Symbiodinium Tridacnidorum Sp. Nov., a Dinoflagellate Common to Indo

Home , Dinoflagellate

This article was downloaded by: [Pennsylvania State University] On: 15 April 2015, At: 10:23 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

European Journal of Phycology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tejp20 Symbiodinium tridacnidorum sp. nov., a dinoflagellate common to Indo-Pacific giant clams, and a revised morphological description of Symbiodinium microadriaticum Freudenthal, emended Trench & Blank Sung Yeon Leea, Hae Jin Jeonga, Nam Seon Kanga, Tae Young Janga, Se Hyeon Janga & Todd C. Lajeunesseb a School of Earth and Environmental Sciences, College of Natural Sciences, Seoul National University, Seoul 151-747, Korea b Department of Biology, 327 Mueller Laboratory, Pennsylvania State University, Click for updates University Park, PA 16802, USA Published online: 13 Apr 2015.

To cite this article: Sung Yeon Lee, Hae Jin Jeong, Nam Seon Kang, Tae Young Jang, Se Hyeon Jang & Todd C. Lajeunesse (2015): Symbiodinium tridacnidorum sp. nov., a dinoflagellate common to Indo-Pacific giant clams, and a revised morphological description of Symbiodinium microadriaticum Freudenthal, emended Trench & Blank, European Journal of Phycology, DOI: 10.1080/09670262.2015.1018336 To link to this article: http://dx.doi.org/10.1080/09670262.2015.1018336

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions Eur. J. Phycol. (2015), 1–18

Symbiodinium tridacnidorum sp. nov., a dinoﬂagellate common to Indo-Paciﬁc giant clams, and a revised morphological description of Symbiodinium microadriaticum Freudenthal, emended Trench & Blank

SUNG YEON LEE1, HAE JIN JEONG1, NAM SEON KANG1, TAE YOUNG JANG1, SE HYEON JANG1 AND TODD C. LAJEUNESSE2

1School of Earth and Environmental Sciences, College of Natural Sciences, Seoul National University, Seoul 151-747, Korea 2Department of Biology, 327 Mueller Laboratory, Pennsylvania State University, University Park, PA 16802, USA

(Received 28 July 2014; revised 16 November 2014; accepted 16 November 2014)

The dinoflagellate genus Symbiodinium contains numerous genetically distinct lineages that appear ‘morphologically cryptic’. However, detailed morphological assessments of plate formulae visible in the motile phase (mastigote) of two distantly related Symbiodinium spp., representing Clades ‘A’ and ‘E’, were recently shown to be different. While there are several formally described species in Clade A, genetic evidence indicates that many more remain uncharacterized. We focused on closely related phylogenetic lineages within this group to examine whether differences in morphology can be used together with genetic and ecological evidence to describe new species. We found fixed differences in nuclear (internal transcribed spacer (ITS) and large subunit rDNA), chloroplast (cp23S) and mitochondrial (cob) gene sequences from cultured and field-collected samples of Symbiodinium microadriaticum (sensu Trench & Blank, 1987) and Symbiodinium sp. associated predominantly with giant clams and Pacific Cassiopea jellyfish (comprising members of the ITS2 A3 lineage, sensu LaJeunesse 2001). Amphiesmal plate tabulations were formulated for the strain of S. microadriaticum (CCMP2464) used by Trench & Blank (1987) in their emended description, and two strains of type A3 (CCMP832 and rt-272) cultured from Indo-Pacific giant clams in the subfamily Tridacninae. The Kofoidian plate formula for type A3 consists of the small plate (x), the elongated amphiesmal vesicle (EAV), 5′, 6a, 8′′,9–11s, a 2-row cingulum, 7′′′ and 3′′′′ (and occasionally 2′′′′), and is different from S. microadriaticum, which has a plate formula of x, EAV, 4′, 5a, 8′′,9–13s, a 2-row cingulum, 6′′′ and 2′′′′. Based on morphological and genetic comparisons, we recognized Symbiodinium tridacnidorum sp. nov., a new Indo-Pacific species. The plate arrangement exhibited by S. microadriaticum appears to be more similar to the distantly related S. natans (also in Clade A). When the tabulations for all three Clade A species are compared with S. voratum (Clade E), the amount of morphological differentiation between species does not correspond to their degree of genetic divergence.

Key words: Cassiopea, Dinoﬂagellates, Kofoidian plate tabulation, Paciﬁc Ocean, Symbiodinium Clade A, taxonomy, Tridacninae Downloaded by [Pennsylvania State University] at 10:23 15 April 2015

INTRODUCTION despite their ecological and economic importance in Dinoﬂagellates in the genus Symbiodinium, commonly marine ecosystems, the taxonomy of Symbiodinium known as ‘zooxanthellae’, are symbiotic with various remains limited because few biologically discrete enti- invertebrates such as corals, sponges, sea anemones, ties have formal binomials. jellyﬁsh, nudibranchs, clams and single-cell hosts After Symbiodinium microadriaticum was initially including ciliates and foraminifera (Trench, 1993; described by Freudenthal (1962), the description was LaJeunesse, 2002; Lobban et al., 2002;Pochonet al., emended by Kevin et al.(1969). Owing to the pre- 2007), although some occur free-living on various sub- vailing dogma which assumed that Symbiodinium was strata and in the water column (Hirose et al., 2008; a monotypic genus (McLaughlin & Zahl, 1966; Kevin Porto et al., 2008;Jeonget al., 2012, 2014; Yamashita et al., 1969; Taylor, 1974), there was little interest in & Koike, 2013). The symbioses that many form with a investigating species diversity further until Loeblich broad diversity of stony corals, order Scleractinia, are & Sherley (1979) attempted to rename the species crucial for building tropical reef ecosystems. However, Zooxanthella microadriatica on the basis that no pre- vious effort had been made to differentiate it from the symbiont cells that Brandt (1881) had examined from Correspondence to: Hae Jin Jeong. E-mail: [email protected]

the radiolarian, Collozoum inerme. Loeblich & Symbiodinium are morphologically nondescript, Sherley (1979) were the first to provide Kofoidian except for obvious differences in cell size. Scanning plate tabulations, based on scanning electron micro- electron microscopy (SEM) allows the resolution of scopy (SEM), for an isolate obtained from the cnidar- Kofoidian plate patterns on the motile cells (masti- ian Cassiopea xamachana, in the Florida Keys, USA gotes) of cultured isolates (Jeong et al., 2014). (isolate 406). They included in their study an environ- Amphiesmal plate formulae are different between S. mental isolate from the temperate northwestern natans (Clade A; Hansen & Daugbjerg, 2009), S. Atlantic Ocean (isolate 395) whose tabulation was voratum (Clade E; Jeong et al., 2014), and S. minutum distinct, yet too similar to justify describing it as a and S. physigmophilum (Clade B; LaJeunesse et al., separate species. 2012; Lee et al., 2014), yet these species are geneti- The revelation that Symbiodinium was speciose cally divergent from each other. It is not known began with Trench & Blank (1987), who introduced whether closely related species (i.e. members of the S goreauii, S. pilosum and S. kawagutii, as well as same clade) differ in the number and arrangement of providing another emendation to S. microadriaticum their amphiesmal plates. An assessment of morpholo- (Trench & Blank 1987; Trench, 2000). The criteria gical variation and evolution among distantly and used in the justification of these additional species closely related Symbiodinium would benefit from relied on differences in cell size and ultrastructure expanding our comparative analyses of thecal mor- including the number of chromosomes, pyrenoids phology, using SEM, when new species are described. and pyrenoid stalks, as well as in the relative volumes In this report, we have characterized the amphies- of chromosomes, nuclei, chloroplasts and mitochon- mal plate morphology and pattern found on S. micro- dria. This work was also the first to utilize molecular adriaticum (Symbiodinium strain CCMP2464, or rt- evidence on photosystem proteins to substantiate 061) isolated from the jellyfish Cassiopea xamachana these claims that the lineages were genetically distinct (ITS2 type A1, sensu LaJeunesse, 2001) because the (Trench & Blank, 1987). Molecular sequence data plate tabulation of this isolate was not described by eventually revealed large genetic differences among Trench & Blank (1987). The genetic identities of the members of this genus, clades of which were desig- isolates Loeblich & Sherley (1979) characterized mor- nated as Clades A, B, C, etc. (Rowan & Powers, phologically and assigned to the species Zooxanthella 1991). The subsequent application of various genetic microadriatica as reported above, were used to methods and markers to identify ecologically distinct resolve their phylogenetic relationships within the lineages facilitated a surge in discovery about pro- genus Symbiodinium. We combined these results cesses underlying the ecology and evolution of these with the morphological characterization of two cul- dinoflagellates (Sampayo et al., 2009; Baums et al., tured strains (CCMP832 and rt-272) isolated from 2014; Thornhill et al., 2014). giant clams in the Pacific Ocean and for which genetic Since the early 1990s, genetic analyses have domi- evidence indicated that they represented a novel spe- nated many aspects of Symbiodinium research cies in Clade A (initially designated ITS2 type A3, (Coffroth & Santos, 2005; Sampayo et al., 2009), sensu LaJeunesse, 2001). The morphological compar- and are the de facto diagnostic to examine ecological ison between S. microadriaticum and isolates from and geographic patterns of diversity (LaJeunesse two giant clam hosts were combined with additional et al., 2010). While there is some contention regarding sequence analyses of the large subunit (LSU) rDNA, how rDNA data are interpreted (Stat et al., 2011; partial chloroplast large subunit (cp23S) and mito- Downloaded by [Pennsylvania State University] at 10:23 15 April 2015 LaJeunesse & Thornhill 2011), the convergence of chondrial cytochrome b (cob). The genetic variation evidence from a combination of several genetic mar- in these markers was analysed from multiple cultures kers can delimit reproductively isolated lineages as well as field-collected specimens from several eco- (LaJeunesse & Thornhill, 2011; Pochon et al., 2012; logical and geographic sources. Finally, we examined Thornhill et al., 2014); and has led to the collective to what extent morphological differences (i.e. plate use of these data in formally describing species tabulations) were concordant with phylogenetic rela- including Symbiodinium spp. that are not in culture tionships among Symbiodinium spp. for which these (LaJeunesse et al., 2012, 2014). However, it is impor- data were available. tant to relate, when possible, these new approaches with more traditional methods for describing dinoflagellate species. MATERIALS AND METHODS While most Symbiodinium are unable to flourish in Cultures and growth conditions of Symbiodinium spp. artificial growth media, there are some that proliferate Symbiodinium microadriaticum (CCMP2464, formally rt- in vitro. Preliminary genetic evidence suggests that 061 sensu Trench & Blank, 1987) and strain CCMP832 many of these strains represent undescribed species were obtained from the National Center for Marine Algae for which a combination of modern and conventional and Microbiota (NCMA) (Table 1). Strain CCMP 2464 of taxonomic approaches can be applied (e.g. Jeong S. microadriaticum was isolated originally from the jelly- et al., 2014). When viewed with light microscopy, fish Cassiopea xamachana in the Florida Keys, USA. A Species of Giant Clam Symbiont 3

