musculature in polychaetes: comparison of myrianida prolifera (syllidae) and sphaerodoropsis sp....

Musculature in polychaetes: comparison of Myrianida prolifera (Syllidae) andSphaerodoropsis sp. (Sphaerodoridae)

Anna Filippova,1 Gunter Purschke,a,2 Alexander B. Tzetlin1 and Monika C. M. Muller2,b

1 Department of Invertebrate Zoology, Moscow State University, Moscow, Russia2 Zoologie, Fachbereich Biologie/Chemie, University of Osnabrueck, Germany

Abstract. The relationship of the polychaete taxa Syllidae and Sphaerodoridae within Phyllo-docida is still unresolved: phylogenetic analyses either show them as sister groups or morewidely separated. The present article aims to provide information about the structure of themuscular system that could be essential for understanding their relationship. A crucial pointis whether the body wall contains circular muscles, which has recently been shown to be ab-sent in more taxa than previously known. The F-actin filaments in members of Myrianidaprolifera (Syllidae) and Sphaerodoropsis sp. (Sphaerodoridae) were labeled with phalloidinand their three-dimensional relationships reconstructed by means of confocal laser scanningmicroscopy. Among the noteworthy differences that emerged between the species are (1)members ofM. prolifera possess four, those of Sphaerodoropsis sp. eight, longitudinal musclestrands; (2) the body wall in M. prolifera contains transverse fibers in a typical, supralongi-tudinal position, while in Sphaerodoropsis sp., corresponding fibers lie beneath the longitu-dinal strands; (3) pro- and peristomium in M. prolifera have no distinct F-actin fibers, whilefive longitudinal pairs and three single transverse muscular fibers shape the anterior end inSphaerodoropsis sp.; (4) the proventricle of M. prolifera comprises primarily radial musclefibers arranged in distinct rows, while in Sphaerodoropsis sp. the axial proboscis consists oflongitudinal and circular fibers and radial fibers are lacking; (5) inM. prolifera, the proximaland distal sections of the two anteriormost pairs of dorsal cirri possess longitudinal myofil-aments, which are separate from the body wall musculature; by contrast, all appendages inSphaerodoropsis sp. do not; (6) both species have bracing muscles: in M. prolifera they arepositioned above the longitudinal fibers, whereas in Sphaerodoropsis sp. they are uniquelypositioned between longitudinal and sublongitudinal transverse fibers. These results do notsupport a sister-group relationship of Syllidae and Sphaerodoridae. In addition,Sphaerodoropsis sp. is yet another example in the list of polychaetes lacking typical circularmuscles in the body wall.

Additional key words: Annelida, phalloidin, F-actin, evolution, proventricle

This article is part of a series of studies re-investi-gating the architecture of the annelid muscular sys-tem. Interest in this system was revitalized by thefinding that a number of polychaete species lack anouter layer of circular fibers (e.g., Filippova et al.2005, 2006; Tzetlin & Filippova 2005; Muller &Worsaae 2006; Purschke & Muller 2006; Ruchel &Muller 2007). These observations are in conflict with

the generally supposed ground pattern of annelidmusculature, which is considered to consist of anouter layer of circular and an inner layer of longitu-dinal muscles (Lanzavecchia et al. 1988; Gardiner1992). Species without circular musculature maypossess transverse fibers that could either be derivedfrom circular fibers or could belong to the parapodialcomplex. For Nereis diversicolor O.F. MULLER, 1776and Aphrodita aculeata LINNE, 1758, Mettam (1967,1971) demonstrated that the ‘‘circular portions’’ ofthe body wall are transverse fibers belonging to theparapodial complex. In Dorvillea bermudensis AKES-

SON & RICE, 1992 and Dorvillea diadema AKESSON,1974, complete rings of transverse muscles were ob-

Invertebrate Biology 129(2): 184–198.

r 2010, The Authors

Journal compilation r 2010, The American Microscopical Society, Inc.

DOI: 10.1111/j.1744-7410.2010.00191.x

aAuthor for correspondence.

E-mail: [email protected]

bCurrent address: Evangelische Akademie Loccum,

PO Box 2158, 31545 Rehburg-Loccum, Germany.

mailto:[email protected]

served in the zone of segment proliferation, whichlater on are disrupted by formation of the parapodiaand its musculature (M.C.M. Muller & T. Hinken,unpubl. data; Purschke & Muller 2006; Bergter et al.2008). This suggests that these circular fibers of D.bermudensis andD. diademamay be transformed intoparapodial muscles as development proceeds.

