Mar Biol (2011) 158:1149–1161

DOI 10.1007/s00227-011-1637-3

ORIGINAL PAPER

Comparison of the statolith structures of Chironex Xeckeri (Cnidaria, Cubozoa) and Periphylla periphylla (Cnidaria, Scyphozoa): a phylogenetic approach

I. Sötje · F. Neues · M. Epple · W. Ludwig · A. Rack · M. Gordon · R. Boese · H. Tiemann

Received: 29 September 2010 / Accepted: 1 February 2011 / Published online: 19 February 2011© Springer-Verlag 2011

Abstract The rhopalia and statocysts of Periphyllaperiphylla (Péron and Lesueur in Ann Mus Hist Nat Mar-seille 14:316–366,1809) and Chironex Xeckeri Southcott(Aust J Mar Freshw Res 7(2):254–280 1956) were exam-ined histologically and showed several homologous charac-teristics. DiVerences in sensory area distribution could beconnected to a slightly diVerent functionality of equilibriumsensing. In P. periphylla, the statoliths (crystals) grow inde-pendently of each other; whereas in C. Xeckeri, one largecrystal covers the smaller ones. The structures of bothstatoliths were examined in detail with single-crystaldiVraction, microtomography and diVraction contrasttomography. The single compact statolith of C. Xeckericonsisted of bassanite as was previously known only for

other rhopaliophoran medusae. An origin area with severalsmall oligocrystals was located in the centre of the cubo-zoan statolith. The origin areas and the accretion of stato-liths are similar in both species. Our results lead to theassumption that the single bassanite statolith of C. Xeckeri(Cnidaria, Cubozoa) is a progression of the scyphozoanmultiplex statolith. It is therefore suggested that the Cubo-zoa are derived from a scyphozoan ancestor and are ahighly developed taxa within the Rhopaliophora.

Introduction

Evident in the fossil records from the Middle Cambrianabout 600 millions years ago, the Cnidaria are one of theoldest animal phyla (Coates 2003). Earliest fossils alreadydisplayed characteristics that are still present in the medusaeof the recent taxa (Adler et al. 2007; Cartwright et al. 2007).Despite more than 100 years of investigation, the phyloge-netic tree and systematics of the Cnidaria are still subject toconsiderable discussion. At present, the most common divi-sion is into the four classes; Anthozoa, Scyphozoa, Cubozoaand Hydrozoa (Schuchert 1993; Werner 1993). Someauthors have, however, introduced an additional class, theStaurozoa (Marques and Collins 2004), while others distin-guish only three classes, grouping Cubozoa with Scyphozoa(Matsumoto 1995; Ruppert et al. 2004). Only the three taxa,the Hydrozoa, Scyphozoa and Cubozoa, develop pelagicmedusa stages with marginal sense organs bearing statocysts(Russell 1970; Werner 1993). While the medusa stage couldhave developed once, twice or three times within the diVer-ent taxa (Thiel 1966; Werner 1973, 1993; Salvini-Plawen1978; Schuchert 1993; Collins 2002) the statocyst, which iscrucial in medusae orientation, must have developedtogether with the pelagic stage.

Communicated by J. Purcell.

I. Sötje (&) · H. TiemannBiocenter Grindel and Zoological Museum, University of Hamburg, Martin-Luther-King-Platz 3, 20146 Hamburg, Germanye-mail: ilka.soetje@zoologie.uni-hamburg.de

F. Neues · M. Epple · R. BoeseInorganic Chemistry, University of Duisburg-Essen, Campus Essen, Universitaetsstraße 5-7, 45117 Essen, Germany

W. Ludwig · A. RackEuropean Synchrotron Radiation Facility, BP220, 38043 Grenoble Cedex, France

W. LudwigInstitut National des Sciences Appliquées de Lyon, 20, Avenue Albert Einstein, 69621 Villeurbanne Cedex, France

M. GordonSchool of Marine and Tropical Biology, James Cook University, McGregor Road, SmithWeld, QLD 4878, Australia

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The Scyphozoa and Cubozoa develop statocysts thatgenerally contain biomineralic crystals (statoliths). Bothtaxa are combined as sister groups within the taxon Rhopa-liophora because of similarities in the medusa formationand the development of marginal sense organs (rhopalia)from tentacles bases (Werner 1975; Straehler-Pohl andJarms 2005). The formation of the hydrozoan medusa andits marginal sense organs are a divergent development,because of their origin as a depression in the velum (Singla1975; Werner 1993; Ax 1995). Evidence for the diVerentialorigin of the marginal sense organs in Rhopaliophora andHydrozoa is supported by a comparison of their statolithmaterial composition. Statoliths of hydrozoans like Obelia,Aglantha and Trachylida are composed of calcium magne-sium phosphate (Singla 1975; Chapman 1985), whereasstatoliths of the rhopaliophoran medusae are composed ofbassanite (calcium sulphate hemihydrate) (Tiemann et al.2002; Becker et al. 2005; Tiemann et al. 2006), which hasbeen described as gypsum in earlier publications (Spangen-berg and Beck 1968; Pollmanns and Hündgen 1981; Vinni-kow et al. 1981; Chapman 1985; Ueno et al. 1995; Uenoet al. 1997).

