Redefinition of the Dinoflagellate Genus Alexandrium Based on Centrodinium: Reinstatement of Gessnerium and Protogonyaulax, and Episemicolon gen. nov. (Gonyaulacales,

The genus Centrodinium contains oceanic and predominantly tropical species that have received little attention. Three species of Centrodinium were examined using thecal plate dissociation, scanning electron microscopy, and molecular sequences. The apical horn of Centrodinium intermedium and C. eminens is formed by the elongation of the fourth apical plate, and a second apical split into two plates. In C. punctatum two apical plates (2 ′ and 4 ′ ) almost completely encircle the apical pore plate (Po), while the contact with the plate 1 ′ in the ventral side is much reduced, and the plate 3 ′ does not reach the Po. Moreover, its left posterior lateral sulcal plate is longer than its right pair, while reversed in the typical Centrodinium spp. The sulcal posterior plate of C. punctatum is located in the left-ventral side below the plates 1 ′′′ and 2 ′′′ , while the sulcal posterior plate located in the right face below the plates 4 and 5 in the typical Centrodinium spp. Phylogenetic analyses based on the small and large subunit of the rRNA gene showed that Centrodinium spp. and Alexandrium affine / A. gaarderae clustered as a sister clade of the Alexandrium tamarense / catenella / fraterculus groups. The clade of the subgenus Gessnerium , and the clade of the type species of Alexandrium, A. minutum , with four divergent species, clustered in more basal positions. The polyphyly of Alexandrium is solved with the split into four genera: (1) Alexandrium sensu stricto for the species of the clade of A. minutum and four divergent species; (2) the reinstatement of the genus Gessnerium for the species of the clade of A. monilatum ; (3) the reinstatement of genus Protogonyaulax for the species of the tamarense / catenella / fraterculus groups, and (4) the new genus Episemicolon gen. nov. for A. affine and A. gaarderae. New combinations in the genera Gessnerium , Protogonyaulax, and Episemicolon are proposed.


Introduction
Dino agellates are ubiquitous protists that play diverse roles in marine ecosystems. Numerous studies are focused on species that are responsible for harmful algal blooms (HABs) in coastal waters. Paralytic shell sh poisoning (PSP) is generally regarded as the most well-known and widespread HAB syndrome, and is associated with toxins produced by certain dinoagellate species in the genus Alexandrium [1]. Whilst neritic species of the planktonic Alexandrium or the epiphytic Gambierdiscus, responsible for toxic events, have received 2. Materials and Methods 2.1. Sampling, Isolation, and Microscopy. Sampling was performed with a phytoplankton net (20 μm mesh size) on the surface waters of the South-Eastern Bay of Biscay, North Atlantic, in August 2017. Samples from two stations at 43°36′ N-1°57′ W and 43°36′ N-2°03′ W are described here. e plankton concentrate was preserved with acid Lugol′s iodine solution to a final concentration of 4% (vol:vol), and kept refrigerated (~3°C). e material was examined with an inverted microscope (Nikon Eclipse TE2000-S, Tokyo) and photographed with a Nikon Digital Sight DS-2 M camera. A er observing the presence of Centrodinium in these two sampling stations, subsamples of the plankton concentrate were treated with small amounts (150-200 μl) of 10% (weight/volume) sodium thiosulfate for removing the iodine. e cells of each species of Centrodinium were micropipetted individually with a fine capillary into a clean chamber filled with autoclaved Milli-Qultrapure water. e same procedure was repeated twice in order to remove any source of contamination. Finally, 30-40 cells of each species were deposited in a 0.2 ml Eppendorf tube filled with absolute.
For plate dissociation, each cell was individually isolated and placed in an Utermöhl chamber with distilled water. Drops of a solution of 5% sodium hypochlorite (commercial bleach solution, 1 : 1 mixture of sodium hypochlorite and Milli-Q water) were added until the split of the thecal plates. In other cases, the theca was squashed by touching it with a fine capillary tube to split the thecal plates. e cell was repeatedly photographed at different stages during the process of splitting the theca with the inverted microscope at 600x magnification.
