Fernando Gómez 2. Purificación López-García and David Moreira

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1 J. Phycol. 47, (2011) Ó 2011 Phycological Society of America DOI: /j x MOLECULAR PHYLOGENY OF DINOPHYSOID DINOFLAGELLATES: THE SYSTEMATIC POSITION OF OXYPHYSIS OXYTOXOIDES AND THE DINOPHYSIS HASTATA GROUP (DINOPHYSALES, DINOPHYCEAE) 1 Fernando Gómez 2 Université Lille Nord de France, Laboratoire d Océanologie et Géosciences, CNRS UMR 8187, Station Marine de Wimereux, 28 Av. Foch, Wimereux, France Purificación López-García and David Moreira Unité d Ecologie, Systématique et Evolution, CNRS UMR 8079, Université Paris-Sud, Bâtiment 360, Orsay Cedex, France The dinophysoid dinoflagellates are currently divided into three families: Amphisoleniaceae, Dinophysaceae (mainly Dinophysis Ehrenb. and Phalacroma F. Stein), and Oxyphysaceae, the latter including only one member, Oxyphysis oxytoxoides Kof. Phalacroma has been recently reinstated separately from Dinophysis, and its amended description is currently restricted to cells whose epithecae were large but <1 4 of the cell length. With the aim of improving the phylogeny of Dinophysales, we obtained 54 new SSU rrna gene sequences of 28 species. Taxon-rich SSU rdna phylogenetic analysis showed that Dinophysales split into two major clades, one containing the Amphisoleniaceae (Amphisolenia F. Stein Triposolenia Kof.) and the other containing the Dinophysaceae. The latter are divided into two wellsupported sister groups, the Dinophysaceae sensu stricto (s.s.) (Dinophysis, Ornithocercus F. Stein, Histioneis F. Stein) and, tentatively, a separate family for the clade of the type and most of the Phalacroma species. Based on combined phylogenies of new SSU rdna and available LSU rdna data, O. oxytoxoides (elongated epitheca, >1 4 of the cell length) branched with a strong support with the type of Phalacroma. We therefore propose Phalacroma oxytoxoides comb. nov. for O. oxytoxoides. Our SSU rdna phylogeny also suggests that the assumed high intraspecific variability of Dinophysis hastata F. Stein hides a number of cryptic species. According to their distinct phylogenetic placement, the forms D. hastata f. phalacromides Jørg. and D. hastata f. uracanthides Jørg. should be erected at the species level. We propose for them the names Dinophysis phalacromoides comb. nov. and Dinophysis uracanthoides comb. nov. Key index words: Amphisolenia; cryptic species; Dinophysiaceae; Dinophysiales; dinophysioid dinoflagellate; Histioneis; Ornithocercus; Oxyphysiaceae; Phalacroma; SSU and LSU rdna phylogeny 1 Received 21 March Accepted 6 October Author for correspondence: fernando.gomez@fitoplancton. com. Abbreviations: bp, base pairs; BV, bootstrap value; s.s., sensu stricto The dinophysoids (see Appendix S1 in the supplementary material for a note on the spelling of the supergeneric names derived from Dinophysis authored by P. C. Silva) are a well-defined order of marine dinoflagellates with 280 recognized species classified in three families: Amphisoleniaceae, Dinophysaceae, and Oxyphysaceae (Fensome et al. 1993, Steidinger and Tangen 1997, Gómez 2005). The cells are laterally compressed with a reduced epitheca and a larger hypotheca consisting of two large plates united by a sagittal serrate suture with a zigzag course (Kofoid and Skogsberg 1928, Tai and Skogsberg 1934, Abé 1967a,b,c, Balech 1967). According to Balech (1980), the dinophysoids are also unusual among the thecate dinoflagellates in that, despite their extreme morphological specialization, their plate arrangement and number are more or less similar in all species and genera, except for the genera Amphisolenia F. Stein and Citharistes F. Stein. The dinophysoid genus Phalacroma F. Stein was morphologically separated from Dinophysis Ehrenb. based mainly on differences in epithecal elevation (Stein 1883, Kofoid and Skogsberg 1928). Dinophysis species have a reduced epitheca, and their anterior cingular list forms a funnel-shaped fan, whereas Phalacroma species have a visible epitheca above an anterior cingular list that is typically narrow and directed horizontally. However, detailed tabulation studies, especially at the sulcus level, did not show any significant difference between Dinophysis and Phalacroma. For this reason and because of their intergrading morphology, Abé (1967b) and Balech (1967) transferred Phalacroma species into Dinophysis. More recently, and based on molecular data, Handy et al. (2009) and Hastrup Jensen and Daugbjerg (2009) observed a deep separation between species of the Phalacroma and Dinophysis lineages. Accordingly, Hastrup Jensen and Daugbjerg (2009) 393

2 394 FERNANDO GÓMEZ ET AL. reinstated the genus Phalacroma and amended its description, which is currently restricted to cells whose epithecae are large but having <1 4 of the cell length. This definition excluded some Phalacroma species with a prominent epitheca (Phalacroma apicatum Kof. et Skogsb. and Phalacroma cf. argus F. Stein) that branched within the other Dinophysaceae clade (Dinophysis, Ornithocercus, Histioneis, Citharistes). Hastrup Jensen and Daugbjerg (2009) divided the Dinophysales into three major clades: one basal clade for Amphisolenia; another clade for species with the classical Phalacroma outline, including the type species of Phalacroma; and a third clade with a variety of subclades (Ornithocercus and Citharistes, Histioneis, some Phalacroma species, and several Dinophysis clusters). These authors reported that Dinophysis was polyphyletic, as it formed four separate clades. This indicated that besides Dinophysis s.s., Dinophysis should be divided into at least three additional new genera. These molecular phylogenetic studies still supported the division of