Critical Evaluation of Characters for Species Identification in The

Home , Carukia, Carukia barnesi, Carukiidae, Carybdeida, Malo (jellyfish), Malo kingi

Plankton Benthos Res 9(2): 83–98, 2014 Plankton & Benthos Research © The Plankton Society of Japan

Critical evaluation of characters for species identiﬁcation in the cubomedusa genus Malo (Cnidaria, Cubozoa, Carybdeida, Carukiidae)

ILKA STRAEHLER-POHL

Zoologisches Institut und Zoologisches Museum, Biozentrum Grindel, Universität Hamburg, Martin-Luther-King-Platz 3, 20146 Hamburg, Germany Received 27 November 2013; Accepted 8 January 2014

Abstract: Medusae of an undetermined species of the carukiid genus Malo were sampled for life cycle research at Port Douglas Marina, Port Douglas, Australia, in 2011. Due to confusing character variations within the specimens, identiﬁcation to species level initially seemed impossible. To resolve their identity, the type material of Malo maxima Gershwin, 2005 and M. kingi Gershwin, 2007 were examined. Comparisons were made of the two, and also with the unknown species. Unexpectedly, no signiﬁcant differences were found between M. maxima and M. kingi. Moreover, all character variations in them were observed as well in the unknown species. Accordingly, M. maxima from West- ern Australia and M. kingi from Queensland are considered to represent populations of the same species. Characters considered both reliable and unreliable for species determination in the genus are discussed, and an emended diagno- sis of Malo is proposed. The earlier name M. maxima is proposed for both populations and for the specimens from Port Douglas.

Key words: Malo kingi, Malo maxima, medusa, morphology, taxonomy

kingi Gershwin, 2007, yet some of the specimens showed Introduction characters used to define the species Malo maxima Gersh- Specimens of an unidentified cubozoan species were win, 2005 from Western Australia. Moreover, all speci- collected in April 2011 at a marina in Port Douglas (Aus- mens bore six eyes per rhopalium rather than two, as de- tralia). The 41 medusae were identified on location as spe- scribed for both M. maxima and M. kingi (Gershwin cies of the order Carybdeida based on the elongated cuboidal 2005a, b, 2007). bell, the single tentacle extending from a single pedalium The purpose of this study was to clarify the identity of arising from each corner of the bell, and rapid locomotion. the collected specimens of Malo by comparing them with Nematocyst bands without distinct protrusions or ‘neck- the type material of M. kingi and M. maxima, and to estab- chieves’ (Underwood & Seymour 2007) excluded them lish which characters are reliable in differentiating species from the genus Carukia, commonly found in the far north- and genera within the group. ern waters of Queensland. From existing taxonomic knowledge, the species was identifiable only to genus, as Materials and Methods Malo Gershwin, 2005. While the highly variable morphology of the medusae precluded further identification at the Species examined time, several of them spawned. Planulae were collected and reared to polyp stage, suggesting that the collected Malo sp.: 20 preserved, immature to mature medusae (7 medusae represented a single species. The sampling loca- mature males, 4 mature females, 9 imature medusae). tion was the same as the type locality of the species Malo Sampling: Medusae, collected for life cycle investigations, sampled by Avril Underwood and Jamie Seymour between Corresponding author: Ilka Straehler-Pohl; E-mail, I.Straehler-Pohl@ 19:30 and 20:00 on 14 April 2011 at Port Douglas Marina web.de (Queensland/Australia), using two 2×1000 Watt underwa- 84 I. Straehler-Pohl ter lights positioned approximately 20 meters apart along the side of the main quay of the marina and suspended in the water with rope to a depth of 50 cm. Individual jellyfish were scooped into a pool net then transferred to 500 mL plastic containers filled with clean seawater. Malo maxima: Holotype Z29940 (Western Australian Mu- seum, Perth), paratypes G 55400 (Queensland Museum, Brisbane) and C 15023 (Northern Territory Museum, Dar- win) were examined at the collections of the Queensland Museum in Townsville and the Museum & Art Gallery of the Northern Territory (NTM) in Darwin. Malo kingi: Paratypes (G 60029 (Queensland Museum, Brisbane) and W 55276 (Tropical Queensland Museum, Townsville) were examined at the collection of the Tropical Queensland Museum in Townsville. The holotype G 317361 (Queensland Museum, Brisbane) was not available during the time of inspection, therefore, the description and the images in Gershwin (2007) were used for comparison. “Gonads” definition Campbell (1974: 142) stated that gonads in most cnidari- ans are not separate organs, as in other animals, in that the germ cells generally are found in interstitial positions of the body tissue which exhibit no reproductive specializa- tion without the germ cells or before they arrive. However, Campbell (1974), Marques & Collins (2004), Bentlage et al. (2010), and Morandini & Marques (2010) are followed in using the term gonad to refer to areas where gametes are formed.