) The culture CCMP832 was originally obtained from the

1987 giant clam Hippopus hippopus living on the Great Barrier Reef, Australia; and isolate rt-272 was cultured from Tridacna gigas from Palau (LaJeunesse, 2001). The cultured isolates 395 and 406 analysed by Loeblich & Sherley (1979) were obtained from the University of Texas at Austin culture collection (UTEX LB 2281 and UTEX LB 2282, respectively). Cultures were grown in L1 seawater medium without silicate (Guillard & Hargraves, 1993), at a temperature of 25°C with continu- ous illumination of 20 μmol photons m−2 s−1 cool white ﬂuorescent light under a 14:10 h light-dark cycle. They were transferred approximately every 2–3 weeks to new d GenBank accession numbers for rDNA 250 ml PC bottles containing fresh media. When culture volumes were large enough to analyse, genetic and morphological characterizations were performed.

Scanning electron microscopy (SEM) Cell size and the analyses of the amphiesmal plate shape, number and arrangement of culture CCMP2464 (S. microadriaticum), CCMP832 and rt-272 were performed by SEM. Cells from dense cultures were fixed for 10 min in osmium tetroxide at a final concentration of 0.3% (w/v) in seawater. Cell collection, dehydration, drying and mounting were performed as described in Kang et al.(2010). (jelly) 1977 FJ823602 AF060896 Trench & Blank ( (giant clam) 1966 HG939435 HG939436 This paper Transmission electron microscopy (TEM) (giant clam) 1982 KF364600 This paper TEM was used for counting the permanently condensed chromosome bodies typical of dinoflagellates. For sample prepara- tion and sectioning, cells from dense cultures were fixed in 2.5% (w/v) glutaraldehyde (final concentration). After 1.5–2 Hippopus hippopus Tridacna gigas Cassiopea xamachana h, cells were concentrated at 1610 x g for 10 min in a Vision Centrifuge VS-5500 (Vision Scientific Company, Bucheon, Korea). Next, the supernatants were discarded, and the pellets were transferred to 1.5 ml tubes and rinsed several times with 0.2 M pH 7.4 sodium cacodylate buffer. Then, the pellets were post fixed with 1% (w/v) osmium tetroxide in deionized water

Great Barrier Reef Palau Florida, USA for 90 min. After ﬁxation, the pellets were embedded in agar. Dehydration was performed via a graded ethanol series (50%, 60%, 70%, 80%, 90%, and 100% ethanol, followed by two changes in 100% ethanol). The agar-embedded pellet was then Downloaded by [Pennsylvania State University] at 10:23 15 April 2015

A3 A3 A1 re-embedded in Spurr’s low-viscosity resin and dried for 3 days at 70 °C. Samples were then serially sectioned (80–100 nm) using an RMC MT-XL ultramicrotome (Boeckeler Instruments Inc., Tucson, Arizona, USA), and stained with 3% (w/v) aqu- eous uranyl acetate followed by lead citrate. Finally, samples were observed with a JEOL-1010 transmission electron micro- scope (JEOL Ltd., Tokyo, Japan). The chromosome number (± 832 CCMP 2464 (rt061) SE) for the strains was determined using these serial sections.

sp. nov. CCMP sp. nov. rt-272 Nucleic acid extraction, PCR amplification, sequencing and phylogenetic analyses Nucleic acids were extracted from 10–15 ml of a dense culture of CCMP832 using the AccuPrep® Genomic DNA Extraction Kit (Bioneer Cooperation, Daejeon, Korea). The fi The list of strains used for morphological analyses, including strain name, rDNA genetic identity, location (LC) and date of original collection, an DNA extracts from cultures and eld-collected samples cor- responding to Symbiodinium microadriatiucum (= type A1), S. pilosum (= type A2) and that of type A3 from the Pacific Table 1. SpeciesSymbiodinium tridacnidorum Symbiodinium tridacnidorum Strain Clade-ITS2 type LC Host Collection date GenBank (ITS) GenBank (LSU) Reference Symbiodinium microadriaticum sequences. Ocean obtained and used in previously published or S. Y. Lee et al. 4

unpublished research were included in subsequent genetic HABITAT: Isolated from the mangrove, or upside- analyses. Details such as culture number, host, depth and down jellyfish Cassiopea xamachana. geographic origin as well as journal citations where particular samples were initially analysed are listed in supplemental Symbiodinium tridacnidorum Lee, Jeong, Kang, & Table S1. A request to acquire DNA from S. natans to LaJeunesse sp. nov. (Figs 4–6, 16–23, 28–35) complete sequencing of the cp23S and cob genes was denied. DIAGNOSIS: Mastigote cells are 9.4–12.9 μmin fi PCR ampli cations for the ITS region of CCMP832 length and 7.5–10.6 μm in width. The cell episome (ITS1, 5.8S and ITS2) were performed using the forward is slightly larger than the hyposome. An EAV is pre- primer Euk1209F (Giovannoni et al., 1988) and the reverse primer ITSR2 (Litaker et al., 2003). All other ITS2 amplifi- sent on the apex. The cingulum consists of 2 rows of – cations used the primer-set and reaction conditions specified pentagonal plates. The cingulum is displaced by 0.06 in Sampayo et al.(2009). Amplifications of the LSU region 0.2 cell lengths and 0.2–0.7 cell widths. Plates are D1/D2 was performed using the forward primer D1RF arranged as x, EAV, 5′, 6a, 8′′,9–11s, 17–19c, 7′′′ (Scholin et al., 1994) and the reverse primer LSUB (Litaker and 2–3′′′′. The 5a plate is hexagonal. The 6′′ plate is et al., 2003), or using the primer set and conditions developed pentagonal. The 2′ plate touches the 4′ plate. The size by Zardoya et al.(1995). For PCR amplification, Solg™ f- of the 5′ plate is similar to that of the 2′ plate. A single, © Taq DNA Polymerase (SolGent Co., Daejeon, Korea) was 2-stalked pyrenoid is present. A type E eyespot is ’ used according to the manufacturer s instructions. The partial located beneath the sulcus in motile cells. A well- chloroplast cp23S and mitochondrial cob genes were ampli- developed PE emerges between the longitudinal and fied and directly sequenced for all cultures and field-collected transverse flagella. The nucleus contains a nucleolus samples according to Zhang et al.(2000) and Zhang et al. – (2005), respectively (Table S1). Sanger sequencing of these and 77 83 condensed chromosomes. plastid markers and the LSU was performed using Big Dye Nucleotide sequences of the large ribosomal subunit 3.1 reagents (Life Sciences) and the products analysed on an rDNA (Accession nos KM816405, KM816406, Applied Biosystems 3730XL instrument. KM972551), ITS2 rDNA (Accession no. The phylogenetic analysis of the ITS (ITS1, 5.8S, ITS2) KM816410), partial chloroplast large subunit, cp23S and LSU rDNA regions of the Symbiodinium strains was (Accession nos KM816407–KM816409) and mito- conducted using MEGA v.4 (Tamura et al., 2007) and chondrial cytochrome b, cob (Accession no. Clustal X2 (Larkin et al., 2007), and the sequences from fi outgroup Symbiodinium from Clades D and E were obtained KM816411) genetically de ne this species. from recently published results (Jeong et al., 2014; HOLOTYPE: A holotype slide labelled USNM stub LaJeunesse et al., 2014). Maximum likelihood (ML) analyses 1251931 of a culture fixed with 0.3% (w/v) osmium were conducted using the RAxML 7.0.4 program with a tetroxide has been deposited in the Protist Type GTRGAMMA model (Stamatakis, 2006). Further, 200 inde- Specimen Slide Collection, US Natural History pendent tree inferences were used to identify the best tree. ML bootstrap values were determined using 1000 replicates. Museum, Smithsonian Institution, Washington, Bayesian analyses were conducted using MrBayes v.3.1 District of Columbia, USA. (Ronquist & Huelsenbeck 2003) in the default GTR + G + I TYPE LOCALITY: Tridacna Reef, Great Barrier model to determine the best available model for the data for Reef, Australia (20°00′00′′S, 149°00′00′′E). each region. For all sequence regions, four independent Markov Chain Monte Carlo runs were performed as HABITAT: Isolated from mantle tissue of the giant described in Kang et al.(2010). clam species Hippopus hippopus. ETYMOLOGY: The specific name ‘tridacnidorum‘ is Downloaded by [Pennsylvania State University] at 10:23 15 April 2015 RESULTS based on the subfamily of bivalves (Family Cardioidea Taxonomic descriptions Lamark, 1809), commonly referred to as giant clams. Symbiodinium microadriaticum Freudenthal 1962, emend. by Trench & Blank 1987, emend. by Lee, Morphology of Symbiodinium microadriaticum Jeong, Kang & LaJeunesse (Figs 1–3, 7–15, 24–27) Motile cells of S. microadriaticum (strain CCMP2464) EMENDATION: This dinoflagellate has a Kofoidian were mushroom shaped, with an episome (epitheca) plate formula of x, EAV, 4′, 5a, 8′′,9–13s, 2 row slightly larger than the hemispherical hyposome cingulum, 6′′′,2′′′′. (hypotheca; Figs 1–3). The cells (n = 30) in log phase Nucleotide sequences of the large ribosomal subunit culturewere8.0–13.8 μminlengthand6.3–11.9 μmin rDNA (Accession no. KM972549), partial chloroplast width, respectively (Table 2). In addition, the ratio of large subunit, cp23S (Accession no. KF740693) and the length to width of the living cells was 1.1–1.9 μm mitochondrial cytochrome b, cob (Accession no. (Table 2). However, when fixed and observed under KF206025) genetically define this species. SEM, the cells were slightly smaller and were 7.6–10.0 μminlengthand5.8–7.7 μminwidth(Table 2). The TYPE LOCALITY: Key Largo, Florida, USA (25°00′ ratio of the length to the width under SEM was 1.2–1.5 00′′N, 80°50′00′′W). μm(Table 2). A Species of Giant Clam Symbiont 5 123

