Zoomorphology (2008) 127:21–36

DOI 10.1007/s00435-007-0045-1

ORIGINAL PAPER

Three-dimensional reconstruction of the central nervous system of Macrobiotus hufelandi (Eutardigrada, Parachela): implications for the phylogenetic position of Tardigrada

Juliane Zantke · Carsten WolV · Gerhard Scholtz

Received: 22 December 2006 / Accepted: 30 September 2007 / Published online: 15 November 2007© Springer-Verlag 2007

Abstract The morphology of the central nervous systemof the tardigrade species Macrobiotus hufelandi was ana-lysed with anti �-tubulin immunostaining in combinationwith confocal-laser-scanning-microscopy and computeraided three-dimensional reconstruction. The brain anatomyis unexpectedly complex with distinct tracts and highlyintermingled nerve Wbres. In contrast to older descriptions,we could not detect a suboesophageal ganglion. Further-more, we found no evidence for a tripartite/three-segmentedbrain organisation. The median part of the brain isdirectly connected to the Wrst pair of trunk ganglia via cir-cumoesophageal connectives. Surprisingly, the four pairedventral ganglia do not show segmental commissures typicalfor the ladder-like nervous system of arthropods. Our Wnd-ings constrain the phylogenetic position of Tardigrada. Themuch simpler organisation of the central nervous system ofTardigrada compared to that of Onychophora and Euarthro-poda and some similarities to the nervous system ofCycloneuralia support a phylogenetic position of Tardi-grada outside an Onychophora/Euarthropoda clade. Thismeans that Tardigrada might be either the sister group to all

other Arthropoda or they are more closely related toCycloneuralia.

Introduction

The phylogenetic relationships of Tardigrada, a group ofsmall metazoans with many unique and autapomorphic fea-tures, have always been controversially discussed (seeRichters and Krumbach 1928; Giribet et al. 1996; Ax 1999;Budd 2001; Nielsen 2001; Kristensen 2003; Jenner andScholtz 2005). Some authors favour close aYnities to thenematodes and their allies whereas other investigationsindicate that tardigrades belong to the arthropods. The latterview is currently more generally accepted. However,whether Tardigrada is the sister group of Euarthropoda, ofOnychophora or of Onychophora plus Euarthropodaremains ambiguous in recent studies. The advent of theEcdysozoa hypothesis (Aguinaldo et al. 1997) put new oilinto the Wre. Because this view allows to interpret the tardi-grade organisation as a kind of intermediate between thatof nemathelminths/cycloneuralians and of arthropods(Schmidt-Rhaesa et al. 1998; Giribet 2003).

The analysis of the nervous system of Tardigrada hasplays an important role in the discussion of the phyloge-netic aYnities of the group (e.g. Hanström 1928; Marcus1929; Kristensen and Higgins 1984a, b; Dewel and Dewel1996, 1997; Nielsen 2001). Previous light microscopicinvestigations of the tardigrade nervous system distin-guish a dorsal cerebral ganglion (supraoesophagealganglion) joined by circumoesophageal connectives to asuboesophageal ganglion and a ladder-type chain of fourpaired ventral trunk ganglia (Doyère 1840; GreeV 1865;Plate 1889; Basse 1905; Thulin 1928; Marcus 1929;Greven 1980, Ramazzotti and Maucci 1983; Wiederhöft

Electronic supplementary material The online version of this article (doi:10.1007/s00435-007-0045-1) contains supplementary material, which is available to authorized users.

J. Zantke · C. WolV · G. Scholtz (&)Humboldt-Universität zu Berlin, Institut für Biologie/Vergleichende Zoologie, Philippstr. 13, 10115 Berlin, Germanye-mail: gerhard.scholtz@rz.hu-berlin.de

Present Address:J. ZantkeDepartment für Evolutionsbiologie, Universität Wien, Althanstr. 14, 1090 Wien, Austria

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and Greven 1996; Dewel and Dewel 1997; Dewel et al.1999). Together with the analysis of the nervous system,cephalic sense organs (Walz 1978; Wiederhöft andGreven 1996) have been described and based on thesedata a tripartite organisation of the tardigrade brain hasbeen suggested by several authors (e.g. Kristensen andHiggins 1984a, b; Dewel and Dewel 1996; Nielsen 2001).This tripartite/three-segmented organisation has beenrelated to the three sections of the euarthropod brain,namely the proto-, deuto-, and tritocerebrum (see Scholtzand Edgecombe 2006) by some authors (Kristensen andHiggins 1984a, b; Nielsen 2001). However, the variousinterpretations of tardigrade brains as being tripartite arevery ambiguous, i.e. diVerent regions and structures havebeen interpreted as corresponding to the three parts of theeuarthropod brain (compare Kristensen and Higgins1984a, b; Dewel and Dewel 1996; Nielsen 2001).

The ventral nerve cord of arthropods and annelids ischaracterised by a rope-ladder like organisation (Scholtz2002; Müller 2006). The paired segmental ganglia are con-nected intrasegmentally by transverse commissures andintersegmentally by longitudinal connectives. These essen-tial elements of a ladder-like central nervous system havealso been described for Tardigrada and are found as generaltextbook knowledge (Kristensen 1982; Brusca and Brusca2002; Ruhberg 2004).

In this study the structure of the nervous system ofMacrobiotus hufelandi C.A.S Schultze, 1833 is describedfor the Wrst time by means of modern morphologicaltechniques such as Xuorescence microscopy, confocal-laser-scanning-microscopy, and computer-aided 3Dreconstruction. Some general aspects of the central nervoussystem of M. hufelandi correspond with older descriptions(Marcus 1929). However, our results reveal new detailsconcerning the internal organisation of the brain, the con-nection of the brain to the ventral chain of ganglia, thequestion of existence of a suboesophageal ganglion, andthe fact that no commissures exist between the ganglia ofthe ventral nerve cord. Furthermore, it is proposed thatganglia and neuropils of the tardigrade brain form a com-plex structure but that there is no indication for a tripar-tite/three-segmented brain comparable to that ofeuarthropods.

Materials and methods

Adult specimens of Macrobiotus hufelandi were extractedfrom moss collected from a roof in Keitum (Sylt), Germanyand maintained at 6°C. The moss pads were regularlymoistened with rainwater. For light microscopy unstainedspecimens were stretched maximally by removing oxygen(see Marcus 1929).

Fixation and permeabilization

For nuclei staining and whole mount antibody staining, thespecimens were Wxed in 3.7% paraformaldehyde in phos-phate-buVered saline (1£ PBS; 1.86 mM NaH2PO4,8.41 mM Na2HPO4, 175 mM NaCl, pH 7.2) for 15–20 minat room temperature. The animals were washed in 1£ PBSseveral times (three times for 5 min, four times for 30 min).In addition, a further Wxation was applied using 2.5% glu-tardialdehyde in 1£ PBS for 25–30 min followed by three5-min and four 30-min washes in 1£ PBS. The Wxation inglutardialdehyde has proved to be more eYcient for theantibody staining of the entire body, whereas the stainingeYciency of formaldehyde Wxed animals was higher eitherin the anterior or posterior part of the body. Before label-ling, specimens were stretched by being placed in distilledwater for 30 min at room temperature. For a better penetra-tion of dyes, Wxed specimens were sonicated for 60–75 s inan Elma ultrasonic bath model Transsonic 310 operating at35 kHz.

Nuclear staining

For staining with the DNA-selective Xuorescent dye Hoe-chst (Bisbenzimide, H33258) specimens were transferredinto Hoechst solution (concentration 100 �g/ml Bisbenzi-mide in 1£ PBS) and incubated for 15 min at room temper-ature. Unbound dye was removed by washing three timesfor 10 min in 1£ PBS. Specimens were mounted on glassslides in DABCO-glycerol (2.5 mg/ml DABCO (1.4 diazo-bicyclo-[2.2.2.]-octane, Merk) in 90% glycerol–PBS).

