were influenced by Quaternary climatic changes. Minuartia laricifolia was divided into four groups with a BAPS cluster analysis of the AFLP data, one group consisted only of subsp. ophiolitica, while three groups were found within subsp. laricifolia: Maritime Alps, north‐western Alps and central Alps. The same groups were recovered in a neighbour‐joining tree, although subsp. ophiolitica was nested within the Maritime Alps group of subsp. laricifolia. Subspecies ophiolitica contained three different chloroplast haplotypes, which were also found in the Maritime Alps group of subsp. laricifolia. Given its high genetic diversity, subsp. ophiolitica appears to have arisen from subsp. laricifolia by vicariance instead of by long‐distance dispersal. Genetic and geographic evidence point to the Maritime Alps populations of subsp. laricifolia as the closest relatives of subsp. ophiolitica. We hypothesize that M. laricifolia was also able to grow on nonserpentine rocks in the northern Apennines during glacial periods when the vegetation was more open, but that only the serpentine‐adapted populations were able to persist until the present due to their competitive exclusion from more favourable habitats.     

Contrasting phylogeographies inferred for the two alpine sister species Cardamine resedifolia and C. alpina (Brassicaceae)

Aim We use Cardamine alpina and C. resedifolia as models to address the detailed history of disjunctions in the European alpine system. These species grow on siliceous bedrock: C. alpina in the Alps and Pyrenees, and C. resedifolia in several mountain ranges from the Sierra Nevada to the Balkans. We explore differentiation among their disjunct populations as well as within the contiguous Alpine and Pyrenean ranges, and compare the phylogeographical histories of these diploid sister species. We also include samples of the closely related, arctic diploid C. bellidifolia in order to explore its origin and post‐glacial establishment. Location European alpine system, Norway and Iceland. Methods We employed amplified fragment length polymorphisms (AFLPs). AFLP data were analysed using principal coordinates analysis, neighbour joining and Bayesian clustering, and measures of diversity and differentiation were computed. Results For the snow‐bed species C. alpina (27 populations, 203 plants) we resolved two strongly divergent lineages, corresponding to the Alps and the Pyrenees. Although multiple glacial refugia were invoked in the Pyrenees, we inferred only a single one in the Maritime Alps – from which rapid post‐glacial colonization of the entire Alps occurred, accompanied by a strong founder effect. For C. resedifolia (33 populations, 247 plants), which has a broader ecological amplitude and a wider distribution, the genetic structuring was rather weak and did not correspond to the main geographical disjunctions. This species consists of two widespread and largely sympatric main genetic groups (one of them subdivided into four geographically more restricted groups), and frequent secondary contacts exist between them. Main conclusions The conspicuously different histories of these two sister species are likely to be associated with their different ecologies. The more abundant habitats available for C. resedifolia may have increased the probability of its gradual migration during colder periods and also of successful establishment after long‐distance dispersal, whereas C. alpina has been restricted by its dependence on snow‐beds. Surprisingly, the arctic C. bellidifolia formed a very divergent lineage with little variation, contradicting a scenario of recent, post‐glacial migration from the Alps or Pyrenees.     

Multilocus Sequence Typing and Further Genetic Characterization of the Enigmatic Pathogen,Staphylococcus hominis

