Complex Colony-Level Organization of the Deep-Sea Siphonophore Bargmannia Elongata (Cnidaria, Hydrozoa) Is Directionally Asymmet

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DEVELOPMENTAL DYNAMICS 234:835–845, 2005

RESEARCH ARTICLE

Complex Colony-Level Organization of the Deep-Sea Siphonophore Bargmannia elongata (Cnidaria, Hydrozoa) Is Directionally Asymmetric and Arises by the Subdivision of Pro-Buds

Casey W. Dunn*

Siphonophores are free-swimming colonial hydrozoans (Cnidaria) composed of asexually produced multicellular zooids. These zooids, which are homologous to solitary animals, are functionally specialized and arranged in complex species-speciﬁc patterns. The coloniality of siphonophores provides an opportunity to study the major transitions in evolution that give rise to new levels of biological organization, but siphonophores are poorly known because they are fragile and live in the open ocean. The organization and development of the deep-sea siphonophore Bargmannia elongata is described here using specimens collected with a remotely operated underwater vehicle. Each bud gives rise to a precise, directionally asymmetric sequence of zooids through a stereotypical series of subdivisions, rather than to a single zooid as in most other hydrozoans. This initial description of development in a deep-sea siphonophore provides an example of how precise colony-level organization can arise, and illustrates that the morphological complexity of cnidarians is greater than is often assumed. Developmental Dynamics 234: 835–845, 2005. © 2005 Wiley-Liss, Inc.

Key words: Bargmannia; siphonophore; Hydrozoa; Cnidaria; directional asymmetry; major transitions; deep sea; colonial animal

Received 2 March 2005; Revised 2 May 2005; Accepted 5 May 2005

INTRODUCTION which asexually produced individuals colonial taxa show intraspecific vari- remain attached and physiologically ability in zooid arrangement and Most studies of animal development integrated throughout their lives gross colony morphology, such that no have focused on the embryonic devel- (Hughes, 2002). There is a diversity of opment of solitary taxa. There are, two colonies are exactly alike (Board- however, other modes of development organizational complexity across colo- man et al., 1973). Other taxa, espe- with different starting and end points nial taxa (Beklemishev, 1969). Some cially those that are pelagic (i.e., that that have remained largely neglected. colonies consist of functionally equiv- live in the water column rather than These include agametic clonal devel- alent zooids (see Table 1 for defini- affixed to a substrate), have invariant opment, in which a new animal arises tions of the specialized terms used colonial organizations that are en- from another animal through fission throughout this report) while others tirely consistent from colony to colony or budding, and colonial development, manifest a marked division of labor of the same species (Mackie, 1986). a variation on clonal development in between zooids (Leuckart, 1851). Most The zooids of most colonial taxa do

Department of Ecology and Evolutionary Biology, Yale University, New Haven, Connecticut Grant sponsor: NSF; Grant number: DEB-0408014. *Correspondence to: Casey W. Dunn, Department of Ecology and Evolutionary Biology, Yale University, PO Box 208106, New Haven, CT 06520-8106. E-mail: [email protected] DOI 10.1002/dvdy.20483 Published online 28 June 2005 in Wiley InterScience (www.interscience.wiley.com).

TABLE 1. Deﬁnitions for Some of the Specialized Terminology Used

Term Definition Bract A gelatinous, shield-like zooid Colonial animal An animal that exists as a series of asexually produced and physiologically integrated zooids; each colony arises from a single zygote and is genetically uniform (baring mutation or fusion with another colony) Cormidium A single iteration of the regularly repeating pattern of zooids found in the siphosome of siphonophores Gastrozooid Polyp specialized for feeding; bearing a single tentacle in siphonophores Gonozooid Specialized polyps that bear the gonophores Gonophore Medusae specialized for reproduction; lacking feeding structures Horn The protuberance within the siphosomal growth zone where the cormidia form Medusa One of two types of cnidarian zooids; familiar solitary medusae include the “true” umbrella- shaped jellyfish Nectophore Medusa specialized for propulsion; lacking feeding and reproductive structures Nectosome The region of a siphonophore colony that bears nectophores Polyp One of two types of cnidarian zooids; solitary cnidarian polyps include Hydra and sea anemones Pneumatophore Gas-filled float at the anterior end of many siphonophores; not a zooid, arises developmentally as an aboral invagination of the embryo (Carre´, 1967) Pro-bud The first bud to arise in the developmental sequence that gives rise to the cormidia of the siphosome Siphosome The region of the colony that bears all zooids except the nectophores Stem The central stalk to which all the zooids are attached; linear in B. elongata; arises developmentally via the elongation of the body column of the first polyp that forms during embryogenesis (Gegenbaur, 1853) Tentaculozooid A zooid that is presumed to be a polyp with an atrophied body and a single hypertrophied tentacle Zooids The units, each of which are homologous to other free living solitary animals, that make up animal colonies; these can be polyps or medusae in cnidarian colonies such as siphonophores

