Eeva M. Soininen

Analysing diet of small herbivores: the efficiency of DNA barcoding coupled with high-throughput pyrosequencing for deciphering the composition of complex plant mixtures

BackgroundIn order to understand the role of herbivores in trophic webs, it is essential to know what they feed on. Diet analysis is, however, a challenge in many small herbivores with a secretive life style. In this paper, we compare novel (high-throughput pyrosequencing) DNA barcoding technology for plant mixture with traditional microhistological method. We analysed stomach contents of two ecologically important subarctic vole species, Microtus oeconomus and Myodes rufocanus, with the two methods. DNA barcoding was conducted using the P6-loop of the chloroplast trn L (UAA) intron.ResultsAlthough the identified plant taxa in the diets matched relatively well between the two methods, DNA barcoding gave by far taxonomically more detailed results. Quantitative comparison of results was difficult, mainly due to low taxonomic resolution of the microhistological method, which also in part explained discrepancies between the methods. Other discrepancies were likely due to biases mostly in the microhistological analysis.ConclusionWe conclude that DNA barcoding opens up for new possibilities in the study of plant-herbivore interactions, giving a detailed and relatively unbiased picture of food utilization of herbivores.

Highly overlapping winter diet in two sympatric lemming species revealed by DNA metabarcoding

Sympatric species are expected to minimize competition by partitioning resources, especially when these are limited. Herbivores inhabiting the High Arctic in winter are a prime example of a situation where food availability is anticipated to be low, and thus reduced diet overlap is expected. We present here the first assessment of diet overlap of high arctic lemmings during winter based on DNA metabarcoding of feces. In contrast to previous analyses based on microhistology, we found that the diets of both collared (Dicrostonyx groenlandicus) and brown lemmings (Lemmus trimucronatus) on Bylot Island were dominated by Salix while mosses, which were significantly consumed only by the brown lemming, were a relatively minor food item. The most abundant plant taxon, Cassiope tetragona, which alone composes more than 50% of the available plant biomass, was not detected in feces and can thus be considered to be non-food. Most plant taxa that were identified as food items were consumed in proportion to their availability and none were clearly selected for. The resulting high diet overlap, together with a lack of habitat segregation, indicates a high potential for resource competition between the two lemming species. However, Salix is abundant in the winter habitats of lemmings on Bylot Island and the non-Salix portion of the diets differed between the two species. Also, lemming grazing impact on vegetation during winter in the study area is negligible. Hence, it seems likely that the high potential for resource competition predicted between these two species did not translate into actual competition. This illustrates that even in environments with low primary productivity food resources do not necessarily generate strong competition among herbivores.

Shedding new light on the diet of Norwegian lemmings: DNA metabarcoding of stomach content

Lemmings are key herbivores in many arctic food webs, and their population dynamics have major impacts on the functioning of tundra systems. However, current knowledge of lemming diet is limited, hampering evaluation of lemming–vegetation interactions. This lack of knowledge is mainly due to methodological challenges, as previously used microhistological methods result in large proportions of poorly resolved plant taxa. We analyzed diets of Norwegian lemmings (Lemmus lemmus) in three different habitats using a new method, DNA metabarcoding of stomach contents. To achieve detailed information on ingested vascular plants, bryophytes, and fungi, we amplified short fragments of chloroplast DNA (for plants; P6 loop of the trnL intron) and nuclear ribosomal DNA (for fungi; ITS1-region). Our results revealed that lemming diets were dominated by grasses, mainly Avenella flexuosa, and mosses, mainly Dicranum spp., but that a variety of other food items were also eaten. Vascular plant composition of the diets differed between heath, meadow, and wetland habitats, whereas bryophyte composition did not. Also, a variety of fungal taxa were retrieved, but as most of the identified taxa belong to micromycetes, they were unlikely to be consumed as food. The role of fungi in the diet of lemmings remains to be investigated. We suggest that there may be substantial variation between habitats and regions in lemming diet.

