MOLECULAR PHYLOGENY OF THE HETEROTROPHIC DINOFLAGELLATES, PROTOPERIDINIUM, DIPLOPSALIS AND PREPERIDINIUM (DINOPHYCEAE), INFERRED FROM LARGE SUBUNIT rDNA 1 Kristin E. Gribble 2 and Donald M. Anderson Woods Hole Oceanographic Institution, Biology Department, MS 32, Woods Hole, Massachusetts 02543 USA The genera Protoperidinium Bergh, Diplopsalis Bergh, and Preperidinium Mangin, comprised of species of marine, thecate, heterotrophic dinoflag- ellates in the family Protoperidinaceae Balech, have had a confused taxonomic history. To elucidate the validity of morphological groupings within the Pro- toperidinium and diplopsalids, and to determine the evolutionary relationships between these and other dinoflagellates, we undertook a study of molecular phylogeny using the D1–D3 domains of the large subunit (LSU) of the rDNA. Based on morphology, the 10 Protoperidinium species examined belonged to three subgenera and five morphological sections. Two diplopsalid species were also included. Single- cell PCR, cloning, and sequencing revealed a high degree of intraindividual sequence variability in the LSU rDNA. The genus Protoperidinium appeared to be recently divergent in all phylogenetic analy- ses. In maximum parsimony and neighbor joining analyses, Protoperidinium formed a monophyletic group, evolving from diplopsalid dinoflagellates. In maximum likelihood and Bayesian analyses, however, Protoperidinium was polyphyletic, as the lenticular, diplopsalid heterotroph, Diplopsalis lenti- cula Bergh, was inserted within the Protoperidinium clade as basal to Protoperidinium excentricum (Paul- sen) Balech, and Preperidinium meunieri (Pavillard) Elbra¨chter fellwithin a separate clade as a sister to the Oceanica and Protoperidinium steidingerae Bale- ch. In all analyses, the Protoperidinium were divid- ed into two major clades, with members in the Oceanica group and subgenus Testeria in one clade, and the Excentrica, Conica, Pellucida, Pyriforme and Divergens sections in the other clade. The LSU rDNA molecular phylogeny supported the histori- cal morphologically determined sections, but not a simple morphology based model of evolution based on thecal plate shape. Key index words: Diplopsalis; heterotrophic dino- flagellate; LSU rDNA; molecular phylogeny; Prep- eridinium; Protoperidinium; SEM; single-cell PCR; taxonomy Abbreviations: DI, deionized water; FSW, filtered seawater; LSU, large subunit; ML, maximum likeli- hood; MP, maximum parsimony; NJ, neighbor- joining The genera Protoperidinium Bergh, Diplopsalis Bergh, and Preperidinium Mangin are comprised of species of marine, thecate, heterotrophic dinoflagel- lates in the family Protoperidinaceae Balech. As rap- torial, often selective feeders on species of large phytoplankton, including diatoms, dinoflagellates (Jacobson and Anderson 1986, Naustvoll 2000), and even zooplankton eggs (Jeong 1996), these het- erotrophs play an important, but poorly defined, role in planktonic trophic dynamics. These genera have had a confused taxonomic history, and until recently, no genetic information has been available to inform species identification or to resolve their evolutionary relationships with one another or with the other dino- flagellates. Protoperidinium is a cosmopolitan genus of more than 200 morphologically defined species (Balech 1974). As summarized here from comprehensive his- torical reviews by Taylor (1976), Abe ´ (1981), Dodge (1982), and references therein, the genus has a long and complex taxonomic history. The genus Peridinium Ehrenberg was established by Ehrenberg in 1830, and originally included not only species now in the Pro- toperidinium but also those in Gymnodinium Stein and Ceratium Schrank. Bergh erected the new genus Pro- toperidinium in 1881, including in it species with distinct sulcal lists, but leaving in the Peridinium species with antapical horns or without sulcal lists (Bergh 1881). Over the next few decades, many other workers di- vided the Peridinium into subgroups based largely on cell shape and girdle configuration until, in 1909, Ko- foid provided the detailed thecal morphological de- scriptions upon which the current thecal classification is based (Kofoid 1909). In 1912, Jo ¨rgensen proposed a system of subdivi- sion of the Peridinium based upon the combinations of the thecal plate patterns of the ventral and dorsal epitheca (Jo ¨rgensen 1912). Species with two epithecal intercalary plates were placed in a new genus, Arch- aeperidinium, while Peridinium retained species with three intercalary plates. Based upon the shape of the first apical (1 0 ) plate, two subgenera were erected, Or- thoperidinium including species with ortho (four sided) 1 0 plates and Metaperidinium including meta (five sided) 1 Received 2 November 2005. Accepted 3 July 2006. 2 Author for correspondence: e-mail kgribble@whoi.edu. 1081 J. Phycol. 42, 1081–1095 (2006) r 2006 by the Phycological Society of America DOI: 10.1111/j.1529-8817.2006.00267.x