Measurements Fig. 1. Schematic drawing of anatomical characters The Malo specimens from Port Douglas were dissected and measurements of an adult medusa of Malo spp., (A) lateral view and (B) subumbrella view. B: bell, BH: bell while the types of Malo kingi and Malo maxima were not; height, Go: gonads, IPD: interpedalial diameter, IRD: although some of the examined specimens had already interrhopalial diameter, iPW: inner pedalial wing, M: been dissected. Measurements of medusae (Fig. 1A, B) manubrium, Mes: mesenterium, NW: nematocyst wart, were taken from flattened specimens as follows: bell height oPW: outer pedalial wing, P: pedalium, PC: pedalial (BH)＝length between bell turn-over (velarium excluded canal, PerL: perradial lappet, RhN: rhopalial niche, Sto: from measurement) and top of apex; interpedalial diameter stomach, T: tentacle, V: velarium, VC: velarial canal. (IPD)＝distance between opposite pedalia (outer pedalial wing edges) at the level of the bell turn-over; interrhopalial diameter (IRD)＝distance between opposite sense niches; Genus Malo Gershwin, 2005 interrhopalial width (IRW)＝distance between adjacent Definition: (after Gershwin 2005b) rhopalia; diagonal bell width (DBW)＝distance between Carybdeidae without gastric phacellae; with frown- opposite pedalia at level of pedalia joining bell. shaped rhopaliar niche ostia; with rhopaliar horns; with two median eyes only, lacking lateral eye spots; with fine, cylindrical, unmodified tentacles; with one stomach pouch Results extension branching into about 4–5 simple to bifurcated velarial canals per octant; with moderately developed perra- Systematics dial mesenteries; pedalial canals lacking prominent thorn. Phylum Cnidaria Verrill, 1865 Type species: Malo maxima Gershwin, 2005 Subphylum Medusozoa Petersen, 1979 Class Cubozoa Werner, 1973 Malo maxima Gershwin, 2005 Order Carybdeida Lesson, 1843 Type location: Western Australia, Kimberley coast, South of Family Carukiidae Bentlage, Cartwright, Yanagihara, Broome, offshore from 80 Mile Beach (19°13.021′S, Lewis, Richards & Collins, 2010 121°15.399′E) and outside Roebuck Bay, Broome (18°4′S, Species identification in genus Malo 85

122°17′E) (Gershwin 2005b). the canals flanking the perradial lappets and the pedalia Inspection of holotype (holotype, Paratype C 15023) or equally complex in all ca- The holotype was in good condition except for the rho- nals (Paratype G 55400). palia. Two of them were cut out and not preserved with the Rhopalial horns in both paratypes were short, but as in rest of the holotype, and are completely missing. the holotype, no general shape could be defined as the Next to the characters listed by Gershwin (2005b; see horns could be either broad or slim, curved inwards or Table 1), two additional morphological features were noted: straight. (1) 3–4 ridges encircled the upper quarter of the pedalial Perradial lappets of Paratype C 15023 bore simple and canals below the knee bend (Fig. 2C), (2) 0–1 nematocyst branched canals protruding from the lappet tips and sides warts were noted on the velarial canal roots (Fig. 2G). (Fig. 2R), while side extensions were missing in Paratype The number of eyes per rhopalia in the holotype could G 55400 (Fig. 2L). not be checked definitively due to the two missing rhopalia. As in the holotype, circular ridges encircling the upper The eye pigments in the remaining rhopalium were almost quarter of the pedalial canals were observed in both in- completely faded, and only the structure of the lower me- spected paratypes (Fig. 2J, O). dian eye was visible, due to its large size, through the rhopalial niche window. Malo kingi Gershwin, 2007 Inspection and comparison of paratypes and holotype Type location: Australia: Far North Queensland, Port (Table 1, Fig. 2A–R) Douglas (16°29′S, 145°27′E), Dickson Inlet (Gershwin 2007) The paratypes were in good condition, but as with the Inspection of paratypes, comparison with holotype and holotype several rhopalia had been cut out and were miss- type material of Malo maxima (Table 2) ing. As a result, further inspection of eye structures was Paratype G 60029 (Fig. 3A) was in good condition. Para- not possible. type W 55276 (Fig. 3G) was much less so; it was examined The paratypes and holotype were similar in their main only partially, and with difficulty, due to its fragility. characters, having a transparent, cubic to pyramidal bell In both paratypes, the main characters were similar to covered with colourless nematocyst warts, long, scalpel- those observed in the holotype by Gershwin (2007) (Table shaped pedalia with straight pedalial canals having rectan- 2). They were also identical to the ones described above for gular knee bends with a short projection and single pinkish Malo maxima (Table 1) and are not repeated here. tentacles. Rhopalial niche orifices were frown-shaped and As in Malo maxima, several differences in the range of located at about 1/6 to 1/5 up the bell rim. All animals had numbers of nematocyst warts on the pedalial outer wings, a velarium with four, triangular perradial lappet pairs and velarial canal roots and perradial lappets, and in the shape 4–5 single-rooted velarial canals per octant. The stomach, of the knee bend projection, were noted during compari- with a very short manubrium, was bag-shaped and con- sons of the paratypes (Table 2). The most obvious differ- nected by well-developed mesenteries to the subumbrella. ences between them were in the shape of the rhopalial The gonads of the immature animal (Paratype C 150023, horns. The rhopalial horns in Paratype C 60029 (Fig. 3D) Fig. 2M) were arrow-head shaped, with the widest part were short, broad, and slightly curved inwards while those above the rhopalial niche. In mature animals, the gonads of Paratype W 55276 (Fig. 3J) differed even within one were large and leaf-like; they overlapped perradially, leav- niche by being either short, broad, and curved outwards or ing a space around the rhopalial niches. long, slim, with a knob-like tip, and straight. The most obvious difference between the paratypes and Both paratypes bore straight tentacles having nearly the the holotype was bell size, being about 40–45% larger in the same width from below the base to the distal ends (Fig. 3C, holotype than in the paratypes. Variations in the numbers I). There were no constrictions, as noted for Malo maxima, of nematocyst warts on the pedalial outer wings, velarial and no ‘halo’ structures as mentioned by Gershwin (2007) canal roots and perradial lappets, in the shape of the knee for the holotype (Table 2). bend projection (triangular or volcano shaped), and in the Circular ridges encircling the upper quarter of the peda- shape of the upper covering scales of the rhopalial niches lial canals as seen in specimens of Malo maxima were also were noted in the paratypes and the holotype (Table 1 and observed in one of the two inspected paratypes (G 60029, Fig. 2). However, the most obvious differences were in the Fig. 3B) of M. kingi. The condition of the pedalia and ten- shape and complexity of velarial canal branching (Fig. 2F, tacle of the second paratype (W 55276) was so poor that G, L, R) and in the shape of the rhopalial horns (Fig. 2D, those structures, if ever present, might have been lost due K, P). to deterioration of the specimen. The 4–5 finger-like velarial canals growing from the sin- The number of eyes could not be checked as eye pig- gle canal root per velarial octant were either broad (para- ments were completely faded. Only the structure of the types) or narrow (holotype), and simple to slightly forked lower median eye was visible, due to its large size, through or branched (holotype, Figs. 2F, G and Paratype C 15023 the rhopalial niche window. Fig. 2R) or multiply forked (Paratype G 55400, Fig. 2L). The complexity of the canal structure was either higher in 86 I. Straehler-Pohl

Table 1. Malo maxima Gershwin, 2005. Character descriptions based on inspections of holotype and paratypes (preserved) and original description of holotype by Gershwin (2005b).

