J. Phycol. 41, 411–420 (2005) r 2005 Phycological Society of America DOI: 10.1111/j.1529-8817.2005.04168.x PHYLOGENY OF DINOFLAGELLATES BASED ON MITOCHONDRIAL CYTOCHROME b AND NUCLEAR SMALL SUBUNIT rDNA SEQUENCE COMPARISONS1 Huan Zhang Department of Marine Sciences, University of Connecticut, Groton, Connecticut 06340 USA Debashish Bhattacharya Department of Biological Sciences and Roy J. Carver Center for Comparative Genomics, University of Iowa, 312 Biology Building, Iowa City, Iowa 52242-1324 USA and Senjie Lin2 Department of Marine Sciences, University of Connecticut, Groton, Connecticut 06340 USA Despite their evolutionary and ecological impor- Key index words: cob; cytochrome b; dinoflagellates; tance, dinoflagellate phylogeny remains poorly phylogeny; rDNA; tertiary endosymbiosis resolved. Here we explored the utility of mitochon- drial cytochrome b (cob) in inferring a dinoflagellate Abbreviations: AU, approximately unbiased; COB, tree and focused on resolving the relationship be- cytochrome b; cob, gene coding for COB; GAPDH, tween fucoxanthin-and peridinin-containing taxa. glyceraldehyde-3-phosphate dehydrogenase; ML, Trees were inferred using cob and small subunit maximum likelihood; MP, maximum parsimony; rDNA alone or in combination as concatenated data mt, mitochondrial; SSU, small subunit and including members of the six major dinoflag- ellate orders. Many regions of the cob DNA or pro- tein and rDNA trees were congruent with support Dinoflagellates (subphylum Dinoflagellata, phylum for the monophyly of Symbiodinium spp. Freud- Dinozoa) are an evolutionarily diverse group of pro- enthal and of the Prorocentrales and the early di- tists that are closely related to the apicomplexan par- vergence of Crypthecodinium cohnii Seligo in Grasse. asites and together with the ciliates form the crown However, these markers provided differing support group Alveolata (Gajadhar et al. 1991, Cavalier-Smith for the monophyly of Pfiesteria spp. Steidinger et 1998). Dinoflagellates play pivotal roles in the marine Burkholder (only supported strongly by rDNA) and ecosystem and are of tremendous economic signifi- of the fucoxanthin dinoflagellates with Akashiwo sp. cance. They are important primary producers, with (Hirasaka) Hansen et Moestrup (Gymnodiniales, their abundance second only to diatoms; they support only supported strongly by the cob data). The ap- coral reef ecosystems through symbiotic associations; proximately unbiased (AU) test was used to assess and they include species that cause harmful algal these results using 13-and 11-taxon (excluding blooms. Dinoflagellates are considered unusual eukar- apicomplexans) backbone maximum likelihood yotes because, among other well-recognized unique trees inferred from the combined cob þ rDNA data. cytological features (Hackett et al. 2004a), they harbor The AU test suggested that our data were insuffi- a highly reduced plastid genome (approximately cient to resolve the phylogenetic position of Symbio- 14 genes, Hackett et al. 2004b) of 1–3 gene minicir- dinium spp. and that the ancestral position of cles (Zhang et al. 1999, Barbrook and Howe 2000, C. cohnii might have resulted from long-branch at- Koumandou et al. 2004), a nuclear-encoded form II traction to the apicomplexan outgroup. We found RUBISCO of alpha-proteobacterial origin (Morse significant support, however, for the association of et al. 1995, Rowan et al. 1996, Zhang and Lin 2003), fucoxanthin dinoflagellates with Akashiwo sp. The and a complex RNA editing machinery (Lin et al. monophyly and relatively derived position of the 2002). The ecological and economic importance of Gymnodiniales in our cob DNA and protein trees dinoflagellates, combined with their unusual genetic/ and in the cob þ rDNA tree is consistent with the genomic traits, renders them an evolutionary group of tertiary endosymbiotic origin of the plastid in high biological interest. fucoxanthin dinoflagellates. Despite the increasing research effort in recent years to study the phylogeny of dinoflagellates and their plastids, many questions remain open regarding 1Received 13 September 2004. Accepted 31 December 2004. the evolution of the ‘‘host’’ cell and the photosynthetic 2Author for correspondence: e-mail [email protected]. organelle (Hackett et al. 2004a). Resolution of these 411 412 HUAN ZHANG ET AL. issues would be greatly aided by the availability of a targeted glyceraldehyde-3-phosphate dehydrogenase resolved dinoflagellate host tree. The small subunit (GAPDH) (unpublished data) of haptophyte origin in (SSU) rRNA gene (rDNA) has been instrumental in the K. brevis nuclear genome. Despite these data, the advancing our understanding of dinoflagellate evolu- monophyly of fucoxanthin-containing taxa has had tion; however, the low resolving power of this