Although the statolith material composition is the samein all examined rhopaliophoran medusae, the structure ofthe scyphozoan and cubozoan statolith appears to diVer.Scyphozoan statocysts mostly consist of an accumulationof single crystals (statoliths) (Spangenberg and Beck 1968;Russell 1970; Arai 1997; Becker et al. 2005), whereasgrown cubozoan medusae bear a single large statolithwithin the statocyst (Chapman 1985; Ueno et al. 1995,1997; Kawamura et al. 2003; Gordon et al. 2004).

ClassiWcation using established morphological featuresis in occasional disagreement regarding the phylogenetictree of the Cnidaria, when compared to classiWcation meth-ods utilizing molecular data. Therefore, it is a matter ofongoing debate whether the class Cubozoa is a sister groupof the Scyphozoa including the order Stauromedusae (Wer-ner 1973; Salvini-Plawen 1978; Schuchert 1993; Straehler-Pohl and Jarms 2005; Straehler-Pohl 2009) or is a sistergroup of the Staurozoa and is, rather remotely, related tothe remaining scyphozoans (Thiel 1966; Collins 2002;Marques and Collins 2004; Collins et al. 2006).

Details of statolith crystallographic structure are a usefuladditional tool when investigating the phylogenetic rela-tionships among diVerent taxa. To date, examinations ofstatoliths from several cubozoan species are mostly donewith light microscopic or scanning electron microscopicmethods (Chapman 1985; Ueno et al. 1995, 1997; Gordonet al. 2004). Therefore, information about the statolith crys-tal structure and formation is currently absent. In the pres-ent investigation, we used more sophisticated methodsincluding single-crystal X-ray diVraction, microtomogra-phy and diVraction contrast tomography to examine and

compare the statoliths of Chironex Xeckeri Southcott (1956)(Chirodropidae, Cubozoa) and Periphylla periphylla (Péronand Lesueur 1809) (Coronatae, Scyphozoa).

Materials and methods

Chironex Xeckeri medusae were collected by hand from theshallow (<0.5 m) sandy foreshores of Hey Point (estuarysite) and Wooldrum Point (coastal site), Weipa, Australia in2007. The interpedalia distance (IPD) of each medusa wasmeasured to the nearest mm, taken to be the distancebetween the midline of alternate pedalia along the line pass-ing through the rhopalia. The four rhopalia of each medusawere removed by pushing a cylindrical stainless steel tubeof 15 mm diameter through the mesoglea surrounding theniche and placed in 90% ethanol. Individual statoliths werelater dissected from the rhopalia using needle point twee-zers, cleaned of all biological material and preserved in90% ethanol.

Morphological and histological examinations of C. Xeckerirhopalia were carried out on material from the “LowerInvertebrates I” collection of the Zoological Museum inHamburg (No. C11193). The medusa was collected byhand by W. N. Hamner in 0.5 m depth at Townsville(Australia) in 1976 and was preserved in 4% formaldehydeWrst and stored in 70% ethanol thereafter. The IPD of theexamined medusa was 33 mm. The rhopalia were dehydratedfurther using ethanol and embedded in paraplast with theintermedium benzylbenzoate. Sections (6 �m) were stainedwith nuclear fast red–aluminium sulphate and light green–orange y (Adam and Czihak 1964).

Specimens of P. periphylla were collected with a conicalring-net, 2 m diameter with 500 �m mesh size (Fraser1968) during cruises with RV Håkon Mosby in Lurefjordand Sognefjord (Norway). For rhopalia, histological exami-nation samples were taken during 1999 and for the statolithexaminations during the years 2003 and 2004. Immediatelyafter capture, the medusa coronal diameter (CD) was mea-sured and the rhopalia were detached and Wxed in 80%ethanol for statolith examinations. Statocyst widths weremeasured and transferred into water-free glycerol on micro-scopic slides before the statoliths were separated from eachother. All statoliths of one statocyst were counted, andwidths of the Wve largest statoliths were measured with aneyepiece micrometre. Examinations were performed withZeiss NeoXuar objectives at 10–40-fold magniWcation.

The examined medusa of P. periphylla had a CD of8.2 mm and a statocyst width of approximately 110 �m.For histological examination, the rhopalia were preservedin 4% formaldehyde/seawater solution immediately aftercollection. Preparation for light microscopy was made bydehydration in acetone and embedding in Spurr (Robinson

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et al. 1985). Sections (0.5 �m) were stained with toluidine-blue–pyronin (Adam and Czihak 1964), examined withZeiss NeoXuar objectives and documented by photomicro-graphs or by drawing using a camera lucida.

