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 19 / 29 to examine more such specimens via combined microscopy and nucleic acid sequencing to bridge our current knowledge gaps in radiolarian life cycles and phylogenetic relationships. Metabarcoding presents a rich, but enigmatic picture of protistan diversity Metabarcoding surveys report on the diversity of molecular sequences in a sample or study area (e.g., [7]), but we cur- rently lack a method for relating these molecular signatures to classical morphospecies concepts, which have been the basic unit of much biological research and theory developed over the last two centuries. These molecular species are a new frontier in diversity research and cannot simply be translated into existing morphological, ecological, or biological spe- cies concepts [3]. Despite its limitations, marine metabarcoding has already transformed our understanding of protistan life [2]. These data have the power to reveal the behavior, functions, and diversity of microscopic organisms on a stagger- ing scale that has not been approachable using traditional methods. Although morphospecies are still considered the “gold standard” for measuring protistan diversity, they are ultimately artificial constructs and not necessarily reliable for all groups. For example, it is well-known in the field of radiolarian systematics that some groups are over-split while other groups are over-lumped. Early literature documenting radiolarian

species richness (e.g., [65]) included sometimes vague descriptions of newly-described taxa and occasionally lacked any illustration or type material, leading to subsequent confusion of morphospecies categories. Since then, much work has been done to improve radiolarian morphological taxonomic concepts (e.g., [66]) and develop radiolarians into reliable biostratigraphic markers that are frequently employed in the age determination of marine sediments [67]. These marker taxa (chronological species) are typically large (>100 μm) with distinct morphological characters, and their elevated bio- stratigraphic importance can lead to them receiving great attention to detail (and potential for subspecies-level splitting). Other taxonomic groups have been chronically under-documented, in part due to their irrelevance for biostratigraphy, relatively small size (<100 μm), and complicated morphological characters, leaving many presumed species-level taxa in open nomenclature or in broad species categories (such as the Lophophaenidae [55]). This essentially leaves us with two moving targets: molecular and morphological species concepts, which compounds the difficulty of reconciling them. Our goal in this long-running reconciliation does not need to be molding one type of data to fit neatly into the other; rather, we can let them work together. Morphological and molecular data are both valuable, as they each can shed light on different aspects of protistan biodiversity, life cycles, and ecological roles. By integrating them we will deepen our overall

understanding of these topics in ways that cannot be achieved with morphological or molecular data alone. As we have shown here, it is not a simple task to merge these types of data, due to factors like intragenomic and intraspecies genetic variation, which manifest very differently across taxa. Our results demonstrate that individual polycystine radiolarian spec- imens usually contain a multitude of ASVs, and their ASV assemblages are inconsistent among specimens belonging to the same morphospecies, even those from the same population. Similar findings of genetic diversity have been observed in other protistan groups as well, suggesting that this phenomenon is widespread, highly complex, and can severely obfuscate diversity metrics in metabarcoding studies (e.g., [39,40]). By adopting a flexible approach to metabarcoding data interpretation that is informed by known characteristics of the target genomes, such as intragenomic variation, copy number, and ploidy, we can begin to build a stronger foundation for new discoveries. Materials and methods Approach This study was designed to characterize 18S rRNA gene sequence variability within morphospecies and isolated speci- mens of polycystine Radiolaria. To accomplish this, live radiolarian specimens belonging to orders Collodaria, Nassellaria, and Spumellaria were collected at sea and processed for both microscopy and long-read nucleic acid circular consensus sequencing. Sequence data were brought through a standard bioinformatic pipeline and customized taxonomic filtering