Mar Biol (2008) 153:351–363 DOI 10.1007/s00227-007-0811-0 RESEARCH ARTICLE Molecular characterization of the zoanthid genus Isaurus (Anthozoa: Hexacorallia) and associated zooxanthellae (Symbiodinium spp.) from Japan James Davis Reimer Æ Shusuke Ono Æ Junzo Tsukahara Æ Fumihito Iwase Received: 18 March 2007 / Accepted: 30 August 2007 / Published online: 27 September 2007 Ó Springer-Verlag 2007 Abstract The zoanthid genus Isaurus (Anthozoa: Hexa- sequenced in order to investigate the molecular phyloge- corallia) is known from both the Indo-Pacific and Atlantic netic position of Isaurus within the order Zoantharia and Oceans, but phylogenetic studies examining Isaurus using the family Zoanthidae. Additionally, obtained sequences molecular markers have not yet been conducted. Here, two and morphological data (polyp size, mesentery numbers, genes of markers [mitochondrial cytochrome oxidase sub- mesogleal thickness) were utilized to examine Isaurus unit I (COI) and mitochondrial 16S ribosomal DNA (mt species diversity and morphological variation. By com- 16S rDNA)] from Isaurus specimens collected from paring our obtained sequences with the few previously southern Japan (n = 19) and western Australia (n = 3) were acquired sequences of genera Isaurus as well as with Zoanthus, Acrozoanthus (both family Zoanthidae), and Palythoa spp. (family Spenophidae) sequences, the phy- Communicated by S. Nishida. logenetic position of Isaurus as sister to Zoanthus within the Family Zoanthidae was suggested. Based on genetic Electronic supplementary material The online version of this data, Isaurus is most closely related to the genus Zoanthus. article (doi:10.1007/s00227-007-0811-0) contains supplementary material, which is available to authorized users. Despite considerable morphological variation (in particu- lar, polyp length, mesentery numbers, external coloration) J. D. Reimer between collected Isaurus specimens, all specimens Research Program for Marine Biology and Ecology, examined are apparently conspecific or very closely related Extremobiosphere Research Center, Japan Agency for Marine-Earth Science and Technology based on molecular data and observed morphological var- (JAMSTEC), 2-15 Natsushima, Yokosuka, iation within colonies. Additionally, obtained internal Kanagawa 237-0061, Japan transcribed spacer of ribosomal DNA (ITS-rDNA) sequences from symbiotic zooxanthellae (Symbiodinium J. D. Reimer Á F. Iwase Biological Institute on Kuroshio, 560 Nishidomari, spp.) from all Isaurus specimens were shown to be subc- Otsuki, Kochi 788-0333, Japan lade C1-related Symbiodinium. J. D. Reimer (&) Department of Marine Science, Biology and Chemistry, Faculty of Science, University of the Ryukyus, Senbaru 1, Nishihara, Okinawa 903-0213, Japan Introduction e-mail: [email protected] The order Zoantharia (Anthozoa: Hexacorallia) currently S. Ono Miyakonojo Higashi High School, Mimata, consists of five recognized families, including two mainly Miyazaki 889-1996, Japan zooxanthellate families (possessing symbiotic zooxanthel- lae of the dinoflagellate genus Symbiodinium); the sand- J. Tsukahara encrusted Sphenopidae, and the non-encrusted Zoanthidae. Department of Developmental Biology, Faculty of Science, Kagoshima University, Korimoto 1-21-35, Species from both families are often seen in sub-tropical Kagoshima, Kagoshima 890-0065, Japan and tropical shallow coral reef habitats worldwide. 123 352 Mar Biol (2008) 153:351–363 Zoanthidae is currently organized into three genera: Zo- Recent genetic studies investigating the genera Zoanthus anthus, Acrozoanthus, and Isaurus. (Reimer et al. 2006a) and Palythoa (Reimer et al. 2006b, Isaurus spp. are known from both the Atlantic and Indo- 2007b) have demonstrated that relatedness in many Pacific, and are generally found in intertidal or shallow zoanthids is difficult to judge based solely on morphology. subtropical and tropical waters. Currently, three species are This is largely due to zoanthids often being very morpho- recognized; I. tuberculatus Gray from Hawaii, Fiji, Aus- logically plastic with regards to their external morphology tralia, East Africa, and the Caribbean (pan-tropical (polyp and colony shape, etc.) (Karlson 1982; Larson and distribution); I. maculatus Muirhead and Ryland described Larson 1982). DNA sequencing and analyses have often from Fiji; and I. cliftoni Gray from western Australia (see Muirhead and Ryland 1985), although 22 species have been historically listed in the literature (Fautin 2006). In Japan, Isaurus spp. are believed to be very rare, and have been reported from only a handful of locations (Uchida 2001; Uchida and Iwase personal communication; see also Fig. 1). The observed rarity of Isaurus may be partially due to its cryptic appearance (see Fig. 2) and/or its