Morphological and Phylogenetic Characterisation of Prorocentrum spinulentum, sp. nov. (Prorocentrales, Dinophyceae), a Small Spiny Species from the North Atlantic

Prorocentrum comprises dinophytes with several unique traits, including the presence of two large thecal plates and apical insertion of flagella. Species delimitation for many small and similar planktonic species is challenging, as SEM analyses and DNA sequence information of type material are rarely available. Based on a strain from the North Atlantic Prorocentrum spinulentum, sp. nov. is described here. Cells were small (9.0–12.8 µm long, 8.5–11.9 µm deep), oval to almost round in lateral view and moderately compressed. The ovoid nucleus was in median or slightly sub-median position on the cells ventral side. The plate surface appeared spiny in light microscopy with thecal pores visible in empty thecae. Electron microscopy revealed plates densely covered by relatively long spines and two size classes of thecal pores. The periflagellar area consisted of 8 platelets, and there was a prominent wing (ca. 1 µm wide and long) on platelet 1. The new species is distinct in DNA trees and embedded in the Prorocentrum shikokuense species group. It differs from the protologues of other small species of Prorocentrum by the unique combination of cell size and shape, the presence of long spines on the thecal plate surface and scattered thecal pores. The thorough morphological description of this species, representing a previously uncharacterised lineage within Prorocentrum, increases and improves our knowledge of the diversity within this important group of planktonic organisms.


Introduction
Prorocentrales (Dinophyceae) have a peculiar morphology characterised by the presence of two major large thecal plates with a distinct sagittal suture, the lack of cingulum and sulcus and an apical flagella insertion (desmokont flagellation). Both flagella arise from one pore in the periflagellar area, which consists of tiny platelets and an additional accessory pore [1,2]. Prorocentrum Ehrenberg is diverse and comprises predominantly marine species with a worldwide distribution [1,2]. Identification of small (<20 µm), planktonic species of Prorocentrum is challenging [3]. They are inconspicuous, and differentiating traits, such as surface ornamentation of thecal plates, number and distribution of thecal pores and the presence/absence of apical projections, are difficult to observe unambiguously in light microscopy (LM). Moreover, most small species of Prorocentrum have been described a long time ago, and thus, detailed information on morphology or DNA sequence data linked to the types, or other original material, are lacking [3].
A good example of taxonomic confusion and ambiguity in Prorocentrum is a group, which contains sequences in molecular phylogenetics assigned to a variety of taxa such as Prorocentrum dentatum F.Stein, Prorocentrum donghaiense D.D.Lu, Prorocentrum obtusidens J.Schiller and Prorocentrum shikokuense Hada. Identity and synonymy of these taxa is  [4,5], but ITS sequence data indicate that all may represent the same species. We agree with Gómez et al. [5] that the few morphological documentations of such strains conform with P. shikokuense (=P. donghaiense). In contrast, both P. dentatum (having a very characteristic shape) and P. obtusidens (being much larger) are morphologically different, and reliable sequence data for these taxa have not yet been obtained. We thus here refer to this group as the P. shikokuense species group.
Different from the old names of Prorocentrum, Prorocentrum nux Puigserver & Zingone has been described using scanning electron microscopy (SEM) [6] but even for this species, the only sequence data available in GenBank are from strain RCC303 (without accompanying morphological documentation) and not from the authentic strain pronap1, from which the type material has been prepared. Prorocentrum ponticum Krakhmalny & Terenko (nom. corr.: ICN Art. 23.5) is also documented using SEM, but no sequence data linked to type material have been gained [7]. In turn, a new clade of Prorocentrum has been identified based on ribosomal RNA (rRNA) sequence data referring to small cells identified as Prorocentrum cf. balticum (Lohmann) A.R.Loeblich, but their morphology is illusive at present [8].
The description of the new small, planktonic species Prorocentrum pervagatum Tillmann, Hoppenrath & Gottschling is based on multiple cultured strains from geographically distant localities and by combining high resolution LM and SEM with DNA sequence information [3]. Only a short time later, another small species, P. thermophilum F.Gómez, Tangcheng Li, Hu.Zhang & Senjie Lin, was described, together with other strains of P. pervagatum (=P. criophilum Gourvil & Gutiérrez-Rodríguez, syn. nov.) [9]. Such studies, combining molecular and morphological approaches, are the prerequisite for a solid taxonomic basis, facilitating a comprehensive comparison and reliable delimitation of other small species of Prorocentrum. Based on a strain isolated from the Celtic Sea (North Atlantic) in 2018, we provide here a next step in the characterisation of another small and new species of Prorocentrum using an integrative taxonomy approach.

