MARINE ECOLOGY PROGRESS SERIES Vol. 244: 17–26, 2002 Published November 29 Mar Ecol Prog Ser Molecular diversity of symbiotic algae at the latitudinal margins of their distribution: dinoflagellates of the genus Symbiodinium in corals and sea anemones A. M. Savage1, M. S. Goodson1, S. Visram1, H. Trapido-Rosenthal2, J. Wiedenmann3, A. E. Douglas1,* 1Department of Biology, University of York, PO Box 373, York YO10 5YW, United Kingdom 2Bermuda Biological Station for Research, Ferry Reach, Bermuda GE01 3Department of General Zoology and Endocrinology, University of Ulm, Albert Einstein Allee 11, 89069 Ulm, Germany ABSTRACT: To investigate the molecular diversity of symbiotic algae at the latitudinal limits of their distribution, the ribosomal RNA gene sequences (rDNA) of the dinoflagellate Symbiodinium in ben- thic Cnidaria (corals, sea anemones etc.) on Bermuda (32°N) and in the Mediterranean and NE Atlantic (35 to 53°N) were analysed. The algae in Bermudian Cnidaria were identified as Symbio- dinium of Phylotypes A, B and C, as previously described for benthic Cnidaria in the Caribbean (12 to 27°N). The algae in every sample of sea anemones (Anemonia spp. and Cereus pedunculatus) in the NE Atlantic and Mediterranean were a previously undescribed group within Phylotype A, possibly endemic to this high latitude region. KEY WORDS: Symbiosis · Symbiodinium · Zooxanthellae · Molecular diversity · Marginal populations Resale or republication not permitted without written consent of the publisher INTRODUCTION been reported in the free-living condition and those that have are invariably close to symbioses (Carlos et Reef-building scleractinian corals and most other al. 1999), suggesting that free-living populations are benthic Cnidaria (e.g. sea anemones, gorgonians) on small and transient (i.e. individual cells of Symbio- shallow water coral reefs bear dinoflagellate algae of dinium derived from symbioses may not persist indefi- the genus Symbiodinium in their tissues. These nitely in the natural environment). symbiotic algae provide the animal with nutrients, The purpose of this study was to investigate the mol- thereby promoting animal growth and reproduction as ecular diversity of Symbiodinium at the latitudinal well as enhancing calcification in scleractinian corals limits of symbioses in Cnidaria. We predicted that (Trench 1993, Gattuso et al. 1999). Coral reefs are Symbiodinium diversity would be low and include restricted to low latitudes, generally 25° N to 25° S, divergent, possibly endemic, forms as a result of iso- where mean winter sea surface temperatures do not lation or adaptation to the high latitude environment. fall below 18°C (Stehli & Wells 1971). At higher lati- Our study area was the Atlantic and the sites were tudes, with greater seasonal variation in temperature Bermuda at 32° N in the West Atlantic, the European and day length, coral reefs and Cnidaria bearing Sym- coast of the Mediterranean at 35 to 43° N, and coastal biodinium are of low species diversity or absent (Cross- waters of the UK, France and Italy at 48 to 53° N. land 1988, Veron 1995). Symbiodinium have rarely Bermuda is the most northerly reef site in the Atlantic Ocean, warmed by eddies of the Gulf Stream (Verrill 1901). Benthic Cnidaria bearing Symbiodinium on *Corresponding author. Email: [email protected] Bermuda are generally accepted as conspecific with © Inter-Research 2002 · www.int-res.com 18 Mar Ecol Prog Ser 244: 17–26, 2002 members of the Caribbean fauna, but they are of much guez-Lanetty et al. 2001) and in the UK, the sea reduced diversity, e.g. of the 62 species of scleractinian anemone Anemonia viridis (= A. sulcata) has Phylo- corals bearing symbiotic algae on Jamaica, 17 have type A (Bythell et al. 1997). been reported on Bermuda (Logan 1988). The benthic cnidarian fauna of coastal waters of the NE Atlantic is different from low latitudes and dominated by taxa MATERIALS AND METHODS lacking Symbiodinium. Exceptionally, sea anemones of the genus Anemonia are widespread and locally The samples and DNA extraction methods. The abundant, for example to 57°N along the west coast of focus of this study was symbioses from Bermuda and Britain (Pax & Müller 1962, A. E. Douglas unpubl. obs.: Europe which are listed in Table 1. The Medi- for consideration of taxonomy of Anemonia species, terranean/UK samples of Anemonia species and the see Wiedenmann et al. 1999, 2000). The diversity of Bermudian species were collected directly from the Symbiodinium in Anemonia species from UK waters natural habitat; Cereus pedunculatus from Brittany and the Mediterranean was determined. For compari- (France) was provided fixed in ethanol by W. West- son, an analysis was conducted of Symbiodinium in a heide (University of Osnabruck). Tropical species were second sea anemone genus, Cereus pedunculatus, also used: DNA samples of the coral Montastraea which is locally common