Invertebrate Biology 123(1): 23–42. ᭧ 2004 American Microscopical Society, Inc.

Cladistic analysis of Medusozoa and cnidarian evolution

Antonio C. Marques1,a and Allen G. Collins2

1 Departamento de Zoologia, Instituto de Biocieˆncias, Universidade de Sa˜o Paulo. C.P. 11461, 05422-970, Sa˜o Paulo, SP, Brazil 2 ITZ, Ecology & Evolution, Tiera¨rtzliche Hochschule Hannover, Bu¨nteweg 17d, 30559 Hannover, Germany

Abstract. A cladistic analysis of 87 morphological and life history characters of medusozoan cnidarians, rooted with Anthozoa, results in the phylogenetic hypothesis (Anthozoa (Hydrozoa (Scyphozoa (Staurozoa, Cubozoa)))). Staurozoa is a new class of Cnidaria consisting of Stau- romedusae and the fossil group Conulatae. Scyphozoa is redefined as including those medu- sozoans characterized by strobilation and ephyrae (Coronatae, Semaeostomeae, and Rhizosto- meae). Within Hydrozoa, Limnomedusae is identified as either the earliest diverging hydrozoan lineage or as the basal group of either Trachylina (Actinulida (Trachymedusae (Narcomedusae, Laingiomedusae))) or Hydroidolina (Leptothecata (Siphonophorae, Anthoathecata)). Cladistic results are highly congruent with recently published phylogenetic analyses based on 18S mo- lecular characters. We propose a phylogenetic classification of Medusozoa that is consistent with phylogenetic hypotheses based on our cladistic results, as well as those derived from 18S analyses. Optimization of the characters presented in this analysis are used to discuss evolu- tionary scenarios. The ancestral cnidarian probably had a sessile biradial polyp as an adult form. The medusa is inferred to be a synapomorphy of Medusozoa. However, the ancestral process (metamorphosis of the apical region of the polyp or lateral budding involving an en- tocodon) could not be inferred unequivocally. Similarly, character states for sense organs and nervous systems could not be inferred for the ancestral medusoid of Medusozoa.

Additional key words: Staurozoa, Scyphozoa, Hydrozoa, Cubozoa

Cnidarian species are often used as model organisms ships among its component groups are known with in evolutionary studies (of development, cell biology, precision (Marques 1996; Nielsen et al. 1996; Jenner gene families, etc.) because Cnidaria diverged rela- & Schram 1999; Nielsen 2001; Collins 2002). tively early in the history of Metazoa. The presump- Prior studies focusing on relationships among cni- tion in these studies is that cnidarian species may ex- darian classes contain many contradictions (Salvini- hibit relatively underived character states that can be Plawen 1978, 1987; Petersen 1990; Bridge et al. 1992; compared with those of other animals, particularly bi- Bridge et al. 1995; Schuchert 1993; Odorico & Miller laterians. As an example, a recent study made infer- 1997; Marques 2001; Nielsen 2001; Collins 2002). ences about the ancestral paired domain (a conserved However, in the last decade Anthozoa has emerged as DNA-binding domain present in developmental con- the most likely sister group of the remaining cnidari- trol genes) in animals based on the condition of Pax ans, first dubbed Tesserazoa (Salvini-Plawen 1978), genes in a hydrozoan cnidarian (Sun et al. 2001). but more commonly known as Medusozoa (Petersen Though character states in cnidarians are possibly an- 1979). Monophyly of the medusozoans is particularly cient, features under study in a given cnidarian species well supported by their shared possession of linear may certainly be derived at some level within Cnidaria mtDNA, in contrast to anthozoans and other metazo- and to assume the opposite could be misleading. Thus, ans (Bridge et al. 1992). To move toward a consensus the utility of comparative studies is enhanced as more view of cnidarian phylogeny that will enlighten further taxa are investigated (Lowe et al. 2002) and as knowl- comparative studies using cnidarians, we present a cla- edge of phylogeny increases. In the case of Cnidaria, distic analysis of 87 morphological and life history neither its position within Metazoa nor the relation- characters to generate a hypothesis of relationships among medusozoan cnidarians. We compare the cla- distic results with those of recent phylogenetic analy- a Author for correspondence. E-mail: [email protected] ses of Medusozoa using molecular characters (Collins 24 Marques & Collins

2002), and present a classification that is consistent not available was coded as a question mark (?) and with phylogenetic hypotheses derived from both anal- non-comparable structures were coded in the matrix as yses. Finally, we make inferences about the evolution N. Polymorphic characters were treated as uncertain- of morphological and life history traits within Cnidaria ties. based on our cladistic analysis. Cladistic analyses were carried out using the branch and bound algorithm of the software PAUP* 4.0 Methods (Swofford 2001). First, we searched for most parsi- monious trees without weighting any of the characters. In our cladistic analysis, we used the class Anthozoa Strict and semi-strict consensus trees were calculated as an outgroup, a position supported by previous mor- from the trees obtained in these primary analyses. phological and molecular studies. As terminal taxa, we Then, a complementary analysis was performed using adopted the class Cubozoa (Werner 1975; Franc successive approximation weighting (Farris 1969, see 1994b), the fossil group Conulatae (Wade 1994), the also Carpenter 1988, 1994) by maximum values of re- 4 orders of Scyphozoa (after Franc 1994a), and the 8 scaled consistency indices (RC). Bootstrap indices subclasses of Hydrozoa (following Bouillon 1994a,b were calculated for 500 replicate searches using both with 2 exceptions). Higher-level hydrozoan taxonomy weighted and unweighted characters, and Bremer sup- has suffered from the legacy of early specialists of port indices were calculated using unweighted char- hydroids and hydromedusae, who developed indepen- acters. dent naming schemes. To be inclusive, we use the names Anthoathecata and Leptothecata (Cornelius Results 1992) rather than relicts of older classifications, such as Anthomedusae, Athecata, Leptomedusae, and The- The unweighted analysis of the data matrix (Appen- cata. As in any parsimony analysis, all terminals are dix 2) yielded 48 trees (14 trees using ‘‘amb-’’ option considered to be monophyletic. Although hydrocorals of PAUP; Lϭ126; CIϭ0.730; RIϭ0.728), for which and velellids are sometimes classified as higher taxo- the strict and semistrict consensus trees have the same nomic groups of Hydrozoa (e.g., Milleporina, Stylas- highly polytomic topology, including only 3 mono- terina, Chondrophora, etc.), these groups are well es- phyletic groups: (1) (Coronatae (Semaeostomeae, Rhi- tablished as part of Anthoathecata, based on their zostomeae)); (2) (Siphonophorae, Anthoathecata); and morphology (e.g., Bouillon 1985; Schuchert 1996) and (3) (Actinulida, Trachymedusae (Narcomedusae, Lain- on molecular evidence (Collins 2002). Hydridae and giomedusae)). The successive weighting analysis re- Otohydra are difficult hydrozoan groups and were con- sulted in 3 fully resolved trees (3 using ‘‘amb-’’; sidered here to be part of Anthoathecata and Actinu- Lϭ76.65; CIϭ0.928; RIϭ0.925), in which Limnome- lida respectively. Neither of these inclusions impact dusae appears in 3 different positions: (1) sister-group our scoring of Anthoathecata and Actinulida. That of all other hydrozoans; (2) sister-group of (Actinulida said, if future work indicates that any of our terminal (Trachymedusae (Narcomedusae, Laingiomedusae))); taxa are not monophyletic, our analyses should be and (3) sister group of (Leptothecata (Siphonophorae, emended accordingly. Anthoathecata)). The phylogenetic hypothesis (strict For each taxon, we scored 87 characters, of which consensus) generated by this cladistic analysis (Fig. 1) 48 were informative (Appendices 1 and 2). Non- suggests that Hydrozoa is the monophyletic sister informative characters either have incomplete docu- group of all other medusozoans. Within Hydrozoa, mentation or were judged to be shared by all terminals. Limnomedusae has an unstable position, whereas the We hope that including these characters here will in- other hydrozoan groups form 2 clades (Actinulida spire further studies. Scoring characters for supraspe- (Trachymedusae (Narcomedusae, Laingiomedusae))) cific taxa requires some generalizations unless one in- and (Leptothecata (Siphonophorae, Anthoathecata)). cludes only characters that are constant for all species Non-hydrozoan medusozoans are also monophyletic, in higher taxa or relies on a phylogenetic hypothesis but Scyphozoa, as traditionally circumscribed is not. of the group that permits optimization of all ancestral Instead, 3 scyphozoan groups form a clade (Coronatae states (Whiting et al. 1997). In general, the character (Rhizostomeae, Semaeostomeae)) that is the sister states scored were represented uniformly in each of the group to the clade ((Stauromedusae, Conulatae) Cu- terminals (see Appendix 1 for exceptions and discus- bozoa). sion). Because of the taxonomic breadth of this study, This cladogram (Fig. 1) represents hypotheses that characters and their states were taken mostly from the are both consistent with and contradictory to various literature. Characters were coded as binary or multi- views of cnidarian relationships that have been pro- state and considered unordered. Information that was posed in the past. Conulatae, which is comprised of Cladistic analysis of Medusozoa 25