PY N

Figs 1–6. Light micrographs of the mastigote (motile), coccoid (spherical) and doublet (dividing) cells from Symbiodinium microadriaticum (Figs 1–3) and S. tridacnidorum sp. nov. (Figs 4–6). Fig. 1. S. microadriaticum mastigote; pyrenoid (PY, white arrowhead), nucleus (N). Fig. 2. S. microadriaticum coccoid. Fig. 3. S. microadriaticum doublet. A single PY is generally located in the centre of the cell. Fig. 4. S. tridacnidorum mastigote; pyrenoid (PY, white arrowhead), nucleus (N). Fig. 5. S. tridacnidorum coccoid. Fig. 6. S. tridacnidorum doublet. All cells contained a reticulated chloroplast located at the periphery of the cell. All scale bars = 2 μm.

The Kofoidian plate formula of S. microadriaticum (Table 2). There were 6 postcingular plates (Table 2; cells was x, EAV, 4′, 5a, 8′′,9–13s, the 2 cingulum Figs 15, 27). The 1′′′,2′′′,4′′′,5′′′ and 6′′′ plates were rows 22–24c, 6′′′, and 2′′′′ (Table 2; Figs 7–15 and 24– quadrangular, but the 3′′′ plate was pentagonal 27). At the cell’s apex, the elongated amphiesmal (Figs 15, 27). Both antapical plates, 1′′′′ and 2′′′′, vesicle (EAV) possessed 6–8 aligned knobs and mea- were pentagonal or hexagonal (Table 2; Figs 15, 27).

Downloaded by [Pennsylvania State University] at 10:23 15 April 2015 sured 1.33–2.65 μm in length and 0.13–0.33 μmin A total of 13 sulcal plates, including 2 S (?) plates and width. This structure was bordered ventrally by the x 1 S.p. plate were located in the sulcus. A peduncle plate and surrounded by apical plates, 2′,3′ and 4′ (PE) was present in the middle of the sulcal plates (Figs 12, 14, 26). Apical plates consisted of a rhom- (Table 2; Figs 7, 11, 24). boid-shaped and relatively large 1′, a quadrangular 2′ plate and hexagonal 3′ plate. Surrounding these were the pentagonal 4′ apical plate and 1a–5a intercalary Morphology of Symbiodinium tridacnidorum plates (Figs 12, 13, 26). The 5 intercalary plates were The motile cells of this culture were mushroom- hexagonal (plates 1a and sometimes 3a) pentagonal shaped, with an episome that was slightly larger than (plates 2a, 5a and sometimes 3a), and heptagonal the hemispherical hyposome (Figs 4–6; Figs S1–S3). – – (plate 4a) (Figs 8 10, 12, 13, 24 26). Of the 8 pre- The cells (n = 30) in log phase growth were found to ′′ ′′ ′′ cingular plates, 1 ,4 and 6 plates were quadrangu- be 9.4–12.9 μm in length and 7.5–10.6 μm in width ′′ ′′ ′′ ′′ ′′ ′′ lar, whereas 2 ,3,4,5,7 and 8 plates were (Table 2). In addition, the ratio of the length of the – pentagonal (Table 2; Figs 7 10, 24, 25). Two wide living cells to the width was 1.1–1.4 μm(Table 2). – cingulum rows consisting of 22 24 pentagonal When ﬁxed and observed under SEM, the cells were amphiesmal plates were located in the centre of the found to be 8.1–10.1 μm in length and 5.6–7.1 μmin – cell. The cingulum was displaced by ~0.1 0.2 times width, and the ratio of the length to width was 1.3–1.5 – the cell length and ~0.4 0.7 times the cell width μm(Table 2). S. Y. Lee et al. 6 Downloaded by [Pennsylvania State University] at 10:23 15 April 2015

Figs 7–15. Scanning electron micrographs of Symbiodinium microadriaticum motile cells. Fig. 7. Ventral view showing the episome, cingulum (C), sulcal plates (s) and hyposome. Fig. 8. Ventral-left lateral view showing the episome, cingulum (C) and hyposome. Fig. 9. Dorsal view showing the episome, cingulum (C) and hyposome. Fig. 10. Ventral-right lateral view showing the episome, cingulum (C) and hyposome. Fig. 11. Antapical-ventral view showing the sulcal plates (s) and peduncle (PE). Fig. 12. Apical view showing the episome and elongated amphiesmal vesicle (EAV) plate. Fig. 13. Apical view showing the dorsal view of episome and EAV plate. Fig. 14. Apical view showing the EAV plate with small knobs (arrowhead). Fig. 15. Antapical view showing the hyposome. All scale bars = 1 μm.

The Kofoidian plate formula of S. tridacnidorum Figs S4–S11). At the cell’s apex, the EAV possessed cells is x, EAV, 5′, 6a, 8′′,9–11s, 2 cingulum rows 7–9 aligned knobs and measured 0.76–1.84 μmin 17–19c, 7′′′ and 3′′′′ (rarely 2′′′′; Table 2; Figs 16–23; length and 0.09–0.37 μm in width (Table 2). This A Species of Giant Clam Symbiont 7 Downloaded by [Pennsylvania State University] at 10:23 15 April 2015

Figs 16–23. Scanning electron micrographs of Symbiodinium tridacnidorum motile cells. Fig. 16. Ventral view showing the episome, cingulum (C), sulcal plates (s), peduncle (PE) and hyposome. Fig. 17. Ventral-left lateral view showing the episome, cingulum (C) and hyposome. Fig. 18. Dorsal view showing the episome, cingulum (C) and hyposome. Fig. 19. Ventral-right lateral view showing the episome, cingulum (C), sulcus (s) and hyposome. Fig. 20. Apical view showing the episome and elongated amphiesmal vesicle (EAV) plate. Fig. 21. Apical view showing the EAV plate with small knobs (arrowhead). Fig. 22. Antapical view showing the hyposome. Fig. 23. Antapical-ventral view showing the sulcal plates (s) and peduncle (PE). All scale bars = 1 μm. S. Y. Lee et al. 8

Figs 24–27. Drawings of Symbiodinium microadriaticum motile cells strain showing the external morphology. Fig. 24. Ventral view. Fig. 25. Dorsal view. Fig. 26. Apical view. Fig. 27. Antapical view. All scale bars = 1 μm.

structure was bordered ventrally by the x plate and usually 3, but sometimes 2, antapical plates (Table 2; surrounded by 4 apical amphiesmal plates (2′,3′,4′ Figs S10, S12, S13). The 1′′′′ and 2′′′′ plates are either

