A New Species of Box Jellyfish (Cnidaria: Tripedaliidae: Tripedalia) from Hong Kong, China (2024)

Associated Data

Supplementary Materials

Abstract

We describe a box jellyfish, Tripedalia maipoensis sp. nov., based onsamples collected from a shrimp pond in Hong Kong. This new species is morphologicallydistinct from other species of the family Tripedaliidae by the following combination ofcharacters: (1) three pedalia at each bell corner; (2) each pedalium with one tentacle;and (3) velarium with forked canals. Phylogenetic analyses based on a concatenated datasetof the 16S, 18S and 28S rRNA genes showthat T. maipoensis sp. nov. is sister to the morphologically similarspecies T. cystophora, but the two species exhibit 17.4% divergence inthe 16S rRNA gene, supporting T. maipoensis sp. nov. asa distinct species. This new species represents the fourth described species ofTripedaliidae, and the first record of the family in Chinese coastal waters.

Keywords: Box jellyfish, Tripedalia, Morphology, Phylogeny, Hong Kong

BACKGROUND

The class Cubozoa, commonly known as box jellyfish due to the cube-shaped bells, consistsof a small group of cnidarians with approximately 50 described species and is well-known tothe public for containing some of the most venomous marine animals in the world (Bentlage etal. 2010; Collins and Jarms 2022). Cubozoa is divided into two monophyletic orders, theCarybdeida and Chirodropida (Bentlage et al. 2010). The two orders can be easilydistinguished, as carybdeids possess only one tentacle per pedalium, whereas chirodropidspossess multiple tentacles per pedalium.

Tripedaliidae is a small family of Carybdeida, with only three described species in twogenera (Copula and Tripedalia). Tripedalia possesses two to three pedalia per bell corner, which differs fromCopula, which has one pedalium per bell corner (Collins et al. 2006;Bentlage and Lewis 2012). Currently Tripedalia is represented by only twospecies: T. binata Moore, 1988 which has two pedalia at each bell corner,and T. cystophora Conant, 1897 which has three pedalia at each bell corner.Although both species inhabit estuarine waters in mangrove areas, T. cystophora has been reported from a much wider geographic range than T.binata. Following its first discovery in Jamaica in 1857, T. cystophora has been widely reported around the tropics and subtropics, including the UnitedStates (Orellana and Collins 2011), Japan (Uchida 1970), Singapore (Kong and Iesa 2021),Mexico (Lasley et al. 2016), Australia (Ekins and Gershwin 2014) and India (Riyas and Kumar2020). In contrast, T. binata is only known from Australia (Underwood etal. 2013) and Thailand (Toshino et al. 2019).

Although box jellyfish are often common animals in coastal habitats, their diversity in theChinese coastal waters is poorly known, with only three species reported, all from thefamily Carybdeidae (Liu 2008). In the summer of three consecutive years from 2020 to 2022,we found box jellyfish from the mangrove habitat of intertidal shrimp ponds (locally knownas gei wai) in Mai Po Nature Reserve, Hong Kong (Fig. S1A), located on theeastern shore of the Pearl River Estuary. Here we report the box jellyfish as a new species,Tripedalia maipoensis sp. nov. (Tripedaliidae). We describe themorphology of this new species and determine its phylogenetic placement based on three genemarkers.

MATERIALS AND METHODS

We collected the samples using a plankton net of 30 cm mouth diameter and 100 μm mesh size(Fig. S1B). In the laboratory, we attempted to rear the box jellyfish and observed theirmating behaviour and early development (Fig. S2; Supplementary Note). We fixed adults formorphological description in 4% formalin solution for three days, then transferred them to90% ethanol for long-term preservation. We preserved adults for molecular studies in 95%ethanol. All specimens were deposited into the Tropical Marine Biodiversity Collections ofthe South China Sea (TMBC), Chinese Academy of Sciences, Guangzhou. We took photographs oflive and preserved specimens using either a Canon EOS5D Mark IV camera with a 100 mm macrolens, or a Canon 700 camera attached to a Motic BA 210 microscope through a phototube. Wemeasured the following characters of the formalin fixed specimens as in Straehler-Pohl(2014) and Acevedo et al. (2019): bell height (BH): distance from apex of the bell to thevelarial turnover; diagonal bell width (DBW): distance between opposite pedalia at level ofpedalia joining bell; interrhopalial width (IRW): distance between adjacent rhopalia; DRT:distance from rhopalium to turnover of bell; pedalia width (PW): distance at the widest partof the pedalia; and pedalia length (PL): distance at the longest part of the pedalia.

