Zoomorphology (2011) 130:85–95 DOI 10.1007/s00435-011-0120-5 ORIGINAL PAPER Three-dimensional analysis of the canal network of an Indonesian Stylaster (Cnidaria, Hydrozoa, Stylasteridae) by means of X-ray computed microtomography Stefania Puce · Daniela Pica · Lucia Mancini · Francesco Brun · Alessandro Peverelli · Giorgio Bavestrello Received: 7 May 2010 / Revised: 19 January 2011 / Accepted: 20 January 2011 / Published online: 11 February 2011  Springer-Verlag 2011 Abstract This study describes the architecture of the coenosteal network in an Indonesian Stylaster species investigated by means of the X-ray computed microtomography (-CT) technique. The 3D approach allowed to characterize all internal cavity structures: a network of thin canals, gastropores, dactylopores, and ampullae. The main feature highlighted by this reconstruction is a dense network of thin canals extended to the entire colony. This network gives rise to and surrounds each cyclosystem. Moreover, the 3D analysis made it possible to study the reciprocal relationship between adjacent cyclosystems and to hypothesize the growth process of the branches of Stylaster sp.: each new cyclosystem buds between the gastropore and the dactylopores of the last formed one. The dactylopores of each cyclosystem are partially derived from the precedent one and are partially newly formed. The thin canals enveloping the dactylopores are actively involved in both the formation of a new gastropore and in the re-establishment of the typical amount of dactylopores in each cyclosystem. This growth process was conWrmed by the scanning electron microscopy (SEM) observations of apical cyclosystems of several specimens. Results indicate that the non-destructive X-ray -CT technique can be fruitfully applied to characterize the coenosteal structures of stylasterids allowing the repetitive study of a specimen by means of virtually inWnite section planes and diVerent kinds of analyses (e.g., channel width and porosity). Keywords Stylasteridae · Stylaster · Canal network · Growth · X-ray microtomography · 3D analysis Introduction Communicated by T. Bartolomaeus. S. Puce (&) · D. Pica · G. Bavestrello DiSMar, Università Politecnica delle Marche, Via Brecce Bianche, 60131 Ancona, Italy e-mail: s.puce@univpm.it L. Mancini · F. Brun Sincrotrone Trieste S.C.P.A., S.S. 14—Km 163.5 in AREA Science Park, 34149 Basovizza, Trieste, Italy F. Brun Department of Industrial Engineering and Information Technology, University of Trieste, Via A. Valerio 10, 34127 Trieste, Italy A. Peverelli Immagini & Computer Snc, Via Don Carlo Riva 4, 20010 Bareggio, Milano, Italy The hydroids belonging to the Stylasteridae produce a massive calcareous skeleton called coenosteum. The colony surface is characterized by pores that have diVerent functions: the gastropores where the gastrozooids are lodged, the dactylopores containing the dactylozooids, and the nematopores housing the nematophores (Cairns 1983). These pores are interconnected by a complex three-dimensional (3D) network of coenosteal canals, which contains the living coenosarc, linking together all the polyps of the colony (Broch 1942; Moseley 1879, 1881). Already Moseley (1881, page 38) stated that “The tortuous canals and pores by which the coenostea of all the Stylasteridae are traversed, are occupied in all the genera alike, in the living condition of the coral, by a series of meshworks of correspondingly branching, twisting, and anastomosing canals, which compose the coenosarc or common body of the compound organism in each case”. Broch (1942) stated 123 86 Zoomorphology (2011) 130:85–95 a non-destructive technique. For example, this technique was employed for the Wne reconstruction of the excavations produced by boring sponges in calcareous substrata (Schönberg and Shields 2008). The aim of this paper is to describe the architecture of the coenosteal network in an Indonesian Stylaster species by using the X-ray -CT technique. Fig. 1 Sketch of the X-ray microtomography set up at the TOMOLAB station at Elettra Synchrotron Light Laboratory the general rule that the coenosteal structure characterized by calcareous lamellae having diVerent extension and width is more prominent in species with a richly developed canal system than in species with a more scantily developed meshwork of canals. The study of the 3D organization of internal branched organs is diYcult. Generally, they are visualized through a reconstruction of serial sections, but this method is extremely laborious and the results are sometimes not precise. Nevertheless, accurate descriptions of the canal system of several species belonging to diVerent genera were provided by Moseley (1879, 1881), who also produced detailed illustrations. This author observed that in many of the examined species, such as Sporadopora dichotoma Moseley 1879, Inferiolabiata labiata (Moseley 1879) (cited as Errina labiata), Stylaster densicaulis Moseley 1879, Stylaster profundus (Moseley 1879) (cited as Allopora profunda), Astya subviridis (Moseley 1879) (cited as Astylus subviridis), the interspaces in the meshwork are larger and wider in the deeper regions of the coenosteum than close to its surface. Commonly, the largest trunks of the meshwork are those which proceed directly from the bases of the zooids and gonophores. Moreover, around the sacs containing the zooids, the canals of the coenosarc have a special radiate disposition. DiVerently from the other species, in the walls of the cyclosystems of S. densicaulis and S. profundus, a series of large canals form a network in which the branches have a general direction parallel to the axis of the gastropore. They form a direct communication between the basis of the dactylozooids and the large canals which spring from the bases of the gastrozooids. After the works by Moseley (1879, 1881), the study of the canal system stopped for 130 years but recently the reconstruction of the canal network of Errina dabneyi (Pourtalès de 1871) from Azores was conducted by a modern method producing etched vacuum-epoxy-casts of the aragonite skeleton (Wisshak et al. 2009). A new promising opportunity is the use of X-ray -CT allowing the reconstruction of 3D images of the sample by 123 Materials and methods The studied samples were collected in the Bunaken Marine Park (North Sulawesi, Indonesia). Stylaster sp., characterized by stout colonies (Fig. 2a), is particularly abundant in the crevices of a vertical face of the reef. The specimens were gathered by diving between 10 and 40 m depth and preserved dried. One dried colony of the studied species was deposited in the Museo di Storia Naturale, Genova, Italy (MSNG 56006). The morphology of the specimens was Wrst observed using the stereomicroscope. Longitudinal sections of coral branches were produced by means of a grinder in order to describe the internal structure. Samples were then prepared for a SEM analysis. Small coral portions were treated with sodium hypochlorite for 10 min, rinsed with distilled water, and dried. The samples were coated with gold–palladium in a Balzer Union evaporator and examined with a Philips XL20 scanning electron microscope. Another sample was investigated by using the X-ray -CT technique. Since X-ray -CT is a non-destructive technique, the dried sample did not need any special preparation procedure. The experiment was performed at the TOMOLAB facility of the Elettra Synchrotron Light Laboratory in Trieste (Italy) (http://www.elettra.trieste.it/ Labs/TOMOLAB). The TOMOLAB station is a conebeam -CT system equipped with a sealed microfocus X-ray tube operating in an energy range from 40 to 130 kV at a maximum current of 300 A. A water-cooled, 12-bit CCD camera (Photonic Science XDI-VHR) was used as detector, consisting of a full-frame CCD imager coupled to a Gadolinium oxysulphide scintillator by a Wber optic taper. This CCD provides a combination of a large Weld of view (max. 50 mm £ 33 mm) with a small pixel size (12.5 m £ 12.5 m). Exploiting the magniWcation eVect oVered by the cone-beam geometry (Kak and Fig. 2 Colony of Stylaster sp. from Bunaken Marine Park a a colony 䉴 in situ, b section of a branch, SEM micrographs of which are shown in c to j, c reticulate-granular texture of coenosteum surface, d a small circular pore, e a large circular pore near the cyclosystem, f elliptical cyclosystem with an adcauline diastema, g cyclosystem showing the gastrostyle tip surrounded by the ring palisade, h gastrostyle, i ampulla, j internal surface of ampulla Zoomorphology (2011) 130:85–95 87 123 88 Slaney 1987), the source-object-detector distance can be varied to achieve a spatial resolution close to the focal spot size of the source (5 m) while imaging samples from a few millimeters to a few centimeters in size. From the tomographic projections acquired at regular angular steps by the CCD camera during a 360 degrees rotation of the sample (Fig. 1), a set of 2D axial slices was reconstructed by the commercial Cobra -Exxim Computing software package. In order to visualize and inspect the 3D structure of the sample, volume renderings were obtained by using the commercial software VGStudio MAX 2.0. The -CT measurements were performed in the following experimental conditions: tube voltage = 70 kV, tube current = 110 A, 750-m-thick Aluminum Wlter, number of projections = 1,800, total scan duration = 2 h, voxel size = 6.7 m. Quantitative analysis was then performed on the reconstructed 3D dataset by using the Pore3D software library (Brun et al. 2010; http://ulisse.elettra.trieste.it/uos/pore3d/ index.html). The Wrst and most important step of the analysis process consists in the segmentation of the images, i.e., the classiWcation of the image voxels (Rosenfeld