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 7 / 29 magnitude [43]. The high degree of variability we observed in radiolarians’ intragenomic richness and ASV abundance structure may underlie some of the apparent ‘species’ richness and relative abundances observed in metabarcoding diver- sity surveys. The prevalence of this situation may extend to other protistan taxa as well. Previous work has documented intrage- nomic variation in Nassellaria and Spumellaria [23,28], and in polycystine radiolarians’ sister clades, the Acantharia [23] and planktonic Foraminifera [44,45]. There is also growing evidence that distally-related protistan clades exhibit intrage- nomic variation (e.g., [42,46,47]), which taken together may partly explain the staggering number of sequence variants observed in marine metabarcoding diversity surveys (i.e., [7]). Intragenomic variation has not been explored for the vast majority of taxa nor is it typically accounted for in richness interpretations of metabarcoding data [48]. It is thus imperative that we gain a better understanding of the magnitude and structure of intragenomic variation across groups, so that we can more accurately apply metabarcoding data to address important questions in ecology, including the unified under- standing and detection of biological units, like individuals, populations, and species. Specimens of the same morphospecies showed varying ASV assemblages Morphospecies of which we were able to collect multiple individual specimens were analyzed for intraspecies genetic

variation. This analysis included a total of 14 morphospecies, and all three polycystine orders were represented. Intraspe- cies genetic variation (here defined as different assemblages of ASVs within specimens of the same morphospecies) was found in every morphospecies we examined, to varying degrees (summarized in Table 1). The most thoroughly-sampled morphospecies in this study was the colonial collodarian, Collosphaera huxleyi (51 specimens). It yielded a combined total of 562 ASVs, with a median of 87 ASVs per specimen (S1 Data). Many of these ASVs (41%; 232) were shared by at least two specimens, and some ASVs (7%; 38) were shared by more than half of specimens. But, only one ASV was shared by every specimen in the dataset (Fig 3) and this ASV was dominant in 48 of 51 specimens (S2 Fig; S1 Data). Generally, there was a positive correlation between the total read abundance of an ASV and the number of specimens that shared it, but this was not always the case, indicating that some specimens yielded high abundances of ASVs that were absent in other specimens (Fig 3). The fact that hundreds of unique sequence vari- ants could be attributed to one morphospecies is surprising, especially given that C. huxleyi exhibits relatively simple and consistent morphological characteristics (S1 Fig) and lacks any known niche differentiation. All of our specimens were collected during summer 2022 in approximately the same location at ~5m depth (see “Mate- rials and methods”). Therefore, temporal or spatial separation cannot easily explain the remarkable magnitude of gene

sequence variation we observed in C. huxleyi specimens. The life cycle of C. huxleyi is poorly understood, but each specimen we collected was colonial, with 10s–100s of skeletonized cells matching the formal species description (sensu Strelkov and Reshetnyak [49]). It was common, however, for colonies to contain cells of varying sizes, sometimes in the process of dividing (also observed by Strelkov and Reshetnyak; Fig 19 in [49]), so we cannot rule out the possibility that differing stages of the colonies or the cells within them could be related to the genetic variation we observed. Alternatively, this intraspecific variation could be due to random neutral mutation that was not related to life stage. Although there was a lack of distinct genetic subgroups in this population (i.e., all specimens shared one, usually dominant, ASV) the fact that some specimens had different dominant ASVs and most ASVs were shared by less than half of specimens, could suggest ongoing genetic drift potentially leading to speciation, including the possible emergence of cryptic species. Either way, the multitude of ASVs observed among the 51 C. huxleyi specimens raises questions as to how to interpret that sequence diversity with respect to the morphologically-defined taxon. The other morphospecies in this study also demonstrated intraspecies genetic variation, but they did so with differing patterns of ASV presence and abundance (Table 1; Figs 4–6). Out of the 14 morphospecies examined, only one (Ptero-

canium praetextum) yielded the same dominant ASV in all specimens, although only 3 specimens could be examined (Fig 5d). There were also two subdominant ASVs found in this species; one was shared by all specimens and the other shared