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 6 / 29 test this hypothesis. Polyploidy has been better documented in other members of Rhizaria, including Acantharia, Phaeo- daria, and Foraminifera [34,35], as well as other protistan groups including ciliates [36] and diatoms [37]. Another non-exclusive explanation for the intragenomic variation we observed could be the presence of high copy numbers of the 18S rRNA gene. Some polycystines (Collodaria) have been shown to have astoundingly high gene copy numbers per cell (estimated at 5,770 ± 1,960 SSU rDNA copies for solitary specimens and 37,474 ± 17,799 for cells iso- lated from colonies [38]), similar to groups such as dinoflagellates and ciliates [39,40,41], although it has been suggested that Nassellaria and Spumellaria have fewer copies [28]. Copy number has been shown to positively correlate with cell length across marine protist clades [38] as well as with intragenomic variation in ciliates [42]. Thus, copy number may have played a role in the high intragenomic richness we observed in Collodaria, and may also partly explain the relatively lower intragenomic richness observed in other polycystine groups that tend to have smaller cell sizes. Regardless of the underlying cause, differences in intragenomic richness and ASV read abundance structure across taxa have major implications for diversity interpretations in metabarcoding studies. High-throughput environmental DNA sequencing yields an immense quantity of data that is broadly representative of diversity in a sample. However, both

the richness and relative abundance of ASVs per specimen was not uniform across major taxonomic groups, so their metabarcoding results should not be interpreted uniformly to avoid misleading conclusions regarding diversity. Global scale genetic surveys of marine protistan species richness have revealed such great diversity of radiolarian molecular taxa [7] that they exceed the number of taxa in morphology-based surveys of similar scale by more than an order of Fig 2. Distribution of relative read abundances among the top 10 ranking ASVs per specimen. Dots represent relative ASV abundances (per- cent 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 ASV relative abundances for each order (Collodaria = pink; Nassellaria = brown; Spumel- laria = green). Data underlying this figure is in S1 Data. https://doi.org/10.1371/journal.pone.0326053.g002