A New Species of Hydrozoan Jellyfish Eutima onahamaensis and New Record of Eutima diademata (Hydrozoa, Leptothecata) from Japan

: The family Eirenidae is one of the major taxa of the order Leptothecata, comprising approximately 80 species from ten genera. In this study, taxonomic investigations, including morphological observations and molecular 16S phylogenetic analyses, were conducted on unknown Eirenidae specimens collected off the coast of Iwaki, Fukushima Prefecture, eastern Japan, in June 2022. The specimens had the following morphological characteristics: marginal warts and tentacular bulbs with lateral cirri and without adaxial papillae, a mouth with simple lips, four simple radial canals, and eight statocysts common to the genus Eutima . However, this species can be distinguished from other species of Eutima by the number of tentacles, number and shape of marginal warts, position of the gonads, and gastric peduncle length. Moreover, the monophyly of the species was evident in the 16S rRNA phylogenetic tree (as indicated by the high bootstrap value of 100%), thereby supporting the validity of the new species. Based on these results, we describe it as a new species, Eutima onahamaensis , for taxonomic stabilization. We also made detailed observations of the morphology and molecular phylogenetic analyses of one of the species newly recorded in Japan: Eutima diademata . A comparative table of the primary diagnostic characteristics of Eutima has been provided. This study provided taxonomic keys for identifying species in the genus Eutima .

Eutima is characterized as follows: Medusa with distinct gastric peduncle; lateral cirri (difficult to observe and often destroyed after fixation); marginal swellings or warts; mouth with simple lips; four simple radial canals; "gonads" on radial canals, either beneath subumbrella or on gastric peduncle or both; with eight (exceptionally twelve) statocysts.Polyps are either single hydranths, erect colonies arising from creeping stolons, or epizootic naked polyps; in nonepizootic forms, hydrocaulus with smooth perisarc, young colonies with cylindrical hydrotheca with diaphragm and a folded pleated operculum formed by convergent flaps not demarcated from the hydrothecal rim (Campanulina type), in older colonies of this type, operculum generally lost and hydrotheca reduced to a perisarcal collar of the type; usually with intertentacular web [1].
To date, two Eutima species, Eutima japonica and Eutima cirrhifera (Kakinuma, 1964), have been reported in Japanese waters [2,20,21].In this study, six specimens of unidentified Eirenidae species were collected off the coast of Iwaki, Fukushima Prefecture, eastern Japan, and off Itoman, Okinawa Prefecture, southern Japan.Our morphological and molecular phylogenetic analyses suggested that these Eirenidae species should be regarded as a new species and a new species record to Japan within the genus Eutima.

Collection and Fixation
Six medusae of Eirenidae specimens were collected from near the water surface (within about 10 m) at Onahama Port, Iwaki, Fukushima Prefecture, eastern Japan (36  20 April 2022 and 4 June 2022.The medusae were captured using a plankton net (mesh size, approximately 0.1 mm, mouth diameter, 30 cm) and a plastic bottle by SCUBA.Four specimens were fixed in 3% formalin in seawater and deposited in the National Museum of Nature and Science, Tsukuba (NSMT).The three specimens (two whole medusa and one tentacle) were preserved in 99.5% ethanol until molecular analysis.Additionally, two specimens of Eutima japonica from Shonan Port, Fujisawa, Kanagawa Prefecture, eastern Japan (35 • 17 ′ 52.40 ′′ N, 139 • 28 ′ 32.20 ′′ E) were used in the molecular phylogenetic analyses (Table 1) [18,[22][23][24].

Morphological Investigation
Taxonomic observations and measurements were conducted on both live and preserved specimens (Figure 1).Medusae were placed and flattened on a glass dish (diameter 50 mm).The umbrella height was measured from the apex of the umbrella to the umbrella margin.The umbrella diameter was measured across the turnover of the exumbrella.Specimens were photographed under a compound microscope (SZ61, OLYMPUS, Tokyo, Japan) using an OLYMPUS OM-D E-M5 MarkII.Measurements were made using methods previously [6] with ImageJ software to the nearest 0.01 mm [25].