Immunocytochemistry

Immunolabelling of microtubules, such as axonal bundlesof neurons, was obtained with a monoclonal anti-acetylated�-tubulin antibody. For immunohistochemical processing,the Wxed and sonicated specimens were Wrst given three10 min washes and four washes of 30 min each in PBT (1£PBS, 0.2% Bovine Serum Albumin, 0.1% Triton X-100)followed by two 30-min washes in PBT + N (5% normalgoat serum). A monoclonal anti-acetylated �-tubulin anti-body, clone 6-11B-1, Sigma diluted at 1/100 in PBT + Nwas applied over night at 4°C. The samples were nextrinsed several times (three times for 5 min and four timesfor 30 min) in PBT followed by two 30-min washes inPBT + N. Next, tardigrades were incubated in goat anti-mouse serum (Sigma) conjugated to Cy3 over night at 4°C.This secondary antibody was diluted 1:200 in PBT + N.After rinsing frequently in PBS (three times for 5 min andsix times for 30 min) specimens were counterstained withXuorescent dye Hoechst (Bisbenzimide, H33258) andmounted in DABCO-glycerol as described above. As negative

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controls, specimens of M. hufelandi were processed with-out incubation in the primary antibody. Such specimensexhibited no detectable Xuorescence beside the autoXuores-cence of the cuticle, claws and buccal tube as visible in thedocumentations. For the applied phalloidin labelling, tardi-grades were washed three times for 5 min and two times for30 min in PBS after Wxation and sonication. Next, sampleswere washed 1 h in PBT followed by incubation in 2 �lphalloidin [TRITC-labelled] (0.1 mg phalloidin/1 mlDMSO) in 1 ml PBS for one hr at room temperature.Unbound dye was removed by washing several times (threetimes for 5 min, four times for 30 min) in PBS. The mountingoccurred as described above. Mounted specimensprocessed for Hoechst, �-tubulin, and phalloidin immuno-reactivity were viewed either with a Zeiss Axioplan 2 Xuo-rescence microscope equipped with a Zeiss digital camera(Axio Cam HRC) or a Leica SP2 confocal laser-scanningmicroscope. Fluorescence microscopic pictures were editedusing Photoshop CS (Adobe). Images emerged from Imagestacks obtained by scanning microscopy were processedand edited using the 3-D reconstruction software IMARIS(Bitplane AG, Zürich). All confocal images are based onstacks of between 50 and 170 optical sections of a z-seriestaken at intervals of 0.2–0.4 �m.

Discrimination between nerve cells and other cell types

To discriminate between nerve cells and muscle cells weapplied a phalloidin staining for visualisation of the muscu-lature especially in the head region (Fig. 1f, g). Two longi-tudinal muscle tracts dominate the dorsal side, in additionto the muscles of the buccal apparatus. A more complexstructure of transverse running muscles as well as longitu-dinal ones is located ventral to the buccal tube. For adetailed description of the musculature system of tardigradesas resolved by phalloidin staining see Schmidt-Rhaesa andKulessa (2007). The double labelling of anti-�-tubulin withHoechst and the comparison with the phalloidin stainingreveals that most of the nuclei dorsal to the buccal tubebelong to nerve cells as well as the nuclei in the region ofthe ventral ganglia. Glia cells are largely ignored in ourinvestigation in reference to the research of Greven andKuhlmann (1972), according to which the nerve tissue ofM. hufelandi possesses only very few glia cells.

Results

The brain

The brain of M. hufelandi is characterized by a complexmeshwork of nerve cell clusters and Wbre bundles ofvarious sizes (Fig. 1b, c). It forms a full circle around the

buccal tube (Figs. 1, 2, 3). Anti-�-tubulin immunoreactivityand Hoechst labelling conWrm the typical lobate appearanceof the tardigrade brain which has been described by Marcus(1929). There are two pairs of caudally directed lobes, theouter dorsolateral lobes which bear the eyes (arrow inFig. 1c) and the more ventral inner lobes (Figs. 1c, 2e). Thelargest part of the brain consists of three paired bilateral-symmetrically arranged neuronal cell clusters (Fig. 1c) andtheir corresponding plexus which are located dorsal and lat-eroventral to the buccal tube forming a saddle-like structure(Fig. 3b). Traditionally this part is called the supraoesopha-geal ganglion. In addition there is a small unpaired ventralcell group forming a mixture of muscles and nerve cells,the ventral cluster (Fig. 1e). Some of the numerous Wbrebundles form large transverse tracts like the dorsal commis-sure, preoral commissure, and postoral commissure whichconnect the lateral halves of the brain (Figs. 2, 3).

The anteriormost of three three pairs of dorso- and ven-trolateral nerve cell arrangements are the anterior cell clus-ters which are connected by a band of nerve cells dorsal tothe buccal tube (Fig. 1c). The most central paired nerve cellgroups are represented by the posterior cell clusters whichagain are joined by a transverse dorsal cell band and whichform the inner lobes of the brain (Figs. 1c, 2e, 3a). The pos-terior- and dorsalmost brain neurons are arranged in thedorsal clusters which form the outer lobes (Figs. 1c, 2e, 3a,b). Double labelling with anti-�-tubulin and Hoechst showsa distinct arrangement of Wbre bundles in the regions corre-sponding to the cell clusters (Fig. 3). Fibres of the posteriorclusters are located more central, Wbres of the anterior clus-ters extend ventrolaterally to the buccal tube (arrow inFig. 2f). A serial of single stacks of anti-�-tubulin immuno-reactivity (Fig. 2a–f) reveals the complex pattern of Wbreslocated in the head dorsal, dorsolateral, lateral, and latero-ventral to the buccal tube. The dorsalmost part of the brainconsists of a median plexus which gives rise to a stronglylabelled longitudinal tract linked to a small ganglion(Figs. 2b, 3a) consisting of Wve to eight cells (data notshown). Various Wbres connect the small ganglion withWbres of the posterior neuropils (Fig. 2b).The median andventral part of the brain is composed of Wbres laterallyarranged to the buccal tube (Figs. 2d, e). Compared to theamount of nerve cell nuclei located dorsal to the buccaltube, there are much less nuclei on the ventral side(Fig. 1d, e).

The few neurons of the ventral cluster form, togetherwith the postoesophageal commissure, the ventral part ofthe circumbuccal brain (Fig. 1e). The ventral cluster com-prises in total around 25–35 nuclei. The muscle stainingwith phalloidin shows quite a complex structure of trans-verse and longitudinal tracts ventral to the buccal tube(Fig. 1g). Hence it is likely that most of the nuclei ventral tothe buccal tube belong to muscle cells. We identiWed at

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�

Fig. 1 Anti-acetylated �-tubulin immunoreactivity in the CNS of thetardigrade Macrobiotus hufelandi complemented with nuclearstaining. Anterior is up. a Double labelling with the DNA-selectiveXuorescent dye Hoechst (blue) and the anti-acetylated �-tubulinantibody (red). Ventral view showing the segmentally repeated fourganglia (gI–gIV). The frame indicates a higher magniWcation of thesecond ganglion pair (one optical section). Nuclei of smaller ganglia(arrow in the fourth segment) and clawglands (cgl) can be seen in theposteriormost part of the legs (arrowhead). Claws (c), cuticle, buccaltube and placoids are autoXuorescent. b Anti-acetylated �-tubulinlabelling from (a). Fibre bundles dorsal to the buccal tube are highlyreactive for anti-�-tubulin (arrow). The Wrst ventral ganglion pair isconnected to the outer lobes of the brain by the outer connective (oco).The four ventral ganglia are linked to each other by paired connectives(co) along the longitudinal axis. Claws, cuticle, buccal tube andplacoids are autoXuorescent. c Dorsal view of the brain (Hoechst),

inverted confocal image. The nuclei of the dorsal brain part arearranged in three cell clusters, the anterior cluster (acl), the dorsalcluster (dcl), and the posterior cluster (pcl). Two bands of nerve cellnuclei link the clusters (arrowhead). Some nuclei are arranged circum-buccal (star). The position of the eye is indicated by arrowhead; Wrstganglion pair (gI). The buccal tube is autoXuorescent. d Ventral viewof the cerebral ganglion indicates a smaller amount of nuclei ventral tothe cerebral ganglion (Hoechst). e Double labelling with Hoechst(blue) and anti-acetylated �-tubulin (red), single stack. Anterior to theWrst ganglion (gI) nuclei form the ventral cluster (dotted circle).A paired neuron was identiWed within the ventral cluster (arrowhead).f Phalloidin staining to mark the body musculature. Dorsal view showstwo longitudinal muscles (arrowhead). g Phalloidin staining, ventralview. Scale bar a, b 50 �m; c, d 20 �m; e, f 30 �m; g 20 �m (see sup-plementary material movie 1)