the Dinophysales into three families: Amphisoleniaceae, Dinophysaceae, and Oxyphysaceae (Hastrup Jensen and Daugbjerg 2009). The latter is restricted to the monotypic genus Oxyphysis Kof., which has a peridinioid appearance, suggesting a possible link between dinophysoids and peridinioids (Kofoid 1926). Nonetheless, based on morphological traits, Abé (1967a) suggested that Oxyphysis might have a common ancestor with the Amphisolenia-Triposolenia line, leaving its phylogenetic relationship unresolved. With the aim of improving the phylogeny of Dinophysales, we obtained 54 new SSU rrna gene sequences of 28 dinophysoid species from several dinophysoid genera, including Amphisolenia, Triposolenia, and Oxyphysis as well as the type of Phalacroma. Their phylogenetic analysis together with that of combined available LSU rdnas showed that Dinophysales split into two major clades, the Amphisoleniaceae (Amphisolenia-Triposolenia) and the Dinophysaceae, the latter including two well-supported groups, the Dinophysaceae s.s. (Dinophysis, Ornithocercus, Histioneis) and Phalacroma species. O. oxytoxoides branched within the Phalacroma, supporting its transfer to this genus. D. hastata F. Stein hides a number of cryptic species, and we propose two of these be renamed D. phalacromoides comb. nov. and D. uracanthoides comb. nov. MATERIALS AND METHODS Sampling and isolation of material. The specimens were collected by slowly filtering surface seawater taken from the end of the pier (depth 3 m) of the Station Marine d Endoume, Marseille ( N, E), from October 2007 to September A strainer with netting of 20, 40, or 60 lm mesh size (Millipore Inc., St. Quentin-Yveline, France) was used to collect the organisms, and the filtered volume varied between 10 and 100 L, according to the concentration of particles. The concentrated sample was examined in Utermöhl chambers at 100 magnification with an inverted microscope (Nikon Eclipse TE200, Nikon Inc., Tokyo, Japan) and was photographed at 200 or 400 magnification with a digital camera (Nikon Coolpix E995). Sampling continued from October 2008 to August 2009 in the surface waters of the port (depth of 2 m) of Banyuls sur Mer, France ( N, E), and from September 2009 to February 2010 in the Bay of Villefranche sur Mer, Ligurian Sea. For the latter location, sampling was performed at a long-term monitoring site called Point B ( N, E; water column depth 80 m) and sporadically at Point C (43 40 N, E; water column depth 600 m). Sampling in double oblique angle was performed with a custom-made conical phytoplankton net (53 lm mesh size, 54 cm diameter and 280 cm length). The samples were prepared with the same procedure described above. The specimens were observed with an inverted microscope (Olympus IX51; Olympus Inc., Tokyo, Japan) and photographed with an Olympus DP71 digital camera. The specimen of O. oxytoxoides was collected by using a strainer with netting of 20 lm aperture from the surface waters of the Étang de Thau at Sète ( N, E) and analyzed following the procedure described above. Étang de Thau is a large semienclosed coastal lagoon on the French Mediterranean coast (75 km 2, mean depth 4.5 m) under the influence of freshwater inputs. In addition, open-water samples were collected from the BOUM (Biogeochemistry from the Oligotrophic to the Ultra-oligotrophic Mediterranean) cruise in the Mediterranean Sea between the Gulf of Lions and Cyprus. Ten liters was collected from the surface with a bucket and filtered by using a strainer of 20 lm netting aperture. The retained material was fixed with absolute ethanol to a final concentration of 50% concentrate seawater sample and 50% ethanol. At the laboratory, the ethanol sample was examined following the procedure described above. In all cases, each specimen was micropipetted individually with a fine capillary into a clean chamber and washed several times in serial drops of 0.2 lm filtered and sterilized seawater (live specimens from coastal waters) or ethanol (ethanol prefixed specimens from open waters). Finally, the specimen was deposited into a 0.2 ml tubes (ABgene; Thermo Fisher Scientific Inc., Courtaboeuf, France) filled with several drops of absolute ethanol. The sample was kept at room temperature and in darkness until the molecular analysis could be performed. PCR amplification of SSU rrna genes and sequencing. The specimens fixed in ethanol were centrifuged (Eppendorf, Hamburg, Germany) gently for 5 min at 504g. Ethanol was then evaporated in a vacuum desiccator and single cells were resuspended directly in 25 ll of Ex TaKaRa buffer (TaKaRa, distributed by Lonza Cia., Levallois-Perret, France). PCRs were performed in a volume of ll reaction mix containing pmol of the eukaryotic-specific SSU rdna primers EK- 42F (5 -CTCAARGAYTAAGCCATGCA-3 ) and EK-1520R (5 - CYGCAGGTTCACCTAC-3 ) (López-García et al. 2001). PCRs were performed under the following conditions: 2 min denaturation at 94 C; 10 cycles of touch-down PCR (denaturation at 94 C for 15 s; a 30 s annealing step at decreasing temperature from 65 C down to 55 C employing a 1 C decrease with each cycle, extension at 72 C for 2 min); 20 additional cycles at 55 C annealing temperature; and a final elongation step of 7 min at 72 C. A nested PCR was then carried out using 2 5 ll of the first PCR products in a GoTaq (Promega, Lyon, France) polymerase reaction mix containing the eukaryotic-specific primers EK-82F (5 -GAAACTGCGAATGGCTC-3 ) and EK- 1498R (5 -CACCTACGGAAACCTTGTTA-3 ) (López-García et al. 2001) and similar PCR conditions as described above. A third, seminested, PCR was carried out using the dinoflagellate specific primer DIN464F (5 -TAACAATACAGGGCATCCAT-3 ) (Gómez et al. 2009) and keeping the reverse primer EK-1498R.

