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

Fernando Gómez; Luis Felipe Artigas

doi:10.1155/2019/1284104

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

Gómez, Fernando;Artigas, Luis Felipe 2019-12-31 00:00:00 Hindawi Journal of Marine Biology Volume 2019, Article ID 1284104, 17 pages https://doi.org/10.1155/2019/1284104 Research Article Redefinition of the Dinoflagellate Genus Alexandrium Based on Centrodinium: Reinstatement of Gessnerium and Protogonyaulax, and Episemicolon gen. nov. (Gonyaulacales, Dinophyceae) 1,2 2 Fernando Gómez and Luis Felipe Artigas Carmen Campos Panisse 3, E-11500 Puerto de Santa María, Spain Université du Littoral Côte d′Opale, Université de Lille, CNRS, UMR 8187, LOG, Laboratoire d′Océanologie et de Géosciences, 32 Av. Foch 62930, Wimereux, France Correspondence should be addressed to Fernando Gómez; fernando.gomez@toplancton.com Received 17 September 2019; Revised 26 October 2019; Accepted 23 November 2019; Published 31 December 2019 Academic Editor: Punyasloke Bhadury Copyright © 2019 Fernando Gómez and Luis Felipe Artigas. ƒis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ƒe genus Centrodinium contains oceanic and predominantly tropical species that have received little attention. ƒree species of Centrodinium were examined using thecal plate dissociation, scanning electron microscopy, and molecular sequences. ƒe 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 le’ posterior lateral sulcal plate is longer than its right pair, while reversed in the typical Centrodinium spp. ƒe sulcal posterior plate of C. punctatum is located in the le’-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 a‹ne/A. gaarderae clustered as a sister clade of the Alexandrium tamarense/catenella/fraterculus groups. ƒe 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. ƒe 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. a‹ne and A. gaarderae. New combinations in the genera Gessnerium, Protogonyaulax, and Episemicolon are proposed. considerable attention, other open-ocean gonyaulacoid dino- 1. Introduction ™agellates remain under-investigated because of the paucity Dino™agellates are ubiquitous protists that play diverse roles of material due to their low densities. ƒe neritic HAB species in marine ecosystems. Numerous studies are focused on spe- of Alexandrium are typically non- or slightly compressed spe- cies that are responsible for harmful algal blooms (HABs) in cies, without horns or spines [2], while the oceanic gonyaula- coastal waters. Paralytic shellsh poisoning (PSP) is generally coid dino™agellates have horns and spines (Ceratocorys spp., regarded as the most well-known and widespread HAB syn- Gonyaulax taylorii, etc.), and/or the cells are o’en ™attened drome, and is associated with toxins produced by certain dino- (i.e. Gonyaulax paci‘ca, [3]). Kofoid [3] described the genus ™agellate species in the genus Alexandrium [1]. Whilst neritic Centrodinium for oceanic species characterized by a high lat- species of the planktonic Alexandrium or the epiphytic erally ™attened cell body with an apical and an antapical horn. Gambierdiscus, responsible for toxic events, have received Kofoid [3] also described the genus Murrayella for three types 2 Journal of Marine Biology of species: globular, biconical, and laterally compressed spe- observing the presence of Centrodinium in these two sampling cies. An account of the taxonomy of Centrodinium and stations, subsamples of the plankton concentrate were treated Murrayella is reported in the Appendix S1 part 1–4, 7 as with small amounts (150–200 μl) of 10% (weight/volume) Supplementary material. Balech [4–6] carried out studies on sodium thiosulfate for removing the iodine. e cells of each Centrodinium and the laterally ﬂattened species Murrayella species of Centrodinium were micropipetted individually with and in 1967 commented on the general resemblance between a ﬁne capillary into a clean chamber ﬁlled with autoclaved Centrodinium and his new species Murrayella mimetica, but Milli-Qultrapure water. e same procedure was repeated he maintained the split of both genera due to the diﬀerences twice in order to remove any source of contamination. in the plate formula following a strict Kofoidian scheme of Finally, 30–40 cells of each species were deposited in a 0.2 ml tabulation. e classiﬁcation of Centrodinium has been a mat- Eppendorf tube ﬁlled with absolute. ter of controversy ([7–9]; see Appendix S1 part 8 as For plate dissociation, each cell was individually isolated Supplementary material). and placed in an Utermöhl chamber with distilled water. In 2012, Gómez [10] classiﬁed Centrodinium in the same Drops of a solution of 5% sodium hypochlorite (commercial subfamily of Alexandrium within the Gonyaulacales. Li et al. bleach solution, 1 : 1 mixture of sodium hypochlorite and [11] reported that Centrodinium punctatum unexpectedly Milli-Q water) were added until the split of the thecal plates. clusters with Alexandrium aﬃne, and consequently the genus In other cases, the theca was squashed by touching it with a Alexandrium was polyphyletic. Li et al. [11] did not propose ﬁne capillary tube to split the thecal plates. e cell was repeat- the split of the genus Alexandrium because they were based edly photographed at diﬀerent stages during the process of only on C. punctatum. e species C. punctatum diﬀers from splitting the theca with the inverted microscope at 600x the typical species of Centrodinium that are fusiform, with an magniﬁcation. elongated and high ﬂattened body, and a smooth thecal sur- For analyses using scanning electron microscopy, a sub- face. Li et al. [11] submitted the sequences as Alexandrium sp. sample was ﬁltered through a 3 μm pore size polycarbonate (GenBank accession numbers MF043217–20), and they did membrane (Millipore Ltd., Middlesex, U.K.). e ﬁlter was not propose the split of Alexandrium as C. puntactum does rinsed three times in Milli-Q water, dehydrated through not represent the typical morphology of the genus graded ethanol series (30%, 50%, 70%, 80%, 90%, 95%, and Centrodinium. Li et al. [11] remarked the need of the study for two steps in 100%). en, the protocol was to immerse the the typical species of Centrodinium before considering the ﬁlter in HMDS (Hexamethyldisilazane, Molekula Limited, generic split of Alexandrium. Newcastle, U.K.) for 30 minutes (twice). e HMDS was evap- In this study, we investigate the morphology of two highly orated by placing the sample overnight under the fume hood. laterally ﬂattened species with apical and antapical horns, Filters were mounted on an aluminium stub, sputter-coated C. eminens and C. intermedium, and also Centrodinium with Au/Pd (Polaron SC7620, Quorum Technologies Ltd., punctatum. We provide the ﬁrst molecular data (SSU and LSU Ashford, U.K.) and observed at 15 kV with a SEM LEO 438 rRNA gene sequences) for the typical species of Centrodinium. VP (Carl Zeiss AG, Oberkochen, Germany). Images were pre- e new morphological and molecular data conﬁrm the poly- sented on a black background using Adobe Photoshop CS3 phyletic character of Alexandrium. We propose the split of (Adobe Systems Inc., San Jose, CA, USA). Alexandrium into four genera that reconciles with the molec- 2.2. DNA Extraction, PCR Ampliﬁcation of rRNA Gene and ular and morphological data, and requires fewer taxonomical innovations. No taxonomical innovations are needed for the Sequencing. Prior to PCR, the sample tube was centrifuged, species comprising the clade that contains the type species of and ethanol was evaporated by placing the tube overnight in a Alexandrium, A. minutum, which remains as Alexandrium s.s. desiccator at room temperature. Genomic DNA was extracted e species of the clades that contain the type species of using Chelex (InstaGeneTM Matrix; Bio-Rad, Hercules, CA, Protogonyaulax and Gessnerium are placed in the revived USA) following protocols adapted from Richlen and Barber genera Protogonyaulax and Gessnerium, respectively. e [12], as outlined in Gómez et al. [13]. e SSU rRNA gene species A. aﬃne and A. gaarderae, closely related to was ampliﬁed using two sets of primers: EukA and 1055R; Centrodinium, need to be transferred into a new erected genus. and 570F and EukB [14]. e D1–D3 domains of the LSU rRNA gene were ampliﬁed using primers D1R and D3Ca [15]. PCR ampliﬁcations were performed in a 25 μl reaction volume containing 1 μl of template DNA (supernatant from each 2. Materials and Methods Chelex extraction), 1 × PCR buﬀer (500 mM KCl and 100 mM 2.1. Sampling, Isolation, and Microscopy. Sampling was Tris–HCl, pH 8.3), 2 mM MgCl , 0.8 mM dNTPs, 0.5 mM of performed with a phytoplankton net (20 μm mesh size) on each primer, and 0.5 U of AmpliTaq DNA Polymerase (Applied the surface waters of the South-Eastern Bay of Biscay, North Biosystems Inc., Foster City, CA, USA). Hot start PCR Atlantic, in August 2017. Samples from two stations at 43°36′ ampliﬁcations were performed in a Mastercycler Nexus thermal N–1°57′ W and 43°36′ N–2°03′ W are described here. e cycler (Eppendorf, Hamburg, Germany) with the following plankton concentrate was preserved with acid Lugol′s iodine cycling conditions for both primer sets: initial denaturation solution to a ﬁnal concentration of 4% (vol:vol), and kept (95°C/5 min); 35 cycles of denaturation (95°C/30 s), annealing refrigerated (~3°C). e material was examined with an (55°C/1 min), and extension (72°C/2 min); ﬁnal extension inverted microscope (Nikon Eclipse TE2000-S, Tokyo) and (72°C/10 min). PCR products were visualized on a 1% agarose photographed with a Nikon Digital Sight DS-2 M camera. Aer gel stained with GelRed (Biotium, Hayward, CA, USA). Journal of Marine Biology 3 Positive PCR products were cloned into vector PCR 2.1 using detailed description of the plate arrangement of C. punctatum, a TOPO TA cloning kit (Invitrogen, Carlsbad, CA, USA). C. intermedium, and C. eminens is available in the Appendix Clones were screened for inserts by PCR ampliﬁcation with S2 as Supplementary material. We describe here the apical, plasmid primers M13F and M13R, and positive clones from sulcal, and antapical plate series. each PCR amplicon were puriﬁed using the Qiaquick PCR e plates 2′ and 4′ almost completely encircled the apical puriﬁcation kit (Qiagen, Hilden, Germany), and sequenced pore plate (Po), while the contact with the plate 1′ in the ven- in both the forward and reverse direction (Euroﬁns MWG tral side was much reduced, and the plate 3′ did not reach the Operon, Ebersberg, Germany). Sequence reads were aligned Po (Figures 1(d)–1(f )). Scanning electron microscopy revealed and assembled in Geneious Pro 11.1.2 (Biomatters, Auckland, a horseshoe-shaped apical pore surrounded by a rim of small New Zealand). e newly generated consensus sequences were marginal pores (Figures 1(q)–1(s)). e sulcal plates are placed deposited in DDBJ/EMBL/GenBank under accession numbers between the anterior sulcal plate (S.a.) in the epitheca and the MK714074–MK714082. posterior sulcal plate (S.p.) near the antapex (Figures 1(g)–1(l), 1(p)). Two small plates known as the anterior and posterior 2.3. Phylogenetic Analyses. SSU– and LSU rRNA gene sequences median plates (S.m.a. and S.m.p.)—one above the other— of Centrodinium spp. were analysed using Basic Local Search occurred below the anterior sulcal and the le and right ante- Tool (BLAST, http://blast.ncbi.nlm.nih.gov/Blast.cgi) against rior lateral plates (S.s.a. and S.d.a.). Two lateral pairs of plates databases in GenBank. e closest matches to these searches were located below, the le and right posterior lateral plates were sequences in the genus Alexandrium (primarily A. aﬃne) (S.s.p. and S.d.p.), with the le plate being longer than the right and the sequences reported as “Alexandrium sp. ZL2017” that pair (Figures 1(h)–1(k)). e sulcal posterior (S.p.) was an were later identiﬁed as Centrodinium punctatum in Li et al. irregular pentagon with length approximately equal to the [11]. Based on these results, rRNA gene sequence data were width (Figures 1(f ), 1(h)). e S.p. plate was displaced towards compiled from similar sequences identiﬁed using BLAST. the le side below the plates 1′′′ and 2′′′ and the le margin Sequence alignments of available SSU– and D1-D2 LSU rRNA joining to the plate 1′′′′ (Figures 1(h), 1(l) and 1(m)). ere gene sequences of Centrodinium spp., representatives of each were two antapical plates with a triangular shape that con- species of Alexandrium, other gonyaulacoid dinoﬂagellates, formed a pointed antapex directed towards the ventral side. and other dinokaryotic dinoﬂagellates were accomplished e ﬁrst antapical (1′′′′) in the le face (Figures 1(h), 1(l)– by Clustal W [16] and the evolutionary history was inferred 1(m)) was slightly smaller than the second antapical plate by using the Maximum Likelihood method based on the (2′′′′) in the right face (Figures 1(f ), 1(i), 1(n)–1(o)). e plate General Time Reversible model with Gamma distributed with 1′′′′ was in contact to S.p. and 2′′′′ plate (Figures 1(h), Invariant sites and the default settings in MEGA7 soware 1(l)–1(n)). [17]. Bootstrap values were obtained aer 1000 replications. e apicomplexan Eimeria tenella (AF026388) was used as 3.2. Morphology of Centrodinium intermedium. e lateral an out group in the SSU– and LSU rRNA gene phylogenies. 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 3. Results (Figure 1(a)). e apical horn of C. intermedium was usually 3.1. Morphology of Centrodinium punctatum. e species shorter than the other species of Centrodinium. Cells were 130– Centrodinium punctatum, C. intermedium, and C. eminens 175 μm long. e depth of the cells (dorso-ventral distance) was were the most abundant (in that order) in the sampling 55–80 μm. e width between the le and right sides is stations in the South-Eastern Bay of Biscay (Figure 1(a)), diﬃcult to measure in these highly laterally ﬂattened cells, providing material for the morphological (plate dissociation with values of about 25–35 μm wide at the cingulum level and SEM) and molecular analyses. A few individuals of (Figures 2(a)–2(c)). e dense poroid ornamentation of the Centrodinium maximum were also found, but in an insuﬃcient theca observed in C. punctatum was missing in C. intermedium, abundance for detailed studies. e sea surface temperatures with only scattered pores, more abundant in the right face of in the two sampling stations ranged from 23.4°C to 23.8°C and the apical horn (Figure 2(d)). e apical horn (~20 μm long) of the salinity from 34.5 to 34.6. Centrodinium punctatum was C. intermedium was a short truncated cone (Figures 2(a)–2(d)). the most abundant species compared with other congeneric e antapical horn was longer (>50 μm) and directed towards taxa. e cells were slightly laterally ﬂattened with a rhomboid the le-ventral side. Consequently, the antapical horn was shape. Cell dimensions were 65–90 μm long, 35–42 μm in a diﬀerent same plane than the main body and the apical depth (dorso-ventral diameter), and 24–34 μm wide (length horn (Figure 2(c)). e antapical horn had a triangular section between the right and le lateral sides) (Figure 1(a)). e with a slight anticlockwise torsion, and three terminal spinules epitheca was conical with a blunt apex. e hypotheca was (Figures 2(m)–2(o)). Each face of the antapical horn had a row conical with a pointed antapex directed towards the ventral of sunken areas with 3-4 small pores (Figure 2(n)). side. In addition to the size variability, the individuals showed e molecular data revealed a very close phylogenetical a diﬀerent degree of development the pointed antapex relationship between C. punctatum and C. intermedium (see (Figure 1(b) and 1(c)). e theca was ornamented with poroids below in Figures 4 and 5). It is commonly assumed that con- (Figures 1(d)–1(f ), 1(l)–1(q)). Centrodinium punctatum had generic species share a similar plate formula. e epithecal a plate formula Po, 4′, 6′′, 6c, 8s+, 5′′′, and 2′′′′. A more plate formula of C. punctatum is Po, 4′, 6′′ or alternatively 3′, 4 Journal of Marine Biology F¨©ª«¬ 1: Light (a–j) and scanning electron (l–s) micrographs of Centrodinium punctatum. (a) Plankton sample with Centrodinium spp. ƒe arrows point the cells of C. punctatum. (b–c) Individuals used for molecular analyses. (d) Partially dissociated theca in ventral view. (e–f ) Epitheca. ƒe insets show the rst postcingular plate and the posterior sulcal plate. (g) Right-ventral face. ƒe inset shows the right antapex. (h) Le’-ventral view. ƒe inset shows the dorsal antapex. (i) Ventral view of the sulcal area. (j-k) Dissociated sulcal plates. (l) Le’-ventral view. (m) Le’ antapex. (n) Le’ face. (o) Right face. (p) Ventral view. (q) Apical view. (r–s) Apex. 1′–4′ = apical plates; 1′′–6′′ = precingular plates; 1′′′–5′′′ = postcingular plates; 1′′′′–2′′′′ = antapical plates; C1–C6 = cingular plates; c.p. = closing, cover platelet or canopy; mp = marginal pores surrounding the apical pore plate; Po = apical pore plate; S.a. = anterior sulcal plate; s.a.p. = pore of the anterior sulcal plate; S.d.a. = right (dexter) anterior lateral sulcal; S.d.p. = right posterior lateral sulcal; S.m.a. = anterior median sulcal; S.m.p. = posterior median sulcal; S.p. = posterior sulcal plate. S.s.a. = le’ (sinister) anterior lateral sulcal; S.s.p. = le’ posterior lateral sulcal. Scale bars (a–q) = 20 μm, (r–s) = 2 μm. 1a, 6′′ in a strict Kofoidian scheme. ƒe species C. interme- conform the apical horn, and the development of these plates dium has an additional plate in the le’ face of the epitheca, hindered that the plates 1′ and 3 ′ reached the apex (Figures and the plate formula in a strict Kofoidian scheme is Po, 2′, 2(d), 2(o)–2(q)). While the plate 4′ was narrow and long, the 2a, 7′′. In contrast to C. punctatum, the apical plates of C. elongation of the plate 2′ resulted in the split into two plates. intermedium were larger than the precingular plates (Figure ƒe formula of the epitheca of C. punctatum and C. interme- 2(d)). ƒe rst apical plate of C. punctatum reached the apical dium is similar (Po, 4′, 6′′), using the labelling 