The IPD of the examined C. Xeckeri medusa was98 mm, and the examined statolith was 878 �m long and464 �m wide. For single-crystal X-ray diVraction, the stato-lith was mounted with instant adhesive on a thin glass Wbre,which was attached to a brass pin and was mounted ontothe heads of the goniometer. The single-crystal X-raydiVraction on C. Xeckeri statoliths was carried out at thediVractometer Siemens SMART three axis goniometer withAPEX II area detector system with a � range of 3.38–29.44°and a completeness of 96.7% (index ranges 9 · h · 9,¡9 · k · 9, ¡7 · l · 8). The diVractometer control soft-ware was Bruker AXS APEX 2 version 3.0/2009, whichwas also used for data reduction and empirical absorptioncorrection. Structure solution and reWnement was carriedout with the Bruker AXS SHELXTL version 2008/4/© 2008software.

For synchrotron X-ray microtomography (SR�-CT) ofboth species and for X-ray diVraction contrast tomography(DCT) of C. Xeckeri ethanol-preserved statocysts, respec-tively, statoliths were dehydrated in acetone and embeddedin Spurr medium (Robinson et al. 1985). The IPD of theexamined C. Xeckeri medusa was 135 mm, and the exam-ined statolith was 830 �m long and 444 �m wide.

The microtomography scans of the P. periphylla rhopa-lia were carried out at beamline BW2 at HASYLAB/DESY(Hamburg, Germany). The CDs of the diVerent examinedmedusae were 8.4 mm and 55 mm and statocyst width230 �m and 560 �m of in accordance. The synchrotronradiation was monochromatized to a) 10 keV and b)14 keV. Projection images were recorded in steps of 0.25from 0 to 180°. The detector was a KX2 instrument (Apo-gee Instruments; 14-bit digitalization at 1.25 MHz,1536·1024 pixel; each 9·9 �m2). For normalization of therecorded data, Xat Weld images of the beam were recordedevery eight projections. Reconstruction was performedusing a Wltered backprojection algorithm (Donath et al.2004). 3D renderings were created with the program VGStudio MAX 1.2. The voxel side length after reconstructionwas 2.06 and 2.10 �m.

The microtomography scan of the C. Xeckeri statolithwas performed at the TopoTomo beamline of the ANKAlight source (Karlsruhe, Germany) using polychromaticillumination (the radiation of the bending magnet sourcewas Wltered by a 1-mm-thick Si and a 0.5-mm-thick BeWlter, resulting in a broad spectrum with the mean energyaround 20 keV) (Rack et al. 2009). The detector employedconsisted a visible-light microscope (OptiquePeter, France)equipped with a 4£ Olympus objective (NA 0.16) and a2.5£ eye-piece. The microscope is used to project the

luminescence image of a 25-�m-thick Eu-doped Lu3Al5O12

(LAG:Eu) scintillating thin Wlm (grown on top of a150-�m-thick Y3Al5O12 (YAG) substrate) onto a PCO 4000CCD camera (PCO AG, Germany—4008 £ 2672 pixels,each 9 �m in size, 5000:1 dynamic range). The spatialresolving power of the detector is 2.5 �m (0.9 �m eVectivepixel size) (Rack et al. 2009). Thousand projection imageswere recorded within a 180° scan. Tomographic recon-struction was done utilizing the PyHST software packageof the European Synchrotron Radiation Facility (Mironeet al. 2009). For the volume renderings, the software pack-age myVGI 1.2 by Volume Graphics GmbH was employed.

The X-ray diVraction contrast tomography (DCT) scanof the C. Xeckeri statolith was performed at beamline ID19beamline of ESRF (Johnson et al. 2008; Ludwig et al.2008; Ludwig et al. 2009). And 360 monochromatic beam(17.6 keV) projection images, each integrated over 1 degreein rotation angle, were recorded on a high-resolution detec-tor system (4.8 mm Weld of view; 2.4 �m pixel size), posi-tioned 5 mm downstream the sample. The size of the X-raybeam was adjusted to about 1 £ 1 mm, illuminating theentire sample at any rotation angle. From the correspondingdirect beam absorption image formed in the central area ofthe detector screen, one can calculate the conventionalabsorption contrast tomogram. Due to the proximity ofsample and detector part of the diVracted beams are cap-tured on the detector. These “diVraction spots” can beapproximated as parallel projections of the grain. Afterbackground correction of the raw images, 380 such diVrac-tion spots were segmented and used for further analysis.Out of the initial 380 spots, 180 Friedel pairs (hkl and -h-k-l reXections) of diVraction spots were identiWed andunambiguously indexed into 10 distinct grains sets, using theunit cell parameters and space group of “bassanite” (Abriel andNesper 1993). Details of the polycrystal index and recon-struction process can be found in (Ludwig et al. 2009).

