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 1 / 29 OPEN ACCESS Citation: Trubovitz S, Sandin MM, Caron DA (2025) Genetic variability in microbial eukaryotes reshapes marine biodiversity assessment in the age of amplicon sequencing. PLoS One 20(6): e0326053. https://doi. org/10.1371/journal.pone.0326053 Editor: Claudia Isabella Pogoreutz, Université de Perpignan: Universite de Perpignan Via Domitia, FRANCE Received: December 2, 2024 Accepted: May 22, 2025 Published: June 20, 2025 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal. pone.0326053 Copyright: © 2025 Trubovitz et al . This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, RESEARCH ARTICLE Genetic variability in microbial eukaryotes reshapes marine biodiversity assessment in the age of amplicon sequencing Sarah Trubovitz 1,2*, Miguel M. Sandin3, David A. Caron1 1 Department of Biological Sciences, University of Southern California, Los Angeles, California, United States of America, 2 Ocean Sciences Department, University of California-Santa Cruz, Santa Cruz, California, United States of America, 3 Institut de Biologia Evolutiva (CSIC-Universitat Pompeu Fabra), Barcelona, Spain * strubovi@ucsc.edu Abstract

Rapidly improving DNA sequencing technology has revolutionized our ability to efficiently survey the biodiversity of microbial life. We are now equipped to inves- tigate protistan richness and community dynamics on scales that would not have been imaginable with traditional observational methods. However, for most taxa the relationship between DNA sequences and morphologically-defined species is poorly understood, and morphology has remained the cornerstone of taxonomy for centuries. To better utilize the wealth of sequence data being collected, we must understand how it relates to entities such as individuals, populations, and species. Here we use a combined microscopy and sequencing approach to unveil the striking intragenomic and intraspecies genetic variation in one group of ecologically-important marine protists, the polycystine Radiolaria. Long-read 18S rRNA gene amplicon data from 173 isolated and morphologically-identified radiolarians showed that the vast majority (90%) yielded multiple sequence variants per specimen. Furthermore, every morphospecies analyzed displayed a range of different genetic signatures. Intraspe- cies genetic variability was expressed as specimens having different assemblages of ASVs, different dominant ASVs, or having no ASVs in common with other specimens of the same morphospecies. By integrating morphological and molecular information, we begin to parse the genetic richness of Radiolaria in ocean environments, as well as illuminate relationships between taxa, and their poorly-known life stages. Our

findings emphasize the need to account for protists’ taxon-specific sequence variabil- ity, particularly their intragenomic and intraspecies genetic variation, in interpreting metabarcoding diversity survey data.