Proc. R . Soc. B | vol. 277 n o. 1680 p p . 345?501 | 7 Feb ruary 2010 7 February 2010 volume 277 . number 1680 . pages 345?501 ISSN 0962-8452 volume 277 number 1680 pages 345?501 7 February 2010 Founded in 1660, the Royal Society is the independent scientific academy of the UK, dedicated to promoting excellence in science Registered Charity No 207043 rspb.royalsocietypublishing.org Published in Great Britain by the Royal Society, 6?9 Carlton House Terrace, London SW1Y 5AG Review articles Reelin and apolipoprotein E receptor 2 in the embryonic and mature brain: effects of an evolutionary change in the apoER2 gene 345 N. B. Myant Research articles Ecological constraints and benefits of philopatry promote group-living in a social but non-cooperatively breeding fish 353 M. Y. L. Wong Vertical transmission as the key to the colonization of Madagascar by fungus-growing termites? 359 T. Nobre, P. Eggleton & D. K. Aanen A look into the invisible: ultraviolet-B sensitivity in an insect (Caliothrips phaseoli ) revealed through a behavioural action spectrum 367 C. A. Mazza, M. M. Izaguirre, J. Curiale & C. L. Ballar? Lower limits of ornithischian dinosaur body size inferred from a new Upper Jurassic heterodontosaurid from North America 375 R. J. Butler, P. M. Galton, L. B. Porro, L. M. Chiappe, D. M. Henderson & G. M. Erickson Evidence for modular evolution in a long-tailed pterosaur with a pterodactyloid skull 383 J. L?, D. M. Unwin, X. Jin, Y. Liu & Q. Ji Costly major histocompatibility complex signals produced only by reproductively active males, but not females, must be validated by a ?maleness signal? in three-spined sticklebacks 391 M. Milinski, S. W. Griffiths, T. B. H. Reusch & T. Boehm Post-glacial redistribution and shifts in productivity of giant kelp forests 399 M. H. Graham, B. P. Kinlan & R. K. Grosberg Sex-specific chemical cues from immatures facilitate the evolution of mate guarding in Heliconius butterflies 407 C. Estrada, S. Yildizhan, S. Schulz & L. E. Gilbert Joint evolution of multiple social traits: a kin selection analysis 415 S. P. Brown & P. D. Taylor Organic preservation of fossil musculature with ultracellular detail 423 M. McNamara, P. J. Orr, S. L. Kearns, L. Alcal?, P. Anad?n & E. Pe?alver-Moll? Words as alleles: connecting language evolution with Bayesian learners to models of genetic drift 429 F. Reali & T. L. Griffiths Helping effort increases with relatedness in bell miners, but ?unrelated? helpers of both sexes still provide substantial care 437 J. Wright, P. G. McDonald, L. te Marvelde, A. J. N. Kazem & C. M. Bishop Juvenile sparrows preferentially eavesdrop on adult song interactions 447 C. N. Templeton, ?. Ak?ay, S. E. Campbell & M. D. Beecher An inducible morphological defence is a passive by-product of behaviour in a marine snail 455 P. E. Bourdeau Stochastic evolutionary dynamics of direct reciprocity 463 L. A. Imhof & M. A. Nowak Urban noise and the cultural evolution of bird songs 469 D. Luther & L. Baptista History-dependent properties of skeletal muscle myofibrils contracting along the ascending limb of the force?length relationship 475 C. Pun, A. Syed & D. E. Rassier Conservative ecological and evolutionary patterns in liverwort?fungal symbioses 485 M. I. Bidartondo & J. G. Duckett Evolution of box jellyfish (Cnidaria: Cubozoa), a group of highly toxic invertebrates 493 B. Bentlage, P. Cartwright, A. A. Yanagihara, C. Lewis, G. S. Richards & A. G. Collins See further with the Royal Society in 2010 ? celebrate 350 years RSPB_277_1680_Cover.qxd 12/09/09 02:39 PM Page 1 Evolution of box jellyfish o rt ich , Th S 6 l M Wa iosc I 9 ty o 07 l cla nd ic hypothesis for the group. Here, we present a com- l g hon 1. INT Althou prising known sion o (e.g. N and m extrem are m the s interes pered museu investi The la nized framew diversi lution elec- seum e was buffer using ing to auto- toGen proto- l 16S SSU) rimers ollins genics lytical Biology, Smithsonian Institution (Suitland, MD, USA). Trace files were assembled in SEQUENCHER (v. 4.8; Gene Codes, MI) and subsequently aligned using MUSCLE (v. 3.7; Edgar 2004). Highly variable, poorly aligned regions were * Autho and Evolutionary Biology, The University of Kansas, 1200 Sunnyside Avenue, Lawrence, KS 66045, USA (bentlage@ku.edu). Electronic supplementary material is available at http://dx.doi.org/10. on December 29, 2009rspb.royalsocietypublishing.orgDownloaded from 1098/rspb.2009.1707 or via http://rspb.royalsocietypublishing.org.Received Acceptedmechanism(s) underlying Irukandji syndrome, (ii) deep divergences between Atlantic and Indo-Pacific clades may be explained by ancient vicariant events, and (iii) sexual dimorphism evolved a single time in concert with complex sexual behaviour. Furthermore, several cubozoan taxa are either para- or poly- phyletic, and we address some of these taxonomic issues by designating a new family, Carukiidae, a new genus, Copula, and by redefining the families Tamoyidae and Tripedaliidae. Lastly, cubozoan species identities have long been misunderstood and the data presented here support many of the recent scientific descriptions of cubozoan species. However, the results of a phylogeographic analysis of Alatina moseri from Hawai?i and Alatina mordens from Australia indicate that these two nominal species represent a single species that has maintained metapopulation cohesion by natural or anthropogenic dispersal. Keywords: Cubozoa; box jellyfish; Irukandji; systematics; biogeography; phylogeography RODUCTION gh Cubozoa is the smallest class of Cnidaria, com- some 50 described box jellyfish species, it is well for several remarkable attributes. From the posses- f complex eyes and associated visual capabilities ilsson et al. 2005), to extraordinary courtship ating behaviour (e.g. Lewis & Long 2005), to e toxicity (e.g. Brinkman & Burnell 2009), there any reasons why cubozoans catch the attention of cientific community and public. Despite this t, studies of cubozoan evolution have been ham- by a paucity of specimens in natural history ms preserved for both morphological and molecular gation, as well as by their perceived lack of diversity. st decade has seen more than a doubling in recog- cubozoan species, but so far a robust phylogenetic ork for investigating the evolution of cubozoan ty has been missing. We present a comprehensive phylogeny for Cubozoa and use it to discuss the evo of venom, life history and biogeography. 