Morphological Investigation
Taxonomic observations and measurements were conducted on both live and preserved specimens (Figure 1).Medusae were placed and flattened on a glass dish (diameter 50 mm).The umbrella height was measured from the apex of the umbrella to the umbrella margin.The umbrella diameter was measured across the turnover of the exumbrella.Specimens were photographed under a compound microscope (SZ61, OLYMPUS, Tokyo, Japan) using an OLYMPUS OM-D E-M5 MarkII.Measurements were made using methods previously [6] with ImageJ software to the nearest 0.01 mm [25].
For nematocyst identification in medusae, squash prepared from fresh tissue was examined under a compound microscope (BX53, OLYMPUS, Tokyo, Japan).The nematocysts were identified using the method previously described by Kubota [10].In total, 90 nematocysts were identified, measured, and counted to determine the abundance of nematocyst types in medusae.Measurements were made using ImageJ to the nearest 0.1 µm [25].For nematocyst identification in medusae, squash prepared from fresh tissue was examined under a compound microscope (BX53, OLYMPUS, Tokyo, Japan).The nematocysts were identified using the method previously described by Kubota [10].In total, 90 nematocysts were identified, measured, and counted to determine the abundance of nematocyst types in medusae.Measurements were made using ImageJ to the nearest 0.1 µm [25].

Molecular Phylogenetic Analysis
Near-complete sequences of the mitochondrial 16S rDNA gene (approximately 600 bp) were used for molecular phylogenetic analysis.Genomic DNA was extracted from the ethanol-preserved tissue of the cultured specimens using the DNeasy Blood and Tissue Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions.The 16S rDNA was PCR-amplified and sequenced with the forward and reverse primer pair TCGACT-GTTTACCAAAAACATAGC and ACGGAATGAACTCAAATCATGTAAG [26], using the following PCR profile: initial denaturation at 94 • C for 5 min; five cycles at 94 • C for 50 s, 45 • C for 50 s, and 72 • C for 60 s; 30 cycles at 94 • C for 50 s, 50 • C for 50 s, and 72 • C for 60 s; and final elongation at 72 • C for 5 min [27].The PCR products were purified using a QIAquick PCR Purification Kit (Qiagen) and sequenced in both directions using an ABI 3730 automated sequencer (Applied Biosystems, Bedford, MA, USA).The new sequences were aligned using MEGAX with built-in ClustalW [28].Phylogenetic analysis and pairwise distance measurements were performed using the maximum likelihood method based on the Kimura 2-parameter model [29], with 1000 bootstrap replications in MEGAX.Five sequences of the Eutima species were deposited in GenBank under the accession numbers LC822404-822408 (Table 1).Description.Mature medusae umbrella flat (Figure 2A), 2-4 mm high and 4-8 mm in diameter.Umbrella apex rounded and mesoglea thickened.Exumbrella smooth and nematocysts sparsely scattered (Figure 2A,B).Manubrium hanging in the umbrella cavity, tubular, reddish-brown in color (Figures 2A and 3A).Gastric peduncle very short and indistinct.Manubrium length is about 1 mm, 1/5 of the length of the umbrella diameter, not extended beyond umbrella margin.Mouth cruciform, with four frilled lips (Figure 3B).