Fig. 2 Anti-acetylated �-tubulin immunoreactivity in the head repre-sented in a series of single scans from dorsal (a) to ventral (f). Anterioris up. a Fibre bundles form a median plexus. A median tract (arrow) isassociated with a small ganglion (star). Fibres curve between the me-dian tract and Wbres of the posterior cluster (arrowhead). Fibres of thedorsal cluster (dcl) expand dorsally. b The dorsal commissure (dcm)connects the Wbres of the posterior clusters (inner lobes) (pcl). c Thenerve 14 (n14) connects the small ganglion (see Fig. 2a) and the innerlobes (the posterior cluster) via two lateral branches (arrowheads). dFocus is still dorsal to the buccal tube. The preoral commissure (prcm)links the Wbre bundles of the posterior clusters (inner lobes). Fibres of

the posterior clusters (see e) emerge and project in posteroventraldirection forming the paired circumbuccal connectives (cco). e Fibresof the cerebral ganglion are morphologically arranged as outer lobes(ol) and inner lobes (il). The circumbuccal connectives extends to theposterior margin of the inner lobe. f Focus is ventral to the buccal tube.Fibre bundles of the circumbuccal connectives extend to the midlineforming the postoral commissure (pocm). Further Wbre bundles of thecircumbuccal connectives run in posterior direction representing theinner connectives (ico). Fibres of the anterior clusters extend ventrally(arrow). Scale bar a 20 �m applies to images b–f

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�

least the somata of one pair of neurons within the ventralcluster (Fig. 1e) which are linked to the circumbuccal con-nectives at the transition to the postoral commissure(Fig. 4a, c). These neurons send their axons in anteriordirection towards the mouth contributing to nerve 11. Moremedial, the postoral commissure gives rise to a pair ofaxonal Wbre bundles that project anteriorly towards themouth characterized as nerve 12 (Figs. 4a, c). The ventralcluster corresponds to the structure that has traditionallybeen named suboesophageal ganglion. However, it does notcontain a large amount of Wbres and it is not connected viaconnectives to the Wrst ventral ganglion (see below).

Dorsal commissure, preoral commissure, circumbuccal ring, and postoral commissure

The brain contains a voluminous transverse tract, the dor-sal commissure. The dorsal commissure connects the Wbreregions of the posterior cell clusters (inner lobes)

(Figs. 2b, 3a, b). In addition to the dorsal commissure, asecond more delicate Wbre bundle, the preoral commis-sure is found dorsal to the buccal tube (Figs. 2d, 3b). Itlies ventral to the dorsal commissure and connects alsothe inner lobes (Fig. 3b). Fibres deriving from the preoralcommissure run anteriorly to the mouth cone and posteri-orly to the stylets muscles. Paired Wbres of the dorsalcommissure project posteroventrally and cross the innerlobes of the brain (Figs. 2d, e). These Wbre tracts pass thebuccal tube laterally while taking course posteroventrally.They form the circumbuccal (circumoesophageal) con-nectives which are the lateral parts of the circumbuccalnerve ring (Figs. 2d, e, 4b). Each circumbuccal connec-tive branches into two delicate Wbre bundles ventrolateralto the buccal tube (Fig. 2f). A transverse Wbre tractbeneath the buccal tube forms the postoral commissure,the ventral part of the circumbuccal ring (Figs. 2f, 4a).The postoral commissure connects the inner lobes viathe circumbuccal connectives. Recently obtained data of

Fig. 3 Anti-acetylated �-tubulin immunoreactivity in the head. Ante-rior is up unless indicated. a Dorsal view of Wbre bundles located in thehead. The small image shows the double labelling with Hoechst (blue)in the same anterior (a) posterior (p) orientation. The dorsal commis-sure (dcm) ranges between Wbres of the posterior clusters (pcl). Fibresof the median tract (arrow) project anteriorly. They are described asnerve 13 (n13) which terminates in the median area of the forehead. Fi-bres of the dorsal commissure proceed in posterior direction, bendanteriorly and extend to the anterior margin of the head representingnerve 4 (n4). Multiple Wbres described as nerve 5 (n5) link Wbres of theanterior (acl) and posterior cluster (pcl). Nerve 6 (n6) emerges from

nerve 5 (n5) and projects to Wbres of the dorsal cluster (dcl) where itbranches brush-like. 45° rotation of the image in anterior directionleads to the projection in Wgure (b). The frame indicates the position of(c). b View from behind showing the course of the dorsal commissure(dcm) and the preoral commissure (prcm). The small image shows thedouble labelling with Hoechst (blue) in the same dorsal (d) ventral (v)orientation. Position of the buccal tube is indicated by an asterisk. cDorsolateral view Nerves 7–10 (n7, n8, n9, n10) emerge from the plex-us of the anterior cluster and project anteriorly to the forehead. Scalebar a, b 20 �m, c 10 �m (see supplementary material movie 2)

Fig. 4 Anti-acetylated �-tubulin immunoreactivity in the head region.Anterior is up. a Ventral view, projection indicates the level of Wbresfrom ventral (v) to dorsal to (d) by colour code. The inner connective(ico) extends between Wbre tracts of the inner lobe and the connectiveof the ventral ganglia chain (co). The outer connective (oco) leads be-tween Wbre tracts of the outer lobe (ol) and peripheral Wbres of the Wrstganglion (gI). Very faint Xuorescent signal marks the outer connectives(arrowhead). The postoral commissure (pocm) is located ventral to thebuccal tube and sends nerves 11 and 12 (n11, n12) in anterior direction

towards the mouth opening. Nerve 1 (n1) is associated with the outerconnective projecting into the Wrst leg. b Ventrolateral view. The innerconnective (ico) arises from the circumbuccal connective (cco) andshows a dorsal-ventral course. The outer connective (oco) is linked tothe outer lobe (ol). c Scheme of (a) and (b). Abbreviations: cco circum-buccal connective; co ventral connective; gI Wrst ganglion; ico innerconnective; n1 nerve 1; n11-n12 nerves 11 and 12; oco outer connec-tive; pocm postoral commissure. Scale bar a 25 �m (see supplementarymaterial movie 3)

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anti-tubulin immunoreactivity in 72 h old embryos haverevealed a circumbuccal nerve ring that is ventrally linkedto paired connectives extending to the posterior end (datanot shown). Paired Wbre tracts, the inner connectives, pro-ceed from the circumbuccal connective and project pos-teroventrally to the Wrst pair of trunk ganglia (Figs. 2f,4b). They continue into the connectives of the ventralcord. These are thus directly connected to the inner lobes(posterior cluster) of the brain by the circumbuccal con-nectives. In addition to the inner connectives, the outerlobes of the brain give rise to the outer connectives whichmerge into the paired plexus of the Wrst ganglia (Fig. 4a,b). A delicate nerve n1 connects the outer connective andthe Wrst leg (Fig. 4a). In summary, the brain is joined withthe ventral chain of ganglia by two paired connectives, (1)the inner connectives originating in the inner brain lobes(posterior cluster) and (2) the outer connectives originat-ing from the outer brain lobes (dorsal cluster) (Fig. 4b).

Head nerves

Marcus (1929) listed and numbered the large head andtrunk nerves of tardigrades. Here we follow his accountwith some modiWcations based on our observations. Nervesn1 to n3 name the paired nerves of ventral segmental gan-glia (see below) (Fig. 5a–g). Nerves n4 to n14 are brainnerves (Figs. 2, 3, 4). Except for the median nerves n13 andn14 all other nerves are paired.

Nerve 4 (n4): originates from cells of the posteriorcluster near the dorsal commissure. It projects posteri-orly, curves anteriorly, and then branches multipletimes and merges into the plexus of the anterior cluster(Fig. 3a).Nerve 5 (n5): exits the posterior cluster in anterolateraldirection. The nerve splits into two branches of whichone passes to the dorsal cluster (see nerve 6); the otherbranch splits into delicate Wbres merging into the ante-rior cluster (Fig. 3a).Nerve 6 (n6): arises from nerve n5, projects dorsally tothe plexus of the dorsal cluster branching in a brush-like manner near the epidermis (Fig. 3a).Nerves 7–10 (n7–n10): exit the plexus of the anteriorcluster and terminate Xanking the epidermis forming ahomogenous Weld (Fig. 3c). Nerves n7 and n8 arelocated centrally, nerves n9 and n10 are arrangedlaterally.Nerve 11 (n11): originates from the postoral commis-sure, extends horizontally in anterior direction inner-vating the mouth cone ventral to the buccal tube(Fig. 4a).Nerve 12 (n12): runs parallel and median to n11(Fig. 4a).

Nerve 13 (n13): is a median nerve emerging from theWbres of the dorsomedian tract (arrow in Fig. 2a). Itpasses the dorsal commissure leading in anterior direc-tion in parallel to the buccal tube to the epidermis ofthe forehead (Fig. 3a).Nerve 14 (n14): is a median nerve which connects theinner lobes and the small dorsomedian ganglion(Fig. 2a) It starts from the inner lobes (posterior clus-ters) as paired Wne Wbre tracts which unite to one nervewhich passes the dorsal commissure ventrally andleads in posterodorsal direction to the dorsomedianganglion (arrowhead in Fig. 2c).

