Journal of Plankton Research academic.oup.com/plankt J. Plankton Res. (2022) 44(6): 846–850. First published online August 18, 2022 https://doi.org/10.1093/plankt/fbac044 BRIEF COMMUNICATION Novel combination coccospheres from Helicosphaera spp indicate complex relationships between species DANIELA STURM 1,2, *, GERALD LANGER3 AND GLEN WHEELER1 1 the marine biological association of the united kingdom, the laboratory, citadel hill, plymouth pl1 2pb, devon, uk, 2 ocean and earth science, university of southampton, university rd, highfield, southampton so14 3zh, hampshire, uk and 3 institute of environmental science and technology (icta), universitat autonoma de barcelona, carrer de les columnes s/n, barcelona 08193, spain *corresponding author: danstu@MBA.ac.uk Received April 28, 2022; editorial decision July 28, 2022; accepted July 28, 2022 Corresponding editor: Lisa Campbell Coccolithophores play an important role in global biogeochemical cycling, but many aspects of their ecology remain poorly understood, including their heteromorphic haplo-diplontic life cycle. The presence of combination coccospheres in environmental samples, which represent a transition between the lightly calcified haploid (HOL) and heavily calcified diploid (HET) life phases, provides crucial evidence linking the two life cycle phases of a particular species. Here, we describe combination coccospheres from the Southern Ocean that show a novel association between Helicosphaera hyalina (HET) and Helicosphaera HOL catilliferus type. The ability of Helicosphaera HET and HOL morphospecies to form multiple different combinations indicates a substantial complexity in the relationships between life cycle phases in this group. The findings suggest recent divergence within the Helicosphaera lineage may have resulted in significant inter- and intra-specific variability, with cryptic speciation in one or both life cycle phases contributing to their ability to form multiple HET/HOL associations. KEYWORDS: coccolithophores; life-cycle; combination-coccosphere; haplo-diplontic; Southern Ocean available online at academic.oup.com/plankt © The Author(s) 2022. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. D. STURM ET AL. NOVEL COMBINATION COCCOSPHERES INTRODUCTION Coccolithophores (Haptophyta) are calcified unicellular phytoplankton that play an important role in biogeo- chemical cycling. They exhibit a haplo-diplontic hetero- morphic life cycle that remains poorly understood (Frada et al., 2019). Coccolithophores exist in either haploid (1N) or diploid (2N) life cycle phases that can each undergo asexual reproduction, although the factors that trigger a switch between life cycle phases remain unclear. Both phases are morphologically distinct, with the heavily cal- cified 2N phase producing the characteristic heterococ- coliths (HET) and the usually lightly calcified 1N phase producing holococcoliths (HOL), which are formed from small rhombohedral crystallites (Young et al., 2003). The HET and HOL life cycle phases exhibit distinct but over- lapping geographical distributions that may allow species to expand their ecological niche (De Vries et al., 2021; Frada et al., 2019). As life cycle transitions are not commonly observed in laboratory cultures, environmental observations of com- bination coccospheres have provided crucial information for the study of coccolithophore life cycles. Combination coccospheres contain coccoliths from the previous and the new life-cycle phase, i.e. both heterococcoliths and holo- coccoliths. Critically, they have allowed researchers to link morphologically distinct cell types that were previously thought to be different taxa, but are in fact the HOL and Fig. 1. Scanning electron micrographs of Helicosphaera from the South- HET phases of the same species (Triantaphyllou et al., ern Ocean. A: HET phase of Helicosphaera hyalina. Note that there is no 2015; Young et al., 2020). However, combination cocco- transverse bar separating two aligned openings of heterococcoliths, as spheres have not been recorded for all species and in some in H. carteri, B: HOL phase of Helicosphaera catilliferus type, C–F: Com- bination coccospheres of H. hyalina (HET) and H. HOL catilliferus type cases relationships between HET and HOL phases have from the Southern Indian Ocean. Note the holococcolith protruding out not been straightforward, indicating that much remains from in between the heterococcoliths in image E. Scale bars 2 μm. to be learnt about coccolithophore life cycles. In this study, we examined the occurrence