Downloaded by [Pennsylvania State University] at 10:23 15 April 2015 and 5′ plates; Figs 20, 30; Fig. S8). There are 6 inter- pentagonal or hexagonal, while the 3′′′′ is hexagonal calary plates. Of these 4 are hexagonal (plates 1a, 3a, when present (Fig. 22; Figs S4–S13). A total of 4a and 5a), one was pentagonal (2a), and one is either between 9–11 sulcal plates including 2 S (?) plates pentagonal-, hexagonal- or heptagonal-shaped and one posterior sulcal plate (S.p.) was observed. An depending on the cell imaged (6a) (Figs 16–20, 28– accessory sulcal plate positioned between where the 30; Figs S4–S8). For the 8 precingular plates, the 1′′ transverse and longitudinal ﬂagella emerge from the plate is invariably quadrangular, the 2′′,5′′,6′′ and 8′′ cell (Table 2; Figs 23, 28; Fig. S11). plates are pentagonal, however the 3′′,4′′ and 7′′ plates Through TEM analysis, the periphery of the cell are variable and either quadrangular or pentagonal interior of S. tridacnidorum (CCMP832) is often (Table 2; Figs 16–20, 28, 29; Figs S4–S8). Two wide occupied by chloroplast lobes. A single pyrenoid cingulum rows consisting of 17–19 pentagonal plates located in the central part of each cell is connected create the groove around the middle of the cell where by 2 stalks to the adjacent chloroplast and surrounded the transverse ﬂagellum lies. The cingulum is dis- by a distinct starch cap (Figs 32, 35). No chloroplast placed in the region of the sulcus grove by ~0.1–0.2 thylakoid lamellae penetrated the pyrenoid. A large times the cell length and ~0.2–0.7 times the cell width number of lipid globules and starch were observed (Table 2). Of the 7 postcingular plates, 1′′′,2′′′,5′′′,6′′′ (Fig. 32). Furthermore, an eyespot (type E), consisting and 7′′′ plates are quadrangular, while the 3′′′ and 4′′′ of multiple layers of rectangular electron-translucent plates are pentagonal (Table 2; Figs 22, 31). There are vesicles or crystalline deposits, was observed in A Species of Giant Clam Symbiont 9

Figs 28–31. Drawings of Symbiodinium tridacnidorum motile cells showing the external morphology. Fig. 28. Ventral view. Fig. 29. Dorsal view. Fig. 30. Apical view. Fig. 31. Antapical view. All scale bars = 1 μm. Downloaded by [Pennsylvania State University] at 10:23 15 April 2015 sectioned mastigote cells (Fig. 33). A mean of 80 ± 3 Table S1). In addition to the ITS2 ‘A3’ sequence, (SE) condensed chromosomes were estimated from several additional sequence variants, A3a, A6 and serial sections of cell nuclei (n=4 cells; Table 2; A3*(‘A3x’sensu Weber 2009) were found to be diag- Fig. 34). nostic of this Symbiodinium lineage (Fig. 36). These sequences were recovered from direct sequencing of PCR products or from screening using denaturing Phylogenetic delineation of Clade A Symbiodinium gradient electrophoresis (DGGE; see Sampayo et al., Concordant data from ribosomal DNA (ITS2 and 2009 for explanation of the method for identifying LSU), mitochondrial cob and chloroplast cp23S those ITS sequences of diagnostic value in organisms sequences show that S. tridacnidorum is a reproduc- with high intragenomic rDNA variation). The culture tively isolated, evolutionarily divergent lineage and CCMP832 is characterized by the A3* ITS2 sequence, therefore distinct from other described Clade A spe- which differentiated from Symbiodinium A3 by a sin- cies (Figs 36, 37, 38). Some sequence variation in gle base substitution (Fig. 36). The LSU sequence of rDNA was found among cultured and field-collected A3* (e.g. CCMP832) also contained two base substi- specimens originating from giant clams in the sub- tutions (Fig. 38). The rDNA variant of CCMP832 family Tradacninae (formerly the family Tridacnidae) matched with a field-collected sample of Tridacna and the upside-down jellyfish (Cassiopea; Figs 36, 38; maxima originating from 12–15 m depth in Palau S. Y. Lee et al. 10

Figs 32–35. Transmission electron micrographs of Symbiodinium tridacnidorum cells. Fig. 32. A transverse section of a mastigote cell from S. tridacnidorum showing the position of the pyrenoid in the middle of cell, the chloroplasts and type E eyespot (stigma) Downloaded by [Pennsylvania State University] at 10:23 15 April 2015 near the cell’s surface, lipid globules and starch. Fig. 33. A magniﬁed view of the type E eyespot consisting of multiple layers of rectangular electron-translucent vesicles, or crystalline deposits. Fig. 34. Serial sectioning through the nucleus showing large number of condensed chromosomes (approximately 80 ± 3). Fig. 35. A magniﬁed view of a single pyrenoid with 2 stalks, located in the central part of each cell and surrounded by a distinct polysaccharide cap. Scale bar = 1 μm for Figs 32–34 and 0.5 μm for Fig. 35.

(Fig. 38) and from samples obtained from the Indo- tridacnidorum. The only exceptions found were West Pacific, West Pacific and Central South Pacific from samples of Cassiopea andromeda collected in (Figs 36, 37; Weber, 2009). Palau. These were differentiated from the others by a The LSU (and cp23S; data not shown) sequences single base substitution in domain V of the cp23S from the two isolates examined by Loeblich & Sherley marker (Fig. 39). (1979) placed them in the genus Symbiodinium. The rDNA (ITS2 and LSU) and plastid (cp23S and Isolate 406 is a strain of S. microadriaticum cob) sequences used to diagnose cultures and field- (Fig. 38), while isolate 395 matched sequences of S. collected samples of S. microadriaticum were invar- voratum obtained from populations in the northwes- iant despite originating from several locations in the tern Pacific(Fig. 38). north central and western Caribbean (Florida Keys, Mitochondrial cob and partial chloroplast cp23S the Mexican Yucatan and Belize), Red Sea (Gulf of sequences were identical for most samples of S. Aqaba) and one culture (KB8) from O’ahu, Hawaii A Species of Giant Clam Symbiont 11

Table 2. Morphological differences among Clade A Symbiodinium in comparison to S. voratum (Clade E) based on electron microscopy.

S. tridacnidorum S. microadriaticum S. natans S. voratum

AP length (μm; living cells) 9.4–12.9 (10.9) 8.0–13.8 (9.8) 9.5–11.5 (10) 10.8–16.2 (13.1) Cell width (μm; living cells) 7.5–10.6 (8.8) 6.3–11.9 (8.1) 7.4–9.0 (8) 7.8–11.5 (9.5) Ratio of length to width (living cells) 1.1–1.4 (1.3) 1.1–1.9 (1.2) Unk. 1.1–1.8 (1.4) AP length (μm; SEM) 8.1–10.1 (9.2) 7.6–10.0 (9.2) 10.38* 8.5–12.4 (10.5) Cell width (μm; SEM) 5.6–7.1 (6.5) 6.3–11.9 (8.1) 8.25* 6.4–9.8 (8.2) Ratio of length to width (SEM) 1.3–1.5 (1.4) 1.2–1.5 (1.3) 1.26* 1.2–1.4 (1.3) EAV length (μm) 0.76–1.84 (1.42) 1.33–2.65 (1.94) 2 1.75–3.09 (2.45) EAV width (μm) 0.09–0.37 (0.18) 0.13–0.33 (0.21) 0.2 0.15–0.27 (0.2) Numbers of aligned knobs on EAV 7–96–8129–13 Cingulum displaced by cell length 0.06–0.20 (0.12) 0.06–0.24 (0.14) 0.23* 0.13–0.21 Cingulum displaced by cell width 0.23–0.70 (0.40) 0.38–0.71 (0.61) 1.0 0.48–0.85 (0.65) Numbers of cingular plates 17–19 22–24 20 17–20 Numbers of sulcal plates 9–11 9–13 Unk. 9 Numbers of apical plates 5 4 4 5 Numbers of intercalary plates 6 5 5 5 Numbers of precingular plates 8 8 8 8 Numbers of postcingular plates 7 6 6 6 Numbers of antapical plates 3 (rarely 2) 2 2 2 No. of chromosomes 80 ± 3 (n=4) 97 ± 2 (n=6)# Unk. 74 ± 1.5 (n=5) Plate formula x, EAV, 5′, 6a, 8′′,9–11s, x, EAV, 4′, 5a, 8′′,9–13s, x, EAV, 4′,5a,8′′,?s, x, EAV, 5′, 5a, 8′′, 9s, 17–19c, 7′′′,2–3′′′′,PE 22–24c, 6′′′,2′′′′,PE ?c, 6′′′,2′′′′,PE 17–20c, 6′′′,2′′′′,PE Reference (1) (1), (2) (3) (4)

Mean values are shown in parentheses. AP, anteroposterior; EAV, elongated amphiesmal vesicle; PE, Peduncle; Unk, Unknown; #Obtained from Trench & Blank (1987). *Obtained from Jeong et al.(2014) (1) This study. (2) Trench & Blank (1987). (3) Hansen & Daugbjerg (2009). (4) Jeong et al.(2014)

(Figs 36–38). Similarly, the sequences of these mar- Morphological comparisons among Clade A kers were identical for each of three cultures of S. Symbiodinium pilosum (type A2; sensu LaJeunesse, 2001) obtained The new description of S. tridacnidorum sp. nov. and from three host genera and two Caribbean locations emended description of S. microadriaticum show that (Figs 36, 38, 39; Table S1). This species is only known the mastigotes of genetically similar species are mor- from opportunistic contaminants during the culturing phologically distinct (Fig. 40). Our analyses of two process, as there are no known ﬁeld-collected samples genetically distinct strains of S. tridacnidorum of S. pilosum (LaJeunesse pers. obs.). obtained from different regions of the Paciﬁc Ocean The phylogenetic reconstructions of Clade A (GBR and Palau) indicate that morphology is stable Symbiodinium based on sequences from chloro- among individuals (i.e. strains) of this species plast and mitochondrial genes corresponded with (Figs 36, 38; Figs S4–S11). rDNA phylogenies (cp23S and cob trees not The size, shape, number and arrangement of shown). Fixed sequence differences in both the amphiesmal plates can also be used to discern Clade