not change positions within the col- and include the longest animals in the try that consistently occur in the same ony, so the geometry and dynamics of world, with colonies of some species direction, and are found throughout the budding process have a direct ef- exceeding 40 m in length (Robison, the Bilateria (Neville, 1976). They in- fect on the arrangement of zooids and 1995). The zooids and colonies of most clude the displacement of the heart to on overall colony shape. Both microen- siphonophores have an organization the left in humans, the well-defined vironment (reviewed by Harvell, that is bilaterally symmetric at a first chirality of most spiraled gastropod 1994) and internal parameters, such approximation (Totton, 1965; see shells, and the consistent asymmetry as the dynamics of gastrovascular Haddock et al., 2005, for a discussion of the nervous system in Caenorha- fluid flow (Blackstone and Buss, 1993; of siphonophore organization and the biditis elegans (Hobert et al., 2002). Dudgeon and Buss, 1996), have been terminology used to describe the ma- The directional asymmetries de- shown to influence the development of jor axes). This is not surprising, as it scribed in the siphonophore systemat- colonies with variable form. To date, has long been known that many cni- ics literature have never been consol- little is known about the developmen- darians show marked bilateral sym- idated and have escaped wider notice. tal mechanisms of those taxa with in- metry (e.g., Delage and Herouard, They do, however, indicate that the variant organization. At the very 1901; Hyman, 1940; Beklemishev, symmetry of at least some cnidarians least, a description of their budding 1969; Martindale et al., 2002). Bilat- can be of the same order as that of the process is required before the mecha- eral organization is not unique to the most derived Bilateria. This raises nisms that generate precise colony- “Bilateria,” the monophyletic group of questions as to how many times direc- level organization can be investigated. animals that includes almost all tional asymmetries have evolved in The siphonophores (Fig. 1), a group model systems, as is often misstated animals, and how old they are. It is of about 160 described species of pe- or implied (e.g., Meinhardt, 2001). still not clear if homologous develop- lagic hydrozoans (Cnidaria), have the The zooids and colonies of many si- mental axes even exist in the Cnidaria highest division of labor between zoo- phonophores are directionally asym- and Bilateria, though expression data ids and the most precise organization metric (e.g., Totton, 1932; Stepan- are largely consistent with the hy- of all colonial animals (Beklemishev, jants, 1967; Pugh and Youngbluth, pothesis that they do (Hayward et al., 1969, p 83). Siphonophores are among 1988; Pugh and Pages, 1997; Map- 2002; Finnerty et al., 2004). The axes the most abundant carnivores of the stone, 2003). Directional asymmetries of siphonophore colonies are labeled oceans’ macroplankton (Pugh, 1984) are deviations from bilateral symme- with the same names as the axes of COLONY-LEVEL DEVELOPMENT OF A SIPHONOPHORE 837

