Experimental quantification of pollen with DNA metabarcoding using ITS1 and trnL.

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 high-throughput 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.

Contribution of plant species to the high N retention capacity of a subalpine meadow undergoing elevated N deposition and warming.

While numerous studies have examined the effect of N deposition on ecosystem N retention, few have analyzed the involvement of plant species and climate warming in this process. We experimentally investigated the effects of increasing N deposition (Nexo) and climate warming on the fate of Nexo in a subalpine meadow and established the involvement of plant species. Using 15N tracer, we tracked Nexo sprayed on the vegetation in belowground and aboveground plant biomasses (AGB) and in bulk soil over three growing seasons. We assessed the Nexo absorption capacity of plant species and the contribution of Nexo to their AGB N pool. The meadow retained a large proportion of Nexo (≈65%, mostly in AGB) for depositions up to four times the background N rate. Nexo present in the meadow compartments in year 2 was still present in year 3, suggesting that the ecosystem was unsaturated after three years of high N input. Nexo retention resulted more from an increase in N concentration in plant tissues than from the increase in AGB. The species-specific Nexo absorption capacity was inversely related to their AGB N concentration. Nexo accounted for up to 40% of total AGB N depending on the species and the N treatments. The contribution of species to ecosystem Nexo retention more contingent on their AGB than on their relative cover in the community, ranked as follows: C. vulgaris (14.0%) > N. stricta (7.0%) > other Poaceae = C. caryophyllea (2.5%) > other Eudicotyledons (1.5%) > non-vascular species = P. erecta > Fabaceae (0.8-0.2%). Climate warming increased AGB and decreased tissue N concentration. No warming-Nexo interaction was observed. Thus, Pyrenean subalpine meadows that have not undergone a decline in plant species richness in recent decades paradoxically display a high potential to sequester atmospheric N deposition.

Pollinator specialization increases with a decrease in a mass-flowering plant in networks inferred from DNA metabarcoding.

How native mass-flowering plants affect the specialization of insects at individual and species levels and the consequences for pollination networks have received much less attention than for mass-flowering crops or alien species and basically remain unexplored.Using existing DNA metabarcoding data on the pollen loads of 402 flower-visiting insects, we assessed the effects of a native mass-flowering plant of high reward quality, the shrub Rhododendron ferrugineum, on pollination networks by investigating: (a) the food niches of individual pollinators and pollinator species and (b) the structure of individual and species networks in subalpine heathland patches with extremely contrasted densities of R. ferrugineum.Relative to its high abundance in high-density patches, the shrub was greatly underrepresented and did not dominate individual's or species' generalized networks, rather individual and species specialization increased with a decrease in R. ferrugineum density. Furthermore, individuals of the more generalist dipteran Empididae species tended to extend exclusive interactions with rare plant species in low-density networks. The same trend was observed in the more specialist Apidea but toward rare species in high-density networks. Our results reveal a quite paradoxical view of pollination and a functional complementarity within networks. Niche and network indices mostly based on the occurrence of links showed that individual pollinators and pollinator species and networks were highly generalized, whereas indices of link strength revealed that species and above all individuals behave as quite strict specialists. Synthesis. Our study provides insights into the status of a native mass-flowering plant in individual's and insect species' food niches and pollination networks. It revealed that a generalist pollinator species can be highly specialized at the individual level and how rare plant species coexisting with mass-flowering plants may nevertheless be visited.

DNA metabarcoding data unveils invisible pollination networks.

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.

Pollen transfer in fragmented plant populations: insight from the pollen loads of pollinators and stigmas in a mass-flowering species.

Pollinator and/or mate scarcity affects pollen transfer, with important ecological and evolutionary consequences for plant reproduction. However, the way in which the pollen loads transported by pollinators and deposited on stigmas are affected by pollination context has been little studied. We investigated the impacts of plant mate and visiting insect availabilities on pollen transport and receipt in a mass-flowering and facultative autogamous shrub (Rhododendron ferrugineum). First, we recorded insect visits to R. ferrugineum in plant patches of diverse densities and sizes. Second, we analyzed the pollen loads transported by R. ferrugineum pollinators and deposited on stigmas of emasculated and intact flowers, in the same patches. Overall, pollinators (bumblebees) transported much larger pollen loads than the ones found on stigmas, and the pollen deposited on stigmas included a high proportion of conspecific pollen. However, comparing pollen loads of emasculated and intact flowers indicated that pollinators contributed only half the conspecific pollen present on the stigma. At low plant density, we found the highest visitation rate and the lowest proportion of conspecific pollen transported and deposited by pollinators. By contrast, at higher plant density and lower visitation rate, pollinators deposited larger proportion of conspecific pollen, although still far from sufficient to ensure that all the ovules were fertilized. Finally, self-pollen completely buffered the detrimental effects on pollination of patch fragmentation and pollinator failure. Our results indicate that pollen loads from pollinators and emasculated flowers should be quantified for an accurate understanding of the relative impacts of pollinator and mate limitation on pollen transfer in facultative autogamous species.

