Octorhopalona saltatrix, a New Genus and Species (Hydrozoa, Anthoathecata) from Japanese Waters

Simple Summary In this study, we describe a new genus and species of hydromedusa belonging to the family Halimedusidae (Hydrozoa, Anthoathecata) that is found off Oarai, Sagami Bay, and Tosa Bay, Japan. This family comprises four species in three genera: Halimedusa, Tiaricodon, and Urashimea. The new genus and species can be distinguished from all other Halimedusidae species by genetic sequences and morphological characteristics. A comparative table of the primary diagnostic characteristics of the genus Halimedusa is provided. In addition, the diagnosis of Halimedusidae was modified. Jellyfish blooms cause serious problems in fishing, industry and public health. This paper contributed to the understanding of ecology and diversity of jellyfish on our planet. Abstract Approximately 300 species of cnidarian jellyfish have been reported in Japanese waters. However, many specimens remain unidentified. In this study, taxonomic investigations, including morphological observations and molecular 16S phylogenetic analyses, were conducted on unknown specimens collected off Oarai, Sagami Bay, and Tosa Bay, Japan. The specimens have the following morphological characteristics: distinct peaks in jelly above the base of the manubrium, a red band on the manubrium, and cylindrical marginal bulbs, each with an abaxial ocellus that is common to the family Halimedusidae. However, the specimens can be distinguished from other Halimedusidae species by their eight radial canals, eight tentacles with numerous stalked nematocyst knobs, and eight nematocyst tracks on the exumbrella. Moreover, molecular phylogenetic analyses revealed that the Kimura two-parameter distance between the specimens and other Halimedusa species was 0.066–0.099, which is considered to represent intergeneric variability. Based on this result, we described it as a new species and established a new genus for taxonomic stabilization. We also emended the diagnostic characters of the family Halimedusidae owing to the establishment of the new genus. Halimedusidae comprises five species in four genera. This paper provides taxonomic keys for the identification of species in the family Halimedusidae.


Introduction
The hydrozoan family Halimedusidae is a small group that includes four species from three genera: Halimedusa Bigelow, 1916;Tiaricodon Browne, 1902; and Urashimea Kishinouye, 1910 [1-3]. Halimedusidae species are distributed in the shallow waters of a range of tropical, subtropical, and mild-temperature localities in the Pacific and Atlantic Oceans [1, [4][5][6]. The species have sexual, planktonic medusae, and asexual benthic polyps in their life cycle [1, 6,7]. Free-swimming medusae are liberated by the budding of small solitary polyps.
Historically, the taxonomy of the family Halimedusidae has been unclear because of the limited differences in morphological characteristics among the genera. The first

Molecular Phylogenetic Analysis
The 16S rDNA gene was used for the molecular phylogenetic analysis because it can effectively discriminate between species in Hydrozoa [14][15][16]. In this study, an approximately 600 bp fragment of mitochondrial 16S rDNA was used for phylogenetic analysis. Genomic DNA was extracted from the 99.5% ethanol-preserved tissue of 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 TCGACTGTTTACCAAAAACATAGC and AC-GGAATGAACTCAAATCATGTAAG [17], respectively, using the following PCR profile: an 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 a final elongation at 72 °C for 5 min [14]. The PCR products were purified using a QIAquick PCR Purification Kit (Qiagen, Germany) and sequenced in both directions using an ABI 3730 automated sequencer (Applied Biosystems, Bedford, MA, USA). The new sequences were aligned using MEGA 6.06 with built-in ClustalW [18]. Phylogenetic analysis and pairwise distance measurements were performed using the maximum likelihood method based on the Kimura 2-parameter model [19], with 1000 bootstrap replications in MEGA 6.06. Six sequences were deposited in GenBank under accession numbers LC653016-653021 for the new genus (Table 1) [14,[20][21][22][23].

