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- EXPERIMENTAL PROCEDURES
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.
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- EXPERIMENTAL PROCEDURES
Most studies of animal development have focused on the embryonic development of solitary taxa. There are, however, other modes of development with different starting and end points that have remained largely neglected. These include agametic clonal development, in which a new animal arises from another animal through fission or budding, and colonial development, a variation on clonal development in which asexually produced individuals remain attached and physiologically integrated throughout their lives (Hughes, 2002). There is a diversity of organizational complexity across colonial taxa (Beklemishev, 1969). Some colonies consist of functionally equivalent zooids (see Table 1 for definitions of the specialized terms used throughout this report) while others manifest a marked division of labor between zooids (Leuckart, 1851). Most colonial taxa show intraspecific variability in zooid arrangement and gross colony morphology, such that no two colonies are exactly alike (Boardman et al., 1973). Other taxa, especially those that are pelagic (i.e., that live in the water column rather than affixed to a substrate), have invariant colonial organizations that are entirely consistent from colony to colony of the same species (Mackie, 1986).
Table 1. Definitions for Some of the Specialized Terminology Used
|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 (Carré, 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|
The zooids of most colonial taxa do not change positions within the colony, so the geometry and dynamics of the budding process have a direct effect on the arrangement of zooids and on overall colony shape. Both microenvironment (reviewed by Harvell, 1994) and internal parameters, such as the dynamics of gastrovascular fluid flow (Blackstone and Buss, 1993; Dudgeon and Buss, 1996), have been shown to influence the development of colonies with variable form. To date, little is known about the developmental mechanisms of those taxa with invariant organization. At the very least, a description of their budding process is required before the mechanisms that generate precise colony-level organization can be investigated.
The siphonophores (Fig. 1), a group of about 160 described species of pelagic hydrozoans (Cnidaria), have the highest division of labor between zooids and the most precise organization of all colonial animals (Beklemishev, 1969, p 83). Siphonophores are among the most abundant carnivores of the oceans' macroplankton (Pugh, 1984) and include the longest animals in the world, with colonies of some species exceeding 40 m in length (Robison, 1995). The zooids and colonies of most siphonophores have an organization that is bilaterally symmetric at a first approximation (Totton, 1965; see Haddock et al., 2005, for a discussion of siphonophore organization and the terminology used to describe the major axes). This is not surprising, as it has long been known that many cnidarians show marked bilateral symmetry (e.g., Delage and Herouard, 1901; Hyman, 1940; Beklemishev, 1969; Martindale et al., 2002). Bilateral organization is not unique to the “Bilateria,” the monophyletic group of animals that includes almost all model systems, as is often misstated or implied (e.g., Meinhardt, 2001).
Figure 1. Simplified schematic overview of a siphonophore. Parts not shown to scale, or in their actual numbers. Some siphonophores lack a pneumatophore, while others do not have a nectosome. Although the nectophores are arranged biserially, they are all attached in a line along one side of the stem.
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The zooids and colonies of many siphonophores are directionally asymmetric (e.g., Totton, 1932; Stepanjants, 1967; Pugh and Youngbluth, 1988; Pugh and Pages, 1997; Mapstone, 2003). Directional asymmetries are deviations from bilateral symmetry that consistently occur in the same direction, and are found throughout the Bilateria (Neville, 1976). They include the displacement of the heart to the left in humans, the well-defined chirality of most spiraled gastropod shells, and the consistent asymmetry of the nervous system in Caenorhabiditis elegans (Hobert et al., 2002).
The directional asymmetries described in the siphonophore systematics literature have never been consolidated and have escaped wider notice. They do, however, indicate that the symmetry of at least some cnidarians can be of the same order as that of the most derived Bilateria. This raises questions as to how many times directional asymmetries have evolved in animals, and how old they are. It is still not clear if homologous developmental axes even exist in the Cnidaria and Bilateria, though expression data are largely consistent with the hypothesis that they do (Hayward et al., 2002; Finnerty et al., 2004). The axes of siphonophore colonies are labeled with the same names as the axes of bilaterian animals (reviewed by Haddock et al., 2005), but it should be noted that this is merely a semantic convenience and no homologies are implied by this nomenclature.
A recent molecular phylogeny (Dunn et al., 2005b) helps organize what is already known about the colony-level organization and development of siphonophores. Siphonophores are divided into two monophyletic groups, the Cystonectae and the Codonophora (Fig. 2). The Cystonectae is a small group of only five valid species, which include Physalia physalis, the familiar Portuguese Man o' War. The embryology of the cystonects is entirely unknown. Totton (1960) described several features of the budding process of mature P. physalis colonies and showed that it is highly derived and fundamentally different than any other siphonophore, including the other cystonects. As such, it is difficult to apply the developmental findings from this species to other taxa.
Figure 2. A rooted siphonophore phylogeny, simplified to show the relationship of the taxa discussed here. Physalia physalis is in the Cystonectae, Abylopsis and Sphaeronectes are in the Calycophorae. This cladogram is based on an analysis by Dunn et al. (2005b) of sequence data from the 16S and 18S ribosomal RNA genes. Support is shown as (Bayesian posterior probability x 100)/(maximum likelihood bootstrap score).