Haeckel (1869b), establishes two growth zones that are responsible for further colony-level development (Fig. 1). These growth zones are the sites of both stem elongation and the budding process that gives rise to new zooids throughout the life of the organism. One growth zone gives rise to the nectosome, a region that bears the propulsive asexual medusae called nectophores. The other growth zone gives rise to the more complex siphosome, a region that contains all other types of zooids, including those for feeding, reproduction, and defense. The zooids of the siphosome are organized along the linear stem in a species-specific repeating pattern, each iteration of which is called a cormidium. The Codonophora contains two his- torically recognized groups of siphonophores (Dunn et al., 2005b). These are the Physonectae, a grade, and the Ca- lycophorae, which is monophyletic and nested within the Physonectae (Fig. 2). There is a large diversity of colony-level organization in the siphosome of the Physonectae, while all of the Calycophorae have a similar siphosomal structure (Bigelow, 1911; Totton, 1954, 1965), which their phylogenetic position indicates is derived and secondarily simplified. Siphoso- mal budding has only been described in detail for two Codonophora species, both of which are calycophorans. Chun (1885) found that each cormidium arises as a single bud in Sphaeronectes gracilis, and Schneider Fig. 1. Simplified schematic overview of a siphonophore. Parts not shown to scale, or in their (1896) described some zooids as aris- actual numbers. Some siphonophores lack a pneumatophore, while others do not have a necto- ing as independent buds in Abylopsis some. Although the nectophores are arranged biserially, they are all attached in a line along one tetragona, though his figures are not side of the stem. completely clear on the matter. In a later review of these studies, Garstang (1946) raised several issues bilaterian animals (reviewed by Had- o’ War. The embryology of the cys- with Schneider’s findings, and con- dock et al., 2005), but it should be tonects is entirely unknown. Totton cluded that the subdivision of buds noted that this is merely a semantic (1960) described several features of the was a general mechanism of colony- convenience and no homologies are budding process of mature P. physalis level development in the Calycopho- implied by this nomenclature. colonies and showed that it is highly rae. He also coined the term “pro-bud” A recent molecular phylogeny (Dunn derived and fundamentally different for the bud that gives rise to the mul- et al., 2005b) helps organize what is al- than any other siphonophore, including tiple zooids of a cormidium. ready known about the colony-level or- the other cystonects. As such, it is diffi- Although it is critical to under- ganization and development of siphono- cult to apply the developmental find- standing the development of the an- phores. Siphonophores are divided into ings from this species to other taxa. cestral Codonophora, the budding pro- two monophyletic groups, the Cystonec- The other monophyletic group, the cess in the physonects has proven tae and the Codonophora (Fig. 2). The Codonophora, contains the bulk of si- particularly problematic to study be- Cystonectae is a small group of only five phonophore species. Their embryolog- cause “there is so much crowding to- valid species, which include Physalia ical development, which was first ob- gether of the siphosomal buds that it physalis, the familiar Portuguese Man served by Gegenbaur (1853) and makes observation very difficult” (Tot- 838 DUNN ton, 1954, p 22). The organization of all zooids within a mature cormidium has not even been described for any physonect. Totton (1965) noted that the siphosomal zooids of physonects arose on a protuberance in the growth zone rather than directly on the stem. Garstang (1946) suggested that each zooid of the physonects arises as an independent bud, but it is not clear how he arrived at this conclusion because he did not name any sources that describe the budding process in detail. The phylogenetic positions and derived colony organizations of the taxa that have been examined to date leave wide gaps in our knowledge of the colony-level development of siphonophores. Descriptions of colony structure and budding in physonects Fig. 2. A rooted siphonophore phylogeny, simplified to show the relationship of the taxa and other cystonects are essential if discussed here. Physalia physalis is in the Cystonectae, Abylopsis and Sphaeronectes are in the we are to understand the evolution, Calycophorae. This cladogram is based on an analysis by Dunn et al. (2005b) of sequence data development, and origin of colony- from the 16S and 18S ribosomal RNA genes. Support is shown as (Bayesian posterior probability level complexity, as well as symmetry, x 100)/(maximum likelihood bootstrap score). of siphonophores as a whole. The complex organization of siphonophores indicates the existence of a are so fragile that their zooids often highly canalized colony-level develop- dissociate during collection and pres- mental mechanism without parallel in ervation (Pugh, 1989; Dunn et al., other animals (Garstang, 1946), and 2005a), and the resulting lack of in- provides an opportunity to explore the tact material has largely precluded evolutionary origins of biological com- the study of the symmetry properties, plexity in a novel context. Haeckel colony-level organization, and devel- (1869a) recognized this, and made ex- opmental processes that make sipho- plicit comparisons between special- nophores interesting in a broader de- ized cells in multicellular organisms velopmental and evolutionary context. and specialized zooids in siphono- Modern advances in oceanographic phore colonies. While complex multi- technology, however, alleviate the col- cellular organisms arose via the pre- lecting problems that limited all precise organization of functionally vious work on siphonophores (Had- specialized cells in space and time, si- dock, 2004). phonophores arose by taking the pro- The present study investigates the cess one step further and organized colony-level organization and develop- functionally specialized multicellular ment of a siphonophore, Bargmannia organisms into precise patterns. In- elongata (Fig. 3), using specimens col- terest has recently been rekindled in lected with a remotely operated un- how new levels of biological organiza- derwater vehicle (ROV) deployed from tion arise (Buss, 1987; Michod, 2000), an ocean-going research ship. The Fig. 3. View of a living Bargmannia elongata and this growing field now often goes general colony form of B. elongata (an colony. Photograph courtesy of Steve Had- under the name “the major transitions elongate siphosomal stem, two growth dock. The entire colony is about 40 cm long. in evolution” (Maynard Smith and zones, multiple identical nectophores, Szathma´ry, 1995). Even so, there has the possession of a gas-filled pneu- only been occasional recent mention of matophore) is plesiomorphic for the RESULTS siphonophores in this context Codonophora (Dunn et al., 2005b), un- Collecting Bargmannia (Mackie, 1963; Winsor, 1971; Gould, like the colony form of the calycoph- elongata 1987; Wilson, 2000), and these ani- orans that have previously been inves- mals have remained poorly known tigated. This makes B. elongata a good Nineteen specimens were collected by and largely forgotten in modern times. departure point for understanding the ROV Tiburon from depths of This is because siphonophores live in colony-level evolution and develop- 350–2,865 m. All collection sites were the open ocean, with many species be- ment, and symmetry properties, of within 337 km of Moss Landing, Cal- ing found only in the deep sea. They other siphonophores. ifornia. Most specimens of Bargman- COLONY-LEVEL DEVELOPMENT OF A SIPHONOPHORE 839

