Nathalie Escaravage

Clonal diversity in a Rhododendron ferrugineum L. (Ericaceae) population inferred from AFLP markers

In the European Alps, Rhododendron ferrugineum can constitute dense populations with almost 100% of cover. The developmental pattern by layering and the resulting complexity of population structure make it difficult to identify distinct clones even by excavation. Therefore genotypic structure of a R. ferrugineum population, in the French Alps, was inferred from AFLP markers. In a first step, we analysed 400 samples using AFLP profiles generated by one selective primer pair. Seventeen bands out of 25 were polymorphic (68%). We identified a total of 32 multilocus genotypes. In a second step, the 32 genotypes were verified by applying two additional primer pairs to the two most distant samples from each genotype. The mean similarity (proportion of band sharing) between pairs of clones was 0.85 (range from 0.52 to 0.94). The spatial distribution of clones showed that vegetative spreading mainly occurred down a slope. Based on an annual shoot mean growth of 2.6 cm/year and the size of the widest clone, we estimated the age of the oldest individual to be at least 300 years. A single genotype can occupy a large surface and sometimes form a dense patch, suggesting that this species adopts a phalanx growth form with limited intermingling of some genets.

Dynamics of genotypic structure in clonal Rhododendron ferrugineum (Ericaceae) populations

Two populations of Rhododendron ferrugineum growing at subalpine level in the Pyrenees (France) were studied in two sites (Bethmale and Mourtis). Identification and delimitation of genets were inferred from amplified fragment length polymorphism (AFLP) markers, along a closure gradient (from meadow to more closed heath) in each site. Surface and age of genets, genotypic diversity (Simpson’s index D), ‘proportion distinguishable’ genotypes and genetic relationships between genets were then estimated. Amplification of the 312 DNA samples with three selective primer pairs gave a mean of 98 detectable peaks (i.e. bands) per sample, with size ranging from 60 to 300 bp. In total 60% (Bethmale) and 70% (Mourtis) of the peaks were polymorphic, and a total of 31 and 23 multilocus genotypes were identified, in Bethmale and Mourtis, respectively. We inferred that pioneer genotypes began arriving 110 years ago mainly over a 40‐year period in the Mourtis meadow, and began about 130 years ago over a 100‐year period in the Bethmale meadow. After this pioneer stage, populations extended vegetatively. Two different patterns of genotypic dynamics can be identified. At Bethmale, population closure could have led to a dramatic loss of genets and to the selection of highly genetically related genotypes. In contrast, at Mourtis, genotypic diversity and genet density did not change fundamentally along the closure gradient. However the range of genetic diversity diminished from the open to the closed situation, suggesting that thinning could have occurred in the past.

Complementarity in mineral nitrogen use among dominant plant species in a subalpine community

The underlying mechanisms that enable plant species to coexist are poorly understood. Complementarity in resource use is among the major mechanisms proposed that could favor species coexistence but is insufficiently documented. In alpine soil, low temperatures are a major constraint for the supply of plant nitrogen. We carried out (15)N labeling of soil mineral N to determine to what extent four major species of a subalpine community compete for N, or develop ionic (NH(4)(+) vs. NO(3)(-)) or temporal complementarity. The Poaceae took up much more (15)N per soil area unit than the ericaceous species, and all species displayed three major strategies in exploiting (15)N: (1) uptake mainly early in the growing season (Vaccinium myrtillus), (2) uptake at a slow and similar rate throughout the growing season (Rhododendron ferrugineum), and (3) uptake at high rates over the growing season (Festuca eskia and Nardus stricta). However, while F. eskia used (15)NH(4)(+) mainly early and (15)NO(3)(-) mainly late in the growing season, the reverse was observed for N. stricta. Taking into account (15)N dilution in soil NH(4)(+) and NO(3)(-) pools, we calculated that NH(4)(+) provided more than 80% of the mineral N uptake in Ericaceae and about 60% in grasses. Together, such ionic and temporal complementarity would reduce competition between species and could be a major mechanism promoting species diversity.

VARIATION OF REPRODUCTIVE TRAITS IN RHODODENDRON FERRUGINEUM L. (ERICACEAE) POPULATIONS ALONG A SUCCESSIONAL GRADIENT

Sexual and vegetative reproduction in the alpine species Rhododendron ferrugineum was studied along a successional sequence (meadow → open heathland → closed heathland) at two sites and in a wet heathland. This study aims to determine (1) the characteristics of sexual reproduction in R. ferrugineum populations (2) when and how these populations develop layering (adventitious rooting) and (3) whether reproductive traits and reproductive strategies develop relative to the degree of population closure and maturity. The variables used to describe sexual reproduction were inflorescence density (per m(2) of Rhododendron cover), number of flowers per inflorescence and per m(2) of Rhododendron cover, and seeds production (per fruit and m(2) of Rhododendron cover). Flowering and fruiting phenologies were also recorded. For describing clonal development, we investigated layering variables such as length and annual growth rate of prostrate stems, rooting occurence and ramet density. The results show that the direction toward which the clones extend is mainly determinated by the topography, and that layering steadily increases with increasing population closure and maturity. Reproductive potential of R. ferrugineum is enormous (0.4–2.4 million seeds m(-2)) but reproductive effort remains low with respect to total biomass of seeds (3–21 g m(-2)). Reproductive effort of R. ferrugineum populations could be reduced as a conterpart of layering development only when the shrub draws more matter and energy in layering stems than to aerial stems. The variations in reproductive traits observed on our sites could be due to primarily to phenotypic response to variable microhabitat features, rather than to genetically deterministic processes.

