Description of three new species of Scolanthus (Cnidaria, Anthozoa, Actiniaria, Edwardsiidae): first records of the genus from Japan

Abstract Scolanthus is one genus of Edwardsiidae, a speciose family of burrowing worm-like sea anemones characterized by lacking a physa-like aboral end and by possessing nemathybomes on the whole body except at the distal end. This genus has been recorded worldwide, but there have been no specimens collected from Japan. In this study, we discovered four Scolanthus species in Japan for the first time: Scolanthusarmatus (Carlgren, 1931) and Scolanthuskopepesp. n. from the Ogasawara Islands, Scolanthusenasp. n. from Ena Bay, Kanagawa, and Scolanthusiseisp. n. from Sugashima Island, Mie.


Introduction
The family Edwardsiidae is characterized by their worm-like bodies and is one of the major taxa in the order Actiniaria. Edwardsiidae contains approximately 90 species (Fautin 2013) and are diagnosed by having eight perfect mesenteries on the first cycle even as adults, while almost all other sea anemones have twelve perfect mesenteries in their first cycle (Carlgren 1949). The mesenterial arrangement of Edwardsiidae has been regarded for a long time as an ancestral character among actinarians as the arrangement is similar to those of several sea anemone larvae (reviewed in Daly 2002a, Uchida andSoyama 2001). However, this view has been challenged by the finding that the simplified mesenterial arrangement of this family may not be an ancestral character but is a secondary adaptation to infaunal life (e.g., Manuel 1981, Daly 2002b. Recently, this hypothesis was reinforced by a molecular phylogenetic study (Rodriguez et al. 2014).
In Japan, 13 species of five genera of the family Edwardsiidae have been reported (Table 1; Yanagi 2006, Sanamyan and Sanamyan 2013. Of these, seven species are morphologically described in taxonomic papers (Carlgren 1921, 1931, 1940, Sanamyan and Sanamyan 2013, Stimpson 1856, Uchida 1932) and the remaining five species have only been reported in field guide books without precise morphological information (Uchida 1965, Uchida andSoyama 2001).
The genus Scolanthus was established by Gosse (1853), who designated Scolanthus callimorphus Gosse, 1853 as the type species by monotypy. However, Scolanthus was synonymized with Edwardsia by Gosse himself (Gosse 1855) and S. callimorphus was transferred to Edwardsia. Later, Manuel (1981) stated that this species did not belong to Edwardsia, and the genus Scolanthus was revived for the species and also came to include two species of the genus Isoedwardsia Carlgren, 1921. England (1987 agreed with this synonymization, and also transferred Edwardsia armata Carlgren, 1931 to Scolanthus (see also Daly 2002b, Daly andLjubenkov 2008). Today, Scolanthus is distinguished from other genera by having nemathybomes and no physa in the aboral end (Manuel 1981, Daly andLjubenkov 2008). Table 1. All Edwardsiidae sea anemones recorded in Japanese waters. Note some researchers recently have advocated that Metedwardsia akkeshi (and the genus Metedwardsia) do not belong to Edwardsiidae (Gusmão et al. 2016).
The study of Edwardsiidae in Japanese waters has not progressed much, and additionally the genus Scolanthus has not been reported from Japan thus far. In this research, we report Scolanthus armatus (Carlgren, 1931) from the Ogasawara Islands, Japan, as well as discover and describe three new species from Japanese waters.

Sample collection and preservation
The specimens were dug out from sandy seafloors using a shovel and a sieve, or from rocky substrates using a chisel, by wading, snorkeling, or scuba diving. Sampling was carried out around Chichijima Island in the Ogasawara Islands, in Ena Bay in Kanagawa, and around Sugashima Island in Mie, Japan (Fig. 1). All sea anemones were kept undisturbed in aquaria for several hours to several days after collection, until they were acclimated and completely spread and elongated their tentacles. Then, these relaxed specimens were anesthetized with magnesium chloride solution or l-menthol. For Scolanthus ena sp. n. and the holotype specimen of S. isei sp. n., the whole body, except for a few tentacles, was fixed in 5% seawater formalin solution. Several tentacles were dissected from the body and preserved in 99.5% ethanol. As for S. armatus and S. kopepe sp. n., the tissue samples were cross-sectional sliced portions of the body instead of tentacles, which are too tiny to dissect exactly.