For analyses using scanning electron microscopy, a subsample was filtered through a 3 μm pore size polycarbonate membrane (Millipore Ltd., Middlesex, U.K.). e filter was rinsed three times in Milli-Q water, dehydrated through graded ethanol series (30%, 50%, 70%, 80%, 90%, 95%, and two steps in 100%). en, the protocol was to immerse the filter in HMDS (Hexamethyldisilazane, Molekula Limited, Newcastle, U.K.) for 30 minutes (twice). e HMDS was evaporated by placing the sample overnight under the fume hood. Filters were mounted on an aluminium stub, sputter-coated with Au/Pd (Polaron SC7620, Quorum Technologies Ltd., Ashford, U.K.) and observed at 15 kV with a SEM LEO 438 VP (Carl Zeiss AG, Oberkochen, Germany). Images were presented on a black background using Adobe Photoshop CS3 (Adobe Systems Inc., San Jose, CA, USA).
Positive PCR products were cloned into vector PCR 2.1 using a TOPO TA cloning kit (Invitrogen, Carlsbad, CA, USA). Clones were screened for inserts by PCR amplification with plasmid primers M13F and M13R, and positive clones from each PCR amplicon were purified using the Qiaquick PCR purification kit (Qiagen, Hilden, Germany), and sequenced in both the forward and reverse direction (Eurofins MWG Operon, Ebersberg, Germany). Sequence reads were aligned and assembled in Geneious Pro 11.1.2 (Biomatters, Auckland, New Zealand). e newly generated consensus sequences were deposited in DDBJ/EMBL/GenBank under accession numbers MK714074-MK714082.

Phylogenetic
Analyses. SSU-and LSU rRNA gene sequences of Centrodinium spp. were analysed using Basic Local Search Tool (BLAST, http://blast.ncbi.nlm.nih.gov/Blast.cgi) against databases in GenBank. e closest matches to these searches were sequences in the genus Alexandrium (primarily A. affine) and the sequences reported as "Alexandrium sp. ZL2017" that were later identified as Centrodinium punctatum in Li et al. [11]. Based on these results, rRNA gene sequence data were compiled from similar sequences identified using BLAST. Sequence alignments of available SSU-and D1-D2 LSU rRNA gene sequences of Centrodinium spp., representatives of each species of Alexandrium, other gonyaulacoid dinoflagellates, and other dinokaryotic dinoflagellates were accomplished by Clustal W [16] and the evolutionary history was inferred by using the Maximum Likelihood method based on the General Time Reversible model with Gamma distributed with Invariant sites and the default settings in MEGA7 so ware [17]. Bootstrap values were obtained a er 1000 replications. e apicomplexan Eimeria tenella (AF026388) was used as an out group in the SSU-and LSU rRNA gene phylogenies.

Morphology of Centrodinium punctatum.
e species Centrodinium punctatum, C. intermedium, and C. eminens were the most abundant (in that order) in the sampling stations in the South-Eastern Bay of Biscay (Figure 1(a)), providing material for the morphological (plate dissociation and SEM) and molecular analyses. A few individuals of Centrodinium maximum were also found, but in an insufficient abundance for detailed studies. e sea surface temperatures in the two sampling stations ranged from 23.4°C to 23.8°C and the salinity from 34.5 to 34.6. Centrodinium punctatum was the most abundant species compared with other congeneric taxa. e cells were slightly laterally flattened with a rhomboid shape. Cell dimensions were 65-90 μm long, 35-42 μm depth (dorso-ventral diameter), and 24-34 μm wide (length between the right and le lateral sides) (Figure 1(a)). e epitheca was conical with a blunt apex. e hypotheca was conical with a pointed antapex directed towards the ventral side. In addition to the size variability, the individuals showed a different degree of development the pointed antapex (Figure 1(b) and 1(c)). e theca was ornamented with poroids ( Figures 1(d)-1(f), 1(l)-1(q)). Centrodinium punctatum had a plate formula Po, 4′, 6′′, 6c, 8s+, 5′′′, and 2′′′′. A more detailed description of the plate arrangement of C. punctatum, C. intermedium, and C. eminens is available in the Appendix S2 as Supplementary material. We describe here the apical, sulcal, and antapical plate series. e plates 2′ and 4′ almost completely encircled the apical pore plate (Po), while the contact with the plate 1′ in the ventral side was much reduced, and the plate 3′ did not reach the Po (Figures 1(d)-1(f)). Scanning electron microscopy revealed a horseshoe-shaped apical pore surrounded by a rim of small marginal pores (Figures 1(q)-1(s)). e sulcal plates are placed between the anterior sulcal plate (S.a.) in the epitheca and the posterior sulcal plate (S.p.) near the antapex (Figures 1(g)-1(l), 1(p)). Two small plates known as the anterior and posterior median plates (S.m.a. and S.m.p.)