Sampling, Cell Isolation, Cultivation
A single strain of Prorocentrum (1-D3) was established from a surface water sample (salinity: 34.9; surface temperature: 19.4 • C) collected at the Celtic Sea (51 • 1.31 N; 9 • 4.21 W) during a cruise aboard the research vessel FS. Heincke in 2018. Single cells were isolated by micropipetting under a stereomicroscope (M5A; Wild, Heerbrugg, Switzerland) and transferred into individual wells of 96-well tissue culture plates (TPP, Trasadingen, Switzerland), each containing 250 µL of K-medium [10] prepared from 0.2 µm sterilefiltered North Sea water diluted 1:10 with filtered seawater from the sampling locality. The original K-medium receipt was slightly modified by replacing the organic phosphorous source by 3.62 µM Na 2 HPO 4 . Plates were incubated at 15 • C under dim light (30 µmol photons m −2 s −1 ) in a controlled environment growth chamber (MIR 252; Sanyo Biomedical, Wood Dale, USA-IL). After 3-4 weeks, the strain was inoculated for batch culture in a 65 mL polystyrene cell culture flask. Growth medium was enriched with nutrients corresponding to 50% of K-medium.
For DNA extraction, cells were collected by centrifugation (5810R; Eppendorf, Hamburg, Germany) in 50 mL centrifugation tubes at 3220× g for 10 min. Cell pellets were transferred to 1 mL microtubes, then again centrifuged (5415; Eppendorf) at 16,000× g for 5 min and stored frozen (−20 • C) for subsequent DNA extraction. In addition, the strain was grown and harvested as described above for lipophilic toxin analysis and stored at −20 • C until use. For each harvest, cell density was determined by settling Lugol-fixed samples and counting >400 cells under an inverted microscope.

Microscopy
Observation of living or fixed cells (formaldehyde: 1% final concentration, or neutral Lugol-fixed: 1% final concentration) was carried out using an inverted microscope (Axiovert 200M; Zeiss, Jena, Germany) and a compound microscope (Axioskop 2; Zeiss), both equipped with epifluorescence and differential interference contrast optics. Light microscopic examination of thecal plates was performed using epifluorescence microscopy of cells stained with calcofluor white [11]. The shape and location of the nucleus was determined after staining of formalin-fixed cells with 4 -6-diamidino-2-phenylindole (DAPI, 0.1 µg mL −1 final concentration) for 10 min. Images were taken either with a digital camera (Axiocam MRc5; Zeiss), or videos were recorded using a digital camera (Gryphax; Jenoptik, Jena, Germany) at full-HD resolution. Single frames were then extracted using Corel Video Studio software (Version X8; Corel, Ottawa, ON, Canada). Cell length and depth of freshly fixed cells (neutral Lugol) from dense but healthy and growing strains (based on stereomicroscopic inspection of living material) during late exponential phase were measured at microscopic magnification of 1000× using the compound microscope and the Axiovision software (Zeiss).
For SEM, cells were collected by centrifugation of 15 mL culture (5810R; Eppendorf) at 3220× g for 10 min. The supernatant was removed and the cell pellet re-suspended in 60% ethanol prepared in a 2 mL microtube with seawater (final salinity ca 13) at 4 • C for 1 h in order to strip off the outer cell membrane. Cells were further collected by centrifugation (5415R, Eppendorf) at 16,000× g for 5 min and re-suspended and fixed in a 60:40 mixture of deionised water and seawater (final salinity ca. 13) with the addition of formaldehyde (1% final concentration) and stored at 4 • C for 3 h. Cells were collected on polycarbonate filters (Millipore Merck; Darmstadt, Germany; 25 mm Ø, 3 µm pore-size) in a filter funnel, in which all subsequent washing and dehydration steps were carried out. A total of eight washing steps (2 mL MilliQ-deionised water each) were followed by a dehydration series in ethanol (30%, 50%, 70%, 80%, 95%, 100%; 10 min each). Filters were dried with hexamethyldisilazane (HMDS), first in 1:1 HMDS:EtOH, followed by twice 100% HMDS and then stored in a desiccator under gentle vacuum. Finally, filters were mounted on stubs, sputter coated (Emscope SC500; Ashford, UK) with gold-palladium and viewed at 10 kV under a SEM (FEI Quanta FEG 200; Eindhoven, The Netherlands). Micrographs were presented on a black background using Photoshop 6.0 (Adobe Systems; San Jose, CA, USA).