and Kak 1982). The segmentation of the coral can be easily obtained by deWning a threshold on the gray (or intensity) values (or levels): voxels having a gray level above the visually assessed threshold are classiWed as object voxels. However, voxels having a gray level below or equal to the threshold still need to be classiWed according to a criterion that cannot be based on the intensity value. In fact, the unclassiWed voxels may represent either the background or the canal system since the physical medium, the air, is the same in both cases. In the present study, the segmentation of the canal network from the background has been performed by means of a suitable application of distance transform and watershed segmentation (Soille 2004). After segmentation, a measure of the sample porosity came straightforward by simply computing the ratio of canal voxels to the total number of object and canal voxels (background voxels were excluded) in the considered Volumes of Interest (VOIs). A more reWned measure such as the width of the channels can also be computed. An interesting and eVective approach for the computation of this parameter is based on the extraction of the curve-skeleton, i.e., a onevoxel thick representation of what can be intuitively thought as the “spine” or the medial axis of the canal network. By scanning the curve-skeleton, it is possible to compute the width of a channel as the mean value of the distance transform along a skeleton branch (Hildebrand and Rüegsegger 1997). The assessment of the porosity and the width of the canals was performed analyzing two VOIs: a small one (0.82 £ 0.68 £ 0.68 mm3) including only thin canals and another one (4.73 £ 4.03 £ 4.03 mm3) including all the types of canals. 123 Zoomorphology (2011) 130:85–95 Results Stylaster sp. description The colonies are generally stout, scarcely branched and multiplanar, up to 5 cm tall and 8 cm wide (Fig. 2a). Each branch is usually constant in diameter, and each tip supports a terminal cyclosystem (Fig. 2a, b). The colonies of this species, belonging to the Stylaster type B (see Cairns 1983), present cyclosystems mainly sympodially arranged but with additional cyclosystems on the anterior and posterior branch faces (Figs. 2a, b, 3a). The coenosteum is whitecream colored and reticulate granular in texture (Fig. 2c). It is traversed in all directions by a network of thin canals (Fig. 3b) that are visible on the coral surface as small circular pores (up to about 50 m) with numerous teeth projecting inward toward the center of pore (Fig. 2c, d). Larger circular pores, without teeth, are also present typically near the cyclosystems (about 70 m) (Fig. 2e). Cyclosystems are usually elliptical, and the number of dactylopores per cyclosystem ranges from 15 to 37 (Figs. 2f, 3a). Cyclosystems generally have an adcauline diastema that has a variable width (Figs. 2f, g, 3a). They generally reach the surface with an angle of about 30–45° (Figs. 2b, 3c, d, 4a). The canal of the gastropore is slightly vase shaped (about 500 m) with an oval base from which the gastrostyle (up to 1 mm) arises (Figs. 2b, h, 3c). At the level of the gastrostyle tip, the ring palisade protrudes from the internal surface of the canal (Figs. 2g, 3c). The ring palisade is composed of a single circular row of regular Xattened elements (Fig. 2g). From the basal portion up to the ring palisade, the canal surface is irregular while, after this point, it is smooth (Figs. 2b, h, 3c, d). The canals of dactylopores (about 70 m) run parallel to that of the gastropore, and they have an oval-circular transversal section reaching the coral surface with drop-shaped openings (Figs. 2f, g, 4a, b). The ampullae are rounded prominences, clustered in groups, and often fused together into large masses (Figs. 2i, j, 3c, d, 4b). Canal network description The 3D reconstruction of the canal system permeating the calcareous coenosteum of Stylaster sp. allowed one to characterize all its structures: a network of thin canals, gastropores, dactylopores, and ampullae (Figs. 3b, c, d, 4a, b). These structures are recognizable by their diameter and shape. The dense 3D network of thin canals uniformly permeates the coenosteum producing almost rectangular meshes (Fig. 4c) that become slightly smaller toward the surface of the coenosteum where the canals appear thinner (Fig. 4c). Sometimes, short larger canals, oriented longitudinally, are observed in the deeper portion of the coenosteum (Fig. 4d). Zoomorphology (2011) 130:85–95 89 Fig. 3 Volume rendering of Stylaster sp. a total volume of a branch, b volume of the canal network without the coenosteum, c–d longitudinal sections of the canal network volume. Ampullae (a); large internal cavity (lic); gastrostyle (gt) 123 90 123 Zoomorphology (2011) 130:85–95 Zoomorphology (2011) 130:85–95 䉳 Fig. 4 Volume rendering of Stylaster