Staphylococcus hominis is a commensal resident of human skin and an opportunistic pathogen. The species is subdivided into two subspecies, S. hominis subsp. hominis and S. hominis subsp. novobiosepticus, which are difficult to distinguish. To investigate the evolution and epidemiology of S. hominis, a total of 108 isolates collected from 10 countries over 40 years were characterized by classical phenotypic methods and genetic methods. One nonsynonymous mutation in gyrB, scored with a novel SNP typing assay, had a perfect association with the novobiocin-resistant phenotype. A multilocus sequence typing (MLST) scheme was developed from six housekeeping gene fragments, and revealed relatively high levels of genetic diversity and a significant impact of recombination on S. hominis population structure. Among the 40 sequence types (STs) identified by MLST, three STs (ST2, ST16 and ST23) were S. hominis subsp. novobiosepticus, and they distinguished between isolates from different outbreaks, whereas 37 other STs were S. hominis subsp. hominis, one of which was widely disseminated (ST1). A modified PCR assay was developed to detect the presence of ccrAB4 from the SCCmec genetic element. S. hominis subsp. novobiosepticus isolates were oxacillin-resistant and carriers of specific components of SCCmec (mecA class A, ccrAB3, ccrAB4, ccrC), whereas S. hominis subsp. hominis included both oxacillin-sensitive and -resistant isolates and a more diverse array of SCCmec components. Surprisingly, phylogenetic analyses indicated that S. hominis subsp. novobiosepticus may be a polyphyletic and, hence, artificial taxon. In summary, these results revealed the genetic diversity of S. hominis, the identities of outbreak-causing clones, and the evolutionary relationships between subspecies and clones. The pathogenic lifestyle attributed to S. hominis subsp. novobiosepticus may have originated on more than one occasion.     

Distribution of bulbil- and seed-producing plants of Poa alpina (Poaceae) and their growth and reproduction in common gardens suggest adaptation to different elevations

• Premise of the study: The European Alps harbor a spatially heterogeneous environment. Plants can be adapted genetically to this heterogeneity but may also respond to it by phenotypic plasticity. We expected the important fodder grass Poa alpina to be adapted to elevation either genetically or plastically. • Methods: We investigated in three elevational common gardens whether growth and reproductive allocation of plants reproducing either by seeds or bulbils suggest adaptation to their elevation of origin and to what extent they can respond plastically to different elevations. Additionally, we analyzed genetic diversity using microsatellites and tested whether seeds are of sexual origin. • Key results: In the field, bulbil-producing plants occurred more often at higher elevations, whereas seed-producing plants occurred more often at lower elevations, but bulbil-producing plants were generally less vigorous in the common gardens. The response of plants to elevational transplantation was highly plastic, and vigor was always best at the highest location. The small genetic differences were not clinally related to elevation of origin, underlining the importance of phenotypic plasticity. Reproductive allocation was, however, independent of elevational treatments. Seed-producing plants had higher genetic diversity than the bulbil-producing plants even though we found that seed-producing plants were facultative apomicts mostly reproducing asexually. • Conclusions: Bulbil-producing P. alpina, showing a fitness cost at lower elevations compared with seed-producing plants, seem better adapted to higher elevations. By means of its two reproductive modes and the capacity to adjust plastically, P. alpina is able to occupy a broad ecological niche across a large elevational range.     

Population genetic structure,genetic diversity,and natural history of the South American species of Nothofagus subgenus Lophozonia (Nothofagaceae) inferred from nuclear microsatellite data

The effect of glaciation on the levels and patterns of genetic variation has been well studied in the Northern Hemisphere. However, although glaciation has undoubtedly shaped the genetic structure of plants in the Southern Hemisphere, fewer studies have characterized the effect, and almost none of them using microsatellites. Particularly, complex patterns of genetic structure might be expected in areas such as the Andes, where both latitudinal and altitudinal glacial advance and retreat have molded modern plant communities. We therefore studied the population genetics of three closely related, hybridizing species of Nothofagus (N. obliqua, N. alpina, and N. glauca, all of subgenus Lophozonia; Nothofagaceae) from Chile. To estimate population genetic parameters and infer the influence of the last ice age on the spatial and genetic distribution of these species, we examined and analyzed genetic variability at seven polymorphic microsatellite DNA loci in 640 individuals from 40 populations covering most of the ranges of these species in Chile. Populations showed no significant inbreeding and exhibited relatively high levels of genetic diversity (H_E = 0.502–0.662) and slight, but significant, genetic structure (R_ST = 8.7–16.0%). However, in N. obliqua, the small amount of genetic structure was spatially organized into three well‐defined latitudinal groups. Our data may also suggest some introgression of N. alpina genes into N. obliqua in the northern populations. These results allowed us to reconstruct the influence of the last ice age on the genetic structure of these species, suggesting several centers of genetic diversity for N. obliqua and N. alpina, in agreement with the multiple refugia hypothesis.     