We collected tissues of the bell corner from three individuals, and extracted genomic DNAusing the PureLinkTM Genomic DNA Mini Kit (Invitrogen, Germany) according to themanufacturer’s protocol. We amplified two nuclear genes (18S ribosomal RNA[18S] and 28S ribosomal RNA [28S]) and one mitochondrialgene (16S ribosomal RNA [16S]) as in Cartwright et al. (2008) and Collinset al. (2008), and sequenced the products bi-directionally at BGI, Shenzhen. We depositedthe nucleotide sequences into NCBI GenBank (accession numbers listed in Table S1). Weretrieved sequences of Tripedaliidae, Carybdeida and Chirodropida from GenBank. Sincesequences of the three genes were not available in GenBank for a T. cystophora specimen from the type location in Costa Rica, we re-assembled the transcriptomeusing the raw reads (accession number SRR8101518) downloaded from GenBank, and obtained thesequences of these genes from the assembled contigs. We filtered the reads using Trimmomaticv.0.38 (Bolger et al. 2014) to remove adapters and low-quality sequences, then assembled thetranscriptome using Trinity v2.8.6 (Grabherr et al. 2011). We identified sequences of16S, 18S and 28S using the BLASTnoption of BLAST®+ v.2.10.1+ (Camacho et al. 2009) to search against those from otherspecimens of T. cystophora retrieved from NCBI. We aligned the multiplesequences using ClustalW (Thompson et al. 1994) under the default settings for each gene andcombined after trimming poorly aligned sequences by trimAl (Capella-Gutiérrez et al. 2009).We then conducted phylogenetic analysis using the Maximum-Likelihood (ML) method based onthe concatenated dataset of three genes. We partitioned the concatenated dataset by gene andapplied ModelFinder (Kalyaanamoorthy et al. 2017), which indicated GTR+F+I to be the mostappropriate model for all three genes. We performed ML analysis using IQ-TREE v1.4.4 (Nguyenet al. 2015) with 1000 ultrafast bootstrap (UFBoot) replicates. For 16S, wealso calculated p-distances –a measure of genetic distance –between the newspecies and other species, as well as between the different specimens of the new speciesusing MEGA v.11 (Tamura et al. 2021).

RESULTS

Family Tripedaliidae Conant, 1897

Genus Tripedalia Conant, 1897

Tripedalia maipoensissp. nov.

(Figs. 1, 2, Fig. S1)

urn:lsid:zoobank.org:act:68BA7074-70C9-400A-9F49-572E6DC3622B

Material examined: TMBC030991 (holotype): adult medusa, female (Fig.1A–B), collected 21 April 2022, preserved in 4% formalin, stored in 90% ethanol.TMBC030992–TMBC031003 (paratypes): adult medusa, one individual each, same sampling locationas holotype, TMBC030992 (male), TMBC030993 (female), TMBC030994 (male), TMBC030995 (female),TMBC030996 (sex cannot be determined), TMBC030997 (male), TMBC030998 (female), TMBC030999(female), TMBC031000 (male), TMBC031001 (male), TMBC031002 (female), TMBC031003 (female),TMBC030992–TMBC030995 collected on 21 April 2022, TMBC030996–TMBC031003 collected in August2020, TMBC030992–TMBC030997 preserved in 4% formalin and stored in 90% ethanol,TMBC030998–TMBC031003 preserved in 95% ethanol.

Type locality: Mai Po Nature Reserve, Hong Kong. 22. 4887°N,114.0417°E.

Etymology: maipoensis is the Latinized form of the typelocality Mai Po.

Diagnosis: Medusa with three pedalia per bell corner, each pedalium withone tentacle, velarium with forked canals.

Description: The bell is transparent, colourless, cuboidal-shaped withsmooth, edges rounded, roof slightly arched, with white nematocyst warts of different shapesand sizes densely scattered from apex to bell margin (Fig. 1A–B, G; Fig. S1C–E). There is apair of gonads at each corner of the bell, extending from stomach rim to velarium in bothsexes (Fig. 1A, Fig. S1C). There are four frown-shaped rhopalial niches, each locatedbetween two bell corners. Each rhopalial niche ostium has a prominent upper covering scale,and a small lower scale (Fig. 1A, C). Each ropalial niche has a rhopalium suspended from theroof with a stalk. Each of the four rhopalia bears six eyes: two lateral pit eyes, twolateral slit eyes, a smaller upper lens eye, and a larger lower lens eye (Fig. 1D). Eachrhopalium also bears a large crystalline statolith below the lower lens eye. The velarium issmooth, without nematocyst warts. The velarial canals are biforked to multi-branched with3–6 branches, with sharp tips (Fig. 1E–F). The velarial canal tips are white in maturespecimens (Fig. 1E). The manubrium extends from the bell roof to 1/2–2/3 bell height (Fig.S1C, E); they are cruciform with four terminal lobes that are free of nematocyst warts (Fig.1H). The stomach is flat, with its four corners each connected to a brush-like gastricphacella (Fig. 1G, Fig. S1F). The pedalia are flat and unbended, each with one pedalialcanal inside (Fig. 1A–B, Fig. S1C–D) and nematocyst warts on the outer surface (Fig. 1B,Fig. S1D). Pedalial canal straight, extending through pedalium, slightly tapering at distalend, knee bend rounded without appendage. Each pedalium bears a single unbranched tentacle.Nematocysts from tentacles are oval-shaped (Fig. 1I). Juvenile medusae are similar withadults in gross morphology. At ~BH 3.2 mm, they developed from two to three pedalia per bellcorner (Fig. S1G–H).