2′ ( α + β) to pore (insert 1′), while in C. intermedium it does not reach the denote the split of the second apical plate in C. intermedium. apex (exsert 1′). When compared to C. punctatum, the main ƒe right side of the epitheca was essentially similar to C. punc- modications of C. intermedium were the elongation of the tatum, where 4′ plate has expanded anteriorly, and then the plates 4′ and 2′ (the latter split into two plates) to conform the 3′ plate did not reach the apex (Figure 2(d)). During the plate apical horn, the di§erent length of the posterior lateral sulcal dissociations, the Po remained attached to the plate 2′β as a plates, and the formation of a tubular antapical horn supported circular structure of about 1 μm in diameter (Figure 2(d)). ƒe at its ventral basis by two triangular plates. ƒe apical plates tiny membranous Po platelet was poorly conserved in the SEM 2′ and 4 ′ of C. intermedium have extended anteriorly to preparations. ƒe very thin plate 2′β appeared crushed against Journal of Marine Biology 5 F¨©ª«¬ 2: Light (a–j) and scanning electron (k–r) micrographs of Centrodinium intermedium. (a) Several individuals. (b) Le’ face. (c) Le’- ventral view. Note the antapical horn oriented toward the le’ side. (d) Le’ and right faces of the same epitheca. ƒe inset shows the apex. (e) Le’ hypotheca. ƒe inset shows the rst antapical plate. ƒe arrowhead points a liform extension. (f ) Right hypotheca. (g) Detail of the sulcal area. (h) Dissociated anterior sulcal plates. ƒe arrowheads point a membranous ™ange. (i) Anterior lateral sulcal plates. (j) Dissociated posterior lateral sulcal plates. (k–l) Ventral view. (m) Right face. (n) Di§erent antapical horns. (o) Le’ face. (q–r) Detail of the apex. 1′–4′ = apical plates; 1′′–6′′ = precingular plates; 1′′′–5′′′ = postcingular plates; 1′′′′–2′′′′ = antapical plates; C1–C6 = cingular plates; Po = apical pore plate; S.a. = anterior sulcal plate; s.a.p. = pore of the anterior sulcal plate; S.d.a. = right (dexter) anterior lateral sulcal; S.d.p. = right posterior lateral sulcal; S.m.a. = anterior median sulcal; S.m.p. = posterior median sulcal; S.p. = posterior sulcal. S.s.a. = le’ (sinister) anterior lateral sulcal; S.s.p. = le’ posterior lateral sulcal plate. Scale bars (a–m, o) = 20 μm, (n, q–r) = 2 μm. the thick plate 4′ (Figures 2(q)–2(r)). ƒe antapex of C. punc- was a pore, the posterior attachment pore, located in this tri- tatum and C. intermedium showed di§erences. ƒe posterior angular plate in the right face (Figure 2(f )), which is a char- hypotheca of C. intermedium was composed of three plates: a acteristic of the posterior sulcal plate of chain-forming tubular plate that conforms the antapical horn and two plates gonyaulacoid dino™agellates. In the le’ face, the rst antapical in the ventral side that acted as a counterfort or backstay. ƒese (1′′′′) was a triangular plate that o’en showed a posterior two triangular plates were slightly laterally inclined towards liform extension (Figure 1(e)). ƒe second antapical plate the le’ face, and the antapical horn was directed towards the (2′′′′) emerged from the dorsal side to conform a tubular le’ and ventral sides (Figures 2(e)–2(f ), 2(k)–2(o)). ƒe most antapical horn (Figures 2(e)–2(f )), with a slight anticlockwise immediate interpretation was that the antapex consists one torsion and three terminal spinules (Figures 2(m)–2(n)). antapical plate that conforms the horn, and two posterior In the sulcal plate series, the anterior sulcal plate (S.a.) was intercalary plates that support the ventral basis of the antapical part of the epitheca, enclosed between the plates 6′′, 1′, and horn. ƒis implies that the posterior sulcal plate (S.p.) was 1′′ and the rst cingular plate (Figures 2(g)–2(h)). ƒere was missing in C. intermedium. In C. punctatum, the S.p. was an a prominent pore in the middle of the plate connected to the irregular pentagon located in the le’-ventral side below the right border by a narrow canal. In some cells, the right poste- plates 1′′′ and 2′′′ (Figures 1(f ), 1(i), 1(l)–1(m)), while the S.p. rior corner of the S.a. showed a membranous ™ange that con- of C. intermedium was triangular and located in the right face nected with the rst cingular plate (Figure 2(h)). ƒe right below the plates 4′′′ and 5′′′ (Figures 2(f ), 2(k)–2(m)). ƒere anterior lateral sulcal plate (S.d.a.) was larger than its le’ pair, 6 Journal of Marine Biology with the shape of an irregular right triangle that resembled the unreported since the original description in 1907. It seems shape of the Sicily Island (Figures 2(g), 2(i)). In C. punctatum, likely that C. elongatum corresponds to a recently divided cell the le posterior lateral sulcal plate (S.d.p.) was longer than of C. maximum or C. eminens (see Appendix S1 part 2 in the its right pair (Figures 1(i), 1(k)), while reversed in C. interme- Supplementary material). dium (Figure 2(j)). e right posterior sulcal plate (S.d.p.) of In the SSU rRNA gene phylogeny, the three species of C. intermedium was the longest of the sulcal series and showed Centrodinium clustered together with high support with C. the shape of a knife, with a reinforcement in the le margin punctatum in a basal position. e Centrodinium spp. clade (Figure 2(j)). e le posterior sulcal plate (S.s.p.) was smaller, clustered with Alexandrium aﬃne, with strong support (BP like a very elongated pentagon that ﬁt in the knife handle 100%) (Figure 4). In the LSU rRNA gene phylogeny, formed by the anterior le margin of the S.d.p. (Figure 2(j)). Centrodinium spp. also clustered with sequences retrieved e morphology of these plates suggests that the overlap from GenBank as Alexandrium aﬃne and A. concavum growth of the S.d.p. has hindered the posterior development (Figure 5). In an additional LSU rRNA tree more reference of the S.s.p. sequences were added from GenBank within the A. aﬃne clade to include sequences identiﬁed as A. aﬃne, A. tamarense, 3.3. Morphology of Centrodinium eminens. In lateral view, and A. concavum (Figure S1 as Supplementary material). e the cells of C. eminens were fusiform and slightly sigmoid strains CAWD51-52 diverged from the other sequences of because the apical horn was slightly directed towards the A. aﬃne. In the SSU- and LSU rRNA gene phylogenies dorsal side, and the antapical horn towards the ventral side. (Figures 4–5), the species of the tamarense/catenella/fratercu- e ventral margin of the epitheca was almost straight. e lus groups of Alexandrium clustered with high support as a dorsal margin was curved in the posterior half and almost sister group to the Centrodinium spp. and A. aﬃne clades. e straight in the anterior half where the apical horn with clade of Alexandrium sensu stricto (s.s.) containing the type, a brunt apex was slightly directed towards the dorsal side A. minutum, and four divergent species (A. diversaporum, (Figure 3(a)). e cells of C. eminens were 182–239 μm long, A. leei, A. margaleﬁi, A. pohangense). e species of the sub- and 31–47 μm in depth (dorso-ventral distance), being less genus Gessnerium, A. monilatum and allied species, formed robust (lower depth), and less ﬂattened than C. intermedium. other clade (Figures 4–5). e apical and antapical horns of C. eminens were longer (Figure 3(a)) than in C. intermedium (Figure 2(a)). e 4. Discussion antapical horn of C. intermedium was very inclined towards the face (Figure 2(c)), while the inclination was almost absent 4.1. Aﬃnities between Centrodinium and Alexandrium. e in C. eminens (Figures 3(a)–3(h)). e plate arrangement molecular data reveal that Centrodinium clusters with strong of C. eminens and C. intermedium was similar, with more support amongst the clades of Alexandrium (Figures 4–5; anterior-posteriorly elongated plates, especially in the apical [11]). Species such as C. punctatum have the same plate series in C. eminens (Figures 3(b)–3(l), 3(t)–3(v)). e two formula of Alexandrium (Figures 1, 6(e)). e most typical plates, 2′ (α + β), resulting of the split of the second apical apical pore plate of Alexandrium has a comma-shaped pore plate remained joined (Figure 3(d)). e distal antapical horn surrounded by marginal pores, and the chain-forming species also showed three spinules (Figure 3(u)). e sulcal series have an anterior attachment pore [18]. e apical pore plate of was fully similar (Figures 3(m)–3(q)). e triangular ﬁrst Alexandrium is larger (>6 μm), and we can easily observe an antapical and the posterior sulcal plates showed a ﬁliform oval or comma-shaped pore. e formation of the apical horn posterior extension (Figures 3(r)–3(s)). e posterior sulcal of Centrodinium implies a reduction of the surface available for plate showed a posterior attachment pore (Figures 3(r)–3(s)). the apical pore plate (<2 μm), and the horseshoe-shaped could In the SEM preparations, some individuals of C. eminens were be a result of the constriction of the oval or comma-shaped in better preservation stage than those of C. intermedium, and pore (Figures 1(r)–1(s), 3(y)–3(z)). some details of the apex were revealed (Figures 3(w)–3(z)). e chain-forming species of Alexandrium have an attach- e apex of C. eminens also collapsed in the SEM preparations ment pore (a.a.p.) in the apical pore plate, and an attachment but in some individuals the membranous apical pore platelet pore (p.a.p.) in the posterior sulcal plate. e cells of a chain and the thin second antapical were not crushed against are interconnected by these pores [18]. In Centrodinium, the the thicker four apical plate. In these cases, a large pore anterior attachment pore is more diﬃcult to observe due to of 1–1.5 μm in diameter was observed devoid of the cover the small size and fragility of the membranous apical platelet, platelet (Figures 3(w)–3(x)). is membranous cover platelet or it may be confused with marginal pores. Hernández- remained in few individuals, with the apical pore surrounded Becerril et al. ([9], their Figure 33) reported a pore in the apex by a few tiny pores (Figures 3(y)–3(z)). that could be the apical pore devoid of the foramen, or alter- 3.4. Molecular Phylogeny. e SSU and LSU rRNA gene natively the anterior attachment pore. e posterior attach- ment pore in the posterior sulcal plate is evident in sequences were obtained from three species of Centrodinium: C. punctatum that is the ﬁrst described laterally ﬂattened C. intermedium and C. eminens (Figures 2(f ), 3(r)–3(s)), and C. pulchrum ([9], their Figure 37). species of the former genus Murrayella; C. intermedium that is the most ﬂattened species of this genus with an oval hypotheca, e sequences of Centrodinium clustered as a sister group to Alexandrium aﬃne (Figures 4 and 5; [11]). at clade and C. eminens which morphology is close to the type species, C. elongatum. It should be noted that the type species remains includes sequences retrieved from GenBank under the names Journal of Marine Biology 7 F¨©ª«¬ 3: Light (a–s) and scanning electron (t–z) micrographs of Centrodinium eminens. (a) Several individuals. (b) Le’ face. (c) Right epitheca. (d) Le’-ventral view. ƒe insets show the sulcus. (e) Dissociated plates of the apical horn. (f–g). Ventral views. ƒe inset shows the sulcus. (h) Dissociated epitheca and hypotheca. (i–m) Several views of the same epitheca. (o, q) Dissociated posterior lateral sulcal plates. (p) Anterior sulcal. (r) Posterior sulcal and rst antapical plate. ƒe arrowhead points a liform extension. (s) Antapical horn. (t) Le’ face. (u–v) Right face. (w–z) Apex. 1′–4′ = apical plates; 1′′–6′′ = precingular plates; 1′′′–5′′′ = postcingular plates; 1′′′′–2′′′′ = antapical plates; C1–C6 = cingular plates; c.p. = closing, cover platelet or canopy; mp = marginal pores surrounding the apical pore plate; Po = apical pore plate; S.a. = anterior sulcal plate; s.a.p. = pore of the anterior sulcal plate; S.d.a. = right (dexter) anterior lateral sulcal; S.d.p. = right posterior lateral sulcal; S.m.a. = anterior median sulcal; S.m.p. = posterior median sulcal; S.p. = posterior sulcal; S.s.a. = le’ (sinister) anterior lateral sulcal; S.s.p. = le’ posterior lateral sulcal plate; Scale bar (a–v) = 20 μm, (w–z) = 2 μm. 8 Journal of Marine Biology Centrodinium eminens FG2 MK714079 Centrodinium intermedium FG4 MK714081 Centrodinium intermedium FG3 MK714080 Centrodinium punctatum FG5 MK714082 Centrodinium punctatum MF043219 86 Centrodinium Centrodinium punctatum MF043220 AY775286 Episemicolon gen. nov. AJ535375 Alexandrium fundyense KF908795 KF908800 100 Alexandrium mediterraneum KF908797 Protogonyaulax Alexandrium tamarense KF908799 Alexandrium australiense KF908802 Alexandrium cohorticula AF113935 Alexandrium fraterculus AY421776 Alexandrium pohangense LN811348 U27498 Alexandrium insuetum AB088298 Alexandrium minutum AY883006 Alexandrium s.s. Alexandrium tamutum AJ535379 Alexandrium ostenfeldii U27500 Alexandrium leei AY641565 Alexandrium diversaporum KF251139 Alexandrium satoanum AY641566 Alexandrium monilatum AY883005 Gessnerium Alexandrium taylorii AJ535385 Alexandrium pseudogoniaulax JF521638 Fukuyoa paulensis KM886379 Pyrrhotriadinium polyedricum KM886380 100 Pyrocystis noctiluca AF022156 Fragilidium subglobosum AF033869 F. duplocampanaeforme KY624502 Pyrodinium bahamense AF274275 100 Gonyaulax spinifera AF022155 82 Gonyaulax polygramma AJ833631 Ceratium hirundinella AY443014 Tripos fusus AF022153 Tripos furca AJ276699 100 Peridinium cinctum EF058243 Peridinium willei EF058250 Polarella glacialis AF099183 Pelagodinium bei U37365 Symbiodinium microadriaticum M88521 100 Gymnodinium catenatum AF022193 Gymnodinium fuscum AF022194 Dinophysis acuta AJ506973 100 Phalacroma rotundatum AJ506975 Scrippsiella trochoidea EF492513 Scrippsiella sweeneyae HQ845331 100 Durinskia dybowskii AF231803 Durinskia agilis JF514516 Karenia mikimotoi AF022195 Karenia brevis DQ847434 Prorocentrum micans EF492511 Prorocentrum minimum Y16238 96 Oxytoxum scolopax KY421376 Corythodinium tessellatum KY421378 Heterocapsa triquetra AF022198 0.05 Heterocapsa rotundata AF274267 Azadinium spinosum JN680857 Azadinium caudatum JQ247701 Eimeria tenella AF026388 F¨©ª«¬ 4: Maximum-likelihood phylogenetic tree of the SSU rRNA gene. Bootstrap support values (BP) >70 are shown. New sequences are highlighted in bold. ƒe scale bar represents the number of substitutions for a unit branch length. A. a‹ne, A. tamarense, and A. concavum. Two sequences from Protogonyaulax (Figure 5, S1).ƒe members of the tamarense/ New Zealand, the strains CAWD51 named A. a‹ne (accession catenella group are responsible for paralytic shellsh poisoning number AY338753) and CAWD52 named A. concavum (acces- (PSP) events. ƒe sxtA gene (saxitoxin biosynthesis pathway sion number AF032348) were identical and diverged from the protein A domain) has been detected in the members of the main group of A. a‹ne. ƒe latter subdivided into two groups, tamarense/catenella group or A. fraterculus. In contrast, PSP one for strains isolated exclusively from Japan and China, and toxicity or the presence of the sxtA gene have not been detected other group for strains from diverse world regions (Figure S1). in A. a‹ne [23] and Centrodinium punctatum [11]. ƒe cells of the strain CAWD52 illustrated in MacKenzie et al. Alexandrium a‹ne is distinguished primarily by the apical [19] corresponded to A. gaarderae as dened by Larsen and pore plate and other di§erences in the sulcal plates. Balech Nguyen-Ngoc [20]. ƒe species Alexandrium a‹ne was rst [18] reported that the apical pore platelet is narrow, long, and described as Protogonyaulax a‹nis [21], and since the earlier fundamentally bullet-shaped. ƒe foramen does not form a molecular phylogenies the sequences of A. a‹ne have always true comma because it is oval and relatively small; it is located diverged from the members of the tamarense/catenella group in the ventral half of the plate and a large and almost circular [22]. ƒe species A. a‹ne and A. gaarderae (non A. concavum connecting pore is dorsal [18]. Alexandrium gaarderae emend. Nguyen-Ngoc & Larsen) clustered as a sister group of (reported as A. concavum) also has a dorsal connecting pore Centrodinium and more distantly related to the clade of [24]. ƒe location of the anterior attachment pore at the dorsal Gonyaulacales Journal of Marine Biology 9 Centrodinium eminens FG2 MK714074 Centrodinium intermedium FG3 MK714075 Centrodinium intermedium FG4 MK714076 Centrodinium punctatum MF043217 Centrodinium Centrodinium punctatum MF043218 Centrodinium punctatum FG6 MK714078 Centrodinium punctatum FG5 MK714077 Alexandrium ane AF318229 Episemicolon gen. nov. Alexandrium ‘concavum’ AF032348 Alexandrium fundyense KF908807 Alexandrium mediterraneum KF908808 Alexandrium tamarense KF908805 Alexandrium paci cum KF908803 Protogonyaulax 100 Alexandrium australiense KF908810 Alexandrium fraterculus KF034859 Alexandrium tropicale AY268613 100 Alexandrium tamiyavanichii AB088267 Alexandrium cohorticula AF174614 Alexandrium margale i AY152708 Alexandrium andersonii JF521621 Alexandrium minutum AY705869 Alexandrium s.s. Alexandrium tamutum AY268618 Alexandrium insuetum AB088248 Alexandrium ostenfeldii AY268601 Alexandrium peruvianum FJ011437 Alexandrium satoanum AY438020 Alexandrium taylorii AB607263 Gessnerium Alexandrium pseudogoniaulax AB088254 Alexandrium hiranoi AY438018 Alexandrium leei AY566184 100 Pyrodinium bahamense AB936757 Pyrodinium bahamense AY154959 100 Pyhrrotriadinium polyedricum JQ247712 Fukuyoa paulensis KM886379 96 Fragilidium subglobosum AF260387 Pyrocystis noctiluca FJ939576 Gonyaulax spinifera EF416284 100 Gonyaulax cf. spinifera AY154960 Gonyaulax polygramma DQ162802 Dinophysis acuta AY277648 Phalacroma rapa EU780655 Pelagodinium bei JN558107 100 Gymnodinium fuscum AF200676 Karenia brevis AY355455 100 Heterocapsa triquetra AF260401 Heterocapsa arctica AY571372 Prorocentrum steidingerae DQ336183 0.1 100 Prorocentrum micans EU780638 Eimeria tenella AF026388 F¨©ª«¬ 5: Maximum-likelihood phylogenetic tree of the D1-D2 domains of the LSU rRNA gene. Bootstrap support values (BP) >70 are shown. New sequences are highlighted in bold. ƒe scale bar represents the number of substitutions for a unit branch length. margin of the apical pore plate is the main diagnostic character C. punctatum as Po, 3′, 1a, 6′′, 6c, 8s, 5′′′, 1p, 2′′′′. ƒese authors of the species A. gaarderae and A. a‹ne [24]. In the other follow a strict Kofoidian scheme of tabulation of the epitheca, species of Alexandrium, the apical pore is comma-shaped and and labelled the apical plate that does not touch the apical the anterior attachment pore lying in the right side. ƒe two pore plate as an intercalary plate. Li et al. [11] misidentied posterior lateral sulcal plates are more or less similar in length the sulcal and hypothecal plates. Li et al. ([11], p. 177, their in the members of the tamarense/catenella group, while the Figure 8(c)) illustrated the right (S.d.p.) and le’ posterior sulcal right posterior sulcal plate is longer than the le’ posterior (S.s.p.) plates with a similar length. Li et al. [11] did not carry sulcal plate in A. a‹ne (Figure 6(d)). ƒis feature is variable out a study using plate dissection, and the sulcal lists were in Centrodinium spp. (Figures 6(e) and 6(f )). ƒe cingulum hiding the morphology of the sulcal plates. ƒese plates have and the sulcus of Centrodinium spp. and A. a‹ne are deeply very di§erent length as revealed in this study (Figure 1(k)) and incised and bordered by pronounced list, and the posterior the plate dissections of C. punctatum by Balech [5, 6]. ƒey le’ margin of the plate 6′′ is reinforced, long and concave labelled the le’ lateral posterior sulcal as the posterior plate, (Figures 1–3, 6(d)–6(f ); [20, 21]). and this induces the subsequent errors in the tabulation of the hypotheca (see Appendix S1 part 6 as Supplementary material). 