-one above the otheroccurred below the anterior sulcal and the le and right anterior lateral plates (S.s.a. and S.d.a.). Two lateral pairs of plates were located below, the le and right posterior lateral plates (S.s.p. and S.d.p.), with the le plate being longer than the right pair (Figures 1(h)-1(k)). e sulcal posterior (S.p.) was an irregular pentagon with length approximately equal to the width (Figures 1(f), 1(h)). e S.p. plate was displaced towards the le side below the plates 1′′′ and 2′′′ and the le margin joining to the plate 1′′′′ (Figures 1(h), 1(l) and 1(m)). ere were two antapical plates with a triangular shape that conformed a pointed antapex directed towards the ventral side. e first antapical (1′′′′) in the le face (Figures 1(h), 1(l)-1(m)) was slightly smaller than the second antapical plate (2′′′′) in the right face (Figures 1(f), 1(i), 1(n)-1(o)). e plate 1′′′′ was in contact to S.p. and 2′′′′ plate (Figures 1(h), 1(l)-1(n)).

Morphology of Centrodinium intermedium.
e lateral flattening of C. intermedium is probably the highest of the genus. e species also differed from the congeneric species in the contour of the hypotheca being oval to semicircular (Figures 2(a)-2(b)), while conical in the other species (Figure 1(a)). e apical horn of C. intermedium was usually shorter than the other species of Centrodinium. Cells were 130-175 μm long. e depth of the cells (dorso-ventral distance) was 55-80 μm.
e width between the le and right sides is difficult to measure in these highly laterally flattened cells, with values of about 25-35 μm wide at the cingulum level (Figures 2(a)-2(c)). e dense poroid ornamentation of the theca observed in C. punctatum was missing in C. intermedium, with only scattered pores, more abundant in the right face of the apical horn ( Figure 2(d)). e apical horn (~20 μm long) of C. intermedium was a short truncated cone (Figures 2(a)-2(d)). e antapical horn was longer (>50 μm) and directed towards the le -ventral side. Consequently, the antapical horn was in a different same plane than the main body and the apical horn ( Figure 2(c)). e antapical horn had a triangular section with a slight anticlockwise torsion, and three terminal spinules (Figures 2(m)-2(o)). Each face of the antapical horn had a row of sunken areas with 3-4 small pores (Figure 2(n)). e molecular data revealed a very close phylogenetical relationship between C. punctatum and C. intermedium (see below in Figures 4 and 5). It is commonly assumed that congeneric species share a similar plate formula. e epithecal plate formula of C. punctatum is Po, 4′, 6′′ or alternatively 3′, Journal of Marine Biology 4 conform the apical horn, and the development of these plates hindered that the plates 1′ and 3′ reached the apex (Figures 2(d), 2(o)-2(q)). While the plate 4′ was narrow and long, the elongation of the plate 2′ resulted in the split into two plates. e formula of the epitheca of C. punctatum and C. intermedium is similar (Po, 4′, 6′′), using the labelling 2′ (α + β) to denote the split of the second apical plate in C. intermedium.
e right side of the epitheca was essentially similar to C. punctatum, where 4′ plate has expanded anteriorly, and then the 3′ plate did not reach the apex (Figure 2(d)). During the plate dissociations, the Po remained attached to the plate 2′β as a circular structure of about 1 μm in diameter (Figure 2(d)). e tiny membranous Po platelet was poorly conserved in the SEM preparations. e very thin plate 2′β appeared crushed against 1a, 6′′ in a strict Kofoidian scheme. e species C. intermedium has an additional plate in the le face of the epitheca, and the plate formula in a strict Kofoidian scheme is Po, 2′, 2a, 7′′. In contrast to C. punctatum, the apical plates of C. intermedium were larger than the precingular plates ( Figure  2(d)). e rst apical plate of C. punctatum reached the apical pore (insert 1′), while in C. intermedium it does not reach the apex (exsert 1′). When compared to C. punctatum, the main modi cations of C. intermedium were the elongation of the plates 4′ and 2′ (the latter split into two plates) to conform the apical horn, the di erent length of the posterior lateral sulcal plates, and the formation of a tubular antapical horn supported at its ventral basis by two triangular plates. e apical plates 2′ and 4′ of C. intermedium have extended anteriorly to was a pore, the posterior attachment pore, located in this triangular plate in the right face ( Figure 2(f)), which is a characteristic of the posterior sulcal plate of chain-forming gonyaulacoid dino agellates. In the le face, the rst antapical (1′′′′) was a triangular plate that o en showed a posterior liform extension (Figure 1(e)). e second antapical plate (2′′′′) emerged from the dorsal side to conform a tubular antapical horn (Figures 2(e)-2(f)), with a slight anticlockwise torsion and three terminal spinules (Figures 2(m)-2(n)).