Downloaded by [Pennsylvania State University] at 10:23 15 April 2015 cob and cp23S resolved S. tridacnidorum and S. A species that were initially recognized by genetic and microadriaticum as evolutionary divergent entities. ecological differences (Table 2). Each species possesses The phylogeny based on the concatenated different numbers of sulcal and cingular plates sequence alignments of chloroplast and mitochon- (Table 2). The number of knobs arranged on the EAV drial markers, along with the D1/D2 region of are similar for S. microadriaticum and S. tridacnidorum LSU, is shown in Fig. 39. (6–8and7–9, respectively), but differ signiﬁcantly from S. natans, which has at least 12 knobs. However, simi- larities and differences in plate formulae do not neces- DISCUSSION sarily correspond to phylogenetic relationships. Clade A Symbiodinium contains phylogenetically and Symbiodinium microadriaticum (CCMP2464) and S. ecologically distinct entities that exhibit unique geo- natans contain the same number of apical, intercalary, graphic distributions (LaJeunesse et al., 2009). This postcingular and antapical plates whereas S. tridacni- group also comprises many species that are both eco- dorum had different totals for these plate types (Fig. 40; logically common and are capable of growth in culture Table 2), making the overall plate arrangement for S. media, which allows for detailed morphological ana- tridacnidorum more distinctive than the plate tabulation lysis of the motile phase and experimental manipula- established for S. voratum (Clade E), a distantly related tion (Hennige et al., 2009). species (Fig. 40). Furthermore, the shapes of speciﬁc S. Y. Lee et al. 12

S. microadriaticum (culture rt-362) Red Sea 36 (ITS2 rDNA) (culture rt-370) Red Sea (culture mac508) Florida Keys 1.0/100 (culture rt-061) [AF333505] Jamaica culture (culture KB8) Hawaii A1 field sample (natural sample FL01-1) Florida Keys 1.0/100 (natural sample PM99-01) Yucatan, Mexico (natural sample Be03-34) Belize

1.0/100 (culture rt-379) [AF333511] Bahamas (natural sample FGB05_05) Flower Gardens, TX A4

[HQ896366] Indonesia Tridacna [AF333507] Palau Mastigias GBR Tridanca crocea Palau Tridacna spp. S. tridacnidorum sp. nov. Enewetak Tridacna Philippines Tridacnids (Baillie et al. 2000) Palau Cassiopea Hawaii Cassiopea (Zan07-23) Zanzibar Aglaophenia sp. (A2-223) [HQ896363] Indonesia (Weber 2009) Papua New Guinea A3a (culture TD1E) [EU449035] Philippines

(Pal09_511) Palau (OF_002; ‘A3x’ Weber 2009) American Samoa A3* (CCMP 832) GBR [KM816410]

[HQ896371] Bird’s Head, Indonesia [HQ896367] Bird’s Head, Indonesia (culture TG3A) [EU449036] Philippines A6 [AY686646] Zamami Island Host: Trindacninae (giant clams) Trindacninae Host:

(culture rt-185) Jamaica 1.0/100 (culture rt-130) Jamaica S. pilosum (A2) 1 change (culture rt-24) Barbados 37

2000 km Downloaded by [Pennsylvania State University] at 10:23 15 April 2015

1,3

1 2 4 4 1, 2 4 5 4 4 A3a 4 A3* 4 2,3 4 4 1 Ballie et al. 2000 4 2 LaJeunesse 2001 4 3 LaJeunesse et al. 2004 4 Weber 2009 5 DeBoer et al. 2012 60E120E 180

Figs 36 & 37. Fig. 36. Maximum parsimony (MP) tree created from the alignment of ITS2 data from Clade A Symbiodinium identified as S. microadriaticum (types A1), S. pilosum (A2), S. tridacnidorum sp. nov. (comprising mostly Indo-Pacific samples from tridacnid clams and Cassiopea sp.) and type A4 found in the Western tropical Atlantic. The numbers above the branches correspond to Bayesian posterior probabilities (left) and MP bootstrap values (right). Fig. 37. A summary of the known geographic distributions for S. tridacnidorum associated with Tridacna maxima across the Indo-Pacific. The known ranges of ITS2 variants A3a and A3* [A3x sensu Weber (2009)] suggest the possibility that genetic structure among geographically distant populations may exist. Numbers correspond to the publications where these distributions were reported. A Species of Giant Clam Symbiont 13

(culture rt-362) Red Sea

(culture rt-370) Red Sea [KF364598] (A1) (culture mac508)

1.0/98 (culture CCMP 2464; rt-061) Florida Keys, USA [KM972549] (isolate 406; Loeblich & Sherley 1979) (culture KB8) Hawaii (natural sample FL01-1) Florida Keys, USA (natural sample PM99-01) Yucatan, Mexico (natural sample Be03-34) Belize S. microadriaticum .90/81 FGB05 05 typeA4 [KF364602] A4 rt-378 typeA4 (CCMP 832) GBR Hippopus hippopus [KM816405] A3*

1.0/ 94 .97/85 (natural sample Pal09-511) Palau T. maxima (natural sample Phu002) Phuket T. maxima A6 (natural sample Phu003) Phuket T. maxima (natural sample Phu004) Phuket T. maxima [KM972551] (natural sample Phu005) Phuket T. maxima sp. nov. (culture rt-272) Palau Tridacna gigas [KM816406] A3 .95/84 (culture rt-77) Hawaii Cassiopeia (culture rt-243) Palau T. derasa (culture rt-174) Enewetak T. maxima 1.0/ 98 (culture EL1) Hawaii Cassiopeia sp. (natural sample Zan07-23) Zanzibar Aglaophenia sp.

(natural sample Zan07-48) Zanzibar Aglaophenia sp. S. tridacnidorum (natural sample Pal14-01) Palau C. andromeda (natural sample Pal14-02) Palau C. andromeda Clade A (natural sample Haw02-80) Hawaii Cassiopeia sp. 1.0/ 100 (culture rt-315) Palau T. squamosa [KM972550] A5

S. natans [EU315917] (culture rt-185) Jamaica [KF740671] 1.0/ 100 (culture rt-130) Jamaica S. pilosum (A2) (culture rt-24) Barbados

S. eurythalpos [KF740686] Clade D 1.0/ 100 S. trenchii [KF740689] S. boreum [KF740688]

Eastern Pacific [KF364605] Clade E 1.0/ 100 (isolate 395) Northwest Atlantic Western Pacific [HF568830] S. voratum Mediterranean [KF364606] 5 changes

Fig. 38. Maximum parsimony (MP) tree Clade A diversity based on 620 bp aligned positions encompassing the D1/D2 domains of the LSU. Species representatives from Clades D and S. voratum (Clade E) were used as outgroup taxa. The branch lengths are proportional to the number of character differences (branch lengths separating Clade A from the other lineages are abbreviated). The Downloaded by [Pennsylvania State University] at 10:23 15 April 2015 numbers associated with branches relate to Bayesian posterior probabilities (left) and MP bootstrap values (right). Posterior probabilities ≥0.8 are shown. Grey arrowheads signify those cultured isolates used for morphological analysis.

plates when compared between species corresponded the genus Symbiodinium (Freudenthal). LaJeunesse only partially with phylogenetic data. For example the (2001) genetically analysed CCMP2464 and desig- 5a and 6′′ plates were distinct for S. tridacnidorum nated it as type A1. From the incomplete description being hexagonal and pentagonal, respectively; whereas provided by Freudenthal (1962), for which the original the shapes of these plates for S. microadriaticum and S. type material is not available, it is not possible to know natans matched with S. voratum, pentagonal and quad- whether the particular culture he based his diagnosis on rangular, respectively. Therefore, while the comprehen- was morphologically and genetically the same as the sive analyses of morphological evidence can deﬁne specimens analysed by Loeblich & Sherley (1979)and these ‘morphologically cryptic’ species, the overlap in by Trench & Blank (1987). However, the entity iso- trait values creates some ambiguity when reconciled lated as CCMP2464 by Trench & Blank (1987)accu- against gene-based phylogenies. rately represents the identity of Symbiodinium found in This work provides morphological analysis of the S. ﬁeld-collected samples of C. xamachana from the microadriaticum strain (CCMP2464, or rt-061) used Greater Caribbean (LaJeunesse, 2002; Fig. 38), and by Trench & Blank (1987) to further emend the not an opportunistic species isolated during the culture description of S. microadriaticum, the type species in process (Santos et al., 2001). S. Y. Lee et al. 14

(culture rt-362) Red Sea (culture rt-370) Red Sea (culture mac508) Florida Keys, U.S.A. LSU 1.0/ 100 (culture rt-061) Jaimaca S. microadriaticum cp23S (culture KB8) Hawaii mt cob (natural sample FL01-1) Florida Keys (natural sample PM99-01) Yucatan, Mexico 1.0/ 100 (natural sample Be03-34) Belize

