ORIGINAL ARTICLE

The evolution of colony-level developmentin the Siphonophora (Cnidaria:Hydrozoa)

Casey W. Dunn & Günter P. Wagner

Received: 17 April 2006 /Accepted: 5 July 2006 / Published online: 16 September 2006# Springer-Verlag 2006

Abstract Evolutionary developmental biology has focusedalmost exclusively on multicellular organisms, but there areother relevant levels of biological organization that haveremained largely neglected. Animal colonies are made upof multiple physiologically integrated and geneticallyidentical units called zooids that are each homologous tosolitary, free-living animals. Siphonophores, a group ofpelagic hydrozoans (Cnidaria), have the most complexcolony-level organization of all animals. Here the colony-level development of five siphonophore species, strategi-cally sampled across the siphonophore phylogeny, isdescribed from specimens collected using deep-sea sub-mersibles and by self-contained underwater breathingapparatus diving. These species include three cystonects,Bathyphysa sibogae, Rhizophysa filiformis, and Rhizophysaeysenhardti, and two “physonects”, Agalma elegans andNanomia bijuga. These data, together with previous find-ings, are analyzed in a phylogenetic framework to recon-struct key features of the history of colony-level organizationand development in the Siphonophora. It is shown thatgonodendra and gastrozooids of the examined cystonectsarise as independent buds directly on the stem, whereasprobud subdivision (the origin of feeding, reproductive, and

other zooids from a single bud) is a synapomorphy of theCodonophora. The origin of probud subdivision is associatedwith the origin of cormidia as integrated units of colonyorganization, and may have allowed for greater morpholog-ical and ecological diversification in the Codonophorarelative to the Cystonectae. It is also found that symmetryis labile in siphonophores, with multiple gains and/or lossesof directional asymmetry in the group. This descriptive workwill enable future mechanistic and molecular studies ofcolony-level development in the siphonophores.

Keywords Major transition in evolution .

Asexual reproduction . Animal colonies . Division of labor .

Functional specialization

Introduction

The siphonophores, a group of pelagic colonial hydrozoans(Cnidaria), include the longest animals in the world(Robison 1995) and are among the most abundantcarnivores of the open ocean (Pugh 1984). Even so, theyhave largely escaped the public eye, and many biologistsare not aware of their existence. Siphonophores have notalways been so obscure. They were of central interest tozoologists of the nineteenth century because of anotherdistinguishing feature—they are the most complex of allcolonial animals. Colonial animals have a life cyclewherein multiple asexually produced zooids, each of whichis homologous to a free-living solitary animal, remainattached and physiologically integrated throughout theirlives. Siphonophores have both the highest degree offunctional specialization between zooids and the greatestprecision of colony-level organization of any group of

Dev Genes Evol (2006) 216:743–754DOI 10.1007/s00427-006-0101-8

Communicated by M. Q. Martindale

C. W. Dunn (*)Kewalo Marine Laboratory, University of Hawaii,41 Ahui St.,Honolulu, HI 96813, USAe-mail: cdunn@hawaii.edu

G. P. WagnerDepartment Ecology and Evolutionary Biology, Yale University,P.O. Box 208106,New Haven, CT 06511, USA

colonial animals (Beklemishev 1969; see Fig. 1 for aschematic of the organization of a siphonophore colony).

There has been much interest in the origin of develop-mental mechanisms and organization at new hierarchicallevels of individuality (Buss 1987; Maynard Smith andSzathmáry 1995). Most of this work has been concernedwith the transition from unicellularity to multicellularity(Bonner 2001) and includes important empirical studies ondiverse multicellular organisms such as Volvox and Dic-tyostelium. It will not be possible, however, to knowwhether phenomena revealed by these studies are uniqueto the transition from unicellularity to multicellularity or aregeneral properties of transitions in biological organizationunless there is an empirical push to characterize develop-ment and morphology at other levels of biologicalorganization. These necessarily include coloniality, andthe extreme form of colonial organization found in siphono-phores promises to be particularly informative.

There have been few studies of the colony-leveldevelopment and organization of siphonophores largely

due to the difficulty of collecting them. Although they areoften large and relatively abundant, siphonophores are alsoextremely fragile, and most species are found only in theopen ocean. There are published accounts of the earlyembryology of several siphonophores (Carré and Carré1993; Gegenbaur 1853; Haeckel 1869), but these studiesstopped short of describing the origins and structure of thegrowth zones that are responsible for the colony-leveldevelopment that continues throughout the life of asiphonophore. The colony-level development of only threespecies has been described (Chun 1885; Dunn 2005;Schneider 1896). These three Codonophora taxa share acommon mode of development called probud subdivision.Probuds arise sequentially within the growth zone, which isalso the site of stem elongation. Each cormidium, the well-defined group of multiple zooids that is reiterated along thelength of the siphosomal stem (Fig. 1), arises from a probudthrough a stereotypical series of subdivisions.