In the sulcal plate series, the anterior sulcal plate (S.a.) was part of the epitheca, enclosed between the plates 6′′, 1′, and 1′′ and the rst cingular plate (Figures 2(g)-2(h)). ere was a prominent pore in the middle of the plate connected to the right border by a narrow canal. In some cells, the right posterior corner of the S.a. showed a membranous ange that connected with the rst cingular plate (Figure 2(h)). e right anterior lateral sulcal plate (S.d.a.) was larger than its le pair, the thick plate 4′ (Figures 2(q)-2(r)). e antapex of C. punctatum and C. intermedium showed di erences. e posterior hypotheca of C. intermedium was composed of three plates: a tubular plate that conforms the antapical horn and two plates in the ventral side that acted as a counterfort or backstay. ese two triangular plates were slightly laterally inclined towards the le face, and the antapical horn was directed towards the le and ventral sides (Figures 2(e)-2(f), 2(k)-2(o)). e most immediate interpretation was that the antapex consists one antapical plate that conforms the horn, and two posterior intercalary plates that support the ventral basis of the antapical horn. is implies that the posterior sulcal plate (S.p.) was missing in C. intermedium. In C. punctatum, the S.p. was an irregular pentagon located in the le -ventral side below the plates 1′′′ and 2′′′ (Figures 1(f), 1(i), 1(l)-1(m)), while the S.p. of C. intermedium was triangular and located in the right face below the plates 4′′′ and 5′′′ (Figures 2(f), 2(k)-2(m)). ere with the shape of an irregular right triangle that resembled the shape of the Sicily Island (Figures 2(g), 2(i)). In C. punctatum, the le posterior lateral sulcal plate (S.d.p.) was longer than its right pair (Figures 1(i), 1(k)), while reversed in C. intermedium (Figure 2(j)). e right posterior sulcal plate (S.d.p.) of C. intermedium was the longest of the sulcal series and showed the shape of a knife, with a reinforcement in the le margin (Figure 2(j)). e le posterior sulcal plate (S.s.p.) was smaller, like a very elongated pentagon that fit in the knife handle formed by the anterior le margin of the S.d.p. (Figure 2(j)). e morphology of these plates suggests that the overlap growth of the S.d.p. has hindered the posterior development of the S.s.p.

Morphology of Centrodinium eminens.
In lateral view, the cells of C. eminens were fusiform and slightly sigmoid because the apical horn was slightly directed towards the dorsal side, and the antapical horn towards the ventral side. e ventral margin of the epitheca was almost straight. e dorsal margin was curved in the posterior half and almost straight in the anterior half where the apical horn with a brunt apex was slightly directed towards the dorsal side ( Figure 3(a)). e cells of C. eminens were 182-239 μm long, and 31-47 μm in depth (dorso-ventral distance), being less robust (lower depth), and less flattened than C. intermedium. e apical and antapical horns of C. eminens were longer (Figure 3(a)) than in C. intermedium (Figure 2(a)). e antapical horn of C. intermedium was very inclined towards the face (Figure 2(c)), while the inclination was almost absent in C. eminens (Figures 3(a)-3(h)). e plate arrangement of C. eminens and C. intermedium was similar, with more anterior-posteriorly elongated plates, especially in the apical series in C. eminens (Figures 3(b)-3(l), 3(t)-3 (v)). e two plates, 2′ (α + β), resulting of the split of the second apical plate remained joined (Figure 3(d)). e distal antapical horn also showed three spinules (Figure 3 In the SEM preparations, some individuals of C. eminens were in better preservation stage than those of C. intermedium, and some details of the apex were revealed (Figures 3(w)-3(z)).
e apex of C. eminens also collapsed in the SEM preparations but in some individuals the membranous apical pore platelet and the thin second antapical were not crushed against the thicker four apical plate. In these cases, a large pore of 1-1.5 μm in diameter was observed devoid of the cover platelet (Figures 3(w)-3(x)). is membranous cover platelet remained in few individuals, with the apical pore surrounded by a few tiny pores (Figures 3(y)-3(z)).