The ventral ganglia

The chain of ventral ganglia comprises four paired gangliawhich are connected by paired connectives along the ante-rior-posterior axis (Fig. 1a). The segmental ganglia arearranged slightly anterior to the corresponding segmentalappendages (Fig. 1a). Nuclei of the four ganglia show abilateral-symmetrical arrangement along the longitudinalaxis comprising also some unpaired nuclei. A higher mag-niWcation of the nuclei of ganglion two is shown in Fig. 1a(inset). The number of nuclei in each ganglion diVerswithin one animal and between individuals. Counting thenuclei of ten individuals revealed that the Wrst ganglionpossesses the largest number of nuclei (between 50 and 75nuclei). The second and the third ganglion comprise aver-agely 28% less nuclei than the Wrst ganglion (between 45and 50 nuclei). In comparison to the Wrst ganglion thefourth shows 55% less nerve cells (15–30 nuclei). The ven-tral connectives contain various Wbre bundles which do notform a homogenous tract (Fig. 5b). The ganglia are clearlyseparated from the connectives which are exclusivelyWbrous and lack perikarya of nerve cells (Fig. 1a). Trans-verse commissures connecting the lateral halves of segmen-tal ganglia could not be detected (Fig. 5b). Each plexuswithin one ganglion is separated from the other. Even in thethird and fourth ganglion where the connectives lie close toeach other we could not identify transverse tracts.

Peripheral nervous system of the trunk

Each of the segmental ganglia gives rise to three pairs ofsegmental nerves extending laterally to the periphery(Fig. 5d). The segmental nerves have a common originwithin the plexus of the ganglia. The rostral nerve n1extends dorsally, the central nerve n2 and the caudal nerven3 lie along the ventral body side terminating in the seg-mental limbs (Fig. 5c). Fibres in the distalmost part of eachlimb, which are linked to the central and caudal nerves, are

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highly reactive for anti-� tubulin (Fig. 5a, e). These Wbrebundles represent small ganglia of unclear function(Fig. 5f). The corresponding nerve cell perikarya are foundin close proximity to the cells of the clawglands in legs oneto three only in the fourth limb pair they are spatially disso-

ciated (Fig. 1a). The nerves of the Wrst ganglion show amodiWed course (Fig. 5a). Rostral Wbres give rise to twotracts of which nerve n1a projects dorsally and merges intothe outer lobes of the brain and nerve n1b innervates theWrst limb (Fig. 5a). Nerve n1a forms the outer connective.

Fig. 5 Anti-acetylated �-tubulin immunoreactivity in the peripheralnervous system. Ventral view anterior is up. a–d Peripheral nerves ofthe Wrst three body segments. e–g Peripheral nerves of the fourth bodysegment. a The Wrst ganglion (gI) sends three segmental nerves (n1, n2,n3). Rostral Wbres branch representing the nerves 1a (n1a) and nerve1b (n1b). Nerve 1a extends dorsally and merges to the outer lobe, itforms the outer connective (oco). Nerve 1b projects to the Wrst leg. Thecentral nerve 2 branches and runs to the periphery of the Wrst leg. Thecaudal nerve 3 (n3) innervates the Wrst leg. Fibres linked to the rostralnerve of the Wrst and second ganglion are highly reactive for anti-�-tubulin (arrowheads). b Higher magniWcation of the second ganglionin (c). No transverse Wbre bundles can be seen which are reactive foranti-�-tubulin. c Second ganglion (gII) with peripheral nerves. Nerve 1

(n1) extends to the dorsal musculature. Nerves 2 and 3 (n2, n3) inner-vate the second leg. d Scheme of the segmental nerves 1a, 1b, 2, 3(n1a, n1b, n2, n3) and outer connective (oco) of the ganglia one to three(gI–gIII). e Inverted confocal image. Fibres of the connectives (co)extend posteriorly across the fourth ganglion (gIV) representing nerve2 (n2) which gives rise to nerve 3 (n3). Nerve 1 (n1) is located near theanus and linked to Wbres which are high reactive for anti-�-tubulin(arrowheads). f Inverted confocal image. Double labelling with Hoe-chst (blue). Nerve 3 (n3) joins the fourth ganglion (gIV) with a smallganglion (arrowhead) showing Wbre bundles reactive for anti-�-tubu-lin. Clawglands (clg) are indicated. g Scheme of the segmental nerves1, 2, 3 (n1, n2, n3) of the fourth ganglion (gIV) with corresponding neu-ropils (arrowhead). Scale bar a 25�m; c 20�m, e, f 25 �m

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A delicate Wbre bundle arises from the central nerve n2 ofthe Wrst ganglion terminating in the Wrst limb (Fig. 5a).Also, the peripheral nerves of the fourth ganglion show amodiWed arrangement (Fig. 5e–g). The ventral connectivesextend posteriorly and give rise to two nerves travelling incaudal direction forming a small plexus with correspondingnuclei (Fig. 5e, f). The paired distal nerve is likely to behomologous to the central nerve n2 of the ganglia one tothree because of its course to the margin of the fourth limb.The paired proximal nerve is likely to be homologous to thecaudal nerve n3. A paired nerve originates from the centralpart of the fourth ganglion leading in posterior directioninnervating the anus (Fig. 5e). Due to the fact that thisnerve does not innervate the limb like the rostral nerves ofganglia one to three, we assume that the central nerve of thefourth ganglion is homologous to the rostral nerves of gan-glia one to three.

Discussion

Modern morphological techniques shed new light on the brain anatomy of tardigrades – all brain commissures are associated with the inner lobes

The general organisation of the nervous system of Macro-biotus hufelandi has been described in detail by Marcus(1929). With the modern morphological techniques appliedby us we basically conWrm many of Marcus’ results. How-ever, in addition our investigation led to new data concern-ing the shape and the internal organisation of thesupraoesophageal ganglion, the position of the brain, andthe connection between the brain and the ventral nervecord.

We identiWed the typical lobate appearance of the braincomprising two outer dorsolateral lobes extending posteri-orly and two smaller inner lobes which lie in a more ventralposition. We could not detect the third unpaired posteriorlobe which has been described by Marcus (1929). The dor-solateral and inner lobes correspond largely to deWnednerve Wbre regions and their grouped cell somata which areconcentrated dorso- and ventrolateral to the buccal tube.Furthermore, we identiWed several tracts and Wbre bundlesin the brain lobes which have not been described before.However, despite the compartmental organisation and theoccurrence of well deWned massive tracts and a complexpattern of Wbre arrangements we could not identify braincentres that correspond to neuropils such as the corporapedunculata (mushroom bodies), olfactory lobes, or a cen-tral body as are typical for arthropod brains (see Loesel2005). This result is in agreement with previous studies ofthe brain of M. hufelandi using transmission electronmicroscopy (Greven and Kuhlmann 1972).

Three transverse Wbre bundles, the dorsal commissure,the preoral commissure, and the postoral commissure, con-nect the left and the right brain hemispheres. All threetransverse tracts are associated with the two (inner) lobes ofthe posterior cluster which form the most central areas ofWbres in the brain. Furthermore, the commissures areclearly deWned along a dorsoventral axis. The dorsal com-missure forms the strongest tract and has also beendescribed by Dewel and Dewel (1996) in Heterotardigrada.The preoral commissure is the second transverse tract lyingdorsal to the buccal tube. Nerves run from the preoral com-missure in anterior and posterior directions towards themouth and the oesophagus. These nerves are assumed to beinvolved in the innervation of the stomatogastric nervoussystem. The postoral commissure, which also sends nervesinto the mouth region, forms the circumbuccal ring togetherwith the anterior sections of the circumbuccal connectives(Fig. 6). A clearly deWned connection between the preoraland the postoral commissure was not detected. Accordingto Dewel and Dewel (1996) four commissures exist in thebrain of Echiniscus viridissimus PeterW, 1956, the dorsalcommissure, the stomatogastric nerve system which con-sists of a dorsal and a ventral commissure encasing the buc-cal tube, and a single suboesophageal commissure.However, more recent investigations of Dewel et al. (1999)describe only one postoral brain commissure in tardigrades.