The examined specimens were deposited in the National Museum of Nature and Science, Tokyo (NSMT).

Preparation of histological sections
Histological sections were made generally following standard protocols of Presnell and Schreibman (1997). The specimens of Scolanthus armatus, S. ena and S. isei were dissected into different tissues (LIST), dehydrated by ethanol and xylene, embeded in paraffin, sliced into serial sections (8-10 µm thick) by using a microtome, mounted on glass slides, and stained with hematoxylin and eosin. Because S. kopepe sp. n. was too small to dissect, whole individuals were used to make sections by following the methods above.

Cnidae observation
Cnidae were observed in the tentacle, actinopharynx, nemathybomes, column and filament; this genus does not have physa, so there are no cnidome data for the physa. Images of the cnidae were obtained by differential interference contrast microscopy (Yanagi et al. 2015). The length and width were measured using the software ImageJ v. 1.49 (Rasband 1997(Rasband -2012. Cnidae nomenclature followed Mariscal (1974).
Description. External anatomy. Column rough, ca. 40 mm in whole length, and ca. 5 mm in width, worm-like form, and the proximal part narrower to some extent. The column consists of capitulum, scapulus and scapus. The most proximal part of column capitulum, distinct, extremely short, whitish and semitransparent, but scapulus and scapus indistinct. The periderm-like cuticle, brownish orange with no pattern in color, covering the whole column except capitulum and tentacle but easily stripped off from epidermis ( Fig. 2A). The scapus beneath periderm semitransparent, with scattered small prominent nemathybomes but no papillae ( Fig. 2A). Aboral end of the column tapered or flattened, not differentiated from scapus, with scattered nemathybomes (Fig. 2, A and 2I). No pedal disk, and no physa or physa-like structure. Tentacles in two cycles, 16 in number, eight in inner and eight in outer cycle (Fig. 6A). All tentacles long and slender, 1.5-2.0 mm in length, the inner tentacles as long as outer ones, transparent or semi-transparent, and capitated on their tip (Fig. 2B). Oral disk ca. 1.5 mm in diameter. Internal anatomy. Eight perfect mesenteries, all macrocnemes. Four dorsal and ventral directives, and four lateral mesenteries not-paired with other macrocnemes, arranged in normal Edwardsia pattern (Fig. 6A). All macrocnemes present along whole length of the body from oral to aboral end, and bearing distinct retractor and parietal muscles. The retractor muscle of lateral mesenteries all ventrally facing (Fig. 6A). Eight tiny microcnemes, without muscles, confined only in distal-most part. Four microcnemes between dorsal directives and dorso-lateral mesenteries, two between dorsoand ventro-lateral mesenteries, and two between ventro-lateral mesenteries and ventral directives. Each tentacle between either exo-or endocoelic. (Fig. 6A). Retractor muscles strongly developed and diffused ( Fig. 2F-H), pennon-like, configured with 15-30 muscular processes, each slightly branched (Fig. 2F). Parietal muscles with approximately 10 muscular processes (Fig. 2F). Actinopharynx short, ca. 2.5 mm in length, no distinct siphonoglyph (Fig. 2C). Tentacular circular muscle indistinct (Fig. 2D) and longitudinal muscle ectodermal and distinct (Fig. 2E). Mesoglea thin in the whole body, less than 100 µm even at the thickest part of body wall ( Fig. 2C-I). Nemathybomes, ca. 100 µm in diameter, protrude from mesoglea on the column including the aboral end (Fig. 2I). Marginal sphincter muscle and basilar muscle absent (Fig. 2C, H). Gonads adjacent to the retractor muscle, relatively long (Fig. 2H). Testes between filament and retractor muscle, but no mature gametes in this specimen.