Results

Periphylla periphylla and Chironex Xeckeri: morphology and histology of the rhopalia

In Periphylla periphylla medusa, four rhopalia were presentat the bell margin, hanging downwards between the cleftsof marginal lappets. The rhopalium consisted of a proximalbulb and a distal statocyst composed of many refractivecrystals (statoliths) (Figs. 1, 4a). The connection of thebasal part of the rhopalium (bulb) and the statocyst wereconstricted and were provided with a thickened and com-pact mesogloea. The statocyst was covered by an epidermalhood that was situated in the area between the bulb and thestatocyst. The epidermis of the hood was a cuboidal epithe-

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lium on the outer side and plate-like on the inner side, withthe latter being diVerentiated at the base of the hood by athickened sensory area. The sensory areas are characterizedby nonmotile kinocilia on their surface. The epidermis ofthe statocyst closely to the hood is diVerentiated into acylindrical sensory area. A second, Xatter sensory areaexisted on the opposite proximal side of the statocyst. Theproximal side of the bulb of the rhopalium was diVerenti-ated into a pseudostratiWed epithelium (with neurons situ-ated apically) and a neuropil. The gastrodermis of the bulbsurrounded the gastrovascular cavity, whereas the statocystwas Wlled with gastrodermis completely. The gastrodermalcells contained many lipid droplets within the basal part ofthe rhopalium and contained the statolith vacuoles withinthe statocyst. The smallest statolith vacuoles were locatedtowards the base of the statocyst.

In Chironex Xeckeri medusa, four rhopalia were presentin epidermal niches located close to the bell margin, andalternating with four groups of tentacles. The rhopalia wereconnected to the roof of the rhopalial niche by a stalk. Eachrhopalium had a compact structure, which included a stato-cyst distally and a basal section within which the eyes werehoused (Figs. 2, 4b). The epidermis of the statocyst regionwas diVerentiated into a sensory area on both sides. A gas-trovascular cavity was located within the basal part of therhopalium (Figs. 3, 4b). The epidermis of this region wasdiVerentiated to a pseudostratiWed epithelium with neuronsand a subjacent neuropil. Balloon cells were apparent nearthe aboral lens eye. The gastrodermis of the statocystenclosed one large region, which contained the statolith.The statocyst was broadly attached to rest of the rhopalium.The gastrodermal layer of the statocyst was enclosed by athin mesogloeal layer, whereas the mesogloea was thickerand more compact in the aboral and distal part of the transi-tion zone (Figs. 3, 4b).

Periphylla periphylla: growth of statocysts and statoliths

The growth of the statocyst as well as the number and growthof the statoliths were examined in medusae of 1.8–185 mm indiameter (Pearson correlation, Fig. 5). Statocyst width (Linearregression, r2 = 0.944, F4.02 = 253.57, P < 0.001) as wellas number of statoliths per statocyst (Linear regression,r2 = 0.976, F4.08 = 245.23, P < 0.001) increased expo-nentially with the growth of medusae. The mean width ofthe Wve largest statoliths increased logarithmically with

Fig. 1 Periphylla periphylla: longitudinal section of the rhopalium,median, photograph, scale bar 100 �m, e-epidermis, g-gastrodermis,gc-gastrovascular cavity, h-hood, m-mesogloea, n-neurons, np-neuro-pil, sa-sensory area, sg-statolith producing gastrodermis, sv-statolithvacuole (statoliths are dissolved during preparation)

Fig. 2 Chironex Xeckeri: light microscopical photograph of a com-plete Wxated (90% ethanol) rhopalium, paramedian view, scale bar100 �m, l-lense, lle-lower lens eye, m-mesogloea, pl-pigment layer,sk-stalk, se-slit eye, st-statolith, ule-upper lens eye

Fig. 3 Chironex Xeckeri: Longitudinal section of the rhopalium,median, diagram, scale bar 100 �m, the gastrovascular channel withinthe stalk is not shown, b-balloon cells, l-lens, e-epidermis, g-gastroder-mis, gc-gastrovascular cavity, m-mesogloea, n-neurons, np-neuropil,pl-pigment layer, rs-rhopalial stalk, sa-sensory area, sg-statolith pro-ducing gastrodermis, ds-dissoved statolith, vl-vitrous layer

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medusa growth (Linear regression, r2 = 0.710, F4.06 = 26.79,P < 0.001).

Chironex Xeckeri: X-ray single-crystal diVraction

The X-ray single-crystal diVraction of the C. Xeckeri stato-lith demonstrated that it consisted of several oligocrystalswith minimum sizes of 50 �m. The crystal structure wascompletely clariWed. The crystals were determined to beoligocrystals, because of the orientation of their crystal lat-tice in various axes. The statolith material consisted of cal-cium sulphate hemihydrate (bassanite) with an empiricalformula CaSO4 · 0.5 H2O with a density of � (calc) of2.711 g cm¡3 at a temperature of T = 183(2) K. The struc-ture of the mineral was conWrmed to be bassanite in accor-dance with the crystallographic literature. The crystal shapewas a plate with � = 0.711 Å, and the crystal system is tri-gonal. The structural parameters of the trigonal cell unitdimensions (crystal system) were reWned to a space groupP 3121 a = 6.952(10), b = 6.952(10) and c = 6.352(3) Å and� = � = 90°, � = 120°, V = 265.810(14) Å3 (Z = 3).