(natural sample Pal14-01) Palau [KM816409, cp23S] (natural sample Pal14-02) Palau (CCMP 832) GBR [KM816407, cp23S; KM816411, cob] 1.0/ 100 (natural sample Pal09-511) Palau (culture rt-272) Palau S. tridacnidorum sp. nov. (natural sample Zan07-23) Zanzibar (natural sample Haw02-80) Hawaii

(culture rt-185) Jamaica (culture rt-130) Jamaica S. pilosum (culture rt-24) Barbados 5 changes

Fig. 39. Maximum parsimony (MP) tree created from concatenated alignments of LSU, cp23S and cob sequences. The differently shaded dot symbols along each branch indicate the relative contribution of ﬁxed differences in each genetic marker that differentiate three species of Clade A. The numbers above the branches indicate the Bayesian posterior probability (left) and MP bootstrap values (right). Grey arrowheads signify the cultured isolates used for morphological analysis. Downloaded by [Pennsylvania State University] at 10:23 15 April 2015

Fig. 40. A comparison of apical and antapical plate morphologies from S. microadriaticum, S. tridacnidorum and S. natans (Clade A), and S. voratum (Clade E). The occurrence of additional plates and plate connections that differ from S. microadriaticum are highlighted in grey. Genetic relationships based on LSU rDNA are drawn to the left. A Species of Giant Clam Symbiont 15

The two isolates described as Zooxanthella microa- et al., 1996;Carloset al., 1999; Baillie et al., 2000; driatica by Loeblich & Sherley (1979) are morpholo- LaJeunesse, 2001; LaJeunesse et al., 2004a, 2004b; gically similar to S. microadriaticum (CCMP2464). Weber, 2009;DeBoeret al., 2012). In the Indian These included strain 406 isolated from Cassiopea Ocean, it occurs in the large stinging hydroid xamachana from the Florida Keys, which had a thecal (Aglaophenia; Figs 36, 38). This symbiont has also plate formula of 5′,5–6a, 9–10′′,8–9s, 20c, 7–8′′′ and been identified and cultured from species of the man- 3′′′′; and strain 395 isolated from a decaying red alga, grove upside-down jellyfish in the genus Cassiopea,a Chondrus crispus, collected off the coast of rhizostomate group in the Class Scyphozoa (Figs 36, Massachusetts, USA, which had a plate formula of 5′, 37; LaJeunesse et al., 2004a, 2004b). Cassiopea andro- 4–6a, 10–11′′,8–9s, 20c, 7–8′′′,and3′′′′.Ourgenetic meda from Palau, a likely native of the Pacific Ocean characterization of these isolates unambiguously region (host genetic data not shown), contained S. tri- resolved their identity. Isolate 406 (given to Loeblich dacnidorum populations, diagnosed by a slightly dis- by Trench’s student, Schoenberg) is likely to be the tinct cp23S sequence variant (Fig. 39). Experiments same isolate later used by Trench & Blank (1987)in with S. tridacnidorum have shown that isolates from their emendation of S. microadriaticum (rt-061 synon- Tridacna spp. (rt-272) can infect and induce strobilation ymous with CCMP2464; Fig. 38). Our plate recon- in the scyphistome of Cassiopea xamachana (Fitt, struction of the isolate from Cassiopea xamachana 1985). The disparate host specificities exhibited by collected from the Florida Keys, USA, differs from this symbiont cannot be explained by host phylogenetic their formulation in the number of precingular and relationships, nor the nature of their associations (e.g. antapical plates. The genus Zooxanthella was created extracellular vs. intracellular), yet suggests that by Brandt (1881) during his studies on the algae living unknown biotic or abiotic factors have initiated host in the radiolarian, Collozoum inerme. However, recent shifts during its evolutionary history (Secord & analysis of radiolarian dinoflagellate symbionts shows Kareiva, 1996). that they are evolutionarily divergent from Three ITS2 sequence variants, A3a, A3* (listed as Symbiodinium and the genus Zooxanthella has been ‘A3x’ in Weber, 2009)andA6 diagnose Symbiodinium replaced with the new genus Brantodinium (Probert populations found only in the tridacnids (Figs 36, 37). et al., 2014). Types A3 and A6 are relatively widespread, but type A3a The free-living strain that Loeblich & Sherley occurs predominantly in tridacnids from the Indo-West (1979) obtained from cold temperate waters of the Pacific and Central Indian Ocean, whereas A3* is com- Northwest Atlantic, isolate 395, corresponds to S. mon in clams from the Southwest and Southcentral voratum (Fig. 38). Their morphological characteriza- tropical Pacific(Fig. 37; Weber, 2009). These different tion was similar to that of Jeong et al.(2014), but there geographic distributions indicate regional differentiation appears to be a difference in how antapical vs. post- exists between some populations of S. tridacnidorum cingular plates were designated by each set of (Weber, 2009). The analysis of microsatellite allelic data researchers. Also, the number of precingular plates can be used in the future to test the possibility of regional estimated by Loeblich & Sherley (1979) was higher endemism/isolation across this widely distributed spe- than that reported by Jeong et al.(2014). However, cies (Pinzón et al., 2011;Baumset al., 2014). The broad these morphological discrepancies might be explained geographic range and genetic variation among popula- by the occasional variation in morphology observed tions of S. tridacnidorum associated with the tridacnids among cells of S. voratum, which were characterized indicate that this particular relationship has probably Downloaded by [Pennsylvania State University] at 10:23 15 April 2015 in the supplementary figure published by Jeong et al. lasted millions of years (Thornhill et al., 2014). (2014). Further, despite these apparent morphological Symbiodinium species designated type A3 (sensu differences, which may stem from differences in tech- lato) are associated with a wide range of cnidarians nique, the large cell size, high latitudinal origin and that occur in the Greater Caribbean region, including free-living habit of isolate 395 are consistent with species of the elk- and stag-horn coral (Acropora), genetic analysis identifying this isolate as S. voratum, shallow colonies of the blushing star coral and not Zooxanthella microadriatica. (Stephanocoenia), finger corals from seagrass beds (Porites porites), the top sides of shallow colonies of boulderstarcorals(Orbicella sp.), and colonies of mat The genetic, ecological and geographic attributes of zoanthids (Zoanthus sp.; LaJeunesse, 2002; Finney described species in Symbiodinium Clade A et al., 2010;Baumset al., 2014). Preliminary popula- Symbiodinium tridacnidorum is probably the most pre- tion genetic and phylogenetic comparison between valent Clade A species from the Indo-Pacific. Over its Pacific and Atlantic Symbiodinium A3 indicate that geographic range of distribution, it is harbored by sev- they are different species (Pinzón et al., 2011; eral kinds of animal. Independent researchers, working LaJeunesse unpubl. data). The A3 associated with in the Indo-West Pacific, have commonly identified Caribbean Acropora spp. was given the provisional (genetically) and cultured S. tridacnidorum from giant name Symbiodinium ‘fitti’ (Pinzón et al., 2011; clams in the molluscan subfamily Tradacninae (Rowan Baums et al., 2014), pending formal description (in S. Y. Lee et al. 16

progress). These genetic data also indicate that type A3 reviewers provided constructive feedback that improved from the Greater Caribbean probably comprises several the quality of this paper. species (Pinzón, 2011); and the product of a minor adaptive radiation of this lineage in the Atlantic FUNDING Ocean following its separation from the Pacific3-4 MYA (Thornhill et al., 2014). This research was supported by the National Research All the genetic variants attributed to S. tridacni- Foundation of Korea (NRF) grant funded by the Korea dorum (i.e. A3, A3a, A3* and A6) have been isolated government (MSIP) (NRF-2010-0020702 and NRF- and maintained in stable culture at one time or another 2012R1A2A2A 01010987), and Management of (Fig. 36). However, the lineages diagnosed as A3 from Marine Organisms Causing Ecological Disturbance and the Greater Caribbean have yet to be successfully cul- Harmful Effect Program of Korea Institute of Marine tured despite numerous attempts (M. A. Coffroth, Science and Technology Promotion (KIMST) of SUNY Buffalo, pers. comm.). This hints at the funda- KIMST award to HJJ. Contributions by T. C. mental differences in physiology between Pacificand LaJeunesse were supported by the Pennsylvania State Atlantic members of the ‘A3’ lineage. Symbiodinium University and the National Science Foundation grants (OCE-09287664 and IOS-1258058). triadacnidorum lives at high densities in the tubular system of digestive diverticula that ramify the mantle tissues of giant clams (Norton et al., 1992). Perhaps SUPPLEMENTARY INFORMATION this capacity for extracellular existence explains why it The following supplementary material is accessible is readily cultured in artificial seawater media (Carlos via the Supplementary Content tab on the article’s et al., 1999; Baillie et al., 2000; LaJeunesse, 2001). onlinepageathttp://dx.doi.org/10.1080/09670262. On several occasions, strains of S. microadriaticum 2015.1018336 have proliferated opportunistically during failed attempts to culture the normal symbiont found in a Supplementary table S1. List of sample ID, type, host, particular host coral (e.g. Orbicella faveolata from the geographic origin, and minimum (Min) and maximum Florida Keys and Stylophora sp. from the Gulf of (Max) depths from which isolates were collected. Aqaba in the Red Sea; Figs 36, 37). Therefore S. micro- Supplementary figs S1–S3. Light micrographs of the adriaticum must exist at low background concentra- mastigote (motile), coccoid (spherical) and doublet tions in the environment, and yet are only known to (dividing) cells from several cultured isolates of occur in high densities from field-collected samples of Symbiodinium tridacnidorum (rt-272) sp. nov. strain. S1. Cassiopea xamachana in the Greater Caribbean S. tridacnidorum mastigote. S2. S. tridacnidorum coc- (Figs 36, 37, 38; LaJeunesse, 2002). This particular coid. S3. S. tridacnidorum doublet. All scale bars = 2 μm. species of upside-down jelly appears to have been Supplementary figs S4–S11. Scanning electron micro- introduced to shallow tropical marine environments graphs of motile cells of Symbiodinium tridacnidorum around the world, but there are no data to indicate (strain rt-272). S4. Ventral view showing the episome, from where it originated (Holland et al., 2004). Future cingulum (C), sulcus (s), peduncle (PE) and hyposome. genetic analyses of C. xamachana specimens collected S5. Ventral-left lateral view showing the episome, cin- outside the Atlantic may show that S. microadriaticum gulum (C), and hyposome. S6. Dorsal view showing the is found in C. xamachana everywhere this host occurs. episome, cingulum (C) and hyposome. S7. Ventral-right Finally, the chromosome numbers counted for S. lateral view showing the episome, cingulum (C), sulcus Downloaded by [Pennsylvania State University] at 10:23 15 April 2015 tridacnidorum (n = 80 ± 3) and S. microadriaticum (s) and hyposome. S8. Apical view showing the episome (97 ± 2; Table 2;Trench&Blank,1987)differtoan and elongated amphiesmal vesicle (EAV) plate. S9. extent that would make them reproductively incompa- Apical view showing the EAV plate with small knobs tible as eukaryotes. The chromosome values for S. (arrowhead). S10. Antapical view showing the hypo- pilosum (78 ± 2) are similar to S. tridacnidorum, but some. S11. Antapical-ventral view showing the sulcus significant sequence divergence at several DNA loci (s) and peduncle (PE). All scale bars = 1 μm. – indicates that they are distantly related (Figs 36 39). Supplementary figs S12–S13. Drawings and micro- Additional analysis of the chromosome number from graphs of the antapical plate of Symbiodinium tridacni- other yet undescribed species in Clade A may offer dorum taken using scanning electron microscopy. S12. insights into genome evolution among the closely and Drawings of the antapical view showing 2 antapical distantly related species that comprise this group. plates. S13. Micrographs of the antapical view showing 2 antapical plates. All scale bars = 1 μm.

