A reappraisal of the taxonomy and biodiversity of the extant coccolithophore genus Palusphaera (Rhabdosphaeraceae, Prymnesiophyceae)

ABSTRACT The genus Palusphaera Lecal-Schlauder emend. R.E. Norris is a distinctive modern coccolithophore that accommodates monomorphic, monothecate coccospheres with one type of spine-bearing heterococcoliths. Having examined a set of scanning electron microscopy images from our collections, we were able to demonstrate that four distinct species of Palusphaera exist in the modern oceans, including the type species Palusphaera vandelii, an informally proposed form, Palusphaera sp. 1 (type robusta) that is herein taxonomically ratified as Palusphaera crosiae sp. nov., and a distinctive morphotype, which is formally described as Palusphaera bownii sp. nov. Biometric analyses and observations on the morphologies of numerous P. vandelii specimens revealed the existence of a further form, and its commonly distinguished coccolith morphotypes are therefore taxonomically separated from Palusphaera vandelii and established as a discrete species, Palusphaera probertii sp. nov.


INTRODUCTION
Coccolithophores are an abundant group of eukaryotic unicellular flagellate phytoplankton in the modern-day oceans, belonging to the phylum Haptophyta, class Prymnesiophyceae. Coccolithophores precipitate a multitude of elaborately shaped scales of calcium carbonate (coccoliths) to form a distinctive mineralized cell covering known as a coccosphere (Wallich 1877;Young et al. 1992). Although microscopic in size, coccolithophores exert a significant influence on both oceanic biogeochemistry and the global carbon cycle (Sigman & Boyle 2000;Hutchins 2011;Monteiro et al. 2016), and exhibit a vast array of coccolith morphologies as a result of a substantial variety of species-level calcite production (e.g. Monteiro et al. 2016).
Coccolithophores are one of the most comprehensively described and reliably identifiable groups of marine phytoplankton (Jordan & Green 1994;Jordan et al. 1995Jordan et al. , 2004, and their taxonomy and taxonomic documentation continue to greatly advance, with approximately 280 morphospecies having already been discovered (Young et al. 2021). This has primarily been a matter of documenting new taxa from plankton assemblages (e.g. Young et al. 2003;) and/or identifying cells during their life cycle transition (Cros et al. 2000; but, more recently, crystallographic properties of coccolith calcite and molecular genetics have been applied to calcareous nannoplankton Hagino et al. 2016) to reinforce the observations on the coccolith morphologies and correspondingly rectify important taxonomic problems. Yet, there are still several cases of enigmatic taxa with inconsistent generic use and thus their taxonomic treatment is becoming increasingly important to tackle.
The family Rhabdosphaeraceae Haeckel (1894) is a diverse group of coccolithophores definitely including six extant genera (Rhabdosphaera Haeckel, Acanthoica Lohmann, Algirosphaera Schlauder emend. R.E. Norris, Cyrtosphaera Kleijne, Discosphaera Haeckel and Palusphaera Lecal-Schlauder emend. R.E. Norris), one exclusively fossil genus (Blackites W.W. Hay & Towe) and, more tentatively, an additional extant genus Solisphaera Bollmann, M.Y. Cortés, Kleijne, J.B. Østergaard & Jer.R. Young, which, however, is excluded from the subsequent discussion, because it differs in numerous ways from the other genera (see details in Bollmann et al. 2006). The family is characterized by the formation of planolith-type coccoliths, with a distinctive rim structure and a more variable central area (Figs 1-4), often including the presence of spines (Aubry 1988;Varol 1989;Kleijne 1992;Young et al. 2003;Probert et al. 2007;van de Locht et al. 2014;Bown et al. 2017; Table 1). The rim is almost always narrow and formed of two cycles of elements: a broad outer/upper cycle of V-units with subradial sutures and a narrow inner/lower cycle of R-units (e.g. Fig. 2; see discussion in Bown et al. 2017) showing strong sinistral obliquity in proximal view. The central area, excluding the process, is typically formed of two cycles of units. First, a radial lath cycle, adjacent to the rim, composed of well-formed laths often with gaps between them, whose ends interdigitate regularly with the elements of the rim. Second, a lamellar cycle of less regular elements. The lamellar cycle is present in all genera, but the radial cycle is absent in Rhabdosphaera and Palusphaera. The central process is the most variable part of the structure, showing a wide range of shapes and morphologies, and is absent from some of the coccolith types on the coccospheres of Acanthoica and Rhabdosphaera.
Within this group, Palusphaera is distinguished from the other genera by forming monomorphic coccospheres with body coccoliths, all bearing spines and lacking a radial cycle. Currently, there is only one formally described species in the genus, Palusphaera vandelii Lecal-Schlauder emend. R.E. Norris, but one other morphotype has been reported informally several times. Here, we also report on two further extant morphotypes of the genus and provide formal descriptions of all currently known forms.