2. MATERIAL AND METHODS (a) Phylogenetic inference A list of specimens used for this study is provided in the tronic supplementary material, table S1, including mu catalogue numbers where vouchers exist. Tentacle tissu preserved in pure EtOH or saturated salt DMSO (Dawson et al. 1998), from which DNA was extracted organic phenol?chloroform extraction protocols accord the procedure outlined inCollins et al. (2008) or using the mated DNA isolation system AutoGenPrep 965 (Au Inc., Holliston,MA,USA) following themanufacturer?s col. Ribosomal genes coding for partial mitochondria (16S) and near-complete nuclear 18S (small subunit; and 28S (large subunit; LSU) were amplified using the p and protocols outlined in Cartwright et al. (2008) and C et al. (2008). PCR products were either sequenced byCo (Houston, TX, USA) or at the Laboratory of Ana r and address for correspondence: Department of Ecologylogeny for our understanding of cubozoan venom evolution, biogeography and life-history evolution. Our phylogenetic hypothesis suggests that: (i) the last common ancestor of Carybdeida probably possessed thea group of highly t Bastian Bentlage1,2,*, Paulyn Ca Cheryl Lewis2, Gemma S. R 1Department of Ecology and Evolutionary Biology Lawrence, K 2NMFS, National Systematics Laboratory, Nationa MRC-153, PO Box 37012, 3Be?ke?sy Laboratory of Neurobiology, Pacific B Honolulu, H 4School of Biological Sciences, The Universi Queensland 4 Cubozoa (Cnidaria: Medusozoa) represents a smal which cause serious human envenomations. Our u has been limited by the lack of a sound phylogenet prehensive cubozoan phylogeny based on ribosoma subunit) and 28S (large subunit) and partial mitoc22 September 2009 26 October 2009 493enes coding for near-complete nuclear 18S (small drial 16S. We discuss the implications of this phy-(Cnidaria: Cubozoa), xic invertebrates wright1, Angel A. Yanagihara3, ards4 and Allen G. Collins2 e University of Kansas, 1200 Sunnyside Avenue, 6045, USA useum of Natural History, Smithsonian Institution, shington, DC 20013, USA iences Research Center, 1993 East-West Road, 6822, USA f Queensland, St Lucia Campus, Brisbane, 2, Australia de of approximately 50 described species, some of erstanding of the evolutionary history of Cubozoa Proc. R. Soc. B (2010) 277, 493?501 doi:10.1098/rspb.2009.1707 Published online 18 November 2009This journal is q 2009 The Royal Society LSU sequences of both cubozoan taxa and outgroup taxa were and SSU, thus leading to higher degrees of nucleotide sat- 494 B. Bentlage et al. Evolution of box jellyfish on December 29, 2009rspb.royalsocietypublishing.orgDownloaded from aligned using MUSCLE, subsequently pruned using GBLOCKS and analysed with ML and MP using the criteria described above. Since the large divergence between ingroup and outgroup for the 16S gene does not allow for reliable alignment, we decided a priori not to analyse this marker using an outgroup. In order to investigate the possibility of strongly supported character conflict among partitions in the combined datasets, we performed an incongruence length difference (ILD) test (Farris et al. 1995a,b) as implemented in PAUP*. The ILD test has often been used as a test of combinability of datasets for phylogenetic analyses (e.g. Cunningham 1997), but interpretation of ILD test results has been the subject of debate (e.g. Barker & Lutzoni 2002). (b) Alatina phylogeography Mitochondrial 16S of seven specimens of Alatina mordens from Osprey Reef (Coral Sea, Queensland, Australia) and 19 specimens of Alatina moseri from Waikiki (O?ahu, HI, USA) were amplified and sequenced using the same tech- niques as above (GenBank nos. GQ506980?GQ507005 associated with USNM voucher specimens). All sequences were aligned using MUSCLE and the beginning and end of the alignment were trimmed to the position at which the nucleotides for every specimen are known. A statistical parsi- mony haplotype network was calculated in TCS (v. 1.21; Clement et al. 2000) using the 95 per cent connection limit criterion and gaps treated as a fifth character state. 3. RESULTS (a) Phylogeny of Cubozoa The partition-homogeneity test, with 100 replicates, could not refute the null hypotheses of congruence among partitions in the combined datasets LSU/SSU with outgroup (p ? 0.38; electronic supplementary material, figure S1) and LSU/SSU/16S without outgroup (p ? 0.56; figure 1), suggesting the absence of strong conflict among partitions. Phylogenetic analyses under both MP (not shown) and ML lead to highly congruent results, and node support is similar under both ML and MP (figure 1; electronic sup- plementary material, figures S1?S6). Both Chirodropida and Carybdeida are monophyletic clades with the root of Cubozoa falling in between the two in the SSU and com- bined LSU/SSU datasets with outgroup (electronic supplementary material, figures S1 and S3). Monophylyremoved from the final alignments using GBLOCKS (v. 0.91b; Castresana 2000) with the default parameters except that allowed gap positions were set to half. Nucleotide sequences were deposited in NCBI GenBank (electronic supplementary material, table S1) and alignments used for analyses were deposited in TreeBASE (treebase.org). In addition to the alignments for each gene, all three were combined into a concatenated alignment. These four align- ments were analysed using maximum parsimony (MP) in PAUP* (Swofford 2003) and maximum likelihood (ML) in RAXML (v. 7.0.3; Stamatakis 2006). MRMODELTEST (v. 2.3; Nylander 2004) was used to evaluate nucleotide sub- stitution models for ML analyses. The concatenated dataset was partitioned by gene for analyses and number of invariant sites and gamma shape parameters were calculated separately for each partition in RAXML. To establish the root of the cubozoan phylogeny, SSU andProc. R. Soc. B (2010)uration that may confound signal at the deeper nodes. The addition of LSU data appears to overcome signal, artificial or otherwise, from 16S and SSU data. Indeed, a combined analysis of all three genes (figure 1) leads to a phylogenetic hypothesis that is most congruent with the LSU dataset. Despite the incongruence in the place- ment of Tripedaliidae between markers, the strongest support for its placement occurs in the combined analysis, where it is recovered as sister to Carybdeidae. (b) Alatina phylogeography The final alignment of Alatina 16S sequences contained 545 sites; of the 28 variable characters, 10 were parsi- mony informative. We found 20 unique haplotypes and uncorrected pairwise distances among haplotypes did not exceed 1.84 per cent. The haplotype network (figure 2) shows that haplotypes of both A. moseri and A. mordens are not reciprocally monophyletic and appear inter-digitated. The most common mt16S haplo- type (n ? 4) was found in two specimens of each A. moseri and A. mordens. 