Fig. 4. The organization of the mature cormidia of Bargmannia elongata (ventral view, anterior up, the left side of the stem faces the right of the page). P, posterior; A, anterior; R, right; L, left. a: Photograph of a length of mature stem. The white mature gonophores can be seen on the gonozooids. The largest structures are the gastrozooids. b: Illustration of the organization of a cormidium. The gastrozooid shown at the top of the illustration belongs to the next cormidium to the anterior, and the gonozooid shown at the bottom of the illustration belongs to the next cormidium to the posterior. Bpl, Blg, Brg, and Br have been dissected away, leaving only their lamellae. S, stem; G, gastrozooid; T, tentaculozooid; R, gonozooid; Br, right lateral bract; Bpl, posterior left lateral bract; Bal, anterior left lateral bract; Brg, right gastrozooid-associated bract; Blg, left gastrozooid-associated bract. The unlabeled buds on the stem were inferred to be reserve bracts. nia elongata were not visibly damaged all the zooids of mature cormidia were gonozooid (anterior left lateral bract, during sampling, and survived the as- attached to the stem independently. Bal) and one to the posterior of the ten- cent to the surface intact. If not dis- The organization of the zooids taculozooid (posterior left lateral bract, turbed, they lasted for up to 2 days in within the cormidia was entirely reg- Bpl). There was only one lateral bract a large volume of sea water (Ͼ6lat ular. The gastrozooid, tentaculozooid, on the right side of the stem (right lat- 4°C in the dark) before sinking to the and gonozooid were arranged from eral bract, Br), and its lamella was bottom of the vessel and disintegrat- posterior to anterior within each cor- longer than those of the bracts on the ing. They would not feed in these ves- midium (Fig. 4). While the gastrozooid left. These three lateral bracts were fur- sels, so dynamic observations of and tentaculozooid were on the ven- ther from the ventral midline of the growth were not possible. Instead, the tral midline, the gonozooid was dis- stem than any of the other zooids. Two developmental process was inferred placed towards the left of the stem. other bracts were located on the ante- from the morphology of the growth The gonozooid was adjacent to the rior side of the gastrozooid, one slightly zones, each of which had a complete gastrozooid of the next younger cor- to the right (right gastrozooid-associ- ontogenetic sequence of developing midium to the anterior, so there was ated bract, Brg) and the other slightly to cormidia. much more space within cormidia the left (left gastrozooid-associated than between them (the stem is highly bract, Blg). The lamellae of the gastro- The Siphosome: Cormidial extensible, so it was not possible to zooid-associated bracts ran mostly Organization make absolute measurements of these along the stem, but also ran a slight Each mature cormidium consisted of a distances). There was an annular con- distance up the peduncle of the gastro- gastrozooid, a tentaculozooid, a gono- striction in the stem at the point of zooid. There was a single small bud to zooid, ﬁve mature bracts, and several attachment of each gastrozooid. the inside (i.e., towards the ventral mid- small buds. This is consistent with the The bracts had to be plucked away in line) of the lamella of each lateral bract, zooid inventory described for this spe- order to make observations of the stem, and a single bud between the lamellae cies by Pugh (1999). All of the gono- but the muscular lamella where each of the two gastrozooid-associated phores of each colony were of the same had been attached was clearly visible. bracts. In some specimens, these buds sex, conﬁrming that this species is dio- There were two lateral bracts attached were mature enough to see that they ecious. Aside from the gonophores, to the left side of the stem in each cor- were reserve bracts. which were borne by the gonozooids, midium, one to the posterior of the All 19 of the Bargmannia elongata 840 DUNN