Arctic Small Rodents Have Diverse Diets and Flexible Food Selection

The ecology of small rodent food selection is poorly understood, as mammalian herbivore food selection theory has mainly been developed by studying ungulates. Especially, the effect of food availability on food selection in natural habitats where a range of food items are available is unknown. We studied diets and selectivity of grey-sided voles (Myodes rufocanus) and tundra voles (Microtus oeconomus), key herbivores in European tundra ecosystems, using DNA metabarcoding, a novel method enabling taxonomically detailed diet studies. In order to cover the range of food availabilities present in the wild, we employed a large-scale study design for sampling data on food availability and vole diets. Both vole species had ingested a range of plant species and selected particularly forbs and grasses. Grey-sided voles also selected ericoid shrubs and tundra voles willows. Availability of a food item rarely affected its utilization directly, although seasonal changes of diets and selection suggest that these are positively correlated with availability. Moreover, diets and selectivity were affected by availability of alternative food items. These results show that the focal sub-arctic voles have diverse diets and flexible food preferences and rarely compensate low availability of a food item with increased searching effort. Diet diversity itself is likely to be an important trait and has previously been underrated owing to methodological constraints. We suggest that the roles of alternative food item availability and search time limitations for small rodent feeding ecology should be investigated. Nomenclature Annotated Checklist of the Panarctic Flora (PAF), Vascular plants. Available at: http://nhm2.uio.no/paf/, accessed 15.6.2012.

Sources of variation in small rodent trophic niche: new insights from DNA metabarcoding and stable isotope analysis

Intraspecific competition for food is expected to increase the trophic niche width of consumers, defined here as their diet diversity, but this process has been little studied in herbivores. Population densities of small rodents fluctuate greatly, providing a good study model to evaluate effects of competition on trophic niche. We studied resource use in five arctic small rodent populations of four species combining DNA metabarcoding of stomach contents and stable isotope analysis (SIA). Our results suggest that for small rodents, the most pronounced effect of competition on trophic niche is due to increased use of secondary habitats and to habitat-specific diets, rather than an expansion of trophic niche in primary habitat. DNA metabarcoding and SIA provided complementary information about the composition and temporal variation of herbivore diets. Combing these two approaches requires caution, as the underlying processes causing observed patterns may differ between methodologies due to different spatiotemporal scales.

Determination of plant silicon content with near infrared reflectance spectroscopy.

Silicon (Si) is one of the most common elements in the earth bedrock, and its continental cycle is strongly biologically controlled. Yet, research on the biogeochemical cycle of Si in ecosystems is hampered by the time and cost associated with the currently used chemical analysis methods. Here, we assessed the suitability of Near Infrared Reflectance Spectroscopy (NIRS) for measuring Si content in plant tissues. NIR spectra depend on the characteristics of the present bonds between H and N, C and O, which can be calibrated against concentrations of various compounds. Because Si in plants always occurs as hydrated condensates of orthosilicic acid (Si(OH)4), linked to organic biomolecules, we hypothesized that NIRS is suitable for measuring Si content in plants across a range of plant species. We based our testing on 442 samples of 29 plant species belonging to a range of growth forms. We calibrated the NIRS method against a well-established plant Si analysis method by using partial least-squares regression. Si concentrations ranged from detection limit (0.24 ppmSi) to 7.8% Si on dry weight and were well predicted by NIRS. The model fit with validation data was good across all plant species (n = 141, R2 = 0.90, RMSEP = 0.24), but improved when only graminoids were modeled (n = 66, R2 = 0.95, RMSEP = 0.10). A species specific model for the grass Deschampsia cespitosa showed even slightly better results than the model for all graminoids (n = 16, R2 = 0.93, RMSEP = 0.015). We show for the first time that NIRS is applicable for determining plant Si concentration across a range of plant species and growth forms, and represents a time- and cost-effective alternative to the chemical Si analysis methods. As NIRS can be applied concurrently to a range of plant organic constituents, it opens up unprecedented research possibilities for studying interrelations between Si and other plant compounds in vegetation, and for addressing the role of Si in ecosystems across a range of Si research domains.