Using metabarcoding to reveal and quantify plant-pollinator interactions

Given the ongoing decline of both pollinators and plants, it is crucial to implement effective methods to describe complex pollination networks across time and space in a comprehensive and high-throughput way. Here we tested if metabarcoding may circumvent the limits of conventional methodologies in detecting and quantifying plant-pollinator interactions. Metabarcoding experiments on pollen DNA mixtures described a positive relationship between the amounts of DNA from focal species and the number of trnL and ITS1 sequences yielded. The study of pollen loads of insects captured in plant communities revealed that as compared to the observation of visits, metabarcoding revealed 2.5 times more plant species involved in plant-pollinator interactions. We further observed a tight positive relationship between the pollen-carrying capacities of insect taxa and the number of trnL and ITS1 sequences. The number of visits received per plant species also positively correlated to the number of their ITS1 and trnL sequences in insect pollen loads. By revealing interactions hard to observe otherwise, metabarcoding significantly enlarges the spatiotemporal observation window of pollination interactions. By providing new qualitative and quantitative information, metabarcoding holds great promise for investigating diverse facets of interactions and will provide a new perception of pollination networks as a whole.

Stamen dimorphism in Rhododendron ferrugineum (Ericaceae): development and function

The function of stamen dimorphism in the breeding system of the alpine shrub Rhododendron ferrugineum was studied in two populations in the French Alps. This species has pentameric flowers with two whorls of stamens: an inner whorl of five long stamens and an outer whorl of short stamens. We studied the development of stamens from buds to mature flowers (measurement of the filament, anther, and style lengths at five successive phenological stages) and compared the size and position of reproductive organs at maturity in control and partially emasculated flowers (removal of long-level stamens) to determine whether the presence of long-level stamens constitutes a constraint for the development of the short-level ones. Stamen dimorphism can be observed early in stamen development, from the bud stage of the year prior to flowering. At this early stage, meiosis had already occurred. Emasculation of the long-level stamens induced the short-level ones to grow longer than in normal conditions. We also performed seven pollination treatments on ten randomly chosen individuals in each population, and the number of seeds following each treatment was recorded. Results from these treatments showed that R. ferrugineum produced spontaneous selfed seeds in the absence of pollinators. However, no seed was produced when short-level stamens were emasculated and pollinators excluded, suggesting that long-level stamens are not responsible for selfing in the absence of pollinators and that reproductive assurance is promoted by short-level stamens.

Isolation and characterization of microsatellite loci in Rhododendron ferrugineum (Ericaceae) using pyrosequencing technology

PREMISE OF THE STUDY Microsatellite primers were developed for Rhododendron ferrugineum (Ericaceae) to evaluate genetic diversity, population genetic structure, and mating system of this self-compatible species. METHODS AND RESULTS The new-generation 454 FLX Titanium pyrosequencing technology was used to isolate 102 novel microsatellite loci. Two multiplex PCR sets were optimized to genotype nine polymorphic microsatellite loci. The level of genetic diversity was assessed in two populations from the Pyrenees (France). The mean number of alleles per locus ranged from 1.5 to 7. The mean observed and expected heterozygosities ranged from 0 to 0.76 and from 0.03 to 0.66, respectively. Cross-species amplification was successful for 13 other Rhododendron species and two additional genera of Ericaceae, with an average of seven pairs of primers amplifying per species. CONCLUSIONS These markers will facilitate further studies on the evolutionary history of the large Ericaceae family and Rhododendron in particular.

Genotypic structure in clonal Rhododendron ferrugineum L. (Ericaceae) populations: origin and maintenance

Genotypic structure of a closed population of the clonal ericaceous shrub Rhododendron ferrugineum is examined in the light of two independent studies previously conducted on this species. In the first study, spatial distribution of genotypes in the closed population was inferred from the amplified fragment length polymorphism (AFLP) procedure. Age of clones was estimated using their spatial extent and the annual growth rate of shoots. In the second study, ramet demography was studied in the three most representative stages of shrub invasion on two different sites, including the site where the population investigated by AFLP grew. The demographic data recorded were the area occupied by ramets and ramet age, and from this information the developmental pattern of Rhododendron populations was determined. Additional data such as genet density and distance between genets were calculated.These two sources of information allow us to propose that all or most of the clones detected in the closed population established at the early successional stage, and that the present genotypic structure was established several hundred years ago, long before the population reached total closure. Hypotheses concerning the future development of this genotypic structure are discussed.

Complex interactions between a legume and two grasses in a subalpine meadow

Interactions between plants are a complex combination of positive and negative interactions, with the net outcome depending on environmental contexts. The more frequent association of Trifolium alpinum (legume) with Festuca eskia than with Nardus stricta (grasses) in many Pyrenean subalpine meadows suggests a differential ability to use nitrogen (N) derived from N(2) fixation. In the field, we investigated the interactions between the legume and grasses and, in the glasshouse, the transfer of (15)N from the legume to the grasses. In one grass-Trifolium mixture, the legume had a strong positive effect on the biomass and N content of the grass as compared to pure grass stands. When both grasses grew together with the legume, only Festuca benefited from the presence of Trifolium but, surprisingly, the benefit decreased with increasing Trifolium abundance. Leaf labeling experiments with (15)N-NH(4)(+) revealed a higher transfer of (15)N from Trifolium to Festuca than to Nardus, suggesting a more direct N pathway between the two species. This more direct pathway could prevent Nardus from benefiting from the legume N in the three-species mixtures. Thus, the positive interactions between N-fixers and nonfixers appear to be largely species-specific and to depend strongly on the species in the plant assemblage.