4. DISCUSSION (a) Phylogenetic analyses and signal Since evolutionary rates differ from gene to gene, some incongruence among topologies using different gene trees is not surprising. Combined analysis of all genes should lead to a better estimate of the evolutionary relationships of taxa compared with single-gene analyses (Gadagkar et al. 2005). We find alignment quality to be much improved for the ingroup when excluding highly divergent outgroup taxa. Thus, combined analysis exclud- ing outgroup taxa (figure 1) should represent the best estimate of evolutionary relationships in Cubozoa to date. Previous phylogenetic analyses of Medusozoa (Collins 2002; Collins et al. 2006) were limited in their sampling within Cubozoa. Increased taxon sampling lends itself to begin investigating several questions concerning the evolution of cubozoan toxicity, behaviour and biogeogra- phy. Further, it becomes clear that the taxonomic framework at the family level sensu Daly et al. (2007) is inconsistent with a phylogenetic approach to taxonomy.of Carybdeida is weakly contradicted in the LSU analysis (electronic supplementary material, figure S2); several deep nodes receive lower support when an outgroup is included in LSU analyses (compare figures S2 and S4, electronic supplementary material). In general, LSU and SSU analyses do not strongly disagree with one another; contradictory relationships are weakly supported (compare figures S2?S5, electronic supplementary material). The fastest evolving marker, 16S (electronic sup- plementary material, figure S6), shows much congruence with both SSU and LSU (electronic sup- plementary material, figures S4 and S5). One point of difference involves Alatinidae and Tripedaliidae. These families group together in the 16S-based phylogeny (elec- tronic supplementary material, figure S6), as well as in SSU-based phylogeny without outgroup (albeit without support; electronic supplementary material, figure S5). This putative clade uniting Alatinidae and Tripedaliidae seems surprising, but may be explained by nucleotide sat- uration. 16S evolves much more rapidly than both LSU C auct hapl A , Aus tralia Austr , USA il il onem pula pedal arybd a bre ea ras G) 2 c M lies rib Evolution of box jellyfish B. Bentlage et al. 495 on December 29, 2009rspb.royalsocietypublishing.orgDownloaded from 64/ 53< 100 < 55/ 97 100 100 100 100 90/ 82 67/ 55 88/ 100 100 100 100 0.1 97/100 100 100 67/76 55 99/ 55 Carybdea Tamoya auct. Chironex yamaguchii Japan ?Chironex sp. Palau Chironex fleckeri Northern Territory Chironex fleckeri Queensland, Aus Chiropsella bronzie Queensland, Chiropsalmus quadrumanus North Carolina Chiropsalmus quadrumanus southern Braz Chiropsalmus quadrumanus southern Braz Tamoya cf. hapl Co Tri C Carybde Carybd Figure 1. Maximum likelihood topology (under GTR ? I ? dataset. The alignment contains 5546 characters (LSU, 329 of which 4369 are invariant and 936 parsimony informative. are indicated on each node; if only one value is given it app less than 50; dark grey, Indo-Pacific; light grey, Atlantic/Ca also occur in the Caribbean.Chiropsalmidae, Tamoyidae and Carybdeidae are prob- ably para- or polyphyletic. Consequently, we amend the diagnoses of several taxa or designate new taxa to establish monophyly (changes reflected in figures and table). How- ever, we choose to leave Chiropsalmidae unchanged, as we are missing several chirodropid genera in our analyses. (b) Toxicity The evolution of venom in Cubozoa is of significant inter- est, as many cubozoans are known to be highly toxic (e.g. Williamson et al. 1996), resulting in major costs to public health and the tourism industry, particularly in Australia (e.g. Bailey et al. 2003). Efforts have led to the character- ization of some venom components of a few cubozoan species (see Brinkman & Burnell 2009 for a review) and the development of an antivenom for the deadly cubozoan Chironex fleckeri (see Currie 2003). In order to enhance data interpretation and risk management, a historical framework providing a clear understanding of species identities and systematics is vital. In addition to retrospective interpretation of venom data, the phyloge- netic framework we present here is relevant for phylogenetic forecasting. That is, close relatives of a highly toxic species are more likely than not highly toxic as well. Toxicity varies from species to species with some being completely harmless to humans while others can cause death within minutes. The chirodropid C. fleckeri is con- sidered the most lethal jellyfish known (Wiltshire et al. 2000). Not surprisingly, its close relative Chironex yamaguchii has caused human fatalities in Japan and the Philippines (Fenner & Williamson 1996; Fenner 1997 Proc. R. Soc. B (2010)arybdea branchi South Africa Carybdeidae a b Carybdeida Tripedaliidae sens. nov. Tamoyidae sens. nov. Carukiidae fam. nov. Alatinidae Chirodropidae Chirodropida Chiropsalmidae 100 99/ 96 53/67 Carybdea auct. xaymacana Western Australia Carybdea auct. xaymacana Panama . rastonii New South Wales, Australia onema North Carolina, USA Malo kingi Queensland, Australia Carukia barnesi Queensland, Australia latina mordens = Alatina moseri Queensland, Australia ?Carybdea marsupialis Puerto Rico tralia alia Morbakka virulenta comb. nov. Japan Gerongia rifkinae Northern Territory, Australia a Netherlands Antilles sivickisi gen. nov., comb. nov. Japan/Australia ia cystophora Indonesia ea arborifera Hawai'i vipedalia Japan tonii South Australia Carybdea xaymacana Panama of the combined nuclear LSU, SSU and mitochondrial 16S haracters; SSU, 1777 characters and 16S: 486 characters), L/MP parametric bootstrap support values (1000 replicates) to both ML and MP. Less than symbol, bootstrap support bean; a, includes SE Atlantic and b, both nominal species(both as Chiropsalmus quadrigatus); Lewis & Bentlage 2009). By contrast, Chiropsalmus and Chiropsella species are considered much less dangerous (but see Bengtson et al. 1991). Differences in toxicity among chirodropids may be explained by differences in the amount of tentacle surface area, and consequently, the amount of venom that can be delivered (see Nagai 2003). Interestingly, an unvouchered tissue specimen from Palau appears to be closely related to C. yamaguchii from Japan, raising questions about the toxicity and identity of this chirodropid. In contrast to the notion that chirodropids represent the most lethal box jellyfishes, haemolytic activity of pur- ified toxin proteins appears lower in C. yamaguchii (Nagai et al. 2002; as C. quadrigatus) than in Alatina sp. (Nagai et al. 2000a; as Carybdea alata) and highest in Carybdea brevipedalia (Nagai et al. 2000b; as C. rastoni [sic]). Simi- larly, lethal doses of venom appear much lower in C. brevipedalia when compared with Alatina sp. and C. yamaguchii (Nagai 2003). Note, however, that these haemolytic assays do not appear to have been standar- dized among treatments, potentially making direct comparisons unreliable. Nonetheless, Bailey et al. (2005) also reported higher haemolytic activities in a species of Carybdea compared with two chirodropid species. However, haemolytic activity does not appear to be the lethal factor in the venoms investigated (Bailey et al. 2005), and haemolytic proteins represent only a frac- tion of the proteins present in cubozoan venom (Chung et al. 2001). Sequencing of haemolytic proteins demonstrated two carybdeid and three chirodropid protein toxins to display 496 