Molecular Phylogeny of Sinophysis: Evaluating the Possible Early 2 3 Q1 Evolutionary History of Dinophysoid Dinoﬂagellates 4 5 M

1 Molecular phylogeny of Sinophysis: Evaluating the possible early 2 3 Q1 evolutionary history of dinophysoid dinoflagellates 4 5 M. HOPPENRATH1,2*, N. CHOME´ RAT3 & B. LEANDER2 6 1 7 Forschungsinstitut Senckenberg, Deutsches Zentrum fu¨r Marine Biodiversita¨tsforschung 8 (DZMB), Su¨dstrand 44, D-26382 Wilhelmshaven, Germany 9 2 10 Departments of Zoology and Botany, University of British Columbia, Canadian Institute 11 for Advanced Research, Program in Integrated Microbial Biodiversity, Vancouver, 12 BC, V67 1Z4, Canada 13 3 14 IFREMER, LER FBN Station de Concarneau, 13 rue de Ke´rose, 29187 15 Concarneau Cedex, France 16 17 *Corresponding author (e-mail: [email protected]) 18 19 20 Abstract: Dinophysoids are a group of thecate dinoflagellates with a very distinctive thecal plate 21 arrangement involving a sagittal suture: the so-called dinophysoid tabulation pattern. Although the number and layout of the thecal plates is highly conserved, the morphological diversity within the 22 group is outstandingly high for dinoflagellates. Previous hypotheses about character evolution 23 within dinophysoids based on comparative morphology alone are currently being evaluated by 24 molecular phylogenetic studies. Sinophysis is especially significant within the context of these 25 hypotheses because several features within this genus approximate the inferred ancestral states 26 for dinophysoids as a whole, such as a (benthic) sand-dwelling lifestyle, a relatively streamlined 27 theca and a heterotrophic mode of nutrition. We generated and analysed small subunit (SSU) 28 rDNA sequences for five different species of Sinophysis, including the type species (S. ebriola, 29 S. stenosoma, S. grandis, S. verruculosa and S. microcephala). We also generated SSU rDNA 30 sequences from the planktonic dinophysoid Oxyphysis (O. oxytoxoides). Temperate and tropical species as well as the complete spectrum of thecal ornamentation within Sinophysis was addressed 31 in our study. The sequences from the Sinophysis species formed a robust monophyletic group that 32 was the sister to a robust clade consisting of all other dinophysoid genera, including Oxyphysis,in 33 some analyses. Although the sister relationship received weak statistical support, this tree topology 34 was consistent with inferences based on comparative morphology. 35 36 37 38 39 Dinophysoids are a morphologically diverse group these features are not understood within a molecular 40 of dinoflagellates with a highly distinctive thecal phylogenetic context. 41 plate pattern involving a sagittal suture (e.g. Of the 12 genera and about 280 species of 42 Kofoid & Skogsberg 1928; Taylor 1976; Fensome dinophysoids recognized today, only one genus 43 et al. 1993). Character evolution within dinophy- is benthic: Sinophysis Nie and Wang (e.g. Hoppen- 44 soids has been hypothesized based on morphology rath 2000; Selina & Hoppenrath 2004; Chome´rat 45 alone (e.g. Tai & Skogsberg 1934; Abe´ 1967a, b, et al. 2009). Sinophysis consists of seven species: 46 c; Taylor 1980; Hoppenrath et al. 2007) and is S. microcephala (the type) and S. canaliculata are 47 only now being evaluated by molecular phyloge- found in tropical habitats and S. ebriola, S. gran- 48 netic studies of ribosomal gene sequences (Handy dis, S. stenosoma, S. minima and S. verruculosa 49 et al. 2009; Hastrup Jensen & Daugbjerg 2009; are found in temperate habitats (Herdman 1924; 50 Go´mez et al. 2011, 2012). Comparisons of extant Nie & Wang 1944; Balech 1956; Quod et al. 51 morpho-species suggest that the ancestral dinophy- 1999; Hoppenrath 2000; Selina & Hoppenrath 52 soids were benthic and consisted of relatively 2004; Chome´rat et al. 2009). Selina & Hoppenrath 53 streamlined cells that subsequently evolved more (2004) and Chome´rat et al. (2009) have previously 54 elaborate extensions of the theca in association addressed the distinctive morphological features 55 with planktonic lifestyles (e.g. expansions of the within this genus. We were interested in using a 56 cingular and sulcal lists in Histioneis and Ornitho- broad sampling of molecular phylogenetic data to 57 cercus). Different habitats, modes of nutrition and test whether the relatively streamlined theca, hetero- 58 levels of toxicity are known in dinophysoids, but trophic mode of nutrition and a benthic life style

From:Lewis, J. M., Marret,F.&Bradley, L. (eds) 2013. Biological and Geological Perspectives of Dinoﬂagellates. The Micropalaeontological Society, Special Publications. Geological Society, London, 199–206. # The Micropalaeontological Society 2013. Publishing disclaimer: http://www.geolsoc.org.uk/pub_ethics 200 M. HOPPENRATH ET AL.