Molecular Phylogeny.
e SSU and LSU rRNA gene sequences were obtained from three species of Centrodinium: C. punctatum that is the first described laterally flattened species of the former genus Murrayella; C. intermedium that is the most flattened species of this genus with an oval hypotheca, and C. eminens which morphology is close to the type species, C. elongatum. It should be noted that the type species remains unreported since the original description in 1907. It seems likely that C. elongatum corresponds to a recently divided cell of C. maximum or C. eminens (see Appendix S1 part 2 in the Supplementary material).
In the SSU rRNA gene phylogeny, the three species of Centrodinium clustered together with high support with C. punctatum in a basal position. e Centrodinium spp. clade clustered with Alexandrium affine, with strong support (BP 100%) (Figure 4). In the LSU rRNA gene phylogeny, Centrodinium spp. also clustered with sequences retrieved from GenBank as Alexandrium affine and A. concavum ( Figure 5). In an additional LSU rRNA tree more reference sequences were added from GenBank within the A. affine clade to include sequences identified as A. affine, A. tamarense, and A. concavum ( Figure S1

Affinities between Centrodinium and Alexandrium.
e molecular data reveal that Centrodinium clusters with strong support amongst the clades of Alexandrium (Figures 4-5; [11]). Species such as C. punctatum have the same plate formula of Alexandrium (Figures 1, 6(e)). e most typical apical pore plate of Alexandrium has a comma-shaped pore surrounded by marginal pores, and the chain-forming species have an anterior attachment pore [18]. e apical pore plate of Alexandrium is larger (>6 μm), and we can easily observe an oval or comma-shaped pore. e formation of the apical horn of Centrodinium implies a reduction of the surface available for the apical pore plate (<2 μm), and the horseshoe-shaped could be a result of the constriction of the oval or comma-shaped pore (Figures 1(r)-1(s), 3(y)-3(z)). e chain-forming species of Alexandrium have an attachment pore (a.a.p.) in the apical pore plate, and an attachment pore (p.a.p.) in the posterior sulcal plate. e cells of a chain are interconnected by these pores [18]. In Centrodinium, the anterior attachment pore is more difficult to observe due to the small size and fragility of the membranous apical platelet, or it may be confused with marginal pores. Hernández-Becerril et al. ([9], their Figure 33) reported a pore in the apex that could be the apical pore devoid of the foramen, or alternatively the anterior attachment pore. e posterior attachment pore in the posterior sulcal plate is evident in C. intermedium and C. eminens (Figures 2(f), 3(r)-3(s)), and C. pulchrum ([9], their Figure 37). e sequences of Centrodinium clustered as a sister group to Alexandrium affine (Figures 4 and 5; [11]). at clade includes sequences retrieved from GenBank under the names  A. a ne, A. tamarense, and A. concavum. Two sequences from New Zealand, the strains CAWD51 named A. a ne (accession number AY338753) and CAWD52 named A. concavum (accession number AF032348) were identical and diverged from the main group of A. a ne. e latter subdivided into two groups, one for strains isolated exclusively from Japan and China, and other group for strains from diverse world regions ( Figure S1). e cells of the strain CAWD52 illustrated in MacKenzie et al. [19] corresponded to A. gaarderae as de ned by Larsen and Nguyen-Ngoc [20]. e species Alexandrium a ne was rst described as Protogonyaulax a nis [21], and since the earlier molecular phylogenies the sequences of A. a ne have always diverged from the members of the tamarense/catenella group [22]. e species A. a ne and A. gaarderae (non A. concavum emend. Nguyen-Ngoc & Larsen) clustered as a sister group of Centrodinium and more distantly related to the clade of Protogonyaulax ( Figure 5, S1). e members of the tamarense/ catenella group are responsible for paralytic shell sh poisoning (PSP) events. e sxtA gene (saxitoxin biosynthesis pathway protein A domain) has been detected in the members of the tamarense/catenella group or A. fraterculus. In contrast, PSP toxicity or the presence of the sxtA gene have not been detected in A. a ne [23] and Centrodinium punctatum [11]. Alexandrium a ne is distinguished primarily by the apical pore plate and other di erences in the sulcal plates. Balech [18] reported that the apical pore platelet is narrow, long, and fundamentally bullet-shaped. e foramen does not form a true comma because it is oval