Origin of Japanese name. New Japanese name: Ogasawara-ashinashi-mushimodoki. "Ogasawara" is the locality name. In Japanese waters, this species has been collected only in the Ogasawara Islands. "Ashinashi-mushimodoki" is short for the Japanese name of this genus (see the etymology of genus).
Remarks. Scolanthus armatus was originally described by Carlgren (1931) as Edwardsia armata when the genus Scolanthus was a junior synonym of Edwardsia. After Scolanthus was revived by Manuel (1981), this species was transferred to Scolanthus by England (1987). This species had no physa-like structure but many nemathybomes in the aboral end. This is the most characteristic feature of Scolanthus, and this feature is not found in Edwardsia. The specimen from the Ogasawara (Bonin) Islands almost completely agrees with the description of Scolanthus armatus in England (1987); e.g. 16 tentacles which are capitated on the tip; rounded or flattened aboral end; scattered nemathybomes extending to the proximal end; strong and diffused retractor muscle (slightly branched muscular processes also correspond to England, 1987;fig. 13). The proportion and size of body is also similar to England's description. There are, however, a few differences in the cnidome; England (1987) mentioned nothing about b-mastigophores in the description; stating that basitrichs and microbasic b-mastigophores could be distinguished by both the large diameter of the capsule and the broadened shaft shape (England 1991). However, both cnidomes are still easily confused and Table 2.   England (1987) to probably be basitrichs. This probability is reinforced by England (1987: table 6): two types of "basitrichs" in filament of S. armatus, but one type of basitrich with apparently broader capsules than the other type while the lengths are not different. In addition, there are very long basitrichs in our specimen, but they are few in number. If the numbers of these long basitrichs are very low, it is possible that England (1987) did not observe them. Moreover, the other cnidae size ranges resemble those of specimens of England (1987).
In conclusion, we identified this specimen as S. armatus because of its similarity in almost all morphological features to the original description of the species. The slight difference observed in the cnidome may be simply individual variation.
Description. External anatomy. Column rough, rugged and uneven, ca. 80 mm in whole length in holotype, and 10-15 mm in width, pipe-like in form both in living (Fig. 3A) and fixed specimen. The most upper part narrower to some extent. The most proximal part of column capitulum, dark brownish semitransparent, and the remaining part to aboral end scapus. The periderm of column orange, with no pattern in color, but thinner on the mesenteries so that the mesenterial line visible through the body wall. Scapus with scattered nemathybomes but no papillae. Aboral end rounded, not differentiated from scapus, with nemathybome ( Fig. 3A, H). No pedal disk, but no physa or physa-like structure. Tentacles slender, no acrosphere, brownish, semitransparent with white patch on each surface (Fig. 3A). Tentacles 20 in number, in two cycles; ten in inner and ten in outer cycle (Fig. 6B), 7.0-10.0 mm in length, longer than oral disk diameter and the inner tentacles shorter than outer ones. Oral disk ca. 5.0 mm in diameter. The mouth swelled and dome-like.
