molecular phylogeny of dinophysoid...

MOLECULAR PHYLOGENY OF DINOPHYSOID DINOFLAGELLATES:THE SYSTEMATIC POSITION OF OXYPHYSIS OXYTOXOIDES AND THE

DINOPHYSIS HASTATA GROUP (DINOPHYSALES, DINOPHYCEAE)1

Fernando Gómez2

Université Lille Nord de France, Laboratoire d¢Océanologie et Géosciences, CNRS UMR 8187, Station Marine de Wimereux,28 Av. Foch, 62930 Wimereux, France

Purificación López-Garcı́a and David Moreira

Unité d’Ecologie, Systématique et Evolution, CNRS UMR 8079, Université Paris-Sud, Bâtiment 360, 91405 Orsay Cedex, France

The dinophysoid dinoflagellates are currentlydivided into three families: Amphisoleniaceae, Dino-physaceae (mainly Dinophysis Ehrenb. and Phalacro-ma F. Stein), and Oxyphysaceae, the latter includingonly one member, Oxyphysis oxytoxoides Kof. Phalac-roma has been recently reinstated separately fromDinophysis, and its amended description is currentlyrestricted to cells whose epithecae were large but1 ⁄ 4 of the cell length)branched with a strong support with the type ofPhalacroma. We therefore propose Phalacroma oxy-toxoides comb. nov. for O. oxytoxoides. Our SSUrDNA phylogeny also suggests that the assumed highintraspecific variability of Dinophysis hastata F. Steinhides a number of cryptic species. According totheir distinct phylogenetic placement, the formsD. hastata f. phalacromides Jørg. and D. hastataf. uracanthides Jørg. should be erected at the specieslevel. We propose for them the names Dinophysisphalacromoides comb. nov. and Dinophysis uracan-thoides comb. nov.

Key index words: Amphisolenia; cryptic species;Dinophysiaceae; Dinophysiales; dinophysioiddinoflagellate; Histioneis; Ornithocercus; Oxyphysi-aceae; Phalacroma; SSU and LSU rDNA phylogeny

Abbreviations: bp, base pairs; BV, bootstrap value;s.s., sensu stricto

The dinophysoids (see Appendix S1 in the sup-plementary material for a note on the spelling ofthe supergeneric names derived from Dinophysisauthored by P. C. Silva) are a well-defined order ofmarine dinoflagellates with 280 recognized speciesclassified in three families: Amphisoleniaceae, Dino-physaceae, and Oxyphysaceae (Fensome et al. 1993,Steidinger and Tangen 1997, Gómez 2005). Thecells are laterally compressed with a reduced epit-heca and a larger hypotheca consisting of two largeplates united by a sagittal serrate suture with a zig-zag course (Kofoid and Skogsberg 1928, Tai andSkogsberg 1934, Abé 1967a,b,c, Balech 1967).According to Balech (1980), the dinophysoids arealso unusual among the thecate dinoflagellates inthat, despite their extreme morphological specializa-tion, their plate arrangement and number are moreor less similar in all species and genera, except forthe genera Amphisolenia F. Stein and Citharistes F.Stein.

The dinophysoid genus Phalacroma F. Stein wasmorphologically separated from Dinophysis Ehrenb.based mainly on differences in epithecal elevation(Stein 1883, Kofoid and Skogsberg 1928). Dinophysisspecies have a reduced epitheca, and their anteriorcingular list forms a funnel-shaped fan, whereasPhalacroma species have a visible epitheca above ananterior cingular list that is typically narrow anddirected horizontally. However, detailed tabulationstudies, especially at the sulcus level, did not showany significant difference between Dinophysis andPhalacroma. For this reason and because of theirintergrading morphology, Abé (1967b) and Balech(1967) transferred Phalacroma species into Dinophy-sis. More recently, and based on molecular data,Handy et al. (2009) and Hastrup Jensen and Daugb-jerg (2009) observed a deep separation between spe-cies of the Phalacroma and Dinophysis lineages.Accordingly, Hastrup Jensen and Daugbjerg (2009)

1Received 21 March 2010. Accepted 6 October 2010.2Author for correspondence: e-mail fernando.gomez@fitoplancton.

com.

J. Phycol. 47, 393–406 (2011)� 2011 Phycological Society of AmericaDOI: 10.1111/j.1529-8817.2011.00964.x

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reinstated the genus Phalacroma and amended itsdescription, which is currently restricted to cellswhose epithecae are large but having

Negative controls without template DNA were used at allamplification steps. Amplicons of the expected size(�1,200 base pairs [bp]) were then sequenced bidirectionallywith primers DIN464F and EK-1498R using an automated 96-capillary sequencer ABI PRISM 3730xl (Cogenics, Meylan,France).

Phylogenetic analyses. The new SSU rDNA sequences werealigned to a large multiple sequence alignment containing1,100 publicly available complete or nearly complete(>1,300 bp) dinoflagellate sequences using the profile align-ment option of MUSCLE 3.7 (Edgar 2004). The resultingalignment was manually inspected using the program ED of theMUST package (Philippe 1993). Ambiguously aligned regionsand gaps were excluded in phylogenetic analyses. Preliminaryphylogenetic trees with all sequences were constructed usingthe neighbor-joining method (Saitou and Nei 1987) imple-mented in the MUST package (Philippe 1993). These treesallowed identifying the closest relatives of our sequencestogether with a sample of other dinoflagellate species, whichwere selected to carry out more computationally intensivemaximum-likelihood (ML) analyses. These were done with theprogram TREEFINDER (Jobb et al. 2004) applying aGTR + C + I model of nucleotide substitution, taking intoaccount a proportion of invariable sites and a C-shapeddistribution of substitution rates with four rate categories.Bootstrap values (BVs) were calculated using 1,000 pseudore-plicates with the same substitution model.