The ability to safely dive using self-contained underwa-ter breathing apparatus (SCUBA) from ships in the openocean (Hamner 1975) and the refinement of midwatersampling devices that can be mounted on submersiblevehicles (Youngbluth 1984) now make it possible to collectmany species of siphonophores intact for the first time.These new tools have enabled us to make the firstcomparative study of siphonophore colony-level organiza-tion and development that draws on taxa sampled across thefull diversity of the group. In conjunction with a recentmolecular phylogeny (Dunn et al. 2005), this allows us toreconstruct the history of major changes in colony-leveldevelopment and organization within siphonophores and tobegin to understand how a diversity of form has beenrealized at this poorly understood level of biologicalorganization. This descriptive work also provides a foun-dation for future molecular and mechanistic studies ofsiphonophore development that are relevant to questionsof general interest regarding the origins and evolution ofindividuality.

Materials and methods

Fresh specimens were collected by manned submersibles,remotely operated vehicles, blue-water diving, and land-based diving. This material was prepared for scanningelectron microscopy (SEM) as previously described byDunn (2005). Preserved siphonophores from Dr. Philip R.Pugh’s collection (most of which had been fixed with 4%formaldehyde in borax-buffered seawater) were also suc-cessfully prepared for SEM despite the fact that somespecimens were decades old. The same protocol was usedas for fresh material, beginning part way through with the500-mM NaCl wash.

Siphosome

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Fig. 1 Schematic overview of siphonophore structure modified fromDunn (2005). The depicted colony is a physonect and possesses apneumatophore (gas-filled float), nectosome (a region with necto-phores, the propulsive zooids), and siphosome (a region with zooidsof mixed functional types). Cystonects lack a nectosome, whereascalycophorans lack a pneumatophore

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The budding sequence of each species was inferredthrough static observations of the ontogenetic sequence ofdeveloping zooids. The organization of zooids along amature portion of the stem was first established. Theregularity of siphonophore organization made it possibleto then determine the identity of immature zooids further tothe anterior based on their relative position.

Results

A note on terminology

Haddock et al. (2005) consolidated and standardized theusage of terms for axes and orientation in siphonophores,and the present article adheres to this clarified scheme. Thenomenclature used to describe the functionally specializedzooids of siphonophores is similar to that used for theHydrozoa in general, but also includes several terms thatare specific to the group (Dunn 2005). The term gonoden-dron is used here to describe any compound reproductivestructure consisting of multiple zooids that are attached tothe stem via a common peduncle. The structure and zooidcomposition of gonodendra vary widely across taxa; it is notat all clear if they are homologous across siphonophores orhave independently arisen multiple times.

Material examined

Specimen data for the examined material are listed inTable 1. Specimens of Apolemia sp. and the cystonects arefrom Philip R. Pugh’s collection. Most Agalma elegansmaterial was collected in the bay at Villefranche-Sur-Mer,France, in the months of March–May in 2003 and 2004; theremaining A. elegans specimens were collected by blue-water diving from the RV Oceanus in the summers of2000–2002 along the east coast of the USA. Nanomiabijuga and Forskalia formosa were both collected inMonterey Bay, CA, and the bay at Villefranche-Sur-Mer,France. These newly acquired specimens have beendeposited in the Yale Peabody Museum (catalog numberscan be found in Table 1). The Lychnagalma utriculariaspecimen quickly deteriorated after being caught and wasnot preserved.

Bathyphysa sibogae

The gastrozooids and gonodendra of Bathyphysa sibogaewere found to be in a uniserial sequence (Fig. 2b). Thegastrozooids first appear as isolated buds at the anterior endof the siphosome and grow a tentacle on their anterior sideas they mature and are carried to the posterior by theelongating stem. The first several gastrozooids at the

anterior end of the sequence (i.e., the youngest gastro-zooids) have no sign of gonodendra between them.Gonodendra arise further to the posterior as isolated budsbetween and in line with the maturing gastrozooids.