4.2. Reclassi‘cation of the Subgenus Gessnerium. An historical ƒe genus Alexandrium is currently a pool of species with account of the taxonomy and nomenclature of Alexandrium signicant di§erences in the plate arrangement [26]. Balech s.l., including Gessnerium and Protogonyaulax, is reported in [18] reported that the species of the subgenus Gessnerium were the Appendix S3 as Supplementary material. ƒe plate formula closer to Pyrrhotriadinium than Alexandrium. ƒe apical pore of Alexandrium is usually reported as Po, 4′, 6′′, 6c, 8s+, 5′′′, plate in Pyrrhotriadinium is totally transverse orientated, while 2′′′′ [25]. It is similar to the plate formula of C. punctatum oblique in Gessnerium [18, 27]. Pyrrhotriadinium lacks the and di§ers from the more ™attened species of Centrodinium accessory sulcal plates, and the two median sulcal plates are in the anterior elongation of the plates 4′ and 2′, and the split separated, while in Gessnerium the accessory plates are in the latter plate. Li et al. [11] reported the plate formula of prominent and the two median sulcal plates are in contact [18]. Gonyaulacales Saca pap C6 pap Sp Sp Sacp 7''(6'') C5 Sda C3 Sp Saca Sdp Ssa ' 2'α 2 α 10 Journal of Marine Biology 4'' (3'') 3' 1' (2') 5'' 4' 3'' (4'') 3'' 4'' 1' (2'') 1' 2'' 1'' 3' Sa (2') 5'' 2' 6'' 6'' 4' Sma (5'') 5'' 1'' 7'' 5''' 1' (6'') Sa Ssa 1''' Sda Saca 6'' 3''' Sdp 3''' Ssp aap Po 2'''' Ssa 4''' 2''' Po Sdp cp 4''' Sp Ssp 2'''' 2''' Sp 1'''' 5''' Sp 1''' 1' 1'''' 5''' Sp 1''' 1' (a) (b) 3' 2' Po 4' 3'' 2'' 4' 4'' 1' 1' 1'' 5'' Sa 1'' 2'' 3' Sa 5'' 6'' 6'' 2' Sda Sma 5'' 2''' Sma 4' 1' Ssa Sda 1'' 2''' 1' Ssa 5''' 1''' 5''' 6'' Sdp 1''' Sdp Ssp Ssp aap 3''' Po cp 3''' Sp 2''' 4''' Sp 2'''' 2''' 2'''' 4''' 1' Sp 1'''' 1'''' Sp 5''' 1''' 5 5''' ''' 1''' (d) (c) 2' 4' 2' 3' 1' 4' 2'β 1' 2'β 4'' 3'' 4' 2'' 4' 3' 3'' 2'' 2' 1'' 5'' 4'' 1'' 6'' 6'' 3' 1' C1 5'' 5'' C2 2'' 3' C1 C2 1' 4' 5'' C5 4'' 3''' C6 6'' 1''' 3'' 1''' 1'' 2''' 3''' 2'' 6'' 1'' 5''' Sa 4''' 5'' 2'' 5''' 3'' 6'' C4 4'' C3 1' C2 C5 C6 C1 1'' 4''' 3''' 1''' 3''' 1''' 4''' 5''' 5''' Sp 2''' 4''' Sa 1'''' 2'''' Sp C3 2''' C4 1''' Sa 2''' Smp 3''' Sd a 1'''' C3 3'' 4''' 2'''' Po Sda C4 Ss p 4'' 2''' C5 2' C2 2'' 3' 1'''' 2'''' 1'''' 2' 4' C5 C2 2'''' Sp Sdp 1' 2'''' 5'' 4' 2'''' 1' 5''' 1''' 1'' C1 C1 C6 6'' Sp C6 (e) (f ) F¨©ª«¬ 6: Line drawings of the ventral, apical and antapical views, apical pore plate and sulcal plates of Alexandrium sensu lato and Centrodinium. (a) Gessnerium monilatum redrawn and modied from Balech (1995). (b) Alexandrium minutum redrawn and modied from Balech (1989, 1995). (c) Protogonyaulax tamarensis redrawn and modied from Balech (1995). (d) Episemicolon a‹ne gen. & comb. nov. (formerly A. a‹ne) redrawn and modied from Balech (1995). (e) Centrodinium punctatum. (f ) Centrodinium eminens. 1′–4′ = apical plates; 1′′–6′′ = precingular plates; 1′′′–5′′′ = p ostcingular plates; 1′′′′–2′′′′ = antapical plates; a.a.p. = anterior attachment pore; C1–C6 = cingular plates; c.p. = closing, cover plate or canopy; p.a.p. = posterior attachment pore; Po = apical pore plate; S.a. = anterior sulcal plate; S.a.c.a. = anterior accessory sulcal; S.a.c.p. = posterior accessory sulcal; S.d.a. = right (dexter) anterior lateral sulcal; S.d.p. = right posterior lateral sulcal; S.m.a. = anterior median sulcal; S.m.p = posterior median sulcal; S.p. = posterior sulcal; S.s.a. = le’ (sinister) anterior lateral sulcal; S.s.p. = le’ posterior lateral sulcal. 3''' 4''' 2''' 5''' 1''' 1'' (1') S s a 6'' (5'') 1'' (1') 3' (4') C4 2'' (1'') cp 1'''' Sp 3' (4') Ssp 2'' (1'') 2' (3') 2''' Smp Smp Sma Smp Smp Ssp Sdp Journal of Marine Biology 11 In Pyrrhotriadinium, the ﬁrst precingular plate, equivalent to long. e anterior and posterior attachment pore is a common the ﬁrst gonyaulacoid apical plate, does not contact the le feature in chain-forming species, but few species of apical plate [18]. e 1′′ plate is pentagonal in Gessnerium Alexandrium s.s. forms chains, and the attachment pores are (Figure 6(a)) and quadrangular in Pyrrhotriadinium, while absent (Figure 6(b), Table 1; [18, 24]). rhomboidal in nearly all the species of the subgenus Alexandrium e genus Protogonyaulax contains species where the ﬁrst (Figures 6(b)–6(c)). is is a precingular plate based on its apical plate is rhomboidal and directly connects to the apical shape and position. e posterior sulcal plate of Gessnerium is pore plate. e posterior sulcal plate is reversed pentagonal, large, longer than wide and prolonged obliquely towards the symmetrical, and longer than wide. ere are numerous posterior right (Figure 6(a)). In the species of the subgenus chain-forming species, and the presence of anterior and pos- Alexandrium, the posterior sulcal plate is relatively smaller and terior attachment pores is a common feature (Figure 6(c), non-oblique ([18]; Figures 6(b)–6(d)). PSP toxicity has not Table 1; [18, 24]). been reported in species of Gessnerium. e new scenario derived on the close relationship of e species Alexandrium margaleﬁi and A. pohangense Centrodinium and the species of Alexandrium s.l. suggests the have the ﬁrst apical plate disconnected from the apical pore reinstatement of the genera Gessnerium and Protogonyaulax, plate, which suggests an aﬃnity with Gessnerium, but this plate and the erection of a new genus for A. aﬃne and A. gaarderae. is quadrangular in these species while pentagonal in e diagnoses of the genera Centrodinium, Alexandrium, Gessnerium [18, 23]. e position of A. margaleﬁi and Gessnerium, and Protogonyaulax need to be amended. e spe- A. pohangense in the molecular phylogenies is unstable, typi- cies Peridinium splendor-maris, type of the genus Blepharocysta, cally is represented as divergent species of the clade of the type, has been interpreted to correspond to an earlier description of A. minutum [23]. ese two species, and other two divergent Alexandrium balechii. Carbonell-Moore [31] submitted a pro- species (A. diversaporum, A. leei) need further research before posal to conserve the name Peridinium splendor-maris as a to propose a change of genus. species Blepharocysta, avoiding the possible transfer of all the species of Alexandrium into Blepharocysta. If the proposal is 4.3. e Generic Split of the Subgenus Alexandrium. Previous rejected, the change does not aﬀect Alexandrium because A. morphological and molecular phylogenetic studies including balechii is now a species of Gessnerium. If the proposal is rec- sequences of Pyrodinium already suggested the reinstatement ommended, no change is applied to Alexandrium. of the Gessnerium at the genus level ([28, 29], Appendix S3 as Supplementary material). With the inclusion of Centrodinium 4.4. Emended Diagnosis of Centrodinium (Figures 6(e)–6(f), spp. in the molecular phylogenies, Alexandrium can no Table 1) longer be considered as a monophyletic genus (Figures 4 and 5). e morphological diﬀerences amongst species of 4.4.1. Centrodinium Kofoid emended Gómez & Artigas. the subgenera Gessnerium (Figure 6(a)) and Alexandrium Gonyaulacoid dinoﬂagellates with diﬀerent degree of lateral (Figures 6(b)–6(d)) are evident, but a split between species ﬂattening, an elongated brunt apex or an apical horn. of the subgenus Alexandrium based on morphology Cingulum deep, median, descending about one cingular is less conspicuous. In the molecular phylogenies, the width, without overhanging. e cingular list at both upper sequences of the subgenus Alexandrium are divided into and lower margins are prominent. e sulcus with list at both two major groups. A group that contains the type species right and le margins. e apical pore plate with a horseshoe- of Alexandrium, A. minimum, and another group split into shaped pore surrounded by small marginal pores. e plate two sister clades: one major clade that contains the type formula is Po, 4′ (2′ α + β), 6′′, 6c, ≥8s, 5′′′, 2′′′′, and the species of Protogonyaulax, P. tamarensis, with members of more ﬂattened species showed a split of the second apical plate, the tamarense/catenella/fraterculus groups, and other major 2′ (α + β). e apical pore plate is mainly surrounded by the clade for Centrodinium and Alexandrium aﬃne/A. gaarderae second and fourth apical plates, while the third apical plate (Figures 4–5). Sequences of the members of the subgenus does not reach the apex. In the less compressed species, the Alexandrium do not cluster as a monophyletic group, ﬁrst apical plate (1′) reaches the apex, whereas in the more unless we consider placing all the species of the subgenus ﬂattened species the 1′ plate does not reach the apex, and the Alexandrium into Centrodinium because Centrodinium 2′ plate is divided into two plates. In all the species, the anterior Kofoid 1907 has the priority over Alexandrium Halim sulcal plate has a distinct pore. e sulcus contains at least 1960. is implies numerous taxonomical innovations, and 8 plates, the two lateral posterior sulcal plates are long. e requires merging species with diﬀerent morphologies into a le plate is longer than the right one in the less compressed single genus. Splitting members of the subgenus Alexandrium species, and vice versa in the more compressed species. In less into three genera reconciles the molecular and morphological compressed species, the antapex is pointed, while in the more data, and requires fewer taxonomical innovations. ﬂattened species the antapical horn derived from a tubular e clade Alexandrium s.s. contains the type species, second antapical plate has terminal spinules. e antapical A. minutum, and other species in which the ﬁrst apical plate horn is supported by two triangular plates, the posterior is rhomboidal and may connect directly or indirectly through sulcal in the right face, and ﬁrst antapical plate in the le face. a thread-like prolongation with the apical pore plate e posterior sulcal plate may contain a posterior attachment (Figure 6(b), Table 1). is feature may vary intraspeciﬁcally pore near the anterior margin. e species of Centrodinium as reported for A. minutum [30]. e posterior sulcal plate is typically inhabit in warm oceans and have chloroplasts. e relatively small, symmetrical or asymmetrical, and wider than species C. punctatum is not toxic. 12 Journal of Marine Biology T 1: Comparison of the morphological and ecological characters of the genera Gessnerium emend., Alexandrium emend., Protogon- yaulax emend., Episemicolon gen. nov., and Centrodinium emend. Gómez & Artigas. Data based on Balech (1995), and this study. Gessnerium Alexandrium Protogonyaulax Episemicolon Centrodinium emend. emend. emend. gen. nov. emend. Non or slightly Non or slightly Non or slightly Non compressed or Moderate to highly Cell compression compressed compressed compressed slightly compressed lateral ﬂattening No No No No Variable Horn or spines Epitheca plate Po, 3′(4′), 7′′ (6′′) Po, 4′ (3′), 6′′ (7′′) Po, 4′, 6′′ Po, 4′, 6′′ Po, 4′, 6′′ formula Shape of apical pore Comma or ﬁshhook Comma Comma Oval or bullet Horse-shoe plate Anterior attachment Right to Po Right to Po Right to Po Dorsal to Po Unreported pore (when present) Shape of the Pentagonal Rhomboidal Rhomboidal Rhomboidal Rhomboidal equivalent gonyaula- coid ﬁrst apical plate Pore in anterior sulcal No No No No Yes Precingular part in No Sometimes Sometimes No No sulcal anterior Le cingular part in the anterior sulcal No No No No Yes plate Median sulcal plates Large Small Small Small Small Accessory sulcal plates Large Small Small Small Small Lateral posterior Right longer than Right and le of Similar or right Right longer than le Variable sulcal plates le similar length longer than le Short, wider than Short, longer than Short, longer than Polygonal or trian- Posterior sulcal plate Large long wide wide gular Absent or incon- Moderate or prom- Moderate Moderate Very prominent Sulcal list spicuous inent Posterior sulcal Typically present Present in Present in Present in Typically absent or in chain-forming chain-forming chain-forming chain-forming attachment (connect- inconspicuous ing) pore species species species species V-shaped anterior margin of the sulcal Prominent or not Inconspicuous Prominent Prominent No posterior plates Variable Rarely Common Variable Variable Chain-forming Plankton, cos- Plankton, cos- Plankton, low abun- Plankton, tropical mopolitan, mopolitan, Plankton, tropical to Habitat dance in open warm to temperate seas bloom-forming in bloom-forming in temperate seas to tropical seas neritic waters neritic waters Paralytic shellﬁsh No Yes Yes No No poisoning (1) Type species: Centrodinium elongatum Kofoid 1907. (vii) Centrodinium intermedium Pavillard 1930. (2) Other species: (viii) Centrodinium maximum Pavillard 1930. (i) Centrodinium biconicum (G. Murray & Whitting (ix) Centrodinium mimeticum (Balech 1967) F.J.R. Taylor 1899) F.J.R. Taylor 1976 [=Murrayella biconica (G. 1976 (=M. mimetica Balech). Murray & Whitting) Pavillard 1931, Pavillardinium (x) Centrodinium ovale (Pavillard 1930) Hernández- biconicum (G. Murray & Whitting) Rampi 1948]. Becerril in Hernández-Becerril et al. 2010 [=M. (ii) Centrodinium complanatum (Cleve 1903) Kofoid ovalis Pavillard, P. ovale (Pavillard) G. De Toni 1936]. 1907 (=Steiniella complanata Cleve). (xi) Centrodinium paciﬁcum (Rampi 1950) F. J. R. Taylor (iii) Centrodinium deﬂexoides Balech 1962. 1976 (=P. paciﬁcum Rampi). (iv) Centrodinium deﬂexum Kofoid 1907. (xii) Centrodinium pavillardii F. J. R. Taylor 1976 [=P. (v) Centrodinium eminens Böhm 1933. intermedium (Pavillard 1916) G. de Toni 1936, M. intermedia Pavillard, non C. intermedium Pavillard (vi) Centrodinium expansum Kofoid & J. R. Michener 1930]. 1911. Journal of Marine Biology 13 (xiii) Centrodinium porulosum Kofoid & J. R. Michener (3) e placement in Alexandrium s.s. needs further research 1911. for the species: Alexandrium diversaporum Sh. Murray et al. 2014, A. leei Balech 1985, A. margaleﬁi Balech 1994, (xiv) Centrodinium pulchrum Böhm 1933 [=C. eminens and A. pohangense A. S. Lim & H. J. Jeong in Lim, Jeong, f. pulchrum (Böhm) J. Schiller 1933]. Kim, & Lee 2015. (xv) Centrodinium punctatum (Cleve 1900) F. J. R. Taylor 1976 [=Steiniella punctata Cleve, M. punctata 4.6. Reinstatement of the Genus Gessnerium (Figure 6(a), Table 1) (Cleve) Kofoid 1907, P. punctatum (Cleve) G. De Toni 1936, M. splendida Rampi 1941, P. splendidum 4.6.1. Gessnerium Halim 1967 ex Halim 1969 emended (Rampi) Rampi 1950]. Gómez & Artigas. Gonyaulacoid dinoﬂagellates without or scarce cell compression, without spines or horns. Cingulum 4.5. Emended Diagnosis of Alexandrium (Figure 6(b), Table 1) deep, median, descending about one cingular width, without overhanging. Apical pore plate is longitudinally oriented. e 4.5.1. Alexandrium Halim 1960 emended Gómez & typical gonyaulacoid ﬁrst apical plate, narrow and rhomboidal Artigas. Gonyaulacoid dinoﬂagellates without or with scarce of Alexandrium s.l. is absent. e anterior right margin of cell compression, and lacking horn or spines. Cingulum the ﬁrst apical is an anterior margin in Gessnerium, and the deep, median, descending about one cingular width, without equivalent plate is pentagonal and never reaches the apical overhanging. e theca is usually smooth, and only ornamented pore plate. is plate is considered the ﬁrst apical plate (Po, in few species. e plate formula is Po, 4′, 6′′, 6c, >8s, 5′′′, 2′′′′. 