Meteorona kishinouyei, a new family, genus and species (Cnidaria, Cubozoa, Chirodropida) from Japanese Waters

Abstract A new family, genus and species of cubozoan box jellyfish belonging to the order Chirodropida is reported from the eastern Japan. Meteorona kishinouyei gen. et sp. n. possesses the following unique morphological characters with respect to other known species in the Chirodropida: having one tentacle per scalpel-like unbranched pedalium and slightly raised unbranched gastric saccules. A comparative table of the primary diagnostic characters of genus and order in the Chirodropida is given. The order Chirodropida is redefined. The family Chiropsellidae is established. Discussion is provided on the implications for these findings on our current understanding of Cubozoan systematics.


Introduction
The order Chirodropida currently comprises 13 species in two families, Chirodropidae and Chiropsalmidae (Gershwin 2006a;Cornelius et al. 2005;Lewis and Bentlage 2009;Bentlage 2013). Chirodropids are characterized by having multiple tentacles extending from a single branching pedalium on each corner of the swimming bell. The first identified species of Chirodropida, Chiropsalmus quadrumanus (Müller, 1859) was described by Müller (1859) (as Tamoya quadrumana). Agassiz (1862) erected the genus, Chiropsalmus Agassiz (1862) and T. quadrumana was subsequently moved to that genus by Haeckel (1880) when he erected the order Chirodropida (as Chirodropidae) as part of Cubomedusae (formerly of the class Scyphozoa). Thiel (1936) erected the family Chiropsalmidae to replace the family Chirodropidae, when at the time it was classified as a monotypic family in the order Cubomedusae. Werner (1973) elevated Cubomedusae to the class Cubozoa that included Chirodropidae and Carybdeidae. Subsequently, Gershwin (2006a) elevated the family Chirodropidae to the order Chirodropida, and resurrected the family Chiropsalmidae. Recent molecular phylogenetic analyses and taxonomic investigations suggested that Chirodropida is a monophyletic order thought to include the paraphyletic Chiropsalmidae (Collins et al. 2006;Collins 2009;Bentlage et al. 2010).
Chirodropids have been reported from a range of tropical, sub-tropical, and mild temperature localities in the Pacific and Atlantic (Gershwin 2006a;Cornelius et al. 2005; Lewis and Bentlage 2009;Bentlage 2013). This group is infamous and well known by local fishermen, divers and bathers as dangerous box jellyfish due to their potentially lethal sting. In particular, envenomation by Chironex yamaguchii poses a serious problem to public health and tourism in Japan, with 100 to 200 stings reported per year, and a total of three fatalities following envenomation in Okinawa, southern Japan (Yamaguchi 1980;Okinawa Prefectural Institute for Health and Environment 1999). Currently, part of the life cycle of only one chirodropid is known: C. fleckeri (Yamaguchi and Hartwick 1980;Hartwick 1991). In an attempt to focus on countermeasures against envenomations, some workers have looked at diurnal migration and seasonal occurrence of Chirodropid medusae, and the potential habitat of polyps has been investigated using light traps (Iwanaga et al. 2005).