MATERIAL AND METHODS
We have examined the scanning electron microscope (SEM) images of extant Palusphaera specimens from our collections. The material used in the present study comes from seawater samples collected in different marine basins (Fig. 5) and from several sampling projects carried out over the last two decades. The methodology used here is analytically explained below in chronological order and according to the sample source. Information about all used samples is given in Table 2.

ATLANTIC AND PACIFIC OCEANS
Water samples were collected aboard the R/V Poseidon during the P233 Expedition B (Knoll et al. 1998) in the Canary Islands, northeastern Atlantic Ocean in September 1997 and during the M68-03 Expedition in the Mauretania upwelling, northeastern Atlantic Ocean onboard the R/V Meteor in April-August 2006 (Koschinsky et al. 2009). Specimens of Palusphaera were also found in seawater samples that were obtained from the Miyake Island, western Pacific Ocean in November 1999 and from the eastern Pacific Ocean in October-December 2004 during the oceanographic cruise BIOSOPE aboard the R/V L'Atalante (Claustre & Sciandra 2004). Samples also originate from surface waters of the South Atlantic Ocean during the Atlantic Meridional Transect cruises AMT16 (Robinson et al. 2006) and AMT18 (Woodward 2009)

Palusphaera vandelii
Body coccoliths that are circular in outline. Spine is styliform, long and tapering with spirally arranged crystal segments near its base.

Palusphaera probertii
Body coccoliths that are broadly elliptical in shape. Spine is styliform and tapering with crystallites parallel oriented to its axis.

Palusphaera crosiae
Body coccoliths that are nearly circular. Spine is styliform and markedly thicker at onethird to one-quarter height from the base of the spine.

Palusphaera bownii
Body coccoliths that are circular in shape. Spine is salpingiform and short.

WESTERN MEDITERRANEAN SEA
Samples have been obtained from the surface waters of the northwestern Mediterranean and Alboran Seas on board the R/V Hesperides during the oceanographic cruise MATER II in September-October 1999 (Font 1999).

ADRIATIC SEA
A time series sample used in the present work comes from the surface waters (5 m water depth) of a coastal station (45° 04.8'N, 13°36.6'E) off Rovinj, Croatia, in the northeastern Adriatic Sea in March 2009 aboard the R/V Villa Velebita (as described in Godrijan et al. 2018).

Filtration and SEM
Seawater samples were obtained via 5-litre Niskin bottles that were attached to CTD rosette samplers, except for samples originating from the Miyake Island that were collected using a bucket. Approximately 1 litre of seawater was filtered through 0.8-μm pore size Whatman Nuclepore polycarbonate track-etched filters of 25 mm diameter apart from P233-B cruise, during which 4 litres of seawater were filtered onto 47-mm cellulose nitrate filters (Sartorius) of 0.45 μm pore size. A low-pressure vacuum pump was used during the filtration process to prevent mechanical damage of the coccosphere specimens. The filters were subsequently ovendried at 40°C and stored in 47-mm Millipore plastic Petri dishes; a portion of each filter was cut out and fixed onto aluminium stubs to be gold-coated. Examination of the samples was then performed on a Phillips XL-30 FEG field emission SEM at the facilities of the Natural History Museum, London.

Terminology and morphology
Morphological descriptions are given based on the guidelines of Kleijne (1992) and . With regards to the terminology of Rhabdosphaeraceae cycles, we follow the scheme proposed by Kleijne (1992), as also used by Young et al. (2003) and Bown et al. (2017). All morphological measurements presented in this study were obtained using the image analysis software programme ImageJ (Schneider et al. 2012).