Molecular Phylogeny
A systematically representative set of prorocentralean and related accessions was compiled from known reference trees, similarly to a previous approach [3]. The taxon sample was enriched by all those accessions showing ultimate similarity to the sequences gained from strain 1-D3, as inferred from BLAST searches [17]. Voucher information is provided in Table S1, which also includes GenBank accessions of Prorocentrum spinulentum (OQ220500, OQ220501), and outgroup details comprising Dinophysales and Gymnodiniales. For alignment, separate matrices of the rRNA operon (i.e., SSU, ITS, LSU) were constructed, aligned using MAFFT v6.502a [18] and then concatenated. The aligned matrices are available as a file named 'spinulentum.nexus' upon request. Phylogenetic analyses were carried out using maximum likelihood (ML) and Bayesian approaches as described previously [19].

Terminology
Terminology of cell orientation, designation of thecal plates and platelets and ornamentation follows Hoppenrath et al. [2] and includes some additions and modifications as discussed by Tillmann et al. [20].

Formal Description
Prorocentrum spinulentum Tillmann, Gottschling & Hoppenrath, sp. nov. (Figures 1-3). Description: Small, photosynthetic, thecate, prorocentroid Dinophyceae with desmokont flagellation; cell shape irregularly oval to round in lateral view or rarely heart-shaped, 9.0-12.8 µm in length, 8.5-11.9 µm in depth, with a mean l/d ratio of 1.1. Cells slightly compressed laterally after division or almost globose in older cells with a broad and transversely striated intercalary band; two reticulate chloroplasts, an oval-shaped nucleus in ventral position and a pusule in apical position close to the flagellar pore. Apical projection visible in LM. Thecal plates covered by elongated spines, with thecal pores of two different size classes, in total about 30 pores per plate, scattered mainly towards the plate margin or arranged in two short lines close to the thecal centre. A cluster of three to four large pores located on the right theca in apical ventral position. Periflagellar area composed of 8 platelets, accessory and large flagellar pores, with projections present on platelets 1, 2, 3, 5, 6 and 8. An elongated conspicuous wing (width and height ca. 1 µm each) running all along the right margin of platelet 1 as the most prominent apical projection.
Holotype: SEM-stub prepared from clonal strain 1-D3 (designated CEDiT2023H159) deposited at the Senckenberg Research Institute and Natural History Museum, Centre of Excellence for Dinophyte Taxonomy, Germany.
Isotypes: Formalin-fixed sample prepared from clonal strain 1-D3 (designated CEDiT2023I160) deposited at the Senckenberg Research Institute and Natural History Museum, Centre of Excellence for Dinophyte Taxonomy, Germany.
Etymology: The epithet (Lat. spinulentus = spinose) reflects the spinose surface of both lateral plates provided by rather long spines (compared to the small cell size).