4′, 6′′, 6c, 10s, 5′′′, 2′′′′) or a precingular plate in a strict e plate 1′ is rhomboidal, narrow, and asymmetrical and can Kofoidian scheme (plate formula Po, 3′, 7′′, 6c, 10s, 5′′′, 2′′′′). be either in direct contact with the apical pore plate or indirectly e le anterior sulcal plate (S.s.a) is large and superﬁcial, connected via a thin suture (thread-like process). Alexandrium while in Alexandrium s.l. is small and sunk into the sulcus. insuetum, has severely reticulated thecal plates and the exsert 1′. ere are two relatively large accessory sulcal plates that e plate 6′′ is usually narrow. e posterior sulcal is relatively are absent or hardly visible in Alexandrium s.l. e right small, wider than long. e apical pore plate contains a comma- posterior lateral sulcal plate (S.d.p.) is long and narrow. e shaped pore. Relatively few chain-forming species, and the posterior sulcal plate (S.p.) is longer than wide, extending attachment pore, if present, is in the lateral right to the apical pore obliquely towards the posterior right. e second antapical plate. A posterior connecting pore is usually absent. e species plate of Gessnerium is lateral, while this plate is more dorsal are typically bloom-forming in eutrophic and/or conﬁned neritic than lateral in Alexandrium s.l. e formation of chains is waters. PSP toxicity has been reported in numerous species. variable amongst the species. e species are more common in warm waters, and rarely reported in cold waters. Paralytic (1) Type species: Alexandrium minutum Halim 1960 (=A. shellﬁsh poisoning has not been associated with the presence ibericum Balech 1985, A. lusitanicum Balech 1985, A. of Gessnerium, but several species are known as ﬁsh-killers angustitabulatum F. J. R. Taylor 1995 nom. illeg.) that produce goniodomin A, spirolide, or hemolytic toxins (2) Other species of the genus Alexandrium: that may be involved in mixotrophy. (i) Alexandrium andersonii Balech 1990. (1) Type species: Gessnerium mochimaense Halim 1967 ex (ii) Alexandrium insuetum Balech 1985. Halim 1969 [=Gessnerium monilatum (J. F. Howell 1953) (iii) Alexandrium ostenfeldii (Paulsen 1904) Balech & A. R. Loeblich 1970]. Tangen 1985 [=Goniodoma ostenfeldii Paulsen, (2) Other species of Gessnerium: Gonyaulax ostenfeldii (Paulsen) Paulsen 1949, Protogonyaulax ostenfeldii (Paulsen) S. Fraga & F. J. (i) Gessnerium balechii (Steidinger 1971) A. R. Sánchez 1985, Heteraulacus ostenfeldii (Paulsen) A. Loeblich & L.A. Loeblich 1979 [=Gonyaulax balechii R. Loeblich 1970, Gessnerium ostenfeldii (Paulsen) Steidinger, Pyrodinium balechii (Steidinger) F. J. R. A. R. Loeblich & L. A. Loeblich 1979, Triadinium Taylor 1976, Alexandrium balechii (Steidinger) ostenfeldii (Paulsen) J. D. Dodge 1976, ?Gonyaulax Balech 1995]. globosa (Braarud 1945) Balech 1971 nom. illeg., (ii) Gessnerium concavum (Gaarder 1954) A. R. Loeblich ?Protogonyaulax globosa (Braarud) F. J. R. Taylor & L. A. Loeblich 1979 [=Goniodoma concava Gaarder, 1979, ?Gonyaulax trygvei M. Parke & J. D. Dodge A. concavum (Gaarder) Balech 1985 emend. Nguyen- in Parke & Dixon 1976, ?Gonyaulax tamarensis Ngoc & Larsen 2004, non Gonyaulax concava M. Lebour var. globosa Braarud 1945, ?Gonyaulax (Gaarder) Balech 1967, nec A. concavum (Gaarder) dimorpha Biecheler 1952]. Balech 1985, auct. non Balech 1995 (=A. gaarderae (iv) Alexandrium peruvianum (Balech & B.R. Mendiola Nguyen-Ngoc & J. Larsen 2004)]. 1977) Balech & Tangen 1985 [=Gonyaulax peruviana (iii) Gessnerium monilatum (J. F. Howell 1953) A. R. Balech & B. R. Mendiola, Protogonyaulax peruviana Loeblich 1970 [=Gonyaulax monilata J. F. Howell, (Balech & B. R. Mendiola) F. J. R. Taylor 1979, ?A. Pyrodinium monilatum (J. F. Howell) F. J. R. Taylor ostenfeldii Paulsen 1904) Balech & Tangen 1985]. 1976, Alexandrium monilatum (J. F. Howell) Balech (v) Alexandrium tamutum Montresor, Beran & U. John 1985, Gessnerium mochimaensis Halim 1967 ex Halim 1969]. 14 Journal of Marine Biology 4.6.2. New Combinations of Gessnerium: 4.7. Reinstatement of the Genus Protogonyaulax (Figure 6(c), Table 1) (i) Gessnerium camurascutulum (L. MacKenzie & K. Todd 2002) F. Gómez & Artigas, comb. nov. 4.7.1. Protogonyaulax F. J. R. Taylor 1979 emended Gómez & Basionym: Alexandrium camurascutulum L. Artigas. Gonyaulacoid dinoﬂagellates without or with scarce MacKenzie & K. Todd (2002, Harmful Algae, 1: cell compression, and lacking horn or spines. Cingulum 296, Figure 1). MacKenzie & Todd [32] designed as deep, median, descending about one cingular width, without holotype the Figures 1, 7, and 13. e Figure 13 also overhanging. e theca is usually smooth, and very rarely includes A. minutum, and the Figures 1 and 7 corre- ornamented. e plate formula is Po, 4′, 6′′, 6c, ≥8s, 5′′′, spond to cells from diﬀerent geographical origins (see 2′′′′. e ﬁrst apical plate (1′) plate is rhomboidal, narrow Article 8.1 of International Code of Nomenclature and asymmetrical and always directly connected to the (I.C.N.) for Algae, Fungi, and Plants). e Figure 1 apical pore plate (Po). e plate 6′′ is usually wide. e (cell from Marlborough Sounds) is designed as type. posterior sulcal plate is longer than wide, with usually two (ii) Gessnerium hiranoi (T. Kita & Fukuyo 1988) F. Gómez ventrally directed anterior prolongations and a connecting & Artigas, comb. nov. Basionym: Alexandrium hira- pore. e Po plate contains a comma-shaped pore, and noi T. Kita & Fukuyo (1988, Bull. Plankt. Soc. Jap. 35: usually an anterior attachment pore in the right lateral side 2, pl. 1 a–k, Figures 1(a)–1(f )). of the apical pore plate. Relatively many chain-forming (iii) Gessnerium foedum (Balech 1990) F. Gómez & species. e species are bloom-forming in eutrophic and/ Artigas, comb. nov. Basionym: Alexandrium foedum or conﬁned neritic waters. Cosmopolitan distribution with Balech (1990, Helgol. Meeresunters. 44: 392, Figures a few species reported from cold waters. Paralytic shellﬁsh 19–33). Synonym: Goniodoma pseudogoniaulax poisoning toxicity events have been reported in numerous sensu Kita et al. 1985. species. (iv) Gessnerium globosum (Nguyen-Ngoc & J. Larsen in (1) Type species: Protogonyaulax tamarensis (M. Lebour Larsen & Nguyen-Ngoc 2004) F. Gómez & Artigas, 1925) F. J. R. Taylor 1979. Basionym: Gonyaulax tama- comb. nov. Basionym: Alexandrium globosum rensis M. Lebour. Homotypic synonyms: Alexandrium Nguyen-Ngoc & J. Larsen in Larsen and Nguyen- tamarense (M. Lebour) Balech 1985, Gessnerium tam- Ngoc (2004, Opera Bot., 140: 93, pl. 7, Figure 8). arense (M. Lebour) A. R. Loeblich & L. A. Loeblich Non Protogonyaulax globosa (Braarud 1945) F. J. R. 1979. Heterotypic synonyms: Gonyaulax tamaren- Taylor 1979. sis var. excavata Braarud 1945, Gonyaulax excavata (v) Gessnerium pseudogoniaulax (Biecheler 1952) (Braarud) Balech 1971, Alexandrium excavatum F. Gómez & Artigas, comb. nov. Basionym: (Braarud 1945) Balech & Tangen 1985. Goniodoma pseudogoniaulax Biecheler (1952, (2) Other species of the genus Protogonyaulax: Bull. Biol. Fr. Belg., Suppl. 36: p. 55, Figures 30–32). Synonyms: Triadinium pseudogoniaulax (i) P rotogonyaulax acatenella (Whedon & Kofoid 1936) (Biecheler) J.D. Dodge 1981, Alexandrium pseu- F. J. R. Taylor 1979 [=Gonyaulax acatenella Whedon dogoniaulax (Biecheler 1952) Horiguchi 1983 ex T. & Kofoid, A. acatenella (Whedon & Kofoid) Balech Kita & Fukuyo 1992. e epithet is oen reported 1985]. as “pseudogonyaulax”. (ii) Protogonyaulax catenella (Whedon & Kofoid 1936) (vi) Gessnerium satoanum (K. Yuki & Fukuyo 1992) F. J. R. Taylor 1979 [=Gonyaulax catenella Whedon F. Gómez & Artigas, comb. nov. Basionym: & Kofoid, A. catenella (Whedon & Kofoid) Balech Alexandrium satoanum K. Yuki & Fukuyo (1992, J. 1985, Gonyaulax washingtonensis Hsu 1967, Phycol., 28: 396, Figures 1–12). Gessnerium catenella (Whedon & Kofoid) A. R. (vii) Gessnerium taylorii (Balech 1994) F. Gómez & Loeblich & L. A. Loeblich 1979]. John et al. [33] pro- Artigas, comb. nov. Basionym: Alexandrium taylorii posed to reject the name Gonyaulax catenella, the Balech (1994, Trans. Amer. Microscop. Soc., 113: basionym of A. catenella, in order to permit usage 219, Figures 7–11). of the more recent name A. fundyense. e proposal (2302) was not recommended, and Prud′homme van Reine [34] reported: “Alexandrium fundyense 4.6.3. e Next Species Do not Belong to Gessneri- and A. catenella are certainly conspeciﬁc, and then um:. Gessnerium acatenella (Whedon & Kofoid 1936) A. R. “catenella” has nomenclatural priority”. Loeblich & L. A. Loeblich 1979 (accepted as Protogonyaulax (iii) Protogonyaulax cohorticula (Balech 1967) F. J. R. acatenella). Gessnerium catenella (Whedon & Kofoid 1936) A. Taylor [=Gonyaulax cohorticula Balech nom. inval., R. Loeblich & L. A. Loeblich 1979 (accepted as Protogonyaulax Gessnerium cohorticula (Balech) A. R. Loeblich & catenella). Gessnerium cohorticula (Balech 1967) A. R. Loeblich L. A. Loeblich 1979, A. cohorticula (Balech 1967) & L. A. Loeblich 1979 (accepted as Protogonyaulax cohortic- Balech 1985]. Balech described the basionym ula). Gessnerium fraterculus (Balech 1964) A. R. Loeblich & lacking Latin description and designation of type. L. A. Loeblich 1979 (accepted as Protogonyaulax fraterculus). Balech described the species under the Zoological Gessnerium tamarense (M. Lebour 1925) A. R. Loeblich & Nomenclature (see Article 45 of I.C.N.). L. A. Loeblich 1979 (accepted as Protogonyaulax tamarensis). Journal of Marine Biology 15 (iv) P rotogonyaulax compressa Fukuyo, K. Yoshida & H. (v) Protogonyaulax tamiyavanichii (Balech 1974) Inoue 1985 [=Alexandrium compressum (Fukuyo, K. F. Gómez & Artigas, comb. nov. Basionym: Yoshida & H. Inoue) Balech 1995]. Alexandrium tamiyavanichii Balech (1994, Trans. Amer. Microscop. Soc., 113: 217, Figures 1–6). (v) Protogonyaulax fraterculus (Balech 1964) F. J. R. Synonym: Protogonyaulax cohorticula (Balech) Taylor 1979 [=Gonyaulax fraterculus Balech 1964 F. J. R. Taylor 1979 sec Kodama et al. 1988. as G. “fratercula”, nom. inval.; Gessnerium fratercu- lus (Balech) A. R. Loeblich & L. A. Loeblich 1979, (vi) Protogonyaulax tropicalis (Balech 1985) F. Gómez & Alexandrium fraterculus (Balech) Balech 1985, Artigas, comb. nov. Basionym: Alexandrium tropicale nom. inval.]. Balech [35] described the basionym Balech in Anderson et al. [1985, Toxic dinoﬂagellates. lacking Latin description and designation of type. Proceedings of the ird International Conference Balech described the species under the Zoological on Toxic dinoﬂagellates. Elsevier, New York, p. 37, Nomenclature (see Article 45 of I.C.N.). Figure 7]. Synonym: Gonyaulax excavata Braarud 1945 sensu Balech (1971, Serv. Hydrogr. Naval, (vi) Protogonyaulax kutnerae (Balech 1979) Sournia Buenos Aires, H 654: 28, pl. 7: Figures 119–124). 1984 (=Gonyaulax kutnerae Balech 1979). (vii) Protogonyaulax leei (Balech 1985) Fukuyo, 4.7.3. e Next Species Do not Belong to Protogonyaulax Pholpunthin & K. Yoshida 1988 (=Alexandrium leei Balech). (i) Protogonyaulax aﬃnis H. Inoue & Fukuyo 1985 (placed (viii) Protogonyaulax phoneus (Wołoszyńska & W. in a new genus, see below). Protogonyaulax dimor- Conrad 1939) F. J. R. Taylor 1979 [=Pyrodinium pha (Biecheler 1952) F. J. R. Taylor 1979 (accepted as phoneus Wołoszyńska & W. Conrad, Gonyaulax Gonyaulax dimorpha Biecheler, may be related to A. phoneus (Wołoszyńska & W. Conrad) F. J. R. ostenfeldii). Protogonyaulax globosa (Braarud 1945) Taylor 1975, Gonyaulax phoneus (Wołoszyńska & F. J. R. Taylor 1979 [accepted as Gonyaulax globosa W. Conrad) Loeblich & A. R. Loeblich 1975, ?A. (Braarud) Balech 1971 nom. illeg., and it may be ostenfeldii (Paulsen 1904) Balech & Tangen 1985]. related to A. ostenfeldii]. Protogonyaulax ostenfeldii e epithet “phoneus” is masculine, while the genus (Paulsen 1904) S. Fraga & F. J. Sánchez 1985 (accepted is feminine. as Gessnerium ostenfeldii). Protogonyaulax peruviana (Balech & B.R. Mendiola 1977) F. J. R. Taylor 1979 4.7.2. New Combinations of Protogonyaulax (accepted as Alexandrium peruvianum or synonym of A. ostenfeldii). (i) Protogonyaulax australiensis (Sh. Murray in John et al. 2014a) F. Gómez & Artigas, comb. nov. Basionym: 4.8. New Erected Genus for the Clade of Alexandrium aﬃne Alexandrium australiense Sh. Murray in John et al. (Figure 6(d), Table 1) 2014a (Protist, 165: 797–798, Figure 8). Synonym: Alexandrium australis Wang et al. 2014 nom. inval. 4.8.1. Episemicolon F. Gómez & Artigas, gen. nov. (1) Diagnosis: (ii) Protogonyaulax fundyensis (Balech 1985) F. Gómez Gonyaulacoid dinoﬂagellate without or scarce cell compression, & Artigas, comb. nov. Basionym: Alexandrium fun- without spines or horns. Cingulum deep, median, descending dyense Balech in Anderson et al. (1985, Toxic dino- about one cingular width, without overhanging. e cingular ﬂagellates. Proceedings of the ird International lists at both upper and lower margin are prominent. e sulcus Conference on Toxic dinoﬂagellates. Elsevier, New with list at both right and le margins. Plate formula Po, 4′, York, p. 37, Figure 18). Prud′homme van Reine [34] 6′′, 6c, ≥8s, 5′′′, 2′′′′. e ﬁrst apical plate is rhomboidal and reported: “Alexandrium fundyense and A. catenella reaches the apical pore plate. e apical pore plate contains are certainly conspeciﬁc, and then “catenella” has an oval or bullet-shaped apical pore, with an attachment pore nomenclatural priority”. lying at the dorsal side. e sulcus contains at least eight (iii) Protogonyaulax mediterranea (U. John in John et al. plates, the two lateral posterior plates are long, and with the 2014a) F. Gómez & Artigas, comb. nov. Basionym: right one longer than the le pair. e posterior sulcal plate is Alexandrium mediterraneum U. John in John et al. right displaced and may contain a marginal attachment pore. 2014a (Protist 165: 795–797, Figure 7). Synonym: Paralytic shellﬁsh poisoning toxicity has not been reported. Alexandrium mediterranis Wang et al. 2014 nom. inval. (2) Etymology: epi- from Ancient Greek “epi” (= on top (iv) Protogonyaulax paciﬁca (Litaker in John et al. of ); semicolon: the punctuation mark (;) from Latin “semi” 2014a) F. Gómez & Artigas, comb. nov. Basionym: (= half ), and Greek “kolon” (= verse, a part of a strophe, col- Alexandrium paciﬁcum Litaker in John et al. 2014a umn) and a mark of punctuation (:). e apical pore and the (Protist, 165: 793–795, Figure 6). John et al. ([36], dorsal attachment pore in the apical pore plate resemble the p. 794) reported “As it is no longer possible to estab- typographic symbol (;). e gender is neuter. lish the identity of the material originally assigned to (3) Type species: Episemicolon aﬃne (H. Inoue & Fukuyo 1985) A. catenella, we have designated a new species name F. Gómez & Artigas, gen. & comb. nov., hic designatus. Basionym: for Group IV isolates - A. paciﬁcum - and have sub- Protogonyaulax aﬃnis H. Inoue & Fukuyo in Fukuyo et al. (1985, mitted a proposal to reject the name Alexandrium Proceedings of the ird International Conference on Toxic dino- catenella (John et al., 2014b)”. e proposal (2302) in ﬂagellates. Elsevier, New York, p. 30, Figure 3(a)–3(c)). Synonyms: John et al. [33] was not recommended [34]. A. aﬃne (H. Inoue & Fukuyo) Balech 1985 nom. inval., A. fukuyoi 16 Journal of Marine Biology Balech in Anderson et al. 1985 nom. inval., A. aﬃne (H. Inoue & Conflicts of Interest Fukuyo) Balech 1995. e authors declare that they have no conﬂicts of interests regarding the publication of this paper. 4.8.2. Other Species. Episemicolon gaarderae (Nguyen-Ngoc & J. Larsen in Larsen & Nguyen-Ngoc 2004 ex F. Gómez & Artigas) F. Gómez & Artigas, comb. nov. Basionym: Gonyaulax Acknowledgments concava Gaarder sensu Balech (1967, Rev. Mus. argent. Cienc. Nat. ′B. Rivadavia′, Hidrobiol., 2, 108–111; plate 6, Figures F. G. was partly supported by a convention (no: 2101893310) 108–116). Synonym: Alexandrium gaarderae Nguyen-Ngoc between CNRS INSU and the French Ministry of Ecology & J. Larsen in Larsen & Nguyen-Ngoc 2004 ex F. Gómez & (MTES) for the implementation of the Monitoring Program Artigas, A. concavum (Gaarder) Balech 1985, non Goniodoma of the European Marine Strategy Framework directive (MSFD) concava Gaarder 1954, nec Goniodoma gaarderae Balech 1980 for pelagic habitats and the descriptor “biodiversity”. Samples nom. inval. According to Index Nominum Algarum (http:// were collected during the IFREMER “Etoile” cruise onboard ucjeps.berkeley.edu/ina/), Nguyen-Ngoc and Larsen [20] did R/V Côtes de la Manche (CNRS-INSU) in the frame of the not provide Latin description and designation of type of A. Joint European Research Infrastructure network for Coastal gaarderae (intended as a new name for Gonyaulax concava sensu Observatory – Novel European eXpertise for coastal observa- Balech 1967, but eﬀectively a new species). We designate as type Tories (JERICO-Next) H2020 INFRAIA project no. 654410. the Figure 4, plate 6, in Larsen & Nguyen-Ngoc ([20, p. 91). We thank I. Puillat, project manager, and P. Lazure, scientiﬁc coordinator of the Etoile cruise. We thank L. Courcot for the SEM assistance. Abbreviations a.a.p.: Anterior attachment pore in apical pore plate Supplementary Materials auct. non: auctorum non (of authors [but] not....), used for misapplied names Supplementary 1. Appendix S1. Taxonomy, synonymy, plate BP: Bootstrap probability arrangement and classiﬁcation of Centrodinium. c.p.: Cover or closing platelet or canopy in the Supplementary 2. Appendix S2. Detailed morphology of apical pore plate Centrodinium spp. HAB: Harmful algal bloom Supplementary 3. Appendix S3. Brief historical account of the I.C.N.: International Code of Nomenclature for taxonomy and nomenclature of Alexandrium sensu lato. algae, fungi, and plants Supplementary 4. Figure S1. Maximum-likelihood phy- LM: Light microscopy logenetic trees of D1–D2 domains of the LSU rRNA gene LSU: Large subunit sequences of selected species of Alexandrium sensu lato and mp: Marginal pore in the apical pore plate Centrodinium spp. with especial focus on the group of A. aﬃne. nom. inval.: nomen invalidum, an invalid name nom. illeg.: nomen illegitimum, an illegitimate name. Po: Apical pore plate References p.a.p.: Posterior attachment pore in the posterior [1] D. M. Anderson, T. J. Alpermann, A. D. Cembella, Y. Collos, sulcal plate E. Masseret, and M. Montresor, “e globally distributed PCR: Polymerase chain reaction genus Alexandrium: multifaceted roles in marine ecosystems PSP: Paralytic shellﬁsh poisoning and impacts on human health,” Harmful Algae, vol. 14, no. 1, rRNA: Ribosomal RNA pp. 10–35, 2012. S.a.: Anterior sulcal plate [2] E. Balech, “e genus Alexandrium or Gonyaulax of the s.a.p.: Pore of the anterior sulcal plate tamarensis group,” Toxic Dinoﬂagellates, in Proceedings of the SEM: Scanning electron microscopy 3rd International Conference on Toxic Dinoﬂagellates, D. M. S.d.a.: Right (dexter) anterior lateral sulcal Anderson, A. W. White, and D. G. Baden, Eds., pp. 33–38, S.d.p.: Right posterior lateral sulcal Elsevier, NY, 1985. S.m.a.: Anterior median sulcal [3] C . A. Kofoid, “Reports on the scientiﬁc results of the expedition S.m.p.: Posterior median sulcal to the Eastern Tropical Paciﬁc, in charge of Alexander Agassiz, S.p.: Posterior sulcal by the U.S. ﬁsh commission steamer “Albatross” from October s.l.: Sensu lato 1904 to March 1905, Lieut. Commander L. M. Garrett, U.S.N., s.s.: Sensu stricto commanding IX. 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Copyright: Copyright © 2019 Fernando Gómez and Luis Felipe Artigas. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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DOI: 10.1155/2019/1284104
