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 18 / 29 Another key finding from the ASV composition of these juvenile radiolarians is that they suggest no change in intrag- enomic richness occurs during ontogeny. The juvenile Acanthodesmidae specimen yielded 2 ASVs, which is within the range of ASV richness found in mature specimens morphologically assigned to the same family (Acanthodesmida- vinculata-01, Acanthodesmida-vinculata-02, and acanthodesmid-sp-01; 1, 2, and 4 ASVs, respectively; Fig 5b). Simi- larly, the mature Spumellaria specimen (classified as lithellid sp. 3) yielded 40 ASVs, while the two juveniles putatively belonging to the same taxon yielded 39 and 22 ASVs each. Thus, mature specimens of both Nassellaria and Spumel- laria appear to have intragenomic variation that is consistent with juvenile individuals, making it improbable that radio- larians accumulate intragenomic variation throughout their life cycles, as ciliates do [59]. This result means that juvenile specimens may be successfully classified based on their ASV composition in precisely the same way as fully-mature specimens in metabarcoding surveys. Metabarcoding surveys therefore have the potential to reveal a more complete picture of radiolarian community composition than microscopy counts or in-situ imaging due to their inherent inclusion of juvenile specimens that are too indistinct to be morphologically identified (see S5 Fig). Our results also suggest that Collodaria do not accumulate genetic diversity during cell division and colony growth,

and different life stages yield comparable genetic signatures. Collodaria colonies have similar magnitudes of intrag- enomic richness as the individual cells which comprise them, as well as specimens with a solitary life habit. Sipho- nosphaera socialis was sampled as whole colonies of 10s–100s of cells (3 specimens) and also as individual cells picked from different colonies (3 specimens). Single-cells yielded a mean of 12.3 ASVs (sd = 0.6), whereas entire colonies yielded fewer ASVs on average (mean = 6) but more variation in ASV richness (sd = 7). Likewise, Collodaria specimens with solitary versus colonial life habits did not show consistently different magnitudes of intragenomic richness. Solitary collodarians yielded a mean of 35 ASVs each, whereas colonial specimens yielded between 16 and 50 ASVs on average (depending on whether the exceptionally-diverse Collosphaera huxleyi was excluded or included, respectively) (Fig 4; S1 Data). We interpret this as support for the hypothesis that Collodaria grow their colonies via binary fission and are made up of individuals that are genetically identical to the originating cell (e.g., [60]). Thus, the genetic richness observed in Collodaria does not appear to be directly related to the number of cells making up their colonies. Sequence information also has importance for understanding the roles of naked (un-skeletonized) solitary morpho- types, which may be analogous across different polycystine groups. Due to the physical resemblance of two Procyttarium

primordialis? (Collodaria) specimens with the putative juvenile Nassellaria and Spumellaria specimens we observed (S5 Fig), it is possible that this morphotype is common to all polycystine orders during an early juvenile life stage, and not only in the Collodaria. These specimens’ ASV signatures placed them across all three polycystine orders; however, the speci- mens were morphologically indistinguishable. They shared a light, semi-transparent central spherical feature surrounded by algal symbiont cells encased in a gelatinous cytoplasm that could appear clear or slightly striated or cloudy. Although each cell contained a collection of symbionts, there was no siliceous skeletal development in any of them. To our knowl- edge, no such morphotype has previously been described in the literature, and we hypothesize that it could be an interme- diate stage between swarmer cell and skeletal formation during which the specimen acquires photosymbionts. This finding is consistent with other observations of polycystine ontogeny that document skeletal growth (e.g., [5]), but may represent an even younger life stage prior to initial skeletal formation. If there were distinguishing morphological features among these putative juvenile specimens, they were not obvious under our shipboard dissecting microscope (S5 Fig). It is also possible that some taxa (at least within the Collodaria) have more than one life stage that can be described as a naked solitary cell with symbionts: once early in ontogeny and again during reproduction. Reproductive swarmer cells have been

reported in Thalassicolla and other solitary collodarian taxa, suggesting that this morphotype could be a reproductive life stage rather than (or in addition to) a juvenile one [12,61]. However, swarmer release has also been reported in colonial Collodaria specimens [62,63], and in fully-skeletonized Nassellaria and Spumellaria [64]. Thus, naked solitary specimens are not the only polycystine morphotype to exhibit evidence of reproductive swarmers. Our findings underscore the need