J. Phycol. 48, 1380–1391 (2012) © 2012 Phycological Society of America DOI: 10.1111/j.1529-8817.2012.01217.x A GENETICS-BASED DESCRIPTION OF SYMBIODINIUM MINUTUM SP. NOV. AND S. PSYGMOPHILUM SP. NOV. (DINOPHYCEAE), TWO DINOFLAGELLATES SYMBIOTIC WITH CNIDARIA1 Todd C. LaJeunesse,2 John E. Parkinson Department of Biology, The Pennsylvania State University, University Park, Pennsylvania, 16802, USA and James D. Reimer Molecular Invertebrate Systematics and Ecology Laboratory, Rising Star Program, Trans-disciplinary Organization for Subtropical Island Studies, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa, 903–0213, Japan Marine Biodiversity Research Program, Institute of Biogeosciences, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2–15 Natsushima, Yokosuka, Kanagawa, 237–0061, Japan Traditional approaches for describing species of Abbreviations: AA, amino acid; cob, cytochrome b; morphologically cryptic and often unculturable cp23S, chloroplast 23S rRNA gene; DGGE, dena- forms of endosymbiotic dinoflagellates are prob- turing-gradient gel electrophoresis; ITS, internal lematic. Two new species in the genus Symbiodinium transcribed spacer Freudenthal 1962 are described using an integra- tive evolutionary genetics approach: Symbiodinium minutum sp. nov. are harbored by widespread tropical Studies on the diversity, physiology, and ecology anemones in the genus Aiptasia; and Symbiodinium of the genus Symbiodinium have spanned nearly four psygmophilum sp. nov. are harbored by subtropical decades. Yet it was not until widespread and fre- and temperate stony corals (e.g., Astrangia, Cladocora, quent coral bleaching began that research into and Oculina) from the Atlantic Ocean and cnidarian-dinoflagellate symbioses expanded almost Mediterranean Sea. Both new species are readily exponentially, with many studies focused on how distinguished from each other by phylogenetic the identity of the symbiont relates to thermal toler- disparity and reciprocal monophyly of several nucleic ance of reef-building corals (e.g., Berkelmans and acid sequences including nuclear ribosomal internal van Oppen 2006, Jones et al. 2008, LaJeunesse et al. transcribed spacers 1 and 2, single copy microsatellite 2009a, 2010b, Fisher et al. 2012). In addition to this flanker Sym15, mitochondrial cytochrome b, and ecological significance, the intracellular nature of the chloroplast 23S rRNA gene. Such molecular these symbiotic associations has also attracted interest evidence, combined with well-defined differences in in developing a cnidarian model system in the study cell size, physiology (thermal tolerance), and ecology of animal–microbe interactions and their cellular (host compatibility) establishes these organisms as biology (Weis et al. 2008). Most research currently distinct species. Future descriptions of Symbiodinium combines DNA sequencing with phylogenetic analy- spp. will need to emphasize genetics-based descrip- ses to assign identity to those symbionts under inves- tions because significant morphological overlap in tigation (for review, see Sampayo et al. 2009). this group obscures large differences in ecology and However, progress connecting genetic diversity to a evolutionary divergence. By using molecular evidence formal nomenclatural framework is hampered by based on conserved and rapidly evolving genes ana- disagreement in the interpretation of observed lyzed from a variety of samples, species boundaries genetic diversity and its taxonomic and ecological are defined under the precepts of Evolutionary and significance (e.g., Correa and Baker 2009, LaJeu- Biological Species Concepts without reliance on an nesse and Thornhill 2011, Stat et al. 2011). arbitrary genetic distance metric. Because ecological Investigations into the species diversity of Symbiodi- specialization arises through genetic adaptations, the nium began in the 1970s using various morphological, Ecological Species Concept can also serve to delimit biochemical, physiological, behavioral, and genetic many host-specific Symbiodinium spp. approaches (Schoenberg and Trench 1980a,b,c). These analyses found significant differences among Key index words: dinoflagellate; species recognition; cultured isolates, suggesting that “zooxanthellae” Symbiodinium; symbiont; taxonomy; zooxanthellae comprised much more than a single species as was previously assumed (Fitt et al. 1981, Blank and Trench 1985, Trench and Blank 1987, Banaszak 1Received 10 February 2012. Accepted 1 June 2012. et al. 1993, Trench 1993). Numerous DNA base sub- 2Author for correspondence: e-mail [email protected]. stitutions in conserved ribosomal and mitochondrial 1380 DELIMITING SPECIES OF SYMBIODINIUM WITH GENETICS 1381 genes from cultured and natural samples provide TABLE 1. List of all binomials (valid and invalid) used in confirmatory evidence that the genus Symbiodinium connection with Symbiodinium. Names in bold are formally described species, while names in quotation marks are likely originated in the Mesozoic Era and comprises nomina nuda (published specific epithets without formal distantly related monophyletic groups, or clades diagnosis). Clade assignments follow LaJeunesse (2001). (Rowan and Powers 1991, 1992, McNally et al. 1994, LaJeunesse 2001, Tchernov et al. 2004, Stern et al. Species Clade Author(s) 2010). Symbiodinium “bermudense” B Banaszak et al. (1993) The basic subdivision of the genus Symbiodinium Symbiodinium “californium” E Banaszak et al. (1993) into clades is well established, but there is dispute Symbiodinium “cariborum” A Banaszak et al. (1993) over how to interpret the genetic and ecological Symbiodinium “corculorum” A Banaszak et al. (1993) diversity observed within them. Complementary Symbiodinium “fitti” A Pinzo´n et al. (2011) Symbiodinium “glynni” D LaJeunesse et al. (2010b) genetic and ecological data indicate that hundreds Symbiodinium goreaui C Trench and Blank (1987) of genetically distinct lineages (i.e., species) of Symbi- Symbiodinium kawagutii F Trench and Blank (1987) odinium may exist (LaJeunesse 2001, Sampayo et al. Symbiodinium linucheae A (Trench and Thinh 1995)* 2009, Finney et al. 2010, LaJeunesse and Thornhill LaJeunesse (2001) 2011). Most have no formal species description and Symbiodinium A Banaszak et al. (1993) “meandrinae” are often named according to letter (signifying Symbiodinium A Freudenthal (1962), clade) and number combinations, and referred to by microadriaticum Trench and Blank (1987) various authors as “subclades,” “types,” “species,” or Symbiodinium A Blank and Huss (1989) “strains.” These alphanumeric taxonomic schemes microadriaticum var. condylactis differ among members of the research community Symbiodinium B LaJeunesse and Trench and create additional taxonomic confusion (e.g., “muscatinei” (2000) LaJeunesse 2001, 2002, van Oppen et al. 2001, Symbiodinium natans A Hansen and Daugbjerg Santos et al. 2003, Fabricius et al. 2004, Stat et al. (2009) 2011). Clearly, nomenclatural clarity and taxonomic Symbiodinium pilosum A Trench and Blank (1987) Symbiodinium “pulchrorum” B Banaszak et al. (1993) stability are greatly needed. Without valid scientific Symbiodinium “trenchi” D LaJeunesse et al. 2005 names, the accurate communication of Symbiodinium diversity, physiology, ecology, and evolution will *First described as belonging to Gymnodinium. remain problematic (Blank and Trench 1986, LaJeu- nesse et al. 2009b). The life cycle of Symbiodinium alternates between ability to directly compare and build off one coccoid and motile phases (Fitt and Trench 1983). another’s cumulative findings. The motile cells possess morphological variations Cnidarian hosts usually harbor monospecific pop- utilized for traditional species description, but the ulations of Symbiodinium comprising a single domi- proper imaging of this stage often requires cultur- nant genotype (Goulet and Coffroth 2003a,b, Pettay ing (currently problematic for most Symbiodinium) and LaJeunesse 2007, 2009, Thornhill et al. 2009, and the meticulous characterization of slight differ- Andras et al. 2011, Pettay et al. 2011, Pinzo´n et al. ences in external and internal morphology (Blank 2011, Wham et al. 2011). Thus, most host individu- 1986, Trench and Blank 1987, Blank and Huss als and/or colonies act as culture vessels, and sam- 1989, Trench and Thinh 1995, Hansen and Daugb- pling from them provides access to relatively jerg 2009). Of the nine phylogenetic clades of purified genotypes or strains (i.e., individual clones) Symbiodinium (designated clades A-I; sensu Rowan of a particular Symbiodinium sp. Multiple genetic anal- and Powers 1991), formal species descriptions using yses may be employed on each sample with the conventional morphological features are published for understanding that there is little contamination clade A (S. microadriaticum Freudenthal 1962, S. pilosum from other genomes (e.g., Sampayo et al. 2009). Trench and Blank 1987, S. natans Hansen and Daugb- Multi-locus data are increasingly used to test for jerg 2009, S. linucheae Trench and Thinh 1995), clade reproductive isolation and genetic divergence C(S. goreaui Trench and Blank 1987), and clade F among morphologically cryptic and closely related (S. kawagutii Trench and Blank 1987, Trench 2000). organisms (e.g., Hausdorf and Hennig 2010, Gazis Nine additional binomials (S. bermudense, S. californium, et al. 2011). It was recently demonstrated that a S. cariborum, S. corculorum, S. fitti, S. glynni, S. meandri- lineage-based approach (sensu Avise and Wollenberg nae, S. muscatinei, S. pulchrorum, S. trenchi) appear in