ACKNOWLEDGEMENTS AUTHOR CONTRIBUTIONS We are grateful for the help of Michele Weber whose SY Lee, HJ Jeong, TC LaJeunesse: original concept, dissertation and consultation provided important drafting and editing manuscript; SY Lee, HJ Jeong, TY insights in the writing of this paper. Two anonymous Jang: culture experiments; SY Lee, NS Kang, SH Jang: A Species of Giant Clam Symbiont 17

electron microscopy; SY Lee, SH Jang, TC LaJeunesse: a new planktonic mixotrophic dinoflagellate Paragymnodinium analysis of molecular data. shiwhaense n. gen., n. sp. from the coastal waters off western Korea: morphology, pigments, and ribosomal DNA gene sequence. Journal of Eukaryotic Microbiology, 57: 121–144. REFERENCES Kevin, J.M., Hall, T.W., McLaughlin, J.J.A. & Zahl, P.A. (1969). Symbiodinium microadriaticum Freudenthal, a revised taxonomic Baillie, B.K., Belda-Baillie, C.A. & Maruyama, T. (2000). description, ultrastructure. Journal of Phycology, 5: 341–350. Conspecificity and Indo-Pacific distribution of Symbiodinium gen- LaJeunesse, T.C. (2001). Investigating the biodiversity, ecology, and otypes (Dinophyceae) from giant clams. Journal of Phycology, 36: phylogeny of endosymbiotic dinoflagellates in the genus 1153–1161. Symbiodinium using the internal transcribed spacer region: in Baums, I.B., Devlin-Durante, M.K. & LaJeunesse, T.C. (2014). New search of a ′′species′′ level marker. Journal of Phycology, 37: insights into the dynamics between reef corals and their associated 866–880. dinoflagellate endosymbionts from population genetic studies. LaJeunesse, T.C. (2002). Diversity and community structure of Molecular Ecology, 23: 4203–4215. symbiotic dinoflagellates from Caribbean coral reefs. Marine Brandt, K. (1881). Ueber das Zusammenleben von Thieren und Biology, 141: 387–400. Algen. Verhandlungen der Physikalischen Gesellschaft zu Berlin, LaJeunesse, T.C. & Thornhill, D.J. (2011). Improved resolution of 1881-82: 22–26. reef-coral endosymbiont (Symbiodinium) species diversity, ecol- Carlos, A.A., Baillie, B.K., Brett, K., Kawaguchi, M. & Maruyama ogy, and evolution through psbA non-coding region genotyping. T. (1999). Phylogenetic position of Symbiodinium (Dinophyceae) PloS ONE, 6: e29013. isolates from tridacnids (Bivalvia), cardiids (Bivalvia), a sponge LaJeunesse, T.C., Thornhill, D.J., Cox, E., Stanton, F., Fitt, W.K. & (Porifera), a soft coral Anthozoa), and a free-living strain. Journal Schmidt, G.W. (2004a). High diversity and host specificity of Phycology, 35: 1054–1062. observed among symbiotic dinoflagellates in reef coral commu- Coffroth, M.A. & Santos, S.R. (2005). Genetic diversity of symbio- nities from Hawaii. Coral Reefs, 23: 596–603. tic dinoflagellates in the genus Symbiodinium. Protist, 156:19–34. LaJeunesse, T.C., Bhagooli, R., Hidaka, M., DeVantier, L., Done, T., DeBoer, T.S., Baker, A.C., Erdmann, M.V., Ambariyanto, Jones, P. Schmidt, G.W., Fitt, W.K. & Hoegh-Guldberg, O. (2004b). R. & Barber, P.H. (2012). Patterns of Symbiodinium distribution in Closely related Symbiodinium spp. differ in relative dominance three giant clam species across the biodiverse Bird’s Head region in coral reef host communities across environmental, latitudinal of Indonesia. Marine Ecology Progress Series, 444:117–132. and biogeographic gradients. Marine Ecology Progress Series, Finney, J.C., Pettay, T., Sampayo, E.M., Warner, M.E., Oxenford, H. 284: 147–161. & LaJeunesse, T.C. (2010). The relative significance of host-habi- LaJeunesse, T.C., Loh, W. & Trench, R.K. (2009). Do introduced tat, depth, and geography on the ecology, endemism and speciation endosymbiotic dinoflagellates ‘take’ to new hosts? Biological of coral endosymbionts. Microbial Ecology, 60: 250–263. Invasions, 11: 995–1003. Fitt, W.K. (1985). Effect of different strains of the zooxanthellae LaJeunesse T.C., Pettay, T., Sampayo, E.M., Phongsuwan, N., Symbiodinium microadriaticum on growth and survival of their Brown, B., Obura, D., Hoegh-Guldberg, O. & Fitt, W.K. (2010). coelenterate and molluscan hosts. Proceedings of the 5th Long-standing environmental conditions, geographic isolation and International Coral Reef Congress, 6: 131–136. host–symbiont specificity influence the relative ecological domi- Freudenthal, H.D. (1962). Symbiodinium gen. nov. and nance and genetic diversification of coral endosymbionts in the Symbiodinium microadriaticum sp. nov., a zooxanthella: genus Symbiodinium. Journal of Biogeography, 37: 785–800. taxonomy, life cycle, and morphology. Journal of Protozoology, LaJeunesse, T.C., Parkinson, J.E. & Reimer, J.D. (2012). A genetics- 9:45–52. based description of Symbiodinium minutum sp. nov. and S. psy- Giovannoni, S.J., DeLong, E.F., Olsen, G.J. & Pace, N.R. (1988). gmophilum sp. nov. (Dinophyceae), two dinoflagellates symbiotic Phylogenetic group-specific oligodeoxynucleotide probes for with Cnidaria. Journal of Phycology, 48: 1380–1391. identification of single microbial cells. Journal of Bacteriology, LaJeunesse, T.C., Wham, D.C., Pettay, D.T., Parkinson, J.E., 170: 720–726. Keshavmurthy, S. & Chen, C. A. (2014). Ecologically differen- Guillard, R.R.L. & Hargraves, P.E. (1993). Stichochrysis immobilis tiated stress tolerant endosymbionts in the dinoflagellate genus is a diatom, not a chysophyte. Phycologia, 32: 234–236. Symbiodinium Clade D are different species. Phycologia, 53: Hansen, G. & Daugbjerg, N. (2009). Symbiodinium natans sp. nov.: 305–319. a free-living dinoflagellate from Tenerife (northeast-Atlantic Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., Ocean). Journal of Phycology, 45: 251–263. McGettigan, P.A., McWilliam, H., Valentin, F., Wallace, I.M., Hennige, S.J., Suggett, D.J., Warner, M.E., McDougall, K.E. & Wilm, A., Lopez, R., Thompson, J.D., Gibson, T.J. & Higgins, Downloaded by [Pennsylvania State University] at 10:23 15 April 2015 Smith, D.J. (2009). Photobiology of Symbiodinium revisited: D.G. (2007). Clustal w and clustal x version 2.0. Bioinformatics, bio-physical and bio-optical signatures. Coral Reefs, 28: 179–195. 23: 2947–2948. Hirose, M., Reimer J. D., Hidaka, M. & Suda, S. (2008). Lee, S.Y., Jeong, H.J., Kang, N.S., Jang, T.Y.,Jang, S.H. & Lim, A.S. Phylogenetic analyses of potentially free-living Symbiodinium (2014). Morphological characterization of Symbiodinium minutum spp. isolated from coral reef sand in Okinawa, Japan. Marine and S. psygmophilum belonging to clade B. Algae, 29:299–310. Biology, 155: 105–112. Litaker, R.W., Vandersa, M.W., Kibler, S.R., Reece, K.S., Stokes, N. Holland, S.B., Dawson, M.N., Crow, G.L. & Hofmann, D.K. (2004). A., Steidinger, K.A., Millie, D.F., Bendis, B.J. & Pigg, R.J. (2003). Global phylogeography of Cassiopea (Scyphozoa: Rhizostomeae): Identification of Pfiesteria piscicida (Dinophyceae) and Pfiesteria- molecular evidence for cryptic species and multiple invasions of the like organisms using internal transcribed spacer-specific PCR Hawaiian Islands. Marine Biology, 145:1119–1128. assays. Journal of Phycology, 39: 754–761. Jeong, H.J., Yoo, Y.D., Kang, N.S., Lim, A.S., Seong, K.A., Lee, S.Y., Lobban, C.S., Schefter, M., Simpson, A.G.B., Pochon, X., Lee, M.J., Lee, K.H., Kim, H.S., Shin, W., Nam, S.W., Yih, W. & Pawlowski, J. & Foissner, W. (2002). Maristentor dinoferus n. Lee, K. (2012). Heterotrophic feeding as a newly identified survival gen., n. sp. a giant heterotrich ciliate (Spirotrichea: Heterotrichida) strategy of the dinoflagellate Symbiodinium. Proceedings of the with zooxanthellae, from coral reefs on Guam, Mariana Islands. National Academy of Sciences USA, 109:12604–12609. Marine Biology, 140:411–423. Jeong, H.J., Lee, S.Y., Kang, N.S., Yoo, Y.D., Lim, A.S., Lee, M.J., Loeblich, A.R. III. & Sherley, J.L. (1979). Observations on the theca Kim,H.S.,Yih,W.H.&LaJeunesse,T.C.(2014).Geneticsand of the mobile phase of free-living and symbiotic isolates of morphology characterize the dinoflagellate Symbiodinium voratum, Zooxanthella microadriaticum (Freudenthal) comb. nov. Journal n. sp. (Dinophyceae) as the sole representative of Symbiodinium clade of the Marine Biological Association of the United Kingdom, 59: E. Journal of Eukaryotic Microbiology, 61:75–94. 195–205. Kang, N.S., Jeong, H.J., Moestrup, Ø., Shin, W.G., Nam, S.W., Park, McLaughlin, J.J. & Zahl, P.A. (1966). Endozoic algae. In Symbiosis J.Y., de Salas, M.F., Kim, K.W. & Noh, J.H. (2010). Description of Volume 1 (Henry, S.M., editor), 257–297. Academic Press, New York. S. Y. Lee et al. 18