59 60 61 62 63 64 65 66 67 68 69 70 Fig. 1. Light micrographs showing the Sinophysis morpho-species represented in the phylogeny: (a) S. microcephala, 71 (b) S. verruculosa,(c) S. ebriola,(d) S. stenosoma and (e) S. grandis. Scale bar ¼ 10 mm. 72 73 74 of Sinophysis species reflects the ancestral con- cloning kit (Invitrogen, Carlsbad, CA, USA) Q2 75 dition of dinophysoids. according to the manufacturer’s recommendations. 76 In order to consider the secondary structure of small subunit (SSU), sequences of 61 operational 77 Material and methods 78 taxonomic units (OTU) were aligned using SINA Q3 // 79 Sand samples and plankton samples were taken aligner (online version v1.2.9, available at http: / / et al. 80 in Canada (British Columbia: Boundary Bay, www.arb-silva.de aligner ) (Pruesse 2007). 81 49800.00′N, 123808.00′W, August 2005 and May The matrix was then analysed by maximum likeli- ′ ′ et al. 82 2007 and Bamfield, 48850.00 N, 125808.00 W, hood (ML) using PHYML v. 3.0 (Guindon Q4 83 June 2006), Germany (Island of Sylt, 55801.80′N, 2010) and Bayesian inference (BI) using Mr Bayes 84 08826.00′E, March 2009 and Wilhelmshaven, v. 3.1.2 (Ronquist & Huelsenbeck 2003). The ′ ′ best-suited nucleotide model was determined using 85 53830.52 N, 08808.42 E, February 2009) and France + 86 (Brittany, 47851.85′N, 04805.05′W; 47847.86′N, jModeltest v. 0.1.1 (Posada 2008). A model GTR ′ ′ ′ ′ + G ¼ 87 03851.09 W; 47847.75 N, 03849.88 W; 47847.43 N, I 4 was chosen for ML analysis and BI (nst 6). 88 03847.30′W, September 2010 and Martinique Branch support was assessed with bootstrap values 89 Island, Caribbean, 14831.53′N, 61805.41′W, March calculated from 1000 pseudoreplicates in ML. For 90 2010). Manually isolated and washed living cells Bayesian analysis, four Markov chains were run 91 were used for either DNA extraction or directly simultaneously for 2 000 000 generations (sampled 92 for polymerase chain reaction (PCR). Cells fixed every 100 generations) in two independent runs. A 93 with Lugol’s solution were washed and bleached majority-rule consensus tree was constructed from 94 with sodium thiosulphate (Auinger et al. 2008). 18 000 post burn-in trees that were used to calculate 95 The sequences from Canadian isolates were gener- the posterior probabilities of the nodes. 96 ated using the methodology described in Hoppen- 97 rath et al. (2009). The sequences from Germany Results and discussion 98 were generated using the same procedure up to the 99 first round of PCR. These samples were finished in Small subunit ribosomal DNA (SSU rDNA) gene 100 France by the protocol described in Chome´rat sequences were obtained for five of the seven 101 et al. (2010, 2011). Following the second round of known Sinophysis species, namely S. microcephala 102 PCR, the amplicons were either directly sequenced (the type species), S. ebriola, S. grandis, S. steno- 103 as described in Chome´rat et al. 2010, or cloned soma and S. verruculosa. Sinophysis microcephala 104 into a pCR 2.1-TOPO vector using the TOPO-TA is one of the two known tropical species with 105 106 107 Fig. 2. Maximum likelihood phylogeny of the dinoflagellates SSU rDNA sequence dataset (61 OTU, 1766 characters). 108 The likelihood value was found to be log lk ¼ 214 235. The tree is rooted using Perkinsus marinus sequence as + + 109 outgroup. Model selected: GTR I G4. Assumed proportion of invariable sites I ¼ 0.366. Rates at variable site assumed to be gamma distributed with shape parameter a ¼ 0.529. Assumed nucleotides frequencies f(A) ¼ 0.25110; 110 ↔ 111 f(C) ¼ 0.18540; f(G) ¼ 0.26003; f(T ) ¼ 0.30347. GTR relative rate parameters: A C ¼ 1.84704; A ↔ G ¼ 4.37972; A ↔ T ¼ 1.61657; C ↔ G ¼ 0.53631; C ↔ T ¼ 9.20255 and G ↔ T ¼ 1.00000. Support above 112 branches: ML bootstraps (1000 pseudoreplicates)/Bayesian posterior probabilities (2 000 000 generations). ‘ + ’ 113 indicates a bootstrap value ,65 or a Bayesian posterior probability ,0.75. Absence of value indicates the existence 114 of the branch in ML (not supported) but an irresolution in BI. Sequences acquired in this study are in bold type. Symbols 115 for geographic origin of isolates: B, Brittany (France); BC, British Columbia (Canada); G, Germany; M, Martinique 116 Island (France). SINOPHYSIS PHYLOGENY 201