Methods
Specimens were collected using a scoop, 170 mm in diameter, at Souma, Onahama and Fujisawa, eastern Japan between 24 August 2011 and 18 October 2013 (Fig. 1). The medusae ware fixed in 5% formalin in seawater, and deposited in the National Museum of Nature and Science, Tsukuba, Japan (NSMT). Prior to fixation, a tentacle subsample of each specimen was preserved in 99.5 % ethanol for DNA extraction. Taxonomic observation and measurements were made on preserved specimens. Measurements were made with digital calipers (CD-20CPX, Mitsutoyo Corporation, Japan) to the nearest 0.01 mm. The following measurements were made according to Gershwin 2005 (Fig. 2): bell height (BH), diagonal bell width (DBW), interrhopalial width (IRW), pedalial width (PW), pedalial canal width (PCW), outer keel width (OKW), inner keel width (IKW), tentacle base width (TBW), velarial width (VW). Sex was determined by examining gonadal tissue under a light microscope. In this study, some of new measurements were added as below: Diagonal exumbrella width (DEW) and diagonal subumbrella width (DSW) were both measured across diagonal base of outer keel or inner keel of pedalia, respectively, on a flattened specimen. Outer keel  length (OKL) and inner keel length (IKL) were both measured from the exumbrella lamella or subumbrellar lamella, respectively, to the tentacle insertion. Rhopalial height (RH) was measured from the base of rhopalial stalk to velarial turnover. Manubrium length (ML) was measured from the base of manubrium to mouth.
For nematocyst identification in the medusae, squashes prepared from fresh tissues were examined under a compound microscope. Nematocysts were identified according to Mariscal (1971Mariscal ( ), Östmann (2000 and Gershwin (2006b). For determination of the abundance of nematocyst types in medusae, roughly 500 nematocysts were identified, measured and counted from two specimens, NSMT-Co1569 and NSMT-Co 1571.
Near complete sequences of the nuclear 18S rDNA genes (approximately 1800 bp) were used for molecular phylogenetic analysis. Genomic DNA was extracted from the 99.5 % ethanol preserved subsampled tentacle tissue of NSMT-Co1569, NSMT-Co1571 and NSMT-Co1572 using the DNeasy Blood and Tissue Kits (Qiagen, Germany) following the manufacturers protocol. 18S rDNA was PCR amplified and sequenced using primers and protocols outlined in Collins et al. (2008). The new sequences were aligned using MEGA 6.06 with built in ClustalW. Phylogenetic analysis and pairwise distance measurements were determined using the maximum likelihood method with 1000 bootstrap replications in MEGA 6.06. All of these sequences have been deposited in DDBJ under accession numbers LC033478-LC033480 for the new species (Table 2).   Description. Adult medusae with smooth exumbrella, with evenly thick mesoglea of rigid consistency (Fig. 3, Fig. 4, Fig. 5). BH about 35 mm and DBW about 50 mm (Table 3). Interradius thickened throughout bell height, with deep median furrow spanning height of bell. Adradial furrows spanning lower two thirds of bell. Coronal indentation shallow just below apex. Exumbrella lacking nematocyst warts or freckles. Gonads attached along entire length of interradial septa, leaf-shaped, not overlapping  along the interradius (Fig. 6A, 7A). Manubrium length about 40% of bell height. Manubrium cruciform with four narrow, lanceolate lips (Fig. 6B).
Gastric saccules unbranched, slightly raised and opaque (Fig. 5D, 6C, 7B). Gastric phacellae horseshoe-shaped in each corner of stomach (Fig. 6D, 7C). Gastric cirri simple and unbranched (Fig. 6E, 7D). Sensory niches four, perradial, U-shaped, with one shallow covering scale above and no lower scale, upper scale with central tongue-shaped flap partially shielding rhopalium, located approximately 1/6 of bell height from velarial turnover (Fig. 6F, G, 7E, F). The rhopaliar niche located in a triangular shaped depression of exumbrella (Fig. 6F, 7E). Subumbrellar rhopalial windows flat (Fig. 6H). Each of the four rhopalia bearing a set of six eyes, with the two median possessing prominent lenses and the four lateral ones adjacent to the lens eyes being pigment pits and slit ( Fig.  6I-K, Fig. 7G, H). A single broad bean shaped statolith located behind each complex eye (Fig. 6I-K). Velarial canals one per octant, dendritic, with only the tips defined along the velarial margin (Fig. 6L, Fig. 7I). Frenulum a single solid, gelatinous struc- ture, extending to near velarial margin (Fig. 6L). Velarial width about 20% of DSW. Pedalia scalpel-like, each bearing one tentacle (Fig. 6M, Fig. 7J). Pedalia about half of bell height, inner keel rounded, about two times the width of pedalial canal. Outer keel width approximately twice that of pedalial canal, inner keel width and outer keel width almost equal. Proximal pedalial canal bend slight volcano-shaped (Fig. 6N, Fig. 7K). Tentacles four, with one per pedalium, base width up to 2.7 mm thick, round in cross section, with nematocyst rings alternate thick and thin in preserved specimens (Fig. 6O,  7L). Color and length of tentacles in living specimens was light brownish (Fig. 3A, B).
Molecular phylogenetics. In the resulting ML tree (Fig. 12), three major monophyletic clades were formed in the order Chirodropida: 1) Chirodropidae (Chironex Habitat and ecology. Holotype specimen of Meteorona kishinouyei was collected near shore in shallow waters of the Matsukawa-ura lagoon, Fukushima prefecture, eastern Japan. Until March 2011, the north part of the lagoon extended to the sea via a man-made channel. However, the channel was destroyed and considerable sea water flowed into the lagoon as a result of the 2011 Tohoku earthquake and tsunami. M. kishinouyei medusae may have been brought into the lagoon on the high waves. Moreover, M. kishinouyei may be a neritic species. Young medusae appeared during August, while adult medusae appeared between August and November. Carybdea brevipedalia was collected together with M. kishinouyei along at Onahama port and Shonan at the water surface in a shallow area (water depth 3-10 m) port during daytime. Polyps of M. kishinouyei may metamorphose into young medusae during summer. However, the polyp stage and life history are unknown.
Etymology. The species is named kishinouyei (noun in apposition) to honor Professor Kamakichi Kishinouye, zoologist and fishery scientist. Kishinouye's meticulous studies and observations have led to many important contributions in the description of cubozoan zoology Differential diagnosis. A comparison of key features of the species of Chirodropida is presented in Table 5. Meteorona kishinouyei can be distinguished from all other chirodropid species by shape of pedalium, gastric saccules and rhopaliar niche. All of the species in the order Chirodropida have branching pedalia, each bearing multiple tentacles. M. kishinouyei has one tentacle per unbranching pedalium like species of the order Carybdeida. Shape of gastric saccules are cock's-comb shaped/grape-cluster-like (Chironex), elongate, tapered, with numerous axial processes, or absent (Chirodropus)

