Olindias deigo sp. nov., a new species (Hydrozoa, Trachylinae, Limnomedusae) from the Ryukyu Archipelago, southern Japan

Abstract A new hydromedusa belonging to the order Limnomedusae is reported from the Ryukyu Archipelago, southern Japan. Olindias deigosp. nov. can be distinguished from other Olindiidae species by the number and color of tentacles. Mature medusae of O. deigosp. nov. were collected to observe the life history, including polyp (hydroid) and medusa formation. A comparative table of the primary diagnostic characters of the genus is provided.

Species of the genus Olindias Müller, 1861 are large hydrozoans with umbrella diameters reaching 10 cm (Kramp 1961). Olindias formosus (Goto, 1903) is a very beautiful jellyfish called the "flower hat jellyfish" and is popular for exhibition in public aquaria worldwide (Yasuda 2003;Patry et al. 2014). Venomous stings by another species, Olindias sambaquiensis Müller, 1861, have been reported around South American seashores, and it is regarded as a venomous jellyfish (Mianzan and Ramírez 1996;Resgalla et al. 2011). To date, a single incidence of lethal envenomation has been documented for O. formosus in Japan (Yasuda 1988;Purcell et al. 2007), which occurs near seashore.
Recently, ten specimens of Olindias were collected from Okinawa Island, southern Japan. In this study, morphology and molecular phylogenetic analyses revealed that the specimens represent a new species of Olindias. Furthermore, we observed and documented the life history of this new species of Olindias.

Collection and fixing
Ten medusae were collected from Kunigami, Motobu, and Nago, Okinawa Prefecture, Ryukyu Archipelago, southern Japan between March 29, 2015 and April 8, 2018 (Fig. 1). The medusae were collected using a dipper net (diameter 20 cm) and plastic bags while scuba diving, or a set net. Additionally, specimens of O. formosus collected from Iwate, Oita and Miyazaki prefectures were used for comparison of morphology and for molecular phylogenetic analyses (Table 1). After preserving a subsample in ethanol (for molecular analysis), collected medusae were fixed in 5% formalin seawater and deposited in the National Museum of Nature and Science, Tsukuba, Japan (NSMT). Part of the tentacles were preserved in 99.5% ethanol for DNA extraction.

Morphological investigations
Morphological observations and measurements were made on living or preserved specimens. Measurements were made with digital calipers (CD-20CPX, Mitutoyo Corporation, Japan) to the nearest 0.01 mm. For nematocyst identification in the medusae, squashes prepared from fresh tissues were examined under a compound microscope (BX53, OLYMPUS, Japan). In this study, the following morphological characters were recorded (Fig. 2): umbrella height, umbrella diameter, number of centripetal canals,   Goto (1903) distinguished exumbrella tentacles, those arising from the exumbrella at any level, from those occurring proximal to the apex -just a short distance from the velum; however, he did not distinguish exumbrella from primary tentacles. In this study, the exumbrella tentacles are defined as tentacles that arise from the black band on the exumbrella, rather than those arising from the margin of the umbrella. Nematocysts were identified according to Östman (2000) from wild and cultured specimens. Measurements were made using ImageJ (NIH, USA) to the nearest 0.1 µm.

Molecular phylogenetic analyses
Near complete sequences of the nuclear 16S rDNA genes (approximately 600 bp) were used for molecular phylogenetic analyses. Genomic DNA was extracted from the 99.5% ethanol preserved tissue of specimens using the DNeasy Blood and Tissue Kit (QIAGEN, Germany) following the manufacturer's protocol. 16S rDNA was PCR amplified and sequenced using primers and protocols outlined in Collins et al. (2008). Unidentified and deposited olindiid sequences in GenBank (Table 1) were used for molecular comparison and to clarify the unidentified Olindias species. The generated sequences were aligned using MEGA 6.06 with built in ClustalW (Tamura et al. 2013). Phylogenetic analysis and pairwise distance measurements were determined using the maximum likelihood method with 1000 bootstrap replications in MEGA 6.06 (Tamura et al. 2013). All sequences have been deposited in DNA Data Bank of Japan (DDBJ) under accession numbers LC508961-LC508970 for the new species (Table 1).