The phylogenetic position of the dinophysoids was analyzedby means of a global alignment of 77 taxa representingsequences of dinoflagellates, including sequences of dinophy-soid species, with representatives of the lineages of theGymnodiniales, Prorocentrales, and Peridiniales. We did notinclude the environmental dinophysoid sequences of Handyet al. (2009) in our final SSU rDNA phylogenetic trees, as theywere short and imposed a limitation in the final number of

positions to be considered in our analysis. Similarly, from the26 new SSU rDNA sequences of identified cells that theseauthors obtained, we only included the 11 sequences that werelonger than 1,300 bp. To compare the position of O. oxytoxoidesin SSU and LSU rDNA phylogenies, we retrieved available LSUrDNA sequences of dinophysoid dinoflagellates from GenBankusing Entrez (http://www.ncbi.nlm.nih.gov/sites/gquery) witha taxonomic query. We reconstructed ML phylogenetic treesfor the LSU rDNA sequences alone or combined with SSUrDNA sequences for the same species using TREEFINDER andthe same substitution model specifications as for the SSU rDNAphylogenetic analysis (see above). Our sequences were depos-ited in GenBank under accession numbers HM853763–HM853816 (see Table S1 in the supplementary material).

RESULTS AND DISCUSSION

Species identification. To amplify and sequenceSSU rRNA genes from key dinophysoid species andcarry out phylogenetic analyses, we collected individ-ual cells of a total of 28 dinophysoid species (TableS1). All cells were individually identified, photo-graphed, and collected under the microscope(Figs. 1–4). In the description of the specimens, wefollow the classification into three families accord-ing to the current taxonomic scheme.

Family Amphisoleniaceae. This family is composed oftwo distinctive genera, Amphisolenia and Triposolenia,characterized by an elongated cell body. We obtainednew SSU rDNA sequences for several species ofthe genus Amphisolenia, including the sequence ofthe type, Amphisolenia globifera F. Stein, as well as

FIG. 1. Light micrographs ofspecimens of Amphisolenia andTriposolenia collected for single-cell PCR analysis. See Table S1 (inthe supplementary material) forthe collection date, location, andaccession numbers. (a–b) Amphi-solenia globifera FG1401. (c) Amphi-solenia schauinslandii FG1163. (d)Amphisolenia sp. FG281. (e–f) Am-phisolenia bidentata FG279. (g–i)Other specimen of Amphisoleniabidentata under epifluorescencemicroscopy. Note the intracellularcoccoid symbionts. (j) Triposoleniabicornis 2 FG1153. (k) T. bicornis 4FG1155. (l) T. bicornis 6 FG1157.(m) T. bicornis 8 FG1159. Scalebars, 20 lm.

MOLECULAR PHYLOGENY OF DINOPHYSALES 395

for the genus Triposolenia. A. globifera, one of thesmaller species of the genus, is characterized by aswelling of the antapical end. The specimen studied(207 lm long, 12 lm wide) showed a swelling of8 lm in diameter with two small spines (Fig. 1, a andb). Amphisolenia schauinslandii Lemmerm., closelyrelated to the type, was larger and showed an inflatedmidbody (390 lm long, 35 lm wide) (Fig. 1c).Another specimen (335 lm long, 19 lm wide;Fig. 1d) resembled Amphisolenia complanata Kof. etSkogsb., but it might represent an incompletely devel-oped specimen and was therefore more difficult toidentify. To avoid misnaming, we generically named itAmphisolenia sp. The largest Amphisolenia specimen

(765 lm long, 21 lm wide) contained green granulesin the central body and was identified as Amphisoleniabidentata Schröd. (Fig. 1, e and f). In other similarspecimens, these green granules appeared to containchl a, as seen under epifluorescence microscopy(Fig. 1, g–i).

While Amphisolenia, especially A. bidentata, wascommon in surface waters, the specimens of Tripo-solenia were preferentially distributed in deep waters.We obtained identical SSU rDNA sequences fromfour live specimens of Triposolenia bicornis Kof. fromthe same sample collected at 400 m depth. Thedimensions of the four cells were identical, with alength from the apex to the tip of the antapical

FIG. 2. Light micrographs of Phalacroma. See Table S1 (in the supplementary material) for the collection date, location, and accessionnumbers of the specimens collected for single-cell PCR analysis. (a) Phalacroma porodictyum FG487. (b–c) P. porodictyum FG510. (d–e)P. porodictyum FG519. (f–g, i) P. porodictyum FG490. (h) Original illustration of P. porodictyum by Stein (1883). (j–m) Live specimen ofP. porodictyum FG1193b. (j) Note the pores in the theca. (l) The arrows indicate the two rows of pores along the cingulum. (n) Phalacromasp. FG517. (o) Phalacroma mitra FG175. (p) P. mitra FG175b. (q) P. mitra FG525. (r) P. mitra FG1179. (s–t) Phalacroma rapa FG1187. (u)Phalacroma favus FG1183. (v–w) P. favus FG1188. The arrows (t, u) indicate the different length of the third rib. (x) Phalacroma doryphorumFG365. (y) P. doryphorum FG641. (z) P. doryphorum FG509. (aa) Phalacroma rotundatum FG366. (ab) P. rotundatum FG365. (ac–ad) Phalacromaparvulum FG503. (ae–af) P. parvulum FG505. (ag) P. parvulum FG326. Scale bars, 20 lm.

396 FERNANDO GÓMEZ ET AL.

extensions of 150 lm, the basis of cell body of45 lm, and the diameter of the neck or the antapi-cal horns of 4 lm. The cell body shapes showedslight differences among the specimens, from trian-gular to more rotund contours (Fig. 1, j–m).

Family Dinophysaceae. For the description of thespecimens of the genera Phalacroma and Dinophysis,we follow the classification into sections proposedby Pavillard (1916) and Jørgensen (1923).