Light Microscopy
Cells were asymmetrically oval to round or rarely heart-shaped in lateral view ( Figure 1). Cell length ranged from 9.0 to 12.8 (mean 10.9 µm ± 0.8 µm, n = 50), and cell depth ranged from 8.5 to 11.9 (mean 10.3 µm ± 0.8 µm, n = 50), with a mean l/d ratio of 1.07. Low magnification observation of living and swimming/turning cells indicated a wide range of length/width ratio ( Figure 1G,H) ranging from ca. 0.6 to 0.9, with widest cells dominating in dense cultures. Wide cells had a broad and transversely striated growth band visible in LM ( Figure 1H,I). Both plates were covered by spines visible in LM ( Figure 1E). Thecal pores were difficult to detect in living or fixed cells in brightfield or differential interference contrast microscopy, but were faintly visible on empty thecae ( Figure 1J) and most easily seen in calcofluor-stained cells under UV excitation ( Figure 1K,L). An apical projection was occasionally visible in LM ( Figure 1B-E). Two yellow-orange, reticulate chloroplasts were arranged parietally adjacent to the thecal plates, and no pyrenoid was observed in LM. A small, round, hyaline, vacuole-like structure, the pusule, was occasionally visible in the anterior area close to the flagellar pore ( Figure 1A,B). Long, rod-like structures, presumably trichocysts, were visible in LM and mainly arranged along the cell's longitudinal axis ( Figure 1F). The nucleus was oval in outline and located in the ventral area of the cell in median or submedian position ( Figure 1M-P). One large flagellar pore and a distinctly smaller accessory pore in apical position could be adumbrated in LM ( Figure 1F) and was clearly visible in calcofluor-stained cells under UV excitation ( Figure 1K-M).

Scanning Electron Microscopy
The two thecal plates were uniformly covered by spines (Figure 2), which ranged from 0.30-0.43 µm (Table 1) in length. At the base of each spine, there were three to five radial extensions ( Figure 2L). Mean areal density of spines ranged from 4.2 to 5.0 µm −2 ( Table 1). The transverse striation of the intercalary band consisted of rows of spines ( Figure 2B-E). Details of the thecal pores could be revealed ( Figure 2I-L). Larger pores were about 0.23 µm in diameter (Table 1) and bordered by a crater-formed rim at the outer plate surface (Figure 2I,L). Small pores were about 0.14 µm in diameter (Table 1) and were also bordered by a small elevated rim ( Figure 2L). A cluster of two to three large pores accompanied by several small pores were characteristically visible on the right thecal plate in apical ventral position ( Figure 2I,J). Other pores were scattered on both thecal plates with two distinct rows of pores close to the plate centre (Figure 2A-D,F). There were about 30 pores per plate (mean: 32 ± 6, range 20-51), only a few (ca. 3-5) of them were large (the exact number was difficult to determine).

Scanning Electron Microscopy
The two thecal plates were uniformly covered by spines (Figure 2), which ranged from 0.30-0.43 µm (Table 1) in length. At the base of each spine, there were three to five radial extensions ( Figure 2L). Mean areal density of spines ranged from 4.2 to 5.0 µm −2 ( Table 1). The transverse striation of the intercalary band consisted of rows of spines (Figure 2B-E). Details of the thecal pores could be revealed ( Figure 2I-L). Larger pores were about 0.23 µm in diameter (Table 1) and bordered by a crater-formed rim at the outer plate surface (Figure 2I,L). Small pores were about 0.14 µm in diameter (Table 1) and were also bordered by a small elevated rim ( Figure 2L). A cluster of two to three large pores accompanied by several small pores were characteristically visible on the right thecal plate in apical ventral position ( Figure 2I,J). Other pores were scattered on both thecal plates with The periflagellar area (Figure 3) was about 2.2 µm deep and 1.5 µm wide (Table 2) and located between both thecal plates in a broadly V-shaped indentation of the right plate ( Figure 2E,I). There were a number of apical projections, which were dominated by a conspicuous wing (Figure 2A-J and Figure 3A-D) of about 1 µm each in width and length ( Table 2). In the periflagellar area, eight platelets (1, 2, 3, 4, 5, 6, 7 and 8) surrounded a flagellar pore (fp) and an accessory pore (ap) (Figure 3). The fp was of irregular oval shape and generally longer than wide (ca. 0.9 µm long, 0.6 µm wide, Table 2) and surrounded by platelets 3, 5, 6 and 8. The ap was smaller (ca. 0.6 µm long, 0.4 µm wide, Table 2) and surrounded by platelets 7 and 8. Platelet 2 was part of the upper rim of the ap but was separated by platelet 7 from the inner ring of the ap ( Figure 3B). Both pores were internally closed by two lip-like structures ( Figure 3C-F).
( Table 2). In the periflagellar area, eight platelets (1, 2, 3, 4, 5, 6, 7 and 8) surrounded a flagellar pore (fp) and an accessory pore (ap) (Figure 3). The fp was of irregular oval shape and generally longer than wide (ca. 0.9 µm long, 0.6 µm wide, Table 2) and surrounded by platelets 3, 5, 6 and 8. The ap was smaller (ca. 0.6 µm long, 0.4 µm wide, Table 2) and surrounded by platelets 7 and 8. Platelet 2 was part of the upper rim of the ap but was separated by platelet 7 from the inner ring of the ap ( Figure 3B). Both pores were internally closed by two lip-like structures ( Figure 3C-F).  lateral side of platelet 1 ( Figure 3A-D). Additional lists on platelet 1 provided the outline of the ap. Other apical projections were present on platelets 2, 3, 5, 6 and 8 ( Figure 3A-D) and were variable in dimensions. Platelet 4 was small, triangular and plain and formed an acuminate ventral termination of the periflagellar area. From an internal view, both fp and ap had a tube-like appearance, and the plate sutures of the periflagellar platelets were revealed ( Figure 3E,F). Schematic drawings of P. spinulentum, including of the periflagellar area, are compiled in Figure 4.