and iden- tity of combination coccospheres in the diverse coccol- represented a novel association between Helicosphaera ithophore communities found within the Great Calcite hyalina (HET) and H. HOL catilliferus type (Fig. 1C–F). Belt (GCB) of the Southern Ocean (Balch et al., 2011). The heterococcoliths possessed well-developed wings in the distal flange and a closed central area (Fig. 1A). Holococcoliths were elliptical with a sharply pointed METHODS central protrusion (Cros and Fortuño, 2002) (Fig. 1B). All combination coccospheres were nearly perfectly Water samples for coccolithophore analysis were collected preserved, suggesting that they do not represent a from the surface mixed layer in February 2020 within a sampling artifact e.g. from collisions between cells. mesoscale meander of the Southern Subtropical Front (STF) in the Southern Indian Ocean (38.5–41.5◦ S, 30.9– 35.5◦ E). We vacuum-filtered 150 mL of seawater, pre- pared samples for scanning electron microscopy (SEM) DISCUSSION and imaged as described in Langer et al. (2021). Helicosphaera are medium to large species with heavily cal- cified heterococcoliths and a global distribution (Šupraha and Henderiks, 2020). There is continued uncertainty RESULTS about the relationships between HET and HOL mor- All identified combination coccospheres (n = 7) belonged photypes within Helicosphaera, as multiple combination to the genus Helicosphaera (Order Zygodiscales) and coccospheres have been described and there does not 847 JOURNAL OF PLANKTON RESEARCH VOLUME 44 NUMBER 6 PAGES 846–850 2022 Fig. 2. Schematic showing the currently known combination coccospheres of the group Helicosphaera spp based on the HOL morphotype nomenclature established by Young and Bown (2014). SEM images of HET H. pavimentum, wallichii, hyalina and HOL types dalmaticus, ponticuliferus and confusus were used from Nannotax3 with permission. Scale bars 2 μm. appear to be a simple 1:1 correlation of HET and HOL combination coccospheres. For clarity, we have used types, as in most other coccolithophore groups (Fig. 2). H. the term morphotypes below to differentiate between wallichii (HET) has been associated with the HOL types cell types, although their exact relationships remain ponticuliferus and catilliferus (Couapel et al., 2009; Young unclear. and Bown, 2014). The weak evidence for an additional H. hyalina HET and H. wallichii HET were initially interaction with HOL type dalmaticus probably represents regarded as varieties of H. carteri HET due to their similar an accidental association (Geisen et al., 2004; Young and morphology (Cros and Fortuño, 2002; Jordan and Green, Geisen, 2002). The HOL type catilliferus has in addi- 1994). Analysis of fossil Helicosphaera populations over tion been found in combination with HET coccoliths the last 15 Myr indicates an evolutionary trend favoring of Helicosphaera carteri, and the HOL type dalmaticus is smaller celled species (like H. hyalina) that may have been also associated with H. pavimentum (HET) (Cros et al., driven by the prevalence of oligotrophic conditions and 2000; Young et al., 2020). Our finding adds the fur- could have contributed to divergence within this lineage ther combination of H. hyalina (HET) and HOL type (Šupraha and Henderiks, 2020). catilliferus. Molecular evidence is now needed to unveil whether These observations render the question whether (1) these Helicosphaera morphotypes represent distinct species, these Helicosphaera morphotypes represent a single species and if they do, when and how they emerged. Sequencing where genetic variability leads to the formation of distinct of the fast-evolving chloroplast gene tufA showed multiple coccolith morphologies, or (2) different Helicosphaera substitutions between H. carteri and H. hyalina, suggesting morphotypes represent diverging species that have not yet a recent pseudocryptic speciation event (Sáez et al., 2003). lost (or have regained) the ability to hybridize (introgres- However, there is currently no genetic information for sive hybridization), enabling the formation of multiple any other Helicosphaera HET or HOL morphotypes that 848 D. STURM ET AL. NOVEL COMBINATION COCCOSPHERES would allow a comparison with H. carteri. Our newly FUNDING described association between H. hyalina and H. HOL This work was supported by the Natural Environmental catilliferus indicates that H. hyalina does in fact belong to Research Council [grant number NE/SO07210/1] as the H. wallichii–carteri species complex. 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