Genus Phalacroma: The SSU rDNA sequencesidentified at the species level available in GenBankare limited to Phalacroma rotundatum (Clap. etLachm.) Kof. et J.R. Michener and Phalacroma rapaF. Stein. In addition to several additional sequencesof these species, we obtained new SSU rDNA

sequences for the type, Phalacroma porodictyum F.Stein, and Phalacroma favus Kof. et J.R. Michener,Phalacroma parvulum (F. Schütt) Jørg., Phalacromamitra F. Schütt, and Phalacroma doryphorum F. Stein,with 2–5 sequences for each species. We also deter-mined the sequence of a nonidentified Phalacromaspecies.

Section Euphalacroma Jørg.: This section includedthe genus type as illustrated by Stein (1883) (Fig. 2h).The contour of the P. porodictyum cells collected inour samples was slightly oval (66 lm long, 62 lmwide) with a dome-shaped epitheca clearly projectingover the margin of the upper cingular list (Fig. 2,a–m). The cells were apochlorotic, and the thecashowed regularly scattered pores and two rows of

FIG. 3. Light micrographs of Dinophysis specimens collected for single-cell PCR analysis and other live or Lugol’s-fixed specimens. SeeTable S1 (in the supplementary material) for the collection date, location, and accession numbers of the sequenced specimens. (a) Din-ophysis caudata FG178. (b) Dinophysis tripos FG56. (c–d) Dinophysis hastata FG1432. (e) Other specimen of D. hastata from the same sample.(f) Note the areolation of the empty theca of D. hastata. (g–h) D. hastata f. uracanthides FG499. (i) D. hastata f. uracanthides FG527. (j) Ori-ginal illustration of D. hastata reproduced from Stein (1883). (k–l) First and second illustration of Dinophysis uracantha in Stein (1883).(m) Dinophysis swezyae in Kofoid and Skogsberg (1928). (n) D. hastata f. uracanthides in Jørgensen (1923). (o) Dinophysis balechii in Norrisand Berner (1970). (p) Dinophysis alata in Jørgensen (1923). (q) Dinophysis uracantha var. mediterranea in Jørgensen (1923). (r) D. hastata f.phalacromides in Jørgensen (1923). (s) Dinophysis odiosa in Pavillard (1930). (t) Dinophysis monacantha in Kofoid and Skogsberg (1928). (u)D. uracantha in Jørgensen (1923). (v) Dinophysis pusilla in Jørgensen (1923). (w) Dinophysis schuettii (smaller form) in Jørgensen (1923).(x) Dinophysis acutissima in Gaarder (1954). (y) Dinophysis reticulata in Gaarder (1954). (z–aa) D. hastata f. phalacromides FG1170. (ab) Din-ophysis odiosa FG176. (ac–ad) D. odiosa FG1429. (ae) Another specimen of D. odiosa. (af–ag) Dinophysis monacantha FG1414. (ah) Ethanol-fixed specimen of D. pusilla FG497. (ai) Ethanol-fixed specimen of D. pusilla FG497. (aj) Live specimen of D. cf. pusilla FG524. (ak) Lu-gol’s-fixed specimen from the NW Pacific Ocean with morphology between D. pusilla and D. balechii. (al) D. uracantha var. mediterranea.The inset shows the two lateral ribs in the antapical spine. (am) D. uracantha. (an) D. schuettii. (ao) Ethanol-fixed specimen of Dinophysiscf. acutissima FG523. (ap) Live specimen of D. acutissima. Scale bars, 20 lm.


pores along the cingulum (Fig. 2l). The list betweenthe first and second ribs was covered with an irregularreticulum, while the region between the second andthird ribs was smooth. In the ventral side of the hy-potheca, there was a linear structure that connectedthe second rib and the cingulum (Fig. 2, a and d).We obtained five SSU rDNA sequences from live andethanol-fixed specimens from the coastal and openMediterranean waters (Table S1).

Unclassified Phalacroma: We were unable to iden-tify one Phalacroma specimen at the species levelbecause information on the left sulcal list was diffi-cult to obtain. The cell was slightly elliptical (46 lmlong, 40 lm wide) with a prominent epitheca ofthe same width as the hypotheca. The generalappearance resembled that of Phalacroma ovumSchütt, but to avoid a possible misnaming, we calledit Phalacroma sp. (Fig. 2n).

Section Podophalacroma Jørg.: Described speciesof this section are characterized by an asymmetricalwedge-shaped hypotheca with a prominent, largeareolation in the theca and a greenish pigmenta-tion. The diagnostic criteria for the species differen-tiation are the shape and size of the hypotheca. Weobserved different species from this section in oursamples. P. mitra specimens (58 lm long, 46 lmwide) had a distinctive broad wedge-shaped hypot-heca. The dorsal side was convex, and the ventralside was more or less straight in the sulcus region,becoming distinctly concave at the posterior end ofthe left sulcal list toward the antapical end. Theepitheca was almost flat with horizontal cingular lists

(Fig. 2, o–r). There is a historical controversy onthe synonymy of P. rapa and P. mitra (Schiller1933), but we observed that P. rapa cells were larger(61 lm long, 83 lm wide) and exhibited a greaterangularity of the ventral margin than thoseof P. mitra when seen in lateral view (Fig. 2, s andt). P. favus (64 lm long, 86 lm wide) differed fromP. rapa principally in the constricted, projectingfingerlike antapex. The length of the third rib ofP. favus was smaller than in P. rapa (Fig. 2, u–w).

Section Urophalacroma Jørg.: We collected cellsbelonging to the type of this section, P. doryphorum,which had a wedge-shaped hypotheca, dome-shapedepitheca, and distinct horizontal cingular list. Theleft sulcal list was well-developed, and the most dis-tinctive character was a nonribbed wide posteriorprojection with a triangular shape (Fig. 2, x–z).

Section Paradinophysis Jørg.: We obtained SSUrDNA sequences of two specimens of P. rotundatumcollected from the same sample. The cell contourwas ellipsoidal, the theca was smooth, and the epit-heca was hardly visible above the cingulum. One ofthe specimens was slightly larger and had a widerhypotheca than the other one (Fig. 2, aa and ab).We also obtained sequences of three specimens ofP. parvulum from open and coastal waters (TableS1). The cells (40 lm long, 33 lm wide) had asmooth theca and showed a regularly round outlinein lateral view. The epitheca was dome-shaped withhorizontal cingular lists (Fig. 2, ac–ag).