3 MOLECULAR PHYLOGENY OF DINOPHYSALES 395 Negative controls without template DNA were used at all amplification steps. Amplicons of the expected size (1,200 base pairs [bp]) were then sequenced bidirectionally with primers DIN464F and EK-1498R using an automated 96- capillary sequencer ABI PRISM 3730xl (Cogenics, Meylan, France). Phylogenetic analyses. The new SSU rdna sequences were aligned to a large multiple sequence alignment containing 1,100 publicly available complete or nearly complete (>1,300 bp) dinoflagellate sequences using the profile alignment option of MUSCLE 3.7 (Edgar 2004). The resulting alignment was manually inspected using the program ED of the MUST package (Philippe 1993). Ambiguously aligned regions and gaps were excluded in phylogenetic analyses. Preliminary phylogenetic trees with all sequences were constructed using the neighbor-joining method (Saitou and Nei 1987) implemented in the MUST package (Philippe 1993). These trees allowed identifying the closest relatives of our sequences together with a sample of other dinoflagellate species, which were selected to carry out more computationally intensive maximum-likelihood (ML) analyses. These were done with the program TREEFINDER (Jobb et al. 2004) applying a GTR + C + I model of nucleotide substitution, taking into account a proportion of invariable sites and a C-shaped distribution of substitution rates with four rate categories. Bootstrap values (BVs) were calculated using 1,000 pseudoreplicates with the same substitution model. The phylogenetic position of the dinophysoids was analyzed by means of a global alignment of 77 taxa representing sequences of dinoflagellates, including sequences of dinophysoid species, with representatives of the lineages of the Gymnodiniales, Prorocentrales, and Peridiniales. We did not include the environmental dinophysoid sequences of Handy et al. (2009) in our final SSU rdna phylogenetic trees, as they were short and imposed a limitation in the final number of positions to be considered in our analysis. Similarly, from the 26 new SSU rdna sequences of identified cells that these authors obtained, we only included the 11 sequences that were longer than 1,300 bp. To compare the position of O. oxytoxoides in SSU and LSU rdna phylogenies, we retrieved available LSU rdna sequences of dinophysoid dinoflagellates from GenBank using Entrez ( with a taxonomic query. We reconstructed ML phylogenetic trees for the LSU rdna sequences alone or combined with SSU rdna sequences for the same species using TREEFINDER and the same substitution model specifications as for the SSU rdna phylogenetic analysis (see above). Our sequences were deposited in GenBank under accession numbers HM HM (see Table S1 in the supplementary material). RESULTS AND DISCUSSION Species identification. To amplify and sequence SSU rrna genes from key dinophysoid species and carry out phylogenetic analyses, we collected individual cells of a total of 28 dinophysoid species (Table S1). All cells were individually identified, photographed, and collected under the microscope (Figs. 1 4). In the description of the specimens, we follow the classification into three families according to the current taxonomic scheme. Family Amphisoleniaceae. This family is composed of two distinctive genera, Amphisolenia and Triposolenia, characterized by an elongated cell body. We obtained new SSU rdna sequences for several species of the genus Amphisolenia, including the sequence of the type, Amphisolenia globifera F. Stein, as well as FIG. 1. Light micrographs of specimens of Amphisolenia and Triposolenia collected for singlecell PCR analysis. See Table S1 (in the supplementary material) for the collection date, location, and accession numbers. (a b) Amphisolenia globifera FG1401. (c) Amphisolenia schauinslandii FG1163. (d) Amphisolenia sp. FG281. (e f) Amphisolenia bidentata FG279. (g i) Other specimen of Amphisolenia bidentata under epifluorescence microscopy. Note the intracellular coccoid symbionts. (j) Triposolenia bicornis 2 FG1153. (k) T. bicornis 4 FG1155. (l) T. bicornis 6 FG1157. (m) T. bicornis 8 FG1159. Scale bars, 20 lm.

4 396 FERNANDO GÓMEZ ET AL. FIG. 2. Light micrographs of Phalacroma. See Table S1 (in the supplementary material) for the collection date, location, and accession numbers 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 of P. porodictyum FG1193b. (j) Note the pores in the theca. (l) The arrows indicate the two rows of pores along the cingulum. (n) Phalacroma sp. 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 doryphorum FG365. (y) P. doryphorum FG641. (z) P. doryphorum FG509. (aa) Phalacroma rotundatum FG366. (ab) P. rotundatum FG365. (ac ad) Phalacroma parvulum FG503. (ae af) P. parvulum FG505. (ag) P. parvulum FG326. Scale bars, 20 lm. for the genus Triposolenia. A. globifera, one of the smaller species of the genus, is characterized by a swelling of the antapical end. The specimen studied (207 lm long, 12 lm wide) showed a swelling of 8 lm in diameter with two small spines (Fig. 1, a and b). Amphisolenia schauinslandii Lemmerm., closely related to the type, was larger and showed an inflated midbody (390 lm long, 35 lm wide) (Fig. 1c). Another specimen (335 lm long, 19 lm wide; Fig. 1d) resembled Amphisolenia complanata Kof. et Skogsb., but it might represent an incompletely developed specimen and was therefore more difficult to identify. To avoid misnaming, we generically named it Amphisolenia sp. The largest Amphisolenia specimen (765 lm long, 21 lm wide) contained green granules in the central body and was identified as Amphisolenia bidentata Schröd. (Fig. 1, e and f). In other similar specimens, these green granules appeared to contain chl a, as seen under epifluorescence microscopy (Fig. 1, g i). While Amphisolenia, especially A. bidentata, was common in surface waters, the specimens of Triposolenia were preferentially distributed in deep waters. We obtained identical SSU rdna sequences from four live specimens of Triposolenia bicornis Kof. from the same sample collected at 400 m depth. The dimensions of the four cells were identical, with a length from the apex to the tip of the antapical