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Abstract

Hindawi Journal of Marine Biology Volume 2019, Article ID 1284104, 17 pages https://doi.org/10.1155/2019/1284104 Research Article Redefinition of the Dinoflagellate Genus Alexandrium Based on Centrodinium: Reinstatement of Gessnerium and Protogonyaulax, and Episemicolon gen. nov. (Gonyaulacales, Dinophyceae) 1,2 2 Fernando Gómez and Luis Felipe Artigas Carmen Campos Panisse 3, E-11500 Puerto de Santa María, Spain Université du Littoral Côte d′Opale, Université de Lille, CNRS, UMR 8187, LOG, Laboratoire d′Océanologie et de Géosciences, 32 Av. Foch 62930, Wimereux, France Correspondence should be addressed to Fernando Gómez; fernando.gomez@toplancton.com Received 17 September 2019; Revised 26 October 2019; Accepted 23 November 2019; Published 31 December 2019 Academic Editor: Punyasloke Bhadury Copyright © 2019 Fernando Gómez and Luis Felipe Artigas. ƒis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ƒe genus Centrodinium contains oceanic and predominantly tropical species that have received little attention. ƒree species of Centrodinium were examined using thecal plate dissociation, scanning electron microscopy, and molecular sequences. ƒe 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 le’ posterior lateral sulcal plate is longer than its right pair, while reversed in the typical Centrodinium spp. ƒe sulcal posterior plate of C. punctatum is located in the le’-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 a‹ne/A. gaarderae clustered as a sister clade of the Alexandrium tamarense/catenella/fraterculus groups. ƒe 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. ƒe 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. a‹ne and A. gaarderae. New combinations in the genera Gessnerium, Protogonyaulax, and Episemicolon are proposed. considerable attention, other open-ocean gonyaulacoid dino- 1. Introduction ™agellates remain under-investigated because of the paucity Dino™agellates are ubiquitous protists that play diverse roles of material due to their low densities. ƒe neritic HAB species in marine ecosystems. Numerous studies are focused on spe- of Alexandrium are typically non- or slightly compressed spe- cies that are responsible for harmful algal blooms (HABs) in cies, without horns or spines [2], while the oceanic gonyaula- coastal waters. Paralytic shellsh poisoning (PSP) is generally coid dino™agellates have horns and spines (Ceratocorys spp., regarded as the most well-known and widespread HAB syn- Gonyaulax taylorii, etc.), and/or the cells are o’en ™attened drome, and is associated with toxins produced by certain dino- (i.e. Gonyaulax paci‘ca, [3]). Kofoid [3] described the genus ™agellate species in the genus Alexandrium [1]. Whilst neritic Centrodinium for oceanic species characterized by a high lat- species of the planktonic Alexandrium or the epiphytic erally ™attened cell body with an apical and an antapical horn. Gambierdiscus, responsible for toxic events, have received Kofoid [3] also described the genus Murrayella for three types 2 Journal of Marine Biology of species: globular, biconical, and laterally compressed spe- observing the presence of Centrodinium in these two sampling cies. An account of the taxonomy of Centrodinium and stations, subsamples of the plankton concentrate were treated Murrayella is reported in the Appendix S1 part 1–4, 7 as with small amounts (150–200 μl) of 10% (weight/volume) Supplementary material. Balech [4–6] carried out studies on sodium thiosulfate for removing the iodine. e cells of each Centrodinium and the laterally ﬂattened species Murrayella species of Centrodinium were micropipetted individually with and in 1967 commented on the general resemblance between a ﬁne capillary into a clean chamber ﬁlled with autoclaved Centrodinium and his new species Murrayella mimetica, but Milli-Qultrapure water. e same procedure was repeated he maintained the split of both genera due to the diﬀerences twice in order to remove any source of contamination. in the plate formula following a strict Kofoidian scheme of Finally, 30–40 cells of each species were deposited in a 0.2 ml tabulation. e classiﬁcation of Centrodinium has been a mat- Eppendorf tube ﬁlled with absolute. ter of controversy ([7–9]; see Appendix S1 part 8 as For plate dissociation, each cell was individually isolated Supplementary material). and placed in an Utermöhl chamber with distilled water. In 2012, Gómez [10] classiﬁed Centrodinium in the same Drops of a solution of 5% sodium hypochlorite (commercial subfamily of Alexandrium within the Gonyaulacales. Li et al. bleach solution, 1 : 1 mixture of sodium hypochlorite and [11] reported that Centrodinium punctatum unexpectedly Milli-Q water) were added until the split of the thecal plates. clusters with Alexandrium aﬃne, and consequently the genus In other cases, the theca was squashed by touching it with a Alexandrium was polyphyletic. Li et al. [11] did not propose ﬁne capillary tube to split the thecal plates. e cell was repeat- the split of the genus Alexandrium because they were based edly photographed at diﬀerent stages during the process of only on C. punctatum. e species C. punctatum diﬀers from splitting the theca with the inverted microscope at 600x the typical species of Centrodinium that are fusiform, with an magniﬁcation. elongated and high ﬂattened body, and a smooth thecal sur- For analyses using scanning electron microscopy, a sub- face. Li et al. [11] submitted the sequences as Alexandrium sp. sample was ﬁltered through a 3 μm pore size polycarbonate (GenBank accession numbers MF043217–20), and they did membrane (Millipore Ltd., Middlesex, U.K.). e ﬁlter was not propose the split of Alexandrium as C. puntactum does rinsed three times in Milli-Q water, dehydrated through not represent the typical morphology of the genus graded ethanol series (30%, 50%, 70%, 80%, 90%, 95%, and Centrodinium. Li et al. [11] remarked the need of the study for two steps in 100%). en, the protocol was to immerse the the typical species of Centrodinium before considering the ﬁlter in HMDS (Hexamethyldisilazane, Molekula Limited, generic split of Alexandrium. Newcastle, U.K.) for 30 minutes (twice). e HMDS was evap- In this study, we investigate the morphology of two highly orated by placing the sample overnight under the fume hood. laterally ﬂattened species with apical and antapical horns, Filters were mounted on an aluminium stub, sputter-coated C. eminens and C. intermedium, and also Centrodinium with Au/Pd (Polaron SC7620, Quorum Technologies Ltd., punctatum. We provide the ﬁrst molecular data (SSU and LSU Ashford, U.K.) and observed at 15 kV with a SEM LEO 438 rRNA gene sequences) for the typical species of Centrodinium. VP (Carl Zeiss AG, Oberkochen, Germany). Images were pre- e new morphological and molecular data conﬁrm the poly- sented on a black background using Adobe Photoshop CS3 phyletic character of Alexandrium. We propose the split of (Adobe Systems Inc., San Jose, CA, USA). Alexandrium into four genera that reconciles with the molec- 2.2. DNA Extraction, PCR Ampliﬁcation of rRNA Gene and ular and morphological data, and requires fewer taxonomical innovations. No taxonomical innovations are needed for the Sequencing. Prior to PCR, the sample tube was centrifuged, species comprising the clade that contains the type species of and ethanol was evaporated by placing the tube overnight in a Alexandrium, A. minutum, which remains as Alexandrium s.s. desiccator at room temperature. Genomic DNA was extracted e species of the clades that contain the type species of using Chelex (InstaGeneTM Matrix; Bio-Rad, Hercules, CA, Protogonyaulax and Gessnerium are placed in the revived USA) following protocols adapted from Richlen and Barber genera Protogonyaulax and Gessnerium, respectively. e [12], as outlined in Gómez et al. [13]. e SSU rRNA gene species A. aﬃne and A. gaarderae, closely related to was ampliﬁed using two sets of primers: EukA and 1055R; Centrodinium, need to be transferred into a new erected genus. and 570F and EukB [14]. e D1–D3 domains of the LSU rRNA gene were ampliﬁed using primers D1R and D3Ca [15]. PCR ampliﬁcations were performed in a 25 μl reaction volume containing 1 μl of template DNA (supernatant from each 2. Materials and Methods Chelex extraction), 1 × PCR buﬀer (500 mM KCl and 100 mM 2.1. Sampling, Isolation, and Microscopy. Sampling was Tris–HCl, pH 8.3), 2 mM MgCl , 0.8 mM dNTPs, 0.5 mM of performed with a phytoplankton net (20 μm mesh size) on each primer, and 0.5 U of AmpliTaq DNA Polymerase (Applied the surface waters of the South-Eastern Bay of Biscay, North Biosystems Inc., Foster City, CA, USA). Hot start PCR Atlantic, in August 2017. Samples from two stations at 43°36′ ampliﬁcations were performed in a Mastercycler Nexus thermal N–1°57′ W and 43°36′ N–2°03′ W are described here. e cycler (Eppendorf, Hamburg, Germany) with the following plankton concentrate was preserved with acid Lugol′s iodine cycling conditions for both primer sets: initial denaturation solution to a ﬁnal concentration of 4% (vol:vol), and kept (95°C/5 min); 35 cycles of denaturation (95°C/30 s), annealing refrigerated (~3°C). e material was examined with an (55°C/1 min), and extension (72°C/2 min); ﬁnal extension inverted microscope (Nikon Eclipse TE2000-S, Tokyo) and (72°C/10 min). PCR products were visualized on a 1% agarose photographed with a Nikon Digital Sight DS-2 M camera. Aer gel stained with GelRed (Biotium, Hayward, CA, USA). Journal of Marine Biology 3 Positive PCR products were cloned into vector PCR 2.1 using detailed description of the plate arrangement of C. punctatum, a TOPO TA cloning kit (Invitrogen, Carlsbad, CA, USA). C. intermedium, and C. eminens is available in the Appendix Clones were screened for inserts by PCR ampliﬁcation with S2 as Supplementary material. We describe here the apical, plasmid primers M13F and M13R, and positive clones from sulcal, and antapical plate series. each PCR amplicon were puriﬁed using the Qiaquick PCR e plates 2′ and 4′ almost completely encircled the apical puriﬁcation kit (Qiagen, Hilden, Germany), and sequenced pore plate (Po), while the contact with the plate 1′ in the ven- in both the forward and reverse direction (Euroﬁns MWG tral side was much reduced, and the plate 3′ did not reach the Operon, Ebersberg, Germany). Sequence reads were aligned Po (Figures 1(d)–1(f )). Scanning electron microscopy revealed and assembled in Geneious Pro 11.1.2 (Biomatters, Auckland, a horseshoe-shaped apical pore surrounded by a rim of small New Zealand). e newly generated consensus sequences were marginal pores (Figures 1(q)–1(s)). e sulcal plates are placed deposited in DDBJ/EMBL/GenBank under accession numbers between the anterior sulcal plate (S.a.) in the epitheca and the MK714074–MK714082. posterior sulcal plate (S.p.) near the antapex (Figures 1(g)–1(l), 1(p)). Two small plates known as the anterior and posterior 2.3. Phylogenetic Analyses. SSU– and LSU rRNA gene sequences median plates (S.m.a. and S.m.p.)—one above the other— of Centrodinium spp. were analysed using Basic Local Search occurred below the anterior sulcal and the le and right ante- Tool (BLAST, http://blast.ncbi.nlm.nih.gov/Blast.cgi) against rior lateral plates (S.s.a. and S.d.a.). Two lateral pairs of plates databases in GenBank. e closest matches to these searches were located below, the le and right posterior lateral plates were sequences in the genus Alexandrium (primarily A. aﬃne) (S.s.p. and S.d.p.), with the le plate being longer than the right and the sequences reported as “Alexandrium sp. ZL2017” that pair (Figures 1(h)–1(k)). e sulcal posterior (S.p.) was an were later identiﬁed as Centrodinium punctatum in Li et al. irregular pentagon with length approximately equal to the [11]. Based on these results, rRNA gene sequence data were width (Figures 1(f ), 1(h)). e S.p. plate was displaced towards compiled from similar sequences identiﬁed using BLAST. the le side below the plates 1′′′ and 2′′′ and the le margin Sequence alignments of available SSU– and D1-D2 LSU rRNA joining to the plate 1′′′′ (Figures 1(h), 1(l) and 1(m)). ere gene sequences of Centrodinium spp., representatives of each were two antapical plates with a triangular shape that con- species of Alexandrium, other gonyaulacoid dinoﬂagellates, formed a pointed antapex directed towards the ventral side. and other dinokaryotic dinoﬂagellates were accomplished e ﬁrst antapical (1′′′′) in the le face (Figures 1(h), 1(l)– by Clustal W [16] and the evolutionary history was inferred 1(m)) was slightly smaller than the second antapical plate by using the Maximum Likelihood method based on the (2′′′′) in the right face (Figures 1(f ), 1(i), 1(n)–1(o)). e plate General Time Reversible model with Gamma distributed with 1′′′′ was in contact to S.p. and 2′′′′ plate (Figures 1(h), Invariant sites and the default settings in MEGA7 soware 1(l)–1(n)). [17]. Bootstrap values were obtained aer 1000 replications. e apicomplexan Eimeria tenella (AF026388) was used as 3.2. Morphology of Centrodinium intermedium. e lateral an out group in the SSU– and LSU rRNA gene phylogenies. 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 3. Results (Figure 1(a)). e apical horn of C. intermedium was usually 3.1. Morphology of Centrodinium punctatum. e species shorter than the other species of Centrodinium. Cells were 130– Centrodinium punctatum, C. intermedium, and C. eminens 175 μm long. e depth of the cells (dorso-ventral distance) was were the most abundant (in that order) in the sampling 55–80 μm. e width between the le and right sides is stations in the South-Eastern Bay of Biscay (Figure 1(a)), diﬃcult to measure in these highly laterally ﬂattened cells, providing material for the morphological (plate dissociation with values of about 25–35 μm wide at the cingulum level and SEM) and molecular analyses. A few individuals of (Figures 2(a)–2(c)). e dense poroid ornamentation of the Centrodinium maximum were also found, but in an insuﬃcient theca observed in C. punctatum was missing in C. intermedium, abundance for detailed studies. e sea surface temperatures with only scattered pores, more abundant in the right face of in the two sampling stations ranged from 23.4°C to 23.8°C and the apical horn (Figure 2(d)). e apical horn (~20 μm long) of the salinity from 34.5 to 34.6. Centrodinium punctatum was C. intermedium was a short truncated cone (Figures 2(a)–2(d)). the most abundant species compared with other congeneric e antapical horn was longer (>50 μm) and directed towards taxa. e cells were slightly laterally ﬂattened with a rhomboid the le-ventral side. Consequently, the antapical horn was shape. Cell dimensions were 65–90 μm long, 35–42 μm in a diﬀerent same plane than the main body and the apical depth (dorso-ventral diameter), and 24–34 μm wide (length horn (Figure 2(c)). e antapical horn had a triangular section between the right and le lateral sides) (Figure 1(a)). e with a slight anticlockwise torsion, and three terminal spinules epitheca was conical with a blunt apex. e hypotheca was (Figures 2(m)–2(o)). Each face of the antapical horn had a row conical with a pointed antapex directed towards the ventral of sunken areas with 3-4 small pores (Figure 2(n)). side. In addition to the size variability, the individuals showed e molecular data revealed a very close phylogenetical a diﬀerent degree of development the pointed antapex relationship between C. punctatum and C. intermedium (see (Figure 1(b) and 1(c)). e theca was ornamented with poroids below in Figures 4 and 5). It is commonly assumed that con- (Figures 1(d)–1(f ), 1(l)–1(q)). Centrodinium punctatum had generic species share a similar plate formula. e epithecal a plate formula Po, 4′, 6′′, 6c, 8s+, 5′′′, and 2′′′′. A more plate formula of C. punctatum is Po, 4′, 6′′ or alternatively 3′, 4 Journal of Marine Biology F¨©ª«¬ 1: Light (a–j) and scanning electron (l–s) micrographs of Centrodinium punctatum. (a) Plankton sample with Centrodinium spp. ƒe arrows point the cells of C. punctatum. (b–c) Individuals used for molecular analyses. (d) Partially dissociated theca in ventral view. (e–f ) Epitheca. ƒe insets show the rst postcingular plate and the posterior sulcal plate. (g) Right-ventral face. ƒe inset shows the right antapex. (h) Le’-ventral view. ƒe inset shows the dorsal antapex. (i) Ventral view of the sulcal area. (j-k) Dissociated sulcal plates. (l) Le’-ventral view. (m) Le’ antapex. (n) Le’ face. (o) Right face. (p) Ventral view. (q) Apical view. (r–s) Apex. 1′–4′ = apical plates; 1′′–6′′ = precingular plates; 1′′′–5′′′ = postcingular plates; 1′′′′–2′′′′ = antapical plates; C1–C6 = cingular plates; c.p. = closing, cover platelet or canopy; mp = marginal pores surrounding the apical pore plate; Po = apical pore plate; S.a. = anterior sulcal plate; s.a.p. = pore of the anterior sulcal plate; S.d.a. = right (dexter) anterior lateral sulcal; S.d.p. = right posterior lateral sulcal; S.m.a. = anterior median sulcal; S.m.p. = posterior median sulcal; S.p. = posterior sulcal plate. S.s.a. = le’ (sinister) anterior lateral sulcal; S.s.p. = le’ posterior lateral sulcal. Scale bars (a–q) = 20 μm, (r–s) = 2 μm. 1a, 6′′ in a strict Kofoidian scheme. ƒe species C. interme- conform the apical horn, and the development of these plates dium has an additional plate in the le’ face of the epitheca, hindered that the plates 1′ and 3 ′ reached the apex (Figures and the plate formula in a strict Kofoidian scheme is Po, 2′, 2(d), 2(o)–2(q)). While the plate 4′ was narrow and long, the 2a, 7′′. In contrast to C. punctatum, the apical plates of C. elongation of the plate 2′ resulted in the split into two plates. intermedium