sp. a shape of a cyclosystem and disposition of dactylopores, b group of ampullae near a cyclosystem, c organization of the thin canals in the network, d short longitudinal larger canal (lc) in the deeper region of the coenosteum, e small internal cavity (sic), f canal network enveloping each dactylopore and radial canals connecting the dactylopores with the gastropore Small lacunar cavities are recorded in the mid-deeper portions of the skeleton (Fig. 4e). A network of thin canals envelops each dactylopore, follows it in its running toward the surface, and progressively becomes more regular (Fig. 4f). From this network, groups of canals start and radially converge toward the base of the gastropore. Moreover, at all levels of the gastropore, perpendicular irregular canals connect the gastropore and its dactylopores at variable distance from one another. At the ring palisade level, the gastropore and its dactylopores generally start to diverge, and the connecting canals become more visible and increase in number (Fig. 4f). The ampullae appear as spherical cavities present on the surface of the coenosteum (Figs. 3c, 4b). In addition, large spherical cavities, that have the same diameter of the ampullae, are recorded in the deeper part of the coenosteum (Fig. 3c, d). They are completely undetectable from the surface. Few canals emerge from the inner face of the ampullae and the large cavities, while many canals emerge from their external side reaching the coral surface (Figs. 2i, j, 3c, 4b). Some other small irregular cavities were also found inside the coenosteum (Fig. 4e). 91 The analysis performed by the Pore3D software library on the aforementioned VOIs, extracted from the X-ray -CT dataset, indicates that the volume occupied by the narrow canals of the network accounts for about 5% of the coenosteum volume. In addition, including also cyclosystems and ampullae, the porosity reaches about 24%. The software estimated also the range of the width of the canals: thin canals 13–56 m, dactylopores 56–79 m, and gastropores up to 600 m (Fig. 5). The analysis of a sequence of the coral’s transverse sections revealed a reciprocal relationship between adjacent cyclosystems (Fig. 6a–i). In a branch, the base of each cyclosystem starts inside the preceding one. The space between the dactylopores and the gastropore of the parent cyclosystem enlarges, and the base of the gastropore of the second one arises from the canal network surrounding the dactylopores (Fig. 6a, b). Therefore, at this level, two gastropores are surrounded by a single ring of dactylopores (Fig. 6c). Proceeding toward the distal part of the branch, a thickening between these two gastropores becomes visible (Fig. 6d) probably due to the anastomosis of many canals which are replaced by several dactylopores (Fig. 6e). The distance between the two gastropores increases, and from the canal network, that envelops the already existing dactylopore ring, other dactylopores add up to reach the typical amount for each cyclosystem (Fig. 6f). The two cyclosystems separate, and from the apical one, the base of the next cyclosystem is already visible (Fig. 6g, h). Sometimes, when the two cyclosystems are almost completely separated, the gastropore base of a third one starts to become visible between them (Fig. 6i). Frequently, one or more dactylopores turn slightly away from their original cyclosystem and reach the surface independently where they are recognizable as large circular pores (Figs. 2e, 6f). Description of the apex of a branch Fig. 5 Volume rendering of the smaller VOI used for the estimation of the porosity and the width of the thin canals with superposition of the extracted curve-skeleton and some of the maximal balls used for the assessment of thickness The observation by SEM of the terminal cyclosystem on the tip of a branch sometimes reveals the presence of a remodeling activity area on the enlarged side between the gastropore and the dactylopore ring (Fig. 7a). In this area, it is sometime possible to observe a small cup with a rough surface and a prominent spiny bottom (Fig. 7b, c). Progressively, this cup becomes a young gastropore producing an incomplete gastrostyle and an irregular ring palisade (Fig. 7d). At this stage, the two gastropores are separated by a thin calcareous septum and are surrounded by a single elliptical ring of dactylopores (Fig. 7e). Successively, new dactylopores are formed in both the septum, starting from the lateral extremities, and between the old dactylopores (Fig. 7e, f). They are recognizable as pores covered by a thin and smooth calcareous layer (Fig. 7f). When a new cyclosystem is completely formed, its growth continues independently from the old 123 92 123 Zoomorphology (2011) 130:85–95 Zoomorphology (2011) 130:85–95 䉳 Fig. 6 Sequence of transversal sections of Stylaster sp. showing the