Present Study Gershwin 2005b (text and Fig. 2B) Paratype C 15023 Paratype G 55400 Holotype Z29940

Bell • transparent • hyaline • cubic to pyramidal • narrower at apex than base • apex, slightly rounded without circular constriction • apex, flattened, no coronal furrow • nematocyst warts, colourless (preserved), round, from apex to velarium, more prominent on apex • nematocyst warts, pale purple (in-live), faded to nearly invisi- ble (preserved), round, on apex and along body walls Size • BH 33 mm • BH 34 mm • BH 48 mm • BH 48 mm • IRDa 26 mm • IRDa 29 mm • IRDa 38 mm • IRWb 21 mm • IRWb 15 mm • IRWb 16 mm • IRWb 20 mm • DBWc 41 mm • DBWc 33 mm • DBWc 33 mm • DBWc 40 mm • IPDd 35 mm • IPDb 35 mm • IPDb 45 mm a distance between opposite rhopalia at the level of the bell turn-over b distance between adjacent rhopalia c distance between opposite pedalia, at level of pedalia joining bell c distance between opposite pedalia, at the level of bell turn-over Pedalia • 4, single, simple • 4, single, unbranched • interradial corners of bell rim • interradial • long, flattened, scalpel-shaped • narrow, scalpel-shaped • ＜1/2 BH • ≥1/2 BH • ＜1/2 BH • ca. 1/2 BH • ≥2x longer than wide • proportion, no data Inner wing • flaring from base to midsection, tapering towards tentacle insertion • moderately flared • slightly ‘over-hanging’ tentacle insertion • ‘overhangs’ tentacle insertion Outer wing • narrower, broader edged than inner wing • narrower than inner wing • nematocyst warts, 0 • nematocyst warts, 2–4, flat, • nematocyst warts, 0–3, flat, • nematocyst patches, 3, small, round, if 4 then equidistant, round, if 3 then equidistant, evenly spaced outer rim, partly rubbed off outer rim, partly rubbed off Pedalia canals • cross-section, square • cross-section, quadrate • straight • straight • width, nearly same from knee bend to distal end, slightly flaring at tentacle insertion • slightly flared at tentacle insertion • projection at rectangular knee • projection at rectangular knee • projection at rectangular knee • short, blunt, obliquely-pointed bend, short, triangular bend, short, volcano-shaped bend, short, volcano-shaped projection at bend • ridges encircling upper quarter • ridges encircling upper quarter • ridges encircling upper quarter • circular canal ridges, not men- below knee bend, 4 below knee bend, 3 below knee bend, 4 tioned Tentacles • 4, single, simple • 4, single, unbranched • whitish to pinkish • whitish to pinkish • cross-section, round, flaring at base • cross-section, round, fine, straight-sided at base • width, unevenly segmented by • width, slightly segmented by • width, slightly segmented by • width, macro-segmented of re- constrictions constrictions in upper region then constrictions in upper region then peated hourglass pattern when nearly same width towards distal nearly same width towards distal contracted end end • nematocyst batteries, narrow rings • banding mostly uneven, in a few parts even Species identification in genus Malo 87

Table 1. (Continued.)

Present Study Gershwin 2005b (text and Fig. 2B) Paratype C 15023 Paratype G 55400 Holotype Z29940

Rhopalial niches • orifice, frown-shaped • orifice, frown-shaped • covering scales, upper, slightly • covering scales, upper, shal- • covering scale shapes differed • covering scales, upper, shallowly ‘M’-shaped, lower, upwards lowly curved downwards, from niche to niche, upper, shal- concave, lower, shallowly convex curved lower, shallowly curved up- low ‘M’-shaped to curved upwards wards, lower, curved shallowly to markedly upwards • 1/6 up from margin • 1/6 up from margin • 1/5 up from margin • 1/5 up from margin • number of eyes per rhopalium, not countable due to faded eye pigments due to preservation, several rhopalia • eyes per rhopalium, 2, median missing (cut out) with lenses, no lateral eye spots • statolith, dissolved due to preservation in formalin, cavity still visible • statolith, no data Rhopalial horns • short (≥1/3 of niche cavity • short (＜1/3 of niche cavity • shape differed from niche to niche • short, thick, straight to somewhat height), broad, slightly curved height), broad or slim, straight or even per niche: short (＜1/3 undulating inwards of niche cavity height) or long (1/2 of niche cavity height), broad or slim, straight or curved Velarium • canal root, 1 per octant, palmate shaped • canal root, 1 per octant Canals • 4–5 canals in all • approx. 4, • broad, finger-like, sometimes • broad, finger-like, multiple • narrow, finger-like, simple to • simple to bifurcated with undu- forked/branched, somewhat forked, sometimes slightly slightly forked or branched, lating edges dendritic, slightly undulating branched, somewhat dendritic, slightly undulating slightly undulating • canals flanking pedalia slightly • all canals equally complex • canals flanking perradial lappets • canal complexity, no data more complex than middle ca- and/or pedalia slightly more nals complex than middle canals • no nematocysts warts on canal • no nematocyst warts on canal • nematocyst patches or warts on • nematocyst warts on canal roots roots canal roots, 0–1 roots, no data Perradial lappet pairs • narrow triangular • narrow • simple and branched canals • single finger-like extensions • single finger-like extensions • nipple-like canals extending dis- protruding from tips and sides from tips from tips and sides (bases) tally, lacking lateral canals • nematocyst warts, 2–3 per lap- • nematocyst warts, 1–2 per lap- • nematocyst warts, 1–2 per lap- • nematocyst warts, 1 per lappet, pet, large, round, correspond- pet, round, equal in size, not pet, round, different in size, not large, gelatinous, raised, not ing in position corresponding in position corresponding in position corresponding in position Structures of digestive systems • manubrium, short (1/5 to 1/6 of BH), four lobed • manubrium, very small, very short (1/6 of BH) • stomach, bag-like, stomach floor densely patterned with gastric rugae (parallel corrugations) • stomach, short, flat (preserved), large, bag-like (in live) • mesenteries, well developed • mesenteries, moderately well developed • no phacellae/gastric filaments • phacellae, absent Gonads • interradial • interradial • interradial • not yet fully developed, large, blunt arrow head-shaped, broader in • large, leaf-like, pleated edges, • large, leaf-like, edges pleated, upper two thirds, sparing rhopalial niche broader in upper two thirds overlapping perradially, sparing (overlapping perradially), spar- rhopalial niche ing rhopalial niche • length from stomach to velarium • length from stomach to velar- • length, entire height of interra- ium into canal roots dial septa, extending into bases of velarial canals • septum, no data • septum, no data • septum, lacking perforation 88 I. Straehler-Pohl