in coastal waters throughout annularis at San Blas, Panama, provided by R. Rowan; the NE Atlantic coastal waters (Manuel 1988). skeleton-free coral blastates in DMSO/NaCl buffer of The chosen approach to investigate the molecular Agaricia fragilis, Favia fragum, Siderastrea siderea diversity of Symbiodinium is sequence variation in and Stephanocoenia intersepta at St. Croix, the Virgin ribosomal RNA genes. Sequence analysis of the small- Islands, provided by J. Bythell; and frozen cores of subunit (SSU) rDNA has revealed 2 broad groups, one the coral Goniastrea aspera from Phuket, Thailand, containing Phylotype A and the other containing Phy- provided by B. E. Brown. lotypes B, C and E (Rowan & Powers 1991a, Rowan & DNA extraction followed the method of Rowan & Knowlton 1995). Phylotype E is known as Phylotype D Powers (1991b) or Wiedenmann et al. (2000). For live by some authors (e.g. LaJeunesse 2001). The various samples of the sea anemones and the benthic jellyfish phylotypes can be discriminated by PCR-RFLP ana- Cassiopeia xamachana, fresh tentacle clippings were lysis of SSU rDNA (e.g. Rowan & Powers 1991b, macerated in ice-cold Zooxanthellae isolation buffer Rowan & Knowlton 1995, Darius et al. 1998, Brown et (ZIB) (0.4 M NaCl, 10 mM Na2 EDTA, 20 mM Tris, al. 2002). Phylogenetic trees constructed from partial 8 mM dithiothreitol, pH 8.2); the fixed Cereus pedun- sequences of the large-subunit (LSU) rRNA gene culatus was soaked in ZIB and minced finely with scis- and complete sequences of the ITS/5.8 S regions are sors before maceration; and for the live and frozen congruent with those obtained with SSU rRNA gene coral samples, tissue was removed from the skeleton (Wilcox 1998, LaJeunesse 2001, Pawlowski et al. using an airbrush and ice-cold ZIB. These treatments 2001). yielded intact zooxanthellae and disrupted animal Of immediate relevance to this study, sequence data tissues, which were separated by centrifugation and have been used to examine the geographical distribu- resuspension in fresh ice-cold ZIB 3 times. All the tion of Symbiodinium phylotypes. Most scleractinian ZIB-prepared material and the samples of fixed coral corals in the Caribbean bear Phylotypes A, B and C blastate from St. Croix were washed in DNAB (0.4 M (varying between species) and corals in the Pacific NaCl, 50 mM Na2 EDTA, pH 8) and incubated over- generally have Phylotype C (Baker & Rowan 1997). night at 50°C with 0.5 mg Proteinase K ml–1 DNAB. Phylotype E has been reported in several coral species The digests were incubated with 1.5% cetyltrimethy- in both the Caribbean and Pacific (e.g. Rowan & lammonium bromide, 1 M NaCl and 1.5 µg glycogen Knowlton 1995, Van Oppen et al. 2001, Toller et al. ml–1 (final concentrations). DNA was extracted in chlo- 2001a, Brown et al. 2002). Phylotypes A, B, C and E of roform and precipitated in ice-cold ethanol with 0.3 M Symbiodinium occur in other benthic Cnidaria (e.g. sodium acetate, pH 6.5. sea anemones, zoanthids, gorgonians) in both the PCR amplification of zooxanthellae rDNA. Three Atlantic and Indo-Pacific (e.g. Rowan & Powers 1991b, regions of the dinoflagellate rRNA gene complex were Coffroth et al. 2001). The few studies of Symbiodinium amplified: SSU rDNA with primers ss5z and ss3z at the latitudinal margins of their distribution include (Rowan & Powers 1991b); 5’-region of LSU rDNA with the demonstration that high latitude populations of primers 24D15F1 and 24D2R1 (Baker et al. 1997); and both Anthopleura elegantissima, a sea anemone on the the complete ITS1-5.8S-ITS2 region using primers Pacific coast of North America, and Plesiastrea versi- msg2 (5'- GTA GGT GAA CCT GCG GAA GGA -3’) pora, a Pacific coral of very wide latitudinal range, and msg3 (5'- TCC TCC GCT TAC TTA TAT GCT have Phylotype B (LaJeunesse & Trench 2000, Rodri- TAA -3’), designed from conserved sequences at Savage et al.: Molecular diversity of Symbiodinium 19 3’-end of SSU and 5’-end of LSU sequences, respec- PTC-100™ thermal cycler (MJ Research) with the ther- tively, of Symbiodinium rRNA genes available in Gen- mal profiles: 28 cycles of 45 s at 94°C, 45 s at 56°C and Bank. The reaction mixtures contained 2 mM dNTPs, 2 min at 72°C for SSU amplifications; 30 cycles of 1 min 0.2 µM primers, 1 × Taq polymerase buffer (Promega) at 94°C, 1 min at 54°C and 2 min at 72°C for LSU ampli- –1 and 0.05 U Taq polymerase µl , with MgCl2 at 1.5 mM fications; and 2 min at 94°C, followed by 35 cycles of for SSU and ITS amplifications and 3.0 mM for LSU 1 min at 56°C, 2 min at 72°C, 1 min at 94°C and then amplification. All PCR reactions were carried out in a 1 min at 56°C, 8 min at 72°C for ITS amplifications. Table 1. Symbioses: collection site and phylotype of Symbiodinium as determined by PCR-RFLP analysis of SSU rDNA (a) Europe Host species Collection sitea Nb Phylotype Anemonia rustica Giglio, Italy 2 A Colliure, France 1 A Anemonia sulcata var.