New Species of Clade B Symbiodinium (Dinophyceae) from the Greater Caribbean Belong to Different Functional Guilds: S
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J. Phycol. 51, 850–858 (2015) © 2015 Phycological Society of America DOI: 10.1111/jpy.12340 NEW SPECIES OF CLADE B SYMBIODINIUM (DINOPHYCEAE) FROM THE GREATER CARIBBEAN BELONG TO DIFFERENT FUNCTIONAL GUILDS: S. AENIGMATICUM SP. NOV., S. ANTILLOGORGIUM SP. NOV., S. ENDOMADRACIS SP. NOV., AND S. PSEUDOMINUTUM SP. NOV.1 John Everett Parkinson2 Department of Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA Mary Alice Coffroth Graduate Program in Evolution, Ecology and Behavior and Department of Geological Sciences, State University of New York at Buffalo, Buffalo, New York 14260, USA and Todd C. LaJeunesse Department of Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA Molecular approaches have begun to supersede as corals (Trench 1993). Despite their ecological traditional morphometrics in the species delineation importance, the formal taxonomic description of of micro-eukaryotes. In addition to fixed differences Symbiodinium species has lagged far behind the in DNA sequences, recent genetics-based recognition of molecular diversity within the system, descriptions within the dinoflagellate genus impeding research efforts (LaJeunesse et al. 2012). Symbiodinium have incorporated confirmatory One major reason for this delay is that most mem- morphological, physiological, and ecological bers of the genus bear striking morphological simi- evidence when possible. However, morphological larities to one another. Of the handful of species and physiological data are difficult to collect from descriptions that exist, earlier work emphasized species that have not been cultured, while the natural morphometrics (Freudenthal 1962, Trench and ecologies of many cultured species remain unknown. Blank 1987, Blank and Huss 1989, Trench and Here, we rely on genetic evidence—the only data Thinh 1995, Hansen and Daugbjerg 2009), while consistently available among all taxa investigated—to recent work has shifted toward greater reliance on describe four new Clade B Symbiodinium species. The genetic evidence (LaJeunesse et al. 2012, 2014, ‘host-specialized’ species (S. antillogorgium sp. nov. 2015, Jeong et al. 2014, Hume et al. 2015, Lee et al. and S. endomadracis sp. nov.) engage in mutualisms 2015). with specific cnidarian hosts, but exhibit differences Traditionally, only dinoflagellate species that in our ability to culture them in vitro. The could be cultured were formally described. How- ecologically ‘cryptic’ species (S. aenigmaticum sp. nov. ever, culturing is a highly selective process (von and S. pseudominutum sp. nov.) thrive in culture, but Wintzingerode et al. 1997), particularly for symbi- their roles or functions in the ecosystem (i.e., niches) otic dinoflagellates (Rowan 1998, Carlos et al. 1999, are yet to be documented. These new species call Santos et al. 2001). Efforts to culture the mutualists further attention to the spectrum of ecological guilds that are numerically dominant in a given host have among Symbiodinium. often failed (Santos et al. 2001, LaJeunesse 2002, Goulet and Coffroth 2003). Although most of these Key index words: Clade B; culture; ecological guilds; evolutionarily derived, ‘host-specialized’ lineages do free-living; species recognition; Symbiodinium; not persist in artificial culture (LaJeunesse et al. symbiosis; taxonomy 2005, Krueger and Gates 2012), they are some of Abbreviations: Cob, cytochrome b, cp23S, chloro- the most abundant and ecologically important Sym- plast 23S rDNA; ITS, internal transcribed spacer biodinium. At the same time, those Symbiodinium that perform well in culture are often low abundance types distinct from the dominant symbiont in hos- The genus Symbiodinium encompasses a diverse pite (Santos et al. 2001, LaJeunesse 2002). They array of unicellular dinoflagellates, many of which might be background endosymbionts, opportunistic participate in mutualisms with cnidarian hosts such parasites, ectosymbionts, or even free-living hetero- trophs temporarily captured in the gastrovascular cavity or surface mucus (LaJeunesse 2002, Jeong 1Received 1 April 2015. Accepted 24 July 2015. 