Unveiling the Diet of Elusive Rainforest Herbivores in Next Generation Sequencing Era? The Tapir as a Case Study

Abstract

Characterizing the trophic relationships between large herbivores and the outstanding plant diversity in rainforest is a major challenge because of their elusiveness. This is crucial to understand the role of these herbivores in the functioning of the rainforest ecosystems. We tested a non-invasive approach based on the high-throughput sequencing of environmental samples using small plant plastid sequences (the trnL P6 loop) and ribosomal ITS1 primers, referred to as DNA metabarcoding, to investigate the diet of the largest neotropical herbivore, the lowland tapir. Sequencing was performed on plant DNA extracted from tapir faeces collected at the Nouragues station, a protected area of French Guiana. In spite of a limited sampling, our approach reliably provided information about the lowland tapir's diet at this site. Indeed, 95.1% and 74.4% of the plant families and genera identified thanks to the trnL P6 loop, respectively, matched with taxa already known to be consumed by tapirs. With this approach we were able to show that two families and eight new genera are also consumed by the lowland tapir. The taxonomic resolution of this method is limited to the plant family and genera. Complementary barcodes, such as a small portion of ITS1, can be used to efficiently narrow identifications down to the species in some problematic families. We will discuss the remaining limitations of this approach and how useful it is at this stage to unravel the diet of elusive rainforest herbivores and better understand their role as engineers of the ecosystem.

Methods for the extraction, storage, amplification and sequencing of DNA from environmental samples

“…Several of the soil-based DNA metabarcoding studies targeted earthworms (Bienert et al 2012;Ficotela et al 2015;Pansu et al 2015a) or large vertebrates . Gut and faecal material, collected in 5% of the studies, has been used to analyse the diets of herbivores (Hibert et al 2013), carnivores and carrion feeders (Calvignac-Spencer et al 2013). Several of the 167 DNA metabarcoding studies analysed pools of invertebrate specimens collected in malaise traps (Yu et al 2012;Ji et al 2013;Liu et al 2013;Yang et al 2014) or from soil samples , while others targeted invertebrate DNA extracted directly from soil (McGee & Eaton 2015) or aquatic habitats (Pochon et al 2013;Cowart et al 2015).…”

“…A majority of studies of plant DNA were based on the trnL UAA intron within the chloroplast transfer RNA, or tRNA gene (e.g. Hibert et al 2013;Parducci et al 2013;Pedersen et al 2013). The DNA of arthropods was most commonly targeted by amplification of mitochondrial cytochrome c oxidase subunit I (COI) DNA (e.g.…”

Faeces of generalist predators as ‘biodiversity capsules’: A new tool for biodiversity assessment in remote and inaccessible habitats

“…8 using gut or faecal DNA in parasites (Schnell et al, 2012), herbivores (Hibert et al, 2013) or detritivores (Calvignac-Spencer et al, 2013b), the estimation of abundance is much more difficult in these cases because many individuals often feed on the same individual prey (or host) without necessarily causing its death. This means that although presence or absence of the prey (or host) species can be accurately estimated (Calvignac-Spencer et al, 2013a), the occurrence of prey DNA in the faeces of parasites, herbivores or detritivores (i.e.…”

Dropping Hints: Estimating the diets of livestock in rangelands using DNA metabarcoding of faeces

“…Annotated sections were then removed, leaving only the ITS2 sequence. Contigs with a read depth of less than 10 were excluded in both genes in the main analysis, following the 10-minimum read depth protocol of Hibert et al (2013) and Gebremedhin et al (2016). We also carried out a supplementary analysis with all the same conditions but at a minimum read depth of 3.…”

“…These new methods have been found to recover greater diversity in faecal samples than traditional ones (Soininen et al 2009). Most studies of diet from faecal material have focused on carnivores , Shehzad et al 2012, however, several studies have shown that the technique can work in herbivores (Soininen et al 2009, Hibert et al 2013, Symondson and Harwood 2014. In this study, we use faecal DNA barcoding analysis to characterize the diets of economically and ecologically significant Dorper and Merino sheep (Ovis aries) and the goat (Capra aegagrus hircus).…”