Fig. 5. Bargmannia elongata siphosomal growth zone. a: Scanning electron micrograph of the siphosomal growth zone (view from left, anterior up, ventral to the left of the page). b: Schematic of the siphosomal growth zone. The site of the youngest pro-buds is indicated with a gray circle. A gray line indicates the path of the developing zooids, which are organization in an ontogenetic sequence. c–i: Cormidia in various stages of development. The gray line indicates the order of the ontogenetic sequence (with the youngest cormidia shown being the closest to the circle). The view is indicated in parentheses for each pane. The orientation of the axes is shown to the right of each row and below each figure. P, posterior; A, anterior; V, dorsal; D, dorsal. j: The inferred lineage diagram of zooid origin. Gastrozooids have been broken away in g–i and most of a to allow for a clear view. The pictured specimens are: a, c: Tib675D5; d,e,f: Tib596SS12; g, h: Tib598SS6; i: Tib595D5. P, pro-bud; F, footbud; F1,F2, daughters of F; F21, F22, daughters of F2; G, gastrozooid; T, tentaculozooid; R, gonozooid; Br, right lateral bract; Bpl, posterior left lateral bract; Bal, anterior left lateral bract; Brg, right gastrozooid-associated bract; Blg, left gastrozooid-associated bract. specimens examined were found to de- The Siphosome: The Growth the horn introduces some nomencla- viate from bilateral symmetry as de- Zone and Budding Sequence tural complexity for describing organi- scribed above (gonozooid displaced to- zational axes. Throughout the present wards the left, two left lateral vs. one The youngest cormidia were found on a report, these axes are employed, in ref- right lateral bract). The probability of protuberance, here called the horn. It erence to the horn, as if the horn were these deviations occurring in the same was curled into a sinister coil of about straightened out into a linear anterior direction due to chance alone is (0.5)n-1, one and a half turns, with a radius on projection of the siphosomal stem. The which in a sample of 19 is less than the order of 0.5 mm, and bore the sipho- outer zooid-bearing surface of the horn 3.82 x 10-6. We can conclude, then, that somal elements along its outer side is continuous with the ventral zooid B. elongata is directionally asymmetric. (Fig. 5a,b). The spiral arrangement of bearing side of the siphosomal stem, COLONY-LEVEL DEVELOPMENT OF A SIPHONOPHORE 841 and is, therefore, referred to as ventral. veloped a bisecting furrow and split developing cormidium, but no cor- The tip of the horn is taken to be ante- into two buds, F1 proximally and F2 midia were observed in the necessary rior, and left and right are defined in distally (Fig. 5e). Each of these split stage of development to determine relation to these two axes (as they are again. this with certainty. This bud could be for the colony as a whole). It was not possible to describe every traced from cormidium to cormidium The siphosomal elements were ar- developing cormidium in detail to the by its location as the proximal-most ranged in a linear ontogenetic se- posterior of this point. While the dis- zooid on the right side, and was found quence, with structures to the poste- tance between cormidia did not in- to mature into the right lateral bract rior being more mature. The ventral crease much, the bulk of the various (Br, Fig. 5g–i). tract of maturing zooids was continu- products of the pro-buds did and they Both of the products of F2, here ous, and there was no boundary or became crowded together. Even after called F21 and F22, were found to be discrete transition zone between the physically breaking away the gastro- intermediate buds that split again zooids on the horn and those on the zooids, which became larger than the into products that are here called F21 rest of the siphosomal stem. Serially other zooids, the developing zooids and F22. The products of F21, which arranged pro-buds (P), all of the same were too densely packed to see how were identified by their position to the type, were found at the anterior end of they were attached relative to each anterior left of F22 and to the right of the horn (Fig. 5c). The least developed other. Entire cormidia at various the anterior left lateral bract bud, pro-buds were at the very tip. In some stages of development had to be re- were found to be the tentaculozooid specimens, the youngest pro-buds moved so that the structure of adja- (T) and the posterior left lateral bract were simple transverse ridges. In cent cormidia still attached to the (Bpl). The tentaculozooid, like the other specimens, no regular ridges stem could be observed. This was most gonozooid, had a very distinctive mor- were seen, and the smallest buds were easily carried out on the dried SEM