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 11 / 29 Results showed that there is indeed greater genetic difference between morphospecies than within them, but the typical p-distances separating intragenomic and intraspecific from interspecific genetic diversity varied widely between taxonomic groups (Fig 7). Among Collodaria, intragenomic variation was expressed by p-distances ranging from 0–0.176 (median: 0.009); Spumellaria had a comparable median intragenomic distance of 0.009 but lower range in values (0–0.037), while Nassellaria specimens showed the lowest median and range (median: 0.001; range: 0–0.032). Intraspecies genetic varia- tion was higher than intragenomic distances for all orders, with median p-distance values of 0.037 for Collodaria, 0.004 for Nassellaria, and 0.013 for Spumellaria. However, orders displayed very different magnitudes of intragenomic and intraspe- cies genetic variation, with collodarian intragenomic distances on-par with the intraspecies distances in Spumellaria and even surpassing intraspecies distances in Nassellaria. Similarly, interspecies genetic variation was higher than intraspe- cies variation for all orders, but p-distance values were substantially different between them (Fig 7). The median p- distance separating Collodaria morphospecies in this study was 0.203 (range: 0.083–0.222), 0.113 (range: 0.026–0.164) for Nassellaria morphospecies, and 0.027 (range: 0.011–0.141) for Spumellaria morphospecies. When these analyses

were performed using the extracted V4-region ASVs (Fig 7b), the same patterns held but p-distances were greater at each hierarchical level. This is presumably because the relatively short (~440 bp) V4 hypervariable region is expected to contain a higher proportion of site differences between ASVs at each hierarchical group level compared the full-length (~1450 bp) ASVs, which are comprised of both conserved and hypervariable regions. These results indicate that p-distances have promise for delimiting polycystine species-level taxa in metabarcoding studies, but appropriate p-distance thresholds vary considerably based on the length and characteristics of the target amplicon regions, and the order-level classification of the ASVs in question. Additional single-specimen sequencing efforts involving more Radiolaria from different taxonomic groups will improve our ability to define p-distance thresholds for estimating species richness with metabarcoding data. These findings highlight the challenge of detecting specific morphospecies of interest in DNA metabarcoding sur- veys using the existing structure of reference sequence databases (i.e., PR2 [4]). Reference sequences derived from isolated specimens of known protistan species are vital for interpreting genetic diversity data. But these individual refer- ence sequences will, in many cases, fail to capture the range of prevalent sequence variants produced by species in the environment. This can lead to overestimation of species richness and evenness in a sample when interpreting molecu-

lar data relative to morphospecies data. If we wish to detect a given morphospecies in a DNA survey, it would be more appropriate to organize reference sequence databases by groups of multiple sequence variants that co-occur within the same specimen, and within specimens of the same morphospecies, thereby explicitly accounting for intraspecies genetic variation. Conversely, an approach that is less rigidly bound by morphospecies concepts may ultimately provide us with a more natural characterization of protistan richness. Work on Foraminifera has shown that cryptic species often occupy narrower niches than the morphospecies to which they belong [51]. This means we risk overestimating species’ biogeo- graphic ranges and niche breadths if we fail to consider their genetic variation and look only through the lens of morphol- ogy. Ecological models predicting future geographic shifts in plankton communities could thus be improved by knowledge of intraspecific genetic variation. Such models utilize species’ presumed habitat preferences to predict geographic shifts in plankton communities (e.g., [52]), but if genetically distinct subgroups of some morphologically-described species have different habitat preferences, then our assumptions about their future geographic shifts may be inaccurate. Therefore, interpreting taxa as assemblages of multiple shared ASVs, likely with more than one possible assemblage per morpho- type, could allow the use of metabarcoding data to shed light on their ecological preferences and roles that might other-

wise remain obscured behind classical morphospecies concepts or ungrouped, discrete ASVs. with a random numeric identifier. The x-axis of each heatmap denotes the ASVs (represented by tick marks) present in each specimen of a morphos- pecies 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 by 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.g004