preference for habitats on rocky shores facing the open ocean that are often difficult to access. The status of Isaurus as separate from Zoanthus is somewhat confused when examining past literature. Many researchers placed nominal Isaurus spp. samples in Zoan- thus (discussed in Muirhead and Ryland 1985), and even in more recent literature the extreme similarity in external morphology between Zoanthus praelongus and I. macula- tus has been noted (Muirhead and Ryland 1985). Isaurus has been defined to be different from Zoanthus by having recumbent, non-erect polyps (although Z. praelongus shares this characteristic with Isaurus) and the presence of tubercules on the polyps (although I. cliftoni does not have tubercules), but otherwise shares many morphological characters [e.g. not sand-encrusted, zooxanthellate, colo- nial, generally ‘‘liberae’’ polyps (see Pax 1910)] with Zoanthus, making phylogenetic placement of this genus as separate to Zoanthus open to speculation. 130 E 140 E KOREA JAPAN Shikoku Danjo Islands Kushimoto Hachijojima Tosashimizu (see inset) EAST CHINA SEA Otsuki (see inset) 30 N PACIFIC OCEAN Yakushima nt urre Odo oC 0 5 km i (North, South) sh Otsuki uro Yo r o n K BUNGO Tosashimizu Kerama Islands STRAIT Oshirigai Hozaki Mizushima Okinoshima Shirigai- Nishidomari- Fig. 2 Various Isaurus morphotypes nominally identified as Isaurus 0 300 Matsubae Matsubae PACIFIC OCEAN km tuberculatus a specimen IOtsH6 in situ at Hozaki site (depth = 4.0 m), b two polyps from different colonies at Oshirgai-Matsubai Fig. 1 Map showing sampling locations of Isaurus specimens from (IOtsOM5 left and IOtsOM6 right) showing polyp size and color Japan examined in this study, with inset showing locations in southern variation in a tank at BIK. c Colony with Isaurus sp. B morphology Shikoku. Locations in large font from this study, locations in small from Oshirigai-Matsubai (IOtsOM4) in a tank at BIK. All scale font previous show previously reported locations of Isaurus (main bars = 1 cm. All images originally from Reimer (2007). Note in in map only) situ images the recumbent, non-erect polyps characteristic of Isaurus 123 Mar Biol (2008) 153:351–363 353 resulted in taxonomic revision of species (Reimer et al. and Kagoshima, Japan (Fig. 1) between August 2004 2006b) and genera (Reimer et al. 2006c). and October 2006 (Table 1), and stored in 100% ethanol However, until now only three single genetic sequences at –20°C at the Biological Institute on Kuroshio (BIK) until from Isaurus spp. have been deposited in GenBank; one further utilization. Despite large variation in polyps mitochondrial (mt) 16S ribosomal DNA (mt 16S rDNA) (external coloration, tubercule shape, polyp dimensions), sequence from I. tuberculatus (AF398919—Burnett most samples were nominally classified as Isaurus tuber- unpublished), and two sequences [mt 16S rDNA culatus sensu Muirhead and Ryland (1985) (Fig. 2a, b). (AY995945) and 12S ribosomal DNA (AY995922)] from Uchida (2001) lists specimens from Tatsukushi, Japan as Sinniger et al. (2005), who examined a single unidentified I. assymmetricus, but I. assymmetricus was included in Isaurus sp. sample of origin unknown acquired from the I. tuberculatus (along with three other putative species) by aquarium trade. These sequences suggest the specimens Muirhead and Ryland (1985). Photographs of I. assym- examined are closely related to the genus Zoanthus. metricus from Tatsukushi are morphogically similar to However, due to the limited number of Isaurus, Zoanthus, I. tuberculatus, and thus we have nominally classified and Palythoa sequences examined in Sinniger et al. (2005), samples from Japan collected here as I. tuberculatus as well the overall lack of research conducted on Isaurus, following Muirhead and Ryland (1985). Two colonies questions remain about the phylogenetic position of Isau- (IOtsOM4, IOtsNM1) were observed to be morphologi- rus within Zoanthidea. cally different [having smaller polyps, different polyp Another method that may aid in characterizing zoo- coloration (see Fig. 2), smaller tubercules, larger (thou- xanthellate zoanthid species is by identifying their sands of polyps) colony sizes] from the majority of symbiotic dinoflagellates from the genus Symbiodinium samples, and were nominally classified as Isaurus sp. B (order Suessiales). This is primarily achieved through (Fig. 2c). Additionally, a sample preserved in formalin sequencing of the internal transcribed spacer of ribosomal (thus no genetic examinations were possible) received from DNA (ITS-rDNA) of Symbiodinium. While some zooxan- Dr. H. Uchida from the Danjo Islands (sample IND1; see thellate zoanthid species are known to host more than one Table 1) (collected 26 October 1983 by F. Iwase) also type of Symbiodinium within individuals