phology that could be used to trace it closely packed at the tip of the horn. specimens, rather than in the hy- from cormidium to cormidium. Soon Static observations of the ontoge- drated material. The initial break after arising, its peduncle elongated netic sequence of siphosomal elements along a given stretch of stem usually slightly. It then formed a swelling and indicated that each pro-bud develops damaged several cormidia, and it was a posterior-facing hook arose at its into a single cormidium. The complex necessary to remove adjacent buds un- distal end. This hook continued to organization of the cormidia of Barg- til an intact cormidium was fully ex- elongate into the tentacle. mannia elongata results from a ste- posed (this became easier as a wider F22 was identified in cormidia at reotypical series of divisions of the gap was opened up). While this strat- various stages of development by its pro-bud into multiple zooid buds. The egy did allow for the detailed descrip- position adjacent to the bud of the zooids of each cormidium were all at- tion of cormidia in various stages of right lateral bract (Br). Shortly to the tached to the stem by a common pe- development, it left gaps in the onto- posterior of the point where it could be duncle early in development, and only genetic series that had to be interpo- first identified, it took on a bi-lobed later in development (i.e., further to lated by keeping track of the relative shape. Further to the posterior, where the posterior) do they come to be at- positions of the various zooids and by the zooids began to become attached tached independently to the stem. It identifying distinctive features of the independently to the stem, two buds was not possible to consistently stage various zooids as they matured. were seen at its former position. These cormidia by counting posteriorly from The two products of the division of were the last two buds to form, and the tip of the horn, as the difference in F1 were found to be the gonozooid (R) their position relative to the other zoo- maturity between adjacent cormidia and the anterior left lateral bract ids indicated that they were the gas- was not the same from specimen to (Bal). They were displaced further to trozooid-associated bracts (Brg, Blg). specimen. Scanning electron micros- the left than the products of F2. The A lineage diagram was constructed copy (SEM) proved to be the most ef- gonozooid could be readily identified from these inferred aspects of the bud- fective tool for examining the earlier by its distinct shape. Not far to the ding process (Fig. 5j). stages of development. The youngest posterior of the point where the gono- Not far to the posterior of where the cormidia (i.e., those at the anterior zooid originated, it became elongate buds of the two gastrozooid-associated end of the horn) were completely ex- and pear-shaped, then elongated fur- bracts formed, there was enough posed and the developmental se- ther and gave rise to the gonophore space between the spreading zooids quence could be observed by compar- buds at its distal end. The anterior left that their organization could be ing each cormidium with its lateral bract bud remained small, and readily observed without breaking neighbors. Proceeding to the poste- did not begin to mature until much away any cormidia (Fig. 5i). The cor- rior, the pro-buds became more elon- later. It was identified in developing midia of specimens that had not been gate and then formed a swelling on cormidia by its position at the base of prepared for SEM could also be exam- the left anterior side of their base. the gonozooid. ined in detail to the posterior of this This swelling enlarged and became A bud at the right base of the devel- point. The bracts did not mature at the footbud (Fig. 5d, F). The portion of oping cormidia was first observed in the same rate as each other. The ma- the pro-bud distal to the footbud was cormidia where the gonozooid was turing right lateral bract was the larg- seen, further to the posterior, to be- taking on its pear-shaped appearance. est in each cormidium, followed in or- come the gastrozooid (G), including its The disposition of this bud suggested der of decreasing size by the posterior single tentacle. The footbud then de- that it arose from the peduncle of the left lateral bract, the gastrozooid-as- 842 DUNN sociated bracts, and finally the anterior left lateral bract. In the final stages of the development of each cormidium, the reserve bract buds formed on the stem just to the inside of the bracts, the annular constriction formed in the stem at the attachment point of each gastrozooid, and the gonophores matured at the distal end of the gonozooid. Every 7th to 10th gastrozooid grew larger and darker than the rest.

