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.

Times Cited: 76International audienceIn the European Alps, Rhonodendron 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

Animal pollination, essential for both ecological services and ecosystem functioning, is threatened by ongoing global changes. New methodologies to decipher their effects on pollinator composition to ecosystem health are urgently required. We compare the main structural parameters of pollination networks based on DNA metabarcoding data with networks based on direct observations of insect visits to plants at three resolution levels. By detecting numerous additional hidden interactions, metabarcoding data largely alters the properties of the pollination networks compared to visit surveys. Molecular data shows that pollinators are much more generalist than expected from visit surveys. However, pollinator species were composed of relatively specialized individuals and formed functional groups highly specialized upon floral morphs. We discuss pros and cons of metabarcoding data relative to data obtained from traditional methods and their potential contribution to both current and future research. This molecular method seems a very promising avenue to address many outstanding scientific issues at a resolution level which remains unattained to date; especially for those studies requiring pollinator and plant community investigations over macro-ecological scales.

The breeding system of the alpine shrub Rhododendron ferrugineum was studied at two sites of a population (site L and site H) in the French Alps. The aims were to estimate the degree of selling and (or) outcrossing and to determine if floral traits (including floral morphology, pollen to ovule ratio, stigma receptivity) may be related to the breeding system of the shrub. First, floral traits (corolla length and anther, and style heights) were measured, the number of pollen tetrads and ovules were counted, and the date of stigma receptivity and anther dehiscence were recorded. Second, five pollinations treatments (unmanipulated flowers, natural selfing, hand selfing, natural outcrossing, and hand outcrossing) were performed on 35 randomly chosen individuals in each site, and after each treatment the number of seeds per fruit was recorded. The results demonstrate that Rhododendron ferrugineum is self-compatible (self-compatibility index = 0.95 and 0.97; auto-fertility index = 0.92 and 0.98; selfing rate = 0.24 and 0.11 at sites L and H, respectively). Indices values are not significantly different between the two sites and facultatively xenogamous (pollen to ovule ratio = 669.5 ± 280.5 and 845.2 ± 246.9 at sites L and H, respectively). Key words: breeding system, Rhododendron ferrugineum, floral morphology, phenology, P:O ratio.

Genetic variation within plant species is determined by a number of factors such as reproductive mode, breeding system, life history traits and climatic events. In alpine regions, plants experience heterogenic abiotic conditions that influence the population's genetic structure. The aim of this study was to investigate the genetic structure and phylogeographic history of the subalpine shrub Rhododendron ferrugineum across the Pyrenees and the links between the populations in the Pyrenees, the Alps and Jura Mountains. We used 27 microsatellite markers to genotype 645 samples from 29 Pyrenean populations, three from the Alps and one from the Jura Mountains. These data were used to estimate population genetics statistics such as allelic richness, observed heterozygosity, expected heterozygosity, fixation index, inbreeding coefficient and number of migrants. Genetic diversity was found to be higher in the Alps than in the Pyrenees suggesting colonization waves from the Alps to the Pyrenees. Two separate genetic lineages were found in both the Alps and Pyrenees, with a substructure of five genetic clusters in the Pyrenees where a loss of genetic diversity was noted. The strong differentiation among clusters is maintained by low gene flow across populations. Moreover, some populations showed higher genetic diversity than others and presented rare alleles that may indicate the presence of alpine refugia. Two lineages of R. ferrugineum have colonized the Pyrenees from the Alps. Then, during glaciation events R. ferrugineum survived in the Pyrenees in different refugia such as lowland refugia at the eastern part of the chain and nunataks at high elevations leading to a clustered genetic pattern.

Although the use of metabarcoding to identify taxa in DNA mixtures is widely approved, its reliability in quantifying taxon abundance is still the subject of debate. In this study we investigated the relationships between the amount of pollen grains in mock solutions and the abundance of highthroughput sequence reads and how the relationship was affected by the pollen counting methodology, the number of PCR cycles, the type of markers and plant species whose pollen grains have different characteristics. We found a significant positive relationship between the number of DNA sequences and the number of pollen grains in the mock solutions. However, better relationships were obtained with light microscopy as a pollen grain counting method compared with flow cytometry, with the chloroplastic trnL marker compared with ribosomal ITS1 and with 30 when compared with 25 or 35 PCR cycles. We provide a list of recommendations to improve pollen quantification. Environmental DNA metabarcoding is a molecular method that consists of investigating environmental DNA samples made of complex mixtures of genomes from numerous organisms 1. Due to new sequencing technologies and bioinformatics tools, metabarcoding has been increasingly used to identify taxa in environmental samples 1 to monitor biodiversity 2-4 , to investigate ecosystem functioning 5 and interaction networks 6-8 , in both aquatic and terrestrial ecosystems. Nevertheless, its reliability in quantitative approaches, which depend on the match between counts of high-throughput sequence reads and the amount of sampled biological material 2 , is still the subject of debate 9,10. While taxon identification can reveal individual diet breadth 11 , species richness, and the composition of habitats 2 , communities 12 and ecological networks 4 , taxon quantification provides knowledge on species evenness in those habitats, communities and diets or on the level of individual or species specialization in networks, all of which is very useful in ecological studies. Research on pollination and knowledge of the quantities of pollen transported by pollinators allow for the estimation of plant-pollinator interaction strength and hence it gives a more realistic representations of networks than those made possible using traditional approaches such as observing visits to plants by pollinators 9,13. Metabarcoding has been used in pollen studies to identify pollen in honey 14-16 , insect loads 6-8,17 , insect nests 18 , airborne samples 19 , and to quantify pollen abundance across various sample types. Several studies found significant positive relationships between pollen abundance (estimated using light microscopy) or pollen DNA quantities, and the abundance or the frequencies of high-throughput sequencing reads in experimental samples 10,16,17,20,21 , airborne samples 22,23 , insect pollen loads 21,24-27 or in brood cells of solitary bees 28. Conversely, other studies found low or no significant pollen-sequence abundance relationships when using ITS2 markers applied to pollen provision in...

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.

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.