Fig. 1. Strict consensus of 3 most parsimonious trees (Lϭ76.65; CIϭ.928; RIϭ.925) resulting from a successive weighting analysis. Apomorphic character states (character number and state above and below nodes respectively) are mapped onto the tree at appropriate nodes, characters optimized using Accelerated Transformation. Homoplastic characters are indicated with open squares.

the fossil genus Conchopeltis and the better known than either is to Coronatae and that Scyphozoa as tra- conulariids, is an extinct group of animals with conical ditionally circumscribed is not monophyletic (Fig. 1). exoskeletons that are square in cross section. They Since the elevation of Cubozoa to class status, cu- have often been thought of as early diverging members bozoans have often been envisioned as the sister group of Cnidaria (cf. Werner 1973a; Salvini-Plawen 1978; of either Hydrozoa (Werner 1973b; Bouillon 1981; Bouillon 1981; Nielsen 2001). Specifically, Conulatae Cornelius 1991; Nielsen 2001) or Scyphozoa (Salvini- has been compared to Coronatae because the apatitic Plawen 1978, 1987; Schuchert 1993). However, the tests of conulates and the chitinous thecae of coronate authors of some earlier analyses concluded, as have scyphozoan polyps are similarly composed of 2 layers, we, that stauromedusans and cubozoans share a rela- a thin outer layer and a relatively thicker inner layer tively close relationship (Haeckel 1879; Uchida 1929; (Werner 1966). Moreover, structures on the midlines Thiel 1966). Our morphological results are very con- and corners of conulates are reminiscent of thorn-like sistent with those based on molecular data, which like- wise suggest that scyphozoans may not be monophy- projections at the interradii and perradii of coronate letic, as stauromedusans branch closer to other polyps (Werner 1966; Van Iten et al. 1996). On the medusozoans (Collins 2002). As for the other scypho- other hand, the mineralized septa of the conulate zoan groups (Coronatae, Semaeostomeae, and Rhizos- Eoconularia loculata exhibit an ontogeny inferred to tomeae), all published hypotheses (to our knowledge) be nearly identical to that observed in developing stau- suggest that the latter 2 groups are more closely related romedusans (Kiderlen 1937; Jerre 1994; Wade 1994). to each other than either is to Coronatae, as derived The only cladistic analysis addressing the question of here. Indeed, both molecular data (Collins 2002) and conulate affinities treated Scyphozoa as a single evo- morphology (Mayer 1910; Uchida 1926; Thiel 1966) lutionary entity and concluded that Conulatae was the have suggested that rhizostomes are derived from sister group to Scyphozoa (Van Iten et al. 1996). Our within semaeostomes, a hypothesis that is not tested analysis supports the hypothesis that Conulatae and by our cladistic analysis because we treat Semaeosto- Stauromedusae are more closely related to each other meae as a terminal taxon. 26 Marques & Collins

ture is revealed as homoplastic in Trachymedusae. The lack of identified autapomorphies for Limnomedusae may indicate that the group is polyphyletic, and in fact, there has been a history of various limnomedusan groups being taxonomically linked to either Trachy- medusae or Anthoathecata. Future analysis, with the subgroups of Limnomedusae treated as terminals, may result in splitting of the group. The remaining hydrozoan groups (Actinulida, Tra- chymedusae, Narcomedusae, and Laingiomedusae) form a clade. Molecular data have not been sampled for Actinulida and Laingiomedusae, but 18S data strongly support the hypothesis that Trachymedusae and Narcomedusae are closely allied (Collins 2002). Thus, our cladistic analysis and molecular data are consistent with Bouillon & Boero (2000), who con- cluded that Actinulida, Trachymedusae, and Narco- medusae are relatively closely related. However, our cladistic analysis and molecular data contradict the Fig. 2. Congruence of our cladistic-based hypotheses (left) placement of Limnomedusae and Laingiomedusae in and hypotheses based on 18S sequence data (right, strict a clade with Leptothecata, Siphonophorae, and An- consensus of most parsimonious and maximum likelihood thoathecata (Bouillon & Boero 2000). Laingiomedusae trees from Collins (2002)). The marker on Conulatae denotes is a particularly problematic group because informa- that the group is extinct. The 3 support indices shown at the tion is lacking on the polyp stage, reproduction, and nodes in our hypothesis are: bootstrap indices using weight- ed and unweighted characters, and Bremer support indices 18S rDNA. As these data become available, they may using unweighted characters. Ͻ indicates a bootstrap index clarify the phylogenetic position of Laingiomedusae that is less than 50. within Hydrozoa. Discussion Within Hydrozoa, our analysis suggests that Hy- Phylogenetic classification droidolina (Leptothecata, Siphonophorae, and An- thoathecata) is a clade, a result corroborated by 18S Ranks may convey important information in the data (Collins 2000, 2002) and by another hypothesis context of a classification based on phylogenetic hy- based on morphological characters (Bouillon & Boero potheses. Designating a new class within a phylum is 2000). However, we did not find the clade Hydroido- a statement that the phylum is more diverse at a fun- medusae in its original sense as comprising all non- damental level than previously appreciated because it siphonophore hydrozoans (Bouillon et al. 1992; and contains an additional group that is historically dis- see Marques 2001) or in its emended form as a clade crete, and correspondingly distinct in its biology, from containing Limnomedusae, Laingiomedusae, Lepto- the other classes that comprise the phylum. It goes thecata, Siphonophorae, and Anthoathecata (Bouillon without saying that the arbitrary and subjective nature & Boero 2000). Because Anthoathecata is a terminal of ranks must also be appreciated. The phylum Cni- taxon, the present analysis does not test the hypothesis daria is generally considered to comprise 4 classes: that Siphonophorae is a subgroup of Anthoathecata Anthozoa, Scyphozoa, Cubozoa, and Hydrozoa. (Schuchert 1996; Marques 2001). Despite the fact that many cnidarian morphological This analysis identifies Limnomedusae as the most characters are quite simple (see Appendix 2) and po- basal group of Hydrozoa, Trachylina, or Hydroidolina. tentially prone to homoplasy, our cladistic analysis has 18S data provided relatively strong support for 1 of yielded phylogenetic hypotheses for Medusozoa that these hypotheses, namely that Limnomedusae (though are quite similar to those based on 18S data (Fig. 2). only 2 species were sampled) is the basal group of a Both data sets support the monophyly of Hydrozoa clade also containing Trachymedusae and Narcome- and agree in suggesting that Scyphozoa (when defined dusae (Collins 2000, 2002). Our analysis identifies just as including Stauromedusae, Coronatae, Semaeosto- a single synapomorphy of Limnomedusae, the pres- meae, and Rhizostomeae, but not Cubozoa) may not ence of an urticant ring, a continuous ring of cnidae be monophyletic. In particular Stauromedusae may be on the umbrellar margin of the medusae, and this fea- more closely related to other medusozoans than to Co- Cladistic analysis of Medusozoa 27