relationships between the cyclosystems. a section of a cyclosystem, b cyclosystem enlargement (ce) between gastropore and its dactylopores, c base of a new gastropore, d thickening between the two gastropores, e new dactylopores arise from the thickening, f new cyclosystem completely separated from the parent one and a dactylopore turning slightly away from its original cyclosystem (d), g base of new gastropore, h the new cyclosystem almost separated, i a third cyclosystem starts to be formed before their separation one. Then, it will produce a new cyclosystem in the same way. Sometimes, this new zooid system may grow between the two cyclosystems before they separate (Fig. 7g). Discussion In this investigation, the X-ray -CT technique has been applied for the Wrst time to the study of the canal system of stylasterid hydrozoans. Results clearly indicate that this technique can be fruitfully applied to compare the coenosteal structures of the stylasterid and probably the milleporid hydrozoans also. The comparison of the values of canal diameters obtained by SEM and by X-ray -CT indicates that the voxel size (6.7 m), used for the -CT analysis, appears to give an optimal spatial resolution allowing one to obtain detailed 3D images. If compared to the vacuumepoxy casting technique, this method allows the repetitive study of a specimen by means of virtually inWnite section planes and diVerent kinds of analyses (e.g., channel width and porosity). Moreover, being non-destructive, this method allows the study of rare museum specimens. Canal network The main characteristic, highlighted by the reconstruction of the canal system of Stylaster sp., is a dense network of thin canals extended to the entire colony (Fig. 3b). This network gives rise to and surrounds each cyclosystem (Fig. 3c, d). Gastrozooids, dactylozooids, and gonophores arise from this diVused network that occupies about 5% of the coenosteum. Instead, the entire canal system permeates about 24% of it. These values represent new data that are useful to compare the canal system of diVerent species. The Hydractiniidae and the Stylasteridae are sister taxa (Lindner et al. 2008; Miglietta et al. 2010) and because of their similarities have been long united in the superfamily Hydractinoidea. The hydractiniid hydrorhiza is generally organized as a bi-dimensional network formed by perisarccovered stolonal tubes in some species merging in an encrusting mat either covered by a layer of perisarc or by a naked coenosarc (Bouillon et al. 2006). Sometimes, the hydrorhizal mat is partially (Hydractinia antonii Miglietta 2006) or completely (Janaria mirabilis Stechow 1921 and 93 Hydrocorella africana Millard 1975) invested by a calcareous skeleton. H. antonii is the unique hydractiniid species showing distally a chitinous skeleton formed by a 3D meshwork and basally a calcareous mass with the center harboring living tissue and from whose openings the polyps protrude (Miglietta 2006). As suggested by Cairns (1984), the most important progress in stylasterid evolution is represented by the inclusion of polyps and gonophores in calciWed tubes and ampullae, respectively. In this way, together with the polyps, also the ancestor hydrorhiza has probably been included in the coenosteum and changed its organization from a typical bi-dimensional to a threedimensional network. For the Wrst time, Moseley (1879, 1881) studied the organization of the canals of eight species by means of sections of decalciWed specimens. He provided numerous drawings representing detailed reconstructions of the canals and their relationships with the zooids and the gonophores. Two of his studied species belong to the genus Stylaster, S. densicaulis, and S. profundus. In both species, he observed the canal meshwork in the wall of the cyclosystem describing two Wne reticulations of smaller canals, one beneath the surface and the other immediately beneath the lining membrane of the gastropore. Between the two Wne reticulations, he also observed a series of larger canals with a general direction parallel to the axis of the gastropore and directly connected with the canals which spring from its base. In the Stylaster sp., considered in the present study, this marked stratiWcation and the larger canals parallel to the gastropore are not detectable, and the size of the canal network decreases gradually from the inner part of the coenosteum to its surface (Fig. 4c, d). In the species described by Moseley (1879, 1881), these large parallel canals are also connected with the bases of the dactylozooids. Each dactylopore consists of a wide upper chamber and a narrow tubular continuation of this, which proceeds parallel with the axis of the gastropore for about half the length of the latter. In the studied species, the dactylopores follow the entire length of the gastropore axis, and they are enveloped