Cephalic nerves and sense organs: cirri and clavae are lost within eutardigrades but corresponding sensory areas remain

Arthrotardigrada among the Heterotardigrada possesseleven head appendages with apparent sensory functionincluding the median cirrus (see Kristensen and Higgins1984a, b). This condition has been considered to be ances-tral within Tardigrada whereas the appendage-less condi-tion in Eutardigrada seems to be derived (Marcus 1929;Ramazzotti and Maucci 1983). M. hufelandi as a represen-tative of the Eutardigrada does not show any cephalicappendages. However, already Walz (1978) identiWed vari-ous sense regions in the head of this species. According toThulin (1928) M. hufelandi possesses 19 nerves in the head.In contrast to this we identiWed 10 paired and 2 unpairednerves (in total 22) which are associated with the innerva-tion of speciWc sense regions in the head.

The branching and attachment of nerves to distinct epi-dermal regions found by us supports the studies of Walz(1978) and Wiederhöft and Greven (1996) on the character-isation of an anterolateral and posterolateral sense region inEutardigrada. Moreover, the present investigation revealsthat each cephalic nerve or deWned nerve region in M. hufe-landi corresponds to a cephalic sense organ of Heterotardi-grada (see Table 1). The identiWed nerve n13 in

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M. hufelandi is attached to the epidermis in a similar posi-tion to the median cirrus in Arthrotardigrada. The mediancirrus of the arthrotardigrade Styraconyx nanoqsunguakKristensen and Higgins 1984 is innervated by a small neu-ropil near the centre of the brain (Kristensen and Higgins1984b). In M. hufelandi the unpaired nerve n13 is corre-spondingly connected to a small triangular ganglion. Thecourse of the paired nerve n4 which runs into the area underthe epidermis of the forehead correlates with the position ofthe internal cirri in Heterotardigrada. Further correspon-dences exist between nerves n7–n10 of the Wbres of theanterior cluster in M. hufelandi and the external cirri andsecondary clavae in Heterotardigrada. The position of thelateral cirri and the primary clavae in Arthrotardigrada Wtwith the location of the nerve n6 and Wbres with the samecourse in M. hufelandi. In summary, it is obvious that thesensory regions of the eutardigrade head correspond to thecephalic sensory appendages of heterotardigrades such ascirri and clavae. If the heterotardigrade situation is consid-ered as plesiomorphic then, with the notable exception ofthe members of the Milnesiidae, the Eutardigrada have apo-morphically reduced their head appendages. In spite of thisreduction, the arrangement of distinct nerves in the head ismaintained and the areas of the appendages became trans-formed to cuticular sensory Welds. This scenario is sup-ported by recent molecular and morphological phylogeneticstudies (Garey et al. 1999; Jørgensen and Kristensen 2004;Nichols et al. 2006). Although a sister group relationshipbetween Heterotardigrada and Eutardigrada is suggested,Milnesiidae is the sister taxon to the remaining eutardi-grades (Nichols et al. 2006).

The connection between the brain and the ventral nerve cord: there is no suboesophageal ganglion

A ventral suboesophageal ganglion connected by acircumoesophageal ring to the dorsal supraoesophageal gan-glion and by paired connectives to the Wrst ventral pair of gan-glia is described as a general feature of the central nervoussystem of Tardigrada (Doyère 1840; Plate 1889; Hanström1928; Thulin 1928; Marcus 1929; Kristensen and Higgins1984a, b; Kinchin 1994; Dewel and Dewel 1997; Dewel et al.

1999; Nielsen 2001). More speciWcally, Dewel et al. (1999)identify neuropils concentrated ventrolateral to the buccal tubewhich form a ventral commissure and which are suggested torepresent the suboesophageal ganglion of the brain.

In contrast to these descriptions, we found that nerve cellnuclei and nerve Wbres in the head of M. hufelandi form acircumbuccal ring whereby the saddle-like supraoesophagealganglion extends lateroventrally to the buccal tube and isassociated with a ventral group of nerve cell nuclei embed-ded in muscle cells, which we call the ventral cluster. Wecan show that in the domain of the ventral cluster there areno massive Wbres or neuropil-like structures ventrolateral orventromedial to the buccal tube. The only structures ventralto the buccal tube that show reactivity for anti-�-tubulin are afew nerve cells in the ventral cluster, the postoral commis-sure and two pairs of nerves (nerves n11 and n12). Hence,the presence of nerve cell nuclei in a ventral position and thepostoral commissure do not indicate the existence of aproper suboesophageal ganglion in M. hufelandi.

According to our investigations the connectives of theventral chain of ganglia continue to the brain in the form ofthe inner connectives which are connected with the circum-buccal connectives lateral to the buccal tube (Fig. 6). Nonerves exist between the Wrst ventral ganglion and the ventralcluster. In addition, we found a connection between the outerlobes of the brain and the Wrst ventral ganglion formed by apair of connectives (outer connectives) (Fig. 6) (see alsoMarcus 1929; Dewel and Dewel 1996). However, in contrastto the older descriptions (see Marcus 1929, Wg. 23) we identi-Wed these outer connectives as being formed by a side-branchof the rostral nerve (n1) of the Wrst ventral ganglion. Sincethe rostral nerves of all ventral ganglia lead to the dorsal side,the connection between the Wrst ganglion and the outer lobesof the brain formed by the outer connective might be a sec-ondary feature due to the posterior extension of the outerlobes. This disputes the scenario of Dewel and Dewel (1996)about the evolution of head segmentation in Tardigrada.

In summary, even though nerve cell nuclei exist ventralto the oesophagus in the head of M. hufelandi they do notform an autonomous ganglion with neuropil. Rather theyrepresent a ventral association of the supraoesophagealganglion. This is supported by embryological investiga-tions. Hejnol and Schnabel (2005), who described thedevelopment of the “suboesophageal ganglion” by growingout of the supraoesophageal ganglion whereas a neuronalprogenitor cell, as in the four ventral ganglia, is not detect-able for the suboesophageal region.

Tardigrade head segmentation – questioning the tripartite/three-segmented brain

As with euarthropod (see Scholtz and Edgecombe 2006) andonychophoran heads (Strausfeld et al. 2006), the structures and

Table 1 Correspondence between the regions of cephalic appendagesin Heterotardigrada and the cephalic nerves in Macrobiotus hufelandi

Cephalic sense organs in Arthrotardigrada (Heterotardigrada)

Nerves in M. hufelandi

Internal cirri n4

External cirri, secondary clavae n7, n8, n9, n10

Lateral cirri, primary clavae n6, Wbres with similar course

median cirrus n13

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32 Zoomorphology (2008) 127:21–36

possible segmentation of the tardigrade head have been thesubject of highly controversial interpretations (Plate 1889;Hanström 1928; Marcus 1929; Kristensen 1982; Kristensenand Higgins 1984a, b; Dewel and Dewel 1996; Wiederhöftand Greven 1996; Nielsen 2001). Some authors interpretthe brain as consisting of only one unit corresponding to theprotocerebrum of arthropods (Richters and Krumbach1926; Hanström 1928; Dewel and Dewel 1996) whereasothers consider the lobate brain organisation as indicativeeither for a bipartite (Plate 1889) or a tripartite brain com-prising a protocerebrum, deutocerebrum and tritocerebrumhomologous to the brain parts of euarthropods (Kristensenand Higgins 1984a, b; Nielsen 2001). Plate (1889) inter-prets the inner lobes as the protocerebrum and the outerlobes as the deutocerebrum—although this view implies theunique association of the eyes with the second head seg-ment. According to this author, a tritocerebrum is not yetpresent as a brain part in tardigrades. The hypothesis of atripartite (three-segmented) brain is mainly based on thethree-lobed structure of the brain (as described by theseauthors, but see above) and the connections of theses lobesto the cephalic sense organs such as eyes, cirri, and clavae.However, the interpretation of what structures represent theprotocerebrum, deutocerebrum, and tritocerebrum diVersbetween the authors. The large dorsolateral lobes whichinnervate the primary clavae, the lateral cirri, and the eyes(if present) are interpreted as the protocerebrum by(Kristensen 1982; Kristensen and Higgins 1984a, b;Nielsen 2001). The deutocerebrum, however, which inner-vates the internal cirri, is described by Nielsen (2001) as a

more ventral, rather compact structure, whereas Kristensenand Higgins (1984a, b) characterize it as paired smaller dor-sal lobes. Nielsen (2001) identiWes a circumoesophagealtritocerebrum which has a postoral commissure, while Kris-tensen and Higgins (1984a, b) described the tritocerebrumas paired lateral lobes that innervate the secondary clavaeand the external cirri. Interestingly enough, Dewel andDewel (1996, 1997) also suggest a tripartite brain based onsimilar evidence as Kristensen and Higgins (1984a, b) andNielsen (2001). However, Dewel and Dewel (1996, 1997)conclude that the tardigrade brain corresponds only to theprotocerebrum of euarthropods. Nevertheless these authorslist a three-segmented brain as a putative synapomorphy fortardigrades and (eu)arthropods (Dewel and Dewel 1997:114). These apparent contradictions in the interpretation ofthe segmental status of the tardigrade brain indicate that theevidence for the claims of a metameric composition of thebrain is much too weak.