The young gastrozooids of B. sibogae have pronouncedlateral ridges known as ptera (Leloup 1936), which give thegastrozooids a bract-like appearance. The striking similarityof these Bathyphysa gastrozooids to “physonect” bractsmay indicate that physonect bracts are in fact modifiedpolyps. Each young gastrozooid also has a lamella extend-ing part way up its posterior side and further anchoring it tothe stem, just as physonect bracts do. This holds the younggastrozooid so that its distal end faces to the posterior.Although conspicuous in young gastrozooids, the ptera andlamellae are absent by the 30th gastrozooid in the examinedspecimen.

Leloup (1936) figured and described the early stages ofdevelopment of Bathyphysa gonodendra. He did not,however, trace the fate of the young buds or describe their

Table 1 Specimens examined

Species Specimen Collection

Bathyphysa sibogae BWP796-20-7 PRPRhizophysa filiformis BWP1077-3 PRP

BWP567-8 PRPBWP566-17 PRPBWP566-16 PRPBWP588-21 PRPBWP442 PRP

Rhizophysa eysenhardti BWP539-13 PRPBWP1611-12 PRPBWP634-6 PRPBWP741-3 PRPBWP778-12 PRPBWP465 PRPBWP465 PRPBWP802-1 PRPBWP1086-6 PRPBWP814-12 PRP

Agalma elegans YPM 36365-36390 YPMNanomia bijuga YPM 36427-36444 YPMApolemia sp. JSLII 1450-SS3 PRPForskalia formosa YPM 36445-36448 YPMLychnagalma utricularia Tiburon 676-D6 (specimen

destroyed)

The numbers given in YPM rows are museum catalog numbers. Thespecimens in PRP’s collection are identified by their modeof collection and a unique identifier.PRP Personal collection of Philip R. Pugh (National OceanographicCentre, UK); YPM the Yale Peabody Museum (New Haven,CT); BWP blue-water plankton series; Tiburon the remotely operatedvehicle Tiburon (Monterey Bay Aquarium Research Institute);JSL II the manned submersible Johnson Sealink II(Harbor Branch Oceanographic Institute)

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later stages of development. The material examined herewas in good condition, and the full ontogenetic sequence ofgonodendron development was described (Fig. 3). Theyoungest gonodendron buds are at first simple, smoothevaginations that protrude from the stem (Fig. 3a). Thesethen elongate and become warty in appearance (Fig. 3b,c).Each wart-like protuberance then elongates and developsinto a gonodendron branch (Fig. 3d–g). The branches donot ramify further, and are all attached directly to the

central style (i.e., the main axis of the gonodendron). Asingle bud first arises on the side of the branch (Fig. 3d).This bud takes on a distinctive shape and matures into anectophore. The portion of the branch distal to thenectophore matures into the palpon that terminates thebranch. Gonophores then form from evaginations that arisealong the branch (Fig. 3e), some of which arise distal to thenectophore. Each branch bears on the order of seven to ninegonophores at maturity. The gonodendra of this species,

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Fig. 3 Developmental sequenceof Bathyphysa sibogae gono-dendron. a–c Young gonoden-dra showing the origin of theside branches as evaginations ofthe main gonodendron axis.d–g Close-ups of isolated sidebranches following the develop-mental stage shown in (c).h Photograph of preservedgonodendron. Scale bar(0.5 mm) applies to a–e, Scalebar (1 mm) applies to f, g; scalebar (2 mm) applies to h. Go,gonophore; N nectophore;P gonopalpon

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Rhizophysa eysenhardti

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ba cUniserial TriserialFig. 2 Schematic illustrationsof the budding sequences oflong-stemmed cystonects (theRhizophysidae). (b, c ventralview) a Rhizophysa eysenhardti(adapted from Kawamura 1910).b Uniserial budding, in whichthe gastrozooids arise in a singleline, and the gonodendra arelater intercalated between them.The tentacles are borne on theanterior side of the gastrozooids.c Triserial budding, in which thegastrozooids arise in two outerrows, and the gonodendra arefound in a row between them.The tentacles are borne on theside of the gastrozooid facingthe ventral midline

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like those of the other Cystonectae, are thought to breakaway from the colony at maturity before releasing theirgametes (Totton 1965, P.R. Pugh, personal communication).

Rhizophysa filiformis

All examined specimens of Rhizophysa filiformis had thesame uniserial development of gastrozooids and gonoden-dra as B. sibogae (Fig. 2b). There were from one to sixgonodendra found between the mature gastrozooids. Thegonodendra develop in the same manner as those of B.sibogae (Fig. 3) and have the same general structure atmaturity. The gastrozooids of R. filiformis do not havepronounced ptera at any stage.