Periphylla periphylla: microtomography scan (SR�-CT) of the statocyst

The microtomography scan showed the distribution of thestatoliths within a rhopalium of P. periphylla (Fig. 6). Theaccumulation of statoliths within the statocyst had a hel-met-like shape with an elongated area in the basal and oralpart of the statocyst (Fig. 6a + d). The basally and aborallylocated part appeared to be shorter. The apical area was

rounded. The basally located area had a depression (arrowsin Fig. 6a, d, c, f) and contained the smallest crystals(Fig. 6b + e). The largest crystals were located in the mostapical part and in the apical periphery. Statoliths of inter-mediate size were located in the centre of the statocyst(Fig. 6c + f). The arrangement of statoliths and the overallshape of the statolith accumulation did not change duringthe growth of the statocyst.

Chironex Xeckeri: microtomography scan (SR�-CT) of the statolith

The statolith was solid and had an overall ellipsoid shape.A basally located V-shaped indentation ran parallel at 50%of its shorter principal axis, whereas the apical side wasrounded. While the statolith surface was typically roughand granular, some areas were smooth and Xattened(Fig. 7a). The cross-section through the shorter axis of thestatolith reconstruction appeared homogenous. Granulescould be seen at the marginal surface where some over-growth of the statolith could also be seen (Fig. 7b).

Chironex Xeckeri: diVraction contrast tomography (DCT) of the statolith

The integrated, monochromatic beam projection images(“topographs”) acquired during the DCT scan clearly showthe presence of one large, dominant crystal, occupyingabout 85% of the entire volume of the statolith. Figure 8ashows one out of the twenty integrated, monochromaticbeam projection images that have been acquired from this

Fig. 4 Diagram of a longitudi-nal section through a rhopalium a Periphylla periphylla, scale bar 50 �m b Chironex Xeckeri scale bar 200 �m e-epidermis, g-gastrodermis, gc-gastrovascular cavity, h-hood, le-lens eye with retina, m-mesogloea, n-neuropil, rl- retinal layer, rn-rhopalial niche, sa-sensory area, sc-statocyst with statolith(s), rhopalium position (arrow cross): ao-aboral, d-distal, o-oral, p-proximal

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crystal. One can distinguish a radial structure which mightindicate the presence of growth sectors diverging in diVer-ent radial directions from a common point of origin, thesupposed position of the initial crystal germ from which thestatolith has grown. Figure 8b shows the diVraction CTreconstruction of the entire statolith, and Fig. 8c shows acut through the diVraction CT reconstruction of the entirestatolith, composed out of 9 distinct crystals. The shape andthe 3D spatial arrangement of some of the smaller crystalsresemble growth sectors too, but their distinct orientationsuggests that these crystals have grown from distinct

germs, located in the central region of the statolith(Fig. 8d).

Discussion

The recent discussion of the systematic order of the Cubo-zoa is based to date on classical morphological and histo-logical features and some molecular data. The presentinvestigation mainly focussed on detailed examinations ofthe statocysts and statoliths of the coronate scyphozoanmedusa P. periphylla and of the chirodropide cubozoanmedusa C. Xeckeri. Additionally, some classical morpho-logical and anatomical features were examined for thecomparison with other rhopaliophoran species. The stato-lith is part of the gravity-sense of the rhopaliophoranmedusa and thus important for the organism orientation.Hence, the statolith should be a conservative characteristicthat could be changed only while preserving its function.Therefore, the statolith appeared to be a good target for theexamination of phylogenetic relationships, especially incombination with new and sophisticated methods, such asSR�-CT. Examinations of statoliths using light micro-scopy and scanning electron microscopy used in previousstudies provide only information regarding the shape andsurface of the statoliths as well as images of an artiWciallyprepared cut surface within the crystal. Sophisticatedmethods used in the present study such as X-ray single-crystal diVraction analysis, microcomputer tomography(�-CT) and X-ray diVraction imaging using a synchrotronbeam line have the advantage of providing a nondestructiveand undisturbed view inside of the statocyst and even insidethe crystal, as well as providing information on statolithcomposition.

The X-ray single-crystal diVraction is a common tech-nique used to identify minerals and to clarify their detailedcrystal structure as well as the atomic spacing, includingbond-lengths, bond-angles and site-ordering information.Results of this technique have shown that the statolith of theC. Xeckeri medusae was composed of the comparablyrare (in the animal kingdom) biomineral bassanite (cal-cium sulphate hemihydrate) as it is occurs only in otherscyphozoans and cubozoans (Tiemann et al. 2002; Beckeret al. 2005; Tiemann et al. 2006; Boßelmann et al. 2007).The structural parameters of the trigonal cell unit werereWned to unit cell dimensions a = 6.952(10), b = 6.952(10)and c = 6.352(3) Å in good agreement with the literaturevalues of a = 6.937 and c = 6.345 Å for bassanite (Abrieland Nesper 1993). Even though the statolith appears as onesolid piece, it did not have a single-crystal structure, becausethe crystal lattice of the sample was not continuous. It was,in contrast, composed of several individually recognizableoligocrystals.

