Characterizing the diet of large herbivores and the determinants of its variation remains a difficult task in wild species. DNA-based techniques have the potential to complement traditional time-consuming methods based on the microhistology of plant cuticle fragments in fecal or rumen samples. Recently, it has been shown that a short chloroplast DNA fragment, the P6 loop of the trnL (UAA) intron, can act as a minimalist barcode. Here, we used the trnL approach with high-throughput pyrosequencing to study diet from feces in a wild herbivore, the alpine chamois (Rupicapra rupicapra) and showed that the fine resolution in plant determination obtained with this method allows exploring subtle temporal shifts and inter-individual variability in diet composition. First, we built a DNA barcoding database of 475 plants species. Seventy-two percent of plant species can be unambiguously identified to species level, 79% to genus level and 100% to family level using the P6 loop. Second, we analysed 74 feces collected from October to November. Based on 47,896 P6 loop sequences, we identified a total of 110 taxa, 96 in October and 76 in November, with a clear diet shift between October and November. We recognized four and two clusters of feces composition in October and November, respectively, revealing different diet categories among individuals within each month. DNA-based diet analysis is faster and more taxonomically precise than studies based on microhistology, and opens new possibilities for analysing plant-herbivore interactions in the wild.

The "niche variation hypothesis" (NVH) predicts that populations with wider niches should display higher among-individual variability. This prediction originally stated at the intra-specific level may be extended to the inter-specific level: individuals of generalist species may differ to a greater extent than individuals of a specialist species. We tested the NVH at intra- and inter-specific levels based on a large diet database of three large herbivore feces collected in the field and analyzed using DNA metabarcoding. The three herbivores (roe deer Capreolus capreolus, chamois Rupicapra rupicapra and mouflon Ovis musimon) are highly contrasted in terms of sociality (solitary to highly gregarious) and diet. The NVH at the intraspecific level was tested by relating, for the same population, diet breadth and inter-individual variation across the four seasons. Compared to null models, our data supported the NVH both at the intra- and inter-specific levels. Inter-individual variation of the diet of solitary species was not larger than in social species, although social individuals feed together and could therefore have more similar diets. Hence, the NVH better explained diet breadth than other factors such as sociality. The expansion of the population niche of the three species was driven by resource availability, and achieved by an increase in inter-individual variation, and the level of inter-individual variability was larger in the generalist species (mouflon) than in the specialist one (roe deer). This mechanism at the base of the NVH appears at play at different levels of biological organization, from populations to communities.

Species diversity in large herbivore communities is often explained by niche segregation allowed by differences in body mass and digestive morphophysiological features. Based on large number of gut samples in fall and winter, we analysed the temporal dynamics of diet composition, quality and interspecific overlap of 4 coexisting mountain herbivores. We tested whether the relative consumption of grass and browse differed among species of different rumen types (moose-type and intermediate-type), whether diet was of lower quality for the largest species, whether we could identify plant species which determined diet quality, and whether these plants, which could be “key-food-resources” were similar for all herbivores. Our analyses revealed that (1) body mass and rumen types were overall poor predictors of diet composition and quality, although the roe deer, a species with a moose-type rumen was confirmed as an “obligatory non grazer”, while red deer, the largest species, had the most lignified diet; (2) diet overlap among herbivores was well predicted by rumen type (high among species of intermediate types only), when measured over broad plant groups, (3) the relationship between diet composition and quality differed among herbivore species, and the actual plant species used during winter which determined the diet quality, was herbivore species-specific. Even if diets overlapped to a great extent, the species-specific relationships between diet composition and quality suggest that herbivores may select different plant species within similar plant group types, or different plant parts and that this, along with other behavioural mechanisms of ecological niche segregation, may contribute to the coexistence of large herbivores of relatively similar body mass, as observed in mountain ecosystems.