Rhizophysa eysenhardti

The origin of the siphosomal elements of Rhizophysaeysenhardti is triserial (Fig. 2c). The buds of the two outerrows give rise to gastrozooids. The gastrozooids arestaggered with respect to each other. The single tentacleof each gastrozooid forms on the side facing the midline.When the gastrozooids are mature, it is not entirely obviousthat they arose in two separate rows, but the alternateattachment of the tentacle to the left and right sides of thegastrozooids is clearly discernable.

The median row of siphosomal elements, found betweenthe left and right rows of gastrozooids, consists solely ofgonodendra. Gonodendron buds are found just as far to theanterior as the gastrozooid buds are. The gonodendron budsare not exactly in phase with the gastrozooid buds, andthere are a variable number of gonodendra between gastro-zooids at maturity (but always at least one). The gonodendraof R. eysenhardti develop in the same manner as those of B.sibogae (Fig. 3).

Agalma elegans

Our observations of the zooid organization of A. elegans(Fig. 4b,c) are consistent with Totton’s (1954, frontispiece—reproduced here as Fig. 4a). Each cormidium consists, fromposterior to anterior, of a large palpon (marked as “B” inTotton’s (1954) frontispiece so it will be referred here as theB-palpon), a female gonodendron with an associatedpalpon, a gastrozooid with multiple gastric palponsattached to its peduncle, and several clusters of maleelements. Each cluster of male elements has a palpon,several male gonophores, and bracts in various stages ofdevelopment. Bracts also are spread along the full lengthof the stem. Each cormidium can span up to 4 cm or moreof the stem, and there are few developing cormidia withinthe growth zone. This makes the inference of the buddingsequence quite difficult in this species because there are

few intermediate stages of cormidial development in eachcolony. It also implies that zooids may be added to thecolony at a very slow rate.

There is not a pronounced horn (sensu, Dunn 2005) inthe siphosomal growth zone of mature specimens of A.elegans, although the tip of the siphosomal growth zonedoes form a slight overhang (Fig. 4d–j, marked “T” in eachpane). Totton (1956) noted a well-developed horn (whichhe called a nectostyle) in the larvae of this species, so itseems to recede as the colony matures. The anteriormost-differentiated cormidium consists of six buds on a commonpeduncle (as seen in cormidium 1 of the specimens figuredin Fig. 4d–g and h–j). The largest of these is a bud that wasinferred to be the gastrozooid based on its rounded shapeand consistent position relative to the other buds. There aretwo buds on the anterior side of the gastrozooid base. Theproximal bud, which was identified at various stages ofdevelopment by its anteriormost position within thecormidia, differentiates into the primary male reproductiveelements. The distal bud on the anterior side of thegastrozooid peduncle differentiates into one, or perhapsseveral, gastric palpons. There is also a bud on the left sideof the gastrozooid base at the same level as the bud of themale reproductive elements. This lateral bud was inferred tobe a bract based on the differentiation of buds in the sameposition in more posterior cormidia. Finally, there are twobuds to the posterior of each young gastrozooid. The onefurthest to the posterior had a characteristic shape evenearly in development that indicated that it was a palpon,and its precocious development and location indicate that itis the B-palpon. Between the B-palpon and the gastrozooidis a bud that in posterior cormidia differentiates into thefemale reproductive elements. The B-palpon grows largerthan all the other zooids of a cormidium by the point wherethe cormidium is second in the sequence. Totton (1954)considered the gastrozooid to be the posterior element ofeach cormidium. In light of the developmental sequence,we differ with him on this point and consider the B-palponto be the posteriormost structure.

In addition to the bract that arises on the left of the baseof each gastrozooid, there is a lateral row of bracts on boththe left and right sides of the stem. These bracts are furtherfrom the ventral midline than any other zooids and are moredeveloped than adjacent bracts arising from the base of thegastrozooids. No buds that could give rise to these lateralbracts were ever observed within the derivatives of theprobud, and the lamellae of these bracts sometimesextended along the stem slightly to the anterior of the tipof the siphosomal growth zone. These findings suggest thatthey arise directly on the side of the stem.

