Heterotrophic flagellates and centrohelid heliozoans from marine waters of Curacao, the Netherlands Antilles

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Abstract

Recent progress in understanding the early evolution of eukaryotes was tied to morphological identification of flagellates and heliozoans from natural samples, isolation of their culture and genomic and ultrastructural investigations. These protists are the smallest and least studied microbial eukaryotes but play an important role in the functioning of microbial food webs. Using light and electron microscopy, we have studied the diversity of heterotrophic flagellates and centrohelid heliozoans from marine waters of Curacao (The Netherlands Antilles), and provide micrographs and morphological descriptions of observed species. Among 86 flagellates and 3 centrohelids encountered in this survey, five heterotrophic flagellates and one сentrohelid heliozoan were not identified even to the genus. Some flagellate protists have a unique morphology, and may represent undescribed lineages of eukaryotes of high taxonomic rank. The vast majority (89%) of identified flagellates is characterized by wide geographical distribution and have been reported previously from all hemispheres and various climatic regions. More than half of the species were previously observed not only from marine, but also from freshwater habitats. The parameters of the species accumulation curve indicate that our species list obtained for the Curacao study sites is far from complete, and each new sample should yield new species.

Introduction

Heterotrophic flagellates and centrohelid heliozoans are the smallest and least studied groups of protists at both the morphological and molecular levels. Heterotrophic flagellates – the collective name for an extremely diverse “hodgepodge” of polyphyletic, colorless protists moving or feeding with flagella at least in one stage of their life cycle (Patterson and Larsen 1991) – are characterized by a significant variety of metabolism and ecology. At the same time, centrohelid heliozoans (Centroplasthelida Febvre-Chevalier and Febvre, 1984) is a monophyletic group of predatory protists, related to haptophyte algae within the supergroup Haptista Cavalier-Smith, 2003 (Burki et al. 2016). Centrohelids are characterized by the presence of surface cytoskeletal structures – siliceous scales or organic spicules, having an important diagnostic value. Heterotrophic flagellates and heliozoans are widespread in different types of freshwater and marine biotopes and play an important role in the functioning of the microbial food webs, thus providing effective pathways for the transformation of matter and energy in aquatic ecosystems (Arndt et al. 2000; Domaizon et al. 2003; Kiss et al. 2009).

Faunistic investigations of heterotrophic flagellates and heliozoans supported with morphological descriptions were especially common in protistological literature in the 90 s – early 2000s (Larsen and Patterson 1990; Lee and Patterson 2000; Lee et al. 2003; Mikrjukov 2001; Patterson and Simpson 1996; Patterson et al. 1993; Tong 1997b, c; Vørs 1992). At present, such studies are relatively rare and have been largely replaced by molecular surveys from bulk microbial community DNA (Geisen et al., 2019; Pawlowski et al., 2016). However, several recent advances in the study of eukaryotic evolution have been specifically tied to morphological identification of flagellates and heliozoans in natural samples, their isolation into culture, and genomic and ultrastructural investigations of such cultures. Morphological recognition and identification (sometimes approximate) of these protists in samples is precisely based on such “classical” studies, and would not have been possible without these. For example, recent recognition, isolation, and investigation of previously understudied or unknown heterotrophic flagellates was essential in addressing major evolutionary problems, such as the origins of photosynthesis and parasitism and the trajectory of plastid spread (Gawryluk et al. 2019; Janouškovec et al. 2015; Tikhonenkov et al. 2020b), the origin of multicellular animals (Hehenberger et al. 2017; Tikhonenkov et al. 2020a), the evolution of mitochondrial genomes, and the rooting of the tree of eukaryotes and clarification of their relationships (Janouškovec et al. 2017; Lax et al. 2018; Strassert et al. 2019). Identification and establishing of clonal cultures of centrohelids was crucial for transcriptomic research and untangling the early diversification of eukaryotes (Burki et al. 2016).

