Assigning the unassigned: A signature-based classification of rDNA metabarcodes reveals new deep-sea diversity

Environmental DNA metabarcoding reveals a vast genetic diversity of marine eukaryotes.
Yet, most of the metabarcoding data remain unassigned due to the paucity of reference
databases. This is particularly true for the deep-sea meiofauna and eukaryotic microbiota,
whose hidden diversity is largely unexplored. Here, we tackle this issue by using unique
DNA signatures to classify unknown metabarcodes assigned to deep-sea foraminifera. We
analyzed metabarcoding data obtained from 311 deep-sea sediment samples collected in
the Clarion-Clipperton Fracture Zone, an area of potential polymetallic nodule exploitation in
the Eastern Pacific Ocean. Using the signatures designed in the 37F hypervariable region of
the 18S rRNA gene, we were able to classify 802 unassigned metabarcodes into 61 novel
lineages, which have been placed in 27 phylogenetic clades. The comparison of new lineages with other foraminiferal datasets shows that most novel lineages are widely distributed
in the deep sea. Five lineages are also present in the shallow-water datasets; however, phylogenetic analysis of these lineages separates deep-sea and shallow-water metabarcodes
except in one case. While the signature-based classification does not solve the problem of
gaps in reference databases, this taxonomy-free approach provides insight into the distribution and ecology of deep-sea species represented by unassigned metabarcodes, which
could be useful in future applications of metabarcoding for environmental monitoring.