Discussion
The description of Prorocentrum spinulentum provides a scientific name for a small, already recognised and early branching subclade [3,9] of the P. shikokuense species group. Strains of this species group are described as having knob-like short spines on the thecal plate surface [4,21,22] and are thus different from P. spinulentum having long spines. Moreover, cells of P. shikokuense are larger and elongate [5] and thus cannot be confused with the smaller and in outline roundish P. spinulentum.
Two strains similar to P. spinulentum (i.e., CCMP1529 and RCC922) have been previously identified as P. cordatum, although the latter species is much larger. As inferred from the protologue of P. cordatum the cells have a surface ornamentation due to fine pores [23] but in SEM, rather long spines on the thecal plates are present [1], being very similar to P. spinulentum. Details of apical structures are not available from the original description of P. cordatum, but in SEM, a characteristic 'double wing' is present on platelet 1 (denoted as double-layered, curved apical collar) as well as spine-like projections (denoted as forked, single, and small teeth) on other periflagellar platelets [24][25][26]. In contrast, P. spinulentum has a characteristically different, wide and long wing on platelet 1.
There are 14 species of Prorocentrum (occasionally introduced under Exuviaella Cienkowski) of small size (<20 µm) with a roundish shape in outline that have already been compiled [3] (see Table 5 and Figure 7 therein). Among those, P. spinulentum differs from Exuviaella equatorialis Hasle (19 µm), Prorocentrum antarcticum (Hada) Balech (15-23 µm) and Prorocentrum rotundatum J.Schiller (16-21 µm) by size. Moreover, it differs in shape from Prorocentrum cornutum J.Schiller, which has a unique posterior horn. Detailed morphological features of all other small species of Prorocentrum [3] and the newly described P. thermophilum, are compiled and contrasted with P. spinulentum in Table 3. With the rather long thecal spines that P. spinulentum shares with P. cordatum only, it can be differentiated from species having a smooth surface (P. nux, P. cordiforme Bursa) or knob-like flat structures on the thecal plates (i.e., P. pervagatum, P. ponticum, P. termophilum; Table 3). Furthermore, P. spinulentum lacks a distinct apical spine and can therefore be differentiated from species with such a spine, including P. cordiforme (described with a short simple spine), P. nanum J.Schiller (described with a short spine), P. spheroideum J.Schiller (described with one solid spine) or P. pomoideum Bursa (described with one main apical tooth) ( Table 3).
There are other small species of Prorocentrum without an apical spine and whose plate surface structure has not been described, and these are more difficult to differentiate from P. spinulentum. The distinct ovate outline of Prorocentrum ovum J.Schiller in lateral view differs from the broad oval to round outline shape of P. spinulentum. Prorocentrum pusillum J.Schiller shares with P. spinulentum the small size and lack of an apical spine, but it has strong lateral compression compared to the broader P. spinulentum. Prorocentrum balticum from the German Baltic Sea [27] is a frequently encountered small species [28][29][30] and has a very similar size compared to P. spinulentum. However, whether such determinations in fact represent the organism is challenging due to the gross similarity of the small species. The original description does not mention presence or absence of an apical spine or other obvious apical projections, and pores or other surface features of the thecal plates are not reported in the protologue as well. However, it can be argued that a spiny plate surface, as it is visible in LM for P. spinulentum, would have been noticed by Hans Lohmann and thus differentiates both species.  