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 5 / 29 ranged from 1–21 (median = 2), and Spumellaria ranged from 1–37 sequence variants per specimen (median = 4) (Fig 1e). Therefore, while short-length V4 ASVs masked a significant portion of the genetic richness captured by full-length ASVs, intragenomic variation was still evident in each polycystine order albeit to a lesser degree. This result suggests that metabarcoding surveys utilizing relatively short-reads of hypervariable regions such as the V4 or V9 (e.g., [7]) are not able to detect the same level of genetic richness that can be revealed with long-read sequence data. The biological significance of protistan ASVs and their relationship to concepts such as specimens, populations, or species, remains a complex open question [3]. Previous work has suggested that ASVs almost certainly capture some intraspecies variation [3,10], but our results expand those findings by demonstrating that radiolarian ASVs can consti- tute a great deal of intragenomic variation as well. The fact that all three radiolarian orders demonstrated intragenomic variation indicates this phenomenon is not limited to species with a colonial life habit nor the presence of multiple nuclei, traits which frequently occur in Collodaria but have not been observed in any Nassellaria or Spumellaria [5,31]. Rather, it is probable that individual nuclei contain intragenomic variation for all groups included in this study (except evidently the

Lophophaenidae, but more research is needed). Furthermore, because the average specimen in our dataset yielded 12 unique ASVs, intragenomic variability is likely a significant source of the total genetic richness being measured in metabar- coding surveys of protistan diversity. The intragenomic variability we observed ranged widely among taxa, even those belonging to the same order. Therefore, while multiple ASVs could indicate multiple individuals (or multiple species) in some taxonomic groups, in others it appears to reflect the intragenomic variation within a single individual. This suggests that accurately interpreting the significance of ASV richness will require taxon-specific knowledge of genomic variability. Next, we examined ASV read abundances within specimens to assess the prominence of intragenomic variants. Thresholds were applied to determine the number of ASVs contributing at least 0.1%, 1%, or 10% of the total reads within each specimen (Fig 1b–d, respectively). These more abundant subsets of ASVs within each specimen presumably reflect their higher frequencies within the genome, potentially having a greater impact on the organism’s function and fitness compared to rarer ASVs [32]. Collodaria specimens yielded a median of 21 ASVs (range: 1–104) that comprised at least 0.1% relative read abundance (Fig 1b). At a minimum of 1% relative read abundance (Fig 1c), this decreased to a median of 5 ASVs per collodarian (range: 1–31), and at 10% (Fig 1d) collodarians exhibited between 1 and 4 ASVs with a median

value of 1 per specimen. Nassellaria and Spumellaria had fewer ASVs per specimen than Collodaria at every relative abundance threshold except for 10%, where Nassellaria exhibited the same median and range as Collodaria, and Spu- mellaria exceeded both groups by 1 ASV in terms of median and range (2 and 5 ASVs, respectively) (Fig 1d). The partitioning of reads among ASVs was investigated in greater detail by examining the relative read abundance of the top ten ranking ASVs per specimen in each order (Fig 2). Nassellaria specimens, on average, had a single, highly- dominant ASV, while a relatively low number of subdominant ASVs within their genomes comprised a minor percentage of reads. Among Nassellaria, the top ranking ASV made up a median of 92% of total reads within each specimen, versus 77% in Collodaria and only 60% in Spumellaria. Compared to the other orders, Spumellaria showed a relatively equitable distribution of reads among their dominant and top few subdominant ASVs. For example, the 2nd rank ASV in Spumel- laria comprised 23% of total reads per specimen on average, whereas 2nd rank ASVs in Collodaria and Nassellaria only made up 5% and 4% of reads per specimen, respectively. Among the extracted V4 region ASVs, the different partitioning patterns between Spumellaria and the other two orders were even more pronounced (S3 Fig). Collodaria and Nassellaria specimens had top ranking V4-ASVs make up 88% and 98% of their total reads (median values), respectively, whereas

Spumellaria specimens only had a median of 61% of total reads in their top-ranking V4-ASV. Based on these findings, we speculate that intragenomic variants in Spumellaria may tend to be more functionally important compared to those in other orders, even though Collodaria yielded the highest numbers of intragenomic variants overall. One possible explanation for the intragenomic variation observed in radiolarians is that they could be polyploid. Past imaging studies have suggested that some polycystines exhibit polyploidy (e.g., [33]); however, we are not aware of any definitive evidence that polyploidy is confirmed or common in polycystine radiolarians, and our dataset is not sufficient to