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 17 / 29 [24], who also showed that Collozoum and Sphaerozoum were polyphyletic, but we speculate that Rhaphidozoum may be polyphyletic as well. ASVs from our Rhaphidozoum specimens did not group in the same clade as the Rhaphidozoum acuferum reference sequences (see Fig 8); however, since only two specimens were collected during this study and few reference sequences were available, more data from this genus is needed to confirm its polyphyly. Our results also suggest that morphospecies concepts of solitary Collodaria may be invalid and instead synonymous with various colonial taxa. Phylogenetic analysis indicated that all solitary Collodaria specimens in this study (morpholog- ically assigned to Thalassicolla nucleata and Procyttarium primordialis?) either shared their dominant ASV with a colonial specimen, or their closest phylogenetic relative was a colonial specimen (Fig 8). One ASV (coll-002) was dominant in 9 specimens, morphologically-classified as Sphaerozoum sp. (colonial), Procyttarium primordialis? (solitary), or Thalassi- colla nucleata (solitary). Because this ASV comprised between 54% and 87% of reads in these specimens, it is not likely the result of environmental contamination or technical error and probably indicates an authentic shared ASV. Additionally, these specimens were picked from three different net tow samples on North Pacific Subtropical Gyre cruise HOT 339, discounting the possibility that their shared dominant ASV is attributable to physical contact during sample collection.

ASV coll-007 was also shared by both solitary and colonial taxa, and ASV coll-012 was dominant in several Thalassi- colla nucleata (solitary) specimens while also being most closely phylogenetically related to the dominant ASV in colonial Rhaphidozoum specimens (Fig 8). We additionally found that the dominant ASV in two Sphonosphaera socialis colonies (coll-003) was taxonomically assigned to the Thalassicolla melacapsa (solitary) reference sequence in PR2 with 99.5% confidence, suggesting these morphospecies may be conspecific. Furthermore, the ASVs mentioned above did not form a monophyletic clade, which is consistent with the findings of Biard et al. [24] that Thalassicollidae is polyphyletic (Fig 8). Together, these results support the hypothesis that solitary Collodaria are conspecific with colonial taxa [24,57,58], rather than a distinct taxonomic group, as they have previously been considered (e.g., [21,22]). Solitary Collodaria should thus no longer be considered valid species in biodiversity surveys, but instead a morphotype that occurs across multiple clades. (3) Informing on radiolarian life stages and development. Our results indicate that some juvenile polycystine specimens lacking skeletal development may be identifiable to the fam- ily level or lower based on their ASV composition and phylogenetic clustering. These specimens were devoid of diagnostic taxonomic characteristics under microscopy, so they may only be distinguishable and possible to classify with sequencing

methods. One putative juvenile specimen was genetically classified to the family Acanthodesmidae based on genetic sim- ilarity, but due to the poor representation of this family in reference sequence databases classification at lower taxonomic levels was not possible (S5 Fig; Fig 5e; S1 Data). Another putative juvenile was assigned to the family Pyloniidae, due to its genetic match to Larcopyle butschlii reference sequences (S5 Fig; Fig 6d). There were two other putative juvenile specimens for which all ASVs were assigned to Spumellaria, but no genetic matches were found below the order level (S1 Data). These juvenile specimens shared their dominant and many subdominant ASVs (19 ASVs shared in total) with a mature spumellarian specimen that was morphologically classified as lithellid sp. 3 (sensu Trubovitz et al. [55]) (Fig 6d; S5 Fig). Therefore, all three specimens likely belong to the same open-nomenclature taxon which, based on its phylogenetic clustering, may comprise a novel Spumellaria clade (S4 Fig). highlighted in green and collapsed for clarity. The number of dominant ASVs clustering in each group is shown with yellow bubbles and the number of specimens per clade, family, and dominant ASV is denoted with blue bubbles. Colored boxes headlined with ASV identifiers depict photographs of all specimens that shared the same dominant ASV, and correspond to the colored highlighting in the expanded Collodaria portion of the phylogenetic tree.

These cases point to examples in which solitary and colonial Collodaria shared the same dominant sequence variant, or in the case of coll-012, a closely related ASV. Such examples are evidence that solitary and colonial specimens, previously described as different morphospecies, may actually repre- sent different life stages of the same taxon. A full version of this phylogenetic tree that depicts all specimens’ dominant ASVs can be found in S4 Fig. Sequence data underlying this tree can be found in S1 Data and S1 Table, and tree parameters are detailed in “Materials and methods”. https://doi.org/10.1371/journal.pone.0326053.g008