Observation of life cycle
Collected male and female medusae were transferred to an aquarium tank (18 × 32 × 22 cm, volume 13 L) to obtain fertilized eggs. Spawning was induced by alternation of light and dark conditions using an LED lamp (8 W) with a blue filter. The medusae were incubated in light between 20:30 and 7:00 and in dark between 7:00 and 20:30. Obtained fertilized eggs were kept in Petri-dishes (diameter 8 cm, height 4 cm) with filtered seawater (5 µm) at about 20 °C in the laboratory at Okinawa Churaumi Aquarium. Artemia nauplii were fed to primary and secondary polyps twice to thrice a week. Full water changes were conducted with filtered seawater (5 µm) about three hours after feeding. Newly detached medusae were kept in Petri-dishes (diameter 8 cm, height 4 cm) with filtered seawater (5 µm) at about 20 °C. Artemia nauplii were fed to the young medusae daily. The medusae that grew to about 4 cm of umbrella diameter were transferred into a tank (38 × 48 × 58 cm, volume 96 L). Juvenile anchovies and krill were fed to the medusae daily. Culture water was replaced with filtered seawater (5 µm) about three hours after feeding.       5D). Different length of black bands elongated from umbrella margin to the apex of exumbrella (Fig. 5F). Centripetal canals about 80 to 100, long and short alternately aligned (Fig. 5D). Long canals reached apex of the umbrella while short ones were half or quarter length that of long canals terminating in tentacles. Some canals connected or branched (Fig. 5D).

Phylum
Tentacles and marginal clubs aligned on the umbrella margin (Figs 3D, 5E). Primary tentacles about 80 to 140, thin, short with distal adhesive pads and cnidocysts in transverse clasps. Color of exumbrella tentacles and primary tentacles pale black with purple and glowing green tips and with black base (Fig. 3D). Number of secondary tentacles about 50, thick, no adhesive pads, cnidocysts in rings, deep-brown in color (Fig. 3D). Contracted secondary tentacle short, coil-like while elongate ones reaching 2 m in length. Exumbrella tentacles about 30 to 60, developing on tip of black bands (Fig. 5F). Shape and color similar to those of primary tentacles (Fig. 3D). Number of  marginal clubs about 170 to 240, rounded, short, whitish in color (Fig. 3D). Statocysts were not found in fixed mature medusae. Life cycle. Fertilization and polyp formation. Spawning occurred in dark conditions. Thousands of fertilized eggs were collected from the bottom of the tank in the early morning (from 8 to 9 am); diameter of blastocysts ~100 µm (Fig. 6A). Blastocysts developed into planulae within two days. Planulae had a pear-shaped body, 70 µm in diameter and 130 µm in length (Fig. 6B); they developed into polyps within 20 days.
The polyps form small colonies by elongation of the stolon, developing into a network (Fig. 6C-F). The hydrorhizae were cylindrical with small egg-shaped or cylindrical hydranths developing on the stolon. The hydranths had an ovoid body, 0.7 mm in length (Fig. 6E). The body was divided in two parts, gastric region (0.3 mm in diameter and 0.5 mm in length) and hypostome (0.2 mm in diameter and 0.2 mm in length). Tentacle single, filiform, 1.7 mm in length (Fig. 6E, F). The hydroid, usually brownish or yellowish, became orange or pink owing to the consumption of Artemia nauplii. Tentacle and hypostome transparent. Budding and development of young medusa. Budding of young medusae was observed after 8 months of polyp formation. Medusa bud formation occurred on stolon (Fig. 7A) at temperatures below 20 °C. The shape of the buds was ovoid and 0.3 mm in diameter (Fig. 7A). Two days after onset of budding, four radial canals and a circular canal appeared, but were obscure (Fig. 7B). Eight days after onset of budding, rudiments of tentacles developed from the bud (Fig. 7C). Fourteen days after onset of budding, the buds enlarged (0.8 mm in diameter) and green fluorescence was observed on the tentacles (Fig. 7D). Fifteen days after onset of budding, the medusa buds detached.
Newly detached medusae had a spherical umbrella translucent in color ( Fig. 8A-C). Umbrella height about 1.6 mm and diameter about 1.5 mm. Exumbrella with tiny nematocysts along entire exumbrella (Fig. 8D). Four simple radial canals from four corners of the stomach (Fig. 8B, D). Statocysts four, enclosed in mesoglea, adjacent to primary tentacles (Fig. 8E). Manubrium long, about 50% that of umbrella height (Fig. 8F). Marginal tentacles of two types (Fig. 8C, G, H). Primary tentacles four, short (about 1 to 2 times that of umbrella diameter) bearing nematocyst clusters on the tips (Fig. 8G). Secondary tentacles two, long (about 5 times that of umbrella diameter) bearing 10 to 20 nematocyst batteries (Fig. 8H). The medusae attached using the tip of the primary tentacles, but adhesive pad was not observed (Fig. 8G). Green fluorescence was observed at the base of tentacles and four corners of the stomach (Fig. 8D-F).
Ninety-day-old medusae were about 10 mm in diameter (Fig. 9A). Umbrella bowl-shaped. Primary and secondary tentacles about 40 and 20, respectively. About 20 centripetal canals were observed. Medusae aged 120-day-old were about 15 mm in diameter (Fig. 9B). White fibrous structures appeared around radial canals. Manubrium elongated and mouth rips developed. Number of primary and secondary tentacles and radial canals not increased much. Medusae aged 150-day-old were about 20 mm in diameter (Fig. 9C). Primary and secondary tentacles about 60 and 20, respectively. About 20 centripetal canals observed. Exumbrella tentacles developed near umbrella margin, but were not observed on the apex of exumbrella. Medusae aged 200-day-old were about 40 mm in diameter (Fig. 9D). Primary and secondary tentacles about 80 and 40, respectively. About 60 centripetal canals were observed. Gonad developed. Exumbrella tentacles developed near the margin of umbrella and the middle part of exumbrella. Medusae aged 240-day-old were about 60 mm in diameter (Fig. 9E). Primary and secondary tentacles about 120 and 40, respectively. About 60 centripetal canals observed. Gonad developed and matured. Spawning observed (Fig. 9E).
Habitat and ecology. Medusae of O. deigo appeared in shallow waters (from 3 to 10 m) during winter and spring in a range of subtropical temperature localities in the Ryukyu Archipelago, southern Japan. The medusae rested on the sandy bottom or in areas with a good slope and movement of water during the daytime while they drifted and swam by extending their tentacles during the night. Thus, the species seems to be nocturnal in behavior. Stinging events attributable to O. deigo have not been reported thus far.
Etymology. The species name comes from the beautiful appearance of the jellyfish. The Japanese name deigo (noun in apposition) means Erythrina variegata which is popular as the "prefectural flower" of Okinawa.
Differential diagnosis. A comparison of key features of the species in the genus Olindias is presented in Table 5. All species of Olindias have four radial canals and numerous centripetal canals; numerous tentacles of two kinds: primary ones issuing above the umbrella  margin, with distal adhesive pads and cnidocysts in transverse clasps and secondary ones on the umbrella margin, no adhesive pads, cnidocyst in rings; gonads with papilliform processes, on radial canals; numerous marginal clubs, statocyst usually in pairs at base of primary tentacles (Bouillon et al. 2006 (Table 5).