Etymology.The specific name "onahamaensis" refers to the Onahama, southern region of Iwaki City, Fukushima Prefecture, which includes the type locality in which the species was found.
Habitat and ecology.Medusae of Eutima onahamaensis sp.n. appeared in shallow waters (5 m depth) in June in a range of cold-temperature localities on the coast of Onahama, Iwaki, Fukushima Prefecture, eastern Japan.The medusae swam by contracting their tentacles and relaxed with extended tentacles (Figure 2A,B).Seasonal occurrence and early life cycle, including embryogenesis, are unknown.
Etymology.The specific name "onahamaensis" refers to the Onahama, southern region of Iwaki City, Fukushima Prefecture, which includes the type locality in which the species was found.
Eutima diademata (Kramp, 1959) New Japanese name.Enaga-konoha-kurage Material examined.NSMT-Co1849; Off Itoman, Itoman, Okinawa Prefecture, western Japan; 26°7′6.41″N, 127°35′53.36″E; 20 April 2022; collector: Sho Toshino.Description.Mature medusae umbrella flat (Figure 5A), 8.7 mm high and 10.4 mm in diameter.Umbrella apex rounded and mesoglea thickened.Exumbrella smooth and nematocysts sparsely scattered (Figure 5A-C).Manubrium hanging in the umbrella cavity, tubular, and whitish in color (Figure 6A,B).Gastric peduncle very long and distinct.Gastric peduncle length about 25 mm, three times the length of umbrella diameter, extended beyond umbrella margin.Mouth cruciform, with four frilled lips.Four radial canals and a circular canal (Figure 6C).Gonads linear, extending along almost the entire length of radial canals and gastric peduncle (Figure 6A).Tentacular bulbs four, swollen, whitish in color (Figure 6D,E).Tentacles nine, filiform, tapering to a tip, length about three times the umbrella diameter (Figure 6F).The four tentacles are arranged perradially and four interradially, but an additional tentacle is included due to malformation.Marginal warts 96, usually 11-13 between successive tentacles (Figure 6G).Statocysts eight (Figure 6G,H).Marginal warts and tentacular bulbs with lateral cirri and without adaxial papillae (Figure 6H).Velum narrow, 1/5 of umbrella diameter.Cnidome.Not examined in this study.Habitat and ecology.Eutima diademata has been reported offshore in Mindanao, Philippines, the Nansha Islands region, the coast of the southeast China Sea, and the Taiwan Strait [30][31][32][33][34].The medusa was abundant in the surface waters along the upwelling region of the southern part of the Taiwan Straits in June [34].In this study, medusae of E. diademata appeared approximately 5 m below the surface in April in a range of subtropicaltemperature localities off Itoman, Okinawa Prefecture, southern Japan.Developmental stages and structural details of medusae have been reported [34].The early life cycle, including embryogenesis and polyp, is unknown.
Etymology.The specific name "diademata" is taken from the Latin word 'diadema,' meaning 'crown.'The name may reflect the shape of a medusa, similar to a crown.