Molecular Phylogenetic Analysis
The 16S rDNA gene was used for the molecular phylogenetic analysis because it can effectively discriminate between species in Hydrozoa [14][15][16]. In this study, an approximately 600 bp fragment of mitochondrial 16S rDNA was used for phylogenetic analysis. Genomic DNA was extracted from the 99.5% ethanol-preserved tissue of 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 TCGACTGTTTACCAAAAACATAGC and ACG-GAATGAACTCAAATCATGTAAG [17], respectively, using the following PCR profile: an 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 a final elongation at 72 • C for 5 min [14]. The PCR products were purified using a QIAquick PCR Purification Kit (Qiagen, Germany) and sequenced in both directions using an ABI 3730 automated sequencer (Applied Biosystems, Bedford, MA, USA). The new sequences were aligned using MEGA 6.06 with built-in ClustalW [18]. Phylogenetic analysis and pairwise distance measurements were performed using the maximum likelihood method based on the Kimura 2-parameter model [19], with 1000 bootstrap replications in MEGA 6.06. Six sequences were deposited in GenBank under accession numbers LC653016-653021 for the new genus (Table 1) [14,[20][21][22][23].

Morphological Investigation
Taxonomic observations and measurements were performed on live and preserved specimens ( Figure 2). Measurements were made using ImageJ [24] to the nearest 0.1 mm. For nematocyst identification in the medusae, squash prepared from fresh tissues was examined under a compound microscope (ECLIPSE Ci, Nikon, Tokyo, Japan). Nematocysts were identified according to a previously described method [1,25]. To determine the abundance of nematocyst types in medusae, approximately 100 nematocysts were identified, measured, and counted in unregistered specimens. Measurements were performed using ImageJ [24] to the nearest 0.1 µm.

Molecular Phylogenetic Analysis
We sequenced six individuals of Octorhopalona saltatrix for the 16S rDNA fragments, in addition to 12 Anthoathecata taxa, for statistical analyses. The maximum likelihood tree constructed for the family Halimedusidae based on the 16S rDNA sequences (

Molecular Phylogenetic Analysis
We sequenced six individuals of Octorhopalona saltatrix for the 16S rDNA fragments, in addition to 12 Anthoathecata taxa, for statistical analyses. The maximum likelihood tree constructed for the family Halimedusidae based on the 16S rDNA sequences ( Figure 3) comprised four major clades formed in the suborder Capitata: (1) Moerisia inkermanica, (2) Tiaricodon orientalis, (3) Urashimea globosa, and (4) Octorhopalona. The family paraphyly of Halimedusidae, including clades 1-3 and 4, was evident in the 16S rDNA phylogenetic tree with high bootstrap values (92%), which supports the validity of the genus. Moreover, Halimedusidae includes M. inkermanica, which is presently classified in the family Moerisiidae.