gene varying levels of support from analyses of nuclear regarding lineage relationships has resulted in an in- and chloroplast SSU rDNA (Tengs et al. 2000, Sal- complete understanding of the major splits among darriaga et al. 2001, de Salas et al. 2003) and remains dinoflagellates despite a taxonomically broad sampling to be substantiated. The cob analyses presented here of data (Saunders et al. 1997, Saldarriaga et al. 2001). allowed us to address the position of fucoxanthin- Recent studies clearly show the advantage of using containing taxa in the dinoflagellate tree. multiple genes for phylogenetic analysis (Pryer et al. 2001, Mattern 2004, Yoon et al. 2004). Mitochondrial MATERIALS AND METHODS (mt) DNA is a useful candidate for phylogeny recon- Algal cultures. Cultures of dinoflagellates and other algae struction for several reasons. First, mt genes are gen- used in this study were obtained from several sources (Table erally conserved although are more variable than 1). The photosynthetic species were grown in f/2 medium, nuclear genes such as SSU rDNA (Avise 1994, Saccone whereas heterotrophic dinoflagellates (Pfiesteria shumwayae et al. 2000, Garesse and Vallejo 2001). Second, the Glasgow et Burkholder, CCMP1827, CCMP1828, and mitochondrion likely emerged at nearly the same time as CCMP1835) were grown with an algal prey (Rhodomonas sp. Karsten CCMP768). Seawater was adjusted to 28 psu for the nucleus (Gray et al. 1999), rendering mt genes de- most species and to 15 psu for Rhodomonas sp., K. micrum,and spite their organellar location appropriate for studying the heterotrophic dinoflagellates. The temperature was eukaryotic host cell phylogeny (Lang et al. 1998, maintained at 15 Æ 11 CforAlexandrium tamarense (Lebour) 1999). Third, some mt genes are closer to a molecu- Balech; 20 Æ 11 CforRhodomonas, K. micrum, Akashiwo sp., lar clock than SSU rDNA (i.e. they have a relatively Ceratium sp. Schrank, and the heterotrophic dinoflagellates; constant mutation rate across taxa) and thus are ideal and 25 Æ 11 CforK. brevis and the two Symbiodinium species for cross-taxa phylogenetic studies (Saccone et al. (CCMP830, CCMP832). Illumination was provided in a 12:12-h light:dark cycle with a photon flux of around 2000). Cytochrome b (cob) is one of the mt genes 100 mmol photons Á m–2 Á s–1. The growth rate was monitored most widely used for phylogenetic and population ge- by microscopic cell counts using a Sedgwick-Rafter chamber netic analyses (Conway et al. 2000, Taylor and Hel- (Phycotech, St. Joseph, MI, USA). lberg 2003). A combination of cob and nuclear genes Sample collection and DNA extraction. Samples were collect- has provided robust phylogenetic trees for alveolates ed when cultures were in the exponential growth phase. and other organisms (Serizawa et al. 2000, Rathore Samples of heterotrophic dinoflagellates were collected after feeding was discontinued for 2 days and few cells of the prey et al. 2001). To date, cob has been studied only for alga Rhodomonas sp. were present. Previous studies had three species of dinoflagellates (Lin et al. 2002), and its shown that Rhodomonas cob and SSU rDNA sequences were utility for inferring the dinoflagellate phylogeny has distinct from those of dinoflagellates. The PCR primers used yet to be explored. Here we cloned and sequenced cob to amplify the dinoflagellates genes did not therefore target for 14 species from six major dinoflagellate orders: the Rhodomonas homologs, even if contaminating DNA was Gonyaulacales, Gymnodiniales, Prorocentrales, Peri- present in the samples (Zhang and Lin 2002). The cells were harvested by centrifugation at 3000 g at 41 C for 20 min. diniales, Suessiales, and Dinamoebales (Pfiesteria and These cell pellets were subjected to DNA extraction in a its relatives). We then used the existing and newly ob- buffer containing 0.1 M EDTA, 1% SDS essentially following tained cob gene sequences to reconstruct the dino- Zhang and Lin (2002). flagellate tree. PCR, cloning, and sequencing. To PCR amplify cob from Based on cob and SSU rDNA analyses, we also at- dinoflagellates, a set of primers was designed from the highly tempted to address the phylogeny of the Gymnodini- conserved regions of this gene based on previous data ales. This clade includes taxa (e.g. Gymnodinium Stein (Zhang and Lin 2002). The primer sequences were Dino- cob1F (forward), 50-ATGAAATCTCATTTACAWWCATAT- sensu stricto, Akashiwo spp.) that, like most photosyn- CCTTGTCC-30, and Dinocob1R (reverse), 50-TCTCT- thetic dinoflagellates, possess peridinin as the main ac- TGAGGKAATTGWKMACCTATCCA-30. The PCR reaction cessory pigment as well as taxa (Karenia spp. Hansen et was done using approximately 50 ng of genomic DNA for Moestrup, Karlodinium micrum [Leadbeater et Dodge] each species, and amplification