Our observations on M. hufelandi conWrm that there aredistinct Wbre and cell somata regions in the brain whichpartly correspond to the lobes already described in the 19thcentury. However, these structures alone cannot be used asevidence for a segmental composition of the brain sinceseveral compartmental areas such as neuropils, cellclusters, lobes, tracts etc. are described for single euarthro-pod brain neuromeres such as the protocerebrum or deut-ocerebrum (e.g. Hanström 1928; Sandeman et al. 1993;Harzsch 2006; Strausfeld et al. 2006). Whether the serialarrangement of cephalic cirri and clavae is indicative forthe fusion of several segments is also problematic since the

Fig. 6 SimpliWed drawing of the CNS and peripheral nerves of thehead and the Wrst trunk segment. a Dorsal view. The circumbuccal ringis formed by the dorsal commissure (dcm), the circumbuccal connec-tives (cco), and the postoral commissure (pocm). The cephalic nervesinnervating sensory Welds in the head are marked by arrowheads. Thecircumbuccal ring is linked to the Wrst ganglion (gI) via the innerconnectives (ico) in the area of the inner lobes (il). The outerconnectives (oco) join the outer lobes (ol) and the Wrst ganglion. The

preoral commissure (prcm) lies ventral to the dorsal commissure con-necting Wbres of the posterior clusters. Nerves visible are: segmentalnerves of the chain of ventral ganglia (n1–n3), head nerves 4–13 (n4–n13). Anterior cluster (acl), dorsal cluster (dcl), posterior cluster (pcl),eyes (e), small limb ganglion (arrow). b Lateral view. Nervous struc-tures are the same as in (a). The position of the ventral cluster is indi-cated by an arrowhead

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occurrence of a median cephalic cirrus suggests that,despite the fact that in the trunk cirri are often segmentallyarranged, cirri occur independent of segmental aYnities.The total lack of limb-like head appendages allows no fur-ther conclusions either. If we assume that the dorsal clusterrepresented the protocerebrum, the posterior cluster thedeutocerebrum and the anterior cluster the tritocerebrum,then all major transverse tracts connecting the brain hemi-spheres were found in the hypothetical deutocerebrum andnone in the hypothetical protocerebrum or tritocerebrum.Furthermore, the hypothetical tritocerebrum had no connec-tion to the ventral nerve cord and the major tracts connect-ing the three brain parts do not allow putting thehypothetical proto-, deuto-, and tritocerebrum in a properanteroposterior sequence (even if, as is occurring in manyeuarthropods, an upfolded anterior brain area is consid-ered). All this would be a very atypical pattern for anarthropod tripartite brain (see Scholtz and Edgecombe2005, 2006; Strausfeld et al. 2006).

In summary, we think that the case for a tardigrade headand brain formed by several metameric units is not con-vincing. On the contrary, our brain anatomy data indicate acompact, unsegmented brain. The compartmentalisation ofbrain areas and the concentration of sensory structuresmight simply reXect functional specialisations rather thanthe fusion of segments. The view of an ametameric brain isconsistent with the results of older and more recent embry-ological studies. Already Marcus (1929) reports that thehead mesoderm is formed by a single pair of coelomicpouches as is the case with the segmental mesoderm in thetrunk. However, the strongest evidence comes from theanalysis of the expression of the segmentation genesEngrailed and Pax3/7 in embryos of the eutardigradespecies Hypsibius dujardini (Doyère 1840) (Gabriel andGoldstein 2007). Both genes show a clear segmental patternwith one major expression domain in the brain region.Pax3/7 is expressed in the segmental ganglia of the trunkand in the brain anlage forming a ring around the stomo-daeum. Engrailed expression occurs in pairs of v-shapedstripes in the posterior region of each trunk segment, as apair of semicircular stripes at the posterior margin of theforming brain, and in some cells in the anterior brain area(Gabriel and Goldstein 2007). In particular, the Engrailedexpression in the head region of H. dujardini resembles theocular/protocerebral Engrailed expression of euarthropods(see Scholtz and Edgecombe 2005: Fig. 2) suggesting thatthe tardigrade brain might correspond to the anteriormostbrain part in euarthropods, the protocerebrum (see alsoDewel and Dewel 1996). The tardigrade brain, however,forms a complete circumoesophageal ring composed ofWbres and nerve cell perikarya. This stands in contrast tothe protocerebrum of euarthropods which is considered tobe a purely praeoesophageal neuromere, and the posterior

boundary of the euarthropod circumoesophageal nerve ringis thought to be composed of deutocerebral and tritocere-bral commissures (Harzsch 2004a; Scholtz and Edgecombe2006).

The ventral ganglia: three segmental nerves and the absence of segmental commissures

To our great surprise we could not detect segmentalcommissures in the four ganglia of the ventral nerve cord inM. hufelandi. Nevertheless, it is obvious from the cellarrangement, the lateral swellings, and the three segmentalnerves that the ganglia are bilaterally formed. We found insome cases nerve Wbres projecting towards the midline inthe ganglia but they never form a complete connectionbetween the two segmental ganglionic plexus. To ourknowledge the absence of segmental commissures has notbeen reported before in tardigrades. Of course, the lack ofcommissures could be due to the method, i.e. we simply didnot detect the Wbres. However, we consider this as beingunlikely given that with the techniques applied we foundvery Wne and detailed Wbre structures in the brain and in theperipheral nervous system. Accordingly, it appears that M.hufelandi does not exhibit commissures in the ventral partof the central nervous system. This absence of segmentalcommissures might be due to the close proximity of the lat-eral halves of the ventral ganglia as has been suggested formillipedes which also lack segmental commissures asadults (Harzsch 2004b) despite the early anlagen of com-missures during development (Dove and Stollewerk 2003).Although the central nervous system of tardigrades is gen-erally described as belonging to the rope-ladder like typecomprising ganglia with connectives and commissures,unambiguous documentation of commissures is almostabsent in the literature. Only Marcus (1929: 40, Wg. 26)describes and depicts segmental commissures in the ventralnerve cord of Hypsibius (Ramazottius) oberhaeuseri (Doy-ère 1840). However, this documentation is far from beingsatisfactory, leaving the possibility open that tardigrades ingeneral lack commissures in the ventral part of the nervoussystem. Whether segmental commissures are transientlyformed during embryonic development is unknown.Accordingly, more investigations on adult neuroanatomyand neurogenesis are badly needed to clarify the issue ofsegmental commissures in Tardigrada.

As is described in the older literature (Marcus 1929)our investigation reveals that each ventral ganglion pairpossesses three large paired segmental nerves (n1 to n3).Our Wndings also conWrm that n1 runs to the dorsal bodyregion (Marcus 1929). In contrast to the previousdescriptions, according to which only n3 projects into thelimb, we found that n3 and n2 connect to the segmentalappendage.

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Phylogenetic implications

Neuroanatomy has been used for phylogenetic inferencesfor many decades (e.g. Holmgren 1916; Hanström 1928;Bullock and Horridge 1965; Sandeman et al. 1993; Harzsch2006). In particular, the recent morphological techniques ofconfocal-laser-scanning-microscopy in combination withcomputer-aided 3-dimensional reconstruction oVer a degreeof anatomical resolution unmatched by previous methods.This allows comparisons at great detail with sometimes sur-prising results (e.g. Fanenbruck et al. 2004; Strausfeld et al.2006; this paper).

Recent phylogenetic analyses of tardigrade aYnitiesalmost universally agree that Tardigrada are part of theArthropoda (e.g. Garey et al. 1996; Giribet et al. 1996;Garey 2001; Mallatt and Giribet 2006). Although in somemolecular and morphological analyses tardigrade represen-tatives sometimes appear outside Arthropoda or cluster withcycloneuralians (e.g. Moon and Kim 1996; Bergström andHou 2001; Giribet 2003; Jenner and Scholtz 2005; Mallatand Griribet 2006; Park et al. 2006), and Ax (1999) consid-ers the relationships of Tardigrada to any other bilateriangroup as unresolved. However, even if one accepts tardi-grade aYnities to arthropods whether tardigrades are the sis-ter group to Euarthropoda (Dewel et al. 1999; Edgecombeet al. 2000; Budd 2001; Maas and Waloszek 2001; Nielsen2001), to Onychophora plus Euarthropoda (see discussion inSchmidt-Rhaesa et al. 1998; Bergström and Hou 2001;Schmidt-Rhaesa 2001) or to Onychophora (e.g. Plate 1889;Mallatt and Giribet 2006) is still under debate.