and relatively small; it is located in the ventral half of the plate and a large and almost circular connecting pore is dorsal [18]. Alexandrium gaarderae (reported as A. concavum) also has a dorsal connecting pore [24]. e location of the anterior attachment pore at the dorsal C. punctatum as Po, 3′, 1a, 6′′, 6c, 8s, 5′′′, 1p, 2′′′′. ese authors follow a strict Kofoidian scheme of tabulation of the epitheca, and labelled the apical plate that does not touch the apical pore plate as an intercalary plate. Li et al. [11] misidenti ed the sulcal and hypothecal plates. Li et al. ( [11], p. 177, their Figure 8(c)) illustrated the right (S.d.p.) and le posterior sulcal (S.s.p.) plates with a similar length. Li et al. [11] did not carry out a study using plate dissection, and the sulcal lists were hiding the morphology of the sulcal plates. ese plates have very di erent length as revealed in this study (Figure 1(k)) and the plate dissections of C. punctatum by Balech [5,6]. ey labelled the le lateral posterior sulcal as the posterior plate, and this induces the subsequent errors in the tabulation of the hypotheca (see Appendix S1 part 6 as Supplementary material). e genus Alexandrium is currently a pool of species with signi cant di erences in the plate arrangement [26]. Balech [18] reported that the species of the subgenus Gessnerium were closer to Pyrrhotriadinium than Alexandrium. e apical pore plate in Pyrrhotriadinium is totally transverse orientated, while oblique in Gessnerium [18,27]. Pyrrhotriadinium lacks the accessory sulcal plates, and the two median sulcal plates are separated, while in Gessnerium the accessory plates are prominent and the two median sulcal plates are in contact [18]. margin of the apical pore plate is the main diagnostic character of the species A. gaarderae and A. a ne [24]. In the other species of Alexandrium, the apical pore is comma-shaped and the anterior attachment pore lying in the right side. e two posterior lateral sulcal plates are more or less similar in length in the members of the tamarense/catenella group, while the right posterior sulcal plate is longer than the le posterior sulcal plate in A. a ne (Figure 6(d)). is feature is variable in Centrodinium spp. (Figures 6(e) and 6(f)). e cingulum and the sulcus of Centrodinium spp. and A. a ne are deeply incised and bordered by pronounced list, and the posterior le margin of the plate 6′′ is reinforced, long and concave (Figures 1-3, 6(d)-6(f); [20,21]).

Reclassi cation of the Subgenus
Gessnerium. An historical account of the taxonomy and nomenclature of Alexandrium s.l., including Gessnerium and Protogonyaulax, is reported in the Appendix S3 as Supplementary material. e plate formula of Alexandrium is usually reported as Po, 4′, 6′′, 6c, 8s+, 5′′′, 2′′′′ [25]. It is similar to the plate formula of C. punctatum and di ers from the more attened species of Centrodinium in the anterior elongation of the plates 4′ and 2′, and the split in the latter plate. Li et al. [11] reported the plate formula of long. e anterior and posterior attachment pore is a common feature in chain-forming species, but few species of Alexandrium s.s. forms chains, and the attachment pores are absent ( Figure 6(b), Table 1; [18,24]). e genus Protogonyaulax contains species where the first apical plate is rhomboidal and directly connects to the apical pore plate. e posterior sulcal plate is reversed pentagonal, symmetrical, and longer than wide.
ere are numerous chain-forming species, and the presence of anterior and posterior attachment pores is a common feature (Figure 6(c), Table 1; [18,24]).
e new scenario derived on the close relationship of Centrodinium and the species of Alexandrium s.l. suggests the reinstatement of the genera Gessnerium and Protogonyaulax, and the erection of a new genus for A. affine and A. gaarderae. e diagnoses of the genera Centrodinium, Alexandrium, Gessnerium, and Protogonyaulax need to be amended. e species Peridinium splendor-maris, type of the genus Blepharocysta, has been interpreted to correspond to an earlier description of Alexandrium balechii. Carbonell-Moore [31] submitted a proposal to conserve the name Peridinium splendor-maris as a species Blepharocysta, avoiding the possible transfer of all the species of Alexandrium into Blepharocysta. If the proposal is rejected, the change does not affect Alexandrium because A. balechii is now a species of Gessnerium. If the proposal is recommended, no change is applied to Alexandrium. (Figures 6(e)-6(f), Table 1)

Centrodinium Kofoid emended Gómez & Artigas.