Fig. 2. Malo maxima Holotype Z 29940: A: adult medusa; B: pedalium; C: pedalial knee bend, note volcano-shaped appendage (blue arrow) at knee bend and ridges encircling canal (white arrows); D: rhopalial niche, note rhopalial horns (arrows); E: tentacle structure; F, G: quadrants of velarium, note side canal of perradial lappet (white arrow) and nematocyst wart on velarial canal root (black arrow) ; Paratype G 55400: H: adult medusa; I: pedalium; J: pedalial knee bend, note ridges encircling canal (white arrows); K: rhopalial niche, note rhopalial horns (arrows); L: part of velarium with perradial lappets (pl). Paratype C 15023: M: adult medusa; N: pedalium; O: pedalial canal knee bend with encircling ridges (arrows); P: rhopalial niche with rhopalial horns (arrows); Q: tentacle structure, note constrictions; R: part of velarium, note side canals of perradial lappets. Abbreviations as follows: go: gonads; pl: perradial lappets; vcr: velarial canal root. Scales＝1 mm. Species identification in genus Malo 89

Table 2. Malo kingi Gershwin, 2007: Character description based on paratypes (preserved) and original description of holotype by Gershwin (2007).

Holotype G 317361 Paratype G 60029 Paratype W 55276 (Gershwin, 2007, text and/or Fig. 1A)

Bell • transparent • transparent • cubic to pyramidal • narrower at apex than base • apex, rounded without circular constriction • apex, evenly rounded • nematocyst warts, colourless (preserved), round, from apex to velarium, more promi- • nematocyst warts, pale purple (in-live), nent on apex round, on apex and along body walls Size • BH 25 mm • BH 24 mm • BH 31 mm • IRD 18 mm • IRD 24 mm • IRW 15 mm • IRW 14 mm • IRW 19 mm • DBW 30 mm • DBW 21 mm • DBW 27 mm • IPD 23 mm • IPD 30 mm Pedalia • 4, single, simple • 2, single, 2, branched (aberration) • interradial corners of bell rim • interradial • long, flattened, scalpel-shaped • long, scalpel-shaped • ＞1/2 BH • ≥1/2 BH (Fig. 1A) • 2x longer than wide • no data • no data proportion Inner wing • flaring from base to midsection, taper- • no data • moderately flared ing towards tentacle insertion • slightly ‘over-hanging’ tentacle insertion • no data • slightly ‘over-hanging’ insertion (Fig. 1A) Outer wing • narrower, broader edged than inner wing • no data • narrower than inner wing • nematocyst warts, 0–3, flat, round, • no data • no nematocyst freckles equidistant, outer rim Pedalia canals • cross-section, square • cross-section, strongly quadrate • straight • straight • width, nearly same from knee bend to distal end, slightly flaring at tentacle insertion • unflared at tentacle insertion • projection at rectangular knee bend, • no projection visible on nearly round • lacking smoothly rounded knee-like short, triangular knee bend bend or upward-pointing thorn • 3 ridges encircling upper quarter below • no ridges visible below knee bend • small hump near base knee bend Tentacles • 4, single • 2, single, 2, single, 2 branched off from lower third of pedalial canal (Fig. 1A) • whitish to pinkish • faintly pink • cross-section, round, flaring at base • cross-section, no data • width, nearly same from below base to distal end, not segmented • segmented like a ‘string of sausages’ at similar intervals, diameter grows distally • nematocyst batteries, narrow rings • nematocyst batteries, halo-like, thin sheet-rings, inserted end on around outer edges of ‘halos’ Rhopalial niches • orifice, frown-shaped • ostium, frown-shaped • covering scales, upper, slightly ‘M’-shaped, lower, upwards curved • covering scales, upper, broadly convex with a shallow ‘W’-shape, lower, upwards curved (Fig. 1A) • 1/5 up from margin • 1/5 up from margin (Fig. 1A) • number of eyes per rhopalium, not countable due to faded eye pigments due to pres- • “eye morphology and statolith shape ervation and due to missing rhopalia not discernable in preserved holotype specimen; lateral eye spots lacking in other specimens (Fig 3D)” • statolith, dissolved due to preservation in formalin, cavity still visible 90 I. Straehler-Pohl

Table 2. (Continued.)