Table 1. Classification of Cnidaria that is consistent with envisioned by Hyman (1940) and Hand (1959). phylogenetic hypotheses based on the cladistic analysis pre- Schuchert (1993) also assumed that the ancestral cni- sented here and analyses of 18S sequence data (Fig. 2.). As darian possessed a pelagic medusoid stage, arguing noted in the text, Limnomedusae may be polyphyletic and that the loss of such a stage in the lineage leading to it is possible (and perhaps likely) that some limnomedusans Anthozoa was a more likely evolutionary event than belong in Trachylina (as indicated by 18S data) whereas oth- was the origin of a complex medusa. On the other ers belong in Hydroidolina. hand, there has also been a long history of numerous Phylum Cnidaria supporters of the alternative, namely that Cnidaria had Class Anthozoa a polypoid ancestor (Haeckel 1879; Brooks 1886; Subphylum Medusozoa Hadzi 1953; Werner 1973a, 1975; Salvini-Plawen Class Staurozoa nov. 1978, 1987), though details of this hypothetical ances- Order Stauromedusae tor are at odds. In particular, a biradial sexual polyp Order Conulatae (extinct) without periderm was considered ancestral by Salvini- Class Cubozoa Plawen (1978, 1987), whereas others (Werner 1973a; Class Scyphozoa Bouillon 1981) viewed the ancestral polyp as tetram- Order Coronatae erous and surrounded by a peridermal tube. Subclass Discomedusae In our analysis, we adopted Anthozoa as an out- Order Semaeostomeae Order Rhizostomeae group in order to root the tree and to produce a work- Class Hydrozoa ing hypothesis for Medusozoa. Simply choosing an Order Limnomedusae outgroup for rooting purposes would not normally al- Subclass Trachylina low for a formal optimization of characters in a cla- Order Actinulida distic analysis. However, a clear consensus view has Order Trachymedusae emerged that Anthozoa is the sister group of Medu- Order Narcomedusae sozoa (Werner 1973a; Salvini-Plawen 1978, 1987; Order Laingiomedusae Bridge et al. 1992; Bridge et al. 1995; Schuchert 1993; Subclass Hydroidolina Kim et al. 1999; Medina et al. 2001; Collins 2002). Order Leptothecata Therefore, character optimization is appropriate. Order Siphonophorae Throughout the remaining discussion when referring Order Anthoathecata to characters and states listed in Appendices 1 and 2, we use the following abbreviations: (ch#, st#). We conclude that our cladistic hypothesis is most ronatae, Semaeostomeae, and Rhizostomeae. In the consistent with the inference that the adult cnidarian present analysis, monophyly of Cubozoa and Antho- ancestor was a sessile animal (ch17, st0) with a polyp zoa is assumed, hypotheses that are supported by mo- form (ch36, st1). Further, if the hypothesis generated lecular studies (Kim et al. 1999; Collins 2002). An by our analysis is correct, then the ancestral polyp advantage of morphological analyses over molecular probably possessed complete septa (ch43, st1or2). studies is that fossil groups can be sampled as well. Whether or not this polyp possessed periderm (ch39) Here, for instance, we find that the fossil conulates are or gastrodermal musculature (ch48) is equivocal. The probably more closely related to the benthic stauro- probable symmetry of the ancestral cnidarian is bira- medusans than they are to other cnidarian groups (cf. dial (ch18, st2), as previously concluded by Salvini- also Wade 1994). In light of the congruence of 18S- Plawen (1978, 1987). This result reinforces the con- and morphology-based hypotheses, we present a clas- clusion drawn from a recent study demonstrating that sification for Cnidaria that includes the recognition of anthozoan cnidarians and bilaterians share molecular a new class, Staurozoa (represented by the sessile stau- mechanisms responsible for body axis formation and romedusans and fossil conulates) and re-circumscribe that these were most likely inherited from a common Scyphozoa as those groups that are characterized by biradial ancestor (Hayward et al. 2002). strobilation and distinctive juvenile medusae known as If the ancestral cnidarian was polypoid as an adult, ephyrae (Table 1). then the addition of a medusa stage (ch49, st1) to the life cycle very likely happened in the lineage leading Character evolution in Cnidaria to Medusozoa. This suggests that a medusa is a syn- Whether the ancestor of cnidarians was a polyp or apomorphy of Medusozoa. However, distinct varia- a medusa (or even an actinula or a planula) has been tions in how medusae develop in the various medu- debated for over a century (Brooks 1886) and is still sozoan subgroups raise the possibility that not all under dispute. A medusoid ancestor of Cnidaria was medusozoan groups share their respective medusae 28 Marques & Collins due to common descent from a medusa-bearing ances- been thought of as complete, resulting in a single tor (Salvini-Plawen 1987). Given our cladistic hypoth- planktonic medusoid adult, though in Tripedalia cys- esis, the ancestral process of medusa development tophora the basal portion of the polyp is sometimes (ch20) in Medusozoa is unclear. In one clade, ((Stau- (only 2%) retained and capable of re-growth and the rozoa, Cubozoa) Scyphozoa), the ancestor appears to production of a subsequent medusa (Werner 1973a). In have formed medusae by metamorphosis of the apical a recent study of the cubozoan Carybdea marsupialis, (ϭ oral) portion of the sessile polyp (ch20, st1). In Stangl et al. (2002) reported striking similarity be- contrast, the ancestral hydrozoan probably produced tween this cubozoan’s metamorphosis and what is ob- medusae through development of lateral buds origi- served in Stauromedusae. In both cases, metamorpho- nating from groups of undifferentiated cells (entoco- sis and the development of adult characteristics are dons) located between ectoderm and endoderm (ch20, largely restricted to the oral end of the polyp (Stangl st0). Although these 2 types of medusa production et al. 2002). A primary difference is that staurome- could have evolved independently, the overall similar- dusans remain sessile and retain the aboral portion of ity in medusa morphology suggests that it is more like- the polyp, whereas these cubomedusans liberate them- ly that one process evolved from the other. At the same selves from the substrate, often with the aboral portion time, the 2 processes are rather different, and it is dif- of the polyp still attached to the apex of the medusa ficult to imagine how one might have evolved from before it is fully resorbed (Stangl et al. 2002). the other. Our cladistic hypothesis also yields equivocal infer- That said, hydrozoan medusa production seems to ences about the ancestral sense organs (ch51, ch69) of be somewhat more complicated and we offer a spec- medusae. The medusoid phase of the ancestor of Stau- ulative scenario describing how hydrozoan-type me- rozoa, Cubozoa, and Scyphozoa probably possessed dusa production may have been derived from apical structures (rhopalioids) that were ontogenetically metamorphosis. One can imagine an early medusozoan transformed into sense organs when tentacles of the lineage with a colonial polyp stage, in which new pol- polyp were resorbed (ch51, st1). These structures ap- yps are laterally budded from existing polyps. If apical parently are secondarily simplified (ch52, st0) in stau- production of medusae subsequently became limited rozoans, perhaps because of their sessile habit. In the to polyps specialized for this process, still later evo- lineage leading to cubozoans, sense organs have in- lution could have resulted in the complete reduction stead become more complex, involving eyes with com- of these specialized polyps. The resulting medusa de- pound lenses (ch52, st2), possibly associated with the velopment would be localized to the lateral portions evolution of intricate behaviors for which some of of polyps, as seen in many hydrozoans. It remains to these species are known. Hydrozoans possess onto- be seen if future studies of the molecular basis of me- genetically very different organs that sense equilibri- dusa development can determine whether medusae and um and/or light (ch69), suggesting that these organs the processes that produce them are homologous in evolved independently of those in staurozoans, cubo- diverse medusozoan groups. zoans, and scyphozoans. Differences in the nerve ring Even within the 2 primary medusozoan clades, life (ch53) between hydrozoans and other medusozoans cycles are dramatically variable. Within Hydrozoa, the may be tied to differences in sensory structures. In polyp stage has apparently been lost in Trachylina many hydrozoans, the nerve ring is most closely as- (ch20, st2), whereas in Hydroidolina, complex colo- sociated with structures involved in swimming (e.g., nies with polymorphism have arisen (ch37, st1) and the velum), whereas in other medusozoans the nervous many hydroidolinans exhibit strong reduction of the system is most intimately connected to the sense or- medusa, including its complete disappearance in Hy- gans (rhopalia). dridae. In Scyphozoa, medusae are produced by stro- Our cladistic hypothesis suggests that the primitive bilation (ch21, st0), when the apical part of the polyp cnidae for cnidarians were probably isorhizas (ch13, transforms (e.g., tentacles are resorbed) concomitant st1), the most morphologically simple nematocysts, with constriction, ultimately resulting in the liberation which are distributed throughout the cnidarian classes. of ephyrae by transverse fission. Evidently, polydisk This stands in accordance with the conclusion of strobilation (ch22, st0) preceded monodisk strobilation Bozhenova et al. (1988), but in contrast to the view (ch22, st1), which is known only in rhizostomes. In that haploneme nematocysts are ancestral for Cnidaria Stauromedusae, the apical transformation takes place (Salvini-Plawen 1978, 1987). Other nematocyst types, without transverse fission (ch21, st1), resulting in a such as microbasic euryteles (ch8) and mastigophores sessile polyp-like adult (Kikinger & Salvini-Plawen (ch9; probably the most primitive nematocyst type 1995). We infer a similar process for Conulatae. In with a shaft in medusozoans) and heterotrichous ani- cubozoans, metamorphosis of the polyp has typically sorhizas (ch14; displaying slight differentiation of the Cladistic analysis of Medusozoa 29 tubule), are probably derived from that primitive cni- than a hydrozoan species because Anthozoa is basal is dome. Nevertheless, nematocyst types may have un- nonsensical. After all, hydrozoan species are part of dergone some parallel evolution, which might explain Medusozoa, which is just as basal within Cnidaria as why nematocyst types correlate well with diet among is Anthozoa. In our view, any cnidarian species is ap- pelagic hydrozoans (Purcell & Mills 1988). That said, propriate for these types of studies so long as both a it is reasonable to suspect that the distribution of cni- hypothesized position for the species within Cnidaria domes across Cnidaria has been strongly influenced by and the relevant character states of other cnidarians are phylogenetic history (Bozhenova et al. 1988). Detailed considered. As additional data for diverse cnidarians investigation of medusozoan nematocysts, focusing on are accumulated, a more complete understanding of those groups which exhibit polymorphism, will help the evolution of features of interest will be achieved. in illuminating the extent of convergent evolution of nematocyst types in Medusozoa. Acknowledgments. This study is derived from part of the When interpreting the results of evolutionary studies Ph.D. dissertation of ACM (University of Sa˜o Paulo, Brazil, that use cnidarians as model organisms, the various October 1993 to June 1997). ACM had help and advice from many people, to whom he is sincerely grateful: D. Amorim, medusozoan groups should be appreciated as being de- P. Gnaspini, A. Migotto, A. Morandini, M. de Pinna, E. rived in many respects (Fig. 1). For instance, it was Schlenz, F. Silveira, and S. Vanin (Universidade de Sa˜o Pau- recently proposed that hydrozoans possess a third tis- lo, Brazil); N. Bernardi and M. Simo˜es (Universidade Es- sue layer that is homologous with the mesoderm of tadual Paulista—Campus Botucatu, Brazil); J. Bouillon bilaterians (Boero et al. 1998). In hydrozoans produc- (Universite´ Libre de Bruxelles, Belgium); D. Bridge, A. Eck- ing a medusa via lateral budding, this third tissue lay- ert (USA); S. Cairns (Smithsonian Institution, USA); D. er—the entocodon—is ephemerally located between Calder (Royal Ontario Museum, Canada); C. Castro and D. ectoderm and endoderm and becomes the striated mus- Pires (Museu Nacional do Rio de Janeiro, Brazil); M. Chris- cle layer lining the subumbrella when the medusa is toffersen (Universidade Federal da Paraı´ba, Brazil); D. Mey- liberated. Based on the relative phylogenetic positions er (University of California, Berkeley, USA); P. Cornelius of Hydrozoa within Cnidaria and Cnidaria within (The Natural History Museum, UK); H. Ferrarezzi (Instituto Butantan; Brazil); †K. den Hartog and W. Vervoort (Natio- Metazoa, the hypothesis that this hydrozoan layer is naal Natuurhistorisch Museum, The Netherlands); D. Miller homologous to the mesoderm of Bilateria would imply (James Cook University, Australia); A. L. Pen˜a Cantero independent losses of mesoderm in numerous lineages, (Universidad de Valencia, Spain); L. von Salvini-Plawen which seems unlikely (Collins 2002). On the other (Universita¨t Wien, Austria); F. Schram (University of Am- hand, recent studies have shown strong similarities be- sterdam, The Netherlands); P. Schuchert (Museum d’Histoire tween expression patterns of genes involved with the Naturelle, Switzerland). A. Migotto, V. Pearse, and referees development of mesoderm and muscles in bilaterians of this paper are gratefully acknowledged for their many and expression patterns of similar genes in a hydro- valuable criticisms, but any mistakes herein are entirely our zoan medusa (Spring et al. 2000, 2002). That similar responsibility. Financial support was provided by the Fun- genes would be involved in the specification of muscle dac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FA- systems in both cnidarians and bilaterians is of great PESP grants 1993/3578-8, 1995/2969-9, 1996/10544-0, 1997/04572-4, 1998/11072-0, 2001/02626-7) and Conselho interest, but not all that surprising because muscles Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico were presumably present in the common ancestor of (CNPq grant 300271/2001-8). AGC has been supported by cnidarians and bilaterians. In the absence of evidence the University of California Marine Council and the Deut- for an entocodon in other cnidarians, this structure sche Forschungsgemeinschaft grant Schi277/10 awarded to probably does not play a central role in understanding B. Schierwater. the origin of bilaterian mesoderm. 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We ogy and Systematics of Colonial Organisms. Larwood G coded gap junction plaques as absent in Cubozoa & Rosen BR, eds., pp. 81–103. Academic Press, London. because their electrophysiological responses are 1984. Stamm Cnidaria, Nesseltiere. In: Lehrbuch der inconsistent with the presence of these junctions speziellen Zoologie. Band I: Wirbellose Tiere, 2 teil: Cap. (Mackie et al. 1984). Germain & Anctil (1996) 4. Cnidaria, Ctenophora, Mesozoa, Plathelminthes, Nem- documented intercellular coupling involving con- ertini, Entoprocta, Nemathelminthes, Priapulida. Kaestner nexin-like proteins at very tiny zones in an antho- VA, ed., pp. 1–305. G. Fischer Verlag, Stuttgart. Werner B, Chapman DM, & Cutress CE 1971. Life cycle of zoan, hinting that gap junctions may be very small Tripedalia cystophora Conant (Cubomedusae). Nature in cnidarians other than hydrozoans. Similar stud- 232: 582–583. ies of other cnidarians could be most fruitful. 1976. Muscular and nervous systems of the cubo- 4. Mesoglea (0—non-cellular; 1—cellular). Cells are polyp (Cnidaria). Experientia 32: 1047–1049. found in the mesoglea of anthozoans (Fautin & Whiting MF, Carpenter JM, Wheeler QD, & Wheeler WC Mariscal 1991), as well as in many coronates, se- 1997. The Strepsiptera problem: phylogeny of the holo- maeostomes, and rhizostomes (Hyman 1940; D. metabolous insect orders inferred from 18S and 28S ri- Chapman 1966; G. Chapman 1966). Although the 34 Marques & Collins