5 MOLECULAR PHYLOGENY OF DINOPHYSALES 397 FIG. 3. Light micrographs of Dinophysis specimens collected for single-cell PCR analysis and other live or Lugol s-fixed specimens. See Table S1 (in the supplementary material) for the collection date, location, and accession numbers of the sequenced specimens. (a) Dinophysis 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) Original 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 Norris and 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) Dinophysis odiosa FG176. (ac ad) D. odiosa FG1429. (ae) Another specimen of D. odiosa. (af ag) Dinophysis monacantha FG1414. (ah) Ethanolfixed specimen of D. pusilla FG497. (ai) Ethanol-fixed specimen of D. pusilla FG497. (aj) Live specimen of D. cf. pusilla FG524. (ak) Lugol 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 Dinophysis cf. acutissima FG523. (ap) Live specimen of D. acutissima. Scale bars, 20 lm. extensions of 150 lm, the basis of cell body of 45 lm, and the diameter of the neck or the antapical horns of 4 lm. The cell body shapes showed slight differences among the specimens, from triangular to more rotund contours (Fig. 1, j m). Family Dinophysaceae. For the description of the specimens of the genera Phalacroma and Dinophysis, we follow the classification into sections proposed by Pavillard (1916) and Jørgensen (1923). Genus Phalacroma: The SSU rdna sequences identified at the species level available in GenBank are limited to Phalacroma rotundatum (Clap. et Lachm.) Kof. et J.R. Michener and Phalacroma rapa F. Stein. In addition to several additional sequences of 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., Phalacroma mitra F. Schütt, and Phalacroma doryphorum F. Stein, with 2 5 sequences for each species. We also determined the sequence of a nonidentified Phalacroma species. Section Euphalacroma Jørg.: This section included the genus type as illustrated by Stein (1883) (Fig. 2h). The contour of the P. porodictyum cells collected in our samples was slightly oval (66 lm long, 62 lm wide) with a dome-shaped epitheca clearly projecting over the margin of the upper cingular list (Fig. 2, a m). The cells were apochlorotic, and the theca showed regularly scattered pores and two rows of

6 398 FERNANDO GÓMEZ ET AL. FIG. 4. Light micrographs of Ornithocercus, Histioneis, and Oxyphysis collected for single-cell PCR analysis. See Table S1 (in the supplementary material) for the collection date, location, and accession numbers. (a) Ornithocercus magnificus FG25 (b) Ornithocercus heteroporus FG324. (c) O. heteroporus FG323. (d) O. heteroporus FG507. (e) O. heteroporus FG506. (f) Ornithocercus quadratus var. quadratus FG1004. (g) O. quadratus var. quadratus FG1174. (h) O. quadratus var. schuettii FG1173. (i) H. cymbalaria FG325. (j) Histioneis longicollis FG1167. (k) H. longicollis FG1168. (l) Histioneis gubernans FG26. (m) Another specimen of H. gubernans. (n) Oxyphysis oxytoxoides FG278. (o) Lugol s-fixed specimen of an undescribed dinophysoid from the Pacific Ocean. Note the elongate epitheca. Scale bars, 20 lm. pores along the cingulum (Fig. 2l). The list between the first and second ribs was covered with an irregular reticulum, while the region between the second and third ribs was smooth. In the ventral side of the hypotheca, there was a linear structure that connected the second rib and the cingulum (Fig. 2, a and d). We obtained five SSU rdna sequences from live and ethanol-fixed specimens from the coastal and open Mediterranean waters (Table S1). Unclassified Phalacroma: We were unable to identify one Phalacroma specimen at the species level because information on the left sulcal list was difficult to obtain. The cell was slightly elliptical (46 lm long, 40 lm wide) with a prominent epitheca of the same width as the hypotheca. The general appearance resembled that of Phalacroma ovum Schütt, but to avoid a possible misnaming, we called it Phalacroma sp. (Fig. 2n). Section Podophalacroma Jørg.: Described species of this section are characterized by an asymmetrical wedge-shaped hypotheca with a prominent, large areolation in the theca and a greenish pigmentation. The diagnostic criteria for the species differentiation are the shape and size of the hypotheca. We observed different species from this section in our samples. P. mitra specimens (58 lm long, 46 lm wide) had a distinctive broad wedge-shaped hypotheca. The dorsal side was convex, and the ventral side was more or less straight in the sulcus region, becoming distinctly concave at the posterior end of the left sulcal list toward the antapical end. The epitheca was almost flat with horizontal cingular lists (Fig. 2, o r). There is a historical controversy on the synonymy of P. rapa and P. mitra (Schiller 1933), but we observed that P. rapa cells were larger (61 lm long, 83 lm wide) and exhibited a greater angularity of the ventral margin than those of P. mitra when seen in lateral view (Fig. 2, s and t). P. favus (64 lm long, 86 lm wide) differed from P. rapa principally in the constricted, projecting fingerlike antapex. The length of the third rib of P. favus was smaller than in P. rapa (Fig. 2, u w). Section Urophalacroma Jørg.: We collected cells belonging to the type of this section, P. doryphorum, which had a wedge-shaped hypotheca, dome-shaped epitheca, and distinct horizontal cingular list. The left sulcal list was well-developed, and the most distinctive character was a nonribbed wide posterior projection with a triangular shape (Fig. 2, x z). Section Paradinophysis Jørg.: We obtained SSU rdna sequences of two specimens of P. rotundatum collected from the same sample. The cell contour was ellipsoidal, the theca was smooth, and the epitheca was hardly visible above the cingulum. One of the specimens was slightly larger and had a wider hypotheca than the other one (Fig. 2, aa and ab). We also obtained sequences of three specimens of P. parvulum from open and coastal waters (Table S1). The cells (40 lm long, 33 lm wide) had a smooth theca and showed a regularly round outline in lateral view. The epitheca was dome-shaped with horizontal cingular lists (Fig. 2, ac ag). Genus Dinophysis: Various sequences of the chloroplast-containing species of Dinophysis s.s. were