Descriptions of many flagellates and heliozoans were made in the late XIX and early XX centuries using imperfect light microscopy. Many of these species lack type material, ultrastructural, and molecular data, and the taxonomy of many species and groups is in need of revision (Lee et al. 2003; Schoenle et al. 2020). Currently, the described diversity of these protists represents only a small fraction of their total species richness in nature (Cavalier-Smith and von der Heyden 2007; Corliss 2002). Metagenomic and metabarcoding sequencing of environmental samples has revealed several lineages representing high levels of hidden diversity: e.g., ribogroups MALV, MAST, MAOP, MAFO, deep‐sea pelagic diplonemids (DSPD), or eupelagonemids (del Campo and Ruiz-Trillo, 2013; del Campo et al., 2015; Guillou et al. 2008; Massana and Pedrós-Alió 2008; Okamoto et al. 2019; de Vargas et al., 2015), as well as a great variety of small new phylogenetic lineages associated with almost all large eukaryotic groups (del Campo et al., 2016; Keeling and Campo, 2017). New species of flagellates and centrohelids are being discovered constantly, which is indicative of a poor state of exploration and insufficient sampling.

At the same time, the issues of the geographical distribution of protists are subject to lively, but stubbornly unresolved debate (Azovsky et al. 2016). Are protists species widespread around the globe, or is their distribution, like those of macroorganisms, more governed by the rules of historical biogeography? There are two alternative points of view on this issue: the Ubiquity Model (Fenchel and Finlay 2004; Finlay et al. 1996) and Moderate Endemicity Model (Foissner 2004, 2008). Additional data are needed to resolve this issue, especially in those regions on the planet that remain unexplored.

Here we describe a microscopical investigation of the species diversity of heterotrophic flagellates and centrohelid heliozoans in marine waters of the coast of Curacao, which has not specifically been investigated previously. Only one species (Choanoeca flexa Brunet et al., 2019) was described from the coastal waters of Curacao (Brunet et al. 2019). Thirty six species of heterotrophic flagellates and one centrohelid heliozoan (Heterophrys myriopoda Archer, 1869) are known from the Caribbean Sea in general (Thomsen and Østergaard 2019; Thomsen et al. 2020; Vørs, 1993b) until recently. We find the diversity of heterotrophic protists on Curacao to be rich and intriguing, including the presence of species potentially important for clarifying previously puzzling evolutionary and ecological questions. We describe a survey of light and electron microscopical studies of the diversity of heterotrophic flagellates and centrohelid heliozoans from marine waters of several locations on Curacao, and provide micrographs and morphological descriptions of observed species, as well as discussing their distribution and potential importance.

Section snippets

Material and methods

Marine water samples were taken from eight locations around the island of Curacao in April 2018. Samples came from several biotopes (from the surface of corals and sponges, Sargassum algae wrings, sand, coral sand, and water column) both nearshore and at depths of 12–25 m (detailed descriptions of sampling points are given in Table 1). From each biotope, several replicates were taken, which were subsequently summarized. A total of 52 samples were investigated microscopically. Water temperature

Results

Eighty-six species and forms of heterotrophic flagellates and three species of centrohelid heliozoans were observed (Table 2) and listed systematically according to the system of eukaryotes by Adl et al. (2019). This system operates hierarchical nameless ranks that ignore endings of clade names. Thus, genera grouped into a clade represent a family (Adl et al., 2019). A system of asterisks was used to identify levels of taxonomic ranks from high to low (the more asterisks, the lower the rank of

Discussion

The morphology of many of the observed species was slightly different from previous descriptions of any protist. Seven flagellate species were identified only to genus level: Thecamonas sp., Colpodella sp., Cyranomonas sp., Goniomonas sp., Petalomonas sp., Ploeotia sp. 1, and Ploeotia sp. 2. These organisms may represent not yet described species of these genera, but further studies are needed. Among the 86 heterotrophic flagellates and 3 centrohelids encountered in this survey (Table 2), five

Author contributions

Light and electron microscopy, species identification, preparation of species descriptions and illustrations, writing of the original draft (KIP, DVT); statistical analysis (DVT); fieldwork and supervision (DVT, PJK); funding acquisition (DVT, PJK); manuscript review and editing (PJK).

Acknowledgments

This work was supported by grant from the Russian Foundation for Basic Research (grant no. 20-34-70049) and carried out within the framework of the project no. АААА-А18-118012690098-5 of the Ministry of Education and Science of the Russian Federation. We thank The Gordon and Betty Moore Foundation for travel support, Mark Vermeij and CARMABI research station for field sampling support, Emma George for help with sample collection, and the anonymous reviewers for their careful reading of our

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