Genus Diagnosis
Halimedusidae, with eight nematocyst tracks in the exumbrella; eight radial canals; eight characteristic perradial and interradial blead-shaped 'smooth peaks' in the mesoglea between the radial canals; eight tentacles, with numerous stalked nematocyst knobs over their entire length; and tentacular bulbs swollen with an abaxial ocellus.
Type species. Octorhopalona saltatrix sp. nov. is designated here. The genus name 'Octorhopalona' is taken from the Greek words 'octo' and 'rhopalon', meaning 'eight' and 'club', respectively; the gender is feminine. The name indicates that the medusa bears eight tentacles that look like eight clubs.    (Figures 4 and 5A-C), 9 mm in height and 9 mm in diameter ( Table 3). The umbrella apex is rounded and the mesoglea is thickened (Figures 4 and 5A). Exumbrella are smooth and nematocysts are sparsely scattered. Nematocysts track eight, perradial and interradial, on the exumbrella about twothirds of the umbrella height (UH). Manubrium hang in the umbrella cavity, quadrilateral bottom, and are light brown or translucent ( Figure 6A). The extended manubrium length was approximately 2 mm and did not extend beyond the umbrella margin. The gonads cover the entire surface of the manubrium, except for the mouth lip. The mouth cruciform has four lips. The stomach has short, sac-like perradial and interradial lobes ( Figure 6B). There are eight characteristic interradial blead-shaped 'smooth peaks' in the mesoglea between the radial canals, rising above the level of the radial canals ( Figure 6A). There are eight radial canals and one circular canal ( Figure 5B,C and Figure 6B,C). The velum is narrow, with a velarial width of 10% of the umbrella diameter (UD) ( Figure 6C). The tentacular bulbs were swollen, each with a dark brown abaxial ocellus ( Figure 6D). There were eight tentacles, with numerous stalked nematocyst knobs in their entire length, approximately the same length as live umbrella height (UH) ( Figure 6E).       The smallest young medusa had a UH of 1.3 mm and a UD of 0.9 mm. The mesoglea at the apex of the exumbrella was thinner than that of adults ( Figure 7A). Nematocyst tracks on the exumbrella were approximately two-thirds of the UH. The manubrium was short, thin, translucent to whitish, and approximately one-third the length of the UH. Its mouth was simple and circular. It possessed eight radial canals and a single circular canal. Eight interradial peaks were absent in the mesoglea between the radial canals ( Figure 7B). The velum was wide, and the velarial width was 20% of the UD ( Figure 7C). The tentacular bulbs were swollen, each with a red-brown abaxial ocellus. There were eight tentacles, pearl-string-like, and 1-3 white or white-brown nematocyst batteries aligned, one-half of the UH. mouth was simple and circular. It possessed eight radial canals and a single circular canal. Eight interradial peaks were absent in the mesoglea between the radial canals ( Figure 7B). The velum was wide, and the velarial width was 20% of the UD ( Figure 7C). The tentacular bulbs were swollen, each with a red-brown abaxial ocellus. There were eight tentacles, pearl-string-like, and 1-3 white or white-brown nematocyst batteries aligned, one-half of the UH. Cnidome. Two nematocyst types were identified and measured (Table 4, Figure 8). Exumbrella: stenoteles. Manubrium: desmonemes and stenoteles. Tentacle: desmonemes and stenoteles. Tentacle bulb: desmonemes and stenoteles.  Cnidome. Two nematocyst types were identified and measured (Table 4, Figure 8). Exumbrella: stenoteles. Manubrium: desmonemes and stenoteles. Tentacle: desmonemes and stenoteles. Tentacle bulb: desmonemes and stenoteles. Habitat and ecology. Medusae of Octorhopalona saltatrix appeared on the surface of shallow waters (5-10 m in depth) off Oarai (eastern Japan) during November and December; in Sagami Bay (western Japan) from May to November; and in Tosa Bay (Western Japan) during November. Polyps of the species have never been found in the wild; however, medusa budding seems to occur between spring and fall in Japanese waters. In some specimens, the mesoglea at the apical part was infested with flukes.
Etymology. The specific name 'saltatrix' is taken from the Latin word 'saltatrix', meaning 'female dancer'. The gender is feminine. The name reflects the medusa swimming like a female dancer using its umbrella and eight tentacles. Habitat and ecology. Medusae of Octorhopalona saltatrix appeared on the surface o shallow waters (5-10 m in depth) off Oarai (eastern Japan) during November and Decem ber; in Sagami Bay (western Japan) from May to November; and in Tosa Bay (Western Japan) during November. Polyps of the species have never been found in the wild; how ever, medusa budding seems to occur between spring and fall in Japanese waters. In some specimens, the mesoglea at the apical part was infested with flukes.
Etymology. The specific name 'saltatrix' is taken from the Latin word 'saltatrix', mean ing 'female dancer'. The gender is feminine. The name reflects the medusa swimming like a female dancer using its umbrella and eight tentacles.

Molecular Phylogenetic Analysis
The paraphyly of Halimedusidae, including Octorhopalona and three species (U. glo bosa, T. coeruleus, and M. intermanica), was evident in the 16S rDNA phylogenetic tree with high bootstrap values (92%), which supports the validity of the new species. The maxi mum-likelihood tree includes Moerisia inkermanica (Moerisiidae), because Tiaricodon was placed in the family Moerisiidae [26]. The tree suggests that M. inkermanica may be derived from within Halimedusidae.