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.

Subalpine Pyrenees received higher nitrogen deposition than predicted by EMEP and CHIMERE chemistry-transport models.

Deposition of reactive nitrogen (N) from the atmosphere is expected to be the third greatest driver of biodiversity loss by the year 2100. Chemistry-transport models are essential tools to estimate spatially explicit N deposition but the reliability of their predictions remained to be validated in mountains. We measured N deposition and air concentration over the subalpine Pyrenees. N deposition was found to range from 797 to 1,463 mg N m(-2) year(-1). These values were higher than expected from model predictions, especially for nitrate, which exceeded the estimations of EMEP by a factor of 2.6 and CHIMERE by 3.6. Our observations also displayed a reversed reduced-to-oxidized ratio in N deposition compared with model predictions. The results highlight that the subalpine Pyrenees are exposed to higher levels of N deposition than expected according to standard predictions and that these levels exceed currently recognized critical loads for most high-elevation habitats. Our study reveals a need to improve the evaluation of N deposition in mountains which are home to a substantial and original part of the world's biodiversity.

High lifetime inbreeding depression counteracts the reproductive assurance benefit of selfing in a mass-flowering shrub.

BACKGROUND: Decreases in mate and/or pollinator availability would be expected to affect the selective pressure on plant mating systems. An increase in self-fertilization may evolve to compensate for the negative effects of pollination failure. However, the benefit of selfing in variable pollination environments depends on the relative fitnesses of selfed and outcrossed progeny. We investigated the potential for selfing to provide reproductive assurance over the lifetime of a long-lived perennial species and its variation between plant patches of various sizes. Patch size is likely to affect mate and pollinator availabilities, thereby affecting pollination success and the rate of selfing. We estimated fruit and seed set, reproductive assurance, self-compatibility, the multilocus patch selfing rate and lifetime inbreeding depression in natural patches of Rhododendron ferrugineum (Ericaceae), a mass-flowering species characterized by considerable patch size variation (as estimated by the total number of inflorescences). RESULTS: Open seed set declined linearly with increasing patch size, whereas pollinator-mediated seed set (emasculated flowers) was not significantly affected. Progeny array analysis indicated that the selfing rate declined with increasing patch size, consistent with greater reproductive assurance in small sparse patches than in large, dense patches. However, fruit set and adult fitness decreased with decreasing patch size, with an estimated mean lifetime inbreeding depression of 0.9 (obtained by comparing F values in adults and progenies). CONCLUSIONS: Lifetime inbreeding depression strongly counteracts the advantage of reproductive assurance due to selfing in this long-lived species. The poor fitness of selfed offspring should counteract any evolution towards selfing, despite its potential to alleviate the negative consequences of pollen limitation. This study highlights the need to estimate lifetime inbreeding depression, together with mating system and pollination parameters, if we are to understand the actual benefit of selfing and avoid the overestimation of reproductive assurance.

Microsatellite marker analysis reveals the complex phylogeographic history of Rhododendron ferrugineum (Ericaceae) in the Pyrenees.

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.

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.

Conservation genetics of the rare Pyreneo-Cantabrian endemic Aster pyrenaeus (Asteraceae).

BACKGROUND AND AIMS: Aster pyrenaeus (Asteraceae) is an endangered species, endemic to the Pyrenees and Cantabrian Mountain ranges (Spain). For its long-term persistence, this taxon needs an appropriate conservation strategy to be implemented. In this context, we studied the genetic structure over the entire geographical range of the species and then inferred the genetic relationships between populations. METHODOLOGY: Molecular diversity was analysed for 290 individuals from 12 populations in the Pyrenees and the Cantabrian Mountains using inter simple sequence repeats (ISSRs). Bayesian-based analysis was applied to examine population structure. PRINCIPAL RESULTS: Analysis of genetic similarity and diversity, based on 87 polymorphic ISSR markers, suggests that despite being small and isolated, populations have an intermediate genetic diversity level (P % = 52.8 %, H(E) = 0.21 ± 0.01, genetic similarity between individuals = 49.6 %). Genetic variation was mainly found within populations (80-84 %), independently of mountain ranges, whereas 16-18 % was found between populations and <5 % between mountain ranges. Analyses of molecular variance indicated that population differentiation was highly significant. However, no significant correlation was found between the genetic and geographical distances among populations (Rs = 0.359, P = 0.140). Geographical structure based on assignment tests identified five different gene pools that were independent of any particular structure in the landscape. CONCLUSIONS: The results suggest that population isolation is probably relatively recent, and that the outbreeding behaviour of the species maintains a high within-population genetic diversity. We assume that some long-distance dispersal, even among topographically remote populations, may be determinant for the pattern of genetic variation found in populations. Based on these findings, strategies are proposed for genetic conservation and management of the species.

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.

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.

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.