Cnidome. Spirocysts (in tentacles), basitrichs (in every tissue), microbasic b-mastigophores (in filament) and microbasic p-mastigophores (in actinopharynx, and filament) ( Etymology. The species epithet is named after the type locality, Ena Bay. The word "ena" is a noun in apposition. Origin of Japanese name: new Japanese name: taruashinashi-mushimodoki. "Taru" is the name of the collector of this new species. Remarks. Scolanthus ena sp. n. has 20 tentacles, as do Scolanthus ignotus (Carlgren, 1920) and S. isei sp. n.: other edwardsiid species have 16 tentacles (England 1987, Daly andLjubenkov 2008, andthis study). Small and large types of basitrichs in nemathybomes of S. ena are far smaller and far larger, respectively, than basitrichs in nemathybomes of S. ignotus (Carlgren 1920, England 1987. Moreover, S. ena sp. n. is 80 mm in body length, approximately three to four times longer than the 20-30 mm of S. ignotus. Scolanthus ena sp. n. is different from S. isei sp. n. in its tentacular arrangement (Fig. 6B, C), structure of column surface (periderm of S. ena sp. n. does not have trichome-like structures [ Fig. 4A] unlike S. isei sp. n.; the nemathybomes of S. ena sp. n. are far more sparse than those of S. isei sp. n.), body size (S. ena sp. nov. is far bigger than S. isei sp. n.), and cnidome (only S. ena sp. n. has microbasic b-mastigophores in their filaments) (see Table 2). In addition, S. isei sp. n. lives in cavities of bare rocks, a different habitat compared to that of S. ena sp. n. (see Remarks of S. isei).
This species is one of the biggest species in the genus Scolanthus: all previously reported species of nominal Scolanthus have bodies less than 80 mm in length (Gosse 1853, McMurrich 1893, Carlgren 1920, Carlgren 1921, Pax 1924, Carlgren 1931, Carlgren 1942, Daly and Ljubenkov 2008. Despite several sample collection surveys at Ena Bay, S. ena sp. n. was collected only once, and no specimens have been collected from any other locality. It is said by local people that the environment of Ena Bay has changed from several decades ago; the bottom of bay was previously rocky, and a muddy flat has formed in recent years by inflow of sediment. Considering some Scolanthus live in rocky habitats compared to other edwardsiids (e.g. S. isei sp. n.), the primary habitat of S. ena sp. n. might be rocky, and perhaps the numbers of individuals have decreased in Ena Bay by recent rapid changes in the environment. It is difficult, however, to examine this hypothesis because Edwardsiidae sea anemones living in or between rocks often cannot be collected easily even if there are many individuals present.
Even though there is only one specimen of S. ena, the character differences from other Scolanthus species make it obvious that this specimen is not a formerly described Scolanthus species. Examination of additional specimens in the future may help better delineate this species.
Description. External anatomy. Column rough, rugged and uneven, ca. 30 mm in whole length in fixed holotype, and 10-12 mm in width, truncated cone-like form both in living and fixed (Fig. 4A) specimen, comparatively tubby form for edwardsiids. Paratype a little small, ca. 18 mm in length and ca. 9 mm in width. Upper part narrower than lower part. No apparent capitulum, all parts of column uniformly scapus. Periderm brownish or whitish, no pattern in color, with trichome-like structure (Fig. 4A), and easily stripped. Column with highly densely scattered nemathybome but no papillae, and the surface on the mesenteries slightly sunken (Fig. 4A). Aboral end rounded, not differentiated from scapus, with nemathybome. No pedal disk, but no physa or physa-like structure (Fig. 4A,  G). Tentacles slender, no acrosphere, completely transparent and white patches or stripes on each surface. Tentacles 20 in number, in two cycles; eight in inner and twelve in outer cycle (Figs 4B, 6C), 5.0-7.0 mm in length, longer than oral disk diameter and the inner tentacles shorter than outer ones. Oral disk ca. 4.0 mm in diameter. Mouth not swollen.