Holotype G 317361 Paratype G 60029 Paratype W 55276 (Gershwin, 2007, text and/or Fig. 1A)

Rhopalial horns • short (＜1/3 of niche cavity height), • shape differed within one niche of the • relatively short, broad, curved inward broad, slightly curved inwards same animal: short (＜1/3 of niche cavity like Viking horns height), broad, curved outwards or long (1/2 of niche cavity height), slim with a knob-like tip, straight Velarium • canal root, 1 per octant, palmate-shaped • canal root, 1 per octant, palmate-shaped Canals • 4–5, finger-like, sometimes forked/branched, somewhat dendritic • 4–5, finger-like, somewhat branched • canals flanking perradial canals/pedalia more complex than middle canals • canals flanking perradial canals/pedalia more complex than middle canals (Fig. 3E) • nematocysts warts on canals or roots, • no nematocysts warts on canals or roots • no nematocyst patches or warts 0–1 Perradial lappets • narrow triangular • narrow triangular • bifurcated canals protruding from tips, single, simple lobations at sides near base • multiple, finger-like extentions from tips • nematocyst warts, 2–4 per lappet, large, round, corresponding or not in position • nematocyst patches, 3–4 per lappet, round, no information given on position Structures of digestive systems • manubrium, short (1/5 of BH), four lobed • manubrium, short • stomach, bag-like, stomach floor densely patterned with gastric rugae • stomach, small, flat (parallel corrugations) • mesenteries, well developed • mesenteries, well developed • no phacellae/gastric filaments • phacellae, lacking Gonads • interradial • interradial • interradial • length from stomach to velarium • length from stomach to velarium into • full length of septa, from stomach into canal roots velarial canals • not yet fully developed, narrow bands • large, leavf-like, broader in upper two • large, leaf-like, overlapping perradially, thirds (overlapping perradially), pleated keyhole-shaped outline around rhopa- edges, sparing rhopalial niche lial niches, pleated edges • septum, no data • septum, no data • septum, lacking perforation

Malo sp. 1/5 up the bell rim. Eye structures had lost all pigments, Sampling location: Australia: Far North Queensland, Port but there had been six eyes per rhopalium (2 median, 2 lat- Douglas (16°29′S, 145°27′E), Marina. eral slit and 2 lateral pit ones) (A. Underwood and J. Sey- Inspection of preserved Malo sp. medusae (Table 3) and mour, pers. comm.). All medusae had a velarium with four comparison with inspected types of Malo maxima (Table 1) triangular perradial lappet pairs and 4–5 single-rooted ve- and Malo kingi (Table 2) larial canals per octant (Figs. 4F, L, M, 5L–N). The very All inspected medusae of Malo spp. (characters of three short manubrium was attached to a stomach that was in all randomly chosen specimens are listed in Table 3) showed cases bag-shaped, bore no gastric phacellae but gastric similar main characters, including (1) a transparent, cubic rugae on the stomach ﬂoor (Fig. 5O) and were connected to pyramidal bell (Figs 4A, G, N) covered with colourless by well-developed mesenteries to the subumbrella. Devel- nematocyst warts (mauve to pale purple in living animals; oping gonads began as a narrow line along the whole sep- A. Underwood, pers. comm.), (2) long, scalpel-shaped tum, later becoming arrowhead-shaped with smooth edges, pedalia with straight pedalial canals (Figs 4B, H, O, 5A–E) covering the gastric pouches from stomach corner to velar- showing rectangular knee bends with a short projection ial canals, and broader in the upper two-thirds than in the (Figs 4C, I, P, 5F, G), and (3) single pinkish tentacles. As in lower third, leaving a space around the rhopalial niches. In Malo maxima and M. kingi, pedalial canals showed 3–4 fully mature animals the gonads were large, leaf-like, and encircling ridges (Figs. 4I, P, 5F) in the upper quarter of overlapped perradially, leaving a space around the rhopa- the canals below the knee bend. The rhopalial niche ori- lial niches. ﬁces were frown-shaped (Fig. 4D, J, Q) and located about As in the type specimens of both Malo maxima and M. Species identification in genus Malo 91

Fig. 3. Malo kingi Paratype G60029: A: medusa; B: pedalium; C: tentacle structure; D: rhopalial niche–note rhopalial horns (arrows); E: pedalial canal knee bend, note triangular appendage (black arrow) and encircling ridges (white arrows); F: quadrant of velarium, note side canals (white arrow) of perradial lappets (pl) and nematocyst wart on velarial canal root (black arrow); Paratype W 55276: G: adult medusa; H: pedalium; I: part of tentacle; J: rhopalial niche, note rhopalial horns. Scales＝1 mm. 92 I. Straehler-Pohl

Table 3. Malo sp. Character description based on examples of 3 randomly selected, preserved animals collected at the Marina of Port Douglas by A. Underwood and J. Seymour in April 2011.

No. 23 No. 43 No. 45

Bell • transparent • nematocyst warts, colourless (mauve in living animals), more prominent on apex than on lateral bell walls • cubic to pyramidal • apex rounded with no horizontal constriction Size • BH 26 mm • BH 26 mm • BH 23 mm • IRD 22 mm • IRD 23 mm • IRD 21 mm • IRW 14 mm • IRW 14 mm • IRW 11 mm • BDW 27 mm • BDW 28 mm • BDW 25 mm • IPD 30 mm • IPD 31 mm • IPD 27 mm Pedalia • 4, single, simple • interradial corners of bell rim • long, flattened, scalpel-shaped • 3/4 BH • ＞1/2 BH • 3/4 BH • ＞2x longer than wide • ＜2x longer than wide • 2x longer than wide Inner wing • flaring from base to midsection, tapering towards tentacle insertion • slightly ‘over-hanging’ tentacle insertion Outer wing • narrower, broader edged than inner wing • nematocyst warts, outer rim, numerous • nematocyst warts, outer rim, numerous • nematocyst warts, outer rim, numerous scattered or 6–8 in a row, flat, round, if scattered or 7–8 in a row, flat, round, scattered 0 or 3–5 in a row, flat, round, not scattered then nearly equidistant, nearly equidistant, nearly equidistant, Pedalia canals • cross-section, square • straight • width, nearly same from knee bend to distal end, slightly flaring at tentacle insertion • projection at rectangular knee bend, • projection at rectangular knee bend, • projection at rectangular knee bend, short, volcano-shaped short, volcano-shaped short, triangular • ridges encircling upper quarter below • ridges encircling upper quarter below • ridges encircling upper quarter below knee bend, 0 knee bend, 3 knee bend, 3 Tentacles • 4, single • whitish to pinkish • cross-section, round, flaring at base • segmented at different intervals (pres- • width, nearly same from below base to distal end ervation artefact) • nematocyst batteries, narrow rings • nematocyst batteries, ‘halo’-like thin • nematocyst batteries, narrow rings when (contracted) sheet-rings, inserted end on around outer contracted, pearl-shaped when relaxed edges of ‘halos’ (preservation artefact) or narrow rings (contracted) Rhopalial niches • orifice, frown-shaped • with or without nematocyst warts on scales (differed also within one animal) • covering scales, upper, slightly ‘M’-shaped, lower, upwards curved, scales • covering scales, upper, slightly ‘W’ or ‘M’-shaped, lower, upwards curved • 1/5 up from margin • 6 eyes per rhopalium in living animal (Underwood, pers. communication), in preserved animal not countable due to faded eye pigments due to preservation • statolith, dissolved due to preservation in formalin, cavity still visible Species identification in genus Malo 93