Discussion and conclusions
Prior to our study, only one olindiid, O. formosus, had been recorded from Japan (Goto 1903). This species was described by Goto (1903) based on specimens collected from Misaki, Kanagawa Prefecture, eastern Japan. The medusae of the species have been reported from warm and cold localities in the Sea of Japan and the Pacific coast of Honshu (Uchida and Uchida 1965), and Jejudo Island, Korea (Park 2006 (Patry et al. 2014) and O. muelleri (identified as Olindias phosphorica (Delle Chiaje, 1841)) (Weill 1936). Polyps form colonies which are stolonal, and hydroids bear a single tentacled hydranth, but lack hydrotheca in O. deigo and O. formosus (Patry et al., 2014) (Table 5). However, polyps of O. muelleri are solitary, and hydranth lacks tentacle but enclosed by hydrotheca. Young medusae of O. deigo resemble those of O. formosus in umbrella sizes and number of tentacles (Patry et al. 2014).
Asexual reproduction and medusa budding of O. deigo were observed at 20 °C. The temperature corresponds with that of winter waters around Okinawa Island (Japan Meteorological Agency 2019). Mature medusae appear between winter and spring in Okinawa. Polyps of O. deigo may produce medusae during fall and winter.
Morphological and molecular phylogenetic analyses in this study provide evidence that Olindias from the Ryukyu Archipelago is a new species. Olindiids are very beautiful and popular but harmful because of their venomous stings (Mianzan and Ramírez 1996;Resgalla et al. 2011). Additional investigations are needed to understand the ecology and distribution of O. deigo.