Fig. 5 Periphylla periphylla. a Increasing statocyst width in relationto medusa diameter, examined individuals n = 56, b increasing stato-lith number in relation to medusa diameter, examined individualsn = 43, c increasing statolith width in relation to medusa diameter,examined individuals n = 45

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Synchrotron-based computed microtomography (SR�CT)using hard X-rays is a useful method to distinguish betweenorganic tissue and minerals located within this tissuebecause of the strong diVerences in their absorption ofX-rays (Bonse and Busch 1996; Beckmann et al. 1999;Tadic et al. 2004; Neues et al. 2007). The great advantageof this method is that it is nondestructive and cuts can beperformed virtually on a three-dimensional dataset. Thismethod has been used previously to show the location andorientation of statoliths within the statocysts of diVerent

scyphozoan species (Becker et al. 2005; Prymak et al.2005; Boßelmann et al. 2007).

X-ray absorption and phase sensitive imaging techniquesreveal diVerences in the local X-ray attenuation coeYcientand in the electron density of the material, respectively.However, both techniques (X-ray single-crystal diVractionand SR�-CT) fail to image the grain structure in mono-phase poly- and oligocrystalline materials, since crystals ofdiVerent orientation generally do not show any diVerence inmaterial properties like electron density. Two-dimensional

Fig. 6 Periphylla periphylla: Microtomography of statoliths of two statocysts from diVerent-sized medusae, a–c CD medusa 8.4 mm, d–e CD medusa 55 mm, a–e Wgures of the com-plete accumulation of statoliths of a statocyst, a + e side view, b + e view on the basal area con-taining the smallest statoliths, c + f longitudinal cut section through the reconstructed stat-ocysts, arrows: basal area con-taining the smallest statoliths, scale bars 100 �m

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projection images of individual crystals can be obtained bya technique known as X-ray diVraction imaging (“topogra-phy”) (Tanner 1976). Here, we have employed an extensionof the X-ray diVraction imaging method to three dimen-sions, termed X-ray diVraction contrast tomography(Johnson et al. 2008; Ludwig et al. 2008, 2009). DCT is asynchrotron X-ray imaging technique combining the con-cepts of X-ray diVraction and tomographic imaging andprovides access to the shape and crystallographic orienta-tion of the grains in poly- and oligocrystalline materials, asit occurred in Chironex Xeckeri statoliths.

The organization of the statocyst itself appears to bediVerent within the Scyphozoa and Cubozoa. The statolithsare surrounded by gastrodermal cells in both examined spe-cies, but in P. periphylla, the whole statocyst was Wlledwith gastrodermis cells that contain the statoliths, whichincrease in number and grow independently, whereas incubomedusae, a thin single-layer of gastrodermis was cov-ering one large compact statolith, which show daily growthrings (Claus 1878; Schäfer 1878; Conant 1898; Spangen-berg 1968, 1976; Russell 1970; Pollmanns and Hündgen1981; Ueno et al. 1995, 1997; Kawamura et al. 2003;

Gordon et al. 2004; Boßelmann et al. 2007; Holst et al.2007). Only the aberrant Tetraplatia volitans has saccularsense organs on the oral sides of the lappets with a single

Fig. 7 Chironex Xeckeri: microtomography. a Complete statolith, thelower side is directed towards the stalk (basally) b cut section throughthe reconstruction of the short axis of the statolith, scale bars 100 �m

Fig. 8 Chironex Xeckeri: a DiVraction topograph of main crystalb–d diVractionCT-images, the colour code represents the individuallyidentiWed crystals b complete statolith c cut through the completestatolith d small crystals within the central part of the statolith, scalebars 100 �m

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statolith (Ralph 1960). This species was determined inolder publications to be a coronate scyphozoan, but theexamination of the smaller and larger subunits of thenuclear ribosome shows that it belongs to the hydrozoantaxon Narcomedusae (Ralph 1960; Collins et al. 2006). Thelatter will be supported by the nonscyphozoan-like statolithstructure (Ralph 1960; Horridge 1969; Singla 1975).

Aside from these diVerences in the organization of thestatocysts of both species, we found fundamental similari-ties regarding the structure of the statoliths themselves.Firstly, the statoliths of C. Xeckeri are comprised of thesame biomineral bassanite as were found in P. periphyllaand all the other examined scyphozoan and cubozoan spe-cies (Tiemann et al. 2002; Becker et al. 2005; Tiemannet al. 2006; Boßelmann et al. 2007). This is a very speciWccharacteristic: Calcium sulphate is a very rare biomineral(Vinnikow et al. 1981; Lowenstam and Weiner 1989) andcalcium sulphate hemihydrate is, to date, known only inmedusae of the taxon Rhopaliophora, among the metazo-ans.