117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 ornamented (areolated) thecal plates with a slightly S. canaliculata, failed. The only temperate species 173 domed epitheca (Fig. 1a). Attempts to sequence the with slight ornamentation (verrucose) of the theca, 174 second tropical species with a similar morphology, S. verruculosa, was isolated from Brittany, France 202 M. HOPPENRATH ET AL.

175 (Fig. 1b). Multiple sequences were obtained from a mixed clade containing seven (I–VII) subgroups. 176 the other three temperate species (Fig. 1c–e) iso- Subclade C-I is Dinophysis s.s., subclade C-V is His- 177 lated from different regions worldwide: S. ebriola tioneis, subclade C-VI is Ornithocercus and Cithar- 178 and S. stenosoma were isolated from Canada and istes and the other clades comprise Dinophysis and 179 France; S. grandis was isolated from Canada, Ger- Phalacroma species. Handy et al. (2009) analysed 180 many and France. All of the Sinophysis species clus- SSU, ITS and LSU rDNA sequences and recovered Q6 181 tered together in a monophyletic group with strong dinophysoid clades corresponding to clades B and 182 statistical support (Fig. 2). Different sequences for C, which included subclades C-I, -V and -VI. 183 identified morpho-species also clustered together, The dinophysoid phylogeny inferred from partial 184 supporting the current species delimitations (Fig. SSU rDNA sequences published by Go´mez et al. 185 2). Two subclades were recognized within the S. (2011) confirmed clades A–C and suggested that 186 grandis clade (Fig. 2): one subclade included the (1) clade A contains also Triposolenia; (2) Oxyphy- 187 isolates from Brittany, France and the second sub- sis branches within the Phalacroma s.s. clade B; 188 clade included the isolates from Canada (from one and (3) clade C contains an additional Dinophysis 189 site) and Germany (from two sites). This is a first subclade. Sinophysis represents a major dinophysoid 190 indication for unrecognized (cryptic) species and lineage, referred to here as ‘clade D’, that forms 191 should motivate further detailed morphological, bio- the sister group to a more inclusive clade consist- 192 geographical and molecular studies on the genus. ing of clades A–C (Fig. 2). Relatively incomplete 193 The phylogenetic positions of S. verruculosa and SSU rDNA sequences from Amphisolenia and 194 S. microcephala were unstable within the Sinophy- Triposolenia were not included in the initial molecu- 195 sis clade in different analyses (not shown). The lar phylogenetic analyses (Fig. 2) in order to maxi- 196 latter species had a very long branch, so its unstable mize the number of unambiguously aligned sites. 197 phylogenetic position is interpreted to reflect meth- Nonetheless, the available SSU rDNA sequences 198 odological artefacts (e.g. long-branch attraction). did not have enough phylogenetic information to 199 Nonetheless, the relatively divergent branch of S. resolve the relationships between the major clades 200 microcephala reflects the divergent morphology of dinophysoids. In the second shorter analysis 201 and habitat of this species. This species differs including Amphisolenia and Triposolenia (Fig. 3), 202 from that of the other analysed (temperate) species the Sinophysis clade branched separately from 203 by a strongly areolated thecal ornamentation and the other dinophysoid taxa. This topology did not 204 a domed epitheca. It is also the only tropical spe- receive statistical support, as shown earlier (Go´mez 205 cies represented in the Sinophysis clade. Additional et al. 2012). Moreover, an Approximately Unbiased 206 sequences from tropical species with a similar