Internal anatomy. Eight perfect mesenteries, all macrocnemes. Four dorsal and ventral directives, and four lateral mesenteries not paired with other macrocnemes (Fig. 6C). All macrocnemes present along whole length of the body, from oral to aboral end, and bearing distinct retractor and parietal muscles. The retractor muscle of lateral mesenteries all ventrally facing (Fig. 6C). Twelve tiny microcnemes, without muscles, confined only in distal-most part. Four microcnemes between dorsal directives and dorso-lateral mesenteries, four between dorso-and ventro-lateral mesenteries, and four between ventro-lateral mesenteries and ventral directives, in unusual arrangement for Edwardsiidae. One tentacle each between exocoels and endocoels (Fig. 6C). Retractor muscles pennonlike, diffused throughout the whole body, smaller next to the actinopharynx (Fig. 4F), but largely developed and almost integrated to gonads in lower part (Fig. 4G). Each muscle pennons consisting of approximately 20-30 single or slightly branched muscular processes (Fig. 4F, G). Parietal muscles with approximately 15-20 muscular processes (Fig. 4G). Actinopharynx short, no distinct siphonoglyphs (Fig. 4F). Both tentacular circular muscle and longitudinal muscle too weakly developed to observe (Fig. 4C, D). Mesoglea thickest in the body wall and aboral end, approximately 200-300 and in some parts over 500 µm thick. However, mesoglea far thinner in actinopharynx and mesenteries (Fig. 4E, F, G), and thinnest in tentacles (Fig. 4C, D). Nemathybomes, around 150 µm in diameter, protruded from body wall (Fig. 4I) in the column but a little buried into the mesoglea in the aboral end (Fig. 4G, J). Marginal sphincter muscle and basilar muscle absent (Fig. 4E, G). Gonads next to retractor muscle, short, and wide (Fig. 4G). Ovary between retractor muscle and filament, and oocytes in gonads of holotype.
Distribution. Southwest coast of Sugashima Island, Mie. Known only from the type locality.
Etymology. The species name was named after Yuji Ise, the collector of both holotype and paratype specimens. Origin of Japanese name: New Japanese name: sugashima-gareba-ashinashi-mushimodoki; "gareba" means rocky seashore, the habitat where this species inhabits.
Remarks. In terms of having 20 tentacles, Scolanthus isei sp. n. resembles S. ignotus and S. ena sp. n., while all other edwardsiid species have 16 tentacles (England 1987, Daly andLjubenkov 2008, andthis study). This species is different from S. ignotus in having two types of basitrichs of different sizes in nemathybomes (Carlgren 1920), and the larger type is far bigger than the basitrichs of S. ignotus. The differences between S. isei sp. n. and S. ena sp. n. are principally regarding the tentacular arrangement and body size (see Remarks of S. ena sp. n.).
Exceptionally for edwardsiids, this species inhabits in the cavities of the underside of boulders, adhering by their aboral end, on rocky seashores. Edwardsiids usually inhabit in sand or mud, and only two species had been reported from other different environments: Edwardsiella andrillae Daly, 2013 living on ice (Daly et al. 2013), and Tempuractis rinkai Izumi, Ise & Yanagi, 2018 living in sponges .
Distribution. Chichijima Island, Ogasawara. Known only from the type locality. Etymology. Most of the specimens were collected from Kopepe Coast. "Kopepe" is the name of the native people that used to live in the Ogasawara Islands, who had emigrated from the Gilbert Islands, now the Republic of Kiribati. The word "kopepe" is a noun in apposition. Origin of Japanese name: New Japanese name: kopepeashinashi-mushimodoki. "Kopepe" is originated from same coast.