Genus Dinophysis: Various sequences of the chlo-roplast-containing species of Dinophysis s.s. were

FIG. 4. Light micrographs ofOrnithocercus, Histioneis, and Oxy-physis collected for single-cell PCRanalysis. See Table S1 (in thesupplementary material) for thecollection date, location, and ac-cession numbers. (a) Ornithocercusmagnificus FG25 (b) Ornithocercusheteroporus FG324. (c) O. heteroporusFG323. (d) O. heteroporus FG507.(e) O. heteroporus FG506. (f) Orni-thocercus quadratus var. quadratusFG1004. (g) O. quadratus var. quad-ratus FG1174. (h) O. quadratus var.schuettii FG1173. (i) H. cymbalariaFG325. (j) Histioneis longicollisFG1167. (k) H. longicollis FG1168.(l) Histioneis gubernans FG26. (m)Another specimen of H. gubernans.(n) Oxyphysis oxytoxoides FG278.(o) Lugol’s-fixed specimen of anundescribed dinophysoid fromthe Pacific Ocean. Note the elon-gate epitheca. Scale bars, 20 lm.


available in GenBank. However, no complete SSUrDNA sequence of any apochlorotic species ofDinophysis was available. In addition to several newsequences of members of Dinophysis s. s., we deter-mined SSU rDNA sequences of several apochlo-rotic species, including different morphotypes ofD. hastata, as well as Dinophysis odiosa (Pavill.) L. S.Tai et Skogsb., Dinophysis monacantha Kof. etSkogsb., Dinophysis pusilla Jørg., and Dinophysis cf.acutissima Gaarder.

Section Homoculus Pavill.: We determined thecomplete SSU rDNA of Dinophysis tripos Gourret.The specimen used was collected from its type local-ity, the Bay of Marseille (Fig. 3a). In addition, weobtained an additional sequence for Dinophysiscaudata Kent (Fig. 3b).

Section Hastata Pavill.: The species of this sectionare apochlorotic and characterized by antapicalspines. We illustrated the single-cell sequenced spec-imens and other specimens to show the differencesamong the species, and we reproduced the originalillustrations of the species and varieties related toD. hastata found in Stein (1883), Jørgensen (1923),Kofoid and Skogsberg (1928), Pavillard (1930),Gaarder (1954), and Norris and Berner (1970) toprovide a reference for the accuracy of our speci-men identifications (Fig. 3, j–y). Due to the histori-cal controversy about the synonymy and supposedintraspecific variability of some species of the sec-tion Hastata, especially the type D. hastata, weextend the description of this section below.

The description of the first member of the sec-tion Hastata, D. hastata, is credited to Stein (1883,pl. 19, fig. 12), who provided a single illustration ofD. hastata (Fig. 3j). Since then, and although therecords under the name D. hastata were numerous,specimens with the same hypotheca contour as inStein’s original description were never observed. Allauthors citing D. hastata assumed that the hypothecaof D. hastata was rounder than the Stein’s specimen.Stein (1883) also described Dinophysis uracantha F.Stein, another Dinophysis species with an antapicalspine. However, under the name D. uracantha, Stein(1883, pl. 20, figs. 21 and 22) provided two illustra-tions that unequivocally corresponded to two differ-ent species based on current morphological criteria(Fig. 3, k and l). The first illustration of D. uracantha(Fig. 3k) was very similar to specimens of D. uracanthaobserved in this study (59 lm length, 49 lm wide)(Fig. 3am), close to Dinophysis swezyae Kof. et Skogsb.(Fig. 3m). The second illustration of D. uracantha(Fig. 3l) showed a specimen with a larger and ovatehypotheca, a very large antapical spine, and a longthird rib extending below the basis of the epitheca.This Stein’s second illustration of D. uracantha hasbeen considered as synonym of D. hastata (Abé1967b). Both Stein’s illustrations of D. uracantha alsodiffered in the reticulation, which was coarser for theillustration used to consider D. uracantha as synonymof D. hastata. Jørgensen (1923) described several

forms of D. hastata and D. uracantha from the openMediterranean Sea. Kofoid and Skogsberg (1928)described two species with antapical spines, D. mona-cantha (Fig. 3t) and D. urceolus Kof. et Skogsb., whichwere further considered as synonyms of D. hastata(Abé 1967b). Kofoid and Skogsberg (1928) proposedthe existence of a high intraspecific variation inD. hastata. Pavillard (1930) remarked the validity ofD. odiosa (Fig. 3s) that was synonymized with D. hasta-ta (Kofoid and Skogsberg 1928, Taylor 1976). Norrisand Berner (1970) described Dinophysis balechii D. R.Norris et L. D. Berner (Fig. 3o) previously consideredone of the small forms of D. hastata.

To clarify the phylogenetic position of D. hastataand other members of this section, we sampledspecimens of the section Hastata that we classifiedinto two different subsections according to theirmorphology: specimens with a Dinophysis-like epit-heca and funnel were considered as ‘‘uracantho-ides,’’ and specimens with flat epitheca andcingular list with a Phalacroma-like cingular list wereincluded in the subgroup ‘‘phalacromoides.’’