7 MOLECULAR PHYLOGENY OF DINOPHYSALES 399 available in GenBank. However, no complete SSU rdna sequence of any apochlorotic species of Dinophysis was available. In addition to several new sequences of members of Dinophysis s. s., we determined SSU rdna sequences of several apochlorotic species, including different morphotypes of D. hastata, as well as Dinophysis odiosa (Pavill.) L. S. Tai et Skogsb., Dinophysis monacantha Kof. et Skogsb., Dinophysis pusilla Jørg., and Dinophysis cf. acutissima Gaarder. Section Homoculus Pavill.: We determined the complete SSU rdna of Dinophysis tripos Gourret. The specimen used was collected from its type locality, the Bay of Marseille (Fig. 3a). In addition, we obtained an additional sequence for Dinophysis caudata Kent (Fig. 3b). Section Hastata Pavill.: The species of this section are apochlorotic and characterized by antapical spines. We illustrated the single-cell sequenced specimens and other specimens to show the differences among the species, and we reproduced the original illustrations of the species and varieties related to D. hastata found in Stein (1883), Jørgensen (1923), Kofoid and Skogsberg (1928), Pavillard (1930), Gaarder (1954), and Norris and Berner (1970) to provide a reference for the accuracy of our specimen identifications (Fig. 3, j y). Due to the historical controversy about the synonymy and supposed intraspecific variability of some species of the section Hastata, especially the type D. hastata, we extend the description of this section below. The description of the first member of the section Hastata, D. hastata, is credited to Stein (1883, pl. 19, fig. 12), who provided a single illustration of D. hastata (Fig. 3j). Since then, and although the records under the name D. hastata were numerous, specimens with the same hypotheca contour as in Stein s original description were never observed. All authors citing D. hastata assumed that the hypotheca of D. hastata was rounder than the Stein s specimen. Stein (1883) also described Dinophysis uracantha F. Stein, another Dinophysis species with an antapical spine. However, under the name D. uracantha, Stein (1883, pl. 20, figs. 21 and 22) provided two illustrations that unequivocally corresponded to two different 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. uracantha observed 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 ovate hypotheca, a very large antapical spine, and a long third rib extending below the basis of the epitheca. This Stein s second illustration of D. uracantha has been considered as synonym of D. hastata (Abé 1967b). Both Stein s illustrations of D. uracantha also differed in the reticulation, which was coarser for the illustration used to consider D. uracantha as synonym of D. hastata. Jørgensen (1923) described several forms of D. hastata and D. uracantha from the open Mediterranean Sea. Kofoid and Skogsberg (1928) described two species with antapical spines, D. monacantha (Fig. 3t) and D. urceolus Kof. et Skogsb., which were further considered as synonyms of D. hastata (Abé 1967b). Kofoid and Skogsberg (1928) proposed the existence of a high intraspecific variation in D. hastata. Pavillard (1930) remarked the validity of D. odiosa (Fig. 3s) that was synonymized with D. hastata (Kofoid and Skogsberg 1928, Taylor 1976). Norris and Berner (1970) described Dinophysis balechii D. R. Norris et L. D. Berner (Fig. 3o) previously considered one of the small forms of D. hastata. To clarify the phylogenetic position of D. hastata and other members of this section, we sampled specimens of the section Hastata that we classified into two different subsections according to their morphology: specimens with a Dinophysis-like epitheca and funnel were considered as uracanthoides, and specimens with flat epitheca and cingular list with a Phalacroma-like cingular list were included in the subgroup phalacromoides. Subsection uracanthoides: This group contained the type of the section, D. hastata. The specimens of this group have a morphology intermediate between the original description of D. hastata (Fig. 3j) and the second of Stein s illustration of D. uracantha (Fig. 3l). We suspect that Stein might have excessively elongated the cell body of D. hastata in his drawing. The members of uracanthoides were characterized by an elliptical cell body and a small epitheca with a funnel-shaped cingular list as in typical Dinophysis s.s. The third rib of the left sulcal list emerged from the lower half of the hypotheca. We obtained SSU rdna sequences for two morphotypes of this kind. The first morphotype was observed in live specimens that were 64 lm long and 51 lm wide (Fig. 3, c f), with a dorsoventral diameter at the base of the funnel (upper girdle list) of 27 lm. Our specimens strongly resembled the original illustration of D. hastata for the antapical spine and coarse left sulcal reticulation (Fig. 3f), and, therefore, we ascribed them to D. hastata (Fig. 3, c f). The second morphotype resembled the description of D. hastata f. uracanthides Jørg. described by Jørgensen (1923) from the Mediterranean Sea (Fig. 3n), for which D. hastata f. uracanthides was smaller than D. hastata and had a narrower and more ventrally deflected antapical spine. In contrast to the specimens of D. hastata, the third rib in the D. hastata f. uracanthides specimens that we observed did not reach the level of the basis of the hypotheca (Fig. 3, g i). We collected two specimens with these characteristics from the same station in the Levantine Basin, which were ethanol-fixed for posterior SSU rdna amplification and sequencing (Table S1). Their cell body was ellipsoidal (46 lm long, 34 lm wide), and the dorsoventral diameter of the base of the funnel (upper girdle list) was 21 lm (Fig. 3, g i). Other members of this subsection are D. balechii (Fig. 3o), D. uracantha var.