Pollen morphology of alpine butterworts (Pinguicula L., Lentibulariaceae)

The pollen morphology of Pinguicula alpina, P. arvetii, P. grandiflora subsp. grandiflora, P. grandiflora subsp. rosea, P. hirtiflora, P. leptoceras, P. poldinii, P. reichenbachiana, and P. vulgaris, belonging to the Alpine flora, was studied.The pollen grains, coming from different populations, were investigated using light microscopy and scanning electron microscopy. The pollen size, the shape (P/E ratio), the number of colpori and the exine ornamentation are, for Pinguicula, important diagnostic characters.Pinguicula pollen grains are medium sized (∼ 30 μm), trinucleate, isopolar, radially symmetric. The shape of the grains is variable from oblate spheroidal to prolate spheroidal and they are (4)-5-9-(10)-zonocolporate. The prevalent ornamentation is rugulate-microreticulate, P. alpina has a rugulate-reticulate ornamentation and only P. hirtiflora has a perforate ornamentation.A pollen key, based on micromorphological data, is presented.     

Limited structure and widespread diversity suggest potential buffers to genetic erosion in a threatened grassland shrub Pimelea spinescens (Thymelaeaceae)

Pimelea spinescens is a critically endangered species of the temperate grasslands of southeastern Australia. Two subspecies are recognised. Subspecies, P. spinescens subsp. spinescens, formerly common and widespread, is found in isolated remnants of previously extensive grasslands. The second subspecies, subsp. pubiflora, is thought to have been historically rare with only two geographically-isolated extant populations. The grassland communities exist now as fragmented remnants representing <1 % of their extent prior to European settlement in the early 1800s. Conservation management strategies for species in these critically endangered ecosystems rely on an understanding of genetic diversity and population structure to ensure long-term evolutionary potential. We used chloroplast DNA (cpDNA) and microsatellite markers to examine the population genetic structure of both subspecies. Analysis of cpDNA revealed 14 haplotypes with high divergence between the single haplotype found in subsp. pubiflora and most remaining haplotypes restricted to subsp. spinescens. Microsatellites also indicated high genetic differentiation between subspecies but little evidence of sub-structuring within either subspecies. Results suggest that seed dispersal has not been as limited as previously thought. In this fragmented habitat, a lack of genetic structure suggests buffers to genetic erosion, with current patterns reflecting genetic diversity prior to fragmentation. Plant longevity and the presence of seed banks may contribute to the maintenance of these patterns, resulting in a lag between fragmentation and genetic erosion. Whilst factors such as longevity and seed banks may be preserving historic genetic diversity, management is required to ensure the maintenance of this diversity into the future.     

Evolution of the Selfing Syndrome in Arabis alpina (Brassicaceae)

Introduction

The transition from cross-fertilisation (outcrossing) to self-fertilisation (selfing) frequently coincides with changes towards a floral morphology that optimises self-pollination, the selfing syndrome. Population genetic studies have reported the existence of both outcrossing and selfing populations in Arabis alpina (Brassicaceae), which is an emerging model species for studying the molecular basis of perenniality and local adaptation. It is unknown whether its selfing populations have evolved a selfing syndrome.

Methods

Using macro-photography, microscopy and automated cell counting, we compared floral syndromes (size, herkogamy, pollen and ovule numbers) between three outcrossing populations from the Apuan Alps and three selfing populations from the Western and Central Alps (Maritime Alps and Dolomites). In addition, we genotyped the plants for 12 microsatellite loci to confirm previous measures of diversity and inbreeding coefficients based on allozymes, and performed Bayesian clustering.

