Arch Microbiol (1988) 149: 515 - 520 Archives of Hicrobielogy 9 Springer-Verlag 1988 Base composition of DNA from symbiotic dinoflagellates: a tool for phylogenetic classification Rudolf J. Blank 1, Volker A. R. Huss 1, and Walter Kersten 2 Institut ffir Botanik und Pharmazeutische Biologic, Universit/it Erlangen-Niirnberg, Staudtstrasse 5, D-8520 Erlangen, Federal Republic of Germany 2 Institut fiir Biochemie der Medizinischen Fakult/it, Universit/it Erlangen-Niirnberg, Fahrstrasse 17, D-8520 Erlangen, Federal Republic of Germany Abstract. DNA of eight endosymbiotic dinoflagellates (zooxanthellae) from seven different host species has been analyzed as to its thermal characteristics and base composi- tion by means of spectrophotometry and high performance liquid chromatography, All algae under investigation contain both methylcytosine and hydroxymethyluracil in addition to the bases typical of nuclear DNA. As a result, melting temperatures are decreased, suggesting lower contents of guanine plus cytosine than actually present. True percentages of guanine plus cytosine plus methylcytosine range from about 43 to 54 mot%. They are unique for the symbionts from different hosts, indicating phylogenetic sep- aration of the taxa comparised within the genus Symbiodi- nium. Key words: Dinofiagellates - DNA composition - Hydroxymethyluracil - Methylcytosine - Speciation - Symbiodinium - Symbiosis - Zooxanthellae Two morphologically distinct forms of dinoflagellates are currently known among eukaryotic endosymbionts in the marine environment. The group of amphidinioid zoo- xanthellae is composed of several species of the genus Amphidinium (Blank and Trench 1986; Trench and Blank 1987), while the gymnodinioid type was for a long time regarded as representing a single pandemic species of symbiotic dinoflagellate (cf. Freudenthal 1962; Taylor 1974), including varieties (cf. Taylor 1984) or nomina nuda (Hollande and Carr~ 1974; Duclaux 1977). It has now been established that gymnodinioid symbionts are also heteroge- neous, and that their species belong to the genus Gymnodi- Offprint requests to: R. J. Blank Abbreviations: dA, deoxyadenosine; dC, deoxycytidine; dG, deoxy- guanosine; dT, deoxythymidine; m5dC, 5-methyldeoxycytidine; hindU, 5-hydroxymethyldeoxyuridine; rC, riboeytidine; Br8G, bromine-8-guanosine; A, adenine; C, cytosine; G, guanine; T, thymine; m5C, 5-methylcytosine; hmU, 5-hydroxymethyluracil; G + C, guanine plus cytosine plus 5-methylcytosine; HPLC, high performance liquid chromatography; T~,, temperature at the midpoint of hyperchromic shift; CTAB, N-cetyl-N,N,N-trimethyl- ammonium bromide; EDTA, ethylenediamine-tetraacetic acid, di- sodium salt; TRIS, tris-(hydroxymethyl)-aminomethane; 1 x SSC, standard saline citrate (0.15 M NaC1 + 0.015 M trisodium citrate, pH 7.0) nium (Spero 1987) as well as to the taxon Symbiodinium (Trench and Blank 1987). Symbiodinium has been recorded free-living outside its hosts only twice (Loeblich and Sherley 1979; Taylor 1983). Studies were performed in situ, or on cultured symbionts in vitro. However, isolated zooxanthellae grow very slowly. It is difficult to bring them into culture, and even harder to bring them into mass culture. Therefore, it is not surprising that little emphasis has been placed on studying their DNA composition. To our knowledge, work including symbiotic dinoflagellates for DNA analyses is very rare (Franker 1970; Rae 1976). Data obtained from these studies have never served as arguments for phylogenetic discussions. Thus, even now the bulk of descriptions of symbiotic dinoflagellates is based on their morphology, with some more sophisticated techniques like behavioral, biochemical and physiological characterizations and karyotyping in tandem with three- dimensional reconstructions employed for the recognition of different species within the genus Symbiodinium (for re- view, see Blank and Trench 1985a, b; Trench and Blank 1987). Besides the need for identifying zooxanthellae from dif- ferent hosts as to their G+C contents, DNA of dino- flagellates in general bears interesting evolutionary aspects due to the occurrence of partially high amounts of methyl- ated bases like methyladenine, methylcytosine and especially hydroxymethyluracyl, substituting the bases customarily present in eukaryotes (cf. Rae and Steele 1978). We have therefore analyzed the DNA base composition of gymnodinioid zooxanthellae from seven different host species of five different orders of marine invertebrates. In order to meet representative selection of symbionts, we stud- ied Symbiodinium-like dinoflagellates from a jellyfish, sea anemones, a zoanthid, and a stony coral, where the algae occur as intracellular cytosymbionts each enclosed within a single host vacuole in nature, as well as giant clams harboring intercellular endosymbionts in their haemal sinuses. Also, care was taken for choosing worldwide dis- tribution of the hosts from which the symbionts had been originally isolated, using algae of invertebrates collected from Caribbean waters as well as from the Indopacific, with one identical host species originating from Palau and the Eniwetok atoll. Our aim was to resolve three questions: (1) would dif- ferent G+C compositions be uncovered in the DNA of zooxanthellae from different hosts; (2) are modified bases substituting for the canonical bases and to what extent; and