Separation of P. probertii sp. nov. from P. vandelii
Our examination of SEM micrographs yielded in total 24 P. vandelii-like coccospheres (14 specimens of P. vandelii and 10 specimens of P. probertii sp. nov.; Table 2). These forms, however, were seen to display considerable variations in their coccolith morphologies and diameters. In order to assess this variability, we subjectively separated specimens with typical P. vandelii characteristics (i.e. circular coccolith outline, larger coccolith size and spine structure with spirally oriented elements) vs specimens that bore coccoliths of noticeably more elliptical shape, smaller size and a spine structure with elements parallel aligned to the long axis. We   Specimens that are not illustrated but were studied are indicated with an asterisk (*).
subsequently performed morphometric analyses on several of these specimens and measured 95 (in total) well-preserved body coccoliths (BCs) in plan view; these allowed a straightforward biometric analysis and correspondingly yielded more objective data (Table S1). There were 35 typical P. vandelii coccoliths from five coccospheres and 60 well-differentiated forms from four wellpreserved coccosphere specimens. The morphometric frequency plots (Fig. 6) indicated a clear bimodal distribution pattern of all studied specimens with an obvious clustering at 1.3 μm to 2.3 μm (mean 2.0 μm) for P. vandelii and a cluster group at 0.9 μm to 1.9 μm (mean 1.4 μm) for all other specimens. This well-defined separation pattern was further supported by our coccolith width data (Fig. 6), with specimens of P. vandelii demonstrating higher width values (>1.5 μm). Hence, two different coccolith size groups could be seen, essentially confirming our initial morphologic grouping, and we therefore taxonomically separated them and proposed them as two discrete taxa.