mor- (AU) test did not reject that Sinophysis and the 207 phology, such as S. canaliculata, would establish other dinophysoid taxa could be monophyletic; 208 whether or not the tropical species had sequences Go´mez et al. (2012) could therefore not dismiss 209 with similar branch lengths (i.e. strong rate hetero- that Sinophysis might be the sister lineage of the 210 geneity) and phylogenetic positions when compared main dinophysoid clade. Sequences from other 211 to the temperate species. A second analysis of an regions of the rDNA operon for Sinophysis species 212 alignment with shorter sequences (Fig. 3) included would provide concatenated alignments that might 213 both Sinophysis taxa published by Go´mez et al. improve phylogenetic resolution within dinophy- 214 (2012). The species identifications for these two soids. Despite several attempts to generate LSU 215 sequences are ambiguous; the reported light micro- rDNA sequences from Sinophysis species, only one 216 graphs do not allow rigorous evaluation. Nonethe- LSU rDNA sequence has been generated so far. 217 less, the S. ‘grandis’ specimen clustered together This sequence (from S. ebriola) was very divergent 218 with our S. ebriola and the S. ‘ebriola’ specimen from the other dinophysoid LSU rDNA sequences 219 had a very unusual morphology and branched as sis- and is considered tenuous because of known pro- 220 ter to S. verruculosa. blems with pseudogenes, heterogeneity within dif- 221 The sequences from the Sinophysis species clus- ferent species of dinoflagellates and heterologous 222 tered as the sister clade to a robust clade consisting copies within specimens (Gribble & Anderson 2007; 223 of all other dinophysoid genera, including Oxyphy- Hart et al. 2007). The current poor resolution of 224 sis (Fig. 2). Although the statistical support for a dinophysoid phylogeny as inferred from ribosomal 225 Dinophysales H.W. Graham clade was weak, this DNA sequences mirrors the past phylogenetic situ- 226 topology makes sense in the light of comparative ation for prorocentroid dinoflagellates (e.g. Hoppen- 227 morphology (e.g. Taylor 1980; Fensome et al. 1993; rath & Leander 2008); therefore, in all likelihood, 228 Selina & Hoppenrath 2004; Chome´rat et al. 2009). analyses of concatenated gene datasets will offer a 229 Q5 Using the LSU rRNA gene, Hastrup Jensen & solution to the current lack of resolution for dinophy- 230 Daugbjerg (2009) divided the Dinophysales into soids (Zhang et al. 2007; Murray et al. 2009). 231 three major subclades: (A) the Amphisolenia clade, A hypothetical framework of character evolution 232 (B) the Phalacroma sensu stricto (s.s.) clade and (C) within the Dinophysales, as reflected in molecular SINOPHYSIS PHYLOGENY 203

233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 Fig. 3. Maximum likelihood phylogeny of the dinoﬂagellates SSU rDNA sequence dataset (63 OTU, 1241 characters). 282 The likelihood value was found to be log lk ¼ 28595.82. The tree is rooted using Perkinsus marinus sequence as + + 283 outgroup. Model selected: GTR I G4. Assumed proportion of invariable sites I ¼ 0.442. Rates at variable site 284 assumed to be gamma distributed with shape parameter a ¼ 0.533. Assumed nucleotides frequencies f(A) ¼ 0.22860; ¼ ¼ ¼ ↔ ¼ 285 f(C) 0.19119; f(G) 0.26924; f(T ) 0.31097. GTR relative rate parameters: A C 1.47340; A ↔ G ¼ 5.60804; A ↔ T ¼ 1.62506; C ↔ G ¼ 0.59617; C ↔ T ¼ 10.68456 and G ↔ T ¼ 1.00000. Support above 286 branches: ML bootstraps (1000 pseudoreplicates)/Bayesian posterior probabilities (2 000 000 generations). ‘ + ’ 287 indicates a bootstrap value ,65 or a Bayesian posterior probability ,0.75. Absence of value indicates the existence of 288 the branch in ML (not supported) but an irresolution in BI. Sequences acquired in this study are in bold type. Symbols for 289 geographic origin of isolates: B, Brittany (France); BC, British Columbia (Canada); G, Germany; M, Martinique Island 290 (France). 204 M. HOPPENRATH ET AL.