Subsection uracanthoides: This group containedthe type of the section, D. hastata. The specimens ofthis group have a morphology intermediate betweenthe original description of D. hastata (Fig. 3j) andthe second of Stein’s illustration of D. uracantha(Fig. 3l). We suspect that Stein might have exces-sively elongated the cell body of D. hastata in his draw-ing. The members of uracanthoides were characterizedby an elliptical cell body and a small epitheca with afunnel-shaped cingular list as in typical Dinophysis s.s.The third rib of the left sulcal list emerged from thelower half of the hypotheca. We obtained SSU rDNAsequences for two morphotypes of this kind. Thefirst morphotype was observed in live specimens thatwere 64 lm long and 51 lm wide (Fig. 3, c–f), with adorsoventral diameter at the base of the funnel(upper girdle list) of 27 lm. Our specimens stronglyresembled the original illustration of D. hastatafor the antapical spine and coarse left sulcal reticu-lation (Fig. 3f), and, therefore, we ascribed them toD. hastata (Fig. 3, c–f). The second morphotyperesembled the description of D. hastata f. uracanthidesJørg. described by Jørgensen (1923) from the Medi-terranean Sea (Fig. 3n), for which D. hastataf. uracanthides was smaller than D. hastata and had anarrower and more ventrally deflected antapicalspine. In contrast to the specimens of D. hastata, thethird rib in the D. hastata f. uracanthides specimensthat we observed did not reach the level of the basisof the hypotheca (Fig. 3, g–i). We collected two speci-mens with these characteristics from the same stationin the Levantine Basin, which were ethanol-fixedfor posterior SSU rDNA amplification and sequen-cing (Table S1). Their cell body was ellipsoidal(46 lm long, 34 lm wide), and the dorsoventraldiameter of the base of the ‘‘funnel’’ (upper girdlelist) was 21 lm (Fig. 3, g–i). Other members of thissubsection are D. balechii (Fig. 3o), D. uracantha var.


mediterranea Jørg. (Fig. 3, q and al), Dinophysis alataJørg. (Fig. 3p), and Dinophysis spinosa Rampi.

Subsection phalacromoides: The members ofphalacromoides are characterized by a flat and widerepitheca. The upper cingular list slightly extendedover the epitheca. The funnel-shaped upper cingu-lar list of previous specimens was lacking, resem-bling that of Phalacroma. Species of this subgroupinclude D. odiosa, first described as Phalacromaodiosum Pavill. As a general trend, the specimens ofphalacromoides were larger and the hypotheca wasmore ovate that in members of uracanthoides. Thethird rib of the sulcal list emerged from the middleof the ventral side of the hypotheca and did notextend beyond the basis of the epitheca. The ant-apical spine was always ventrally deflected. We iden-tified three morphotypes having thesemorphological characteristics, those correspondingto D. odiosa, D. hastata f. phalacromides Jørg., andD. monacantha.

D. odiosa is one of the most common species ofthe section Hastata in the coastal MediterraneanSea. However, it is rarely cited in the MediterraneanSea (Gómez 2003), very likely because it is incor-rectly reported as D. hastata. Its cell body was some-what truncate anteriorly, fairly narrowly roundedposteriorly (Fig. 3, ab–ae), while the cell contour ofD. hastata was ovoid (Fig. 3, c–f). The epitheca of D.odiosa was flat, wider than the greatest height ofepitheca. The anterior cingular list showed numer-ous radial ribs. The third rib was longer and moreposteriorly deflected in D. hastata than in D. odiosa(Fig. 3, ab–ae). We obtained sequences from twospecimens of D. odiosa from different locations(Marseille and Villefranche sur Mer). They were75 lm long and 59 lm wide, with a dorsoventraldiameter of the base of the upper cingular list of50 lm (Fig. 3, ab–ad). The second morphotype cor-responded to Dinophysis hastata f. phalacromides Jørg.described from the Mediterranean Sea by Jørgensen(1923) (Fig. 3r). We collected a live specimen thatwas the largest observed for this section (79 lmlong, 62 lm wide, with a dorsoventral diameter ofthe base of the upper cingular list of 41 lm; Fig. 3,z and aa). The third morphotype of this subsectioncorresponded to D. monacantha (Fig. 3t). The lackof citations of this species is likely due to the factthat it has been considered a synonym of D. hastata(Abé 1967b) and consequently pooled as D. hastata.D. monacantha was the smallest species observed forthis subsection (67 lm long, 50 lm wide, base ofthe upper cingular list, 38 lm in diameter) and itsgeneral morphology resembled that of a smallD. odiosa with a less flat and wider epitheca (Fig. 3,af and ag).

Subsection Pusilla: The members of this groupcontained the smallest species of the sectionHastata. Jørgensen (1923) described D. pusilla as asmall species with rotund hypotheca (28 lm wide),prominent funnel-shaped upper cingular list, and a

well-developed left sulcal list (Fig. 3v). The antapicalspine with a prominent rib was slightly ventrallydeflected and emerged from the posterior-ventralregion of the hypotheca. We obtained SSU rDNAsequences from two ethanol-fixed specimens col-lected in the open Mediterranean Sea. Both were30 lm wide, with a dorsal-ventral diameter of thefunnel base of 14 lm. One of the specimens showeda rotund hypotheca, with a straight third rib(Fig. 3ah). This morphology unequivocally corre-sponded to D. pusilla. The second specimen wasslightly larger, with an ovate contour of the epithecaand the third rib of the sulcal list deflected posteri-orly. A single prominent rib was projected anteriorlyfrom the cingulum (Fig. 3ai). We also provided theillustration of a live specimen (28 lm long, 26 lmwide, diameter of funnel base 8 lm) (Fig. 3aj) andof a Lugol’s-fixed specimen from the Pacific Oceanwith a morphology that was intermediate betweenD. pusilla (Fig. 3v) and D. balechii (Fig. 3o). Weascribed the species Dinophysis schuettii G. Murray etWhitting (Fig. 3, w and an) to this group.