The male bud comes to be attached to the stemindependently from the gastrozooid, gastric palpon, andgastrozooid-associated bract, and the female bud comes to

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Fig. 4 Agalma elegans—false coloring indicates zooid identity. Ante-rior faces the top of the page, unless noted otherwise. a Left lateral viewof the mature colony (adapted from Totton 1954). The bracts, whichsheathe the siphosome, have not been colored. b Photographic collageshowing the growth zone and young siphosomal stem of an anesthetizedliving specimen. Ventral view. Note that both the female gonodendronand the female-associated palpon have been colored orange, and thatthe male gonophore and male associated bract and palpon buds arecolored blue. Buds whose fate could not be assigned with confidencehave been left uncolored. c Schematic of a mature cormidium. Ventralview. The lateral bracts are not shown in their actual position along the

stem relative to the other zooids. d–g SEM of the growth zone ofspecimen 1, shown from four different views. h–j SEM of the growthzone of specimen 2, shown from three different views. In d–j, the tip ofthe growth zone is labeled with a T, and the gastrozooids and B-palponsare numbered according to the cormidium they belong to, beginningwith the first differentiated cormidium. Gastrozooids 1–2 and B-palpon3 have been broken away (h–j). Additionally, gastrozooid 3 has beenbroken away (i). Where young gastrozooids were broken away, the budsof the gastric palpons, and sometimes the left bracts, also broke off withthem. A Anterior; D dorsal; L left; P posterior; R right; V ventral

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be attached to the stem independently from the B-palpon(this is most easily seen in Fig. 4i). Bracts later bud at thefoot of the B-palpon. After the female bud separates fromthe B-palpon, it takes on a bilobed shape (as in cormidium3 of Fig. 4i,j), and further to the posterior, one lobe wasseen to be the female-associated palpon and the other thefemale gonodendron (not shown). These are attached to thestem independent from each other at maturity. Close tothe growth zone, there is only one male cluster percormidium. In cormidia slightly to the posterior, it can beseen that a new male cluster arises directly on the stem,separate from and to the anterior of the original one. Thisprocess repeats several times, with new male clusters beingadded anterior to the existing ones.

There are multiple gastric palpons attached to thepeduncle of gastrozooids in mature cormidia. It was notdetermined if these arise by subdivision of the single gastricpalpon bud that arise within the growth zone or if eachgastric palpon arises as an independent bud on the peduncleof the gastrozooid. There are more gastric palpons incormidia further to the posterior.

Large specimens of A. elegans have an irregularorganization of zooids in their posterior cormidia, but inall examined cases, the irregularity was consistent withzooid loss. In these specimens, cormidia close to the growthzone still have the regular organization originally describedby Totton (1954) and reconfirmed here.

The nectosomal growth zone of a single specimen wasexamined using SEM. It was found that each youngnectophore had a small bud on the posterior side of itspeduncle. A similar bud has previously been found in thenectosomal growth zone of Bargmannia elongata (Dunn2005).

Nanomia bijuga

Totton (1965, Fig. 35) figured the organization of zooidswithin a cormidium of N. bijuga. He found that eachcormidium consisted of a gastrozooid and a series ofpalpons, all attached independently to the stem. A femalegonodendron and a cluster of male gonophores flankedeach palpon and alternated sides from palpon to palpon.Totton noted that secondary palpons, also with maleand female structures at their base, were sometimesintercalated between the primary palpons in maturecormidia.

Our own observations of N. bijuga confirm Totton’searlier findings. In addition, we are also able to describe thebudding sequence by which the zooids of this species arise.There is a pronounced horn within the siphosomal growthzone, and each cormidium arises as a simple probud closeto its tip (Fig. 5d). The bulk of the probud gives rise to thegastrozooid, with most of the other zooids arising on the

anterior side of its peduncle. A palpon and two flankingbracts are the first such zooids (Fig. 5e). Additional bractsare added on both sides of the gastrozooid peduncle, withthe youngest (i.e., the smallest) being the most distal(Fig. 5f). Additional palpons bud on the anterior side ofthe primary palpon, and all palpons initially share acommon base and form a fanlike structure (Fig. 5g). Theoriginal palpon is the most distal in this structure, and thesmallest (youngest) is the most proximal. A lateral bractforms on each side of each palpon as it matures.

The zooids of successively more mature cormidiacome to be spread out along the stem such that eachzooid is eventually attached to the stem independently(Fig. 5h,i). The bracts that arose on the gastrozooidpeduncle come to be arranged in two lateral rows thatflank all the other zooids (the lamellae of the left row ofbracts can be seen in Fig. 5i). The oldest (i.e., largest andmost proximal) bract to arise on the gastrozooidpeduncle comes to be located furthest to the anterior,and the youngest (i.e., the smallest and most distal)remains the closest to the gastrozooid. As the palponsspread out, the youngest (i.e., the smallest and mostproximal) comes to be located the furthest to theanterior of the cormidium, and the first palpon to arise(i.e., the largest and most distal) remains closest to thegastrozooid. The lateral bracts associated with eachpalpon move away from the ventral midline and cometo be arranged in rows just inside of the rows of bractsthat arose on the gastrozooid peduncle. Kawamura(1911) had already noted that the bracts of this speciesare arranged in two rows along each side of the stem.