Remarks. This species resembles not only Scolanthus armatus but also S. scamiti Daly & Ljubenkov, 2008, S. triangulus Daly & Ljubenkov, 2008, S. curacaoensis (Pax, 1924, S. nidarosiensis (Carlgren, 1942) and S. callimorphus Gosse, 1853 in terms of having 16 tentacles (Gosse 1853, Manuel 1981, England 1987, Daly and Ljubenkov 2008. Scolanthus kopepe sp. n. is similar to S. armatus, and both are found around the same island. Scolanthus kopepe sp. n., however, is morphologically distinguishable by several points as below: S. kopepe sp. n. is smaller than S. armatus, one-third to a half in length and onefifth to one-third in width (the specimen of S. armatus is even far bigger than living S. kopepe sp. n.); S. kopepe sp. n. has brownish red stripe from ventral side to dorsal side on oral disk and white patch on capitated tentacle tip, both features are not present on S. armatus; the number of muscular processes of both retractor muscles and parietal muscles of S. kopepe sp. n. are far fewer than those of S. armatus (Figs 2G, H, 5E, G;England 1987); moreover, the S. kopepe sp. n. holotype has two types of basitrichs in the tentacles, actinopharynx and nemathybomes while S. armatus has only one type ( Table 2). The body of S. kopepe sp. n. is slender and uniform in width (both in living and preserved specimens) while that of S. scamiti is stout and increasing in width toward to the aboral end (Daly and Ljubenkov 2008). Besides, the basitrichs in the nemathybomes of S. kopepe sp. n. are two types while those of S. scamiti are only one type (Table 2). Scolanthus kopepe sp. n. is 15-25 mm in body length while S. triangulus has a maximum body length of 11 mm Figure 7. Cnidae of Scolanthus species. A-E S. armatus (NSMT-Co 1609) A1 spirocyst in tentacle A2 basitrich in tentacle B1 basitrich in actinopharynx B2 microbasic b-mastigophore in actinopharynx C basitrich in nemathybome D basitrich in column E1 small basitrich in filament E2 large basitrich in filament E3 microbasic b-mastigophore in filament F-I S. ena sp. n. (NSMT-Co 1610) F1 spirocyst in tentacle F2 basitrich in tentacle G1 basitrich in actinopharynx G2 microbasic p-mastigophore in actinopharynx H1 small basitrich in nemathybome H2 large basitrich in nemathybome I1 basitrich in filament I2 microbasic b-mastigophore in filament I3 microbasic p-mastigophore in filament J-M S. isei sp. n. (NSMT-Co 1611) J1 spirocyst in tentacle J2 small basitrich in tentacle J3 large basitrich in tentacle K1 basitrich in actinopharynx K2 microbasic b-mastigophore in actinopharynx L1 small basitrich in nemathybome L2 large basitrich in regular tentacles M1 small basitrich in filament M2 large basitrich in filament M3 microbasic p-mastigophore in filament N-R S. kopepe sp. n. (NSMT-Co 1614) N1 spirocyst in tentacle N2 small basitrich in tentacle N3 large basitrich in tentacle O1 small basitrich in actinopharynx O2 large basitrich in actinopharynx P1 small basitrich in nemathybome P2 small basitrich in nemathybome Q basitrich in column R1 basitrich in filament R2 microbasic b-mastigophore in filament. (Daly and Ljubenkov 2008), smaller than S. kopepe sp. n. Furthermore, basitrichs of S. triangulus are over 63 µm (Daly and Ljubenkov 2008;table 3), larger than both types of basitrichs of S. kopepe sp. n. Basitrichs in the nemathybomes of S. callimorphus are of only one type and are over 60 µm in length (Manuel 1981, p 265), while S. kopepe sp. n. has two types of basitrichs in the nemathybomes and both of them are less than 60 µm in length. Scolanthus curacaoensis has far larger body, 45 mm in length (Pax 1924), and has a well-developed, circumscribed retractor muscle and rounded distinct parietal muscle (Pax 1924;Figs 4, 5) while S. kopepe sp. n. has diffused and undeveloped retractor and indistinct parietal muscle. Scolanthus nidarosiensis lives in the deep sea of a cold region (125-150 m depth of Norway; Carlgren 1942) in contrast to S. kopepe sp. n., which lives in shallow waters in the subtropical region. The retractor muscles of S. nidarosiensis are well developed and muscle processes were obviously branching (Carlgren 1942; fig. 71) while those of S. kopepe sp. n. are far less developed with simple processes. Moreover, nemathybomes of S. nidarosiensis contain longer basitrichs than the large basitrichs of S. kopepe sp. n., and S. nidarosiensis has only one type of basitrich (Carlgren 1942) while S. kopepe sp. n. has two types of basitrichs.
Scolanthus kopepe sp. n. usually lives in coral sand, and prefers dark environments under large rocks.