The Nectosome Bargmannia elongata has multiple identical nectophores (Pugh, 1999) that are all attached in a line on one side of the stem, though they come to be arranged biserially through the ﬂexing of their peduncles (Figs. 1, 3). The nectophores were found to be attached to the opposite side of the stem as the siphosomal zooids (i.e., dor- sally). The nectosomal growth zone Fig. 6. Scanning electron micrograph of the Bargmannia elongata nectosomal growth zone (view was relatively simple (Fig. 6). The from left, anterior up, dorsal to the right of the page). The arrow indicates one of the buds that form youngest nectophores (i.e., those fur- to the posterior of each nectophore. N, developing nectophore. thest to the anterior) were only slight protrusions, which, further to the pos- elongata arises as a single pro-bud at origin of cormidia via pro-bud subdivi- terior, became elongate and then the tip of the horn, and the structure sion in taxa outside of the Calycopho- formed a peduncle. A second bud was of the cormidium is established rae raises the possibility that it is a found on the posterior side of each through the stereotypic subdivision of general mechanism of development in nectophore peduncle. This bud was this bud into multiple zooids. The the Codonophora, rather than being not noted in the mature portion of the name “pro-bud subdivision” is sug- restricted to the Calycophorae as nectosome, which contained only nec- gested for this mechanism of cor- Garstang believed. Only one species tophores and no other types of zooid. midial development. The shape and has been investigated here, so this hy- orientation of some young pro-buds pothesis cannot be tested until colony- (those that arose as ridges) suggests level development is described in more DISCUSSION that they may be generated by buck- taxa sampled across the Codonophora. The Structure of the Growth ling caused by uneven growth on the While most mature zooids of the zones and Colony-Level curved surface of the horn. Recent physonects are attached indepen- Development in Bargmannia studies indicate that a biophysical dently to the stem, the zooids of each mechanism of this type may initiate calycophoran cormidium are closely elongata the leaf primordia of angiosperms on associated and usually attached by a The present study has found that the the curved surface of the apical mer- common peduncle (Totton, 1965). This siphosomal zooids of Bargmannia istem (Green et al., 1996; Dumais and difference in mature organization may elongata arise on a protuberance Steele, 2000). Morphogenesis result- be what led Garstang to incorrectly within the siphosomal growth zone. ing from the uneven physical stresses infer that physonect buds arose inde- Totton (1965, p 36) suggested that this that can be associated with growth on pendently on the stem. The calycopho- was the general case for physonect si- a curved surface may prove to be im- ran organization of zooids could be de- phonophores. Haeckel (1888, p. 279) portant in very different biological rived via paedomorphosis from the originally described this protuberance systems. type of developmental found in B. in Athorybia, but called it the “necto- The mechanism of zooid formation elongata if zooids were to fail to style” because he mistook it as part of by pro-bud subdivision observed here spread out along the stem after differ- the nectosome rather than as an inte- in Bargmannia elongata is consistent entiation. gral feature of the siphosomal growth with Chun’s (1885) observations of The nectosomal growth zone of zone. To avoid the mistaken connota- budding in the calycophoran Spha- Bargmannia elongata did not have a tions of this original name, the term eronectes gracilis, but contradicts the pronounced horn. There was a small “horn” has instead been used through- assertion made by Garstang (1946) bud immediately to the posterior of out the present report. that all the zooids of physonects arise each developing nectophore, though it Each cormidium of Bargmannia as independent buds on the stem. The did not grow into a mature zooid. The COLONY-LEVEL DEVELOPMENT OF A SIPHONOPHORE 843