2Author for correspondence: e-mail [email protected]. et al. 2014, LaJeunesse et al. 2015). As these Editorial Responsibility: S. Lin (Associate Editor) lineages are difficult to characterize ecologically, but 850 NEW SYMBIODINIUM SPECIES FROM DIFFERENT GUILDS 851 clearly occupy distinct niches compared to classically recently begun the process of speciation and those mutualistic host-specialized species, we refer to them that are farther along. as ‘cryptic’ (sensu LaJeunesse 2001). Here, we use a combination of genetic concor- Population genetic analyses have shown that sex- dance at multiple phylogenetic markers and some ual recombination occurs within but not between ecological information to test the hypothesis that sympatric Symbiodinium populations with distinct four Symbiodinium Clade B lineages are genetically ecological distributions (LaJeunesse et al. 2010, isolated and represent distinct species. Two of the 2014, Thornhill et al. 2014). Such patterns reflect focal lineages associate with cnidarian hosts and reproductive isolation and serve as a powerful appear host-specialized. The first, which has an ITS2 basis for species delineation (LaJeunesse et al. sequence corresponding to type B1 (sensu LaJe- 2014). However, developing microsatellite markers unesse 2001), is mutualistic with sea plumes in the can be resource-intensive, and their utility may be genus Antillogorgia. The second, corresponding to restricted to a few closely related lineages. In the type B7 (sensu LaJeunesse 2002) is mutualistic with absence of population genetic markers, the com- stony corals in the genus Madracis (Diekmann et al. bined analysis of more conservative, unlinked phy- 2003, Pettay and LaJeunesse 2007). The former logenetic markers spanning nuclear, shows a degree of culturability while the latter has mitochondrial, and plastid genomes can effectively not been successfully brought into unialgal culture distinguish evolutionarily divergent lineages (Sam- using current techniques and media. The other two payo et al. 2009). Recombination within species focal lineages thrive in culture but to date have not results in different genealogies among unlinked been detected in host tissues using conventional loci, while between species, drift removes ancestral genetic approaches, and therefore exhibit cryptic, alleles, producing concordant genealogies. Thus, potentially free-living ecologies. The first is closely species can be delimited if they exhibit congruent related to type B23 (sensu Finney et al. 2010). The reciprocal monophyly or nondiscordance at multi- second is closely related to S. minutum (with which ple unlinked loci (Sampayo et al. 2009, Leliaert it shares ITS2 type B1; LaJeunesse et al. 2012). et al. 2014). We recently used this multi-gene approach to delineate S. minutum and S. psygmophilum, two Clade MATERIALS AND METHODS B Symbiodinium (LaJeunesse et al. 2012). These taxa Specimen collection. Tissues from the host cnidarians Antillo- exhibited clear differentiation and reciprocal mono- gorgia bipinnata, A. elisabethae and Madracis spp. were collected phyly across multiple genomic loci, indicating a lack from the Florida Keys, Barbados, Belize and/or Curacßao to of sexual recombination which is consistent with the target symbionts with host-specialized associations. Some of Biological Species Concept (Mayr 1942, Avise and the samples had been used in an earlier study (Finney et al. Wollenberg 1997). Several additional lines of evi- 2010) and were previously screened for the presence of Clade dence were also available, though not all of them B Symbiodinium using denaturing gradient gel electrophoresis of the ITS region as described by Sampayo et al. (2009). New are needed to describe species. S. minutum and samples were similarly screened. Whole tissues were preserved S. psygmophilum were known to function as common in high-salt, 20% DMSO buffer (Seutin et al. 1991) and host-specialized symbionts, so their ecologies and stored at À20°C until processed for molecular analysis. biogeographies had been well studied and deter- Cultures and cell size analysis. Monoclonal cultures of Clade mined to be distinct (satisfying the Ecological Spe- B Symbiodinium isolated from western Atlantic cnidarian hosts cies Concept; Van Valen 1976). They were readily were acquired from the Robert K. Trench collection and the maintained in culture, allowing for physiological Buffalo Undersea Reef Research Culture Center (BURRCC) collection. Cultures were maintained in liquid media (ASP8- experiments that revealed further functional differ- A; Ahles 1967) and kept in incubators delivering 80– À À ences in thermal tolerance. Cell size disparities sug- 120 lmol photons Á m 2 Á s 1 photosynthetically active radia- gested morphological divergence reflective of tion on a 14:10 L:D photoperiod under Philips fluorescent evolutionary divergence. Indeed, subsequent elec- tubes (Koninklijke Philips Electrons, Amsterdam, the Nether- tron microscopy identified differences in amphies- lands) at a temperature of 26°C. mal plate tabulations between the species (Lee et al. Cultures were photographed during log phase growth 2014). Because ecological and morphological data under bright-field illumination at a magnification of 400–1,0009 using an Olympus BX61 compound microscope have thus far corresponded to concordant phyloge- (Olympus Corp., Tokyo, Japan) with a Jenoptik ProgRes CF netic data from multiple