were larger than the precingular plates (Figure ƒe formula of the epitheca of C. punctatum and C. interme- 2(d)). ƒe rst apical plate of C. punctatum reached the apical dium is similar (Po, 4′, 6′′), using the labelling 2′ ( α + β) to pore (insert 1′), while in C. intermedium it does not reach the denote the split of the second apical plate in C. intermedium. apex (exsert 1′). When compared to C. punctatum, the main ƒe right side of the epitheca was essentially similar to C. punc- modications of C. intermedium were the elongation of the tatum, where 4′ plate has expanded anteriorly, and then the plates 4′ and 2′ (the latter split into two plates) to conform the 3′ plate did not reach the apex (Figure 2(d)). During the plate apical horn, the di§erent length of the posterior lateral sulcal dissociations, the Po remained attached to the plate 2′β as a plates, and the formation of a tubular antapical horn supported circular structure of about 1 μm in diameter (Figure 2(d)). ƒe at its ventral basis by two triangular plates. ƒe apical plates tiny membranous Po platelet was poorly conserved in the SEM 2′ and 4 ′ of C. intermedium have extended anteriorly to preparations. ƒe very thin plate 2′β appeared crushed against Journal of Marine Biology 5 F¨©ª«¬ 2: Light (a–j) and scanning electron (k–r) micrographs of Centrodinium intermedium. (a) Several individuals. (b) Le’ face. (c) Le’- ventral view. Note the antapical horn oriented toward the le’ side. (d) Le’ and right faces of the same epitheca. ƒe inset shows the apex. (e) Le’ hypotheca. ƒe inset shows the rst antapical plate. ƒe arrowhead points a liform extension. (f ) Right hypotheca. (g) Detail of the sulcal area. (h) Dissociated anterior sulcal plates. ƒe arrowheads point a membranous ™ange. (i) Anterior lateral sulcal plates. (j) Dissociated posterior lateral sulcal plates. (k–l) Ventral view. (m) Right face. (n) Di§erent antapical horns. (o) Le’ face. (q–r) Detail of the apex. 1′–4′ = apical plates; 1′′–6′′ = precingular plates; 1′′′–5′′′ = postcingular plates; 1′′′′–2′′′′ = antapical plates; C1–C6 = cingular plates; Po = apical pore plate; S.a. = anterior sulcal plate; s.a.p. = pore of the anterior sulcal plate; S.d.a. = right (dexter) anterior lateral sulcal; S.d.p. = right posterior lateral sulcal; S.m.a. = anterior median sulcal; S.m.p. = posterior median sulcal; S.p. = posterior sulcal. S.s.a. = le’ (sinister) anterior lateral sulcal; S.s.p. = le’ posterior lateral sulcal plate. Scale bars (a–m, o) = 20 μm, (n, q–r) = 2 μm. the thick plate 4′ (Figures 2(q)–2(r)). ƒe antapex of C. punc- was a pore, the posterior attachment pore, located in this tri- tatum and C. intermedium showed di§erences. ƒe posterior angular plate in the right face (Figure 2(f )), which is a char- hypotheca of C. intermedium was composed of three plates: a acteristic of the posterior sulcal plate of chain-forming tubular plate that conforms the antapical horn and two plates gonyaulacoid dino™agellates. In the le’ face, the rst antapical in the ventral side that acted as a counterfort or backstay. ƒese (1′′′′) was a triangular plate that o’en showed a posterior two triangular plates were slightly laterally inclined towards liform extension (Figure 1(e)). ƒe second antapical plate the le’ face, and the antapical horn was directed towards the (2′′′′) emerged from the dorsal side to conform a tubular le’ and ventral sides (Figures 2(e)–2(f ), 2(k)–2(o)). ƒe most antapical horn (Figures 2(e)–2(f )), with a slight anticlockwise immediate interpretation was that the antapex consists one torsion and three terminal spinules (Figures 2(m)–2(n)). antapical plate that conforms the horn, and two posterior In the sulcal plate series, the anterior sulcal plate (S.a.) was intercalary plates that support the ventral basis of the antapical part of the epitheca, enclosed between the plates 6′′, 1′, and horn. ƒis implies that the posterior sulcal plate (S.p.) was 1′′ and the rst cingular plate (Figures 2(g)–2(h)). ƒere was missing in C. intermedium. In C. punctatum, the S.p. was an a prominent pore in the middle of the plate connected to the irregular pentagon located in the le’-ventral side below the right border by a narrow canal. In some cells, the right poste- plates 1′′′ and 2′′′ (Figures 1(f ), 1(i), 1(l)–1(m)), while the S.p. rior corner of the S.a. showed a membranous ™ange that con- of C. intermedium was triangular and located in the right face nected with the rst cingular plate (Figure 2(h)). ƒe right below the plates 4′′′ and 5′′′ (Figures 2(f ), 2(k)–2(m)). ƒere anterior lateral sulcal plate (S.d.a.) was larger than its le’ pair, 6 Journal of Marine Biology with the shape of an irregular right triangle that resembled the unreported since the original description in 1907. It seems shape of the Sicily Island (Figures 2(g), 2(i)). In C. punctatum, likely that C. elongatum corresponds to a recently divided cell the le posterior lateral sulcal plate (S.d.p.) was longer than of C. maximum or C. eminens (see Appendix S1 part 2 in the its right pair (Figures 1(i), 1(k)), while reversed in C. interme- Supplementary material). dium (Figure 2(j)). e right posterior sulcal plate (S.d.p.) of In the SSU rRNA gene phylogeny, the three species of C. intermedium was the longest of the sulcal series and showed Centrodinium clustered together with high support with C. the shape of a knife, with a reinforcement in the le margin punctatum in a basal position. e Centrodinium spp. clade (Figure 2(j)). e le posterior sulcal plate (S.s.p.) was smaller, clustered with Alexandrium aﬃne, with strong support (BP like a very elongated pentagon that ﬁt in the knife handle 100%) (Figure 4). In the LSU rRNA gene phylogeny, formed by the anterior le margin of the S.d.p. (Figure 2(j)). Centrodinium spp. also clustered with sequences retrieved e morphology of these plates suggests that the overlap from GenBank as Alexandrium aﬃne and A. concavum growth of the S.d.p. has hindered the posterior development (Figure 5). In an additional LSU rRNA tree more reference of the S.s.p. sequences were added from GenBank within the A. aﬃne clade to include sequences identiﬁed as A. aﬃne, A. tamarense, 3.3. Morphology of Centrodinium eminens. In lateral view, and A. concavum (Figure S1 as Supplementary material). e the cells of C. eminens were fusiform and slightly sigmoid strains CAWD51-52 diverged from the other sequences of because the apical horn was slightly directed towards the A. aﬃne. In the SSU- and LSU rRNA gene phylogenies dorsal side, and the antapical horn towards the ventral side. (Figures 4–5), the species of the tamarense/catenella/fratercu- e ventral margin of the epitheca was almost straight. e lus groups of Alexandrium clustered with high support as a dorsal margin was curved in the posterior half and almost sister group to the Centrodinium spp. and A. aﬃne clades. e straight in the anterior half where the apical horn with clade of Alexandrium sensu stricto (s.s.) containing the type, a brunt apex was slightly directed towards the dorsal side A. minutum, and four divergent species (A. diversaporum, (Figure 3(a)). e cells of C. eminens were 182–239 μm long, A. leei, A. margaleﬁi, A. pohangense). e species of the sub- and 31–47 μm in depth (dorso-ventral distance), being less genus Gessnerium, A. monilatum and allied species, formed robust (lower depth), and less ﬂattened than C. intermedium. other clade (Figures 4–5). e apical and antapical horns of C. eminens were longer (Figure 3(a)) than in C. intermedium (Figure 2(a)). e 4. Discussion antapical horn of C. intermedium was very inclined towards the face (Figure 2(c)), while the inclination was almost absent 4.1. Aﬃnities between Centrodinium and Alexandrium. e in C. eminens (Figures 3(a)–3(h)). e plate arrangement molecular data reveal that Centrodinium clusters with strong of C. eminens and C. intermedium was similar, with more support amongst the clades of Alexandrium (Figures 4–5; anterior-posteriorly elongated plates, especially in the apical [11]). Species such as C. punctatum have the same plate series in C. eminens (Figures 3(b)–3(l), 3(t)–3(v)). e two formula of Alexandrium (Figures 1, 6(e)). e most typical plates, 2′ (α + β), resulting of the split of the second apical apical pore plate of Alexandrium has a comma-shaped pore plate remained joined (Figure 3(d)). e distal antapical horn surrounded by marginal pores, and the chain-forming species also showed three spinules (Figure 3(u)). e sulcal series have an anterior attachment pore [18]. e apical pore plate of was fully similar (Figures 3(m)–3(q)). e triangular ﬁrst Alexandrium is larger (>6 μm), and we can easily observe an antapical and the posterior sulcal plates showed a ﬁliform oval or comma-shaped pore. e formation of the apical horn posterior extension (Figures 3(r)–3(s)). e posterior sulcal of Centrodinium implies a reduction of the surface available for plate showed a posterior attachment pore (Figures 3(r)–3(s)). the apical pore plate (<2 μm), and the horseshoe-shaped could In the SEM preparations, some individuals of C. eminens were be a result of the constriction of the oval or comma-shaped in better preservation stage than those of C. intermedium, and pore (Figures 1(r)–1(s), 3(y)–3(z)). some details of the apex were revealed (Figures 3(w)–3(z)). e chain-forming species of Alexandrium have an attach- e apex of C. eminens also collapsed in the SEM preparations ment pore (a.a.p.) in the apical pore plate, and an attachment but in some individuals the membranous apical pore platelet pore (p.a.p.) in the posterior sulcal plate. e cells of a chain and the thin second antapical were not crushed against are interconnected by these pores [18]. In Centrodinium, the the thicker four apical plate. In these cases, a large pore anterior attachment pore is more diﬃcult to observe due to of 1–1.5 μm in diameter was observed devoid of the cover the small size and fragility of the membranous apical platelet, platelet (Figures 3(w)–3(x)). is membranous cover platelet or it may be confused with marginal pores. Hernández- remained in few individuals, with the apical pore surrounded Becerril et al. ([9], their Figure 33) reported a pore in the apex by a few tiny pores (Figures 3(y)–3(z)). that could be the apical pore devoid of the foramen, or alter- 3.4. Molecular Phylogeny. e SSU and LSU rRNA gene natively the anterior attachment pore. e posterior attach- ment pore in the posterior sulcal plate is evident in sequences were obtained from three species of Centrodinium: C. punctatum that is the ﬁrst described laterally ﬂattened C. intermedium and C. eminens (Figures 2(f ), 3(r)–3(s)), and C. pulchrum ([9], their Figure 37). species of the former genus Murrayella; C. intermedium that is the most ﬂattened species of this genus with an oval hypotheca, e sequences of Centrodinium clustered as a sister group to Alexandrium aﬃne (Figures 4 and 5; [11]). at clade and C. eminens which morphology is close to the type species, C. elongatum. It should be noted that the type species remains includes sequences retrieved from GenBank under the names Journal of Marine Biology 7 F¨©ª«¬ 3: Light (a–s) and scanning electron (t–z) micrographs of Centrodinium eminens. (a) Several individuals. (b) Le’ face. (c) Right epitheca. (d) Le’-ventral view. ƒe insets show the sulcus. (e) Dissociated plates of the apical horn. (f–g). Ventral views. ƒe inset shows the sulcus. (h) Dissociated epitheca and hypotheca. (i–m) Several views of the same epitheca. (o, q) Dissociated posterior lateral sulcal plates. (p) Anterior sulcal. (r) Posterior sulcal and rst antapical plate. ƒe arrowhead points a liform extension. (s) Antapical horn. (t) Le’ face. (u–v) Right face. (w–z) Apex. 1′–4′ = apical plates; 1′′–6′′ = precingular plates; 1′′′–5′′′ = postcingular plates; 1′′′′–2′′′′ = antapical plates; C1–C6 = cingular plates; c.p. = closing, cover platelet or canopy; mp = marginal pores surrounding the apical pore plate; Po = apical pore plate; S.a. = anterior sulcal plate; s.a.p. = pore of the anterior sulcal plate; S.d.a. = right (dexter) anterior lateral sulcal; S.d.p. = right posterior lateral sulcal; S.m.a. = anterior median sulcal; S.m.p. = posterior median sulcal; S.p. = posterior sulcal; S.s.a. = le’ (sinister) anterior lateral sulcal; S.s.p. = le’ posterior lateral sulcal plate; Scale bar (a–v) = 20 μm, (w–z) = 2 μm. 8 Journal of Marine Biology Centrodinium eminens FG2 MK714079 Centrodinium intermedium FG4 MK714081 Centrodinium intermedium FG3 MK714080 Centrodinium punctatum FG5 MK714082 Centrodinium punctatum MF043219 86 Centrodinium Centrodinium punctatum MF043220 AY775286 Episemicolon gen. nov. AJ535375 Alexandrium fundyense KF908795 KF908800 100 Alexandrium mediterraneum KF908797 Protogonyaulax Alexandrium tamarense KF908799 Alexandrium australiense KF908802 Alexandrium cohorticula AF113935 Alexandrium fraterculus AY421776 Alexandrium pohangense LN811348 U27498 Alexandrium insuetum AB088298 Alexandrium minutum AY883006 Alexandrium s.s. Alexandrium tamutum AJ535379 Alexandrium ostenfeldii U27500 Alexandrium leei AY641565 Alexandrium diversaporum KF251139 Alexandrium satoanum AY641566 Alexandrium monilatum AY883005 Gessnerium Alexandrium taylorii AJ535385 Alexandrium pseudogoniaulax JF521638 Fukuyoa paulensis KM886379 Pyrrhotriadinium polyedricum KM886380 100 Pyrocystis noctiluca AF022156 Fragilidium subglobosum AF033869 F. duplocampanaeforme KY624502 Pyrodinium bahamense AF274275 100 Gonyaulax spinifera AF022155 82 Gonyaulax polygramma AJ833631 Ceratium hirundinella AY443014 Tripos fusus AF022153 Tripos furca AJ276699 100 Peridinium cinctum EF058243 Peridinium willei EF058250 Polarella glacialis AF099183 Pelagodinium bei U37365 Symbiodinium microadriaticum M88521 100 Gymnodinium catenatum AF022193 Gymnodinium fuscum AF022194 Dinophysis acuta AJ506973 100 Phalacroma rotundatum AJ506975 Scrippsiella trochoidea EF492513 Scrippsiella sweeneyae HQ845331 100 Durinskia dybowskii AF231803 Durinskia agilis JF514516 Karenia mikimotoi AF022195 Karenia brevis DQ847434 Prorocentrum micans EF492511 Prorocentrum minimum Y16238 96 Oxytoxum scolopax KY421376 Corythodinium tessellatum KY421378 Heterocapsa triquetra AF022198 0.05 Heterocapsa rotundata AF274267 Azadinium spinosum JN680857 Azadinium caudatum JQ247701 Eimeria tenella AF026388 F¨©ª«¬ 4: Maximum-likelihood phylogenetic tree of the SSU rRNA gene. Bootstrap support values (BP) >70 are shown. New sequences are highlighted in bold. ƒe scale bar represents the number of substitutions for a unit branch length. A. a‹ne, A. tamarense, and A. concavum. Two sequences from Protogonyaulax (Figure 5, S1).ƒe members of the tamarense/ New Zealand, the strains CAWD51 named A. a‹ne (accession catenella group are responsible for paralytic shellsh poisoning number AY338753) and CAWD52 named A. concavum (acces- (PSP) events. ƒe sxtA gene (saxitoxin biosynthesis pathway sion number AF032348) were identical and diverged from the protein A domain) has been detected in the members of the main group of A. a‹ne. ƒe latter subdivided into two groups, tamarense/catenella group or A. fraterculus. In contrast, PSP one for strains isolated exclusively from Japan and China, and toxicity or the presence of the sxtA gene have not been detected other group for strains from diverse world regions (Figure S1). in A. a‹ne [23] and Centrodinium punctatum [11]. ƒe cells of the strain CAWD52 illustrated in MacKenzie et al. Alexandrium a‹ne is distinguished primarily by the apical [19] corresponded to A. gaarderae as dened by Larsen and pore plate and other di§erences in the sulcal plates. Balech Nguyen-Ngoc [20]. ƒe species Alexandrium a‹ne was rst [18] reported that the apical pore platelet is narrow, long, and described as Protogonyaulax a‹nis [21], and since the earlier fundamentally bullet-shaped. ƒe foramen does not form a molecular phylogenies the sequences of A. a‹ne have always true comma because it is oval and relatively small; it is located diverged from the members of the tamarense/catenella group in the ventral half of the plate and a large and almost circular [22]. ƒe species A. a‹ne and A. gaarderae (non A. concavum connecting pore is dorsal [18]. Alexandrium gaarderae emend. Nguyen-Ngoc & Larsen) clustered as a sister group of (reported as A. concavum) also has a dorsal connecting pore Centrodinium and more distantly related to the clade of [24]. ƒe location of the anterior attachment pore at the dorsal Gonyaulacales Journal of Marine Biology 9 Centrodinium eminens FG2 MK714074 Centrodinium intermedium FG3 MK714075 Centrodinium intermedium FG4 MK714076 Centrodinium punctatum MF043217 Centrodinium Centrodinium punctatum MF043218 Centrodinium punctatum FG6 MK714078 Centrodinium punctatum FG5 MK714077 Alexandrium ane AF318229 Episemicolon gen. nov. Alexandrium ‘concavum’ AF032348 Alexandrium fundyense KF908807 Alexandrium mediterraneum KF908808 Alexandrium tamarense KF908805 Alexandrium paci cum KF908803 Protogonyaulax 100 Alexandrium australiense KF908810 Alexandrium fraterculus KF034859 Alexandrium tropicale AY268613 100 Alexandrium tamiyavanichii AB088267 Alexandrium cohorticula AF174614 Alexandrium margale i AY152708 Alexandrium andersonii JF521621 Alexandrium minutum AY705869 Alexandrium s.s. Alexandrium tamutum AY268618 Alexandrium insuetum AB088248 Alexandrium ostenfeldii AY268601 Alexandrium peruvianum FJ011437 Alexandrium satoanum AY438020 Alexandrium taylorii AB607263 Gessnerium Alexandrium pseudogoniaulax AB088254 Alexandrium hiranoi AY438018 Alexandrium leei AY566184 100 Pyrodinium bahamense AB936757 Pyrodinium bahamense AY154959 100 Pyhrrotriadinium polyedricum JQ247712 Fukuyoa paulensis KM886379 96 Fragilidium subglobosum AF260387 Pyrocystis noctiluca FJ939576 Gonyaulax spinifera EF416284 100 Gonyaulax cf. spinifera AY154960 Gonyaulax polygramma DQ162802 Dinophysis acuta AY277648 Phalacroma rapa EU780655 Pelagodinium bei JN558107 100 Gymnodinium fuscum AF200676 Karenia brevis AY355455 100 Heterocapsa triquetra AF260401 Heterocapsa arctica AY571372 Prorocentrum steidingerae DQ336183 0.1 100 Prorocentrum micans EU780638 Eimeria tenella AF026388 F¨©ª«¬ 5: Maximum-likelihood phylogenetic tree of the D1-D2 domains of the LSU rRNA gene. Bootstrap support values (BP) >70 are shown. New sequences are highlighted in bold. ƒe scale bar represents the number of substitutions for a unit branch length. margin of the apical pore plate is the main diagnostic character C. punctatum as Po, 3′, 1a, 6′′, 6c, 8s, 5′′′, 1p, 2′′′′. ƒese authors of the species A. gaarderae and A. a‹ne [24]. In the other follow a strict Kofoidian scheme of tabulation of the epitheca, species of Alexandrium, the apical pore is comma-shaped and and labelled the apical plate that does not touch the apical the anterior attachment pore lying in the right side. ƒe two pore plate as an intercalary plate. Li et al. [11] misidentied posterior lateral sulcal plates are more or less similar in length the sulcal and hypothecal plates. Li et al. ([11], p. 177, their in the members of the tamarense/catenella group, while the Figure 8(c)) illustrated the right (S.d.p.) and le’ posterior sulcal right posterior sulcal plate is longer than the le’ posterior (S.s.p.) plates with a similar length. Li et al. [11] did not carry sulcal plate in A. a‹ne (Figure 6(d)). ƒis feature is variable out a study using plate dissection, and the sulcal lists were in Centrodinium spp. (Figures 6(e) and 6(f )). ƒe cingulum hiding the morphology of the sulcal plates. ƒese plates have and the sulcus of Centrodinium spp. and A. a‹ne are deeply very di§erent length as revealed in this study (Figure 1(k)) and incised and bordered by pronounced list, and the posterior the plate dissections of C. punctatum by Balech [5, 6]. ƒey le’ margin of the plate 6′′ is reinforced, long and concave labelled the le’ lateral posterior sulcal as the posterior plate, (Figures 1–3, 6(d)–6(f ); [20, 21]). and this induces the subsequent errors in the tabulation of the hypotheca (see Appendix S1 part 6 as Supplementary material). 