Stylobates birtlesi sp. n., a new species of carcinoecium-forming sea anemone (Cnidaria, Actiniaria, Actiniidae) from eastern Australia

Abstract We describe a new species of carcinoecium-forming sea anemone, Stylobates birtlesi sp. n., from sites 590–964 m deep in the Coral Sea, off the coast of Queensland, Australia. An anemone of this genus settles on a gastropod shell inhabited by a hermit crab, then covers and extends the shell to produce a chitinous structure termed a carcinoecium. Stylobates birtlesi sp. n. is symbiotic with the hermit crab Sympagurus trispinosus (Balss, 1911). The nature of marginal sphincter muscle and nematocyst size and distribution distinguish Stylobates birtlesi sp. n. from other species in the genus. The four known species of Stylobates are allopatric, each inhabiting a separate ocean basin of the Indo-West Pacific. We also extend the known range of Stylobates loisetteae in the Indian Ocean off the coast of Western Australia.


Introduction
Stylobates Dall, 1903, of family Actiniidae Rafi nesque, 1815, is an exclusively deepwater genus of sea anemones in which three species are known: Stylobates aeneus Dall, 1903, from Hawai'i and Guam in the Pacifi c Ocean, Stylobates cancrisocia (Carlgren, 1928a), from the Indian Ocean off Africa, and Stylobates loisetteae Fautin, 1987, from the Indian Ocean off Western Australia. We describe Stylobates birtlesi sp. n. from specimens collected in the Coral Sea off the northeastern coast of Australia. In addition, we report previously unpublished localities for Stylobates loisetteae.
A distinctive feature of Stylobates is the chitinous carcinoecium it produces; a carcinoecium is a shell-like structure inhabited by a hermit crab. Carcinoecia are produced by bryozoans (e.g. Vermeij 1993) as well as many species of cnidarians (e.g. Williams and McDermott 2004), including hydrozoans (e.g. Millard 1975), zoanthids (e.g. Muirhead et al. 1986, Ates 2003 and sea anemones (e.g. Carlgren 1928a, b, Ross 1971, 1984, Dunn and Liberman 1983, Daly et al. 2004. Of anemones that attach to gastropod shells inhabited by hermit crabs, animals of some species (e.g. those belonging to Calliactis) form a thin layer of chitin over the shell. Th ose of other species (e.g. those belonging to Stylobates and Paracalliactis) not only cover but extend the shell, producing a carcinoecium. Th e ability to form a carcinoecium is a convergent attribute of anemones (Gusmão and Daly 2010); anemones possessing this ability belong to four families (Daly et al. 2004).
Th e genus and species Stylobates aeneus were described by Dall (1903, p. 61), who was initially under the impression that the shell of "fl exible, horny consistency," which was inhabited by a hermit crab and covered by a sea anemone, was that of a gastropod. He later corrected his mistake, recognizing that "Th ese specimens were secretions from the bases of the Actinias" (Dall 1919, p. 80). Nonetheless, the holotype of Stylobates aeneus Dall, 1903, consisting only of the carcinoecium, is still housed in the mollusc collection of the United States National Museum of Natural History (USNM). Carlgren (1928a) described Isadamsia cancrisocia as a new genus and species of carcinoecium-forming anemone, making no reference to Dall (1903). Dunn et al. (1981) synonymized the genera Isadamsia and Stylobates. According to International Code of Zoological Nomenclature Article 12.2.8 (International Commission on Zoological Nomenclature 1999), having been published before 1931, the name Stylobates aeneus is available for the anemone because it is a "description of the work of an organism." Th e allopatric distribution that we found for the four species of Stylobates is similar to that of other deep-sea invertebrates in the tropical Indo-West Pacifi c, such as scleractinian corals and squat lobsters.

Methods
Th e holotype and fi ve paratypes of Stylobates birtlesi sp. n. were trawled by ORV Franklin during the Cidaris I expedition on the northeastern continental slope of Queens-land in 1986(Anonymous 1986. Th e holotype was photographed live (Figure 1a, b) within a few minutes of being brought on deck. Four paratypes were trawled by RV Soela off the northeast coast of Queensland and one voucher was trawled by FRV Iron Summer off the southeast coast of Queensland. All specimens were preserved in 70% ethanol. Specimens of Stylobates loisettae were collected on the RV Southern Surveyor expedition to the northwestern coast of Australia in 2007.
Cnida preparations were made from the tentacles, mesenterial fi laments, actinopharynx, and column by smashing tissue with water under a coverslip. Preparations were examined using diff erential interference (Nomarski) optics at 1000×. For each tissue type, the length and width were measured for each type of cnida. Representative cnidae were photographed using an Olympus digital camera. Histological sections were stained with Gomori trichrome (Menzies 1959