The present study is the Wrst to apply immunocytochem-ical techniques to the study of tardigrade neuroanatomy,and accordingly it is diYcult to generalise our Wndings forthe taxon Tardigrada as a whole (see above). However, aputative scenario considering the competing views of theArticulata (e.g. Scholtz 2002) and Ecdysozoa (e.g. Giribet2003) hypotheses might be allowed with respect to the dataat hand.

If the Ecdysozoa hypothesis is correct, then the absenceof commissures in M. hufelandi might also represent a char-acter state resembling the condition of cycloneuralians,where apart from lateral commissures to our knowledge nocommissures connecting the midventral nerve cords of pria-pulids, kinorhynchs, and nematodes have been reported (e.g.Storch 1991; Wright 1991; Nebelsick 1993; White et al.1986) whereas commissures, although in a diVerent pattern(see Ruhberg 2004; Mayer and Harzsch 2007; Whitington2007), occur in euarthropods and onychophorans. Further-more, the type of unsegmented but a circumoesophagealring forming brain found by us in M. hufelandi resemblesthe condition in cycloneuralians, which also exhibit a ringshaped brain around the oesophagus—although the detailedorganisation of the cycloneuralian brain with its typical

arrangement of cell bodies, neuropil, and cell bodies as threelayers and the absence of an enlarged supraoesophageal partwith lobe-like neuropils is diVerent (Wright 1991; Storch1991; Nebelsick 1993; Neuhaus 1994; Schmidt-Rhaesaet al. 1998; Nielsen 2001; Ax 2001). Moreover, the apparentabsence of typical arthropod brain neuropil regions such asmushroom bodies, central complex etc. is shared by Tardi-grada and Cycloneuralia. Greven and Kuhlmann (1972)report further similarities between the nervous system of M.hufelandi and nematodes such as the low number of gliacells and the absence of a perilemma surrounding the gan-glia. Depending on the polarisation of the characters sharedbetween tardigrades and cycloneuralians these charactersmight either represent apomorphies supporting cycloneura-lian aYnities of Tardigrada or they are plesiomorphies (see,for instance, Nielsen 2001, who considers a cricumoesopha-geal brain as part of the protostome ground pattern) withrespect to onychophorans and euarthropods which wereretained by the Tardigrada.

Under the assumption that Articulata is a valid taxon andthat Tardigrada are related to Arthropoda, the lack of com-missures in M. hufelandi would be an evolutionary losssince commissures occur in annelids, onychophorans, andeuarthropods (Scholtz 2002; Müller 2006; Strausfeld et al.2006). If the position of tardigrades as sister group to aclade comprising onychophorans and euarthropods isassumed, the absence of clearly deWned segmental gangliain adult Onychophora (Schürmann 1995; Eriksson et al.2003; Ruhberg 2004; Mayer and Harzsch 2007; Whitington2007) would be a secondary feature.

In summary, the characters of the central nervous systemof M. hufelandi, in particular the lack of a tripartite/three-segmented brain, speak in favour of a phylogenetic positionof Tardigrada outside of a clade comprising Onychophoraand Euarthropoda, irrespective of the Articulata or Ecdyso-zoa hypotheses. However, whether Tardigrada are the sistergroup to Onychophora plus Euarthropoda or whether theyshow closer aYnities to Cycloneuralia remains to be tested.In any case, this preliminary consideration already revealsthat further use of more neuroanatomical data of Tardigradawill contribute to our understanding of tardigrade phyloge-netic aYnities in a more global approach considering avariety of morphological and molecular data.

Acknowledgments We thank Greg Edgecombe for critical commentsand the members of the group “Vergleichende Zoologie” at the Hum-boldt-Universität zu Berlin for many discussions and practical advices.

References

Aguinaldo AMA, Turbeville JM, Linford LS, Rivera MC, Garey JR,RaV RA, Lake JA (1997) Evidence for a clade of nematodes,arthropods and other moulting animals. Nature 387:489–493

123

Zoomorphology (2008) 127:21–36 35

Ax P (1999) Das System der Metazoa II. Ein Lehrbuch der phylogene-tischen Systematik. Fischer Verlag, Stuttgart

Ax P (2001) Das System der Metazoa III. Ein Lehrbuch der phylogene-tischen Systematik. Fischer Verlag, Stuttgart

Basse A (1905) Beiträge zur Kenntnis des Baues der Tardigraden.Z wiss Zool 80:259–281

Bergström J, Hou X-G (2001) Cambrian Onychophora or xenusians.Zool Anz 240:237–245

Brusca R, Brusca GJ (2002) Invertebrates. Sinauer Associates, Sunder-land

Budd GE (2001) Tardigrades as “stem- group arthropods”: the evi-dence from the Cambrian fauna. Zool Anz 240:265–279

Bullock TH, Horridge GA (1965) Structure and function in the nervoussystem of invertebrates. WH Freeman, San Francisco

Dewel RA, Dewel WC (1996) The brain of Echiniscus viridissimus Pe-terW, 1956 (Heterotardigrada): a key to understanding the phylo-genetic position of tardigrades and the evolution of the arthropodhead. Zool J Linn Soc 116:35–49

Dewel RA, Dewel WC (1997) The place of tardigrades in arthropodevolution. In: Fortey RA, Thomas RH (eds) Arthropod Relation-ships. Chapman and Hall, London, pp 109–123

Dewel RA, Budd GE, Castano DF, Dewel WC (1999) The organisationof the suboesophageal nervous system in tardigrades: insights intothe evolution of the arthropod hypostome and tritocerebrum. ZoolAnz 238:191–203

Dove H, Stollewerk A (2003) Comparative analysis of neurogenesis inthe myriapod Glomeris marginata (Diplopoda) suggests moresimilarities to chelicerates than to insects. Development130:2161–2171

Doyère L (1840) Mémoire sur les Tardigrades. Ann Sci Nat 14:269–361

Edgecombe GD, Wilson GDF, Colgan DJ, Gray MR, Cassis G (2000)Arthropod cladistics: combined analysis of histone H3 and U2 sn-RNA sequences and morphology. Cladistics 16:155–203

Eriksson BJ, Tait NN, Budd GE (2003) Head development in the Ony-chophoran Euperipatoides kanagrensis with particular referenceto the central nervous system. J Morph 255:1–23

Fanenbruck M, Harzsch S, Wägele J-W (2004) The brain of the Rem-ipedia (Crustacea) and an alternative hypothesis on their phyloge-netic relationships. Proc Nat Acad Sci USA 101:3868–3873

Gabriel WN, Goldstein B (2007) Segmental expression of Pax3/7 andEngrailed homologs. Dev Genes Evol 217:421–433

Garey JR (2001) Ecdysozoa: The relationship between Cycloneuraliaand Panarthropoda. Zool Anz 240:321–330

Garey JM, Krotec M, Nelson DR, Brooks J (1996) Molecular analysissupports a tardigrade-arthropod association. Invert Biol 115:79–88

Garey J, Nelson DR, Mackey L, Li J (1999) Tardigrade phylogeny:Congruency of morphological and molecular evidence. Zool Anz238:205–210

Giribet G, Carranza S, Baguñà J, Riutort M, Ribera C (1996) Firstmolecular evidence for the existence of a Tardigrada + Arthrop-oda clade. Mol Biol Evol 13:76–84

Giribet G (2003) Molecules, development and fossils in the study ofmetazoan evolution; Articulata versus Ecdysozoa revisited. Zool-ogy 106:303–326

GreeV R (1865) Ueber das Nervensystem der Bärthierchen, Arctisco-ida C.A.S. Schultze (Tardigraden Doyère) mit besondererBerücksichtigung der Muskelnerven und deren Endigungen. ArchMikrosk Anat 1:101–122

Greven H (1980) Die Bärtierchen. Ziemsen Verlag, WittenbergGreven H, Kuhlmann D (1972) Die Struktur des Nervengewebes von

Macrobiotus hufelandi C.A.S. Schultze (Tardigrada). Z Zellforsch135:131–146

Hanström B (1928) Vergleichende Anatomie des Nervensystems derwirbellosen Tiere unter Berücksichtigung seiner Funktion.Springer, Berlin