Taxonomy
Division Haptophyta D.J. Hibberd (1972) ex Edvardsen & Eikrem in Edvardsen et al. (2000) Class Prymnesiophyceae D.J. Hibberd (1976) emend. Cavalier-Smith et al. (1996) Order Syracosphaerales W.W. Hay (1977) emend. Jer.R. Young et al. (2003) Family Rhabdosphaeraceae Haeckel (1894) Genus Palusphaera Lecal-Schlauder emend. R.E. Norris EMENDED DESCRIPTION: Coccosphere monomorphic and monothecate, with only spine-bearing BCs and no differentiated circum-flagellar coccoliths. BCs flat, with two rim cycles, an outer/upper cycle with nearradial sutures, an inner/lower cycle showing strong sinistral obliquity in proximal view. The radial lath cycle shown by many other Rhabdosphaeraceae is absent; instead, the lamellar cycle fills the central area. The lamellar cycle of tabular elements shows dextrogyral obliquity in distal view and laevogyral obliquity in proximal view. Central process is a spine, variable in shape but without a collar; proximal side with a central pore surrounded by several (usually three) angular nodes. TYPE SPECIES: Palusphaera vandelii Lecal-Schlauder (1966) emend. R.E. Norris (1984). The original author published under the name Lecal. EMENDED DESCRIPTION: Coccosphere monomorphic, spherical but usually seen collapsed, composed of c. 50 BCs. BCs planoliths with a subcircular to circular outline; rim thin with typical Rhabdosphaeraceae rim structure. Lamellar cycle fills central area, convex on distal side and concave on the proximal side. Lamellar cycle elements rod shaped, overlapping and displaying dextrogyral curvature on the distal side and laevogyral in proximal view (e.g. Fig. 10). Central process with a central pore in proximal view, usually surrounded by two to three small nodes; distal side with a thin and long spine, formed of numerous spirally arranged elements at the base becoming less spiral upwards. Spine is styliform-shaped without a collar. DESCRIPTION: Coccosphere, monomorphic and spherically shaped, but usually seen collapsed, composed of c. 45-60 BCs. BCs broadly elliptical to subcircular in plan view, with narrow rims and nearly flat bases. Rim with typical Rhabdosphaeraceae rim structure. Central area filled by a lamellar cycle of numerous rod-shaped, slightly overlapping crystal segments; no radial cycle is observed. Central process bears a thin, hollow, styliform spine with no collar; spine structure composed predominantly of numerous elongate elements arranged parallel to the axis of the spine. The spine is remarkably long compared to the coccosphere diameter. The proximal planolith side has three robust angular nodes around the central pore.  Fig. 7. Complete coccosphere. Fig. 8. Specimen with coccoliths that bear planolith bases with usually three nodes in proximal side (arrow). Fig. 9. Collapsed coccosphere with circular coccoliths; inner rim cycle is typically wider in proximal side (arrow). Fig. 10. Detailed view of Fig. 9; arrow shows the laevogyral curvature of the crystal elements of the lamellar cycle, observed in proximal view. Scale bar = 2 μm. Fig. 11. Disarticulated coccoliths bearing spines with spirally arranged crystal segments, highly visible towards the central process (arrows).
DESCRIPTION: Coccosphere, monomorphic and generally seen collapsed but probably spherical in shape, with c. 45-60 BCs. BC bases broadly elliptical in outline and slightly concavo-convex. Rim shows typical Rhabdosphaeraceae rim structure. Central area filled by the lamellar cycle; no radial cycle is present. Central process is a long, tapering spindleshaped spine. Maximum thickness is at one-third to one-quarter of the height and it tapers to a fine point. The spine is formed from robust laths, c. 1.0 μm × 0.15 μm at the base, becoming smaller towards the tip; they abutt neatly to form a 6-to 8-μm sided smooth hollow structure.   Fig. 12. Holotype and complete coccosphere specimen. Scale bar = 2 μm. Fig. 13. Detailed view of a specimen with broadly elliptical planoliths showing a central pore and three angular nodes on the proximal side (a) and clockwise imbrication on the distal side (b); broken spine (c) is long and thin, formed of crystallites arranged parallel to the spine axis. Scale bar = 2 μm. Fig. 14. Collapsed coccosphere. Scale bar = 5 μm. Fig. 15. Incomplete coccolith in distal view; crystal elements show dextrogyral curvature (arrow). Scale bar = 1 μm. Fig. 16. Detailed view of a broadly elliptical coccolith, small in size, and its broken styliform process. The lath-like crystal segments of the spine are arranged parallel to its long axis (see arrow). Scale bar = 1 μm.
typical Rhabdosphaeraceae rim structure. Central area, filled by lamellar cycle, with overlapping tabular elements showing clockwise curvature in distal view. The radial cycle is absent. Central process is a trumpetshaped spine without a collar, formed of vertically elongate elements arranged in spiralling series; the distal end is formed of blockier elements, but these appear to have developed from the spiral elements of the spine. Trumpet aperture wider than the basal spine width. On the proximal side of the coccolith there is a central pore surrounded by two to three nodes.
HOLOTYPE: 302-04 (specimen illustrated in Fig. 22 REMARKS: We have only found two coccospheres of this species and to our knowledge no other specimens have been published. Nonetheless, this is a distinctive form and clearly separate from any other coccolithophore, so we are confident it is a discrete species. The species shows possible affinities to several genera. The trumpet-like spines resemble those of Discosphaera, but the base lacks a radial cycle, and the spine shows a structure that is not seen in Discosphaera. The spine structure is closer to that of Rhabdosphaera, but in Rhabdosphaera coccospheres are dimorphic with both spine-bearing and non-spinebearing BCs. Finally, the species resembles Palusphaera in being monomorphic and lacking a radial cycle. In addition, the rows of the laminar cycle elements show the same curvature (clockwise in distal view) as that of Palusphaera coccoliths and the opposite of that shown by Rhabdosphaera coccoliths. Therefore, we place the species in Palusphaera.

