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.