The male and female gonodendra arise at the baseof the palpons just inside of each palpon-associatedbract (Fig. 5i). Additional clusters, each consisting of apalpon, a male gonodendron, a female gonodendron, andbracts, are added directly on the stem at the anterior endof each cormidium after all zooids have spread out.Clusters may also sometimes be added between existingpalpons.

There is a single tentacle on the anterior side of eachgastrozooid. Likewise, each palpon has a palpacle (as thetentacle of a palpon is called) on its anterior side.Tentacles and palpacles form as simple evaginations. Thebud of the gastrozooid tentacle can be seen in Fig. 5f–h,and the base of the tentacle, which has been broken away,can be seen in Fig. 5i. The palpacle rudiments can be seenin Fig. 5i. Some palpons also have a large basal swelling ontheir anterior side just distal to the palpacle.

Apolemia sp.

One specimen of Apolemia, belonging to the sameundescribed species as “Apolemia 3” in the molecular

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phylogeny of Dunn et al. (2005), was prepared forSEM. The siphosomal growth zone was extremely dense,with many tightly packed cormidia. There was a pro-nounced horn, and the young cormidia close to the tipwere regular in organization and appeared to arise byprobud subdivision (data not shown). The buddingsequence and mature organization of cormidia were notdetermined.

Forskalia formosa

SEM of the siphosomal growth zone of F. formosa indicatesthat the zooids arise by probud subdivision (not shown),although we did not describe the exact budding sequence.The mature cormidia consist of a posterior gastrozooid, apalpon, and a gonodendron bearing several gonopalponsand gonophores (Fig. 6). This is consistent with previous

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Fig. 5 Nanomia bijuga. Falsecoloring indicates zooid identity.Anterior faces the top of thepage in all panes. a Maturecolony (adapted from Kawamura1911). The stem is twisted, butthe view is generally lateral.b Photographic collage showingthe growth zone and youngcormidia of an anesthetized liv-ing specimen. The stem istwisted such that the youngestcormidia are shown from theirleft side, and the older (posteri-or) cormidia are shown fromtheir right side. c Schematic of asingle mature cormidium. Ven-tral view. The lateral bracts arenot shown in their actual posi-tion along the stem relative tothe other zooids. d–i SEMs ofthe youngest cormidia, shownfrom their left side in an onto-genetic sequence (from anteriorto posterior). The largest bractshave been removed, leavingonly their lamellae. d The threeyoungest cormidia at the tip ofthe horn; all other panes showonly one cormidium. D Dorsal;L left; R right; V ventral

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descriptions of the cormidia of other species of Forskalia(Pugh 2003). The gastrozooids bear multiple bracts alongtheir peduncles, and there are several small buds andjuvenile bracts adjacent to the gastrozooid. The peduncle ofthe palpon does not significantly elongate. The gonodendrawe observed were immature, so it was not possible todescribe their organization in detail. It was clear that thegonodendron arises from the base of the palpon. Thepeduncle of the gonodendron appears to be an evaginationof the stem, which at first bears only a single gonopalpon.Gonophores and further gonopalpons are added later indevelopment.

SEM of the nectosomal growth zone revealed a simplebud on the posterior base of the peduncle of each youngnectophore, as was the case for A. elegans.

Lychnagalma utricularia

We were able to make preliminary notes on theorganization of the cormidia of L. utricularia, althoughwe have not yet looked at the siphosomal growth zone. Wefound that the gastrozooids are borne on long peduncles, ashad been noted previously (Pugh and Harbison 1986). Wealso found that there is a palpon at the distal end of thispeduncle just proximal to the gastrozooid. This gastricpalpon is associated with at least one bract, and severalother bracts are scattered along the length of the peduncle.Palpons, male and female gonodendra, and bracts areattached to the stem between the gastrozooid peduncles.There were no palpons within gonodendra of either sex.Gastrozooids alternate with large palpons close to thegrowth zone.

Discussion

Siphosomal elements arise independentlyin the Cystonectae

Gastrozooids and gonodendra arise independently on thestem in the three long-stemmed cystonects examined here.The arrangement of these elements can be either uniserialor triserial (Fig. 2). The independent origin of thegonodendra and gastrozooids, as well as the variablenumber of gonodendra between gastrozooids within aspecimen, bring into question the existence of cormidia ina biologically relevant organizational sense in these taxa.