character appears to be somewhat variable, e.g., 1968), which are very similar to desmonemes, the semaeostome genus Cyanea contains species were reported in a subsequent investigation of the either with or without cellular mesoglea, we have same species, and the majority of actinulid species scored cellular mesoglea as present in Semaeos- investigated do not possess desmonemes (Bouillon tomeae and Rhizostomeae. In stauromedusans, cu- 1985). bozoans, and hydrozoans, mesoglea is non-cellular 10. Mastigophores (0—absent; 1—present). Absent in (Clark 1878; Conant 1898; D. Chapman 1966; G. Stauromedusae, Coronatae, Rhizostomeae, and Chapman 1966; Werner 1973a, 1979; Thomas & Semaeostomeae (Lesh-Laurie & Suchy 1991; Edwards 1991). Schuchert 1993), but present in Cubozoa (Lesh- 5. Collagen structure (0—homotrimerous; 1—heter- Laurie & Suchy 1991; Franc 1994b), Anthozoa otrimerous). Collagen of Stomolophus (Rhizosto- (Fautin & Mariscal 1991), Laingiomedusae meae) is heterotrimerous, resembling that of ver- (Bouillon 1985), Leptothecata, Siphonophorae, tebrates, and is the only report of this type of and Anthoathecata. In Actinulida, Bouillon (1985) collagen for invertebrates (Miura & Kimura 1985; reported microbasic mastigophores in 2 species, Lesh-Laurie & Suchy 1991). Collagen of Antho- quoting Clausen (1967) and Swedmark & Teissier zoa is homotrimerous (Lesh-Laurie & Suchy (1967), but the original records of those authors 1991); we found no information on collagen struc- appear to us to be euryteles, not mastigophores. ture for other cnidarians. Rare in Limnomedusae (Bouillon 1985), and scored as polymorphic. Cnidome 11. Basitrichous isorhizas (0—absent; 1—present). Present in Anthozoa (Picken & Skaer 1966; Fautin 6. Cnidae (0—absent; 1—present). & Mariscal 1991), but absent from Cubozoa, Co- 7. Stenoteles (0—absent; 1—present). Present in Cu- ronatae, Semaeostomeae, and Rhizostomeae bozoa (Werner 1984; Hartwick 1991) and hydro- (Lesh-Laurie & Suchy 1991; Schuchert 1993). zoans generally (Schuchert 1993). Rare in Filifera Also scored as absent from Stauromedusae, (Anthoathecata), but present in capitate anthoath- though 1 species is an exception (Hirano 1986). ecatans, Siphonophorae (Carre´&Carre´ 1994), Among hydrozoans, several groups do not possess Actinulida, and Trachymedusae (Bouillon 1985). basitrichous isorhizas (Actinulida, Trachymedu- Absent from Anthozoa. sae, Narcomedusae, Laingiomedusae, and Siphon- 8. Euryteles (0—absent; 1—present). Documented in ophorae) whereas others do (Leptothecata). An- Stauromedusae, Coronatae, Semaeostomeae, Rhi- thoathecata scored as polymorphic because zostomeae (Calder 1983; Lesh-Laurie & Suchy basitrichous isorhizas are widely distributed in 1991), and Cubozoa (Schuchert 1993; Franc Capitata, but rare to absent in Filifera. Scored as 1994b). Absent in Anthozoa (Schuchert 1993). absent in Limnomedusae even though basitrichous Among hydrozoans, euryteles are present in Tra- isorhizas were recorded for 1 species (Nagao chymedusae (except in the family Petasiidae), 1969; Kubota 1976). In Kubota (1976), identifi- Limnomedusae, Anthoathecata (Bouillon 1985), cation of the nematocyst is inconsistent with the and Siphonophorae (Clausen 1967: 368). In Lep- illustrations. tothecata, euryteles are present in a few species 12. Apotrichous isorhizas (0—absent; 1—present). (Weill 1934; Russel 1940; Bouillon 1985) and al- Reported for the narcomedusan families Aegini- though these occurrences may be homoplastic, dae and Cuninidae (Bouillon 1985). they are scored as polymorphic here. Euryteles 13. Holotrichous or atrichous isorhizas (0—absent; scored as polymorphic for Actinulida; have been 1—present). Two distinct nematocyst types, hol- described in some species of Actinulida (Swed- otrichous and atrichous isorhizas, are combined mark & Teissier 1967; Clausen 1967), but pub- here because when studied by optical microscopy, lished figures are difficult to distinguish from sten- their differences may not be perceptible. Present oteles. for Anthozoa, Stauromedusae, Cubozoa, Corona- 9. Desmonemes (0—absent; 1—present). Appear to tae, Semaeostomeae, Rhizostomeae (Calder & Pe- be absent from non-hydrozoan cnidarians. Among ters 1975; Calder 1983; Rifkin & Endean 1983, hydrozoans, they are present in Anthoathecata 1988; Fautin & Mariscal 1991; Lesh-Laurie & Su- (both Filifera and Capitata), Siphonophorae (Carre´ chy 1991; Franc 1994b), and widely distributed & Carre´ 1994), and possibly in a species of Ac- among hydrozoan groups (Bouillon 1985; Carre´ tinulida (Clausen 1967). The record for Actinulida & Carre´ 1994). is doubtful because only aspiroteles (Lacassagne 14. Heterotrichous anisorhizas (0—absent; 1—pre- Cladistic analysis of Medusozoa 35