B. Bentlage et al. Evolution of box jellyfish on December 29, 2009rspb.royalsocietypublishing.orgDownloaded from amoderate amount of divergence (Nagai 2003; Brinkman& Burnell 2009). Despite reported differences among toxin protein sequences within Cubozoa, secondary structure models suggest at least two shared structural motifs that may be related to cytolytic activity (Brinkman & Burnell 2009 and references cited therein). Thus far no homolo- gous protein outside of Cubozoa has been identified, suggesting that cubozoan venoms may contain a novel and unique family of proteins (Brinkman & Burnell 2009). Several cubozoan species are known to cause a set of symptoms called Irukandji syndrome. Initially, Irukandji syndrome was attributed to Carukia barnesi whose sting causes a sharp prickling sensation without visible injury (Barnes 1964). Systemic effects are delayed by minutes to hours and include severe low back pain, progressing to limb cramping, nausea, vomiting, headache, restless- ness and ?a feeling of impending doom? (Barnes 1964; Fenner 2006). Despite strong systemic effects Irukandji syndrome caused by C. barnesi is not considered life- threatening (Barnes 1964). Since its original description, the syndrome has been reported from, or attributed to, other cubozoans: Morbakka (Fenner et al. 1985), Tamoya (Morandini & Marques 1997), Malo (Gershwin Figure 2. Statistical parsimony network for mitochondrial 16S sequences of Alatina spp. from Hawai?i (A. moseri) and Australia (A. mordens) as calculated by TCS under the 95% connection limit criterion. Gaps were treated as a fifth character state. Lines represent one mutational step; small hollow circles correspond to inferred alleles that have not been sampled. The area of each respective solid circle reflects the number of alleles represented; the smallest solid circles represent a single allele. Grey circle, Waikiki, Honolulu, Hawai?i; black circle, Osprey Reef, Coral Sea, Australia. Proc. R. Soc. B (2010)2005a, 2007), Alatina (Yoshimoto & Yanagihara 2002 (as C. alata); Gershwin 2005b; Little et al. 2006) and Gerongia (Gershwin & Alderslade 2005). Usually Irukandji syndrome in cubozoans other than Carukia is referred to as Irukandji-like syndrome (or as Morbakka syndrome by Morbakka; Fenner et al. 1985). Irukandji-like syndrome shares the same basic symptoms of classic Irukandji syndrome, but may be less severe in some species or even more severe in others. For example, a more severe case causing a fatality off North Queens- land, Australia, was attributed to C. barnesi?s close relative Malo kingi (Fenner & Hadok 2002; Gershwin 2007; but see Bailey 2003). Thus far, very little is known about the mechanism(s) underlying Irukandji syndrome. Thorough toxicological studies of Irukandji- causing species from disparate clades should clarify the function and nature of the syndrome. While disparate clades in Cubozoa contain Irukandji- causing species, all are part of Carybdeida. Irukandji syndrome is particularly well documented for species of Tamoyidae sensu Daly et al. (2007). Interestingly, Tamoya consistently falls outside Tamoyidae (figure 1; electronic supplementary material, figures S1?S6) and is easily distinguishable from its other genera, Carukia, Malo, Gerongia and Morbakka. Hence, we amend the meaning of Tamoyidae Haeckel, 1880 to contain all those carybdeid medusae that possess frown-shaped rho- paliar niche ostia lacking rhopaliar horns (type genus Tamoya Mu?ller, 1859). We propose the new family Caru- kiidae, with type genus Carukia Southcott, 1967, to contain those carybdeids that lack gastric filaments and possess frown-shaped rhopaliar niche ostia with rhopaliar horns (genera Carukia, Malo Gershwin, 2005, Gerongia Gershwin & Alderslade, 2005 and Morbakka Gershwin, 2008). Both Tamoyidae sens. nov. and Carukiidae branch before Carybdeidae and Tripedaliidae, while Ala- tinidae represents the earliest diverging carybdeid clade (?Carybdea marsupialis may be misidentified; see below). This topology suggests that the last common ancestor of Carybdeida probably possessed the mechanism(s) under- lying Irukandji syndrome (figure 3). Further, the ability to cause Irukandji syndrome may have been lost in the line- age leading to Carybdeidae and Tripedaliidae (figure 3; a species of Carybdea was linked to Irukandji syndrome (Little et al. 2006), but this attribution appears uncon- firmed (Gershwin 2006a)). A syndrome described as Irukandji-like may be caused by a couple of non- cubozoan species (e.g. Fenner et al. 1996 (Stomolophus nomurai ); Fenner 1998 (Gonionemus and Physalia)), but homology will remain obscure until the mechanism(s) underlying the syndrome are clarified. Even with a robust phylogeny, several problems hamper cubozoan venom studies. Difficulties in extract- ing venom, the use of whole tentacle tissue instead of isolated nematocysts and contradictory results among research groups (Brinkman & Burnell 2009 and refer- ences cited therein) need to be addressed. Furthermore, toxins have been reported to differ among different body parts of specimens and possibly different ontogen- etic stages (Brinkman & Burnell 2009 and references cited therein). Finally, taxonomic uncertainties and resulting misidentification may impede toxicological research. For example, the number of bioactive proteins isolated from C. marsupialis from the Mediterranean da me ry ene Evolution of box jellyfish B. Bentlage et al. 497 on December 29, 2009rspb.royalsocietypublishing.orgDownloaded from (Rottini et al. 1995) differs from that of the same nominal species from the Caribbean (Sanchez-Rodriguez et al. 2006) leading to the interpretation of intraspecific venom variation. However, true C. marsupialis from the Mediterranean is easily distinguishable from its congeners in the Caribbean (i.e. Carybdea xaymacana and C. auct. xaymacana) by their gastric phacellae. The existence of C. marsupialis in the Caribbean is most probably an example of taxonomic confusion. (c) Courtship behaviour Our working hypothesis with increased taxon sampling supports the preliminary finding that Carybdea sivickisi is more closely related to Tripedalia cystophora than it is Carybdei courtship behaviour loss of Irukandji/ Irukandji-like syndrome (?) Irukandji/Irukandji-like syndro ovovivipa Ca ry bd ei da e Tr ip ed al iid ae Ta m o yi da e Figure 3. Trends in toxicity and life-history evolution; phylogto any species of Carybdea (Collins 2002; Collins et al. 2006). To retain monophyly of Carybdea, we designate the new genus Copula to accommodate Carybdea sivickisi Stiasny, 1926; the name is in reference to the well- documented courtship behaviour and sexual dimorphism (see below). We amend the meaning of Tripedaliidae Conant, 1897 to contain all carybdeids that display sexual dimorphism of the gonads, produce spermato- phores and in which at least the males possess subgastral sacs/seminal