Sexual Dimorphism: Female gonad ‘wings’ are slender, extending from thestomach to the velarium; whitish to light yellow in live specimens (Fig. 1A–B). Male gonad‘wings’ are broad with a rounded top, extending from the stomach to the velarium; whitish inlive specimen (Fig. S1C).

Measurements: n = 8 adults. Mean BH 12.5 (± 3.3) mm, meanDBW 15.2 (± 6.9) mm, mean IRW 1.4 (± 0.7) mm, mean DRT 2.7 (± 1.3) mm, mean PL 4.9 (± 1.1)mm, mean PW 1.5 (± 0.5) mm.

Distribution and ecology: The new species is currently known only from thetype locality, but may also occur in the adjacent waters of the Pearl River Estuary as theponds are connected to the estuary through a tidal channel. The new species was found everyyear since 2020 in some of the brackish water intertidal shrimp ponds of Mai Po NatureReserve during April and May, and also extending to June in 2021 when water temperatureranged from 20°C to 29°C, and salinity from 5.8 psu to 18 psu. The new species was recordedin water channels flanked by Common Reed (Phragmites australis) ormangroves of mainly Kandelia obovata mixed with the golden leather fernAcrostichum aureum.

Genetic analyses: A 567 base pair (bp) fragment of 16S,1734 bp fragment of 18S and 1809 bp fragment of 28S wasused in the genetic analyses. Phylogenetic analyses based on the combined dataset of threegenes strongly supported the monophyly of Tripedalia (Fig. 2). Specimens ofT. maipoensis sp. nov. were grouped to a distinctive clade and sister tothe T. cystophora group comprising specimens from Costa Rica and Indonesiawith strong support (UFBoot = 100, Fig. 2). The Tripedalia clade is thensister to Copula sivickisi (Fig. 2). For16S, the intraspecific p-distance of T.maipoensis sp. nov. for 16S is 0.1%, while the interspecificp-distance between T. maipoensis sp. nov. and T.cystophora is 17.5%. The percentage of genetic distance for 16S between T. maipoensis sp. nov. and Copula sivickisi is 26.6%, while the value between T. cystophora and C.sivickisi is 28.3%.

DISCUSSION

Tripedalia maipoensis sp. nov. and T. cystophora can beeasily distinguished from T. binata, which bears two pedalia per bellcorner. The new species differs from T. cystophora by having forkedvelarial canals, which are unforked in T. cystophora. At ~2.3 mm bellheight, some juvenile medusae bear two pedalia per bell corner while others bear three,indicating that the third pedalium emerged in a narrow time window during the development.

The ML analysis based on a concatenated dataset of the 16S, 18S and 28S reveals an overall tree topology that is consistent withthe cubozoan phylogenetic relationships revealed in a previous study (Bentlage et al. 2010).Members of the family Tripedaliidae form a well-supported clade and is sister toCarybedeidae (UFBoot = 100, Fig. 2). The monophyly of Tripedalia isstrongly supported. Acevedo et al. (2019) has suggested that 16S sequencescan be useful in determing the genetic distances among members of Carybedeidae. Theinterspecific genetic distance threshold of 16S ranges from 10.8% to 33.9%among species (Acevedo et al. 2019). Our results show a p-distance of 17.5%between the two species in 16S, compared to a much lower intra-specificp-distance in both T. maipoensis sp. nov. (0.1%) andT. cystophora (1.1%), supporting T. maipoensis sp. nov.as a distinct species. Unfortunately, there is no genetic data available for T.binata, nor any available specimen of T. binata for molecularwork in the current study, therefore the relationships between this species and the othertwo species remains unclear.

CONCLUSIONS

In this study, we reported the new species Tripedalia maipoensis sp. nov.collected from an intertidal shrimp pond in the Mai Po Nature Reserve, Hong Kong. Althoughthe box jellyfish family Tripedaliidae has been widely reported from tropical andsubtropical waters, this is the first record of it in Chinese coastal waters. We describedthe species with both morphological and molecular data. Future investigations along Chinesecoasts are warranted to improve our understanding of the distribution of this new speciesand the diversity of Cubozoa.

Supplementary materials

Acknowledgments

This work and the new species name were registered with ZooBank underurn:lsid:zoobank.org:pub:C72E93E5-1AD6-400F-A0C6-97BF5B8FDBFF. This work was supported byLantau Conservation Fund (RE-2020-22) and Environmental and Conservation Fund (34/2019). Wethank Dr. Cynthia Yau (The Hong Kong University of Science and Technology) and Dr. LisaGershwin for initial identification, Dr. Simon Sin (The University of Hong Kong) and Dr.Anthony Lau (Lingnan University) for initial molecular work, and Miss Yixuan Li (Hong KongBaptist University) for help in sample collection and photography. Special thanks to theAgriculture, Fisheries and Conservation Department, the Government of the Hong Kong SpecialAdministrative Region of the People’s Republic of China for the continuous support of theoverall management of Mai Po Nature Reserve.

Footnotes

References