Table. 3. (Continued.)

No. 23 No. 43 No. 45 Rhopalial horns • short (＜1/4–1/3 of niche cavity • shape differed from niche to niche or • short (1/3 of niche cavity height), slim, height), broad, slightly curved inwards, within one niche: short (＞1/3 of niche slightly curved inwards, with a knob- with a knob-shaped tip cavity height) or long (1/2 of niche cav- shaped tip ity height), slim, straight to slightly curved outwards; one horn was bent outwards in extreme going back down towards niche Velarium • canal root, 1 per octant, palmate-shaped Canals • 4–5 canals in all • 1–2, simple finger-like (undulating • 3–4 simple finger-like (straight appear- • 2–4 simple finger-like (straight to undu- appearance), 2–3 multiple branched ance), partly lobate, 1–2 multiple lating appearance), 0–4 multiple (‘pinetree’-like appearance), lobate branched branched, lobate • canals flanking perradial canals/pedalia more complexly branched than middle canals • nematocysts warts on canal root, 0–1 • nematocysts warts on canal root, 1–2 Perradial lappets • narrow triangular • simple to bifurcated canals protruding • simple canals protruding from tips • simple to bifurcated canals protruding from tips and laterally from tips and laterally • nematocyst warts, 1–3 per lappet, large • nematocyst warts, 1–3 per lappet, large • nematocyst warts, 1–2 per lappet, large or rarely small, round or rarely small, round or rarely small, round Structures of digestive systems • manubrium, very short (1/6–1/5 of BH), four lobed • stomach, bag-like, stomach floor densely patterned with gastric rugae (parallel corrugations) • mesenteries, well developed • no phacellae/gastric filaments Gonads • interradial • large, leaf-like, pleated edges • length from stomach to velarium • not yet fully developed kingi, differences amongst the medusae were noted in the Discussion numbers of nematocyst warts on the outer wing of the pedalia (Figs. 44B, H, O, 5A–E), on the velarial canal roots Comparisons of the holotypes and paratypes of Malo (Figs. 4F, L, M, 5L, M), and on the perradial lappets, as maxima and M. kingi revealed that the described differ- well as in the number and location of perradial lappet side ences between the two species are not obvious and can be canals (Figs. 4F, 5L, N). considered intraspecific variations, e.g. numbers of nema- The shape of the rhopalial horns varied from short (1/4– tocyst warts on the outer pedalial wing [Malo maxima: 1/3 of niche cavity height) to long (1/2 of niche cavity mean 3–4, range 1–10 (Gershwin 2005b); Malo kingi: 0 to height), slim to broad, straight to curved inwards or out- several (Gershwin 2007)] and nematocyst warts on the per- wards; the tip was sometimes knob-like and sometimes not radial lappets [Malo maxima: mean 2, range 1–4 (Gersh- (Figs. 4D, J, Q, 5H–K). win 2005b); Malo kingi: range 3–4 (Gershwin 2007)]. The shape of the velarial canals varied even within dif- Pedalia of Malo sp. were armed with nematocyst warts. ferent quarters of the same medusa from simple finger-like The warts were either numerous and scattered, or arranged to multi-branched (Figs. 4L, 5M). The number of simple or in a row of 3–8 warts which were nearly equidistant on the more complex canals differed from animal to animal, but edge of the outer wings. Those on the outer wings appeared the outer canals flanking the perradial lappets or the peda- to be more numerous in young medusae, and they seemed lia tended to be more complex than the middle canals to disappear in later developmental stages. This might ex- (Figs. 4L, M, 5M, N). plain the absence of nematocyst warts on the pedalial wings of the holotype of M. kingi (Gershwin 2007). The 1–3 nematocyst warts on the perradial lappets of Malo sp. were within the range seen in M. maxima and M. kingi. 94 I. Straehler-Pohl

Fig. 4. Malo sp. Animal no. 23: A: medusa; B: pedalium, note small, scattered nematocyst warts (arrow), C: pedalial canal knee bend; D: rhopalial niche, note rhopalial horns (arrows); E: tentacle structure, note constrictions; F: octant of velarium, note side canals of perradial lappets (white arrow) and nematocyst wart on velarial canal root (black arrow); Animal no. 43: G: medusa; H: pedalium, note large, equidistant nematocyst warts (arrows); I: pedalial canal knee bend, note encircling ridges (arrows); J: rhopalial niche, note rhopalial horns (arrows); K: tentacle structure, note ‘halo’-like rings of nematocyst batteries; L: quadrant of velarium, note nematocyst wart on velarial canal root (arrow); Animal no. 45: M: quadrant of velarium, note nematocyst warts on velarial canal roots (arrows); N: medusa; O: pedalium, note different sizes of nematocyst warts (arrow) at the very edge of the outer wing; P: pedalial canal knee bend, note encircling ridges (arrows); Q: rhopalial niche, note rhopalial horns (arrows); R: tentacle structure without any abnormal constrictions or ‘halos’. Scales＝1 mm. Species identification in genus Malo 95