Subsection acutissima: Gaarder (1954) describedtwo species, D. acutissima (Fig. 3x) and Dinophysisreticulata Gaarder (Fig. 3y), characterized by anelongated hypotheca with a pronounced antapexthat resembled the morphology of Dinophysisdiegensis Kof. The distinctive character of this specieswas a short antapical spine. Species with these char-acteristics were not included in the sections estab-lished by Pavillard (1916) and Jørgensen (1923).Norris and Berner (1970) reported D. reticulataamong the members of the D. hastata group, butthey did not mention D. acutissima. To the best ofour knowledge, the only record after the firstdescription corresponded to Nguyen et al. (2008).These authors placed D. acutissima in the groupdoryphorum with P. doryphorum as type. We disagreewith this view, and we preferred to place D. acutiss-ima as a member of the section Hastata. We illus-trated a live specimen of D. acutissima (60 lm long,38 wide, diameter of funnel base 28 lm) (Fig. 3ap).As far as we know, this was the first record for theMediterranean Sea (Gómez 2003). We obtained theSSU rDNA sequence from one ethanol-fixed speci-men from the open Ionian Sea (Table S1). This eth-anol-fixed specimen of D. acutissima was 60 lm longand 40 lm wide, and the dorsal-ventral diameter ofthe funnel base was of 24 lm (Fig. 3ao).

Genus Ornithocercus: This ornamented hetero-trophic genus is characterized by a highly developedcingular chamber formed by the cingular list, whichharbors unicellular cyanobacteria. The left sulcal listof these species is also highly developed, with ribsor keels that emerged from the hypotheca. Weobtained the SSU rDNA sequence of Ornithocercusmagnificus F. Stein, the type species collected fromthe type locality, the NW Mediterranean Sea(Fig. 4a). We also obtained sequences of four speci-mens of Ornithocercus heteroporus Kof. from live


(Fig. 4, b and c; Table S1) and ethanol-fixed speci-mens from different locations of the MediterraneanSea (Fig. 4, d and e; Table S1) and of specimens oftwo varieties of Ornithocercus quadratus var. quadratusKof. et Skogsb. (Fig. 4, f and g) and var. schuettiiKof. et Skogsb. (Fig. 4h).

Genus Histioneis: Only one complete SSU rDNAsequence of Histioneis was available in GenBank,derived from a specimen that was not identified tothe species level, illustrated in dorsoventral view inthe original publication (Handy et al. 2009), whichmade difficult its posterior identification. We deter-mined four SSU rDNA sequences of three speciesidentified at the species level. The type of Histioneis,Histioneis remora F. Stein, has been very scarcelyrecorded, and the few existing records are doubtful.Stein’s illustration did not allow defining the typespecies. Stein (1883) also provided under the nameHistioneis cymbalaria F. Stein three illustrations thatunequivocally corresponded to three separate spe-cies. We obtained the sequence of a specimenstrongly resembling one of the Stein’s illustrationsof H. cymbalaria. Hence, although this species hasbeen named either Histioneis depressa J. Schiller byTaylor (1976) or H. cymbalaria by Balech (1988),our specimen has been ascribed to H. cymbalaria fol-lowing Gómez (2007) (Fig. 4i). We sequenced SSUrDNAs from two specimens of Histioneis longicollisKof. collected from the Bay of Villefranche sur Meron two consecutive days (Fig. 4, j and k). Both speci-mens were identical in size (83 lm maximumlength, and the width of the hypotheca was 28 lm)with a peculiar yellow-greenish brightness of the sul-cal list, although they slightly differed in internalornamentation of the left sulcal list. The morphol-ogy corresponded to Histioneis sublongicollis Halimdescribed from the Bay of Villefranche. FollowingGómez (2007) H. sublongicollis was considered asynonym of H. longicollis. The other sequencedspecimen belonged to the Histioneis gubernansgroup (Gómez 2007), closely related to Histioneisstriata Kof. et J. R. Michener, and was ascribed toH. gubernans F. Schütt (Fig. 4l). Another live speci-men of H. gubernans is illustrated for comparison(Fig. 4m).

Family Oxyphysaceae. O. oxytoxoides is the onlymember of this family. No specimen was observedin the coastal or open Mediterranean Sea. Weobtained the SSU rDNA sequence from a specimen(58 lm long, 20 lm wide) collected in the brackishwaters of the Thau lagoon at Sète, south of France(Fig. 4n). In addition to Oxyphysis, a nondescribedspecies from the Pacific Ocean with an elongatedepitheca is shown (Fig. 4o).

Molecular phylogeny. We constructed ML treesfrom a global alignment of dinoflagellate SSU rDNAsequences using other alveolates as outgroup. Allthe sequences of representative dinophysoid dinofla-gellates emerged within a strongly supported (BV of94%) monophyletic clade (Fig. 5). This clade

branched within a large dinoflagellate group com-posed of taxa of the orders Gymnodiniales, Peridini-ales, and Prorocentrales. However, this relationshipwas poorly supported (BV of 62%). Within thisgroup, symmetric species of Prorocentrum Ehrenb.(Prorocentrum lima Ehrenb., Prorocentrum concavumFukuyo, and P. levis M. A. Faust, Kibler, Vandersea,P. A. Tester et Litaker) branched as sister group ofthe dinophysoid clade, although without support(BV

markers with different levels of conservation, stress-ing its reliability.

Internal SSU rDNA phylogeny of the three majordinophysoid groups. Amphisoleniaceae: The species ofAmphisolenia and Triposolenia formed two highly sup-ported groups (BV of 86% and 100%, respectively).However, the relative branching order of the differ-ent Amphisolenia species was weakly supported,although the tree suggested that the two morpho-logically closely related species A. globifera andA. schauinslandii were sister (BV of 68%), withA. bidentata branching in a basal position (Fig. 6).

Phalacroma: Although several nodes within thePhalacroma clade were not well resolved, itappeared that this clade was subdivided into twosubclades: one for the type, P. porodictyum, andP. favus, P. rotundatum, P. doryphorum, and Oxyphysis(BV of 81%); and a second one containing

P. parvulum, P. mitra, and P. rapa (BV of 55%). Wecalled the former Phalacroma subclade because itcontained the type, and the second Rapa becauseP. rapa was the first described species in that subc-lade (Fig. 6).

