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 8 / 29 by two out of three specimens. P. praetextum thus showed a relatively low level of intraspecific genetic variability, though specimens did not have identical ASV compositions. By contrast, other morphospecies showed higher degrees of genetic variation than P. praetextum. In Procyttarium primordialis? (Fig 4e), some specimens yielded different dominant ASVs (dark blue shades in heatmap), although any ASV that was dominant in one specimen was at least present in the others (light blue shades in heatmap). Even greater genetic variation was observed in Collosphaera huxleyi (S2 Fig), Disolenia zanguebarica (Fig 4b), and Euchitonia elegans-furcata (Fig 6c). For these species, the dominant ASV in some specimens was entirely absent in others (white color in heatmaps). Most morphospecies in this study included some specimens that were not linked by any shared ASVs at all, appear- ing to constitute separate genetic subgroups. This was the case for Acrosphaera murrayanna (Fig 4a), Siphonosphaera socialis (Fig 4c), Thalassicolla nucleata (Fig 4e; but see caveats regarding Thalassicolla nucleata in subsequent section), Pterocorys zancleus (Fig 5a), Acanthodesmia vinculata (Fig 5b), Eucyrtidium hexagonatum (Fig 5c), Dictyocoryne pro- funda (Fig 6a), Dictyocoryne truncatum (Fig 6a), and Didymocyrtis tetrathalamus (Fig 6b). These morphospecies each contained at least one representative specimen, or group of specimens, that had an entirely different set of ASVs than

other specimens. These cases in which no ASV was shared across all representatives of a morphospecies could be indic- ative of cryptic species. Notably, the morphological subspecies of Didymocyrtis tetrathalamus (D. t. tetrathalamus and D. t. coronatus; Fig 6b) were not found to be genetically distinct, as they shared some ASVs. Therefore, while Didymocyrtis tetrathalamus contained multiple genetic subgroups, the subgroups did not appear to directly correspond to the morpho- logical distinctions defining the two subspecies. Instead, their morphological differences may be attributable to a gene other than 18S or potentially an epigenetic mechanism. Genetic subgroups have previously been reported in another spumellarian morphospecies, Spongotrochus glacialis, but in that case genetic differences corresponded with habitat par- titioning and a minor difference in skeletal morphology (spine length) [30]. These examples demonstrate that intraspecies genetic variation was the norm rather than the exception among Col- lodaria, Nassellaria, and Spumellaria. Moreover, a single dominant ASV was rarely shared among all representatives of a given morphospecies (only 1 out of 14 species) and most morphospecies (9 out of 14) included some specimens with no Table 1. Categories of intraspecies genetic variation observed in this study. Morphospecies (# specimens) The same ASV was dom- inant in all specimens Some or all specimens had different dominant ASVs, but at least 1 ASV was shared by all There was no ASV shared by every specimen of the morphospecies

Collodaria Acrosphaera murrayanna (6) X Collosphaera huxleyi (51) X Disolenia zanguebarica (2) X Procyttarium primordialis? (3) X Siphonosphera socialis (6) X Thalassicolla nucleata (10) X Nassellaria Acanthodesmia vinculata (2) X Eucyrtidium hexagonatum (2) X Pterocanium praetextum (3) X Pterocorys zancleus (19) X Spumellaria Dictyocoryne profunda (6) X Dictyocoryne truncatum (3) X Didymocyrtis tetrathalamus (10) X Euchitonia elegans-furcata (5) X https://doi.org/10.1371/journal.pone.0326053.t001