Gonyaulacoid dinoflagellates with different degree of lateral flattening, an elongated brunt apex or an apical horn. Cingulum deep, median, descending about one cingular width, without overhanging. e cingular list at both upper and lower margins are prominent. e sulcus with list at both right and le margins. e apical pore plate with a horseshoeshaped pore surrounded by small marginal pores. e plate formula is Po, 4′ (2′ α + β), 6′′, 6c, ≥8s, 5′′′, 2′′′′, and the more flattened species showed a split of the second apical plate, 2′ (α + β). e apical pore plate is mainly surrounded by the second and fourth apical plates, while the third apical plate does not reach the apex. In the less compressed species, the first apical plate (1′) reaches the apex, whereas in the more flattened species the 1′ plate does not reach the apex, and the 2′ plate is divided into two plates. In all the species, the anterior sulcal plate has a distinct pore. e sulcus contains at least 8 plates, the two lateral posterior sulcal plates are long. e le plate is longer than the right one in the less compressed species, and vice versa in the more compressed species. In less compressed species, the antapex is pointed, while in the more flattened species the antapical horn derived from a tubular second antapical plate has terminal spinules. e antapical horn is supported by two triangular plates, the posterior sulcal in the right face, and first antapical plate in the le face. e posterior sulcal plate may contain a posterior attachment pore near the anterior margin. e species of Centrodinium typically inhabit in warm oceans and have chloroplasts. e species C. punctatum is not toxic.
In Pyrrhotriadinium, the first precingular plate, equivalent to the first gonyaulacoid apical plate, does not contact the le apical plate [18]. e 1′′ plate is pentagonal in Gessnerium (Figure 6(a)) and quadrangular in Pyrrhotriadinium, while rhomboidal in nearly all the species of the subgenus Alexandrium (Figures 6(b)-6(c)). is is a precingular plate based on its shape and position. e posterior sulcal plate of Gessnerium is large, longer than wide and prolonged obliquely towards the posterior right (Figure 6(a)). In the species of the subgenus Alexandrium, the posterior sulcal plate is relatively smaller and non-oblique ( [18]; Figures 6(b)-6(d)). PSP toxicity has not been reported in species of Gessnerium. e species Alexandrium margalefii and A. pohangense have the first apical plate disconnected from the apical pore plate, which suggests an affinity with Gessnerium, but this plate is quadrangular in these species while pentagonal in Gessnerium [18,23]. e position of A. margalefii and A. pohangense in the molecular phylogenies is unstable, typically is represented as divergent species of the clade of the type, A. minutum [23]. ese two species, and other two divergent species (A. diversaporum, A. leei) need further research before to propose a change of genus.

e Generic Split of the Subgenus Alexandrium.
Previous morphological and molecular phylogenetic studies including sequences of Pyrodinium already suggested the reinstatement of the Gessnerium at the genus level ( [28,29], Appendix S3 as Supplementary material). With the inclusion of Centrodinium spp. in the molecular phylogenies, Alexandrium can no longer be considered as a monophyletic genus (Figures 4  and 5). e morphological differences amongst species of the subgenera Gessnerium (Figure 6(a)) and Alexandrium (Figures 6(b)-6(d)) are evident, but a split between species of the subgenus Alexandrium based on morphology is less conspicuous. In the molecular phylogenies, the sequences of the subgenus Alexandrium are divided into two major groups. A group that contains the type species of Alexandrium, A. minimum, and another group split into two sister clades: one major clade that contains the type species of Protogonyaulax, P. tamarensis, with members of the tamarense/catenella/fraterculus groups, and other major clade for Centrodinium and Alexandrium affine/A. gaarderae (Figures 4-5). Sequences of the members of the subgenus Alexandrium do not cluster as a monophyletic group, unless we consider placing all the species of the subgenus Alexandrium into Centrodinium because Centrodinium Kofoid 1907 has the priority over Alexandrium Halim 1960. is implies numerous taxonomical innovations, and requires merging species with different morphologies into a single genus. Splitting members of the subgenus Alexandrium into three genera reconciles the molecular and morphological data, and requires fewer taxonomical innovations.