4.2. Reclassi‘cation of the Subgenus Gessnerium. An historical ƒe genus Alexandrium is currently a pool of species with account of the taxonomy and nomenclature of Alexandrium signicant di§erences in the plate arrangement [26]. Balech s.l., including Gessnerium and Protogonyaulax, is reported in [18] reported that the species of the subgenus Gessnerium were the Appendix S3 as Supplementary material. ƒe plate formula closer to Pyrrhotriadinium than Alexandrium. ƒe apical pore of Alexandrium is usually reported as Po, 4′, 6′′, 6c, 8s+, 5′′′, plate in Pyrrhotriadinium is totally transverse orientated, while 2′′′′ [25]. It is similar to the plate formula of C. punctatum oblique in Gessnerium [18, 27]. Pyrrhotriadinium lacks the and di§ers from the more ™attened species of Centrodinium accessory sulcal plates, and the two median sulcal plates are in the anterior elongation of the plates 4′ and 2′, and the split separated, while in Gessnerium the accessory plates are in the latter plate. Li et al. [11] reported the plate formula of prominent and the two median sulcal plates are in contact [18]. Gonyaulacales Saca pap C6 pap Sp Sp Sacp 7''(6'') C5 Sda C3 Sp Saca Sdp Ssa ' 2'α 2 α 10 Journal of Marine Biology 4'' (3'') 3' 1' (2') 5'' 4' 3'' (4'') 3'' 4'' 1' (2'') 1' 2'' 1'' 3' Sa (2') 5'' 2' 6'' 6'' 4' Sma (5'') 5'' 1'' 7'' 5''' 1' (6'') Sa Ssa 1''' Sda Saca 6'' 3''' Sdp 3''' Ssp aap Po 2'''' Ssa 4''' 2''' Po Sdp cp 4''' Sp Ssp 2'''' 2''' Sp 1'''' 5''' Sp 1''' 1' 1'''' 5''' Sp 1''' 1' (a) (b) 3' 2' Po 4' 3'' 2'' 4' 4'' 1' 1' 1'' 5'' Sa 1'' 2'' 3' Sa 5'' 6'' 6'' 2' Sda Sma 5'' 2''' Sma 4' 1' Ssa Sda 1'' 2''' 1' Ssa 5''' 1''' 5''' 6'' Sdp 1''' Sdp Ssp Ssp aap 3''' Po cp 3''' Sp 2''' 4''' Sp 2'''' 2''' 2'''' 4''' 1' Sp 1'''' 1'''' Sp 5''' 1''' 5 5''' ''' 1''' (d) (c) 2' 4' 2' 3' 1' 4' 2'β 1' 2'β 4'' 3'' 4' 2'' 4' 3' 3'' 2'' 2' 1'' 5'' 4'' 1'' 6'' 6'' 3' 1' C1 5'' 5'' C2 2'' 3' C1 C2 1' 4' 5'' C5 4'' 3''' C6 6'' 1''' 3'' 1''' 1'' 2''' 3''' 2'' 6'' 1'' 5''' Sa 4''' 5'' 2'' 5''' 3'' 6'' C4 4'' C3 1' C2 C5 C6 C1 1'' 4''' 3''' 1''' 3''' 1''' 4''' 5''' 5''' Sp 2''' 4''' Sa 1'''' 2'''' Sp C3 2''' C4 1''' Sa 2''' Smp 3''' Sd a 1'''' C3 3'' 4''' 2'''' Po Sda C4 Ss p 4'' 2''' C5 2' C2 2'' 3' 1'''' 2'''' 1'''' 2' 4' C5 C2 2'''' Sp Sdp 1' 2'''' 5'' 4' 2'''' 1' 5''' 1''' 1'' C1 C1 C6 6'' Sp C6 (e) (f ) F¨©ª«¬ 6: Line drawings of the ventral, apical and antapical views, apical pore plate and sulcal plates of Alexandrium sensu lato and Centrodinium. (a) Gessnerium monilatum redrawn and modied from Balech (1995). (b) Alexandrium minutum redrawn and modied from Balech (1989, 1995). (c) Protogonyaulax tamarensis redrawn and modied from Balech (1995). (d) Episemicolon a‹ne gen. & comb. nov. (formerly A. a‹ne) redrawn and modied from Balech (1995). (e) Centrodinium punctatum. (f ) Centrodinium eminens. 1′–4′ = apical plates; 1′′–6′′ = precingular plates; 1′′′–5′′′ = p ostcingular plates; 1′′′′–2′′′′ = antapical plates; a.a.p. = anterior attachment pore; C1–C6 = cingular plates; c.p. = closing, cover plate or canopy; p.a.p. = posterior attachment pore; Po = apical pore plate; S.a. = anterior sulcal plate; S.a.c.a. = anterior accessory sulcal; S.a.c.p. = posterior accessory sulcal; S.d.a. = right (dexter) anterior lateral sulcal; S.d.p. = right posterior lateral sulcal; S.m.a. = anterior median sulcal; S.m.p = posterior median sulcal; S.p. = posterior sulcal; S.s.a. = le’ (sinister) anterior lateral sulcal; S.s.p. = le’ posterior lateral sulcal. 3''' 4''' 2''' 5''' 1''' 1'' (1') S s a 6'' (5'') 1'' (1') 3' (4') C4 2'' (1'') cp 1'''' Sp 3' (4') Ssp 2'' (1'') 2' (3') 2''' Smp Smp Sma Smp Smp Ssp Sdp Journal of Marine Biology 11 In Pyrrhotriadinium, the ﬁrst precingular plate, equivalent to long. e anterior and posterior attachment pore is a common the ﬁrst gonyaulacoid apical plate, does not contact the le feature in chain-forming species, but few species of apical plate [18]. e 1′′ plate is pentagonal in Gessnerium Alexandrium s.s. forms chains, and the attachment pores are (Figure 6(a)) and quadrangular in Pyrrhotriadinium, while absent (Figure 6(b), Table 1; [18, 24]). rhomboidal in nearly all the species of the subgenus Alexandrium e genus Protogonyaulax contains species where the ﬁrst (Figures 6(b)–6(c)). is is a precingular plate based on its apical plate is rhomboidal and directly connects to the apical shape and position. e posterior sulcal plate of Gessnerium is pore plate. e posterior sulcal plate is reversed pentagonal, large, longer than wide and prolonged obliquely towards the symmetrical, and longer than wide. ere are numerous posterior right (Figure 6(a)). In the species of the subgenus chain-forming species, and the presence of anterior and pos- Alexandrium, the posterior sulcal plate is relatively smaller and terior attachment pores is a common feature (Figure 6(c), non-oblique ([18]; Figures 6(b)–6(d)). PSP toxicity has not Table 1; [18, 24]). been reported in species of Gessnerium. e new scenario derived on the close relationship of e species Alexandrium margaleﬁi and A. pohangense Centrodinium and the species of Alexandrium s.l. suggests the have the ﬁrst apical plate disconnected from the apical pore reinstatement of the genera Gessnerium and Protogonyaulax, plate, which suggests an aﬃnity with Gessnerium, but this plate and the erection of a new genus for A. aﬃne and A. gaarderae. is quadrangular in these species while pentagonal in e diagnoses of the genera Centrodinium, Alexandrium, Gessnerium [18, 23]. e position of A. margaleﬁi and Gessnerium, and Protogonyaulax need to be amended. e spe- A. pohangense in the molecular phylogenies is unstable, typi- cies Peridinium splendor-maris, type of the genus Blepharocysta, cally is represented as divergent species of the clade of the type, has been interpreted to correspond to an earlier description of A. minutum [23]. ese two species, and other two divergent Alexandrium balechii. Carbonell-Moore [31] submitted a pro- species (A. diversaporum, A. leei) need further research before posal to conserve the name Peridinium splendor-maris as a to propose a change of genus. species Blepharocysta, avoiding the possible transfer of all the species of Alexandrium into Blepharocysta. If the proposal is 4.3. e Generic Split of the Subgenus Alexandrium. Previous rejected, the change does not aﬀect Alexandrium because A. morphological and molecular phylogenetic studies including balechii is now a species of Gessnerium. If the proposal is rec- sequences of Pyrodinium already suggested the reinstatement ommended, no change is applied to Alexandrium. of the Gessnerium at the genus level ([28, 29], Appendix S3 as Supplementary material). With the inclusion of Centrodinium 4.4. Emended Diagnosis of Centrodinium (Figures 6(e)–6(f), spp. in the molecular phylogenies, Alexandrium can no Table 1) longer be considered as a monophyletic genus (Figures 4 and 5). e morphological diﬀerences amongst species of 4.4.1. Centrodinium Kofoid emended Gómez & Artigas. the subgenera Gessnerium (Figure 6(a)) and Alexandrium Gonyaulacoid dinoﬂagellates with diﬀerent degree of lateral (Figures 6(b)–6(d)) are evident, but a split between species ﬂattening, an elongated brunt apex or an apical horn. of the subgenus Alexandrium based on morphology Cingulum deep, median, descending about one cingular is less conspicuous. In the molecular phylogenies, the width, without overhanging. e cingular list at both upper sequences of the subgenus Alexandrium are divided into and lower margins are prominent. e sulcus with list at both two major groups. A group that contains the type species right and le margins. e apical pore plate with a horseshoe- of Alexandrium, A. minimum, and another group split into shaped pore surrounded by small marginal pores. e plate two sister clades: one major clade that contains the type formula is Po, 4′ (2′ α + β), 6′′, 6c, ≥8s, 5′′′, 2′′′′, and the species of Protogonyaulax, P. tamarensis, with members of more ﬂattened species showed a split of the second apical plate, the tamarense/catenella/fraterculus groups, and other major 2′ (α + β). e apical pore plate is mainly surrounded by the clade for Centrodinium and Alexandrium aﬃne/A. gaarderae second and fourth apical plates, while the third apical plate (Figures 4–5). Sequences of the members of the subgenus does not reach the apex. In the less compressed species, the Alexandrium do not cluster as a monophyletic group, ﬁrst apical plate (1′) reaches the apex, whereas in the more unless we consider placing all the species of the subgenus ﬂattened species the 1′ plate does not reach the apex, and the Alexandrium into Centrodinium because Centrodinium 2′ plate is divided into two plates. In all the species, the anterior Kofoid 1907 has the priority over Alexandrium Halim sulcal plate has a distinct pore. e sulcus contains at least 1960. is implies numerous taxonomical innovations, and 8 plates, the two lateral posterior sulcal plates are long. e requires merging species with diﬀerent morphologies into a le plate is longer than the right one in the less compressed single genus. Splitting members of the subgenus Alexandrium species, and vice versa in the more compressed species. In less into three genera reconciles the molecular and morphological compressed species, the antapex is pointed, while in the more data, and requires fewer taxonomical innovations. ﬂattened species the antapical horn derived from a tubular e clade Alexandrium s.s. contains the type species, second antapical plate has terminal spinules. e antapical A. minutum, and other species in which the ﬁrst apical plate horn is supported by two triangular plates, the posterior is rhomboidal and may connect directly or indirectly through sulcal in the right face, and ﬁrst antapical plate in the le face. a thread-like prolongation with the apical pore plate e posterior sulcal plate may contain a posterior attachment (Figure 6(b), Table 1). is feature may vary intraspeciﬁcally pore near the anterior margin. e species of Centrodinium as reported for A. minutum [30]. e posterior sulcal plate is typically inhabit in warm oceans and have chloroplasts. e relatively small, symmetrical or asymmetrical, and wider than species C. punctatum is not toxic. 12 Journal of Marine Biology T 1: Comparison of the morphological and ecological characters of the genera Gessnerium emend., Alexandrium emend., Protogon- yaulax emend., Episemicolon gen. nov., and Centrodinium emend. Gómez & Artigas. Data based on Balech (1995), and this study. Gessnerium Alexandrium Protogonyaulax Episemicolon Centrodinium emend. emend. emend. gen. nov. emend. Non or slightly Non or slightly Non or slightly Non compressed or Moderate to highly Cell compression compressed compressed compressed slightly compressed lateral ﬂattening No No No No Variable Horn or spines Epitheca plate Po, 3′(4′), 7′′ (6′′) Po, 4′ (3′), 6′′ (7′′) Po, 4′, 6′′ Po, 4′, 6′′ Po, 4′, 6′′ formula Shape of apical pore Comma or ﬁshhook Comma Comma Oval or bullet Horse-shoe plate Anterior attachment Right to Po Right to Po Right to Po Dorsal to Po Unreported pore (when present) Shape of the Pentagonal Rhomboidal Rhomboidal Rhomboidal Rhomboidal equivalent gonyaula- coid ﬁrst apical plate Pore in anterior sulcal No No No No Yes Precingular part in No Sometimes Sometimes No No sulcal anterior Le cingular part in the anterior sulcal No No No No Yes plate Median sulcal plates Large Small Small Small Small Accessory sulcal plates Large Small Small Small Small Lateral posterior Right longer than Right and le of Similar or right Right longer than le Variable sulcal plates le similar length longer than le Short, wider than Short, longer than Short, longer than Polygonal or trian- Posterior sulcal plate Large long wide wide gular Absent or incon- Moderate or prom- Moderate Moderate Very prominent Sulcal list spicuous inent Posterior sulcal Typically present Present in Present in Present in Typically absent or in chain-forming chain-forming chain-forming chain-forming attachment (connect- inconspicuous ing) pore species species species species V-shaped anterior margin of the sulcal Prominent or not Inconspicuous Prominent Prominent No posterior plates Variable Rarely Common Variable Variable Chain-forming Plankton, cos- Plankton, cos- Plankton, low abun- Plankton, tropical mopolitan, mopolitan, Plankton, tropical to Habitat dance in open warm to temperate seas bloom-forming in bloom-forming in temperate seas to tropical seas neritic waters neritic waters Paralytic shellﬁsh No Yes Yes No No poisoning (1) Type species: Centrodinium elongatum Kofoid 1907. (vii) Centrodinium intermedium Pavillard 1930. (2) Other species: (viii) Centrodinium maximum Pavillard 1930. (i) Centrodinium biconicum (G. Murray & Whitting (ix) Centrodinium mimeticum (Balech 1967) F.J.R. Taylor 1899) F.J.R. Taylor 1976 [=Murrayella biconica (G. 1976 (=M. mimetica Balech). Murray & Whitting) Pavillard 1931, Pavillardinium (x) Centrodinium ovale (Pavillard 1930) Hernández- biconicum (G. Murray & Whitting) Rampi 1948]. Becerril in Hernández-Becerril et al. 2010 [=M. (ii) Centrodinium complanatum (Cleve 1903) Kofoid ovalis Pavillard, P. ovale (Pavillard) G. De Toni 1936]. 1907 (=Steiniella complanata Cleve). (xi) Centrodinium paciﬁcum (Rampi 1950) F. J. R. Taylor (iii) Centrodinium deﬂexoides Balech 1962. 1976 (=P. paciﬁcum Rampi). (iv) Centrodinium deﬂexum Kofoid 1907. (xii) Centrodinium pavillardii F. J. R. Taylor 1976 [=P. (v) Centrodinium eminens Böhm 1933. intermedium (Pavillard 1916) G. de Toni 1936, M. intermedia Pavillard, non C. intermedium Pavillard (vi) Centrodinium expansum Kofoid & J. R. Michener 1930]. 1911. Journal of Marine Biology 13 (xiii) Centrodinium porulosum Kofoid & J. R. Michener (3) e placement in Alexandrium s.s. needs further research 1911. for the species: Alexandrium diversaporum Sh. Murray et al. 2014, A. leei Balech 1985, A. margaleﬁi Balech 1994, (xiv) Centrodinium pulchrum Böhm 1933 [=C. eminens and A. pohangense A. S. Lim & H. J. Jeong in Lim, Jeong, f. pulchrum (Böhm) J. Schiller 1933]. Kim, & Lee 2015. (xv) Centrodinium punctatum (Cleve 1900) F. J. R. Taylor 1976 [=Steiniella punctata Cleve, M. punctata 4.6. Reinstatement of the Genus Gessnerium (Figure 6(a), Table 1) (Cleve) Kofoid 1907, P. punctatum (Cleve) G. De Toni 1936, M. splendida Rampi 1941, P. splendidum 4.6.1. Gessnerium Halim 1967 ex Halim 1969 emended (Rampi) Rampi 1950]. Gómez & Artigas. Gonyaulacoid dinoﬂagellates without or scarce cell compression, without spines or horns. Cingulum 4.5. Emended Diagnosis of Alexandrium (Figure 6(b), Table 1) deep, median, descending about one cingular width, without overhanging. Apical pore plate is longitudinally oriented. e 4.5.1. Alexandrium Halim 1960 emended Gómez & typical gonyaulacoid ﬁrst apical plate, narrow and rhomboidal Artigas. Gonyaulacoid dinoﬂagellates without or with scarce of Alexandrium s.l. is absent. e anterior right margin of cell compression, and lacking horn or spines. Cingulum the ﬁrst apical is an anterior margin in Gessnerium, and the deep, median, descending about one cingular width, without equivalent plate is pentagonal and never reaches the apical overhanging. e theca is usually smooth, and only ornamented pore plate. is plate is considered the ﬁrst apical plate (Po, in few species. e plate formula is Po, 4′, 6′′, 6c, >8s, 5′′′, 2′′′′. 