Discussion and conclusions
Meteorona kishinouyei is most likely to be confused with the carybdeid Carybdea brevipedalia from eastern and northern Japan. Kishinouye (1891) described two cubozoan species, C. brevipedalia and Carybdea latigenitalia, collected from Shima (Mie prefecture, western Japan) and Hitachi (Ibaraki prefecture, eastern Japan) respectively. Box jellyfish with similar descriptions have been reported as Carybdea rastoni or Carybdea mora (Kramp 1961;Gershwin and Gibbons 2009). However, recent molecular phylogenetic analyses and taxonomic investigations suggest that C. rastoni reports from Japan should be regarded as C. brevipedalia (Bentlage et al. 2010;Bentlage and Lewis 2012). However, the account and drawing of C. latigenitalia in the original description agree well with M. kishinouyei: dendritic velarial canals, well developed mouth lips, half-moon shaped phacellae, very wide gonads and one tentacle per unbranching long pedalium (Kishinouye 1891). However, Uchida (1929) suggest that C. latigenitalia should be regarded as C. rastonii. Unfortunately, it is likely that the material investigated for the original description of C. latigenitalia was lost. Additionally, as the name has not been in common usage, and it no longer fits the description of a species of the genus Carybdea, there is no justification for/obligation to resurrect the species name (ICZN 1999). Two families, Chirodropidae and Chiropsalmidae are currently classified in the order Chirodropida (Gershwin 2006a) defined by Gershwin (2006a) as: Cubozoa with branched pedalia bearing numerous tentacles; with or without gastric saccules. However, M. kishinouyei has one tentacle per unbranched pedalia and leaf-like gonads. We therefore propose emending the order Chirodropida as follows: Order Chirodropida Haeckel, 1880, sens. emend.

Diagnosis.
Cubozoa with or without gastric saccules; with branched or unbranched pedalium; with a triangular shaped depression of exumbrella surrounded rhopaliar niche; with a triangular shaped perradial lappet and highly divergent branching velarial canals.
Based on our maximum likelihood study, M. kishinouyei and Chiropsella bronzie appear to be the closest relatives in the currently understood cubozoan phylogenetic relationships. Moreover, Meteorona and Chiropsella have some common morphological characters such as the unbranched gastric saccule, rhopaliar flap and slight volcanoshaped pedalial canal bend. However, the shape of pedalium and number of tentacles differs between the two species.