The classical subdivision into sections representedby two or more species as Paradinophysis (P. rotunda-tum, P. parvulum) and Podophalacroma (P. mitra,P. rapa, P. favus) is not supported by the moleculardata, since species of same section appeared in dif-ferent subclades of Phalacroma. In fact, P. rotundatumbranched in the subclade Phalacroma, close toP. porodictyum and O. oxytoxoides with relatively goodsupport (BV of 86%), whereas P. parvulum branchedin the subclade Rapa. P. mitra and P. rapa were sis-ters in the subclade Rapa (BV of 97%). Surprisingly,despite the strong morphological resemblance,P. favus is distantly related to other members of

FIG. 5. Maximum-likelihoodphylogenetic tree of dinoflagel-late SSU rDNA sequences, basedon 1,166 aligned positions. Namesin bold represent sequencesobtained in this study. Numbers atnodes are bootstrap values (values

Podophalacroma, and it branched closely related toP. porodictyum in the Phalacroma subclade.

Dinophysaceae: Our phylogenetic analysis did notprovide a robust resolution of all genera in theDinophysaceae clade, which showed large variationsof evolutionary rate, as depicted by the extremedifferences of branch lengths (Fig. 6). Ornithocercusand Histioneis had short branches and emerged in abasal position with respect to Dinophysis s.s.,although with moderate support (BV of 68%). Allthe Ornithocercus sequences were almost completelyidentical, with the exception of O. magnificus FTL83(Handy et al. 2009), probably because of a fewsequence errors. Sequences of the two O. quadratusvarieties (var. schuettii and var. quadratus) were iden-

tical. This extreme conservation of the SSU rDNAwithin the genus Ornithocercus made this markerinappropriate to resolve the relationships amongthe corresponding species.

The sequences of Histioneis formed a group thatappeared to be more closely related to Ornithocercusthan to Dinophysis. Histioneis cymbalaria emerged atthe base of H. longicollis and H. gubernans (BV of67%). However, the poor internal resolution for thisgenus made it premature to draw conclusions aboutthe Histioneis systematics only on the basis of theSSU rDNA phylogeny.

The chloroplast-containing species of Dinophysis,the so-called Dinophysis s.s. that contained the typespecies, formed a well-supported monophyletic

FIG. 6. Maximum-likelihood phylogenetic tree of Dinophysales SSU rDNA sequences, based on 1,166 aligned positions. Names in boldrepresent sequences obtained in this study. Numbers at nodes are bootstrap values (values

clade. The other species of Dinophysis belonging tothe section Hastata, apochlorotic with an antapicalspine, branched in a basal position to the membersof Dinophysis s.s. Our molecular phylogeny stronglysupports D. odiosa and D. monacantha as separatespecies, given the large divergence between theirsequences (Fig. 6). Likewise, the sequences of thetwo forms of D. hastata, f. phalacromides and f. ura-canthides, were very divergent from that of D. hasta-ta, so that these forms also deserve to beconsidered as two separate species. The species ofthe section Hastata were relatively distant fromDinophysis s.s. in all the SSU rDNA phylogenies.However, the internal relationships between themembers of this section were unstable and poorlysupported in different molecular phylogenies. Thistrend is probably due, at least partly, to theextreme differences of evolutionary rate in thesespecies, with short-branching ones such as D. pusillaand D. cf. acutissima, and very long-branching onessuch as D. monacantha and D. odiosa. D. hastata f.uracanthides branched as sister of the Dinophysis s.s.,making the Hastata paraphyletic (Fig. 6). Neverthe-less, this finding was weakly supported (BV of62%), and we cannot exclude that the paraphyly ofthe Hastata reflected a long-branch attraction arti-fact due to the long branches of certain members.The only well-supported subgroup is composed ofD. odiosa, D. monacantha, D. pusilla, Dinophysis cf.acutissima, and D. hastata f. phalacromides (BV of97%) (Fig. 6). Within this group, there was a veryshort distance between Dinophysis cf. acutissima, withan elongated and antapically pointed hypotheca,and D. pusilla. This relationship suggested that theshape of the hypotheca is a morphological charac-ter highly variable among very closely related spe-cies and, consequently, of relatively small value as aphylogenetic marker of the species.

Nomenclatural considerations. The placement of O.oxytoxoides closely related to the type species of Phalac-roma in the SSU and LSU rDNA phylogenies supportsthe transfer of O. oxytoxoides into Phalacroma.

Phalacroma oxytoxoides (Kof.) F. Gómez, P. López-Garcı́a et D. Moreira, comb. nov.

Basionym: Oxyphysis oxytoxoides Kof. (Kofoid 1926,p. 205, pl. 18).

The type of Dinophysis, Dinophysis acuta Ehrenb.,formed a well-defined clade within the Dinophysiss.s. with other chloroplast-containing species of Din-ophysis. In contrast, the sequences of D. hastata f.phalacromides and D. hastata f. uracanthides, as wellas those of other apochlorotic members of the Din-ophysis hastata group, branched very distantly fromthe type of Dinophysis, which might support theirinclusion in different genera. However, the even-tual split of Dinophysis and the erection of a newgenus for the members of the D. hastata group arepremature. Additional information from othermolecular markers would be required to obtainmore robust phylogenies that would eventually vali-date this claim. Nevertheless, the molecular dataclearly demonstrated that the assumed high intra-specific variability of D. hastata hid a number ofcryptic species. In fact, species such as D. odiosa orD. monacantha, previously considered as synonymsof D. hastata (Abé 1967b, Taylor 1976), appearedto be distant species on the basis of their high SSUrDNA sequence divergence (Fig. 6). Likewise, ourmolecular phylogenetic analysis showed that thetwo forms D. hastata f. phalacromides and D. hastataf. uracanthides described by Jørgensen (1923) docorrespond to separate phylogenetic species. Conse-quently, we propose to erect these forms at the spe-cies level as follows:

Dinophysis phalacromoides (Jørg.) F. Gómez, P.López-Garcı́a et D. Moreira, comb. nov.

Basionym: Dinophysis hastata F. Stein f. phalacro-mides Jørg. (Jørgensen 1923, pp. 30–1, fig. 41).