Additionally, there are similarities regarding the crystal-lographic structure of the statoliths. The statoliths ofP. periphylla increase in number with the age of the medusa,which results also in an increased width of the whole stato-cyst. The length and width of each statolith increases alsowith the age of the medusa, shown on the size of the Wvelargest crystals. The development mode of the statocyst iscomparable to the development of the statocyst of the scy-phozoan medusa Rhizostoma octopus (Holst et al. 2007).This leads to the conclusion that the statocyst of the scy-phozoan medusa grows by producing new, small statoliths,which grow independently and continuously by precipita-tion of new bassanite layers during their lifetime. A similarprecipitation of biomineral is described for the statolith ofthe Cubozoa. Several authors described, via light micro-scope, growth rings in ground statoliths of Carybdearastoni and Chiropsella bronzei described as Chiropsalmusquadrigatus (Ueno et al. 1995, 1997; Kawamura et al.2003; Gordon et al. 2004). These growth rings were notrecognizable with single-crystal diVraction or SR�-CT, whichmade it likely that they are caused by embedded organicmaterial, as is described for the biominerals in other taxa(Wilt and Ettensohn 2007).

The distribution of diVerent-sized statoliths (crystals) ofP. periphylla within the statocyst was shown by SR�-CT.The depression area with the smallest crystals was deWnedas the origin area of the statoliths. New statoliths developedin the basal gastrodermal cells with active nuclei and withvery small vacuoles (Figs. 1, 6). The crystals increased insize by continuous precipitation of bassanite; therefore thelargest (oldest) crystals were located in the apical periph-ery, whereas the smaller (younger) crystals were locatedmore in the centre. We found that the centre of the

V-shaped indentation of the C. Xeckeri statolith was compara-ble to the origin area of the P. periphylla statoliths. Thecompact statolith of C. Xeckeri enclosed in this area, a col-lection of small oligocrystals that were overgrown by onelarge oligocrystal as shown by DCT. The Xattened areas ofthe outer oligocrystal detected by SR�-CT could be inter-preted as crystal faces of the bassanite. The origin area isdescribed by Ueno et al. (1995), Gordon et al. (2004) as thecentre of the statolith. Growth rings, which are apparent inthe area of the outer large crystal, cannot be diVerentiated inthis area (Ueno et al. 1995). Additionally, examinations ofvery young medusae of Carybdea sp. (Carybdeidae) showthat their statocysts also contain several hexagonal crystalssimilar to the statocysts of scyphozoan medusae. Thesecrystals consolidate into a compact mass later during theirfurther development (Tiemann et al. 2006).

Cubozoa and Scyphozoa are separated in two classes dueto diVerences during medusa formation and due to morpho-logical features of the polyp and the medusa (Werner1976). Even though the arguments for establishing the classCubozoa are endorsed by several authors (Arneson andCutress 1976; Yamaguchi and Hartwick 1980), it is notaccepted by all authors (Matsumoto 1995; Ruppert et al.2004). Some authors assume a sister group relationshipbetween the Stauromedusae and the Cubozoa based onribosomal RNA data, and the assumption that follicle cellsare unique within the Stauromedusae (Marques and Collins2004). Examinations of gonads of the coronate medusaP. periphylla, however, show that follicle cells also exist inother scyphozoan taxa (Tiemann and Jarms 2010). Otherauthors suggest the Cubozoa as sister group of the Scypho-zoa with common ancestors and have established the com-mon taxon Rhopaliophora. Based on morphological andanatomical features they conclude that the Scyphozoa andCubozoa have common ancestors and are sister groups,closer related to each other than to other cnidarians (Schuc-hert 1993; Ax 1995). Some authors even assume that themetamorphosis of the Cubozoa might be derived secondar-ily from a monodisc strobilation mode, because of regener-ating polyp remnants after medusa development inCarybdea marsupialis (Thiel 1966; Straehler-Pohl andJarms 2005; Straehler-Pohl 2009). As a result, the rhopaliaof the Scyphozoa and Cubozoa have been suggested byseveral authors to be homologous and synapomorphic char-acteristics (Thiel 1966; Schuchert 1993; Ax 1995). A mainreason for this conclusion is the equivalent developmentalmechanisms seen during medusa formation, especiallybecause the bases of the polyp tentacles develop into therhopalia during strobilation (Scyphozoa) or metamorphosis,respectively (Cubozoa) (Werner et al. 1971; Calder 1973,1982; Werner 1975, 1993; Hofman et al. 1978; Laska-Mehnert 1985; Schuchert 1993; Arai 1997; Stangl et al.2002).

