eDNA metabarcoding shows highly diverse but distinct shallow, mid-water, and deep-water eukaryotic communities within a marine biodiversity hotspot

As the impact of human activities continues to move beyond shallow coastal waters into deeper ocean layers, it is fundamental to describe how diverse and distinct the eukaryotic assemblages from the deep layers are compared to shallow ecosystems. Environmental DNA (eDNA) metabarcoding has emerged as a molecular tool that can overcome many logistical barriers in exploring remote deep ocean areas. We analyzed shallow water samples (<30 m) collected via SCUBA diving and adjacent deeper samples (mid-water 30-150 m, deep-water >200 m) obtained with Niskin samplers within 16 locations in a recognized hotspot of marine biodiversity (Gulf of California, Mexico). We sequenced an eDNA metabarcoding library targeting a fragment of the COI gene of eukaryotes. We demonstrated that the diversity of operational taxonomic units (OTUs) did not peak at shallow coastal regions and that the mid-water and deep-water benthic and pelagic samples had similar levels of biodiversity compared to shallow sites, but detected a significant vertical zonation between shallow and deeper habitats. Our results suggest that the deep refugia hypothesis, which posits that deep environments protect biodiversity during environmental changes, enabling species to survive and repopulate shallower regions, is not supported for most taxa and only applies to about a third (34.9%) of the 5,495 OTUs identified that were shared between the shallow and deeper layers. In comparison, the rest of the taxa were exclusive to either shallow (30.8%) or deeper zones (34.28%). The observation that mid-water and deep-water benthic and pelagic communities were as rich but quite distinct as shallow communities supports extending spatial management and conservation tools to deeper habitats to include a significant fraction of unique phylogenetic and functional diversity.