DISCUSSION
The genus Palusphaera is a distinctive extant coccolithophore, known only from the heterococcolith-bearing phase, and based on our observations it now comprises four discrete taxa   Fig. 17; arrow indicates the part of the spine being markedly thicker at the one-third to one-quarter height from the base. Fig. 19. Complete coccosphere with subcircular planoliths. Fig. 20. Detailed view of Fig. 19; planoliths with (a) a relatively wide outer rim cycle, (b) a styliform spine formed at the centre of the lamellar cycle in distal side, and (c) three robust angular nodes surrounding the central pore in proximal side. Scale bar = 2 μm. Fig. 21. Side view of styliform processes of disarticulated spine-bearing planoliths. Scale bar = 1 μm.
( Fig. 25), namely, P. vandelii, P. probertii sp. nov., P. crosiae sp. nov. and P. bownii sp. nov., which show a range of shared morphological characteristics. All of these species possess monomorphic and monothecate coccospheres composed of spine-bearing BCs, without differentiated circum-flagellar coccoliths. The BCs consist of solid circular to elliptical planolith bases with a rim and an imbricate central area (see Fig. 25; Table 1). The rim, as in all typical Rhabdosphaeraceae (e.g. Fig. 1; Kleijne 1992;Bown et al. 2017) is formed of two cycles: an outer and upper cycle with radial sutures and an inner and lower cycle with strongly oblique sutures, showing sinistral obliquity in proximal view. Unlike Acanthoica, Algirosphaera, Cyrtosphaera and Discosphaera, there is no radial lath cycle present. Instead, the central area is closed by the lamellar cycle of subrectangular, slightly overlapping crystallites. In proximal view, these can be seen to be arranged in rows showing laevogyral curvature; i.e. they appear bent to the left as they run from the centre to the rim; the proximal surface of the central process typically shows a central pore that is surrounded by a limited number of nodes, usually two to three (e.g. Fig. 20). In distal view, lamellar cycle segments show dextrogyral curvature but this is less obvious due to overlap of elements. The central elements of the lamellar cycle extend outwards to form an imbricate spine in distal view. The spines are formed of butting segments that disintegrate into separate elements (e.g. Figs 16, 18, 21), and they do not bear collars at their bases. Palusphaera is most obviously separated from Rhabdosphaera by being monomorphic; all of the BCs have spines, whereas in Rhabdosphaera there is an outer layer of non-spine-bearing coccoliths. In addition, however, our observations suggest that two other features consistently separate the four Palusphaera species from the two extant Rhabdosphaera species. First, spines in Rhabdosphaera species are robust solid structures formed of intergrown elements, whereas those of Palusphaera are delicate hollow structures formed of directly abutting elements. Second, the lamellar cycle elements in Rhabdosphaera show the opposite sense of curvature to those in Palusphaera; i.e. they show laevogyral curvature on the distal side and dextrogyral on the proximal side.
Consequently, the observations reported in our study conclusively supported a proposal to emend the description of both Palusphaera and Palusphaera vandelii. In parallel, based on the well-differentiated subset of taxa within the Rhabdosphaeraceae, our results provided the evidence needed to formally maintain Palusphaera separable from its sister taxa and consider it a clearly diagnosable and taxonomically valid genus. Vaulot and Masanobu Kawachi for their assistance in obtaining seawater samples and/or with the scanning electron microscope analyses. We gratefully acknowledge Jelena Godrijan for providing the sample from the Adriatic Sea used in this study. Special thanks are due to Giles Miller for his assistance in cataloguing our material at the Natural History Museum London, UK. We also extend our gratitude to the editor and the two anonymous reviewers for their insightful suggestions that greatly improved the quality of the article. The work contains data supplied by the Natural Environment Research Council.

FUNDING
This work is a contribution to the EC-TMR research project CODENET, Coccolithophorid Evolutionary Biodiversity and Ecology Network (Contract No. ERB-FRMX-CT97-0113). OAA was supported by the UK Natural Environment Research Council under Grant number NE/S007474/1.  Fig. 22. Holotype. Collapsed coccosphere bearing nearly circular planolith bases with outer rim cycles that are broader on the distal side (arrow a) than on the proximal side (arrow b); the central process displays a short trumpet-shaped process (arrow c). Fig. 23. Collapsed specimen bearing trumpet-shaped spines with spirally arranged crystal segments (see arrow) extending outwards forming distinct apertures. Fig. 24. Detailed view of Fig. 23 showing the ultrastructure of the spine aperture (arrows) and the dextrogyral curvature of lamellar elements in distal view.