Prorocentrum spinulentum thus differs from all other similar species of Prorocentrum by its unique combination of cell size, shape and type of plate surface structures (i.e., spines). Moreover, P. spinulentum has a nucleus in median or sub-median position on the cell's ventral side. Shape and/or position of the nucleus is rarely reported for the small species, but if available, the more subspherical or spherical nucleus (P. pervagatum [3], P. thermophilum [9], P. cordatum [36], P. nux [6]) has a posterior position. Other structural details outlined for P. spinulentum are the presence of two size classes of thecal (and presumably trichocyst) pores, which similarly have been reported for other species of the species complex, such as P. cf. balticum, P. pervagatum and P. thermophilum [3,8,9]. However, large pores of P. spinulentum (0.23 µm in diameter, Table 1) may be slightly smaller than large pores of P. pervagatum (0.26-0.34 µm [3]) or P. thermophilum (0.3 µm [9]). Strikingly, the few available SEM studies of related P. shikokuense report from small pores only (with a size of 0.15 µm corresponding to the small pores of P. spinulentum), which are irregularly distributed on the thecal plates, and explicitly deny size classes of thecal pores [4]. However, the plate surface structure of P. spinulentum hampers an unequivocal identification of large pores being rare (and that is why the quantification for P. spinulentum of thecal pores per plate did not differentiate small and large pores). More detailed, high-magnification SEM may uncover the presence or absence of large thecal pores in the P. shikokuense species group more reliably.
A number of small, in outline roundish species of Prorocentrum, namely P. spinulentum (this work, Figure 2I) and P. pervagatum, P. thermophilum, P. cf. balticum and P. nux [3,6,8,9], shares the presence of a distinct, short row of large pores in apical position on the right thecal plate. However, this trait has not yet been explicitly reported for the P. shikokuense species group or P. cordatum, but more detailed SEM observations of the periflagellar area are needed to better assess the phylogenetic importance of this trait. The periflagellar area of P. spinulentum is very similar in number (i.e., eight) and arrangement of the platelets to P. cordatum [38][39][40] and P. pervagatum [3]. However, P. pervagatum but not P. spinulentum has a distinct spine on platelet 6, and P. spinulentum but not P. pervagatum has a characteristic broad wing on platelet 1. A very similar arrangement of the periflagellar platelets is also shared between P. shikokuense ( [4], as P. obtusidens) and P. spinulentum, and both species have a similar broad wing on platelet 1 as the dominant periflagellar structure. However, a subdivision of platelet 5 is present in P. shikokuense but absent in P. spinulentum. For P. thermophilum, knowledge of the periflagellar area is rather diffuse ("pores, surrounded by folded structures" [9]), and the number and arrangement of the periflagellar platelets remains unclear. The same is true for the preliminary and incomplete morphological description of P. cf. balticum [8]. More detailed SEM studies of more strains and species are needed to finally evaluate the suitability of such differences to distinguish the great diversity seen in the small species of Prorocentrum.
In conclusion, the recent descriptions of three new, small, planktonic species of Prorocentrum, namely P. pervagatum, P. thermophilum and P. spinulentum herein, provide a major step towards an unambiguous application of scientific names in Prorocentrum but with the clarification of many still unresolved and predominantly old names of Prorocentrum, there is a long way to go, preferably by using an epitypification approach based on material from the corresponding type localities [41,42].