Harzsch S (2004a) The tritocerebrum of Euarthropoda: a “non-droso-philocentric” perspective. Evol Dev 6:303–309

Harzsch S (2004b) Phylogenetic comparisons of serotonin-immunore-active neurons in representatives of the Chilopoda, Diplopoda,and Chelicerata: implications for arthropod relationships. J Mor-ph 259:198–213

Harzsch S (2006) Neurophylogeny: architecture of the nervous systemand a fresh view on arthropod phylogeny. Integr Comp Biol46:162–194

Hejnol A, Schnabel R (2005) The Eutardigrade Thulinia stephaniaehas an indeterminate development and the potential to regulateearly blastomere ablations. Development 132:1349–1361

Holmgren N, (1916) Zur vergleichenden Anatomie des Gehirns vonPolychaeten, Onychophoren, Xiphosuren, Arachniden, Crustac-een, Myriapoden und Insekten. Kunglig Svenska Vetensk AkadHandl Stockholm 56:1–303

Jørgensen A, Kristensen RM (2004) Molecular phylogeny of Tardigra-da- investigation of the monophyly of Heterotardigrada. Mol PhylEvol 32:666–670

Jenner RA, Scholtz G (2005) Playing another round of metazoan phy-logenetics: Historical epistemology, sensitivity analysis, and theposition of Arthropoda within the Metazoa on the basis of mor-phology. In: Koenemann S, Jenner RA (eds) Crustacea andArthropod Relationships. CRC Press, Boca Raton, pp 355–385

Kinchin IM (1994) The Biology of Tardigrades. Portland Press,London

Kristensen RM (1982) The Wrst record of cyclomorphosis in Tardigra-da based on a new genus and species from Arctic meiobenthos. ZZool Syst Evolutionsforsch 20:249–270

Kristensen RM (2003) Comparative morphology: do the ultrastructuralinvestigations of Loricifera and Tardigrada support the clade Ec-dysozoa? In: Legakis A, Sfenthourakis S, Polymeni R, Thessalou-Legaki M (eds) The New Panorama of Animal Evolution: Pro-ceedings XVIII International Congress of Zoology. Pensoft,SoWa, pp 467–477

Kristensen RM, Higgins RP (1984a) A new family of Arthrotardigrada(Tardigrada: Heterotardigrada) from the Atlantic coast of Florida,USA. Trans Am Microsc Soc 103:295–311

Kristensen RM, Higgins RP (1984b) Revision of Styraconyx (Tardi-grada: Halechiniscidae) with descriptions of two new speciesfrom Disko Bay, West Greenland. Smithson Contrib Zool391:1–40

Loesel R (2005) Editorial - The arthropod brain: retracing six hundredmillion years of evolution. Arthropod Struct Dev 34:207–209

Maas A, Waloszek D (2001) Cambrian derivatives of the early arthro-pod stem lineage, pentastomids, tardigrades and lobopodians—an‘Orsten’ perspective. Zool Anz 240:451–459

Marcus E (1929) Tardigrada. In: Bronn HG (ed) Klassen und Ordnun-gen des Tierreichs. Akademische Verlagsgesellschaft, Leipzig,pp 1–609

Mallatt J, Giribet G (2006) Further use of nearly complete 28S and 18SrRNA genes to classify Ecdysozoa:37 more arthropods and a kin-orhynch. Mol Phyl Evol 40:772–794

Mayer G, Harzsch S (2007) Immunolocalization of serotonin in Ony-chophora argues against segmental ganglia being an ancestral fea-ture of arthropods. BMC Evol Biol 7:118

Moon SY, Kim W (1996) Phylogenetic position of the Tardigradabased on the 18S ribosomal RNA gene sequences. Zool J LinnSoc 116:61–69

Müller MCM (2006) Polychaete nervous systems: Ground patterns andvariations-cLS microscopy and the importance of novel charac-teristics in phylogenetic analysis. Integr Comp Biol 46:125–133

Nebelsick M (1993) Introvert, mouth cone, and nervous system of Ech-inoderes capitatus (Kinorhyncha, Cyclorhagida) and implicationsfor the phylogenetic relationships of Kinorhyncha. Zoomorphol-ogy 113:211–232

123

36 Zoomorphology (2008) 127:21–36

Neuhaus B (1994): Ultrastructure of alimentary canal and body cavity,ground pattern, and phylogenetic relationships of the Kinorhyn-cha. Micro Mar 9:61–156

Nichols PB, Nelson DR, Garey JR (2006) A family level of analysis oftardigrade phylogeny. Hydrobiologia 558:53–60

Nielsen C (2001) Animal Evolution: Interrelationships of the LivingPhyla. Oxford University Press, Oxford

Park J-K, Rho HS, Kristensen RM, Kim W, Giribet G (2006) Firstmolecular data on the phylum Loricifera – An investigation intothe phylogeny of Ecdysozoa with emphasis on the positions ofLoricifera and Priapulida. Zool Sci 23:943–954

Plate L (1889) Beiträge zur Naturgeschichte der Tardigraden. ZoolJahrb Anat 3:487–550

Ramazzotti G, Maucci W (1983) Il Philum Tardigrada. Mem Ist ItalIdrobiol 41:1–1012

Richters F, Krumbach TH (1926) Tardigrada. In: Kükenthal W, Krum-bach TH (eds) Handbuch der Zoologie 3. Berlin und Leipzig, pp1–68

Ruhberg H (2004) Onychophora, Stummelfüßer. In: Westheide W,Rieger R (eds) Spezielle Zoologie. Spektrum Akademischer Ver-lag, Heidelberg, pp 420–428

Sandemann DC, Scholtz G, Sandemann RE (1993) Brain evolution indecapod Crustacea. J Exp Zool 265:112–133

Schmidt-Rhaesa A (2001) Tardigrades—are they really miniaturizeddwarfs? Zool Anz 240:549–555

Schmidt-Rhaesa A, Kulessa J (2007) Muscular architecture of Milne-sium tardigradum (Tardigrada) and other eutardigrades. Zoomor-phology (in press)

Schmidt-Rhaesa A, Ehlers U, Bartolomaeus T, Lemburg C, Garey JR(1998) The phylogenetic position of the Arthropoda. J Morph238:263–285

Scholtz G (2002) The Articulata hypothesis- or what is a segment? OrgDivers Evol 2:197–215

Scholtz G, Edgecombe GD (2005) Heads, Hox and the phylogeneticposition of trilobites. In: Koenemann S, Jenner R (eds) Crustacea

and Arthropod Relationships. CRC Press, Boca Raton, pp 139–165

Scholtz G, Edgecombe GD (2006) The evolution of arthropod heads:reconciling morphological, developmental and palaeontologicalevidence. Dev Genes Evol 216:395–415

Schürmann FW (1995) Common and special features of the nervoussystem of Onychophora: a comparison with Arthropoda, Annel-ida, and some other invertebrates. In: Breidbach O, Kutsch W(eds) The Nervous Systems of Invertebrates: An Evolutionary andComparative Approach. Birkhäuser, Basel, pp 139–158

Storch V (1991) Priapulida. In: Harrison FW (ed) Microscopic Anat-omy of Invertebrates, vol 4, Wiley-Liss, New York, pp 333–350

Strausfeld NJ, Strausfeld CM, Stowe S, Rowell D, Loesel R (2006)The organization and evolutionary implications of neuropils andtheir neurons in the brain of the onychophoran Euperipatoidesrowelli. Arthropod Struct Dev 35:169–196

Thulin G (1928) Über die Phylogenie und das System der Tardigraden.Hereditas 11:207–266

Walz B (1978) Electron microscopic investigation of cephalic senseorgans of the Tardigrade Macrobiotus hufelandi C.A.S. Schultze.Zoomorphology 89:1–19

Wiederhöft H, Greven H (1996) The cerebral ganglia of Milnesium tar-digradum Doyère (Apochela, Tardigrada): three dimensionalreconstruction and notes on their ultrastructure. Zool J Linn Soc116:71–84

White JG, Southgate E, Thomson JN, Brenner S (1986) The structureof the nervous system of the nematode Caenorhabditis elegans.Phil Trans R Soc B 314:1–340

Whitington PM (2007) The evolution of arthropod nervous systems:insights from neural development in the Onychophora and Myr-iapoda. In: Kaas JH (eds) Evolution of Nervous Systems, vol 1,theories, development, invertebrates. Elsevier, Amsterdam, pp317–336

Wright KA (1991) Nematoda. In: Harrison FW (ed) Microscopic Anat-omy of Invertebrates, vol 4. Wiley-Liss, New York, pp 111–195

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