Evaluating 18S Phylogenetic Placement Accuracy to Uncover Hidden Diversity in Non‐Bilaterian Animals

ABSTRACT

Small subunit ribosomal RNA (SSU rRNA)—18S in eukaryotes—is a universally present gene that was central in resolving ancient relationships in early molecular phylogenies. Nowadays, despite multi‐locus phylogenomics dominating, SSU rRNA sequences still serve as a molecular identifier of biodiversity. Phylogenetic placement uses a reference phylogeny and a given evolutionary model to annotate taxonomically environmental DNA, enabling a more accurate identification of divergent sequences that may belong to unknown lineages. Here, we first created the largest dataset of 18S sequences belonging to non‐Bilateria metazoans by curating the sequences found in PR2 database. We then used it as a case study to test the performance of common 18S‐based barcodes (V4, V9, and full‐length 18S) within a phylogenetic placement framework. We found that the V9 region generally lacks sufficient phylogenetic signal for reliable placements. The V4 region is accurate when the environmental diversity is represented in the reference tree but struggles with divergent lineages. Full‐length 18S overcomes short‐read limitations and emerges as the most robust option to uncover major clades. Finally, we apply phylogenetic placement to empirical environmental 18S data. We observe geographical variation in non‐bilaterians communities and recover a putative clade sister to Ctenophora based on long‐sequence barcodes.