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 9 / 29 shared ASVs at all, suggesting separate genetic subgroups that may reflect cryptic species (Table 1). Polycystine mor- phospecies therefore cannot be easily delimited based on their 18S rDNA sequences alone, because each one contains uncharted genetic richness and an unknown number of genetic subgroups with different ASV compositions. One way to begin to untangle the degree of genetic differentiation in polycystine morphospecies is to compare the genetic distance within species to the genetic distance between species. Characterizing these genetic distance thresholds could provide an approach for estimating species richness in environmental metabarcoding studies, when morphological species counts are not possible. We quantified radiolarian intragenomic, intraspecies, and interspe- cies genetic variation by computing mean p-distances (calculated in MEGA 12 [50]). P-distance is the proportion of sites with non-matching bases across all positions of two aligned sequences. Mean p-distance provides a metric of the average degree of difference between all sequence pairs comprising a pre-defined group (e.g., between all ASVs within a specimen or a morphospecies) [50]. Both full-length ASV data and the extracted V4 hypervariable region sequence variants were used to compute intragenomic, intraspecies, and interspecies p-distances (Fig 7a and 7b, respectively). Fig 3. Intraspecies genetic variation in Collosphaera huxleyi. Each bubble denotes one ASV; the size of the bubble reflects the total read abun-

dance of that ASV across all C. huxleyi specimens collected in this study (natural log). The position of bubbles along the y-axis represents the percent of C. huxleyi specimens that contained a given ASV (with ≥3 reads). Only one ASV was shared by 100% of specimens (top left), and this ASV also had the greatest number of total reads. ASV bubbles are colored with semi-transparent blue fill; the less transparent the blue color appears, the more ASVs were shared by a similar percent of C. huxleyi specimens. The solid blue region at the bottom of the plot reflects the high number of ASVs present in only one specimen (330 ASVs) and their wide-ranging read abundances within those specimens (bubble sizes). Embedded photos show a typical live colony specimen (top) and skeletal remains of three cells from a colony (bottom; scale bar = 50μm). The data underlying this plot can be found in S1 Data, and visualized as a heatmap in S2 Fig. https://doi.org/10.1371/journal.pone.0326053.g003