vesicles (see Hartwick 1991). Species of Carybdea Pe?ron & Lesuer, 1810, the sole genus within Carybdeidae Gegenbauer, 1857, can readily be differentiated from all other cubozoans by their posses- sion of heart-shaped rhopaliar niche ostia (see Gershwin 2005b for diagnoses of Carybdea and Carybdeidae). The new genus Copula is defined to contain tripedaliids that possess adhesive pads on the exumbrellar apex with which they attach themselves to substrates when resting (see Hartwick 1991); its type species is Copula sivickisi (Stiasny, 1926). Tripedaliid life histories are unique among Cubozoa and Cnidaria. In Copula sivickisi, a mature male and female engage in sexual activity by entangling their tenta- cles. While swimming as a couple, the male brings its oral opening close to that of the female and produces a Proc. R. Soc. B (2010)spermatophore that is ingested by the female (Lewis & Long 2005; Lewis et al. 2008). The subsequent gestation period spans some 2?3 days after which an embryo strand is released into the water column (Lewis & Long 2005; Lewis et al. 2008). Sexual dimorphism of medusae and similar courtship behaviour were documented by Werner (1973) for T. cystophora, but he did not observe fertiliza- tion, gestation or embryo release. However, in contrast to the production of an embryo strand, T. cystophora seems to release free-swimming planulae (Conant 1898). Species of both Carybdeidae and Alatinidae appear to be ovoviviparous and eggs are fertilized internally after female medusae have taken up sperm released into the water column by males during spawning aggregations (Studebaker 1972; Arneson 1976). Neither courtship Chirodropida unique toxin (s) reproduction mode? neritic external fertilization pelagic Ca ru ki id ae A la tin id ae Ch iro dr op id ae Ch iro ps al m id ae Ch iro ps al m id ae tic relationships follow figure 1.behaviour nor sexual dimorphism appears to be present in these two families. Further, embryos are released within minutes to hours after fertilization (Studebaker 1972; Arneson 1976). To our knowledge, reproductive strategies of both Tamoyidae sens. nov. and Carukiidae remain undocumented. In Chirodropida, Yamaguchi & Hartwick (1980) reported external fertilization for medu- sae of both C. fleckeri and Chiropsella bronzie (as C. quadrigatus). While information from Carukiidae and Tamoyidae are needed, it appears that internal fertiliza- tion is a synapomorphy of Carybdeida. Further, we suggest that sexual dimorphism evolved a single time concomitant with complex sexual behaviour (figure 3). (d) Biogeography In general, cubozoan distributions are not well documen- ted on intermediate geographical scales (e.g. provinces, states or countries) owing to a lack of sampling, which hampers biogeographic enquiries at this scale (see Bentlage et al. 2009 for a possible strategy to address this issue). However, on larger scales (e.g. ocean basins) several patterns emerge in light of our results. In particu- lar, we uncovered numerous deep divergences among Indo-Pacific and Atlantic clades (figure 1). 498 B. Bentlage et al. Evolution of box jellyfish on December 29, 2009rspb.royalsocietypublishing.orgDownloaded from In Chirodropida, the genus Chiropsalmus is exclusively Atlantic, whereas the confamilial Chiropsella is from the Indo-Pacific groups with the exclusively Indo-Pacific Chironex (family Chirodropidae). Similarly, what had been recognized as Tamoyidae (Tamoyidae sens. nov. plus Carukiidae) can also be divided geographically: Tamoyidae is restricted to the Atlantic and the described species of Carukiidae are known from Australia and Japan, but probably range throughout the Indo-Pacific (Cleland & Southcott 1965; B. Bentlage 2009, unpublished notes). The pattern within Alatinidae and Tripedaliidae is unclear owing to limited taxon sampling. Both sampled species of Tripedaliidae, Copula sivickisi and T. cystophora, can be found in all three oceans and future studies should seek to determine if these species are truly circumtropical or flocks of regional species. Our densest sampling is in Carybdea, but unfortu- nately, relationships among Indo-Pacific and Atlantic/ Caribbean taxa lack support, so it is unclear whether there are deep divergences separating lineages into exclusively Atlantic/Caribbean and Indo-Pacific clades (figure 1). Nonetheless, integrating phylogeny and taxo- nomic investigations suggests that Carybdea spp. are more restricted in their geographical distributions than has been recognized by most workers. For example, C. xaymacana has been sampled from both the Caribbean and Western Australia, but deep divergence indicates crypticism in this nominal species. Similarly, Carybdea rastonii has traditionally been viewed as having a wide dis- tribution with occurrence records from South Australia, Hawai?i and Japan among others. Our sampling shows that this is also a case of numerous species being united under the same name. Examination of the specimens suggests that C. rastonii can be distinguished morphologi- cally (Gershwin & Gibbons 2009; B. Bentlage 2009, unpublished notes). Rather than having cryptic species in the sense that they are indistinguishable morphologi- cally, this appears to be a case in which species have been proposed historically (C. rastonii Haacke, 1886 (South Australia), C. brevipedalia1 Kishinouye, 1891 (Japan) and Carybdea arborifera Maas, 1897 (Hawai?i)) but subsequently synonymized and/or disregarded. Discovering that widespread nominal Carybdea spp. represent geographically isolated species assemblages indicates that these medusae do not exchange genetic material across large bodies of open water. Hence, we suppose that speciation in the genus is largely driven by vicariance. Dispersal events, however, cannot be ruled out as a source to account for diversification. For instance, C. arborifera probably arose inHawai?i after long-range dis- persal; islands have existed at the present position of the Hawaiian Islands from at least the late Paleocene onwards, but were always remote (Carson & Clague 1995). The inability to cross open ocean habitats is most likely a widespread phenomenon in Cubozoa, as most species appear to inhabit near shore habitats above the continen- tal shelves (i.e. the neritic zone). Considering this, deep divergences between Atlantic and Indo-Pacific clades of Cubozoa may be explained by ancient vicariant events. Unfortunately fossil jellyfishes are rare, leading to uncer- tainty in dating cladogenetic events (Cartwright & Collins 2007). However, fossils that possibly represent cubozoans have been discovered from the upper JurassicProc. R. Soc. B (2010)(Quadrumedusina quadrata Haeckel, 1869), upper Car- boniferous (Anthracomedusa turnbulli Johnson & Richardson 1968) and the middle Cambrian (Cartwright et al. 2007). Given their neritic habitat, cubozoans prob- ably diversified as a result of plate movements in concert