8 400 FERNANDO GÓMEZ ET AL. mediterranea Jørg. (Fig. 3, q and al), Dinophysis alata Jørg. (Fig. 3p), and Dinophysis spinosa Rampi. Subsection phalacromoides: The members of phalacromoides are characterized by a flat and wider epitheca. The upper cingular list slightly extended over the epitheca. The funnel-shaped upper cingular list of previous specimens was lacking, resembling that of Phalacroma. Species of this subgroup include D. odiosa, first described as Phalacroma odiosum Pavill. As a general trend, the specimens of phalacromoides were larger and the hypotheca was more ovate that in members of uracanthoides. The third rib of the sulcal list emerged from the middle of the ventral side of the hypotheca and did not extend beyond the basis of the epitheca. The antapical spine was always ventrally deflected. We identified three morphotypes having these morphological characteristics, those corresponding to D. odiosa, D. hastata f. phalacromides Jørg., and D. monacantha. D. odiosa is one of the most common species of the section Hastata in the coastal Mediterranean Sea. However, it is rarely cited in the Mediterranean Sea (Gómez 2003), very likely because it is incorrectly reported as D. hastata. Its cell body was somewhat truncate anteriorly, fairly narrowly rounded posteriorly (Fig. 3, ab ae), while the cell contour of D. hastata was ovoid (Fig. 3, c f). The epitheca of D. odiosa was flat, wider than the greatest height of epitheca. The anterior cingular list showed numerous radial ribs. The third rib was longer and more posteriorly deflected in D. hastata than in D. odiosa (Fig. 3, ab ae). We obtained sequences from two specimens of D. odiosa from different locations (Marseille and Villefranche sur Mer). They were 75 lm long and 59 lm wide, with a dorsoventral diameter of the base of the upper cingular list of 50 lm (Fig. 3, ab ad). The second morphotype corresponded to Dinophysis hastata f. phalacromides Jørg. described from the Mediterranean Sea by Jørgensen (1923) (Fig. 3r). We collected a live specimen that was the largest observed for this section (79 lm long, 62 lm wide, with a dorsoventral diameter of the base of the upper cingular list of 41 lm; Fig. 3, z and aa). The third morphotype of this subsection corresponded to D. monacantha (Fig. 3t). The lack of citations of this species is likely due to the fact that it has been considered a synonym of D. hastata (Abé 1967b) and consequently pooled as D. hastata. D. monacantha was the smallest species observed for this subsection (67 lm long, 50 lm wide, base of the upper cingular list, 38 lm in diameter) and its general morphology resembled that of a small D. odiosa with a less flat and wider epitheca (Fig. 3, af and ag). Subsection Pusilla: The members of this group contained the smallest species of the section Hastata. Jørgensen (1923) described D. pusilla as a small species with rotund hypotheca (28 lm wide), prominent funnel-shaped upper cingular list, and a well-developed left sulcal list (Fig. 3v). The antapical spine with a prominent rib was slightly ventrally deflected and emerged from the posterior-ventral region of the hypotheca. We obtained SSU rdna sequences from two ethanol-fixed specimens collected in the open Mediterranean Sea. Both were 30 lm wide, with a dorsal-ventral diameter of the funnel base of 14 lm. One of the specimens showed a rotund hypotheca, with a straight third rib (Fig. 3ah). This morphology unequivocally corresponded to D. pusilla. The second specimen was slightly larger, with an ovate contour of the epitheca and the third rib of the sulcal list deflected posteriorly. A single prominent rib was projected anteriorly from the cingulum (Fig. 3ai). We also provided the illustration of a live specimen (28 lm long, 26 lm wide, diameter of funnel base 8 lm) (Fig. 3aj) and of a Lugol s-fixed specimen from the Pacific Ocean with a morphology that was intermediate between D. pusilla (Fig. 3v) and D. balechii (Fig. 3o). We ascribed the species Dinophysis schuettii G. Murray et Whitting (Fig. 3, w and an) to this group. Subsection acutissima: Gaarder (1954) described two species, D. acutissima (Fig. 3x) and Dinophysis reticulata Gaarder (Fig. 3y), characterized by an elongated hypotheca with a pronounced antapex that resembled the morphology of Dinophysis diegensis Kof. The distinctive character of this species was a short antapical spine. Species with these characteristics were not included in the sections established by Pavillard (1916) and Jørgensen (1923). Norris and Berner (1970) reported D. reticulata among the members of the D. hastata group, but they did not mention D. acutissima. To the best of our knowledge, the only record after the first description corresponded to Nguyen et al. (2008). These authors placed D. acutissima in the group doryphorum with P. doryphorum as type. We disagree with this view, and we preferred to place D. acutissima as a member of the section Hastata. We illustrated 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 the Mediterranean Sea (Gómez 2003). We obtained the SSU rdna sequence from one ethanol-fixed specimen from the open Ionian Sea (Table S1). This ethanol-fixed specimen of D. acutissima was 60 lm long and 40 lm wide, and the dorsal-ventral diameter of the funnel base was of 24 lm (Fig. 3ao). Genus Ornithocercus: This ornamented heterotrophic genus is characterized by a highly developed cingular chamber formed by the cingular list, which harbors unicellular cyanobacteria. The left sulcal list of these species is also highly developed, with ribs or keels that emerged from the hypotheca. We obtained the SSU rdna sequence of Ornithocercus magnificus F. Stein, the type species collected from the type locality, the NW Mediterranean Sea (Fig. 4a). We also obtained sequences of four specimens of Ornithocercus heteroporus Kof. from live

9 MOLECULAR PHYLOGENY OF DINOPHYSALES 401 (Fig. 4, b and c; Table S1) and ethanol-fixed specimens from different locations of the Mediterranean Sea (Fig. 4, d and e; Table S1) and of specimens of two varieties of Ornithocercus quadratus var. quadratus Kof. et Skogsb. (Fig. 4, f and g) and var. schuettii Kof. et Skogsb. (Fig. 4h). Genus Histioneis: Only one complete SSU rdna sequence of Histioneis was available in GenBank, derived from a specimen that was not identified to the species level, illustrated in dorsoventral view in the original publication (Handy et al. 2009), which made difficult its posterior identification. We determined four SSU rdna sequences of three species identified at the species level. The type of Histioneis, Histioneis remora F. Stein, has been very scarcely recorded, and the few existing records are doubtful. Stein s illustration did not allow defining the type species. Stein (1883) also provided under the name Histioneis cymbalaria F. Stein three illustrations that unequivocally corresponded to three separate species. We obtained the sequence of a specimen