sent). Common in Semaeostomeae and less so in zoa, symmetry is variable (reviewed by Salvini- Rhizostomeae (Calder 1983). Absent from Coron- Plawen 1978), but a bilateral planula symmetry atae and Stauromedusae (Calder 1977, 1983; that persists in the polyp or transforms to biradial Lesh-Laurie & Suchy 1991), unreported for An- is common. We score this character as biradial for thozoa, and absent in Cubozoa, with 1 known ex- Anthozoa and await further resolution of antho- ception (Franc 1994b). For hydrozoans, absent in zoan phylogeny, which should clarify the plesiom- Actinulida, Trachymedusae, Narcomedusae, and orphic condition of this character for Anthozoa Laingiomedusae (Bouillon 1985). For Limnome- and Cnidaria as a whole. dusae and Leptothecata, present in only 1 species of each (Bouillon 1985), and we considered them Reproduction and development absent for these taxa. The character was scored as polymorphic for Siphonophorae and Anthoathe- 19. Sexual condition (0—hermaphroditic; 1—gono- cata because the nematocyst is present only in choric). This variable character requires general- some subgroups of these taxa (Bouillon 1985; izations for the terminals. We considered a taxon Carre´&Carre´ 1994). to be gonochoric when this condition is present in 15. Birhopaloids (0—absent; 1—present). Most likely more than just a few isolated species. Within Cni- an autapomorphy of Siphonophorae (Carre´&Car- daria, Siphonophorae is among the rare hermaph- re´ 1994). roditic groups, though hermaphroditism is not 16. Rhopalonemes (0—absent; 1—present). May be necessarily simultaneous in the majority of species an autapomorphy of Siphonophorae (Carre´&Car- (Kirkpatrick & Pugh 1984). In other hydrozoans, re´ 1994). the predominant condition is gonochorism (see ex- ceptions in Bouillon 1994b). Life habit and symmetry 20. Location of medusa formation (0—lateral, bud- ding from an entocodon; 1—apical/oral; 2—direct 17. Life habit (0—benthic adults; 1—planktonic development without polyp stage). Strobilation in adults). Despite considerable variation within Coronatae, Semaeostomeae, and Rhizostomeae some groups, all taxa were scored as planktonic produces medusae at the apical ends of polyps, as adults except for Anthozoa, Stauromedusae, with very few exceptions (e.g., Pelagia). Trans- and Conulatae, which is considered benthic in formation of the stauromedusan polyp to the adult light of taphonomic data (Simo˜esetal. 2000) form involves a transformation of the oral end of 18. Symmetry (0—radial; 1—radial tetramerous; 2— the polyp (Kikinger & Salvini-Plawen 1995). A biradial). Scoring this character is difficult because similar process of metamorphosis and develop- of considerable variation within our terminal taxa, ment of adult characteristics largely toward the often within the same individual depending on the oral end of the polyp is observed in cubozoans body parts or life-cycle stages being considered (Stangl et al. 2002). In many hydrozoan groups, (e.g., radial canals and gonads or polyp and me- medusae form on the lateral portions of polyps dusa). For the medusoid phases, all our terminal and involve the entocodon, whereas in others the taxa are radially tetramerous or probably derived medusa is produced by direct development with- from such a state, as indicated by the number and out any polyp stage. Information is missing for disposition of radial canals, and/or oral arms, and/ Laingiomedusae because the polyp stage, if it ex- or gonad location, with a few exceptions (e.g., ists, is unknown (Bouillon & Boero 2000). Salvini-Plawen 1987). Similarly, cnidarian planu- 21. Type of apical medusa formation (0—strobilation; lae are typically bilateral. Therefore, scoring of 1—metamorphosis without transverse fission). this character across our terminal taxa applies to Strobilation involves transverse fission and is typ- the respective polypoid stages. Stauromedusans, ical only of Coronatae, Semaeostomeae, and Rhi- cubozoans, coronates, semaeostomes, and rhizos- zostomeae. In both Stauromedusae and Cubozoa, tomes are all radial-tetramerous (D. Chapman adult medusae are produced by a metamorphosis 1966; Lesh-Laurie & Suchy 1991), whereas hy- which involves the resorbtion of polyp tentacles drozoan polyps, in the groups that have them, are and the development of new tentacles and which basically radial. However, 2 primary tentacles de- is not accompanied by transverse fission (Werner velop early in the ontogeny of polyps in many et al. 1971; Kikinger & Salvini-Plawen 1995; medusozoan groups (Salvini-Plawen 1978). Stangl et al. 2002). Therefore, these groups are also biradial, and we 22. Strobilation type (0—polydisk; 1—monodisk). score them as polymorphic (0/2 or 1/2). In Antho- 23. Oocyte development (0—oocytes develop without 36 Marques & Collins