with eustatic sea-level fluctuations; the splitting of Pangea could have provided the setting for this. In contrast to the general pattern described above, we have evidence for a pelagic cubozoan. The genus Alatina is represented with several nominal species in the Pacific (see Gershwin 2005b) from which we sampled A. moseri Mayer, 1906 and A. mordens Gershwin, 2005 from or nearby their type localities (Hawai?i and the Coral Sea, respectively). We found no genetic divergences corre- sponding to geographical locality; in fact both ?species? share at least one 16S haplotype (figure 2). Additionally, no clear pattern differentiating the two populations exists. Rather, the haplotype network reflects a well-mixed popu- lation with regular gene flow. Inspection of specimens (including Hawaiian-type material USNM 22308, 22311 and 29632) and study of its original description (Mayer 1906) demonstrates that A. moseri has been present in Hawai?i at least since the beginning of the twentieth century. The initial discovery of Alatina spp. in Australia seems not as well documented as is true for many marine invertebrates from this continent. In contrast to other cubozoans, Alatina spp. live at or close to the edge of the continental shelf (Arneson&Cutress 1976; as C. alata) and have been obtained from great water depths before (e.g. Morandini 2003; as C. alata). It seems that A. moseri can only be encountered in shallow waters several days after the full moon (e.g. Thomas et al. 2001; Yanagihara et al. 2002; both as C. alata) when individuals congregate to spawn; the same is true for A. mordens (T. Carrette & J. Seymour 2008, personal communication). Furthermore, it has been suggested that individuals of Alatina spp. live up to 12 months (Arneson & Cutress 1976). Hence, it seems quite possible that Alatina spp. have an oceanic lifestyle and are able to maintain cohesive metapopulations across ocean basins. Our investigation of historic specimens demonstrates that A. moseri was present in Hawai?i more than a century ago. An early introduction of A. moseri into Hawai?i is poss- ible, but it seems unlikely that this would have occurred from the Coral Sea, given that ship traffic from Australia to Hawai?i was probably low at the time and the observed genetic signal would suggest multiple introductions rather than a single one. Considering the possible effect World War II naval traffic had on the spread of marine organisms (e.g. Coles et al. 1999), A. moseri may conversely have been introduced into the Coral Sea. Indeed, it is conceivable that A. moseri was introduced into the Coral Sea from Hawai?i and prior to that into Hawaiian waters from yet another location. However, considering the life cycle of A. moseri (and the synonymous A. mordens), we find dis- persal by natural means a more viable explanation of the pattern we observe. Investigation of additional Alatina spp. may show that some of these also represent artificial taxonomic units. (e) Carybdea marsupialis: a model organism misidentified? We recover C. marsupialis together with A. moseri as the sister group to the remaining carybdeids. This placement from a polyp culture at the museum of the University of Hamburg, Germany. To our knowledge these Rico, and considering the placement of C. marsupialis as a close relative of a member of Alatinidae, it is poss- Evolution of box jellyfish B. Bentlage et al. 499 on December 29, 2009rspb.royalsocietypublishing.orgDownloaded from of C. brevipedalia. We gratefully acknowledge J. Seymour and T. Carrette who provided many of the samples included in our analyses. Thanks to A. M. Nawrocki for collecting a specimen and helping with multi-threaded ML analyses. Two anonymous reviewers provided constructive criticisms that improved the manuscript significantly. Collections and technical staff of the National Museum of Natural History and the National Systematics Lab of NOAA?s Fisheries Service provided assistance with handling and cataloguing voucher specimens, extraction of some DNA and amplification and sequencing of some genetic markers. Funding was provided through US NSF Assembling the Tree of Life grant EF-053179 to P.C., A.G.C. and D. Fautin and a PADI Foundation grant to B.B. ENDNOTE 1The name C. brevipedalia is not in widespread usage, but its original description and type locality (Kishinouye 1891) demonstrate that it is the senior synonym of the name C. mora Kishinoye, 1910, recently used as valid in Gershwin (2006b) and Gershwin & Gibbons (2009). REFERENCES Arneson, A. C. 1976 Life history of Carybdea alata Reynaud, 1830 (Cubomedusae). M.S. thesis, University of Puerto Rico, Mayagu?ez, Commonwealth of Puerto Rico, USA. Arneson, A. C. & Cutress, C. E. 1976 Life history of Caryb- dea alata Reynaud, 1830 (Cubomedusae). In Coelenterateible that the culture in Hamburg actually contains the polyp stage of a species of Alatina rather than Carybdea. Since this particular culture has served as the stock for several important experiments on Carybdea develop- ment (e.g. Stangl et al. 2002; Fisher & Hofmann 2004; Straehler-Pohl & Jarms 2005), it is vital to con- firm the identification of the polyps by either rearing medusae to adulthood or collecting fresh material from the Caribbean for genetic comparisons. Inclusion of C. marsupialis from close to its type locality in Italy in future phylogenetic studies should also help shed light on this issue. We thank the following for assistance in the field, facilitation of museum studies, donation of specimens and/or sharing of knowledge: A. C. Arneson, the Australian Volunteer Coastguard Association?Townsville Flotilla, K. Bayha, T. Beukes, T. Coffer, Jen Collins, John Collins, M. Daly, M. N. Dawson, M. Ekins, N. M. Evans, P. J. Fenner, L. Gershwin, B. Gillan, G. C. Gray, the Great Barrier Reef Marine Park Authority, L. Gusma?o, R. Helm, J. O?Keefe, M. Kawahara, S. Keable, S. Kubota, D. Lindsay, A. E. Migotto, A. C. Morandini, P. & L. Richards, R. Satterlie, the Two Oceans Aquarium. M. Migita and the Watanuki family kindly translated the original descriptionpolyps were originally obtained by B. Werner some 40 years ago in La Paguera, Puerto Rico (A. C. Arneson 2008, personal communication) and used for life cycle studies (e.g. Werner et al. 1971; Straehler-Pohl & Jarms 2005). Alatina spp. can be found in Puertoappears surprising considering the stark morphological differences between Carybdea spp. and Alatina spp. (compare Gershwin & Gibbons 2009 with Gershwin 2005b). Specimens of C. marsupialis for this and other studies (Collins 2002; Collins et al. 2006) are derivedProc. R. Soc. B (2010)ecology and behavior (ed. G. O. Mackie), pp. 227?236. New York, NY: Plenum Press. Bailey, P. M. 2003 Fatal envenomation by jellyfish causing Irukandji syndrome. Med. J. Aust. 178, 139?140. Bailey, P. M., Little, M., Jelinek, G. A. & Wilce, J. A. 2003 Jellyfish envenoming syndromes: unknown toxic mechan- isms and unproven therapies. Med. J. Aust. 178, 34?37. Bailey, P. M., Bakker, A. J., Seymour, J. E. & Wilee, J. A. 2005 A functional comparison of the venom of three Aus- tralian jellyfish?Chironex fleckeri, Chiropsalmus sp., and Carybdea xaymacana?on cytosolic Ca2?, haemolysis and Artemia sp. lethality. Toxicon 45, 233?242. (doi:10. 