There are five valid species of cystonects in total, two ofwhich were not examined. Of these, preliminary observa-tions indicate that the siphosomal elements of Bathyphysaconifera may arise in a uniserial pattern (P.R. Pugh,personal communication). Totton (1960) made an excellentdescription of the gross organization of Physalia physalis(the Portuguese Man o’ War), the only cystonect that lacksa long stem. Although his description of colony-leveldevelopment is incomplete, it is sufficient to determine thatbudding in this species is substantially different from that inthe long-stemmed cystonects examined here.

Probud subdivision is a derived, shared modeof development for the Codonophora

The present study has described the origin of cormidia byprobud subdivision in A. elegans and N. bijuga, andestablished that it is the mode of cormidial developmentin F. formosa and an undescribed species of Apolemia. Thephylogenetic distribution of these taxa, and of those forwhich previous descriptions of colony-level developmentare available, indicates that probud subdivision is asynapomorphy of the Codonophora (Fig. 7).

Small buds were found to the posterior of youngnectophores in both of the Codonophora taxa whosenectosomal growth zones were examined (A. elegans andF. formosa). Such a bud was previously found in thenectosome of B. elongata (Dunn 2005), and the necto-phores of Apolemia are known to alternate with nectosomalpolyps (Totton 1965). These findings indicate that theorganization of the nectosome is more complex than hadpreviously been appreciated. It may be that the nectosomearose by tandem duplication of the siphosome andsubsequent simplification.

The developmental underpinnings of organizationaldiversity in the Codonophora

The molecular phylogeny (Dunn et al. 2005) suggests asingle transition from dioecy to monoecy within the

1 mm

A

PD V

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Gonopalpon

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Fig. 6 Schematic of a single cormidium of Forskalia formosa shownfrom the right side. Multiple bracts are attached along the gastrozooidpeduncle (not shown). The gonodendron is immature. A Anterior;D dorsal; L left; P posterior; R right; V ventral

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Codonophora (Fig. 7), although the relevant nodes are notwell supported, and this hypothesis should be treatedprovisionally until more data are available. Within adioecious species, colonies of different sexes are organiza-

tionally the same and differ only in the sex of thegonophores (Dunn 2005). Monoecious colonies are morecomplex in that both male and female gonophores must beorganized along the stem. In the Calycophorae, each

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Cystonectae

“Physonectae” Calycophorae

Codonophora

Origin ofsiphosome,pneumatophore

Newly examined taxaPreviously examined taxa

Bayesian posterior > 0.90

Origin ofnectosome,pro-bud subdivision

Cormidial paedomorphy;Loss ofpneumatophore

Reiteration ofanterior-mostcormidial element

Transition fromdioecy tomonoecy

Phylogeny Key

Each cormidiumarises as a single pro-bud

Gastrozooids and gonodendra arise as seperate buds

Gonodendra buds subdivide into reproductive zooids

Gastrozooid bud

Cormidium

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The zooids of eachcormidium arise by subdivision of the pro-bud

The zooids of each cormidium spread out along the stem

Each cormidiumarises as a single pro-bud

The zooids of eachcormidium arise by subdivision of the pro-bud

The zooids of each cormidium remain in a cluster

Pro-bud

A

DV

P

Fig. 7 Hypothesized phylogenetic reconstruction of the history ofseveral important transitions in siphonophore colony-level organi-zation. Those taxa that have been examined in sufficient detail todetermine the presence or absence of probud subdivision areindicated with boxes. The rooted molecular phylogeny (using datafrom 16S and 18S) is from Dunn et al. (2005). The topology of thetree presented here differs from that in Dunn et al. (2005) in that the

basal polytomy in the sister group to Apolemia has been resolved toshow the monoecious species as being monophyletic. The previousmolecular data were consistent with this topological hypothesis, butdid not favor it more than other hypotheses where monoecy arosemore than once. Bathyphysa sibogae was not included in themolecular phylogenetic analysis; in this figure, it is shown with theother cystonects

752 Dev Genes Evol (2006) 216:743–754

cormidium bears only male or female gonophores (Totton1965). Within the monoecious “physonects”, each cormi-dium bears male and female gonophores. The organiza-tion and developmental origins of the male and femaleelements are very different in the two monoecious“physonects” examined in detail here, so better descrip-tions of colony-level organization in other monoecious“physonects” will need to be made before it is possibleto make specific hypotheses regarding the origin (ororigins) of monoecy.

