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 14 / 29 Intraspecies genetic variation has clear implications for the study of modern and future protistan biodiversity and biogeography, but it can also reshape our understanding of their fossil record, including historical responses to climate change. In paleontological investigations genetic information is typically not available, and morphology is the primary data used to characterize protists’ evolutionary and ecological responses to past environmental shifts (e.g., [53,54,55]). If morphologically-defined species contain differing degrees of genetic variation, possibly including cryptic species, then the impact of some morphospecies extinction or origination events in the fossil record could have had broader ecologi- cal significance than previously believed. As intraspecies variation becomes better understood for each morpho- taxonomic group, we may be able to better estimate the impacts of macroevolutionary events throughout the group’s history. Insights gained from morpho-molecular relationships Long-read metabarcoding provides a unique opportunity to examine both biodiversity and phylogenetic relationships with the same dataset [56]. Most metabarcoding studies utilize short reads of ~300–500 base pairs, making them heav- ily reliant on matches to full-length reference sequences for interpretation of evolutionary relationships between taxa. Because the length of our reads spanned most of the 18S rRNA gene (~1450 base pairs), we were able to construct a

phylogeny from our data that directly informs on evolutionary relationships between ASVs and is decoupled from refer- ence sequence similarity (S4 Fig). In this way, we investigated the connection between morphological taxonomy, total ASV composition, and the phylogenetic relationships of specimens’ dominant ASVs, similar to the approach used by others [24,25,26]. Our results variously revealed that genetic information can in some cases: (1) support morphospecies concepts, (2) indicate taxonomic concepts in need of revision, or (3) potentially inform on radiolarians’ poorly-understood life stages. (1) Supporting morphospecies concepts. One example of morphospecies supported by genetic information was found in the Spumellaria genus, Dictyocoryne. Although D. profunda and D. truncatum have similar morphological characters (Fig 6a), our results showed that they are indeed genetically distinct. All of the dominant ASVs in D. truncatum specimens branched separately from any of the dominant ASVs in D. profunda specimens (S4 Fig). Furthermore, none of the dominant ASVs found in the D. truncatum specimens were present in any D. profunda specimens, and vice versa (Fig 6a). This finding suggests that ASV data can be used to successfully distinguish them in a metabarcoding survey. The specimens assigned to Dictyo- coryne sp. had only partially-developed skeletons (either due to breakage or incomplete growth), making it difficult to determine their species identity from morphology (S1 Fig). Based on their patterns of shared ASVs and phylogenetic

clustering, however, we were able to infer that Dictyocoryne sp. specimens #1–4 belong to D. profunda rather than D. truncatum (Fig 6a; S4 Fig). This exemplifies a case in which molecular data can be used to assist in species classifi- cation, even when morphological characters cannot be observed. There was one ASV present in very low abundance in Dictyocoryne-truncatum-03 (5 reads) that was also present as a subdominant ASV in three D. profunda specimens. However, due to the extremely low number of reads in Dictyocoryne-truncatum-03, we interpret this ASV as environ- mental contamination that was inadvertently sequenced (see “Materials and methods”), rather than an authentically shared ASV between the two Dictyocoryne species. as Spumellaria and Pyloniidae based on the identity of their matching reference sequences (>99% confidence). The x-axis of each heatmap denotes the ASVs (represented by tick marks) present in each specimen of a morphospecies or group of morphotaxa. Relative read abundances of ASVs per specimen are indicated by the shade of blue (light = low abundance, dark = high abundance); ASVs not present in a particular specimen are indicated as white background. Representative micrographs of morphotaxa are shown on the right of each heatmap. All data underlying this figure can be found in S1 Data. Photos of every specimen can be found in S1 Fig. https://doi.org/10.1371/journal.pone.0326053.g006