An assessment of environmental metabarcoding protocols aiming at favouring contemporary biodiversity in inventories of deep-sea communities

1. The abyssal seafloor covers more than 50% of planet Earth and is a large reservoir of still mostly undescribed biodiversity. It is increasingly under target of resource-extraction industries although being drastically understudied. In such remote and hard-to-access ecosystems, environmental DNA (eDNA) metabarcoding is a useful and efficient tool for studying biodiversity and implementing environmental impact assessments. Yet, eDNA analysis outcomes may be biased towards describing past rather than present communities as sediments contain both contemporary and ancient DNA. 2. Using commercially available kits, we investigated the impacts of five molecular processing methods on DNA metabarcoding biodiversity inventories targeting prokaryotes (16S-V4V5), unicellular eukaryotes (18S-V4), and metazoans (18S-V1, COI). As the size distribution of ancient DNA is skewed towards small fragments, we evaluated the effect of removing short DNA fragments via size-selection and ethanol reconcentration using DNA extracted from 10 g of sediment at five deep-sea sites. We also compare communities revealed by DNA and RNA co-extracted from 2 g of sediment at the same sites. 3. Results show that removing short DNA fragments does not affect alpha and beta diversity estimates in any of the biological compartments investigated. Results also confirm doubts regarding the possibility to better describe live communities using environmental RNA (eRNA). With ribosomal loci, RNA, while resolving similar spatial patterns than co-extracted DNA, resulted in significantly higher richness estimates, supporting hypotheses of increased persistence of ribosomal RNA (rRNA) in the environment and unmeasured bias due to over-abundance of rRNA and RNA release. With the mitochondrial locus, RNA detected lower metazoan richness and resolved less spatial patterns than co-extracted DNA, reflecting high messenger RNA lability. Results also highlight the importance of using large amounts of sediment (≥10 g) for accurately surveying eukaryotic diversity. 4. We conclude that DNA should be favoured over RNA for logistically realistic, repeatable, and reliable surveys, and confirm that large sediment samples (≥10 g) deliver more complete and accurate assessments of benthic eukaryotic biodiversity and that increasing the number of biological rather than technical replicates is important to infer robust ecological patterns.