accessory cells; 1—oocytes develop with accessory condition that appears to be unique in Cnidaria cells; 2—oocytes develop within follicles; 3—oo- (Lesh-Laurie & Suchy 1991). cytes develop with uptake of somatic or other germ 31. Glandular cells in the planula (0—absent; 1—pre- line cells). Oocytes develop with accessory cells in sent). Histological studies of cnidarian planulae anthozoans, semaeostomes, and rhizostomes (Eck- are not common and much remains to be learned elbarger & Larson 1993; Eckelbarger 1994). Ova- from future studies. Glandular and nerve cells are ries of Stauromedusae, in which oocytes develop lacking in the planulae of Haliclystus (Otto 1976, within follicles, appear to be uniquely complex 1978) and Cassiopeia (Martin & Chia 1982), in among cnidarians, though only 1 species has so far Stauromedusae and Rhizostomeae, respectively, been investigated (Eckelbarger & Larson 1993). but they are present in semaeostomes (Widersten Hydrozoans differ in the development of their oo- 1968). Within Hydrozoa, anthoathecatan planulae cytes, which take up nutrients from other cells, in- appear to be best studied and reveal glandular and cluding degenerating oocytes (Campbell 1974; Tar- nerve cells (Martin & Chia 1982; van de Vyver dent 1985; Eckelbarger 1994). 1994; Bouillon & Boero 2000). For Anthozoa, 24. Spermatophores (0—absent; 1—present). Known Fautin & Mariscal (1991) report that all cell types only for some cubozoans (Werner 1973b; Bouillon present in the adult were already in the planula 1994a; Franc 1994b). (nematocysts, sometimes spirocysts, supporting 25. Location of gonads (0—gastrodermis; 1—epider- cells, secretory, nervous, and sensory). mis). In non-hydrozoans, the gonads are of gas- 32. Nerve cells in the planula (0—absent; 1—pre- trodermal origin and location (Fautin & Mariscal sent). See comments for ch31. Nerve cells have 1991; Bouillon 1994a). In hydrozoans, the gonads been observed in the planulae of limnomedusan are located in the epidermis (Bouillon 1994a; species (Bouillon & Boero 2000). Nielsen 2001), although for many species the go- 33. Relationship between axes of planula and adult nads originate in the gastrodermis (Tardent 1985). (0—oral-aboral axis in the adult derived from the In most species of Narcomedusae, the gonads longitudinal axis of the planula; 1—oral-aboral seem to originate in the epidermis, although in axis in the adult derived from the transverse axis some species (Pegantha clara and Solmaris fla- of the planula). The oral-aboral axis found in adult vescens) the gonads are of gastrodermal origin narcomedusans develops from the transverse axis (Bigelow 1909; Bouillon 1987). of the planula, presumably a unique characteristic of this group (Bouillon 1987). This character is Siphonophore features probably not known for most taxa. 26. Nectosome (0—absent; 1—present). Present in si- phonophores other than those in Cystonectae. Post-planula 27. Pneumatophore (0—absent; 1—present). Present 34. Ephyrae (0—absent; 1—present). Information is in siphonophores other than those in Physonectae. scarce for Coronatae (Lesh-Laurie & Suchy 1991), but it appears that only coronates, semaeostomes, Planula and rhizostomes have ephyrae in their life cycles 28. Planula in the life cycle (0—absent; 1—present). (Thiel 1966). Absent only from actinulids, which have direct de- 35. Actinula (0—absent; 1—present). Stages de- velopment (Swedmark & Teissier 1966; Bouillon scribed as actinulae occur sparsely among hydro- 1985; Clausen & Salvini-Plawen 1986). zoan groups, in the superfamily Tubularioidea (Pe- 29. Planula ciliation (0—absent; 1—present). Typi- tersen 1990), in Trachymedusae (Thomas & cally ciliated. In Stauromedusae, the planula of Edwards 1991), and in Narcomedusae. Although only 1 species has been investigated and was they are morphologically similar, the anthoathe- found not to be ciliated (Otto 1976, 1978). Al- catan actinula (present in Tubularioidea) is not though Thiel (1966) and Franc (1994a) considered considered homologous to those of Trachymedu- the character generalized for Stauromedusae, the sae and Narcomedusae, because in anthoatheca- universality of this and other planula characters in tans the actinula precedes the polyp whereas in the group is currently unknown. Trachymedusae and Narcomedusae it precedes the 30. Number of endodermal cells of the planula (0— medusa (Petersen 1990; Bouillon 1994b). Here we variable; 1—constant, nϭ16). The endodermis of refer to the actinula that precedes the medusa. The the stauromedusan planula possesses 16 support- name Actinulida also refers to the similarity be- ing cells arranged linearly (Otto 1976, 1978), a tween representatives of this group and the actin- Cladistic analysis of Medusozoa 37

ula stage of Trachymedusae and Narcomedusae, basal area of polyp or to podocysts; 2—present but homology in this case is uncertain. and covering most of polyp). According to 36. Polypoid phase (0—absent; 1—present). A pol- Salvini-Plawen (1978) and Lesh-Laurie & Suchy ypoid phase (a body structure consisting of a col- (1991), periderm is secreted only by some coro- umn and an aperture originating from the blasto- nates among the traditional scyphozoans. How- pore surrounded by tentacles) is present in all ever, D. Chapman (1966) reported a vestigial cu- major cnidarian groups other than Actinulida and ticle in scyphistomae of Aurelia and pointed out Trachymedusae. A polyp-like form is present in that groups with podocysts, such as Semaeosto- some narcomedusans, but this structure is almost meae and Rhizostomeae, necessarily have peri- certainly not homologous to the polyps of other derm involved in forming the resistant structure cnidarian groups (Bouillon 1987). Information (D. Chapman 1966). A small basal periderm has about the life cycle of laingiomedusans is un- been reported for cubopolyps (Werner 1984; known (Bouillon 1978, 1985), though polyp-like Bouillon 1994b). No indication of periderm has forms have been found appearing from medusoid been reported for stauromedusans. Among hydro- budding (J. Bouillon, pers. comm.)—a possible zoans, polyps and colonies of many Limnome- link between this group and the Narcomedusae. dusae, Leptothecata, and Anthoathecata produce Within Semaeostomeae, Leptothecata, and An- perisarc, which is similar to the periderm of scy- thoathecata, some members do not have a polyp phozoans (Thomas & Edwards 1991). Some si- phase, but in each of these cases, the lack probably phonophores also may produce a kind of ‘‘peri- represents a secondary loss. Stauromedusae was sarc’’ that extends over most of the organism (cf. scored as possessing a polyp phase because the Mackie 1960). In the fossil group Conulatae, a adult form is separated ontogenetically from the periderm was present (Salvini-Plawen 1978). An- juvenile polyp-like form by a distinct metamor- thozoans secrete no periderm. phosis. 40. Podocysts (0—absent; 1—present). Resting stages 37. Polymorphic polyps (0—absent; 1—present). appearing in unfavorable conditions are found in Common in Leptothecata, Siphonophorae, and Semaeostomeae and Rhizostomeae (Lesh-Laurie Anthoathecata (Bayer & Owre 1968; Kirkpatrick & Suchy 1991). Also present in a small number & Pugh 1984), and also present in some members of anthoathecatans (D. Chapman 1966; Petersen of Limnomedusae (Bouillon 1994b), which was 1990); we consider them to be secondarily de- scored as polymorphic. Anthozoa is also scored as rived. Information concerning cubopolyps is still polymorphic because octocorals have polymor- indefinite (Schuchert 1993). phic polyps. 41. Structure of polyp tentacles (0—hollow; 1—sol- 38. Desmocytes (0—absent; 1—present). Desmocytes id). Hollow in anthozoans, cubozoans, and the tra- are hardened dead cells ontogenetically derived ditional scyphozoan groups (Schuchert 1993). In from secretory epidermal cells (Marcum & Diehl hydropolyps, the character is not uniform: hollow 1978) linking cuticle to mesoglea. Common in in Limnomedusae, chord-like in Leptothecata many sessile cnidarians (D. Chapman 1966; Lesh- (scored as solid), and polymorphic in Anthoathe- Laurie & Suchy 1991). Documented in a number cata (Petersen 1990). of semaeostomes (He´rouard 1911; Widersten 42. Number of tentacular whorls (0—one; 1—two or 1966; Lesh-Laurie & Suchy 1991), but appear to more). Polyp tentacles in 2 or more whorls occur be absent in cubozoans, rhizostomes, and perhaps broadly only in Anthoathecata (Petersen 1990), coronates (Lesh-Laurie & Suchy 1991, and refer- which we score as polymorphic. Some groups ences therein). Stauromedusans have supporting within Anthozoa (e.g., Ceriantharia) and Stauro- cells resembling desmocytes (Lesh-Laurie & Su- medusae (Stylocoronella variabilis; see Kikinger chy 1991), but are scored here as absent. Des- & Salvini-Plawen 1995) also have 2 whorls of ten- mocytes are common in anthozoans and hydro- tacles, but this condition is not representative. zoans (Fautin & Mariscal 1991; Lesh-Laurie & 43. Septa in polyp (0—absent; 1—gastrodermal folds Suchy 1991). Even in a siphonophore, a non- present; 2—present). Except in polyps of Cubozoa sessile hydrozoan, Mackie (1960) described des- and Hydrozoa, the gastrovascular cavity is divided mocytes between the stem and the float of Phy- by septa. Salvini-Plawen (1987) argued that cu- salia. Homology of desmocytes across Cnidaria bopolyps exhibit reduced septa and septal mus- has been doubted (Tidball 1982; Fautin & Mar- cles; these septa are gastrodermal folds in which iscal 1991; Thomas & Edwards 1991). the mesoglea is never present (e.g., in Tripedalia, 39. Periderm (0—absent; 1—present but limited to the Chapman 1978, also Lesh-Laurie & Suchy 1991). 38 Marques & Collins