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The anatomy of the rhopalia of some scyphozoan andcubozoan medusae e.g. of the genera Periphylla, Para-phyllina, Nausithoe, Chrysaora, Cotylorhiza, Aurelia,Carybdea and Tripedalia have been examined by severalauthors (Claus 1878; Hertwig and Hertwig 1878; Schäfer1878; Hesse 1895; SchewiakoV 1889; Conant 1898; Ber-ger 1900; VanhöVen 1900, 1902; Maas 1903; Bigelow1910; Russell 1970; Pollmanns and Hündgen 1981; Skoghet al. 2006). In our study, the rhopalia of P. periphylla andC. Xeckeri had a club-like shape. The rhopalium ofC. Xeckeri was the more compact and stunted, as is typicalfor cubozoans. The rhopalium of P. periphylla, however,was longer, more slender and bore a hood, as is typical forscyphozoans. The anatomy of the rhopalia of both exam-ined species corresponded with the anatomical structure ofother rhopaliophoran species regarding the gastrodermalchannel and the thick epidermis with an agglomeration ofneurones in the bulb, as well as the position of the stato-cyst in the apical part. Also comparable in both specieswas the distally and aborally located thickened mesogleoaof the statocyst that is described also by Conant (1898),VanhöVen (1902). This could have an important role inmechanical Wxation of the statocyst on the base of therhopalium.

The rhopalia are assumed to serve as equilibrium orgravity sensors also because of the close relationshipbetween the hair-cell neurites (which serve as mechanore-ceptors) and the statocyst (Horridge 1966; Tardent and Sch-mid 1972; Hündgen and Biela 1982; Spangenberg et al.1994; Spangenberg et al. 1996; Holtmann and Thurm 2001;Nakanishi et al. 2009). As described for other rhopaliopho-ran species, the statocysts in P. periphylla and C. Xeckerideveloped within a terminal cyst of the gastrodermislocated in an identical position in close contact to the proxi-mally located neuroepithelium of the apical area of therhopalium (Conant 1898; Haeckel 1879; Hertwig and Her-twig 1878; Laska and Hündgen 1984; Maas 1903; Russell1970; SchewiakoV 1889; Satterlie 2002; Satterlie andNolan 2001; VanhöVen 1902). Newer information aboutsensing in cnidarian rhopalia is often related to light per-ception and conduction, whereas information about gravity-sensing is more restricted (Horridge and MacKay 1962;Horridge 1969, 1971; Hündgen and Biela 1982; Laska andHündgen 1982, 1984; Spangenberg 1991; Spangenberget al. 1994, 1996; Nakanishi et al. 2009). Therfore, the mul-tilayered neuroepithelium has been connected to the devel-opment of eyes (Claus 1878; Martin 2004; Garm et al.2006; Skogh et al. 2006; Ekström et al. 2008; O’Connoret al. 2009), but could also be involved in processing andtransmitting information about specimen position (Nakani-shi et al. 2009). This is even more likely because it devel-oped in a similar manner in P. periphylla medusae, whichlacks an eye.

Both examined species had (similar to other rhopalio-phorans) two sensory epithelia that could be connected tomechanosense reception in homologous positions on thedistal and proximal side of their rhopalia. These sensoryareas are partially described as touchplates in scyphozoans(Claus 1878; Conant 1898; Hesse 1895; Bigelow 1910;Thiel 1936; Horridge 1969; Russell 1970; Pollmanns andHündgen 1981; Chapman 1985; Nakanishi et al. 2009). Thefunctionality of gravity-sensing would have to be slightlydiVerent in the two taxa: On one hand, a statocyst that wasmovable against the bulb of the rhopalium with a hood ascounterpart in P. periphylla, and on the other hand, a rhopa-lium with an immotile attached statocyst that was movableagainst the stalk using the stalk itself or even the rhopaliarniche as counterpart in C. Xeckeri. This led to the assump-tion that the statolith had, in both cases, an indirect role ingravity-sensing. Supporting this claim, at least in Tripeda-lia, is the lack of neurons contacting the crystal directly(Garm pers. communication).

The anatomical comparison of the P. periphylla andC. Xeckeri rhopalia demonstrated a coincident organizationoverall. Furthermore, the crystallographic structure of thestatoliths, in particular, supports the conclusion that themarginal sense organs in Scyphozoa and Cubozoa arehomologous structures. This lead to the hypothesis that theCubozoa are a highly developed group of the Rhopalio-phora with roots connected to scyphozoan ancestors.

Acknowledgments We are grateful to HASYLAB at DESY,Hamburg and ANKA, Karlsruhe as well as the European SynchrotronRadiation Facility in Grenoble (France) for generous allocation ofbeamtime. For technical assistance and reconstruction of the microto-mography scans at DESY, we thank Felix Beckmann and Julia Herzen.Paulina Kämpfe and Henning Urch we thank for assistance duringimage recording at DESY. For assistance in specimen collection, wethank Jamie Seymour of TASRU (JCU) and grants from the LionsFoundation, National Geographic, Australian Geographic, Cairns CityCouncil, Cardwell City Council, Smart State QLD, JCUPRS & GRSand Rio Tinto.

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