Multi-marker metabarcoding for tardigrade diversity studies in temperate deciduous forests
Lasse Topstad1, Roberto Guidetti2, Markus Majaneva1, Aina M. Aspaas1 & Torbjørn Ekrem1
1Department of Natural History, NTNU University Museum, Trondheim, Norway.
2Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy
Was: "Tardigrade diversity assessment using morphology, DNA barcoding and multi-marker metabarcoding"
•Large scale assessments of microscopic invertebrates using metabarcoding are now more common
•These methods have so far not been evaluated for their applicability and proficiency on the phylum Tardigrada.
•Our study attempts to fill this gap by comparing data obtained through metabarcoding and traditional sampling. Furthermore, we will assess the two methods’ ability to capture tardigrade alpha- and beta diversity between different microhabitats
1.Samples of moss, lichen and litter
2.Samples homogenized and subsampled for traditional morphology, and for eDNA extraction with subsequent metabarcoding using three markers (COI universal, COI specific and 18S specific)
3.Bioinformatic pipeline in vsearch, swarm, mothur and blastn with downstream analysis in R
Metabarcoding consistently captured equal or higher diversity of tardigrade species (MOTUs matching reference sequences above 97% similarity) than traditional sampling methods. The difference in assemblage of species inhabiting moss, lichen and litter showed similar patterns for both methods, but metabarcoding contained higher variance.
•The MDS plot of the metabarcode data includes cryptic species and MOTUs assigned to «Tardigrada». Thus, with lower taxonomic resolution, the lower goodness of fit is explained.
•Metabarcoding of eDNA successfully amplifies a wide range of tardigrade species. We detected representatives of most major families of terrestrial tardigrades, indicating that metabarcoding is a suitable tool for documentation of diversity in this group, even if sequences cannot be matched to species in a reference library.
•The discrepancy in species detection among the markers, as well as between traditional and eDNA methods, could be due to PCR bias, but is more likely the result of incomplete reference databases.