Molecular Phylogenetics
We sequenced Eutima onahamaensis, E. diademata, E. japonica, and the nine Leptothecata taxa for statistical analyses using the 16S rDNA fragments.The maximum likelihood tree constructed for the family Eirenidae based on the 16S rDNA sequences (Figure 7) comprised five major clades formed by the family Eirenidae: (1) Tima, (2) Eugymnanthea, (3) Eutima, (4) Eutonina, and ( 5) Eirene.Eutima onahamaensis clustered in the subclade Eu- Cnidome.Not examined in this study.Habitat and ecology.Eutima diademata has been reported offshore in Mindanao, Philippines, the Nansha Islands region, the coast of the southeast China Sea, and the Taiwan Strait [30][31][32][33][34].The medusa was abundant in the surface waters along the upwelling region of the southern part of the Taiwan Straits in June [34].In this study, medusae of E. diademata appeared approximately 5 m below the surface in April in a range of subtropicaltemperature localities off Itoman, Okinawa Prefecture, southern Japan.Developmental stages and structural details of medusae have been reported [34].The early life cycle, including embryogenesis and polyp, is unknown.
Etymology.The specific name "diademata" is taken from the Latin word 'diadema', meaning 'crown'.The name may reflect the shape of a medusa, similar to a crown.

Morphological Investigation
A comparison of the key features of species in the genus Eutima (two and four tentacled species) is presented in  The Kimura two-parameter distance was 0.064-0.167between Eutima onahamaensis n. sp. and other Eutima, 0.124-0.205between E. onahamaensis and other Eirenidae genera (Eirene, Eugymnanthea, Eutonina and Tima), 0.009-0.011between Eutima diademata from Japan and E. diademata from Taiwan, and 0.000 between Eutima japonica from Japan and E. japonica (MW066348) (Table S1).
Prior to our study, two Eutima species, Eutima japonica and E. cirrhifera, were reported in Japanese waters [2,20,21].Additionally, Eutima japonica is classified into four types, northern, southern, intermediate, and transitional, based on the following morphological characteristics: number of tentacles, length of manubrium, and presence of lateral cirri [10].Eutima onahamaensis tends to resemble those of the intermediate form of Eutima japonica.The intermediate form of the species was originally described by Kubota as Eucheilota intermedia (Kubota, 1984) based on specimens from Zagashima Island, Ago Bay, Mie Prefecture, Japan [39].This form has been reported on Tsushima and Okinawa Islands [39,40,46].Eutima onahamaensis and the intermediate form of E. japonica have short, indistinct peduncles on the manubrium and cruciform oral lips.However, E. onahamaensis has a larger number of marginal warts (39-55 vs. 20-24) and long gonads (half the length of the gonads in E. japonica).The distributions of the two species did not overlap.
Eutima diademata was described as Eucheilota diademata by Kramp from Candos Bay, Mindanao, Philippines, based on a single immature specimen [30].Additionally, Guo et al. described Eutima krampi by observing developmental stages and structural details of medusae from the southern part of the Taiwan Strait [34].Guo et al. regarded that E. diademata is the young medusa of E. krampi because of the following morphological characters: absence of gonads; 2 opposite, perradial tentacles with 1 pair of lateral cirri and without black spot; 29 marginal warts with 1 pair of cirri and each with a distinct black spot on the extreme tip; with 8 statocysts; without gastric peduncle.Given the result of morphological observation, Eutima krampi is regarded as a junior synonym of E. diademata [2].The morphological inspection of E. diademata from Japan agrees well with the morphological description of the mature stage by Guo et al. [34].

Molecular Phylogenetic Analysis
The paraphyly of Eutima, including Eutima onahamaensis and seven species (E.curva, E. diademata, E. gegenbauri, E. gracilis, E. japonica, E. levuka, and E. viridula), was evident in the 16S rDNA phylogenetic tree with high bootstrap values, which supports the validity of the new species.

Conclusions
Our morphological and molecular phylogenetic analyses suggested that the specimens collected off the coast of Iwaki, Fukushima Prefecture, eastern Japan, are new species belonging to the genus Eutima.Additionally, we first reported Eutima diademata from Japan.The medusae of Eutima onahamaensis sp.n. appeared in the shallow waters of Japan in June.However, polyps of the species have not been detected in the wild.Polyps of Eutima species have been found inside bivalve molluscs, Barbatia virescens (Reeve, 1844), Dendostrea sandvichensis (G.B. Sowerby II, 1871), Magallana gigas (Thunberg, 1793), Mytilus edulis Linnaeus, 1758, Mytilus galloprovincialis Lamarck, 1819, Mizuhopecten yessoensis [12,46,47,53].Further investigations, including polyps and their life cycles, are necessary to gain insight into the ecology of the new species.

Figure 7 .
Figure 7. Maximum likelihood tree (under GTR + G + I) for 12 Leptothecata taxa based on the mitochondrial 16S rDNA data set.The scale bar indicates branch length in substitutions per site.Nodal support values are presented as the ML bootstrap value; only values >50% are shown.

Figure 7 .
Figure 7. Maximum likelihood tree (under GTR + G + I) for 12 Leptothecata taxa based on the mitochondrial 16S rDNA data set.The scale bar indicates branch length in substitutions per site.Nodal support values are presented as the ML bootstrap value; only values >50% are shown.

Table 1 .
Taxa included in the phylogenetic analyses and GenBank accession numbers for the sequences.Sequences obtained in this study are in bold.

Table 4 .
Morphology of Eutima in previous and present studies.