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The other monophyletic group, the Codonophora, contains the bulk of siphonophore species. Their embryological development, which was first observed by Gegenbaur (1853) and 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 historically recognized groups of siphonophores (Dunn et al., 2005b). These are the Physonectae, a grade, and the Calycophorae, 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. Siphosomal 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 (1896) described some zooids as arising as independent buds in Abylopsis tetragona, though his figures are not completely clear on the matter. In a later review of these studies, Garstang (1946) raised several issues with Schneider's findings, and concluded that the subdivision of buds was a general mechanism of colony-level development in the Calycophorae. He also coined the term “pro-bud” for the bud that gives rise to the multiple zooids of a cormidium.
Although it is critical to understanding the development of the ancestral Codonophora, the budding process in the physonects has proven particularly problematic to study because “there is so much crowding together of the siphosomal buds that it makes observation very difficult” (Totton, 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 and other cystonects are essential if we are to understand the evolution, development, and origin of colony-level complexity, as well as symmetry, of siphonophores as a whole.
The complex organization of siphonophores indicates the existence of a highly canalized colony-level developmental mechanism without parallel in other animals (Garstang, 1946), and provides an opportunity to explore the evolutionary origins of biological complexity in a novel context. Haeckel (1869a) recognized this, and made explicit comparisons between specialized cells in multicellular organisms and specialized zooids in siphonophore colonies. While complex multicellular organisms arose via the precise organization of functionally specialized cells in space and time, siphonophores arose by taking the process one step further and organized functionally specialized multicellular organisms into precise patterns. Interest has recently been rekindled in how new levels of biological organization arise (Buss, 1987; Michod, 2000), and this growing field now often goes under the name “the major transitions in evolution” (Maynard Smith and Szathmáry, 1995). Even so, there has only been occasional recent mention of siphonophores in this context (Mackie, 1963; Winsor, 1971; Gould, 1987; Wilson, 2000), and these animals have remained poorly known and largely forgotten in modern times. This is because siphonophores live in the open ocean, with many species being found only in the deep sea. They are so fragile that their zooids often dissociate during collection and preservation (Pugh, 1989; Dunn et al., 2005a), and the resulting lack of intact material has largely precluded the study of the symmetry properties, colony-level organization, and developmental processes that make siphonophores interesting in a broader developmental and evolutionary context. Modern advances in oceanographic technology, however, alleviate the collecting problems that limited all previous work on siphonophores (Haddock, 2004).
The present study investigates the colony-level organization and development of a siphonophore, Bargmannia elongata (Fig. 3), using specimens collected with a remotely operated underwater vehicle (ROV) deployed from an ocean-going research ship. The general colony form of B. elongata (an elongate siphosomal stem, two growth zones, multiple identical nectophores, the possession of a gas-filled pneumatophore) is plesiomorphic for the Codonophora (Dunn et al., 2005b), unlike the colony form of the calycophorans that have previously been investigated. This makes B. elongata a good departure point for understanding the colony-level evolution and development, and symmetry properties, of other siphonophores.
Figure 3. View of a living Bargmannia elongata colony. Photograph courtesy of Steve Haddock. The entire colony is about 40 cm long.
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All specimens were collected by the Monterey Bay Aquarium Research Institute's remotely operated underwater vehicle Tiburon. These specimens, as well as space to work on them aboard the RV Western Flyer, were graciously provided by Dr. Steven Haddock. The material was collected in Monterey Bay, California, and adjacent waters, on cruises in March of 2002, July of 2003, May of 2004, and October of 2004.
Intact specimens were stored at 4°C until they were processed. A portion of the stem containing the siphosomal growth zone and the apical portion of the siphosome was excised from each of the specimens and transferred to a smaller vessel, where it was anaesthetized by adding 4°C isotonic magnesium chloride (7.5% MgCl2 · 6H20 in distilled water) to about 1/3 of the total volume. Once relaxed, the tentacles were cut away and all mature bracts and nectophores were plucked off with forceps. The remaining portion of the stem was pinned out in a dish lined with the clear silicone elastomere Sylgard 184 (Dow Corning). Nectosomal growth zones were isolated in a similar way.
Notes and photographs were made from this anaesthetized material. It was then fixed by adding several drops of 50% glutaraldehyde while still pinned out. This proved critical to prevent the stem from contracting and twisting. After 0.5–1 h, the specimen was transferred to a tube with 2% glutaraldehyde in sea water and stored at 4°C.
Back on land the specimens were rinsed with 500 mM sodium chloride and the regions of interest dissected out. Some were photographed under a dissecting microscope, and others were further prepared for scanning electron microscopy (SEM). SEM specimens were fixed on ice for 0.5–1 h with the following fixative: 1% osmium tetroxide, 2.5 mM calcium chloride, 500 mM sodium chloride, 50 mM sodium cacodylate (pH 7.8). They were then rinsed 3 times (10 min each) with ice cold buffer containing 500 mM sodium chloride and 50 mM sodium cacodylate (pH 7.8), and dehydrated as follows (15 min per step, ethanol diluted with distilled water): 70% ethanol, 80% ethanol, 90% ethanol, and 3 times with 100% ethanol. Specimens were dried in a critical point drier (Polaron), sputter coated with gold (EMS 550x), and photographed using an ISI-SS40 scanning electron microscope. It was sometimes necessary to dissect these prepared specimens to determine the later stages of zooid differentiation. The gastrozooids were easily broken away with a hypodermic needle attached to a micromanipulator, leaving their peduncles and all associated buds. Entire cormidia were likewise removed at several locations to get a complete view of neighboring zooids.