1016/j.toxicon.2004.10.013) Barker, F. K. & Lutzoni, F. M. 2002 The utility of the incon- gruence length difference test. Syst. Biol. 51, 625?637. (doi:10.1080/10635150290102302) Barnes, J. H. 1964 Cause and effect in Irukandji stingings. Med. J. Aust. 1, 897?904. Bengtson, K., Nichols, M. M., Schnadig, V. & Ellis, M. D. 1991 Sudden death in a child following jellyfish enveno- mation by Chiropsalmus quadrumanus. Case report and autopsy findings. J. Am. Med. Assoc. 266, 1404?1406. (doi:10.1001/jama.266.10.1404) Bentlage, B., Peterson, A. T. & Cartwright, P. 2009 Inferring distributions of chirodropid box-jellyfishes (Cnidaria: Cubozoa) in geographic and ecological space using ecological niche modeling. Mar. Ecol. Prog. Ser. 384, 121?133. (doi:10.3354/meps08012) Brinkman, D. L. & Burnell, J. N. 2009 Biochemical and mol- ecular characterisation of cubozoan protein toxins. Toxicon 54, 1162?1173. (doi:10.1016/j.toxicon.2009.02.006) Carson, H. L. & Clague, D. A. 1995 Geology and biogeogra- phy of the Hawaiian Islands. In Hawaiian biogeography? evolution on a hot spot archipelago (eds W. L. Warren & V. A. Funk), pp. 14?29. Washington, DC: Smithsonian Institution Press. Cartwright, P. & Collins, A. G. 2007 Fossils and phylogenies: integrating multiple lines of evidence to investigate the origin of early major metazoan lineages. Integr. Comp. Biol. 47, 744?751. (doi:10.1093/icb/icm071) Cartwright, P., Halgedahl, S. L., Hendricks, J. R., Jarrard, R. D., Marques, A. C., Collins, A. G. & Lieberman, B. S. 2007 Exceptionally preserved jellyfishes from the middle Cambrian. PLoS ONE 2, e1121. (doi:10. 1371%2Fjournal.pone.0001121) Cartwright, P., Evans, N. M., Dunn, C. W., Marques, A. C., Miglietta, M. P., Schuchert, P. & Collins, A. G. 2008 Phy- logenetics of Hydroidolina (Cnidaria: Hydrozoa). J. Mar. Biol. Assoc. UK 88, 1663?1672. (doi:10.1017/ S0025315408002257) Castresana, J. 2000 Selection of conserved blocks from mul- tiple alignments for their use in phylogenetic analysis. Mol. Biol. Evol. 17, 540?552. Chung, J. L., Ratnapala, L. A., Cooke, I. M. & Yanagihara, A. A. 2001 Partial purification and characterization of a hemolysin (CAH1) from Hawaiian box jellyfish (Carybdea alata) venom. Toxicon 39, 981?990. (doi:10. 1016/S0041-0101(00)00237-3) Cleland, J. B. & Southcott, R. V. 1965 Injuries to man from marine invertebrates in the Australian region. National Health and Research Council, Special Report Series 12, 282 p., Canberra, Australia. Clement, M., Posada, D. & Crandall, K. 2000 TCS: a com- puter program to estimate gene genealogies. Mol. Ecol. 9, 1657?1660. (doi:10.1046/j.1365-294x.2000.01020.x) Coles, S. L., DeFelice, R. C., Eldredge, L. G. & Carlton, J. T. 1999 Historical and recent introductions of non- indigenous marine species into Pearl Harbor, Oahu, Hawaiian Islands. Mar. Biol. 135, 147?158. (doi:10. 1007/s002270050612) 500 B. Bentlage et al. Evolution of box jellyfish on December 29, 2009rspb.royalsocietypublishing.orgDownloaded from Collins, A. G. 2002 Phylogeny of Medusozoa and the evol- ution of cnidarian life cycles. J. Evol. Biol. 15, 418?432. (doi:10.1046/j.1420-9101.2002.00403.x) Collins, A. G., Schuchert, P., Marques, A. C., Jankowski, T., Medina, M. & Schierwater, B. 2006 Medusozoan phylo- geny and character evolution clarified by new large and small subunit rDNA data and an assessment of the utility of phylogenetic mixture models. Syst. Biol. 55, 97?115. (doi:10.1080/10635150500433615) Collins, A. G., Bentlage, B., Lindner, A., Lindsay, D., Haddock, S. H. D., Jarms, G., Norenburg, J. L., Jankowski, T. & Cartwright, P. 2008 Phylogenetics of Trachylina (Cnidaria: Hydrozoa) with new insights on the evolution of some problematical taxa. J. Mar. Biol. Assoc. UK 88, 1673?1685. (doi:10.1017/ S0025315408001732) Conant, F. S. 1898 The Cubomedusae. Mem. Biol. Lab. Johns Hopkins Univ. 4, 1?61. Cunningham, C. W. 1997 Can three incongruence tests pre- dict when data should be combined? Mol. Biol. Evol. 14, 733?740. Currie, B. J. 2003 Marine antivenoms. J. Toxicol. Clin. Toxicol. 41, 301?308. (doi:10.1081/CLT-120021115) Daly, M. et al. 2007 The phylum Cnidaria: a review of phy- logenetic patterns and diversity three hundred years after Linneaeus. Zootaxa 1668, 127?182. Dawson, M. N., Raskoff, K. A. & Jakobs, D. A. 1998 Field preservation of marine invertebrate tissue for DNA analyses. Mol. Mar. Biol. Biotechnol. 7, 145?152. Edgar, R. C. 2004 MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucl. Acids Res. 32, 1792?1797. (doi:10.1093/nar/gkh340) Farris, J. S., Ka?llersjo?, M., Kluge, A. G. & Bult, C. 1995a Constructing a significance test for incongruence. Syst. Biol. 44, 570?572. Farris, J. S., Ka?llersjo?, M., Kluge, A. G. & Bult, C. 1995b Testing significance of incongruence. Cladistics 10, 315?319. (doi:10.1111/j.1096-0031.1994.tb00181.x) Fenner, P. J. 1997 The global problem of cnidarian (jellyfish) stinging. M.D. thesis, University of London, London, UK. Fenner, P. J. 1998 Dangers in the ocean: the traveller and marine envenomation. J. Travel Med. 5, 135?141. (doi:10.1111/j.1708-8305.1998.tb00487.x) Fenner, P. J. 2006 Jellyfish responsible for Irukandji syn- drome. Q. J. Med. 99, 802?803. (doi:10.1093/qjmed/ hcl104) Fenner, P. J. & Hadok, J. C. 2002 Fatal envenomation by jel- lyfish causing Irukandji syndrome. Med. J. Aust. 177, 362?363. Fenner, P. J. & Williamson, J. A. 1996 Worldwide deaths and severe envenomation from jellyfish stings. Med. J. Aust. 165, 658. Fenner, P. J., Fitzpatrick, P. F., Hartwick, R. J. & Skinner, R. 1985 ?Morbakka?, another cubomedusan. Med. J. Aust. 143, 550?555. Fenner, P. J., Williamson, J. A. & Burnett, J. W. 1996 Clinical aspects of envenomation by marine animals (Abstract). Toxicon 34, 145 (full article available from www.marine-medic.com.au/pages/articles/pdf/parisArticle. pdf). (doi:10.1016/0041-0101(96)83656-7) Fischer, A. B. &Hofmann, D. K. 2004 Budding, budmorpho- genesis, and regeneration in Carybdea marsupialis Linnaeus, 1758 (Cnidaria: Cubozoa). In Developments in hydrobiology?coelenterate biology 2003 (eds D. G. Fautin, J. A. Westfall, P. Cartwright, M. Daly & C. R. Wyttenbach), pp. 331?337. Dordrecht, The Netherlands: Springer. Gadagkar, S. R., Rosenberg, M. S. & Kumar, S. 2005 Inferring species phylogenies from multiple genes: conca- tenated sequence tree versus consensus gene tree. J. Exp. Zool. 304B, 64?74. (doi:10.1002/jez.b.21026)Proc. R. Soc. B (2010)Gershwin, L. 2005a Two new species of jellyfishes (Cnidaria: Cubozoa: Carybdeida) from tropical Western Australia, presumed to cause Irukandji Syndrome. Zootaxa 1084, 1?30. Gershwin, L. 2005b Carybdea alata auct. and Manokia stias- nyi, reclassification to a new family with description of a new genus and two new species. Mem. Qld Mus. 51, 501?523. Gershwin, L. 2006a Jellyfish responsible for Irukandji syn- drome. Q. J. Med. 99, 801?802. (doi:10.1093/qjmed/ hcl105) Gershwin, L. 2006b Nematocysts of the Cubozoa. Zootaxa 1232, 1?57. Gershwin, L. 2007 Malo kingi: a new species of Irukandji jellyfish (Cnidaria: Cubozoa: Carybdeida), possibly lethal to humans, from Queensland, Australia. Zootaxa 1659, 55?68. Gershwin, L. & Alderslade, P. 2005 A new genus and species of box jellyfish (Cubozoa: Carybdeidae [sic]) from tropical Australian waters. Beagle, Rec. Mus. Art Galler. North. Territ. 