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 25 / 29 S2 Fig. Heatmap illustrating ASV presence and abundance in all 51 Collosphaera huxleyi specimens analyzed in this study. Specimen names are shown on the y-axis, corresponding to specimen names in S1 Data and S2 Fig. The x-axis denotes individual ASVs (represented by tick marks) present in each specimen of the morphospecies. Relative read abundances of ASVs per specimen are indicated by the shade of blue (light = low abundance, dark = high abundance); ASVs not present in a particular specimen are indicated as white background. Representative micrographs of C. huxleyi are shown below the heatmap. (PDF) S3 Fig. Distribution of relative read abundances among the top 10 ranking V4-ASVs per specimen. Dots represent relative V4-ASV abundances (percent of total reads yielded by each specimen) at each designated rank (1st–10th) for all 173 specimens. Boxplots denote the median, interquartile range, maximum, and minimum values (outliers excluded) of V4-ASV relative abundances for each order (Collodaria = pink; Nassellaria = brown; Spumellaria = green). Data underlying this figure is in S1 Data. (PDF) S4 Fig. Expanded phylogenetic tree showing the dominant ASVs of all 173 specimens. Tip labels indicate the morphological taxonomy of specimens with a given dominant ASV, as well as the number of specimens that shared the same dominant ASV. ASV identifiers correspond with those in S1 Data. Polycystine radiolarian reference sequences

downloaded from PR2 are denoted with black text. Green text indicates new ASVs obtained from specimens in this study. Parameters used to generate this tree are described in “Materials and methods.” Accession numbers for the polycystine reference sequences and acantharian outgroup sequences can be found in S1 Table. (PDF) S5 Fig. Putative juvenile specimen morphologies across all polycystine Radiolaria orders, photographed live under darkfield and brightfield microscopy (shipboard dissecting scope; Leica MZAPO). Each box displays spec- imens that shared dominant ASVs and are presumably intraspecific, and/or have a dominant ASV that closely matches a reference sequence in PR2 or GenBank. Background colors indicate the polycystine order determined based on ASV taxonomic assignments (pink = Collodaria; brown = Nassellaria; green = Spumellaria). These solitary specimens lacked any siliceous skeletonization needed for morphological taxonomic classification, but molecular sequencing revealed taxonomic identities in all three orders. This morphotype is therefore hypothesized to be a previously-undescribed juvenile life stage that has similar characteristics in Collodaria, Nassellaria, and Spumellaria. (PDF) S6 Fig. Bioinformatic flowchart, summarizing each step of the data processing and analysis workflow used in this study. The purple box denotes the type of input data, blue boxes show intermediate steps in the workflow, and green boxes indicate the main results discussed in this study. (PDF)

S1 Data. Occurrence chart of all long-read and V4 region ASVs per specimen and their read abundances. There are individual sheets for Collodaria, Nassellaria, and Spumellaria specimens, titled either “full-length ASVs” or “V4-only” respectively. The first column of each sheet lists the ASV identifiers (as discussed in the text and displayed in figures). The second column gives the full ASV sequence. The third column, “Taxon,” provides the taxonomic assignment of each ASV based on PR2 reference taxonomy, and the “Confidence” column lists the confidence level of each molecular taxonomic assignment on a scale from 0–1. Subsequent columns give the specimen morphologi- cal taxonomic assignments with numeric identifiers (as referenced throughout in the manuscript text and in figures), and the absolute number of reads of each ASV produced by individual specimens. All ASVs that matched the family