Genetic variability in microbial eukaryotes reshapes marine biodiversity assessment in the age of amplicon sequencing

PLOS One | https://doi.org/10.1371/journal.pone.0326053 June 20, 2025 26 / 29 assignment of each morpho-specimen are included, even those with <3 reads per specimen. The sheets containing extracted V4-regions of each ASV indicate which of the original long-read sequences had unique V4 regions ver- sus identical V4 matches with other sequences (causing their read counts to be merged in the occurrence chart). Each merged V4 sequence is labelled “merged” in the ASV_ID column; all other V4 ASVs retain the same IDs as their long-read counterparts. For the analyses in this study, ASVs with <3 reads in each specimen were removed to account for potential contamination (see “Materials and methods”), but the data in raw form is provided here to enable maximum flexibility in future use. (XLSX) S2 Data. P-distances quantifying genetic variation within and between specimens and morphospecies. The first sheet lists intra-specimen mean p-distances (number of base differences per site averaged across all sequence pairs comprising each specimen). P-distances between the groups of ASVs comprising each morphospecies are pro- vided in the matrices in second sheet of the dataset; calculations within each taxonomic order were computed sepa- rately. Likewise, intra-morphospecies and inter-morphospecies p-distances are given in the third and fourth sheets, respectively. When a specimen or morphospecies only yielded a single ASV, p-distance could not be calculated (“N/A” cells in the spreadsheets). Only specimens morphologically-classified to the species level were included in p-distance analyses.

(XLSX) S1 Table. List of accession numbers for all PR2 reference sequences included in the phylogenetic trees (Fig 8 and S4 Fig). (XLSX) Acknowledgments The authors would like to thank the Hawaiian Ocean Time-series (HOT) program for allowing ST to sail as a guest sci- entist and collect samples on cruises HOT 338 and HOT 339. We also thank Jennifer Beatty and Samantha Gleich for collecting Collodaria specimens for this study on SCOPE-PARAGON II. Author contributions Conceptualization: Sarah Trubovitz, David A. Caron. Data curation: Sarah Trubovitz. Formal analysis: Sarah Trubovitz, Miguel M. Sandin. Funding acquisition: Sarah Trubovitz, David A. Caron. Investigation: Sarah Trubovitz. Methodology: Sarah Trubovitz, Miguel M. Sandin. Project administration: David A. Caron. Resources: David A. Caron. Supervision: David A. Caron. Validation: Miguel M. Sandin. Visualization: Sarah Trubovitz, Miguel M. Sandin. Writing – original draft: Sarah Trubovitz. Writing – review & editing: Sarah Trubovitz, Miguel M. Sandin, David A. Caron.