Fig. 5. Anatomical variations of Malo sp. Pedalia A, B: animal no. 23; C, D: animal no. 17; E: animal no. 42; F, G: pedalial canal knee bend of animal no. 43, note encircling ridges (F: arrows) and blunt triangular appendage (G: arrow); rhopalial win- dows with rhopalial horns (arrows): H: animal no. 42, I, J, K: animal no. 43; quadrant of velarium, note side canals of perradial lappets (white arrows) and nematocyst warts on perradial lappets (black arrows): L: animal no. 17; M: animal no. 42, N: animal no. 14; O: cut open part of stomach lacking phacellae or gastric ﬁlaments – note the parallel corrugations; P: developing rhopalium of an intermediate metamorphosis stage, note developing eye spots (arrows). Abbreviations as follows: pc: pedalial canal; pl: perradial lappets; rh: rhopalium; st: statolith; scales＝1 mm. 96 I. Straehler-Pohl

Rhopalial horns were as variable in Malo sp. as in the 20 mm (Carrette et al. 2012). Significant differences in bell other two described species. They were mostly short in im- sizes in adult animals might be due either to access to richer mature specimens, indicating that their development may food resources, or to the fact that animals may become ma- continue with bell growth. ture before reaching maximal bell height and be able to re- Variations in the complexity of velarial canals were de- generate gonads to spawn more than one time as observed scribed in Malo maxima as “may or may not have side in Copula sivickisi (Stiasny, 1926) (unpublished data). branches” (Gershwin 2005b, p. 18), and in Malo kingi as The second apparent difference between inspected spec- having “ . . . complexly branched velarial canals, i.e., with imens of Malo maxima and M. kingi relates to the structure lateral diverticula, whereas in others the canal branches of the tentacles. In the holotype of M. kingi, tentacles are are more digitiform” (Gershwin 2007, p. 62). Gershwin segmented and nematocyst batteries are described as (2007, p. 62) concluded that the “taxonomic significance of “ . . . halo-like rings encircling tentacle; nematocysts in- these differences, if any, is not at this time well understood”. serted end-on into edges of rings, radiating outward; . . . ” Such diverse shapes of the velarial canals were also ob- (Gershwin 2007, p. 60). Tentacles of preserved specimens served in Malo sp. of M. maxima are slightly to markedly segmented (Gersh- While the characters listed above reveal no clear differ- win 2005b) as in M. kingi, but they lack ‘halo’-like nemato- ences between Malo maxima and M. kingi, there are two cyst batteries. Such segmentation, as well as the ‘halo’-like characteristics that might initially appear to distinguish structure, were also observed in specimens of Malo sp. populations of Malo from the two Australian coasts into (Fig. 4E, K) that were inadequately preserved. Preserva- two species: One is the huge bell size of the holotype of M. tion of specimens can sometimes be inadequate under field maxima, which is more than 1.5 times larger than that of conditions, leading to deformities in preserved material the holotype of M. kingi and about 1.9 to 2.0 times higher from maceration processes and from gas development pro- than bell sizes of the paratypes of M. kingi. However, the duced by autolysis within tentacle canals (personal obser- bell size of the holotype of M. kingi lies within the size vation). Therefore, such segmentations and ‘halos’ are con- range of the inspected paratypes of M. maxima. While the sidered preservation artefacts and considered not useful in bell sizes of the specimens of Malo sp. are much smaller species determination or characterization. than the holotypes of M. maxima or M. kingi, they are still It seems that all these variations, including the different within the range of mature paratypes of both species (Ger- shapes of rhopalial canals, velarial canals, bell sizes, and shwin 2005b: Malo maxima: ca. 23–35 mm; Gershwin numbers of nematocysts on various parts of the medusa, 2007: Malo kingi: ca. 23–30 mm). Bell size might therefore are characters that change from individual to individual be quite variable in the species, as in Carukia barnesi and from growth stage to growth stage. They are not con- Southcott, 1967. The holotype of C. barnesi (female) has a sidered reliable as characters for species determination, at bell height of 14 mm (Southcott 1967, Gershwin 2005b) least not in the genus Malo and especially not in the Aus- while recently collected adult medusae had bell sizes be- tralian populations. Bentlage & Lewis (2012) described an- tween 18 mm (Fig. 6; unpublished data) and more than other species of Malo, M. filipina, from the Philippines having three distinguishing characters: a long appendage at the pedalial canal knee bend (in contrast to the very short, blunt, ascending projection of the Australian species), 3–4 instead of 4–5 velarial canals per root, and a different cni- dome. These characters might be the only reliable features to separate species within the genus. According to Gershwin (2005b, 2007), species of Malo possess no slit and no pit eyes on the lateral sides of the rhopalium. Medusae of Malo sp. (A. Underwood, pers. comm.), and our observations on early life cycle stages of the species (Underwood et al. unpublished data; Fig. 5P), revealed that six eyes per rhopalium develop during medusa production (two median lens eyes, two pit eyes and two slit eyes) instead of only two median lens eyes as described for the other two species referred to the genus (Gershwin 2005a, b, 2007). A closer look at images of the rhopalia of M. kingi [fig. 3D in Gershwin (2007)] shows that at least the structure of a lateral slit eye is still visible. In addition, in the image of the rhopalium of M. maxima [fig. 3D in Gershwin (2005b)], the structures of both the slit Fig. 6. Mature, preserved medusa of Carukia barnesi and the pit eye are still visible. I agree with the comment from Double Island, North Queensland. by Bentlage & Lewis (2012, p. 2605) that “ . . . the eye