Apolemiidae, which are sister to all spective, could help differentiate be- case, then it may also be that the left/ other Codonophora (Fig. 2), have ma- tween these two hypotheses. right patterning mechanisms respon- ture polyps adjacent to each necto- sible for directional asymmetries are phore (Totton, 1965). No structures Directional Asymmetry in also shared by the Bilateria and Cni- other than nectophores have been daria, and that these mechanisms are Siphonophores found in the nectosome of other sipho- much older than currently believed. nophores until the present study. If Directional asymmetries are common Alternatively, siphonophores and the the small nectosomal buds of B. elon- within the Bilateria, and have long Bilateria may have independently gata are vestigial polyps, then the been of interest to comparative biolo- gained mechanisms for establishing most parsimonious reconstruction of gists (Palmer, 2004). They are also directional asymmetries. The appar- the history of the nectosome would in- medically important because about 1 ent lack of directional asymmetries in dicate that the common ancestor of all in 5,000 humans are born with varia- other cnidarians would seem to favor Codonophora possessed a nectosome tions from normal directional asym- the latter hypothesis. But, as for si- with both polyps and nectophores. metries that can seriously impact phonophores, directional asymmetries This would have important implica- health (Casey and Hackett, 2000). may already have been described in tions for the origin of the nectosome, There has been much recent progress the systematics literature of other or- indicating that it was originally more in understanding the developmental ganisms but remained overlooked. complex than previously believed. It mechanisms that initiate and control Cnidarians that are radially or bilat- may even have arisen as a tandem directional asymmetries in the Bilat- erally symmetric at maturity may also duplication of the siphosome. eria (reviewed by Hamada et al., 2002; have cryptic developmental direc- There has been increasing interest Levin, 2005). Determining the evolu- tional asymmetries that are not evi- in clonal budding, including molecular tionary history of directional asymme- dent at a morphological level. The directionally asymmetric development characterizations of clonal budding in tries, a prerequisite for learning how of Bargmannia elongata agrees with cnidarians (e.g., Smith et al., 1999; to relate findings from one model de- other recent findings (Spring et al., Hobmayer et al., 2000; Spring et al., velopmental system to another, would 2002; Kortschak et al., 2003; Martin- 2002; Reinhardt et al., 2004) and uro- be greatly assisted by understanding dale et al., 2004; Kusserow et al., chordates (e.g., Tiozzo et al., 2005). the symmetry properties of the common ancestor of the Bilateria. This re- 2005) that indicate that cnidarians The budding process presented here quires looking at the symmetry prop- have a greater degree of genetic, de- differs from that of these other colo- erties of outgroups to the Bilateria, velopmental, and morphological com- nial animals in that one bud gives rise such as the Cnidaria. plexity than is usually acknowledged to multiple functionally specialized The present study has found that in the contemporary literature. zooids. Previous studies of other hy- colonies of Bargmannia elongata are drozoans have characterized differen- directionally asymmetric, and that tial gene expression between polyp EXPERIMENTAL this directional asymmetry arises and medusa buds in Podocoryna car- PROCEDURES through the consistent directionality nea (Spring et al., 2002; Bridge et al., in the subdivision of the pro-bud. All specimens were collected by the 2004) and between functionally spe- Freeman (1983) demonstrated that Monterey Bay Aquarium Research In- cialized polyps in Hydractinia symbi- the site of origin of the first cleavage stitute’s remotely operated underwa- olongicarpus (Cartwright et al., 1999). furrow in the siphonophores Nanomia ter vehicle Tiburon. These specimens, This is directly relevant to under- bijuga (which was misidentified as as well as space to work on them standing the instantiation and differ- Nanomia cara) and Muggiaea atlan- aboard the RV Western Flyer, were entiation of functionally specialized tica corresponds to the oral end of the graciously provided by Dr. Steven zooids in siphonophores, but does not planula. He also showed that the Haddock. The material was collected address how one bud can give rise to plane of bilateral symmetry in M. at- in Monterey Bay, California, and ad- multiple zooids. It may be that pro- lantica corresponds to the plane of the jacent waters, on cruises in March of bud subdivision arose through the first cleavage, so that one blastomere 2002, July of 2003, May of 2004, and evolutionary fusion of multiple sites of forms the left side of the colony and October of 2004. zooid budding, which would be similar the other the right side. His findings Intact specimens were stored at 4°C to the fusion of zooid and shoot bud- present an obvious starting point for until they were processed. A portion of ding zones that has been proposed to the examination of the embryological the stem containing the siphosomal have occurred in some benthic hydro- mechanisms that establish directional growth zone and the apical portion of zoans (Marfenin and Kosevich, 2004). asymmetry in siphonophores. the siphosome was excised from each Alternatively, the pro-bud (or perhaps Gene expression data are consistent of the specimens and transferred to a the footbud) may have originally given with the hypothesis that cnidarians smaller vessel, where it was anaesthe- rise to a single zooid, but now goes and bilaterians share some anterior/ tized by adding 4°C isotonic magne- ⅐ through a process of fission early in its posterior and perhaps even dorsal/ sium chloride (7.5% MgCl2 6H20in development. A survey of colony-level ventral patterning mechanisms (e.g., distilled water) to about 1/3 of the to- development in other siphonophores Shenk et al., 1993; Hayward et al., tal volume. Once relaxed, the tenta- may reveal variability in zooid bud- 2002; Wikramanayake et al., 2003; cles were cut away and all mature ding that, with a phylogenetic per- Finnerty et al., 2004). If this is the bracts and nectophores were plucked 844 DUNN off with forceps. The remaining por- helpful critiques have been invalu- phonophora, Physonectae). Bull Mar Sci tion of the stem was pinned out in a able. Thanks to Erika Edwards, Terri 76:699–714. dish lined with the clear silicone elas- Williams, and Mark Martindale for Dunn CW, Pugh PR, Haddock SHD. 2005b. Molecular phylogenetics of the Siphono- tomere Sylgard 184 (Dow Corning). reading earlier drafts of this article, phora (Cnidaria), with implications for Nectosomal growth zones were iso- and for the helpful comments of two the evolution of functional specializa- lated in a similar way. anonymous reviewers. This work tion. Syst Biol (in press). Notes and photographs were made has been supported by the NSF Grad- Finnerty JR, Pang K, Burton P, Paulson D, Martindale MQ. 2004. Origins of bilat- from this anaesthetized material. It uate Research Fellowship, the NSF eral symmetry: Hox and dpp expression was then fixed by adding several Doctoral Dissertation Improvement in a sea anemone. Science 304:1335– drops of 50% glutaraldehyde while Grant DEB-0408014, and NSF Grant 1337. still pinned out. This proved critical to DEB-9978131 A000 (the Hydrozoan Freedman G. 1983. Experimental studies prevent the stem from contracting and PEET Grant, thanks to Cliff Cunning- on embryogenesis in hydrozoans (Tra- chylina and Siphonophora) with direct twisting. After 0.5–1 h, the specimen ham). Thanks also to the crews of development. Biol Bull Mar Biol Lab was transferred to a tube with 2% glu- ROV Tiburon and RV Western Flyer, Woods Hole 165:591–618. taraldehyde in sea water and stored at and to Barry Piekos for SEM assis- Garstang W. 1946. 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