Except for Cubozoa, we score simply the presence Rhizostomeae (Moore & Harrington 1956; D. or absence of septa, rather than considering the Chapman 1966). many characters that could be derived from them (e.g., the arrangement of muscle bundles), because Medusae of difficulty in determining character states and judging homology. These characters apply to an adult life phase that 44. Hydrotheca/gonotheca (0—absent; 1—present). typically follows an intermediate polyp stage. Al- Unique to Leptothecata. Similar structures occur though homology between cnidarian medusoid phases in other hydrozoans, e.g., families Stylasteridae has long been debated, the strong morphological sim- and Milleporidae, but these are not constituted by ilarity of various medusae leads us to score these char- perisarc. acters across the medusozoan groups. Anthozoa has no 45. Stomodeum (0—absent; 1—present). Stomodeum, comparable phase and is scored accordingly through- corresponding to the pharynx, is unique to Antho- out. zoa. The structure called a scyphopharynx in some scyphozoans (discussed in Lesh-Laurie & Suchy 49. Medusoid phase (0—absent; 1—present). 1991) is probably not homologous to the antho- 50. Pedalium of coronate type (0—absent; 1—pre- zoan pharynx, nor is it widely distributed. sent). This type of pedalium, present only in Co- 46. Organization of the nervous system (0—in 1 or 2 ronatae, is part of the umbrella, and should not be nets; 1—with nerve rings). Cubopolyps have 2 confused with the pedalia of Cubozoa, which are nerve rings near the oral cone, 1 in the gastro- the thickened bases of tentacles or tentacle bunch- dermis and 1 in the epidermis, a condition unique es (Thiel 1966). Cf. ch56. among cnidarian polyps (Lesh-Laurie & Suchy 51. Rhopalia/rhopalioids (0—absent; 1—present). 1991; Bouillon 1994a). Cf. ch77. Hollow structures ontogenetically derived from 47. Canal system (0—absent; 1—present). Werner tentacles are present in Cubozoa, Coronatae, Se- (1973a) noted a canal system in the polyp of a maeostomeae, and Rhizostomeae, in which they coronate and pointed out similar canal systems in are associated with statocysts and photoreceptors. scyphopolyps of Semaeostomeae and Rhizosto- Rhopalia of cubozoans contain strikingly complex meae. Canals also occur in Stauromedusae and sensory structures, including ocelli with corneas, Cubozoa (Thiel 1966). There are no indications of vitreous body, lenses, and retinas (Pearse & Pearse any canal system in the well-known polyps of Hy- 1978). The simpler hollow structures of Stauro- drozoa and Anthozoa. medusae are known as rhopalioids or anchors, and 48. Gastrodermal musculature (0—not organized in have a similar ontogeny involving the reduction bunches; 1—organized in bunches of gastroder- of primary tentacles. Statocysts of Trachymedusae mal origin; 2—organized in bunches of ectoder- and Narcomedusae are also of tentacular origin, mal origin). The gastrodermal musculature among but they do not appear to be homologous with anthozoans is organized into longitudinal bundles rhopalia because they are not associated with pho- of gastrodermal origin, except for Ceriantharia, in tosensitive structures. which the musculature is organized in longitudinal 52. Complexity of rhopalium/rhopalioid (0—rhopa- layers, with retractor muscles occurring in the lioids; 1—rhopalia; 2—rhopalia with complex mesenteries (Fautin & Mariscal 1991). In hydro- eyes). Cf. ch51. polyps, the musculature consists of a layer of lon- 53. Nerve ring(s) (0—absent; 1—one; 2—two). Scor- gitudinal epidermal muscular fibers and circular ing of this character follows Schuchert (1993). gastrodermal fibers (Werner et al. 1976; Lesh- The medusae of Stauromedusae, Coronatae, Se- Laurie & Suchy 1991; Thomas & Edwards 1991). maeostomeae, and Rhizostomeae have 2 epider- In Stauromedusae, Coronatae, Semaeostomeae, mal nerve nets connected in marginal centers or and Rhizostomeae, polyps have 4 main muscles of ganglia (Lesh-Laurie & Suchy 1991). Hydrozoan ectodermal origin, in an interradial position, and medusae have 2 nerve rings, 1 internal and the located in the mesoglea (Thiel 1966). Cubopolyps other external (Thomas & Edwards 1991) in Lim- also possess intramesogleal muscles organized in nomedusae, Trachymedusae, Leptothecata, Si- bunches of ectodermal origin, though the number phonophorae (2 rings in the nectophore, Thomas is not fixed. Fossil conulates with 4 opercular flaps & Edwards 1991; Carre´&Carre´ 1994), and An- can be inferred to have had 4 muscle bundles with thoathecata. Cf. ch77. a position topologically equivalent to those of 54. Gastric filaments (0—absent; 1—present). Present Stauromedusae, Coronatae, Semaeostomeae, and in the adult stages of Stauromedusae, Cubozoa, Cladistic analysis of Medusozoa 39

Coronatae, Semaeostomeae, and Rhizostomeae tomeae (2 species) are made of CaSO4 (Chapman (Thiel 1966; Bouillon 1994a). 1985; Lesh-Laurie & Suchy 1991). Chapman 55. Coronal muscle (0—well developed; 1—marginal (1985) also noted that the ratio of Mg to Ca differs and tiny). The coronal muscle of medusae is re- between Limnomedusae and Leptothecata, possi- sponsible for swimming, and according to Lesh- bly indicating independent origins. The character Laurie & Suchy (1991), the muscles of Stauro- is non-comparable for Stauromedusae, Laingiome- medusae, Cubozoa, Coronatae, Semaeostomeae, dusae, Siphonophorae, and Anthoathecata because Rhizostomeae, and Hydrozoa have the same lo- members of these groups do not have statocysts, cation, structure, and arrangement of myofibrils, and consequently lack statoliths. Statolith com- despite some structural differences, e.g., the pres- position can be related to physiological factors, ence of a velum in Hydrozoa. A coronal muscle but the character is included to inspire future in- occurs in all hydrozoan groups, including Siphon- vestigations. ophorae (Carre´&Carre´ 1994). In Stauromedusae 62. Septa (0—absent; 1—present). Septa in the gas- this musculature is present as a tiny subumbrellar trovascular cavity of the medusa occur in Stau- marginal strip (Gwilliam 1960; Bayer & Owre romedusae, Cubozoa, and Coronatae (though di- 1968), or even absent (as in Stylocoronella, Sal- minutive) (Thiel 1966). Comparable septa are vini-Plawen, pers. comm.). absent in Semaeostomeae, Rhizostomeae (Thiel 56. Pedalium of the cubozoan type (0—absent; 1— 1966), and Hydrozoa. Because the location of gas- present). As stated above (ch50), the pedalia of tric filaments should be influenced by the presence Coronatae and Cubozoa are homonyms rather than or absence of septa (Thiel 1966), we have avoided homologous structures. double weighting of the character by not scoring 57. Velum (0—absent; 1—present). A circular mem- characters related to its position. brane of tissue projecting toward the center of the 63. Septal shape (0—straight; 1—y-shaped). The sep- bell opening acts in the swimming of some me- ta of Stauromedusae, Cubozoa, and Conulatae are dusae. The velum comprises 2 epidermal epithelia, y-shaped (Kiderlen 1937; Thiel 1966; Jerre 1994). one subumbrellar and the other exumbrellar, with These septa often form a claustrum, a lamella con- a mesolamella in between. It is connected to the stituted by 2 endodermal layers, located between subumbrella by a non-muscular zone containing 2 contiguous septa in medusoids (see Thiel 1966). the nerve rings. This structure is characteristic of 64. Radial canals (0—absent; 1—present; 2—present most hydrozoan groups; a few exceptions (e.g., and complex). Absent in Stauromedusae, but pre- Anthoathecata Milleporidae) appear to be charac- sent in Coronatae (in a simple form) and in Se- ter losses. Actinulida also lacks a velum (Bouillon maeostomeae and Rhizostomeae (in complex mor- 1985), but it is scored as non-comparable because phological arrangements). In Semaeostomeae, the of its diminutive umbrella. canals ramify, join, and even extend into the mar- 58. Umbrellar margin (0—smooth and continuous; ginal lobules, tentacles, and statocysts. In Rhizos- 1—lobed). The umbrellar margin in Coronatae, tomeae, the morphology is still more complicated, Semaeostomeae and Rhizostomeae is lobed, as the canals anastomose in a wide zone (see Thiel whereas that in Stauromedusae and Cubozoa is not 1966 and references therein). In Cubozoa, canals (Thiel 1966). Among Hydrozoa, medusa margins are absent. However, cubozoans have gastrovas- are lobed in Narcomedusae and Laingiomedusae cular spaces that topologically correspond to ra- (Laingia jaumotti with 4 lobes and Kantiella en- dial canals. We score the character as polymor- igmatica with 8 lobes; Bouillon 1978). phic, states 0 and 1, to reflect our uncertainty 59. Tentacles (0—absent; 1—present). Present at the about whether these spaces do or do not constitute umbrella margin in all groups of Medusozoa ex- radial canals. In Hydrozoa, radial canals are absent cept Rhizostomeae (Thiel 1966) and Siphonopho- in Actinulida and Narcomedusae, but present in rae. all other groups (Bouillon 1994a; Carre´&Carre´ 60. Tentacular bulbs (0—absent; 1—present). Scoring 1994). of this character is based on data from Bouillon 65. Circular canal (0—absent; 1—partially present; & Boero (2000). 2—fully present). Absent in Stauromedusae, Cu-

