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 24 / 29 due to their lack of sufficient reads (<5 reads) belonging to taxonomically-appropriate ASVs. It is interpreted that any apparent amplification was due to off-target environmental DNA that may have come into contact with the specimen prior to or during sample collection, and the target DNA coincidentally failed to properly extract or amplify from the specimen. A similarly-designed study [28] found that environmental contamination can contribute exogenous sequence data when amplifying isolated radiolarian cells, even after cleaning and prey digestion. Thus, while we made our best efforts to remove contamination from every specimen in this study via stringent sample preparation and bioinformatic filtering by both read abundance and ASV taxonomic assign- ment (described above), we cannot absolutely guarantee that every ASV we document here originated from within the target specimen. To compare the genetic richness information provided by our long-read ASVs with commonly used V4 hypervariable region ASVs, we extracted V4 sequences from each full-length ASV. V4 regions were identified by aligning all sequences with MAFFT [74] using lingsi parameters, then searching for matches to the general eukaryotic primers TAR-EukF1 (5’-CCAGCASCYGCGGTAATTCC-3’)/ TAR-EukR3 (5’-ACTTTCGTTCTTGATYRA-3’) [76] using AliView software [68]. ASVs that had identical V4 regions were merged for the purpose of building V4-only occurrence charts (S1 Data; sheets

4–6) and exploring V4-only intragenomic variation (Fig 1, e–h) and intra- and inter-species variation (Fig 7b). After merg- ing identical V4 sequences, there was a total of 604 unique V4 sequences in the dataset; 417 assigned to Collodaria, 70 to Nassellaria, and 117 to Spumellaria. We computed p-distances within and between specimens and morphospecies to provide quantitative metrics of intrag- enomic and intraspecies genetic variation. P-distance is the proportion of sites that are different between two aligned sequences being compared. We used MEGA (Molecular Evolutionary Genetic Analysis) 12 software [50] to calculate the mean p-distances within and between groups of ASVs belonging to individual specimens, and comprising each morphos- pecies. The MEGA analysis option “Compute within group mean distance” was used to obtain average intragenomic and intraspecies p-distances within each polycystine order, and the option “Compute between group mean distance” provided average genetic distances between specimens and morphospecies (Fig 7; S2 Data). Distance calculations were per- formed separately on full-length ASVs and V4-region ASVs for comparison. Only specimens that were morphologically- identified to the species level were included in p-distance calculations, to avoid mixing unequal taxonomic levels in the same analysis. ASV richness, presence, and abundance patterns within specimens, morphospecies, and orders were assessed and visualized in R (v4.3.1, “Beagle Scouts”) [77] using the dplyr [78] and ggplot2 [79] packages. ASVs for each of the 173

specimens were ranked based on decreasing relative abundance per specimen in base R. The most abundant ASV in each specimen was used to construct a maximum likelihood phylogenetic tree in RAxML-NG using the same parameters as the initial tree described above, and was visualized in FigTree v1.4.4 [80] (Fig 8; S4 Fig). This phylogenetic tree was used to infer relationships among specimens collected for this study with previously-published radiolarian sequences as a framework. A flowchart summarizing all bioinformatic processing and analytical steps is given in S6 Fig. Supporting information S1 Fig. Plates of all 173 isolated specimens used in analyses: living and skeletal. Photos taken of live specimens in darkfield and brightfield with the shipboard dissecting scope have been cropped and adjusted for white balance and con- trast when necessary. Photos taken of skeletal remains with the compound microscope are unedited. Specimens collected on the SCOPE-PARAGON II cruise were not photographed live, so only photos of skeletal remains are included. Other specimens that only have one photo either could not be photographed on board the ship due to high rocking motion, or they broke beyond recognition during/after DNA extraction and could only be identified from their live photos. (PDF)