Results and Discussion

Plants from the three selfing populations had markedly smaller flowers, less herkogamy and lower pollen production than plants from the three outcrossing populations, whereas pistil length and ovule number have remained constant. Compared to allozymes, microsatellite variation was higher, but revealed similar patterns of low diversity and high Fis in selfing populations. Bayesian clustering revealed two clusters. The first cluster contained the three outcrossing populations from the Apuan Alps, the second contained the three selfing populations from the Maritime Alps and Dolomites.

Conclusion

We conclude that in comparison to three outcrossing populations, three populations with high selfing rates are characterised by a flower morphology that is closer to the selfing syndrome. The presence of outcrossing and selfing floral syndromes within a single species will facilitate unravelling the genetic basis of the selfing syndrome, and addressing which selective forces drive its evolution.     

Morphological and genetic diversity within Pilosella hoppeana aggr. (Asteraceae) in Italy and taxonomic implications

Morphological variation, ploidy level and genetic diversity have been studied on 10 populations of the Pilosella hoppeana aggr. from the Alps, Abruzzo, Calabria and Sicily. Chromosome counts showed that the plants from Abruzzo and those from Sicily are tetraploid (2n = 36); they are assigned to P. hoppeana subsp. macrantha. The plants from the Alps (P. hoppeana subsp. hoppeana) and those from Calabria are diploid. The Calabrian populations, previously included in P. hoppeana subsp. macrantha, are shown to belong to a separate species, P. leucopsilon. The principal component analysis, based on 25 morphological characters, allowed distinguishing clearly four groups. An allozyme study using 10 enzyme systems revealed 7 polymorphic loci with a total of 20 alleles, some of them exclusive at regional level, others shared between populations showing similar morphological features. The genetic differentiation between populations was relatively high. The obtained dendrogram supports recognition of the morphologically defined taxa.     

Genetic diversity and geographic distribution of variation of Hordeum murinum as revealed by retroelement insertional polymorphisms in Iran

Hordem murinum is a widespread weedy/wild species, growing in different ecological conditions in Iran. Populations of H. murinum subsp. glaucum (2n = 2x = 14) and subsp. leporinum (2n = 4x, 6x = 28, 42) are found in this region. Inter-retroelement amplified polymorphism (IRAP) markers were used to analyse the genetic diversity of 57 accessions of H. murinum from different regions of Iran, and to examine patterns of diversity related to the taxonomy and geography. Eight IRAP primer combinations amplified a total of 241 distinct DNA fragments sized 150-1400 bp, from which 236 (97.9%) were polymorphic. On average, each primer combination amplified about 30.12 fragments (ranged from 23 to 34) in PCR. The patterns of genetic diversity were closely correlated with taxonomic groups, ploidy levels and geographic origin. Along with the high genetic diversity, three geographic sub-genepools were evident, 1: in the North-Northeast region along the Alborz Mountains, 2: in the West-Northwest region along the Zagros Mountains, and 3: in the Central — Southern region. The genetic diversity in diploids was higher than polyploids. Also genetic diversity in W-NW region along the Zagros Mountains was considerably higher than that of the other regions.     

Genetic Variability and Population Structure of Disanthus cercidifolius subsp. longipes (Hamamelidaceae) Based on AFLP Analysis

Disanthus cercidifolius subsp. longipes is an endangered species in China. Genetic diversity and structure analysis of this species was investigated using amplified fragments length polymorphism (AFLP) fingerprinting. Nei''s gene diversity ranged from 0.1290 to 0.1394. The AMOVA indicated that 75.06% of variation was distributed within populations, while the between-group component 5.04% was smaller than the between populations-within-group component 19.90%. Significant genetic differentiation was detected between populations. Genetic and geographical distances were not correlated. PCA and genetic structure analysis showed that populations from East China were together with those of the Nanling Range. These patterns of genetic diversity and levels of genetic variation may be the result of D. c. subsp. longipes restricted to several isolated habitats and “excess flowers production, but little fruit set”. It is necessary to protect all existing populations of D. c. subsp. longipes in order to preserve as much genetic variation as possible.     