strongly resembling one of the Stein s illustrations of H. cymbalaria. Hence, although this species has been named either Histioneis depressa J. Schiller by Taylor (1976) or H. cymbalaria by Balech (1988), our specimen has been ascribed to H. cymbalaria following Gómez (2007) (Fig. 4i). We sequenced SSU rdnas from two specimens of Histioneis longicollis Kof. collected from the Bay of Villefranche sur Mer on two consecutive days (Fig. 4, j and k). Both specimens were identical in size (83 lm maximum length, and the width of the hypotheca was 28 lm) with a peculiar yellow-greenish brightness of the sulcal list, although they slightly differed in internal ornamentation of the left sulcal list. The morphology corresponded to Histioneis sublongicollis Halim described from the Bay of Villefranche. Following Gómez (2007) H. sublongicollis was considered a synonym of H. longicollis. The other sequenced specimen belonged to the Histioneis gubernans group (Gómez 2007), closely related to Histioneis striata Kof. et J. R. Michener, and was ascribed to H. gubernans F. Schütt (Fig. 4l). Another live specimen of H. gubernans is illustrated for comparison (Fig. 4m). Family Oxyphysaceae. O. oxytoxoides is the only member of this family. No specimen was observed in the coastal or open Mediterranean Sea. We obtained the SSU rdna sequence from a specimen (58 lm long, 20 lm wide) collected in the brackish waters of the Thau lagoon at Sète, south of France (Fig. 4n). In addition to Oxyphysis, a nondescribed species from the Pacific Ocean with an elongated epitheca is shown (Fig. 4o). Molecular phylogeny. We constructed ML trees from a global alignment of dinoflagellate SSU rdna sequences using other alveolates as outgroup. All the sequences of representative dinophysoid dinoflagellates emerged within a strongly supported (BV of 94%) monophyletic clade (Fig. 5). This clade branched within a large dinoflagellate group composed of taxa of the orders Gymnodiniales, Peridiniales, and Prorocentrales. However, this relationship was poorly supported (BV of 62%). Within this group, symmetric species of Prorocentrum Ehrenb. (Prorocentrum lima Ehrenb., Prorocentrum concavum Fukuyo, and P. levis M. A. Faust, Kibler, Vandersea, P. A. Tester et Litaker) branched as sister group of the dinophysoid clade, although without support (BV <50%). Concerning the internal phylogeny of the Dinophysales, this general phylogenetic tree provided good support for the sister relationship of the genera Amphisolenia and Triposolenia (BV of 88%), whereas the rest of species emerged within a strongly supported group (BV of 99%) (Fig. 5). To obtain a more resolved view of the phylogenetic relationships among the Dinophysales, we constructed a SSU rdna data set restricted to these species. The corresponding phylogenetic tree (Fig. 6) was rooted with the clade of Amphisoleniaceae (according to the results of the previous general analysis containing other dinoflagellates, Fig. 5). The other major clade, containing all the remaining dinophysoids, was divided into two well-supported large subclades: one for species with the classical Phalacroma morphology, which also included O. oxytoxoides (BV of 91%), and a second one for the species of Dinophysis, Ornithocercus, and Histioneis (BV of 100%). Our phylogenetic tree did not support the consideration of O. oxytoxoides as the only member of its own family and even as a separate genus from Phalacroma. On the contrary, it emerged well nested among the Phalacroma species (BV of 81%), closely related to the type of Phalacroma (Fig. 6). To test the robustness of this unexpected placement of O. oxytoxoides within the clade containing the type of Phalacroma, we constructed an LSU rdna tree using the relatively large sampling of dinophysoid sequences available at GenBank (including O. oxytoxoides: EF613359). The LSU rdna phylogeny (Fig. S1 in the supplementary material) also supported the late emergence of O. oxytoxoides among the Phalacroma species, with even stronger support than the SSU rdna (BV of 89%). Finally, we carried out a combined phylogenetic analysis of concatenated SSU and LSU rdna sequences for the dinophysoid species for which sequences of both markers are available. The SSU + LSU rdna tree (Fig. 7) provided very strong support for the inclusion of O. oxytoxoides within the genus Phalacroma (BV of 100%), more precisely within a subclade clustering the Phalacroma type, P. porodictyum, and P. doryphorum (BV of 100%). SSU and LSU rdnas have very contrasted degrees of variability in the dinophysoid species. For example, the sequence identity between O. oxytoxoides and P. porodictyum was 99.27% for the SSU rdna and 93.82% for the LSU rdna. Therefore, the phylogenetic position of O. oxytoxoides as a member of the genus Phalacroma retrieved support from

10 402 FERNANDO GÓMEZ ET AL. FIG. 5. Maximum-likelihood phylogenetic tree of dinoflagellate SSU rdna sequences, based on 1,166 aligned positions. Names in bold represent sequences obtained in this study. Numbers at nodes are bootstrap values (values <50% are omitted). Accession numbers are provided between brackets. The scale bar represents the number of substitutions for a unit branch length. markers with different levels of conservation, stressing its reliability. Internal SSU rdna phylogeny of the three major dinophysoid groups. Amphisoleniaceae: The species of Amphisolenia and Triposolenia formed two highly supported groups (BV of 86% and 100%, respectively). However, the relative branching order of the different Amphisolenia species was weakly supported, although the tree suggested that the two morphologically closely related species A. globifera and A. schauinslandii were sister (BV of 68%), with A. bidentata branching in a basal position (Fig. 6). Phalacroma: Although several nodes within the Phalacroma clade were not well resolved, it appeared that this clade was subdivided into two subclades: one for the type, P. porodictyum, and P. 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%). We called the former Phalacroma subclade because it contained the type, and the second Rapa because P. rapa was the first described species in that subclade (Fig. 6). The classical subdivision into sections represented by two or more species as Paradinophysis (P. rotundatum, P. parvulum) and Podophalacroma (P. mitra, P. rapa, P. favus) is not supported by the molecular data, since species of same section appeared in different subclades of Phalacroma. In fact, P. rotundatum branched in the subclade Phalacroma, close to P. porodictyum and O. oxytoxoides with relatively good support (BV of 86%), whereas P. parvulum branched in the subclade Rapa. P. mitra and P. rapa were sisters in the subclade Rapa (BV of 97%). Surprisingly, despite the strong morphological resemblance, P. favus is distantly related to other members of