Depending on the phylogenetic hypothesis consid-ered, the annelid stem species is either seen oligo-chaete-like without parapodia and head appendages,or polychaete-like with well-developed parapodiaand head appendages (see Bartolomaeus et al. 2005,for discussion). In the former scenario, Clitellata aresister to a clade Polychaeta and the simple-bodied,oligochaete-like polychaetes, named Scolecida, forma basal group in the latter (e.g., Rouse & Fauchald1997; Rouse & Pleijel 2001), a pattern not recoveredin molecular analyses (e.g., McHugh 1997; Roussetet al. 2007; Struck et al. 2007, 2008) and questionedby morphological data as well (Westheide 1997;Purschke 2002; Bartolomaeus et al. 2005). Finally,the hypothesis of an oligochaete-like annelid stemspecies goes back to the ideas of Clark (1962, 1964,1981) regarding a burrowing form as last commonancestor of the annelids. This view is inevitably cor-related with the organization of the body wall mus-culature, because circular muscles are definitelyneeded for peristaltic burrowing, whereas such mus-cles are not necessarily essential for moving via pa-rapodia as shown by many examples (Tzetlin et al.2002a,b; Tzetlin & Filippova 2005). The repeatedfindings of polychaetes without or with only weaklydeveloped circular musculature raises the question:which pattern is apomorphic and which is plesio-morphic, and therefore must belong to the groundpattern of the entire group?

To the long list of species lacking circular fibers(e.g., Mettam 1967, 1971; Storch 1968; Hermans1969; Gardiner & Rieger 1980; see Tzetlin et al.2002a,b; Tzetlin & Filippova 2005) several speciesbelonging to different taxa have recently been added:Chrysopetalidae (Dysponetus pygmaeus LEVINSEN,1879, Tzetlin et al. 2002a,b; Chrysopetalum, Tzetlinet al. 2002a), Magelonidae (Magelona cf. mirabilisO.F.MULLER, 1858) and Spionidae (Prionospio cirrif-eraWIREN, 1883) (Filippova et al. 2005), Dorvilleidae(Dorvillea kastjani TZETLIN, 1980, Filippova et al.2006; D. diadema, Bergter et al. 2008; D. bermuden-sis, M.C.M. Muller & T. Hinken, unpubl. data, seePurschke & Muller 2006), and Nerillidae (Muller &Worsaae 2006). The latter studies applied a combi-nation of F-actin labeling and confocal laser scan-ning microscopy (cLSM), a combination of methodswhich has already been proven to be a very valuable

tool for 3D reconstructions of myo- and neuroanat-omy, especially in small animals (Mollers & Muller2001;Muller & Schmidt-Rhaesa 2003;Muller & Ster-rer 2004; Muller et al. 2004; Wanninger et al. 2005;Bergter et al. 2007, 2008; Bolanos & Litvaitis 2009;Hunnekuhl et al. 2009; Neves et al. 2009). These in-vestigations may be complemented by transmissionelectron microscopy (TEM), especially if the natureof certain structures labeled appears to be uncertain.

In the present study, these methods were applied toinvestigate and three-dimensionally reconstruct themusculature of Myrianida prolifera O.F. MULLER,1788 (Syllidae) and Sphaerodoropsis sp. HARTMAN &FAUCHALD, 1971 (Sphaerodoridae). One aim was todemonstrate the presence or absence of circular mus-cles in the body wall. Another aim was to compareprecisely the species of these two taxa in order to lookfor structures which could prove to be synapomor-phic, and thus to test their discussed relationship (seeRouse & Pleijel 2001; Aguado & San Martin 2009).Among others, one character of special importance isthe proventricle in the foregut, which—based onevaluation of the pertinent literature—has been re-garded to be present in these two taxa (see Fauchald& Rouse 1997). The term ‘‘proventricle’’ was appliedby Rudermann (1911) for the muscular part of thepharynx in Ephesia gracilis (5Sphaerodorum fla-vum), suggesting a high degree of similarity with thesyllid proventricle. However, in Sphaerodoridae it isnot known whether it represents a muscular axialpharynx, as typical for many Phyllodocida, or showsspecific correspondences with the proventricle ofSyllidae. Consequently, coding a proventricle, aspresent in phylogenetic analyses, Sphaerodoridaeand Syllidae may group together. Generally, thisgroup is not well supported, still leaving the phylo-genetic position of Sphaerodoridae as uncertain(Rouse & Pleijel 2001). As stated by Aguado &Rouse (2006) a comparison of this structure in thesetwo families is clearly needed; and as a first step theproventricle is studied with cLSM in the present in-vestigation.

Methods

Specimens ofMyrianida prolifera 5Autolytus pro-lifer (O.F. MULLER, 1788) were collected in the phytalof the North Sea Island of Helgoland, Germany, andfrom pontoons in a marina at Morgat, France; thoseof Sphaerodoropsis sp. were collected from subtidalmud in 10–30m depth from Kandalaksha Bay at theWhite Sea, Russia.

