Environmental filtering along a bathymetric gradient: A metabarcoding meta-analysis of free-living nematodes.

Identifying and understanding patterns of biological diversity is crucial at a time when even the most remote and pristine marine ecosystems are threatened by resource exploitation such as deep-seabed mining. Metabarcoding provides the means through which one can perform comprehensive investigations of diversity by examining entire assemblages simultaneously. Nematodes commonly represent the most abundant infaunal metazoan group in marine soft sediments. In this meta-analysis, we compiled all publicly available metabarcoding datasets targeting the 18S rRNA v1-v2 region from sediment samples to conduct a global-scale examination of nematode amplicon sequence variant (ASV) alpha diversity patterns and phylogenetic community structure at different depths and habitats. We found that nematode ASV richness followed a parabolic trend, increasing from the intertidal to the shelf, reaching a maximum in the bathyal and decreasing in the abyssal zone. No depth- or habitat-specific assemblages were identified as a large fraction of genera were shared. Contrastingly, the vast majority of ASVs were unique to each habitat and/or depth zone; genetic diversity was thus highly localized. Overwhelmingly, nematode ASVs in all habitats exhibited phylogenetic clustering, pointing to environmental filtering as the primary force defining community assembly rather than competitive interactions. This finding stresses the importance of habitat preservation for the maintenance of marine nematode diversity.

Phylogenetic clustering and rarity imply risk of local species extinction in prospective deep-sea mining areas of the Clarion-Clipperton Fracture Zone.

An understanding of the forces controlling community structure in the deep sea is essential at a time when its pristineness is threatened by polymetallic nodule mining. Because abiotically defined communities are more sensitive to environmental change, we applied occurrence- and phylogeny-based metrics to determine the importance of biotic versus abiotic structuring processes in nematodes, the most abundant invertebrate taxon of the Clarion-Clipperton Fracture Zone (CCFZ), an area targeted for mining. We investigated the prevalence of rarity and the explanatory power of environmental parameters with respect to phylogenetic diversity (PD). We found evidence for aggregation and phylogenetic clustering in nematode amplicon sequence variants (ASVs) and the dominant genus Acantholaimus, indicating the influence of environmental filtering, sympatric speciation, affinity for overlapping habitats and facilitation for community structure. PD was associated with abiotic variables such as total organic carbon, chloroplastic pigments equivalents and/or mud content, explaining up to 57% of the observed variability and providing further support of the prominence of environmental structuring forces. Rarity was high throughout, ranging from 64 to 75% unique ASVs. Communities defined by environmental filtering with a prevalence of rarity in the CCFZ suggest taxa of these nodule-bearing abyssal plains will be especially vulnerable to the risk of extinction brought about by the efforts to extract them.

Metabarcoding free-living marine nematodes using curated 18S and CO1 reference sequence databases for species-level taxonomic assignments.

High-throughput sequencing has the potential to describe biological communities with high efficiency yet comprehensive assessment of diversity with species-level resolution remains one of the most challenging aspects of metabarcoding studies. We investigated the utility of curated ribosomal and mitochondrial nematode reference sequence databases for determining phylum-specific species-level clustering thresholds. We compiled 438 ribosomal and 290 mitochondrial sequences which identified 99% and 94% as the species delineation clustering threshold, respectively. These thresholds were evaluated in HTS data from mock communities containing 39 nematode species as well as environmental samples from Vietnam. We compared the taxonomic description of the mocks generated by two read-merging and two clustering algorithms and the cluster-free Dada2 pipeline. Taxonomic assignment with the RDP classifier was assessed under different training sets. Our results showed that 36/39 mock nematode species were identified across the molecular markers (18S: 32, JB2: 19, JB3: 21) in UClust_ref OTUs at their respective clustering thresholds, outperforming UParse_denovo and the commonly used 97% similarity. Dada2 generated the most realistic number of ASVs (18S: 83, JB2: 75, JB3: 82), collectively identifying 30/39 mock species. The ribosomal marker outperformed the mitochondrial markers in terms of species and genus-level detections for both OTUs and ASVs. The number of taxonomic assignments of OTUs/ASVs was highest when the smallest reference database containing only nematode sequences was used and when sequences were truncated to the respective amplicon length. Overall, OTUs generated more species-level detections, which were, however, associated with higher error rates compared to ASVs. Genus-level assignments using ASVs exhibited higher accuracy and lower error rates compared to species-level assignments, suggesting that this is the most reliable pipeline for rapid assessment of alpha diversity from environmental samples.