Norton, J.H., Shepherd, M.A., Long, H.M. & Fitt, W.K. (1992). The analysis of a fragment of the LSU rRNA gene. Journal of zooxanthellal tubular system in the giant clam. Biological Bulletin, Phycology, 30:999–1011. 183: 503–506. Secord, D. & Kareiva, P. (1996). Perils and pitfalls in the host Pinzón, J.H. (2011). Phylogenetics, population genetics and ecology specificity paradigm. BioScience, 46: 448–453. to understand the evolution of coral-algal mutualisms. Stamatakis, A. (2006). RAxML-VI-HPC: maximum likelihood- Dissertation, Penn State University, pp. 153. based phylogenetic analyses with thousands of taxa and mixed Pinzón, J.H., Devlin-Durante, M.K., Weber, X.M., Baums, I.B. & models. Bioinformatics, 22: 2688–2690. LaJeunesse, T.C. (2011). Microsatellite loci for Symbiodinium A3 Stat, M., Bird, C.E., Pochon, X., Chasqui, L., Chauka, L.J., (S. fitti) a common algal symbiont among Caribbean Acropora (stony Concepcion, G.T., Logan, D., Takabayashi, M., Toonen, R.J. & corals) and Indo-Pacific giant clams (Tridacna). Conservation Gates, R.D. (2011). Variation in Symbiodinium ITS2 sequence Genetics Resources, 3:45–47. assemblages among coral colonies. PloS ONE, 6: e15854. Pochon, X., Garcia-Cuestos, L., Baker, A.C., Castella, E. & Tamura, K., Dudley, J., Nei, M. & Kumar, S. (2007). MEGA4: Pawlowski, J. (2007). One-year survey of a single Micronesian molecular evolutionary genetics analysis (MEGA) software v. reef reveals extraordinarily rich diversity of Symbiodinium types in 4.0. Molecular Biology and Evolution, 24: 1596–1599. sorited foraminifera. Coral Reefs, 26: 867–882. Taylor, D.L. (1974). Symbiotic marine algae; taxonomy and biolo- Pochon, X., Putnam, H.M., Burki, F. & Gates, R.D. (2012). gical fitness. In Symbiosis in the Sea (Vernberg, W.B., editor), 245– Identifying and characterizing alternative molecular markers for 262. University of South Carolina Press, Columbia. the symbiotic and free-living dinoflagellate genus Symbiodinium. Thornhill, D.J., Lewis, A., Wham, D.C. & LaJeunesse, T.C. (2014). PloS ONE, 7: e29816. Host-specialist lineages dominate the adaptive radiation of reef Porto, I., Granados, C., Restrepo, J.C. & Sanchez, J.A. (2008). coral endosymbionts. Evolution, 68: 352–267. Macroalgal-associated dinoflagellates belonging to the genus Trench, R.K. (1993). Microalgal-invertebrate symbioses: a review. Symbiodinium in Caribbean reefs. PloS ONE, 3: e2160. Endocytobiosis Cell Research, 9: 135–175. Probert, I., Siano, R., Poirier, C., Decelle, J., Biard, T., Tuji, A., Trench, R.K. (2000). Validation of some currently used invalid Suzuki, N. & Not, F. (2014). Brandtodinium gen. nov. and B. names of dinoflagellates. Journal of Phycology, 36: 972. nutricula comb. Nov. (Dinophyceae), a dinoflagellate commonly Trench, R.K. & Blank, R.J. (1987). Symbiodinium microadriaticum found in symbiosis with polycystine radiolarians. Journal of Freudenthal, S. goreauii sp. nov., S. kawagutii sp. nov., and S. Phycology, 50: 388–399. pilosum sp. nov.: Gymnodinioid dinoflagellate symbionts of mar- Ronquist, F. & Huelsenbeck, J.P. (2003). MrBayes 3: Bayesian phylo- ine invertebrates. Journal of Phycology, 23: 469–481. genetic inference under mixed models. Bioinformatics, 19: 1572– Weber, M.X. (2009). The biogeography and evolution of 1574. Symbiodinium in giant clams (Tridacnidae). PhD Dissertation, Rowan, R. & Powers, D.A. (1991). A molecular genetic classifica- University of California, Berkeley. tion of zooxanthellae and the evolution of animal-algal symbiosis. Yamashita, H. & Koike, K. (2013). The genetic identity of free- Science, 251: 1348–1351. living Symbiodinium obtained over a broad latitudinal range in the Rowan, R., Whitney, S.M., Fowler, A. & Yellowlees, D. (1996). Japanese coast. Phycological Research, 61:68–80. Rubisco in marine symbiotic dinoflagellates: Form II enzymes in Zardoya, R., Costas, E., Lopez-Rodas, V., Garrido-Pertierra, A. eukaryotic oxygenic phototrophs encoded by a nuclear multigene & Bautista, J.M. (1995). Revised dinoflagellate phylogeny family. Plant Cell, 8:539–553. inferred from molecular analysis of large subunit ribosomal Santos, S.R., Taylor, D.J. & Coffroth, M.A. (2001). Genetic com- RNA gene sequences. Journal of Molecular Evolution, 41: parisons of freshly isolated vs. cultured symbiotic dinoflagellates: 637–645. implications for extrapolating to the intact symbiosis. Journal of Zhang, Z., Green, B.R. & Cavalier-Smith, T. (2000). Phylogeny of Phycology, 37: 900–912. ultra-rapidly evolving dinoflagellate chloroplast genes: a possible Sampayo, E., Dove, S. & LaJeunesse, T.C. (2009). Cohesive mole- common origin for sporozoan and dinoflagellate plastids. Journal cular genetic data delineate species diversity in the dinoflagellate of Molecular Evolution, 51:26–41. genus Symbiodinium. Molecular Ecology, 18: 500–519. Zhang, H., Bhattacharya, D. & Lin, S. (2005). Phylogeny of dino- Scholin, C.A., Herzog, M., Sogin, M. & Anderson, D.M. (1994). flagellates based on mitochondrial cytochrome b and nuclear small Identification of group- and strain-specific genetic markers for subunit rDNA sequence comparisons. Journal of Phycology, 41: globally distributed Alexandrium (Dinophyceae). II. Sequence 411–420. Downloaded by [Pennsylvania State University] at 10:23 15 April 2015