61. Statolith composition (0—MgCaPO4; 1—CaSO4). bozoa (though there is a morpho-physiologically Statoliths of hydrozoans (at least in 7 species of similar structure in the velarium), and Coronatae, Leptothecata and 1 of Trachymedusae) are com- but partially present in Semaeostomeae and Rhi-

posed of MgCaPO4, whereas those of Coronatae, zostomeae (Thiel 1966). In Hydrozoa, a circular Rhizostomeae, Cubozoa (1 species), and Semaeos- canal is absent in Actinulida, but present in other 40 Marques & Collins

groups with some variation. For instance, narco- bell-shaped, more or less narrowing in the oral- medusans have a system in which the circular ca- aboral axis. However, the shape in Stauromedusae nal is arranged around the peronia and tentacular is pyramidal, in Cubozoa cubic, and in Actinulida bases (Bouillon 1985, 1994b), a condition similar actinuloid (Mayer 1910; Thiel 1966). to that seen in Laingiomedusae (Bouillon 1978). 72. Shape of horizontal cross-section (0—circular; 66. Velarium (0—absent; 1—present). Occurs only in 1—quadrate, i.e., with 4-parted symmetry). A Cubozoa. It has a gross structure similar to that of quadrate horizontal cross-section is seen in Stau- the velum, but is entirely of subumbrellar origin romedusae, Cubozoa, and Conulatae. (Hyman 1940). 73. Urticant rings (0—absent; 1—present). Urticant 67. Coronal furrow (0—absent; 1—present). Present rings are rings of cnidae in the basal area of the only in Coronatae (Mayer 1910). umbrellar margin and are present in some Lim- 68. Gonadal location (0—manubrium; 1—radial ca- nomedusae (P. Shuchert, pers. comm.) and Tra- nals). In Hydrozoa, members of Narcomedusae chymedusae (Bouillon 1994b). (Bouillon 1985), Laingiomedusae (Bouillon 1978, 74. Oral arms with suctorial mouths (0—absent; 1— 1985), Siphonophorae (Kirkpatrick & Pugh 1984), present). Present in Rhizostomeae, whose 8 fused and Anthoathecata (Bouillon 1994b) have gonads oral arms are ontogenetically derived from the 4 on the manubrium, though sometimes gonads ex- oral lobules of the manubrium of the ephyrae tend along the radial canals. Following the topo- (Thiel 1966). logical equivalencies of Clausen (1967), the area 75. Tentacular insertion (0—umbrellar margin; 1— of the gastric tube of Actinulida, where actinulids away from the umbrellar margin). Tentacles most bear gonads, corresponds to the manubrium of commonly arise at the umbrellar margin, but never other medusae (Clausen 1967; Bouillon 1985). In in Narcomedusae and Laingiomedusae, in which Limnomedusae, gonads are located on radial ca- the tentacular insertion is near the apex of the um- nals (except in Limnocnida, an African and Indian brella (Bayer & Owre 1968; Bouillon 1978, freshwater group). In most trachymedusans, go- 1985). Rhizostomes and siphonophores were nads are also located on the area of the radial ca- scored non-comparable because their medusoids nals, but some species have them on the pseudo- do not have tentacles. manubrium (Bayer & Owre 1968), dubbed pseudo 76. Manubrium (0—absent; 1—present). Members of because the radial canals are present. In Leptothe- Narcomedusae often lack a manubrium (cf. Bayer cata, gonads are also on the radial canals (Bouillon & Owre 1968). However, at least 1 species has a 1994b), with a few exceptions. In Cubozoa, go- conspicuous manubrium, which even extends out- nads occur along the interradial septa (Mayer side the bell (Cunina proboscidea, Bouillon 1910), an area equivalent to where the radial ca- 1994b). We score this character absent for Nar- nals are in those medusae that possess them. comedusae, while anticipating more complete in- 69. Statocysts (0—absent; 1—endodermal; 2—ecto- formation in the future. dermal). Stauromedusae, Laingiomedusae (Bouil- 77. Nervous system organization (0—GFNN absent; lon 1994a), Siphonophorae (Carre´&Carre´ 1994), 1—GFNN present). Coronates, semaeostomes, and Anthoathecata lack statocysts. The statocysts and rhizostomes have 3 basic components in the of Coronatae, Semaeostomeae, and Rhizostomeae nervous system: a giant fiber nerve net (GFNN), are reported to be of endodermal origin (Mayer which transmits pulses to the marginal ganglion 1910). In Hydrozoa, statocysts of endodermal or- and to the swimming muscle; a diffuse nerve net igin are present in Limnomedusae, Actinulida, (DNN), in which neurons are located in the sub- Trachymedusae, and Narcomedusae, whereas Lep- umbrellar epidermis, oral arms, and tentacles (if tothecata is the only group to have statocysts of present); and marginal centers, which include the exclusively ectodermal origin (Bouillon 1985, marginal ganglion and the rhopalium (Horridge 1994b). 1956; Lesh-Laurie & Suchy 1991). No GFNN is 70. Perradial ‘‘mesenteries’’ (0—absent, 1—present). present in Stauromedusae (Gwilliam 1960; Pas- The so-called perradial mesenteries are triangular sano 1982). In cubozoans, there is evidence of pouches adjacent to the base of the stomach and DNN fibers organized in a diffuse system, as well located along the radial canals of many species of as of a GFNN organized in the subumbrellar epi- Anthoathecata (Petersen 1990). dermis and in the nerve ring (Werner et al. 1976; 71. Adult medusoid shape (0—bell; 1—pyramidal; Chapman 1978; Satterlie 1979; Satterlie & Spen- 2—cubic; 3—actinuloid). Adults of medusozoans cer 1979; Passano 1982; Lesh-Laurie & Suchy are medusae in most groups, and are typically 1991). Hydrozoan medusae also have DNN fibers, Cladistic analysis of Medusozoa 41

as well as ganglia (Thomas & Edwards 1991), and 81. Ocelli (0—absent; 1—present). Present in the me- a GFNN also occurs in Anthoathecata (e.g., Po- dusae of Cubozoa, Coronatae, Semaeostomeae, lyorchis, Stomotoca, Amphinema: Chapman 1974; and Rhizostomeae. Although stauromedusans gen- Mackie & Singla 1975; Anderson & Mackie 1977; erally do not have ocelli as adults, some interstitial Singla 1978a; Spencer 1979; Thomas & Edwards forms do appear to possess them (Blumer et al. 1991), Leptothecata (e.g., Obelia, Chapman 1995), and we score the character as polymorphic 1968), Limnomedusae (e.g., Limnocnida, Bouillon for the group. In Hydrozoa, ocelli are generally 1956), Trachymedusae (e.g., Aglantha: Singla not present (Bouillon 1978, 1985, 1994b; Carre´& 1978b), and Siphonophorae (e.g., Nanomia, Carre´ 1994), except in Anthoathecata and rarely Mackie 1973). in Leptothecata (Bouillon 1994b). 78. Structure of medusa tentacles (0—hollow; 1—sol- 82. Peripheral canal system (0—absent; 1—present). id). Hollow primary or secondary tentacles are Present only in Narcomedusae and Laingiomedu- present in Cubozoa, Stauromedusae, and Se- maeostomeae. In Hydrozoa, hollow tentacles oc- sae, this is a limited circular canal encircling per- cur in Limnomedusae (Bouillon 1994a) and Lep- onia and tentacular bases (Bouillon 1978). tothecata (except for Obelia and perhaps 83. Umbrellar furrow (0—absent; 1—present). Pre- Dipleurosoma pacificum) (Bouillon 1994b). Ten- sent only in Laingiomedusae (Bouillon 1978). tacles are solid in Coronatae, Actinulida, Laingi- 84. Development of the umbrella (0—fully developed; omedusae, and Narcomedusae (Thiel 1966; Bouil- 1—aboral cone). The aboral cone occurs only in lon 1985). In Trachymedusae, the majority of Actinulida (Clausen 1967). species have solid tentacles and determine the 85. Number of tentacular whorls (0—one whorl; 1— scoring, despite species with hollow tentacles in two whorls). Actinulids have 2 tentacular whorls Geryonidae (Bouillon 1994a, 1994b). Anthoathe- as adults, 1 aboral and 1 subaboral (Clausen cata is polymorphic for this character. 1967). Two whorls of tentacles are also present in 79. Tentacular morphology (0—straight tentacles; 1— the interstitial Armohydra, an unusual member of tentacles with an angular inflection). The tentacles Limnomedusae, which we code as polymorphic of Laingia jaumotti and Kantiella enigmatica for this character. (Laingiomedusae) have a right-angle inflection 86. Velar canals (0—absent; 1—present). Present in close to where the tentacles meet the umbrella (Bouillon 1978a). the velarium of cubozoans, varying in number and 80. Peronia (0—absent; 1—present). A peronium, morphology (Franc 1994b). No equivalent struc- found only in Narcomedusae and Laingiomedu- tures are known in other medusozoans. sae, is a structure formed when the tentacular ec- 87. Frenulae (0—absent; 1—present). Frenulae are toderm extends up to the umbrellar margin, and supporting structures of the velarium, perradially lines the umbrellar furrow with cnidae (Bouillon located in Cubozoa (Franc 1994b). No equivalent 1978a). structures are known in other medusozoans. 42 Marques & Collins

Appendix 2