pig- Species identification in genus Malo 97 ment may fade in fixative and lead to inaccurate counts helped in all matters concerning the collection and photo (Bentlage 2010) . . . ”. Moreover, Carrette et al. (2014) ob- equipment. I would also like to thank the staff of the Mu- served the same phenomenon in Alatina cf. moseri. There- seum & Art Gallery of the Northern Territory, especially fore, I suggest amending the genus definition, in accor- Suzanne Horner and Kelly Rau, for their help within the dance with Bentlage et al. (2010) and Bentlage & Lewis collection and for making it possible to work in the collec- (2012), to: tion after very short notice. Jane Fremont from the West Genus Malo: Carukidae with tentacles with pearl- Australian Museum in Perth provided holotype material shaped nematocyst batteries lacking neckerchief-shaped and sent it to Townsville. I would also like to thank PD extensions; with one velarial canal root branching into Gerhard Jarms of the Zoological Institute and Museum, about 3–5 simple to branched velarial canals per octant; University of Hamburg, Germany, for his kind allowance two rows of one to four perradial lappet warts. of me to use his facilities at any time. A molecular analysis comparing Malo maxima, M. kingi and M. sp. was not possible as all examined specimens References were initially preserved in formalin. Gershwin (2005a) performed an 18S rDNA analysis of the ‘Broome Irukandji’ Bentlage B (2010) Carybdea alata auct. (Cubozoa): rediscovery population (M. maxima) of Western Australia and the of the Alatina grandis type. Zootaxa 2713: 52–54. ‘Pseudo Irukandji’ population (M. kingi) from Queensland. Bentlage B, Cartwright P, Yanagihara AA, Lewis C, Richards When checking those data (Gershwin 2005a: p. 196–200, GS, Collins AG (2010) Evolution of box jellyfish (Cnidaria: appendix 3. Partial 18S rDNA Alignment) there was no re- Cubozoa), a group of highly toxic invertebrates. Proc R Soc B markable difference between the two groups. 277: 493–501. From the above mentioned results, I conclude that Malo Bentlage B, Lewis C (2012) An illustrated key and synopsis of maxima and M. kingi are two populations of one species. the families and genera of carybdeid box jellyfishes (Cnidaria: Based on the principle of priority (Article 23, ICZN 1999) Cubozoa: Carybdeida), with emphasis on the “Irukandji family” Malo maxima is the earlier name and is therefore adopted (Carukiidae). J Nat Hist 46: 2595–2620. as the binomen for the populations of both Western Austra- Campbell RD (1974) Cnidaria. In: Reproduction of marine inver- lia and Queensland, including Malo sp.; M. kingi is to be tebrates, Volume I: Acoelomate and pseudocoelomate metazo- ans (eds Giese AC, Pearse JS). Academic Press, London, Am- considered its junior synonym. sterdam, New York, pp. 133–199. A key to the developmental stages of medusae from the Carrette T, Straehler-Pohl I, Seymour J (2014) Early life history newly detached stage to maturity, as provided by Uchida of Alatina cf. moseri populations from Australia and Hawaii (1926: fig. 50) and Uchida (1929: figs. 74–78), Fuentes et al. with implications for taxonomy (Cubozoa: Carybdeida, Alati- (2011: table 4) or Straehler-Pohl et al. (2011: tables 3a–g, nidae). PLoS One, 9(1): 1–8. doi: 10.1371/journal.pone.0084377. plates 4–5) is important for species indentification, espe- Carrette TJ, Underwood AH, Seymour JE (2012) Irukandji syn- cially for the younger stages of any scyphozoan or cubo- drome: a widely misunderstood and poorly researched tropical zoan species. As not all developmental stages of Malo marine envenoming. Diving Hyperb Med 42: 214–223. maxima are yet available, this will have to remain an im- Fuentes V, Straehler-Pohl I, Atienza D, Franco I, Tilves U, Gentile portant task for a future project. M, Acevedo M, Olariaga A & Gili J-M (2011) Life cycle of the jellyfish Rhizostoma pulmo (Scyphozoa: Rhizostomeae) and its Acknowledgements distribution, seasonality and inter-annual variability along the Catalan coast and the Mar Menor (Spain, NW Mediterranean). I thank Ass. Prof. Dr. J. Seymour for hosting me in his Mar Biol 158: 2247–2266. lab, and Teresa Carrette MSc for hosting me for three Gershwin L-A (2005a) Taxonomy and Phylogeny of Australian weeks in her home. I have to thank Prof. André C. Moran- Cubozoa. PhD Thesis. Townsville, Queensland: School of Ma- dini for giving helpful advice concerning taxonomic mat- rine Biology and Aquaculture, James Cook University. ters and manuscript contents. My gratitude is expressed to Gershwin L-A (2005b) Two new species of jellyfishes (Cnidaria: Prof. Dale Calder for checking and correcting the wording Cubozoa: Carybdeida) from tropical Western Australia, pre- and grammar of the draft version. I thank also an anony- sumed to cause Irukandji Syndrome. Zootaxa 1084: 1–30. mous reviewer for helpful advice to improve the manu- Gershwin L-A (2007) Malo kingi: A new species of Irukandji jel- script and suggestions for future investigations. Gratitude lyfish (Cnidaria: Cubozoa: Carybdeida), possibly lethal to hu- is extended to Avril Underwood MSc for contacting muse- mans, from Queensland, Australia. Zootaxa 1659: 55–68. ums on my behalf, for organising the transportation of ho- ICZN (1999) International Code of Zoological Nomeclature. lotypes to the Queensland Museum and for lending me ITZN, London, 306 pp. specimens of Malo sp. for thorough observations. I am Marques AC, Collins AG (2004) Cladistic analysis of Medusozoa grateful to the staff of the Queensland Museum in Towns- and cnidarian evolution. Invertebr Biol 123: 23–42. ville, especially Dr. Carden Wallace, who made the visit to Morandini AC, Marques AC (2010) Revision of the genus Chrys- aora Péron & Lesueur, 1810 (Cnidaria: Scyphozoa). Zootaxa the museum possible, Barbara Done, Dr. Kevin J. Tilbrook 2464: 1–97. and Dr. Paul R. Muir who gave me a friendly welcome and Southcott RV (1967) Revision of some Carybdeidae (Scyphozoa: 98 I. Straehler-Pohl

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