by a thin reticulation that connects them with the gastropore (Figs. 3c, d, 4a, f). Moseley (1879, 1881) recorded a similar radial connection in S. profundus but not in S. densicaulis. In Stylaster sp., the dactylopores of each cyclosystem are partially derived from the parent cyclosystem and are partially newly formed. When the new and the old cyclosystems start to diverge, the newly formed dactylopores are split between them. The scarce number and the diverging course of the new dactylopores of the old cyclosystem result externally in a diastema or in a series of widely separated dactylopores (Fig. 2f, g). Moreover, the large circular pores scattered on the surface originate from a dactylopore turning slightly away from their original cyclosystem (Figs. 2e, 6f). 123 94 Zoomorphology (2011) 130:85–95 Fig. 7 SEM micrographs of the apical growth of Stylaster sp. a remodeling activity area (arrow) in a lateral enlargement between the gastropore and the dactylopore ring, b small cup in the apical position, c small cup with a rough surface and a prominent spiny bottom, d short gastropore with an incompletely formed gastrostyle and irregular ring palisade, e two cyclosystems surrounded by a single ring of dactylopores and separated by a thin calcareous septum, f new dactylopores covered by a thin and smooth calcareous layer, g new cyclosystem between two old ones before their complete separation Reproductive structures (Figs. 2i, 4b). Below the coral surface, but never in correspondence with the area bearing the grouped ampullae, the 3D analysis permitted the recording of other similar round cavities. Owing to the use of dried material for this study, it The observation of the surface of Stylaster sp. revealed the presence of prominent ampullae organized in small groups 123 Zoomorphology (2011) 130:85–95 was impossible to verify the content of the cavities and to deWne their role. However, they generally have a typical diameter and shape of the ampullae (Fig. 3c, d). Some other small irregular cavities were also found (Fig. 4e). In S. densicaulis, similar lacunar cavities were reported by Moseley (1881). He also observed the same structures in Sporadopora dichotoma aYrming that they “probably represent spaces occupied in an earlier condition of the coral by gonophores”. Wisshak et al. (2009), explaining the skeletal reorganization of Errina dabneyi, noted that the old ampullae are Wlled by carbonate or remain open but embedded while new active ampullae are formed closer to the surface. Moreover, several authors suggest that the ampullae do not go to waste and, in this way, they are used repeatedly (Broch 1942; Moseley 1881; Ostarello 1973). In light of these suggestions, the small irregular cavities of Stylaster sp. may be considered ampullae waiting to be completely Wlled or simply anastomosed canals. Colony growth The 3D analysis of the coenosteal structure allowed the study of the reciprocal relationship between adjacent cyclosystems and to hypothesize the growth process of the coral branches: each new cyclosystem buds between the gastropore and the dactylopores of the last formed one. This hypothesis was conWrmed by the SEM observations of apical cyclosystems of several specimens where diVerent steps of growth were recognized, starting from a small remodeling area. Puce et al. (2010) observed that in the early stages of the development of a Stylaster species, the calcium carbonate dissolution appears to be the normal process in cyclosystem formation. The skeletal architecture undergoes modiWcation during the growth. As the branches get thicker, the formerly superWcial meshwork of narrow canals is located deeper inside where wider canals are observed. Accordingly, Wisshak et al. (2009) observed that some of the central canals of E. dabneyi become enlarged by dissolution of skeletal material and, at the same time, other canals are Wlled by aragonite re-precipitates. The new canals are formed by a secondary dissolution of the calcareous substance (Broch 1942), and probably, the cells composing the stolon nets running in the canals are responsible for this skeletal plasticity (Puce et al. 2010). In particular, the observations carried out in this work suggest that the canals enveloping the dactylopores are actively involved in both the formation of a new gastropore and the re-establishment of the typical amount of dactylopores in each cyclosystem. Acknowledgments This work was Wnancially supported by Ministero degli AVari Esteri (Grande Rilevanza). The comments provided by Dr. Helmut Zibrowius and an anonymous referee greatly improved the manuscript. 95 References Bouillon J, Gravili C, Pagès F, Gili JM, Boero F (2006) An introduction to hydrozoa. Mémoir Mus Natl Hist Nat 194 Broch H (1942) Investigations on Stylasteridae (Hydrocorals). 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