Haplotype and ribotype diversity of Saxifraga cuneifolia s. l. (Saxifragaceae)

Saxifraga cuneifolia L. (sect. Gymnopera, Saxifragaceae) is a plant distributed in the main mountain ranges of southern Europe, from the eastern Pyrenees to the eastern Carpathian. Currently, two subspecies are recognized based on morphological characteristics: S. cuneifolia subsp. cuneifolia grows in the Maritime Alps and North Apennines and S. cuneifolia subsp. robusta is located in the remaining area of distribution. A more delicate form and a smaller number of flowers in S. cuneifolia subsp. cuneifolia are the morphological characteristics that differentiate this subspecies from the widespread S. cuneifolia subsp. robusta. To explore the genetic diversity and the subspecific geographic patterns of S. cuneifolia s. l. we conducted a molecular study of nuclear and plastid sequences. Samples of S. cuneifolia s. l. have been analysed throughout the distribution area of this species. Our results, based on nuclear (ITS) and plastid (rbcL, trnL–F, and psbA–trnH) markers, showed a genetic characterization of both subspecies presenting discriminant haplotypes and ribotypes that confirm the current subspecific systematics.     

Infraspecific classification reflects genetic differentiation in the widespread Petunia axillaris complex: A comparison among morphological,ecological, and genetic patterns of geographic variation

Plants are subjected to natural selection during expansion of their geographic range or when facing changes in environmental conditions, which in turn may affect phenotypic diversity. Studies on geographic phenotypic variation provide insight into the evolutionary processes and have long contributed to better understanding of diversification within and among species. Petunia axillaris is widely distributed in temperate South America, occurring throughout the Pampas region. The current taxonomy of this species recognizes three allopatric subspecies, which occupy nearly adjacent territories, according to floral morphology: P. axillaris subsp. axillaris, P. axillaris subsp. parodii, and P. axillaris subsp. subandina. In this study, we sampled the three P. axillaris subspecies and used both molecular markers (from the plastid and nuclear genomes) and morphological measurements to investigate how genetic diversity and morphological variation correlate to ecological variables and the geographic context. We used different forms of Mantel tests (partial and correlograms) to investigate the geographic distribution patterns in distinct types of similarity/dissimilarity among the populations and their relationships. We also modeled the morphological variation in P. axillaris as a function of the genetic marker frequencies in the populations. We found that the morphological differences leading to the recognition of different subspecies of P. axillaris reflect historical processes of isolation and that adaptation to different ecological conditions faced by each lineage is perhaps not merely a consequence of phenotypic plasticity. These findings suggest that differences between subspecies could represent an incipient speciation stage.     

Comparative pollen morphology of Turkish species of <Emphasis Type="Italic">Petrorhagia</Emphasis> (Caryophyllaceae) and its systematic implications

Pollen morphology of 10 taxa from Turkey, four of them endemic, belonging to the genus Petrorhagia (Caryophyllaceae), P. alpina subsp. alpina, P. alpina subsp. olympica, P. cretica, P. dubia, P. hispidula, P. lycica, P. pamphylica, P. peroninii, P. prolifera and P. saxifraga, has been investigated using light (LM) and scanning electron (SEM) microscopy. Pollen morphology differences among these taxa have been determined. The pollen type of investigated taxa is polyporate (12–22-porate), sphaeroidal, the exine exhibits a tectate structure and a microechinate ornamentation. Petrorhagia alpina subsp. alpina has the smallest pollen grain diameter (18.57 μm) and P. dubia the largest one (37.80 μm). The number of pores ranges from 12 (in P. dubia and P. saxifraga) to 22 (in P. alpina subsp. alpina and P. alpina subsp. olympica and P. hispidula) with a minimum pore diameter of 2.37 μm in P. alpina subsp. alpina and a maximum pore diameter of 4.23 μm in P. peroninii. The exine thickness ranges from 1.73 μm in P. saxifraga to 3.78 μm in P. pamphylica. In this study, the systematic implications are discussed in the light of palynological results.