291 phylogenetic studies to date, is presented in characterized by a small epitheca, very narrow cin- 292 Figure 4. Additional clades in the main clade C gular lists, the left sulcal list (LSL) bent to the right 293 (Dinophysis s.l. and Phalacroma s.l.) have been lateral side covering the sulcus and oblique cell flat- 294 published but were not included here because taxo- tening with the sulcus on the ‘right’ lateral side. 295 nomical revisions are needed to clarify the morpho- Prior to this study, clade A (Amphisolenia and Tri- 296 logical features characteristic for each clade. This posolenia) has been shown as the earliest diverging 297 hypothesis should be viewed as a baseline that can dinophysoid clade (relative to all other dinophysoid 298 be tested and refined with additional molecular species). These genera are also characterized by a 299 phylogenetic studies, different molecular markers small epitheca and very narrow cingular lists. The 300 and additional morphological studies that revise hypotheca is elongated, the arrangement of the 301 current taxonomic schemes. The Sinophysis clade two small ventral hypothecal plates (H1 & H4) is 302 (major clade D, Fig. 2) can be interpreted as the distinctive and the LSL is narrow. Photosynthetic 303 sister group to the lineage including all other dino- endosymbionts have also been acquired in this line- 304 physoid taxa. The heterotrophic, benthic genus is age. These two lineages taken together suggest that 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 Fig. 4. Hypothetical framework of character evolution within Dinophysales as reflected in molecular phylogenetic 344 studies to date. The division into four major subclades follows Hastrup Jensen & Daugbjerg (2009): (a) Amphisolenia 345 clade; (b) Phalacroma s.s. clade; (c) a mixed clade containing subgroups; and (d) the Sinophysis clade from this study. 346 A dash on a linage denotes a synapomorphy listed above. Arrows denote where the evolutionary events – listed behind 347 asterisks – may have occurred. Some characteristic features for the genera and the hypothetical common ancestor are 348 listed. LSL, left sulcal list; H1, first hypothecal plate; H4, fourth hypothecal plate. SINOPHYSIS PHYLOGENY 205

349 the most common ancestor of dinophysoid dinofla- We thank M. Loir for sampling in Martinique Island and 350 gellates was most likely heterotrophic with a very L. Medlin for suggesting a better alignment method. This 351 small epitheca, with narrow cingular and sulcal lists work was supported by a postdoctoral research salary to 352 and with the synapomorphic characters: (1) sagittal MH from the Deutsche Forschungsgemeinschaft (grant 353 (serrate) suture over the whole cell; (2) flagella Ho3267/1-1) and operating funds from the National Science Foundation – Assembling the Tree of Life (NSF 354 emerging from one pore; and (3) rhabdosome- #EF-0629624) and the National Science and Engineering 355 like organelles. Research Council of Canada (NSERC 283091-09). BSL 356 The distinctive morphological characters present is a fellow of the Canadian Institute for Advanced 357 in the other dinophysoid clades are summarized in Research, Program in Integrated Microbial Biodiversity. 358 Figure 4. Whether shape and size of the epitheca; The EGIDE association is acknowledged for providing a 359 the general morphology of the LSL (size, shape, grant allowing one of the authors (MH) to stay for one 360 ribs, etc.); the size, shape and direction of the cingu- month at Ifremer’s laboratory in Concarneau, France. 361 lar lists; and the micromorphology of the ventral This study was partly funded by the Contrat de Projet ´ ´ ` 362 area (small plates and pores) are of taxonomic and Etat–Region Souchotheque de Bretagne (PIDETOX project). 363 phylogenetic value remain to be determined. The 364 possibility of convergent evolution within the dino- 365 physoids and the polyphyly of morphologically 366 defined genera (e.g. Dinophysis) are still uncertain- References 367 ties. On the other hand, the branching order of Abe´, T. H. 1967a. The armored Dinoflagellata: II. Proro- 368 Oxyphysis as the sister lineage to Phalacroma rotun- centridae and Dinophysidae (A). Publication Seto 369 data has been confirmed in our study. However, the Marine Biological Laboratory, 14, 369–389. 370 very recent transfer of Oxyphysis into the genus Pha- Abe´, T. H. 1967b. The armored Dinoflagellata: II. Proro- 371 lacroma (Go´mez et al. 2011) was premature in our centridae and Dinophysidae (B) – Dinophysis and its 372 opinion. Significant morphological differences exist allied genera. Publication Seto Marine Biological Lab- 373 between Oxyphysis and Phalacroma and there is oratory, 15, 37–78. Abe´ 374 still the possibility that Phalacroma s.s., as under- , T. H. 1967c. 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