Question: Resources quality and quantity are both important determinants of habitat use for large herbivores. We aim to understand how these two variables vary throughout the growing season in sub-alpine grasslands. How do productivity and phenology (quality) of different plant communities within a landscape vary over time? Do productivity and phenology co-vary? Which environmental constraints or growth form composition best explain differences in productivity and phenology among plant communities?Location: Calcareous sub-alpine grasslands, the external Alps, France.Methods: We assessed how productivity (above-ground biomass) and phenology varied with date, slope, radiation and altitude among seven plant communities. Then we explored (1) co-variation among maximum biomass, the date of maximum biomass and average community flowering date for each plant community, and (2) whether these variables were related to snow regime or growth form composition.Results: Temporal dynamics of biomass and phenology varied markedly among plant communities. More productive communities reached their maximum biomass later. Flowering occurred after the biomass peak, except for one plant community. However, the later the biomass peak, the shorter the lag to flowering peak. The timing of flowering and date of maximum biomass were best explained by differences in snow regimes among communities. The impact of growth form composition was marginal.Conclusions: Plant communities are under strong time constraints to grow and flower. Snowmelt regime plays a crucial role at one end of the growing season, while time left for fruiting and maturing is likely to influence the other end. Sub-alpine grassland communities are highly heterogeneous in productivity, timing of maximum productivity and flowering phenology, creating small-scale variability in the forage quality and quantity available to large herbivores.

We reviewed data on the diets of mouflon (Mediterranean island populations Ovis gmelini musimon and introduced hybridized populations Ovis gmelini musimon × Ovis sp.) from 33 field studies (comprising 51 independent data points suitable for analysis) to detect general patterns in the botanical composition of the diet and identify ecological factors explaining its variation. We expected mouflon, generally classified as grazers, to include botanical entities other than grass in their diet, especially when they are forced to do so by low resource availability, and in certain seasons. Diet composition was investigated based on samples of rumen content and faeces. We combined these data with environmental characteristics at each site using a co‐inertia analysis. As expected, grass often constituted the highest proportion in the diet (in 28 of the 51 data points) and represented on average 35% (range = 0–91%) of mouflon diet, confirming the importance of this food for the species. However, referring strictly to commonly used thresholds (>75% or >90%) shows that the classification of mouflon as grazers could be questioned. Indeed, forbs and shrubs constituted 24% (range: 0–93%) and 16% (range: 0–55%) of their diet, respectively, so that mouflon should at least be considered as variable grazers. Forbs represented a high percentage of the overall diet in the Kerguelen Archipelago, southern Indian Ocean (autumn and winter: 73%) and Teide National Park, Canary Islands, Spain (autumn and winter: 83%), whereas shrubs represented a high proportion of the overall diet in Mediterranean areas (19%). Diet composition varied with spatio‐temporal variation in forage availability (documented as habitat related or seasonal variation), confirming that mouflon are able to feed on a large variety of plants. Further investigations concerning both digestive morphology and consequences of the inclusion of browse in the diet on population dynamics of mouflon are needed to understand the persistence of this species over a wide range of habitats despite a potential mismatch between its digestive ability and its observed diet.

It is a challenge to find effective methods to estimate biomass over a large range of biomass values in diverse plant communities, such as typically found in mountain grasslands. We compared the performance of three non-destructive methods for estimating plant biomass (3D quadrat: a point quadrat method, plate meter: a measure of physical volume, and visual estimation: a component of the BOTANAL method) in mountain grasslands. We tested whether: (1) all methods performed equally in terms of linearity of estimations over a large range of biomass and (2) under and over-estimations of biomass were related to specific plant compositions. We estimated plant biomass in 30 plots, for which real plant biomass was measured by destructive sampling. We accounted for the significant effect of heteroscedasticity (which was significant for all three methods) when testing for the linearity of the relationship between real biomass and biomass estimates. The plate meter displayed non-linearity, being insensitive to variation of biomass at low biomass values. BOTANAL and the 3D quadrat yielded linear relationships, with BOTANAL providing better estimates of real biomass (greater R 2 ). Specific floristic compositions (e.g. presence of Deschampsia cespitosa, Chaerophyllum sp., and abundant small forbs) explained underestimation and overestimation of biomass estimates for the plate meter and 3D quadrat while BOTANAL was insensitive to floristic composition. In heterogeneous grasslands, BOTANAL appeared to be the most appropriate method, given its linear relationship with real biomass over the whole range of biomass and its low residual variation.

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