The events following probud subdivision differ some-what between the “Physonectae” and the Calycophorae.In the “Physonectae”, the zooids within a cormidiumspread out along the stem, whereas in the Calycophorae,the zooids of a cormidium remain attached to the stemby a common peduncle. The molecular phylogenyindicates that the calycophoran state is derived (Fig. 7).This derived calycophoran arrangement could have arisenby cormidial paedomorphosis, whereby cormidial devel-opment is truncated before the expansion of zooids alongthe stem, although the zooids within the cormidia maturefully.

Siphosomal budding has now been described in threephysonect species, B. elongata, A. elegans, and N. bijuga.Agalma elegans and N. bijuga both belong to theAgalmatidae sensu stricto and are more closely related toeach other than to B. elongata (Dunn et al. 2005). Thecormidial organizations of A. elegans (Fig. 4c) and N.bijuga (Fig. 5c) are quite different. They are, however,developmentally similar in that some zooids are intercalatedat well-defined locations within the cormidium and arisedirectly on the stem. In both of these species, theintercalated structures are reiterations of the anteriormostderivative of the probud (this is the cluster consisting of apalpon, bracts, and male gonophores in A. elegans and thecluster consisting of a palpon, male and female gonophores,and bracts in N. bijuga). Even when zooids arise directly onthe stem outside of the growth zone, they do so at well-defined locations within the cormidia.

Directional asymmetry of siphonophore colonies

Dunn (2005) described the directional asymmetry of thecormidia of B. elongata, noting that many other descrip-tions of directional asymmetry are scattered throughout thesiphonophore systematics literature. No directional asymmet-ries were identified in any of the Cystonectae examined here.Within the Codonophora, no directional asymmetries werefound in N. bijuga, but there was a bract on the left side ofeach gastrozooid in A. elegans. Although data on directionalasymmetries are still sparse, these findings suggest thatdirectional asymmetry has been gained and/or lost multipletimes within the siphonophores.

Conclusions

Comparisons between the Codonophora and Cystonectae,the broadest contrast that can be made among extantsiphonophores, reveal some key differences in colony-levelorganization and development. The same two reiteratedelements, the gastrozooids and gonodendra, are present inall long-stemmed cystonects. These elements arise inde-pendently, and there are a variable number of gonodendrabetween gastrozooids. The gonodendra, which consist ofthree types of zooids, are the most complex structures in thecolony. The gonodendra arise by subdivision of thegonodendron bud, but this subdivision has nothing to dowith the organizational context in which the gastrozooidsarise. In the Codonophora, the organizational context of allsiphosomal zooids is established by the process of probudsubdivision that gives rise to cormidia. The cormidialorganization of the Codonophora is extremely diverseacross species (with respect to both the types of zooidspresent and the position of the zooids relative to eachother), although it is highly consistent within species.

There is little known ecological diversity within thelong-stemmed Cystonectae (all species are piscivorous),and there are only five extant cystonect species in total(P. physalis, the fifth species, being clearly morphologicallydistinct but still poorly known). The great morphologicaldiversity of the Codonophora is accompanied by largeecological diversity (with respect to prey and habitat), andthe Codonophora are much more speciose than their sistergroup, the Cystonectae. These patterns in diversity make ittempting to speculate that probud subdivision may havebeen a key colony-level developmental innovation thatopened up a complex morphospace that is inaccessible toother colonial animals. Among other things, this morpho-space includes an increased number of functionally spe-cialized zooids and greater flexibility in the way thesezooids are organized relative to each other.

It is possible that the organizational mechanisms at playin probud subdivision did not arise de novo but wereco-opted from zooid development. The development of azooid from a bud (whether that bud arises independentlyor by probud subdivision) requires the specification offields that specify the location of various zooid structures.It may be that these already-existing fields are used in theprobud to specify where the different zooids will ariseduring subdivision. Future mechanistic and molecularstudies of colony-level and zooid-level development inthe siphonophores will provide an opportunity to test thishypothesis.

Acknowledgements This work was supported by a mini-PEETgrant from the Society for Systematic Biology, a National ScienceFoundation Graduate Research Fellowship, and a National ScienceFoundation (NSF) doctoral dissertation improvement grant (DEB-

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0408014). Thanks to S.H.D. Haddock, J. Godfrey, L. Madin, C.Sardet, and C. Carré for their assistance with collecting siphono-phores. E. Edwards provided valuable comments on the text. Specialthanks to P. Pugh for making his specimens available for this work, forproviding feedback on the manuscript, for checking some of theobservations presented here, and, in particular, for our manyilluminating conversations.

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