Shrub patch configuration at the landscape scale is related to diversity of adjacent herbaceous vegetation

Background: Patterns of shrub patches reflect the long-term effects of several environmental drivers; the same drivers may also affect adjacent herbaceous vegetation. The identity of such drivers may be indicated by associating patterns of shrubs and adjacent vegetation. However, such associations have rarely been examined. Aim: Assess the hypothesis that configuration of shrub patches is related to the diversity of adjacent herbaceous vegetation, through the impact of variation in herbivory and/or growing conditions. Methods: Multivariate analyses of the relationship between patch configuration of tall shrubs and diversity of adjacent herbaceous vegetation, across two tundra river catchments in Norway. Results: Configuration of shrub patches was clearly related to α and β diversity of adjacent herbaceous vegetation, independent of concurrent herbivore abundance (estimated from faeces during the study), or growing conditions. Highly-shredded shrub patches (i.e. fragmented patches with high edge density) were associated with grazing-tolerant species and low species richness. Conversely, little-shredded tall shrub patches were associated with grazing-sensitive shrub saplings. This indicates change in herbivory on a timescale longer than that covered by our faeces data. Conclusions: Consistent co-variation of shrub and herbaceous patches in tall shrub tundra supports the hypothesis of impacts of a shared driver. Interpretation of this co-variation points to long-term influence of herbivory as the shared driver.

Food selection by barnacle geese (Branta leucopsis) in an Arctic pre-breeding area

Detailed patterns of food selection by pre-breeding barnacle geese (Branta leucopsis) were investigated in Vårsolbukta, western Spitsbergen, Svalbard, in moss-dominated vegetation. This habitat is favoured by geese during the early Arctic spring when grass abundance is low. Grass is more profitable food than moss in terms of nutrient content and digestibility, and a five-fold higher proportion of grass in geese faeces compared with other vegetation indicated that geese selected grass in spite of its low availability compared with moss. As profitability may also depend on various properties that enhance searching efficiency, we studied the effects of grass tiller size, density and patchiness on goose selectivity in an experiment comparing ungrazed vegetation with vegetation where geese were allowed to feed for controlled periods of time. Large (two-leaved) tillers provided more biomass than small (one-leaved) tillers. The abundance of ungrazed large tillers relative to small tillers was lower in the grazed plots than in the ungrazed control plots, indicating that the geese preferred large tillers. Grass tiller density or spatial tiller distribution did not affect the degree of selectivity of geese for grass. Thus, we suggest that the feeding strategy of the geese in the early Arctic spring is primarily driven by the degree of tiller conspicuousness, as determined by tiller size. Furthermore, we suggest that an intense time allocation to feeding and an enhanced quality of grasses compared with mosses enabled geese to profit from feeding on the scarce grasses.

Complementary use of density estimates and hunting statistics: different sides of the same story?

The use of hunting statistics for population monitoring has often been criticized because such data are affected by several sources of error. Still, for many harvested populations, hunting statistics are the only available data source and cautious use of such data may be valuable for management. Here we assessed to what extent long-term monitoring of Svalbard rock ptarmigan spring densities and hunting statistics (bag size and proportion of juveniles in the hunting bag) reflect similar population fluctuations. We found a decreasing trend in both bag size and proportion of juveniles in the bag, but no trend in ptarmigan spring densities. However, annual fluctuations of ptarmigan spring density and bag size were correlated. Together, these time series indicate that both population abundance (bag size) and recruitment (proportion of juveniles in the bag) are decreasing, but the reproductive component fraction (density of territorial males) is not yet compromised. This biological interpretation remains, however, uncertain due to lack of hunting effort data. Monitoring programs using hunting statistics should therefore critically discuss and evaluate what the hunting statistics reflect and fine-tune the hunter data collection to obtain maximum biological relevance. Still, our results illustrate that the combination of population estimates and hunting statistics can provide more nuanced information about the population status than the density estimates alone.

Not only mosses: lemming winter diets as described by DNA metabarcoding

The temporal dynamics of most tundra food webs are shaped by the cyclic population dynamics of lemmings. While processes during winter may be behind the recent disruptions of lemming cycles, lemming winter ecology is poorly known. We present here the first DNA metabarcoding data on the winter diet of Norwegian lemmings (Lemmus lemmus), based on feces collected after a winter of population increase. Prostrate willows, mosses, and graminoids dominated the species winter diet, indicating that the conventional idea of lemmings as moss-specialists should be revised. The behavior of lemming-plant models in theoretical studies is conditional on the assumptions of mosses being their main winter food item. As shrubs have been excluded from the framework of these models, incorporating them in future modeling studies should nuance our understanding on how plants affect lemmings. We also sampled diet of a few individuals found dead on top of the snow. These individuals had relatively empty stomachs and had, prior to death, relied heavily on mosses. This apparent lack of abundant good quality indicates spatial heterogeneity in local food availability during the population increase phase.