Family Actiniidae Rafi nesque, 1815
Genus Stylobates Dall, 1903 Stylobates Dall, 1903: p. 62 Isadamsia Carlgren, 1928a General. Because Dall (1903) had been under the impression that the carcinoecium upon which he based his description was that of a gastropod, the fi rst description of the anemone was by Carlgren (1928a) for Isadamsia cancrisocia from the east coast of Africa. Carlgren (1928aCarlgren ( , 1949 is the only person to have defi ned the genus, and his defi nition was based on the single species he knew. We update the defi nition of Stylobates to incorporate information from all four known species.
Deep-sea Actiniidae with very wide pedal disc that covers a gastropod shell inhabited by a hermit crab. Anemone pedal disc secretes carcinoecium. Column smooth, thinwalled. Marginal sphincter muscle endodermal, circumscribed, palmate or pinnate. Tentacles hexamerously arranged; fewer than mesenteries at base. Longitudinal muscles of tentacles and radial muscles of oral disc ectodermal. Mesenteries of 5-6 orders; those of lowest orders complete and sterile, those of highest orders incomplete and fertile. Retractor muscles weak, diff use; parietobasilar and basilar muscles distinct.
Column: Not cylindrical: wraps around gastropod shell so column much longer on one side than on diametrically opposite side. Smallest specimen with shortest side 4 mm, longest side 50 mm. Largest specimen with shortest side 15 mm, longest side 90 mm. Smooth, thin. Fosse shallow. Live specimens light pink, body wall translucent (Figure 1a, b); preserved specimens beige. Mesenterial insertions visible through body wall; white in live specimens ( Figure 1a) and preserved specimens.
Oral disc: Oriented toward substrate in life, over umbilicus area (Figure 1c, d). Disc and mouth circular ( Figure 1e); disc exposed and mouth agape in all specimens examined. Ectodermal musculature radial.
Orientation: Directive axis in line with spire of shell, parallel to parietal wall of aperture (dotted red line, Figure 1e).
Tentacles: Beige, slightly darker than column, no pattern. Relatively narrow, tip terete. 96 to more than 200 in largest specimens; at margin, in 3 or 4 cycles. Not of uniform length: shortest ones (1-4 mm) on directive axis, at end of one siphonoglyph, beside longest ones (3-9 mm); tentacle length grades between them around oral disc ( Figure 1e). Ectodermal musculature longitudinal (Figure 2).
Marginal sphincter muscle: Well developed, circumscribed, palmate (Figure 3a, b). Mesenteries and internal anatomy: Two siphonoglyphs visible in most specimens; actinopharynx ribbed, darker beige than column. Mesenteries to fi ve orders ( Figure 4a); thin, each with oral but no marginal stoma. Retractor muscles diff use (Figure 4b). Parietobasilar muscle with short free penon. Sexes presumably separate: three females, one male examined. First three orders complete and sterile, rest incomplete and fertile (Figure 4a).
Carcinoecium: Shape similar to that of dextral, trochoid gastropod shell. Aperture with simple arced elliptical outer lip, fairly straight parietal wall along what would be termed the columella in a gastropod. Bronze color, becomes chalky out of liquid.
Cnidae: Cnidom: Spirocysts, basitrichs, microbasic p-mastigophores. Table 1 lists distribution and size of cnidae; Figure 5 depicts each cnida type. Th e largest specimen (MTQ G57580) possessed small basitrichs (e) and (f) in the actinopharynx, and small basitrichs (h) in the column that were not found in other specimens.
Habitat: Mud and rocks, 590-694 m. Distribution: From Coral Sea of northern Queensland to southern Queensland coast ( Figure 6).
Etymology. Th is species is named for R Alastair Birtles of James Cook University, Townsville, who, with the late P Arnold (MTQ) and M Pichon (Australian Institute of Marine Science), collected this species and photographed it alive. Cnidae of all four species of Stylobates, given as range in length X width of undischarged capsules in μm (outlier measurements in parentheses). n = number of capsules measured, N = ratio of number of animals in which that type of cnida was found to the number of animals examined (where data are available). Frequency of cnida type indicated by the following: ++ very common, + common, -sporadic. Letters in parentheses correspond to images in Figure 5 for S. birtlesi sp. n. 21.3-29.5 X 3.9-5.7 n=27 18.9-23.0 X 4.1-5.7 n=12 23.6-32.2 X 3.5-6.2 n=50 N=7/7 Source:

S. birtlesi
Th is study Dunn et al. 1981Dunn et al. 1981Fautin 1987

Diff erential diagnosis
Tables 1 and 2 present the major attributes of the four known species of Stylobates. Stylobates birtlesi sp. n. diff ers from the others in size and distribution of some of its nematocysts (Table 1), and the nature of the marginal sphincter muscle (Figure 3a, b). Compared to Stylobates birtlesi sp. n., Stylobates aeneus and Stylobates cancrisocia do not possess basitrichs (b) in the tentacles nor basitrichs (l) in the mesenterial fi laments; Stylobates aeneus lacks basitrichs (i) and Stylobates cancrisocia lacks basitrichs (j) in the mesenterial fi laments. Th e clearest distinguishing feature of Stylobates birtlesi sp. n. is the possession of an endodermal circumscribed marginal sphincter muscle in which the lamellae are arranged in a palmate fashion (Figure 3a, b). Th is is clearly diff erent to the other three species, which all possess an endodermal circumscribed marginal sphincter muscle in which the lamellae are arranged in a pinnate fashion (Figure 3c-e). Gross morphology of Stylobates birtlesi sp. n. is similar to that of Stylobates aeneus and Stylobates cancrisocia in position and size of oral disc, and size and arrangement of tentacles. Tentacles of Stylobates birtlesi sp. n. (maximum length 9 mm) are shorter than those of Stylobates loisetteae (maximum length 20 mm). Th e tentacles of Stylobates loisetteae, in contrast to those of other species, are more or less the same length around the oral disc, and the marginal tentacles are longer than the discal ones. Th e tentacles of Stylobates aeneus and Stylobates cancrisocia are arranged like those of Stylobates birtlesi sp. n., the longest and shortest ones beside each other (Figure 1e). Diameter of the oral disc of Stylobates birtlesi sp. n. (15-40 mm) is similar to that of Stylobates aeneus and Stylobates cancrisocia, but less than that of Stylobates loisetteae (to 55 mm). Th e position of the oral disc of Stylobates birtlesi sp. n. is near the aperture of carcinoecium, like in Stylobates aeneus and Stylobates cancrisocia, whereas that of Stylobates loisetteae is on the side of the ultimate whorl of the carcinoecium, away from the aperture.

Discussion
Th e four species of Stylobates are distributed allopatrically (Figure 6), in what Cairns (2007) identifi ed as separate biogeographical regions based on distributions of deepwater scleractinian corals. Stylobates birtlesi sp. n. occurs in the Coral Sea off the Queensland coast of Australia (southwestern Pacifi c region); Stylobates aeneus is known from Hawai'i and Guam (central Pacifi c region); Stylobates loisetteae occurs in the Indian Ocean off the northwest coast of Australia (southeastern Indian Ocean region); and Stylobates cancrisocia is known from the Indian Ocean off east Africa (southwestern  (Baba et al. 2008). Uchida and Soyama (2001) reported Isadamsia sp. J from Japan; that locality is consistent with the distribution of Stylobates aeneus. Doumenc (1975) reported Isadamsia cancrisocia in the North Atlantic at 3360-3600 m. We are dubious about this identifi cation (and do not include the record in Figure 6) because all records for the occurrence of Stylobates are from the Indo-West Pacifi c and at shallower depths.
Carcinoecium-forming anemones of genera characterized by a mesogleal sphincter muscle are known from the Atlantic: for example, Paracalliactis consors (Verrill, 1882) occurs off the northeast coast of the United States at depths of 2085-2665 m, and A specimen in the Invertebrate Zoology collection of WAM (catalog number Z31227) of an anemone that laid down some chitinous material on the gastropod shell to which it is attached is from the same region and depth as Stylobates loisetteae off the coast of Western Australia, and has an endodermal sphincter, but does not belong to Stylobates, either. Th is anemone diff ers from Stylobates in that its pedal disc does not cover the whole shell; the chitinous material does not form a carcinoecium; the column is more or less cylindrical and is much thicker than that of Stylobates; and the contracted oral disc creates a collar at the margin.
Hermit crabs form symbioses with about 100 species of cnidarians (Williams and McDermott 2004). Th ey occur shallow and deep, in tropical and temperate seas. Th e hermit crab is thought to be protected by its cnidarian symbiont (e.g. Ross 1971, McLean and Mariscal 1973, Bach and Herrnkind 1980, Brooks 1988, 1989, Brooks and Gwaltney 1993; possible benefi ts to the cnidarian include transport (Balss 1924, Ross 1974), a fi rm substrate for attachment (Brooks and Mariscal 1986), and access to food collected by the hermit crab (Ross 1984).
Many carcineocium-forming species occur in the deep sea, where calcium carbonate (the mineral of mollusc shells) is highly soluble (Correns 1955), resulting in a limited supply of shells (Balss 1924) and rarity of large shells. A hermit crab living in a chitinous carcinoecium need not change shells as it grows, nor will the carcinoecium dissolve (Dunn et al. 1981). In symbioses not involving a carcinoecium, more than one anemone may be attached to a gastropod shell inhabited by a hermit crab, whereas in the Stylobates/Sympagurus system, one anemone is associated with one hermit crab. In the most thorough account of this association, Dunn et al. (1981) considered it to be obligate for the anemone (which receives food, transport, and substrate) and facultative for the hermit crab.

Conclusion
We describe Stylobates birtlesi sp. n., a new species of deep-sea anemone associated with the hermit crab Sympagurus trispinosus (Balss, 1911), from specimens collected in the Coral Sea off the Queensland coast of Australia. Stylobates birtlesi sp. n. diff ers from the other three known species of Stylobates in some aspects of its nematocysts, and in having a palmate marginal sphincter muscle (in the others it is pinnate). Th e four species of Stylobates are allopatrically distributed in the deep Indo-West Pacifi c Ocean, a pattern similar to those of deep-sea scleractinian corals and squat lobsters. and Mark Salotti from WAM and Steve Coles and Holly Bolick from the Bishop Museum, Hawai'i for specimen loans. Th is research was made possible with funding from ABRS to .