11 MOLECULAR PHYLOGENY OF DINOPHYSALES 403 FIG. 6. Maximum-likelihood phylogenetic tree of Dinophysales SSU rdna sequences, based on 1,166 aligned positions. Names in bold represent sequences obtained in this study. Numbers at nodes are bootstrap values (values <50% are omitted). The sequences of the type species are highlighted in gray shaded boxes. Accession numbers are provided between brackets. The scale bar represents the number of substitutions for a unit branch length. Podophalacroma, and it branched closely related to P. porodictyum in the Phalacroma subclade. Dinophysaceae: Our phylogenetic analysis did not provide a robust resolution of all genera in the Dinophysaceae clade, which showed large variations of evolutionary rate, as depicted by the extreme differences of branch lengths (Fig. 6). Ornithocercus and Histioneis had short branches and emerged in a basal position with respect to Dinophysis s.s., although with moderate support (BV of 68%). All the Ornithocercus sequences were almost completely identical, with the exception of O. magnificus FTL83 (Handy et al. 2009), probably because of a few sequence errors. Sequences of the two O. quadratus varieties (var. schuettii and var. quadratus) were identical. This extreme conservation of the SSU rdna within the genus Ornithocercus made this marker inappropriate to resolve the relationships among the corresponding species. The sequences of Histioneis formed a group that appeared to be more closely related to Ornithocercus than to Dinophysis. Histioneis cymbalaria emerged at the base of H. longicollis and H. gubernans (BV of 67%). However, the poor internal resolution for this genus made it premature to draw conclusions about the Histioneis systematics only on the basis of the SSU rdna phylogeny. The chloroplast-containing species of Dinophysis, the so-called Dinophysis s.s. that contained the type species, formed a well-supported monophyletic

12 404 FERNANDO GÓMEZ ET AL. FIG. 7. Maximum-likelihood phylogenetic tree of Dinophysales LSU and SSU rdna sequences, based on 1,980 aligned positions. Numbers at nodes are bootstrap values (values <50% are omitted). The sequences of the type species are highlighted in gray shaded boxes. Accession numbers are provided between brackets. The scale bar represents the number of substitutions for a unit branch length. clade. The other species of Dinophysis belonging to the section Hastata, apochlorotic with an antapical spine, branched in a basal position to the members of Dinophysis s.s. Our molecular phylogeny strongly supports D. odiosa and D. monacantha as separate species, given the large divergence between their sequences (Fig. 6). Likewise, the sequences of the two forms of D. hastata, f. phalacromides and f. uracanthides, were very divergent from that of D. hastata, so that these forms also deserve to be considered as two separate species. The species of the section Hastata were relatively distant from Dinophysis s.s. in all the SSU rdna phylogenies. However, the internal relationships between the members of this section were unstable and poorly supported in different molecular phylogenies. This trend is probably due, at least partly, to the extreme differences of evolutionary rate in these species, with short-branching ones such as D. pusilla and D. cf. acutissima, and very long-branching ones such as D. monacantha and D. odiosa. D. hastata f. uracanthides branched as sister of the Dinophysis s.s., making the Hastata paraphyletic (Fig. 6). Nevertheless, this finding was weakly supported (BV of 62%), and we cannot exclude that the paraphyly of the Hastata reflected a long-branch attraction artifact due to the long branches of certain members. The only well-supported subgroup is composed of D. odiosa, D. monacantha, D. pusilla, Dinophysis cf. acutissima, and D. hastata f. phalacromides (BV of 97%) (Fig. 6). Within this group, there was a very short distance between Dinophysis cf. acutissima, with an elongated and antapically pointed hypotheca, and D. pusilla. This relationship suggested that the shape of the hypotheca is a morphological character highly variable among very closely related species and, consequently, of relatively small value as a phylogenetic marker of the species. Nomenclatural considerations. The placement of O. oxytoxoides closely related to the type species of Phalacroma in the SSU and LSU rdna phylogenies supports the transfer of O. oxytoxoides into Phalacroma. Phalacroma oxytoxoides (Kof.) F. Gómez, P. López- García 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 Dinophysis s.s. with other chloroplast-containing species of Dinophysis. In contrast, the sequences of D. hastata f. phalacromides and D. hastata f. uracanthides, as well as those of other apochlorotic members of the Dinophysis hastata group, branched very distantly from the type of Dinophysis, which might support their inclusion in different genera. However, the eventual split of Dinophysis and the erection of a new genus for the members of the D. hastata group are premature. Additional information from other molecular markers would be required to obtain more robust phylogenies that would eventually validate this claim. Nevertheless, the molecular data clearly demonstrated that the assumed high intraspecific variability of D. hastata hid a number of cryptic species. In fact, species such as D. odiosa or D. monacantha, previously considered as synonyms of D. hastata (Abé 1967b, Taylor 1976), appeared to be distant species on the basis of their high SSU rdna sequence divergence (Fig. 6). Likewise, our molecular phylogenetic analysis showed that the two forms D. hastata f. phalacromides and D. hastata f. uracanthides described by Jørgensen (1923) do correspond to separate phylogenetic species. Consequently, we propose to erect these forms at the species level as follows: Dinophysis phalacromoides (Jørg.) F. Gómez, P. López-García et D. Moreira, comb. nov. Basionym: Dinophysis hastata F. Stein f. phalacromides Jørg. (Jørgensen 1923, pp. 30 1, fig. 41). Dinophysis uracanthoides (Jørg.) F. Gómez, P. López-García et D. Moreira, comb. nov. Basionym: Dinophysis hastata F. Stein f. uracanthides Jørg. (Jørgensen 1923, pp. 30 1, fig. 40). Reconciling morphological and molecular data. The detailed studies on the tabulation carried out by Tai and Skogsberg (1934), Abé (1967a,b,c), Balech (1967, 1988), and Norris and Berner (1970) showed that the plate arrangement and number are more or less similar in all dinophysoid species and genera, even at the level of the small sulcal plates. Abé (1967a) observed some differences in the tabulation for the genera Amphisolenia, Triposolenia, and Oxyphysis when compared with the other dinophysoids. All authors agreed on the establishment of the family Amphisoleniaceae for Amphisolenia and Triposolenia (Abé 1967a, Balech 1977, 1980). Our molecular phylogenetic analysis including SSU rdna sequences of Triposolenia and the type of Amphisolenia, which appear as monophyletic and occupy a basal position