21, 27?36. Gershwin, L. & Gibbons, M. J. 2009 Carybdea branchi, sp. nov., a new box jellyfish (Cnidaria: Cubozoa) from South Africa. Zootaxa 2088, 41?50. Hartwick, R. F. 1991 Observations on the anatomy, behav- iour, reproduction and life cycle of the cubozoan Carybdea sivickisi. Hydrobiologia 216/217, 171?179. (doi:10.1007/BF00026459) Kishinouye, K. 1891 Zwei neue Medusen von Charybdea (Ch. brevipedalia n. sp., Ch. latigenitalia n. sp.). Dobutsugaku Zasshi 3, 437?440 (in Japanese with German diagnoses). Lewis, C. & Bentlage, B. 2009 Clarifying the identity of the Japanese Habu-kurage, Chironex yamaguchii, sp. nov. (Cnidaria: Cubozoa: Chirodropida). Zootaxa 2030, 59?65. Lewis, C. & Long, T. A. F. 2005 Courtship and reproduction in Carybdea sivickisi (Cnidaria: Cubozoa). Mar. Biol. 147, 477?483. (doi:10.1007/s00227-005-1602-0) Lewis, C., Kubota, S., Migotto, A. E. & Collins, A. G. 2008 Sexually dimorphic cubomedusa Carybdea sivickisi (Cni- daria: Cubozoa) in Seto, Wakayama, Japan. Publ. Seto Mar. Biol. Lab. 40, 1?8. Little, M., Pereira, P., Carrette, T. & Seymour, J. 2006 Jelly- fish responsible for Irukandji syndrome. Q. J. Med. 99, 425?427. (doi:10.1093/qjmed/hcl057) Mayer, A. G. 1906 Medusae collected in the Hawaiian Islands by the steamer Albatross in 1902. Bull. US Fish Comm. 1903 23, 1131?1143. Morandini, A. C. 2003 Deep-Sea medusae (Cnidaria: Cubo- zoa, Hydrozoa and Scyphozoa) from the coast of Bahia (western south Atlantic, Brazil). Mitt. Hamb. Zool. Mus. Inst. 100, 3?25. Morandini, A. C. & Marques, A. C. 1997 ?Morbakka? syn- drome: first report of envenomation by Cubozoa (Cnidaria) in Brazil. In VII Congreso Latino-Americano sobre Ciencias do Mar, Resumos Expandidos, vol. 2, pp. 188?189, Oceanographic Institute of the University of Sa?o Paulo (IO-USP), Santos, Brazil. Nagai, H. 2003 Recent progress in jellyfish toxin study. J. Health Sci. 49, 337?340. (doi:10.1248/jhs.49.337) Nagai, H., Takuwa-Kuroda, K., Nakao, M., Sakamoto, B., Crow, G. L. & Nakajima, T. 2000a Isolation and charac- terization of a novel protein toxin from the Hawaiian box jellyfish (sea wasp) Carybdea alata. Biochem. Biophys. Res. Commun. 275, 589?594. (doi:10.1006/bbrc.2000.3352) Nagai, H., Takuwa-Kuroda, K., Nakao, M., Ito, E., Miyake, M., Noda, M. & Nakajima, T. 2000b Novel proteinaceous toxins from the box jellyfish (sea wasp) Carybdea rastoni. Biochem. Biophys. Res. Commun. 275, 582?588. (doi:10. 1006/bbrc.2000.3353) Nagai, H., Takuwa-Kuroda, K., Nakao, M., Oshiro, N., Iwanga, S. & Nakajima, T. 2002 A novel protein toxin from the deadly box jellyfish (sea wasp, Habu-kurage) Chiropsalmus quadrigatus. Biosci. Biotechnol. Biochem. 66, 97?102. (doi:10.1271/bbb.66.97) Nilsson, D., Gislen, L., Coates, M. M., Skogh, C. & Garm, A. 2005 Advanced optics in a jellyfish eye. Nature 435, 201?205. (doi:10.1038/nature03484) Nylander, J. A. A. 2004 MRMODELTEST v. 2. Uppsala University. Program distributed by the author. Rottini, D., Gusmani, L., Parovel, E., Avian, M. & Patriarca, P. 1995 Purification and properties of a cytolytic toxin in venom of the jellyfish Carybdea marsupialis. Toxicon 33, 315?326. (doi:10.1016/0041-0101(94)00174-7) Sanchez-Rodriguez, J., Torrense, E. & Segura-Puertas, L. 2006 Partial purification and characterization of a novel neurotoxin and three cytolysins from box jellyfish (Caryb- dea marsupialis) nematocyst venom. Arch. Toxicol. 80, 163?168. (doi:10.1007/s00204-005-0023-7) Stamatakis, A. 2006 RAxML-VI-HPC: maximum likeli- hood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22, 2688?2690. (doi:10.1093/bioinformatics/btl446) Stangl, K., Salvini-Plawen, L. V. & Holstein, T. W. 2002 Sta- ging and induction of medusa metamorphosis in Carybdea marsupialis (Cnidaria, Cubozoa). Vie Milieu 52, 131?140. Straehler-Pohl, I. & Jarms, G. 2005 Life cycle of Carybdea marsupialis Linnaeus, 1758 (Cubozoa, Carybdeidae) reveals metamorphosis to be a modified strobilation. Mar. Biol. 147, 1271?1277. (doi:10.1007/s00227-005- 0031-4) Thomas, C. S., Scott, S. A., Galanis, D. J. & Goto, R. S. 2001 Box jellyfish (Carybdea alata) in Waikiki: their influx cycle plus the analgesic effect of hot and cold packs on their stings to swimmers at the beach: a randomized, placebo-controlled, clinical trial. Hawaii Med. J. 60, 278. Werner, B. 1973 Spermatozeugmen und Paarungsverhalten bei Tripedalia cystophora (Cubomedusae). Mar. Biol. 18, 212?217. (doi:10.1007/BF00367987) Werner, B., Cutress, C. E. & Studebaker, J. P. 1971 Life cycle of Tripedalia cystophora Conant (Cubomedusae). Nature 232, 582?583. (doi:10.1038/232582a0) Williamson, J., Fenner, P., Burnett, J. & Rifkin, J. 1996 Venomous and poisonous marine animals: a medical and biological handbook. Sydney, Australia: NSW University Press. Wiltshire, C. J., Sutherland, S. K., Fenner, P. J. & Young, A. R. 2000 Optimization and preliminary characterization of venom isolated from 3 medically important jellyfish: the box (Chironex fleckeri), Irukandji (Carukia barnesi), and blubber (Catostylus mosaicus) jellyfish. Wilderness Environ. Med. 11, 241?250. Yamaguchi, M. & Hartwick, R. 1980 Early life history of the sea wasp, Chironex fleckeri (class Cubozoa). In Develop- ment and cellular biology of coelenterates (eds P. Tardent & R. Tardent), pp. 11?16. Amsterdam, The Netherlands: Elsevier/North-Holland Biomedical Press. Yanagihara, A. A., Kuroiwa, J., Oliver, L., Chung, J. & Kunkel, D. 2002 Ultrastructure of a novel eurytele nema- tocyst of Carybdea alata Reynaud (Cubozoa, Cnidaria). Cell Tissue Res. 308, 307?318. (doi:10.1007/s00441- Evolution of box jellyfish B. Bentlage et al. 501 on December 29, 2009rspb.royalsocietypublishing.orgDownloaded from Carybdea marsupialis. M.S. thesis, University of Puerto Rico, Mayagu?ez, Commonwealth of Puerto Rico, USA. Swofford, D. L. 2003 PAUP*. Phylogenetic analysis using parsimony (*and other methods), version 4. Sunderland, MA: Sinauer Associates.Proc. R. Soc. B (2010)Yoshimoto, C. M. & Yanagihara, A. A. 2002 Cnidarian (coelenterate) envenomations in Hawai?i improve following heat application. Trans. R. Soc. Trop. Med. Hyg. 96, 300?303. (doi:10.1016/S0035-9203(02) 90105-7)Studebaker, J. P. 1972 Development of the cubomedusa, 002-0545-8)