Dinophysis uracanthoides (Jørg.) F. Gómez, P.López-Garcı́a et D. Moreira, comb. nov.

Basionym: Dinophysis hastata F. Stein f. uracanthidesJørg. (Jørgensen 1923, pp. 30–1, fig. 40).

Reconciling morphological and molecular data. Thedetailed studies on the tabulation carried out by Taiand Skogsberg (1934), Abé (1967a,b,c), Balech(1967, 1988), and Norris and Berner (1970) showedthat the plate arrangement and number are moreor less similar in all dinophysoid species and genera,even at the level of the small sulcal plates. Abé(1967a) observed some differences in the tabulationfor the genera Amphisolenia, Triposolenia, and Oxyphy-sis when compared with the other dinophysoids. Allauthors agreed on the establishment of the familyAmphisoleniaceae for Amphisolenia and Triposolenia(Abé 1967a, Balech 1977, 1980). Our molecular phy-logenetic analysis including SSU rDNA sequences ofTriposolenia and the type of Amphisolenia, whichappear as monophyletic and occupy a basal position

FIG. 7. Maximum-likelihood phylogenetic tree of DinophysalesLSU and SSU rDNA sequences, based on 1,980 aligned positions.Numbers at nodes are bootstrap values (values

within the Dinophysales, supports their considerationas a separate family.

Abé (1967a, p. 378) reported that ‘‘comparingOxyphysis with Amphisolenia, one will notice that theventral area of Oxyphysis, covered largely by the pos-terior sulcal plate, coincides with the same plate ofAmphisolenia, not only in its shape but also in thestructural relationships to the fission suture and thelonger right ventral hypothecal plate. This findingsuggests clearly a close phylogenetic affinity betweenthem.’’ Oxyphysis was thus placed in Amphisolenia-ceae (Loeblich 1982, Taylor 1987). Nevertheless,since Sournia (1984) established the family Oxy-physaceae, the most extended practice in recentclassifications has been to consider O. oxytoxoides asthe only member of its own family (Steidinger andTangen 1997, Hastrup Jensen and Daugbjerg 2009).However, Balech (1988, p. 201) observed that theplate arrangement of Oxyphysis appeared to be closeto that of Dinophysis (he considered Phalacroma as asynonym) and placed it within the family Dinophys-aceae. Our molecular data support Balech’s view. Amorphological intermediate between Phalacromaand Oxyphysis might be represented by a dinophy-soid specimen with an elongate epitheca from theGulf of Mexico illustrated by Balech (1967, pp. 92–3, figs. 32–37). We provide the first micrograph ofthat nondescribed species from Lugol’s-fixed mate-rial collected the tropical western Pacific Ocean(Fig. 4o).

O. oxytoxoides as well as other Phalacroma species,such as P. rotundatum, feed on ciliates by suckingthe prey cytoplasm by myzocytosis (Hansen 1991,Inoue et al. 1993). Oxyphysis seems to be specializedon tintinnid preys with elongated loricae. Inoueet al. (1993) observed that the distinctive elongatedepitheca of Oxyphysis intruded into the lorica of theciliate preys. It can be speculated that Oxyphysis hasrecently diverged morphologically to adapt to itsparticular prey. The change in its general appear-ance would be associated with a change in the dis-position of the plates when compared with theother Phalacroma species. The elongation of the cellbody would also imply a modification in the disposi-tion and shape of the thecal plates that has classi-cally been used to support the placement in aseparate family.

A morphological character commonly used forthe generic separation in dinophysoid dinoflagel-lates is the height of the epitheca in Dinophysis andPhalacroma (Kofoid and Skogsberg 1928, Hallegraeffand Lucas 1988). Recently, Hastrup Jensen andDaugbjerg (2009) used this criterion for the delimi-tation of Phalacroma, which they restricted to cellspossessing large epithecae but 1 ⁄ 4 of the cell length, inthe Phalacroma subclade closely related to the typeof Phalacroma. Consequently, the description ofPhalacroma should be amended. Most Phalacroma

species form a well-supported clade in SSU rDNAphylogenies, supporting the erection of a separatefamily for the Phalacroma clade using either thename Oxyphysaceae, since Oxyphysis is a synonym ofPhalacroma, or new family name, tentatively ‘‘Phala-cromaceae.’’ However, this change would need aclear definition of diagnostic characters for themembers of this putative separate family. In addi-tion to difficulties in the classification into familiesand genera, even the classification into sectors isnot supported by the molecular data. The membersof Urophalacroma (P. mitra, P. rapa, and P. favus) aremorphologically similar species (Fig. 2, o–w), but,surprisingly, P. favus branched in a subclade differ-ent from P. mitra and P. rapa (Fig. 6). This exempli-fies the difficulties to find stable morphologicalcharacters for the establishment of families, genera,or even sections within the dinophysoid dinoflagel-lates.

This is a contribution to the project DIVERPLAN-MED sup-ported by a postdoctoral grant to F. G. of the Ministerio Espa-ñol de Educación y Ciencia #2007-0213. We acknowledgefinancial support from the French CNRS and the ANR Biodi-versity program (ANR BDIV 07 004-02 ‘Aquaparadox’). Wethank A. Nowaczyk for the BOUM samples and P. C. Silva forhis comments on nomenclature.

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Supplementary material

The following supplementary material is avail-able for this article:

Appendix S1. A note on the spelling of thesupergeneric names derived from Dinophysisauthored by P. C. Silva.

Figure S1. Maximum likelihood phylogenetictree of Dinophysales LSU rDNA sequences, basedon 862 aligned positions.

Table S1. List of new SSU rDNA sequences ofDinophysales used for the phylogenetic analysis.Accession numbers, geographic origin, and col-lection dates are provided.

This material is available as part of the onlinearticle.

Please note: Wiley-Blackwell are not responsi-ble for the content or functionality of any supple-mentary materials supplied by the authors. Anyqueries (other than missing material) should bedirected to the corresponding author for thearticle.


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