Morphological Investigation
A comparison of the key features of the Halimedusidae genera is presented in Table  5. Octorhopalona saltatrix can be distinguished from all other Halimedusidae species by the number of tentacles and radial canals, as well as the shape of the tentacles. All the species in the family Halimedusidae have a bell-shaped umbrella, interradial peaks in the subum brella, a red band on the manubrium, and abaxial ocelli on the basal tentacle bulbs. There are four tentacles (Tiaricodon and Urashimea) [3][4][5][6][26][27][28][29][30][31], four of which are perradial, and ten to eleven are interradial (Halimedusa) [1,9]. The tentacles are moniliform (Halimedusa and Tiaricodon) and numerous stalked nematocyst knobs cover their entire length (Oc torhopalona and Urashimea). All Halimedusidae species have four radial canals and fou

Molecular Phylogenetic Analysis
The paraphyly of Halimedusidae, including Octorhopalona and three species (U. globosa, T. coeruleus, and M. intermanica), was evident in the 16S rDNA phylogenetic tree with high bootstrap values (92%), which supports the validity of the new species. The maximumlikelihood tree includes Moerisia inkermanica (Moerisiidae), because Tiaricodon was placed in the family Moerisiidae [26]. The tree suggests that M. inkermanica may be derived from within Halimedusidae.

Morphological Investigation
A comparison of the key features of the Halimedusidae genera is presented in Table 5. Octorhopalona saltatrix can be distinguished from all other Halimedusidae species by the number of tentacles and radial canals, as well as the shape of the tentacles. All the species in the family Halimedusidae have a bell-shaped umbrella, interradial peaks in the subumbrella, a red band on the manubrium, and abaxial ocelli on the basal tentacle bulbs. There are four tentacles (Tiaricodon and Urashimea) [3][4][5][6][26][27][28][29][30][31], four of which are perradial, and ten to eleven are interradial (Halimedusa) [1,9]. The tentacles are moniliform (Halimedusa and Tiaricodon) and numerous stalked nematocyst knobs cover their entire length (Octorhopalona and Urashimea). All Halimedusidae species have four radial canals and four interradial peaks in the subumbrella; however, only the Octorhopalona has eight radial canals and eight interradial peaks in the subumbrella. Therefore, we propose to amend the family Halimedusidae as follows:
Diagnosis. Anthoathecata, with four or eight radial canals, a low peduncle, and distinct interradial or interradial and perradial peaks in the jelly above the base of the manubrium; red band on the manubrium, gonads extending out from the manubrium as lobes below the upper portions of the radial canals, but without mesenteries; mouth with lips lined by a row of sessile nematocyst clusters; four perradial hollow tentacles, or four perradial tentacles and four interradial single tentacles, or groups of hollow tentacles; cylindrical marginal bulbs, each with an abaxial ocellus.

Conclusions
Our morphological and molecular phylogenetic analyses suggest that these specimens collected off Oarai, Sagami Bay, and Tosa Bay, Japan are a new genera and new species belonging to the family Halimedusidae. This species appears in shallow waters in Japan during May and December. Polyps of this species have never been found in the wild. Additional sampling is required to understand their ecology and development.
Three genera, Halimedusa, Tiaricodon, and Urashimea are currently classified in the family Halimedusidae, defined by [1] as: Anthomedusae with four radial canals, with low peduncle and distinct interradial peaks in jelly above base of the manubrium; gonads extending out from the manubrium as lobes below the upper portions of the four radial canals, but without mesenteries; quadratic mouth with lips lined by a row of sessile nematocyst clusters, with four perradial hollow tentacles or with four perradial tentacles and four interradial groups of hollow tentacles; and possessing cylindrical marginal bulbs each with an abaxial ocellus. However, Octorhopalona saltatrix has eight tentacles, eight radial canals, and four interradial and four perradial peaks. Therefore, we propose to amend the family Halimedusidae as discussed. Funding: This research was financially supported by the Japan Fund for Global Environment and JSPS KAKENHI (grant numbers JP18K14791 and 21K15158, awarded to S. Toshino).
Data Availability Statement: All datasets collected and analyzed during the current study are available from the corresponding author upon request.