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 3 / 29 relatively large size (~20–250 μm for solitary taxa and typically mm–cm for colonial taxa) permits simultaneous morpho- logical and molecular analyses, and their diverse array of siliceous skeletal structures facilitates high-resolution morpho- logical groupings. Additionally, radiolarians play integral roles in marine food webs and biogeochemical cycles, which necessitates a better understanding of their biodiversity. They are abundant in ocean water columns from the surface to abyss, from the tropics to the poles [11], occupying many ecological niches as mixotrophs, herbivores, predators, and detritivores [5,12]. They have also been shown to contribute significantly to the biological ocean carbon pump [13,14,15,16,17,18,19] and the silicon cycle [19,20]. Despite their importance, only a small fraction of polycystine radi- olarian morphospecies have been linked to their genetic sequence information [21,22,23,24,25,26,27]. In addition, very few studies have explored radiolarians’ intragenomic variation [23,28] (which in this manuscript refers to gene sequence variation within the genome of an individual specimen), or potential cryptic species [29,30] (which refers to genetically distinct subgroups within a morphologically-defined species). Here we build on emerging genetic information for polycys- tine Radiolaria by combining morphological and molecular analysis of 173 isolated specimens collected from the North

Pacific Subtropical Gyre. We employ long-read 18S rDNA amplicon data to investigate the genetic variability within individ- ual specimens and morphospecies, explore phylogenetic relationships among taxa, and discuss the implications of these findings for deciphering protistan diversity datasets in the age of amplicon sequencing. Results and discussion The dataset Our dataset included 18S rDNA long-read amplicon sequence variants (ASVs) from 173 polycystine radiolarian specimens belonging to 30 morphologically classified taxa. DNA from each individual was extracted, amplified, and underwent circular-consensus sequencing with Pacific Biosciences technology while morphology-based tax- onomy was assigned via microscopy. After bioinformatic processing, a total of 1,224 unique ASVs met criteria for analysis: a minimum of three reads in at least one specimen and taxonomically assigned to the same family as the morphologically-identified specimen they came from (see “Materials and methods”). The specimens in our dataset represented all three polycystine radiolarian orders: Collodaria (95 specimens), Nassellaria (36 specimens), and Spumellaria (42 specimens). Out of these, the Collodaria displayed the highest genetic richness (836 ASVs) despite including the lowest number of morphotaxa (9). Conversely, Nassellaria specimens had the lowest genetic richness (187 ASVs) but highest number of morphotaxa (11). Spumellaria specimens were comprised of 201 total ASVs from 10 morphotaxa. Taxonomic names of all specimens are listed in “Materials and methods,” photographic documenta-

tion is provided in S1 Fig, and ASV occurrence charts in S1 Data. Individual radiolarians contained multiple ASVs After stringent taxonomy and abundance-based vetting of ASV results (see “Materials and methods”), we found that 156 out of 173 radiolarian specimens (90%) exhibited multiple ASVs, with a median number of 12 ASVs per specimen (Fig 1a). This indicates that intragenomic variation was pervasive among the specimens we analyzed; however, the number of ASVs per specimen varied by taxonomic order. Collodaria specimens showed the greatest genetic richness, with a median of 32 ASVs per specimen and a relatively large range in values (1–141 ASVs per specimen) (Fig 1a). Nassellaria and Spumellaria had fewer ASVs per specimen, with median values of 3 and 5.5, respectively. Nassellarian intragenomic richness ranged from 1–57 ASVs and Spumellaria ranged from 1–40 ASVs per specimen (Fig 1a). The only family that did not yield multiple ASVs per specimen was Lophophaenidae (Nassellaria); Lophophaena hispida and Peromelissa thorac- ites each contained a single ASV (S1 Data). When the hypervariable V4 region was extracted from each long-read ASV (see “Materials and methods”), intrage- nomic richness of specimens in each taxonomic order was effectively reduced by half. There was a median number of 6 V4-region sequence variants per specimen, with Collodaria showing a range of 1–57 variants (median = 16), Nassellaria