4′, 6′′, 6c, 10s, 5′′′, 2′′′′) or a precingular plate in a strict e plate 1′ is rhomboidal, narrow, and asymmetrical and can Kofoidian scheme (plate formula Po, 3′, 7′′, 6c, 10s, 5′′′, 2′′′′). be either in direct contact with the apical pore plate or indirectly e le anterior sulcal plate (S.s.a) is large and superﬁcial, connected via a thin suture (thread-like process). Alexandrium while in Alexandrium s.l. is small and sunk into the sulcus. insuetum, has severely reticulated thecal plates and the exsert 1′. ere are two relatively large accessory sulcal plates that e plate 6′′ is usually narrow. e posterior sulcal is relatively are absent or hardly visible in Alexandrium s.l. e right small, wider than long. e apical pore plate contains a comma- posterior lateral sulcal plate (S.d.p.) is long and narrow. e shaped pore. Relatively few chain-forming species, and the posterior sulcal plate (S.p.) is longer than wide, extending attachment pore, if present, is in the lateral right to the apical pore obliquely towards the posterior right. e second antapical plate. A posterior connecting pore is usually absent. e species plate of Gessnerium is lateral, while this plate is more dorsal are typically bloom-forming in eutrophic and/or conﬁned neritic than lateral in Alexandrium s.l. e formation of chains is waters. PSP toxicity has been reported in numerous species. variable amongst the species. e species are more common in warm waters, and rarely reported in cold waters. Paralytic (1) Type species: Alexandrium minutum Halim 1960 (=A. shellﬁsh poisoning has not been associated with the presence ibericum Balech 1985, A. lusitanicum Balech 1985, A. of Gessnerium, but several species are known as ﬁsh-killers angustitabulatum F. J. R. Taylor 1995 nom. illeg.) that produce goniodomin A, spirolide, or hemolytic toxins (2) Other species of the genus Alexandrium: that may be involved in mixotrophy. (i) Alexandrium andersonii Balech 1990. (1) Type species: Gessnerium mochimaense Halim 1967 ex (ii) Alexandrium insuetum Balech 1985. Halim 1969 [=Gessnerium monilatum (J. F. Howell 1953) (iii) Alexandrium ostenfeldii (Paulsen 1904) Balech & A. R. Loeblich 1970]. Tangen 1985 [=Goniodoma ostenfeldii Paulsen, (2) Other species of Gessnerium: Gonyaulax ostenfeldii (Paulsen) Paulsen 1949, Protogonyaulax ostenfeldii (Paulsen) S. Fraga & F. J. (i) Gessnerium balechii (Steidinger 1971) A. R. Sánchez 1985, Heteraulacus ostenfeldii (Paulsen) A. Loeblich & L.A. Loeblich 1979 [=Gonyaulax balechii R. Loeblich 1970, Gessnerium ostenfeldii (Paulsen) Steidinger, Pyrodinium balechii (Steidinger) F. J. R. A. R. Loeblich & L. A. Loeblich 1979, Triadinium Taylor 1976, Alexandrium balechii (Steidinger) ostenfeldii (Paulsen) J. D. Dodge 1976, ?Gonyaulax Balech 1995]. globosa (Braarud 1945) Balech 1971 nom. illeg., (ii) Gessnerium concavum (Gaarder 1954) A. R. Loeblich ?Protogonyaulax globosa (Braarud) F. J. R. Taylor & L. A. Loeblich 1979 [=Goniodoma concava Gaarder, 1979, ?Gonyaulax trygvei M. Parke & J. D. Dodge A. concavum (Gaarder) Balech 1985 emend. Nguyen- in Parke & Dixon 1976, ?Gonyaulax tamarensis Ngoc & Larsen 2004, non Gonyaulax concava M. Lebour var. globosa Braarud 1945, ?Gonyaulax (Gaarder) Balech 1967, nec A. concavum (Gaarder) dimorpha Biecheler 1952]. Balech 1985, auct. non Balech 1995 (=A. gaarderae (iv) Alexandrium peruvianum (Balech & B.R. Mendiola Nguyen-Ngoc & J. Larsen 2004)]. 1977) Balech & Tangen 1985 [=Gonyaulax peruviana (iii) Gessnerium monilatum (J. F. Howell 1953) A. R. Balech & B. R. Mendiola, Protogonyaulax peruviana Loeblich 1970 [=Gonyaulax monilata J. F. Howell, (Balech & B. R. Mendiola) F. J. R. Taylor 1979, ?A. Pyrodinium monilatum (J. F. Howell) F. J. R. Taylor ostenfeldii Paulsen 1904) Balech & Tangen 1985]. 1976, Alexandrium monilatum (J. F. Howell) Balech (v) Alexandrium tamutum Montresor, Beran & U. John 1985, Gessnerium mochimaensis Halim 1967 ex Halim 1969]. 14 Journal of Marine Biology 4.6.2. New Combinations of Gessnerium: 4.7. Reinstatement of the Genus Protogonyaulax (Figure 6(c), Table 1) (i) Gessnerium camurascutulum (L. MacKenzie & K. Todd 2002) F. Gómez & Artigas, comb. nov. 4.7.1. Protogonyaulax F. J. R. Taylor 1979 emended Gómez & Basionym: Alexandrium camurascutulum L. Artigas. Gonyaulacoid dinoﬂagellates without or with scarce MacKenzie & K. Todd (2002, Harmful Algae, 1: cell compression, and lacking horn or spines. Cingulum 296, Figure 1). MacKenzie & Todd [32] designed as deep, median, descending about one cingular width, without holotype the Figures 1, 7, and 13. e Figure 13 also overhanging. e theca is usually smooth, and very rarely includes A. minutum, and the Figures 1 and 7 corre- ornamented. e plate formula is Po, 4′, 6′′, 6c, ≥8s, 5′′′, spond to cells from diﬀerent geographical origins (see 2′′′′. e ﬁrst apical plate (1′) plate is rhomboidal, narrow Article 8.1 of International Code of Nomenclature and asymmetrical and always directly connected to the (I.C.N.) for Algae, Fungi, and Plants). e Figure 1 apical pore plate (Po). e plate 6′′ is usually wide. e (cell from Marlborough Sounds) is designed as type. posterior sulcal plate is longer than wide, with usually two (ii) Gessnerium hiranoi (T. Kita & Fukuyo 1988) F. Gómez ventrally directed anterior prolongations and a connecting & Artigas, comb. nov. Basionym: Alexandrium hira- pore. e Po plate contains a comma-shaped pore, and noi T. Kita & Fukuyo (1988, Bull. Plankt. Soc. Jap. 35: usually an anterior attachment pore in the right lateral side 2, pl. 1 a–k, Figures 1(a)–1(f )). of the apical pore plate. Relatively many chain-forming (iii) Gessnerium foedum (Balech 1990) F. Gómez & species. e species are bloom-forming in eutrophic and/ Artigas, comb. nov. Basionym: Alexandrium foedum or conﬁned neritic waters. Cosmopolitan distribution with Balech (1990, Helgol. Meeresunters. 44: 392, Figures a few species reported from cold waters. Paralytic shellﬁsh 19–33). Synonym: Goniodoma pseudogoniaulax poisoning toxicity events have been reported in numerous sensu Kita et al. 1985. species. (iv) Gessnerium globosum (Nguyen-Ngoc & J. Larsen in (1) Type species: Protogonyaulax tamarensis (M. Lebour Larsen & Nguyen-Ngoc 2004) F. Gómez & Artigas, 1925) F. J. R. Taylor 1979. Basionym: Gonyaulax tama- comb. nov. Basionym: Alexandrium globosum rensis M. Lebour. Homotypic synonyms: Alexandrium Nguyen-Ngoc & J. Larsen in Larsen and Nguyen- tamarense (M. Lebour) Balech 1985, Gessnerium tam- Ngoc (2004, Opera Bot., 140: 93, pl. 7, Figure 8). arense (M. Lebour) A. R. Loeblich & L. A. Loeblich Non Protogonyaulax globosa (Braarud 1945) F. J. R. 1979. Heterotypic synonyms: Gonyaulax tamaren- Taylor 1979. sis var. excavata Braarud 1945, Gonyaulax excavata (v) Gessnerium pseudogoniaulax (Biecheler 1952) (Braarud) Balech 1971, Alexandrium excavatum F. Gómez & Artigas, comb. nov. Basionym: (Braarud 1945) Balech & Tangen 1985. Goniodoma pseudogoniaulax Biecheler (1952, (2) Other species of the genus Protogonyaulax: Bull. Biol. Fr. Belg., Suppl. 36: p. 55, Figures 30–32). Synonyms: Triadinium pseudogoniaulax (i) P rotogonyaulax acatenella (Whedon & Kofoid 1936) (Biecheler) J.D. Dodge 1981, Alexandrium pseu- F. J. R. Taylor 1979 [=Gonyaulax acatenella Whedon dogoniaulax (Biecheler 1952) Horiguchi 1983 ex T. & Kofoid, A. acatenella (Whedon & Kofoid) Balech Kita & Fukuyo 1992. e epithet is oen reported 1985]. as “pseudogonyaulax”. (ii) Protogonyaulax catenella (Whedon & Kofoid 1936) (vi) Gessnerium satoanum (K. Yuki & Fukuyo 1992) F. J. R. Taylor 1979 [=Gonyaulax catenella Whedon F. Gómez & Artigas, comb. nov. Basionym: & Kofoid, A. catenella (Whedon & Kofoid) Balech Alexandrium satoanum K. Yuki & Fukuyo (1992, J. 1985, Gonyaulax washingtonensis Hsu 1967, Phycol., 28: 396, Figures 1–12). Gessnerium catenella (Whedon & Kofoid) A. R. (vii) Gessnerium taylorii (Balech 1994) F. Gómez & Loeblich & L. A. Loeblich 1979]. John et al. [33] pro- Artigas, comb. nov. Basionym: Alexandrium taylorii posed to reject the name Gonyaulax catenella, the Balech (1994, Trans. Amer. Microscop. Soc., 113: basionym of A. catenella, in order to permit usage 219, Figures 7–11). of the more recent name A. fundyense. e proposal (2302) was not recommended, and Prud′homme van Reine [34] reported: “Alexandrium fundyense 4.6.3. e Next Species Do not Belong to Gessneri- and A. catenella are certainly conspeciﬁc, and then um:. Gessnerium acatenella (Whedon & Kofoid 1936) A. R. “catenella” has nomenclatural priority”. Loeblich & L. A. Loeblich 1979 (accepted as Protogonyaulax (iii) Protogonyaulax cohorticula (Balech 1967) F. J. R. acatenella). Gessnerium catenella (Whedon & Kofoid 1936) A. Taylor [=Gonyaulax cohorticula Balech nom. inval., R. Loeblich & L. A. Loeblich 1979 (accepted as Protogonyaulax Gessnerium cohorticula (Balech) A. R. Loeblich & catenella). Gessnerium cohorticula (Balech 1967) A. R. Loeblich L. A. Loeblich 1979, A. cohorticula (Balech 1967) & L. A. Loeblich 1979 (accepted as Protogonyaulax cohortic- Balech 1985]. Balech described the basionym ula). Gessnerium fraterculus (Balech 1964) A. R. Loeblich & lacking Latin description and designation of type. L. A. Loeblich 1979 (accepted as Protogonyaulax fraterculus). Balech described the species under the Zoological Gessnerium tamarense (M. Lebour 1925) A. R. Loeblich & Nomenclature (see Article 45 of I.C.N.). L. A. Loeblich 1979 (accepted as Protogonyaulax tamarensis). Journal of Marine Biology 15 (iv) P rotogonyaulax compressa Fukuyo, K. Yoshida & H. (v) Protogonyaulax tamiyavanichii (Balech 1974) Inoue 1985 [=Alexandrium compressum (Fukuyo, K. F. Gómez & Artigas, comb. nov. Basionym: Yoshida & H. Inoue) Balech 1995]. Alexandrium tamiyavanichii Balech (1994, Trans. Amer. Microscop. Soc., 113: 217, Figures 1–6). (v) Protogonyaulax fraterculus (Balech 1964) F. J. R. Synonym: Protogonyaulax cohorticula (Balech) Taylor 1979 [=Gonyaulax fraterculus Balech 1964 F. J. R. Taylor 1979 sec Kodama et al. 1988. as G. “fratercula”, nom. inval.; Gessnerium fratercu- lus (Balech) A. R. Loeblich & L. A. Loeblich 1979, (vi) Protogonyaulax tropicalis (Balech 1985) F. Gómez & Alexandrium fraterculus (Balech) Balech 1985, Artigas, comb. nov. Basionym: Alexandrium tropicale nom. inval.]. Balech [35] described the basionym Balech in Anderson et al. [1985, Toxic dinoﬂagellates. lacking Latin description and designation of type. Proceedings of the ird International Conference Balech described the species under the Zoological on Toxic dinoﬂagellates. Elsevier, New York, p. 37, Nomenclature (see Article 45 of I.C.N.). Figure 7]. Synonym: Gonyaulax excavata Braarud 1945 sensu Balech (1971, Serv. Hydrogr. Naval, (vi) Protogonyaulax kutnerae (Balech 1979) Sournia Buenos Aires, H 654: 28, pl. 7: Figures 119–124). 1984 (=Gonyaulax kutnerae Balech 1979). (vii) Protogonyaulax leei (Balech 1985) Fukuyo, 4.7.3. e Next Species Do not Belong to Protogonyaulax Pholpunthin & K. Yoshida 1988 (=Alexandrium leei Balech). (i) Protogonyaulax aﬃnis H. Inoue & Fukuyo 1985 (placed (viii) Protogonyaulax phoneus (Wołoszyńska & W. in a new genus, see below). Protogonyaulax dimor- Conrad 1939) F. J. R. Taylor 1979 [=Pyrodinium pha (Biecheler 1952) F. J. R. Taylor 1979 (accepted as phoneus Wołoszyńska & W. Conrad, Gonyaulax Gonyaulax dimorpha Biecheler, may be related to A. phoneus (Wołoszyńska & W. Conrad) F. J. R. ostenfeldii). Protogonyaulax globosa (Braarud 1945) Taylor 1975, Gonyaulax phoneus (Wołoszyńska & F. J. R. Taylor 1979 [accepted as Gonyaulax globosa W. Conrad) Loeblich & A. R. Loeblich 1975, ?A. (Braarud) Balech 1971 nom. illeg., and it may be ostenfeldii (Paulsen 1904) Balech & Tangen 1985]. related to A. ostenfeldii]. Protogonyaulax ostenfeldii e epithet “phoneus” is masculine, while the genus (Paulsen 1904) S. Fraga & F. J. Sánchez 1985 (accepted is feminine. as Gessnerium ostenfeldii). Protogonyaulax peruviana (Balech & B.R. Mendiola 1977) F. J. R. Taylor 1979 4.7.2. New Combinations of Protogonyaulax (accepted as Alexandrium peruvianum or synonym of A. ostenfeldii). (i) Protogonyaulax australiensis (Sh. Murray in John et al. 2014a) F. Gómez & Artigas, comb. nov. Basionym: 4.8. New Erected Genus for the Clade of Alexandrium aﬃne Alexandrium australiense Sh. Murray in John et al. (Figure 6(d), Table 1) 2014a (Protist, 165: 797–798, Figure 8). Synonym: Alexandrium australis Wang et al. 2014 nom. inval. 4.8.1. Episemicolon F. Gómez & Artigas, gen. nov. (1) Diagnosis: (ii) Protogonyaulax fundyensis (Balech 1985) F. Gómez Gonyaulacoid dinoﬂagellate without or scarce cell compression, & Artigas, comb. nov. Basionym: Alexandrium fun- without spines or horns. Cingulum deep, median, descending dyense Balech in Anderson et al. (1985, Toxic dino- about one cingular width, without overhanging. e cingular ﬂagellates. Proceedings of the ird International lists at both upper and lower margin are prominent. e sulcus Conference on Toxic dinoﬂagellates. Elsevier, New with list at both right and le margins. Plate formula Po, 4′, York, p. 37, Figure 18). Prud′homme van Reine [34] 6′′, 6c, ≥8s, 5′′′, 2′′′′. e ﬁrst apical plate is rhomboidal and reported: “Alexandrium fundyense and A. catenella reaches the apical pore plate. e apical pore plate contains are certainly conspeciﬁc, and then “catenella” has an oval or bullet-shaped apical pore, with an attachment pore nomenclatural priority”. lying at the dorsal side. e sulcus contains at least eight (iii) Protogonyaulax mediterranea (U. John in John et al. plates, the two lateral posterior plates are long, and with the 2014a) F. Gómez & Artigas, comb. nov. Basionym: right one longer than the le pair. e posterior sulcal plate is Alexandrium mediterraneum U. John in John et al. right displaced and may contain a marginal attachment pore. 2014a (Protist 165: 795–797, Figure 7). Synonym: Paralytic shellﬁsh poisoning toxicity has not been reported. Alexandrium mediterranis Wang et al. 2014 nom. inval. (2) Etymology: epi- from Ancient Greek “epi” (= on top (iv) Protogonyaulax paciﬁca (Litaker in John et al. of ); semicolon: the punctuation mark (;) from Latin “semi” 2014a) F. Gómez & Artigas, comb. nov. Basionym: (= half ), and Greek “kolon” (= verse, a part of a strophe, col- Alexandrium paciﬁcum Litaker in John et al. 2014a umn) and a mark of punctuation (:). e apical pore and the (Protist, 165: 793–795, Figure 6). John et al. ([36], dorsal attachment pore in the apical pore plate resemble the p. 794) reported “As it is no longer possible to estab- typographic symbol (;). e gender is neuter. lish the identity of the material originally assigned to (3) Type species: Episemicolon aﬃne (H. Inoue & Fukuyo 1985) A. catenella, we have designated a new species name F. Gómez & Artigas, gen. & comb. nov., hic designatus. Basionym: for Group IV isolates - A. paciﬁcum - and have sub- Protogonyaulax aﬃnis H. Inoue & Fukuyo in Fukuyo et al. (1985, mitted a proposal to reject the name Alexandrium Proceedings of the ird International Conference on Toxic dino- catenella (John et al., 2014b)”. e proposal (2302) in ﬂagellates. Elsevier, New York, p. 30, Figure 3(a)–3(c)). Synonyms: John et al. [33] was not recommended [34]. A. aﬃne (H. Inoue & Fukuyo) Balech 1985 nom. inval., A. fukuyoi 16 Journal of Marine Biology Balech in Anderson et al. 1985 nom. inval., A. aﬃne (H. Inoue & Conflicts of Interest Fukuyo) Balech 1995. e authors declare that they have no conﬂicts of interests regarding the publication of this paper. 4.8.2. Other Species. Episemicolon gaarderae (Nguyen-Ngoc & J. Larsen in Larsen & Nguyen-Ngoc 2004 ex F. Gómez & Artigas) F. Gómez & Artigas, comb. nov. Basionym: Gonyaulax Acknowledgments concava Gaarder sensu Balech (1967, Rev. Mus. argent. Cienc. Nat. ′B. Rivadavia′, Hidrobiol., 2, 108–111; plate 6, Figures F. G. was partly supported by a convention (no: 2101893310) 108–116). Synonym: Alexandrium gaarderae Nguyen-Ngoc between CNRS INSU and the French Ministry of Ecology & J. Larsen in Larsen & Nguyen-Ngoc 2004 ex F. Gómez & (MTES) for the implementation of the Monitoring Program Artigas, A. concavum (Gaarder) Balech 1985, non Goniodoma of the European Marine Strategy Framework directive (MSFD) concava Gaarder 1954, nec Goniodoma gaarderae Balech 1980 for pelagic habitats and the descriptor “biodiversity”. Samples nom. inval. According to Index Nominum Algarum (http:// were collected during the IFREMER “Etoile” cruise onboard ucjeps.berkeley.edu/ina/), Nguyen-Ngoc and Larsen [20] did R/V Côtes de la Manche (CNRS-INSU) in the frame of the not provide Latin description and designation of type of A. Joint European Research Infrastructure network for Coastal gaarderae (intended as a new name for Gonyaulax concava sensu Observatory – Novel European eXpertise for coastal observa- Balech 1967, but eﬀectively a new species). We designate as type Tories (JERICO-Next) H2020 INFRAIA project no. 654410. the Figure 4, plate 6, in Larsen & Nguyen-Ngoc ([20, p. 91). We thank I. Puillat, project manager, and P. Lazure, scientiﬁc coordinator of the Etoile cruise. We thank L. Courcot for the SEM assistance. Abbreviations a.a.p.: Anterior attachment pore in apical pore plate Supplementary Materials auct. non: auctorum non (of authors [but] not....), used for misapplied names Supplementary 1. Appendix S1. Taxonomy, synonymy, plate BP: Bootstrap probability arrangement and classiﬁcation of Centrodinium. c.p.: Cover or closing platelet or canopy in the Supplementary 2. Appendix S2. Detailed morphology of apical pore plate Centrodinium spp. HAB: Harmful algal bloom Supplementary 3. Appendix S3. Brief historical account of the I.C.N.: International Code of Nomenclature for taxonomy and nomenclature of Alexandrium sensu lato. algae, fungi, and plants Supplementary 4. Figure S1. Maximum-likelihood phy- LM: Light microscopy logenetic trees of D1–D2 domains of the LSU rRNA gene LSU: Large subunit sequences of selected species of Alexandrium sensu lato and mp: Marginal pore in the apical pore plate Centrodinium spp. with especial focus on the group of A. aﬃne. nom. inval.: nomen invalidum, an invalid name nom. illeg.: nomen illegitimum, an illegitimate name. Po: Apical pore plate References p.a.p.: Posterior attachment pore in the posterior [1] D. M. Anderson, T. J. Alpermann, A. D. Cembella, Y. Collos, sulcal plate E. Masseret, and M. Montresor, “e globally distributed PCR: Polymerase chain reaction genus Alexandrium: multifaceted roles in marine ecosystems PSP: Paralytic shellﬁsh poisoning and impacts on human health,” Harmful Algae, vol. 14, no. 1, rRNA: Ribosomal RNA pp. 10–35, 2012. S.a.: Anterior sulcal plate [2] E. 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Redefinition of the Dinoflagellate Genus Alexandrium Based on Centrodinium: Reinstatement of Gessnerium and Protogonyaulax, and Episemicolon gen. nov. (Gonyaulacales, Dinophyceae)

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

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

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

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