e clade Alexandrium s.s. contains the type species, A. minutum, and other species in which the first apical plate is rhomboidal and may connect directly or indirectly through a thread-like prolongation with the apical pore plate (Figure 6(b), Table 1). is feature may vary intraspecifically as reported for A. minutum [30]. e posterior sulcal plate is relatively small, symmetrical or asymmetrical, and wider than 4.6. Reinstatement of the Genus Gessnerium (Figure 6(a) ere are two relatively large accessory sulcal plates that are absent or hardly visible in Alexandrium s.l. e right posterior lateral sulcal plate (S.d.p.) is long and narrow. e posterior sulcal plate (S.p.) is longer than wide, extending obliquely towards the posterior right. e second antapical plate of Gessnerium is lateral, while this plate is more dorsal than lateral in Alexandrium s.l. e formation of chains is variable amongst the species. e species are more common in warm waters, and rarely reported in cold waters. Paralytic shellfish poisoning has not been associated with the presence of Gessnerium, but several species are known as fish-killers that produce goniodomin A, spirolide, or hemolytic toxins that may be involved in mixotrophy.
(1)  (Figure 6(b), Table 1 e plate 1′ is rhomboidal, narrow, and asymmetrical and can be either in direct contact with the apical pore plate or indirectly connected via a thin suture (thread-like process). Alexandrium insuetum, has severely reticulated thecal plates and the exsert 1′. e plate 6′′ is usually narrow. e posterior sulcal is relatively small, wider than long. e apical pore plate contains a commashaped pore. Relatively few chain-forming species, and the attachment pore, if present, is in the lateral right to the apical pore plate. A posterior connecting pore is usually absent. e species are typically bloom-forming in eutrophic and/or confined neritic waters. PSP toxicity has been reported in numerous species. (Figure 6(c) Gómez & Artigas. Gonyaulacoid dinoflagellates without or with scarce cell compression, and lacking horn or spines. Cingulum deep, median, descending about one cingular width, without overhanging. e theca is usually smooth, and very rarely ornamented. e plate formula is Po, 4′, 6′′, 6c, ≥8s, 5′′′, 2′′′′. e first apical plate (1′) plate is rhomboidal, narrow and asymmetrical and always directly connected to the apical pore plate (Po). e plate 6′′ is usually wide. e posterior sulcal plate is longer than wide, with usually two ventrally directed anterior prolongations and a connecting pore.

Reinstatement of the Genus Protogonyaulax
e Po plate contains a comma-shaped pore, and usually an anterior attachment pore in the right lateral side of the apical pore plate. Relatively many chain-forming species. e species are bloom-forming in eutrophic and/ or confined neritic waters. Cosmopolitan distribution with a few species reported from cold waters. Paralytic shellfish poisoning toxicity events have been reported in numerous species.

Episemicolon F. Gómez & Artigas, gen. nov. (1) Diagnosis:
Gonyaulacoid dinoflagellate without or scarce cell compression, without spines or horns. Cingulum deep, median, descending about one cingular width, without overhanging. e cingular lists at both upper and lower margin are prominent. e sulcus with list at both right and le margins. Plate formula Po, 4′, 6′′, 6c, ≥8s, 5′′′, 2′′′′. e first apical plate is rhomboidal and reaches the apical pore plate. e apical pore plate contains an oval or bullet-shaped apical pore, with an attachment pore lying at the dorsal side. e sulcus contains at least eight plates, the two lateral posterior plates are long, and with the right one longer than the le pair. e posterior sulcal plate is right displaced and may contain a marginal attachment pore. Paralytic shellfish poisoning toxicity has not been reported.
(2) Etymology: epi-from Ancient Greek "epi" (= on top of); semicolon: the punctuation mark (;) from Latin "semi" (= half), and Greek "kolon" (= verse, a part of a strophe, column) and a mark of punctuation (:). e apical pore and the dorsal attachment pore in the apical pore plate resemble the typographic symbol (;). e gender is neuter.