For cLSM, the organisms were anesthetized for10min in 8%MgCl2 solution and subsequently fixed

Musculature of syllid and sphaerodorid polychaetes 185

Invertebrate Biologyvol. 129, no. 2, spring 2010

on ice overnight in 4% paraformaldehyde in0.15molL�1 phosphate-buffered saline (PBS; pH7.4) containing 12% sucrose. After rinsing and stor-age in PBS with traces of NaN3, specimens were pre-incubated in PBT (PBS with 0.1% Triton X-100) for1 h and then incubated in a fluorescein isothiocyan-ate-labeled phalloidin solution (5mL of 3.3mmolL�1

solution in 100mL PBS). Subsequently, they werewashed several times with PBS. Specimens were em-bedded in Citiflour (Plano, Wetzlar, Germany) be-tween two cover slips to allow observations of eachspecimen from opposite sides. They were investigatedwith Zeiss (Jena, Germany) LSM 410 and Zeiss Pas-cal 5 cLSM. Z-stacks were projected into maximum-intensity pixel images. The 3-D arrangement ofstained structures can be directly deduced from thecolors, which follow the spectral light from red (5pe-ripheral structures) to dark blue (5 central struc-tures). In Sphaerodoropsis sp., three specimens, andin M. prolifera, three complete specimens as well astwo dissected single segments from an additionalspecimen, were analyzed.

For TEM, animals were relaxed in 8% MgCl2 forB15min and then fixed in a solution containing su-crose, picric acid, paraformaldehyde, and glutaralde-hyde buffered with sodium phosphate (SPAFG afterErmak & Eakin 1976). After 2.5 h at 41C, they werewashed in a 0.075molL�1 phosphate buffer (pH 7.2,7 changes, 2.5 h). Specimens were postfixed in buf-fered 1% OsO4 (pH 7.2, 1 h), dehydrated in a gradedethanol series, and embedded in a mixture containingeither Epon 812, or PolyBed 812 (Polysciences Inc.,Warrington, PA, USA) and Araldite (Fullam Inc.,NY, USA). Ultrathin sections were cut with a dia-mond knife on a Leica (Wetzlar, Germany) UltracutE, collected on single-slot grids coated with Piolo-form (0.3% in CH3Cl) support films. Staining wascarried out in a Leica EM Stain with uranylacetate(Leica Ultrostain 1 or in 5% uranylacetate in H2O)for 25min at 481C and lead citrate (Leica Ultrostain2) for 6min at 201C. Sections were examined withZeiss (Oberkochen, Germany) EM 109 and 902Aelectron microscopes. Images were either taken onconventional films or recorded electronically with aslow scan CCD camera. Image adjustment (onlybrightness and contrast) was carried out with AdobePhotoshop 7.0 and arrangement of plates withAdobe Illustrator 11.0 (Adobe Systems Inc., SanJose, CA, USA).

The nomenclature used here was introduced byFilippova et al. (2005): abbreviations for the bodysections—prostomium (pr), palps (p), pharynx (ph),parapodia (pp), body wall (bw), anal cirrus (ac)—arefollowed by orientation as well as location of the

muscles, e.g., ‘‘ma/pl’’ indicates ‘‘median antenna,proximal longitudinal muscles.’’ To keep the abbre-viations as short as possible, a character for ‘‘muscle’’is generally omitted. Furthermore, all muscles ori-ented perpendicular to the anterior–posterior bodyaxis are termed ‘‘transverse muscles.’’ Only trans-verse fibers encircling the entire body as a completering outside the longitudinal musculature (supralon-gitudinal) will be called ‘‘circular muscles.’’

Results

Head region and associated appendages

M. prolifera. The prostomium bears the palps, onemedian, and a pair of dorsolateral antennae, the per-istomium, one pair of ventral and one pair of dorsaltentacular cirri, the second segment chaetae, and onepair of dorsal parapodial cirri, with the same ar-rangement for the following segments (Fig. 1A). Inall of these appendages, a short basal, and a long andslender distal part, can be distinguished. Whereas thebases are penetrated by thick muscle fibers originat-ing from the body wall (Figs. 1C, 2A), the latter partscontain only filigree fibers, which are more or lesslongitudinally oriented (Fig. 2A). A distinct gap be-tween the two parts indicates termination of the mus-cle fibers and articulation of the thin ones. TEMobservations show that the F-actin staining in thedistal regions of the antennae and tentacular cirri isdue to delicate muscle cells (Fig. 3A–D). The fibersare embedded basiepithelially in the epidermal tissueand are located close to the central nerve (Fig. 3A,B).The muscle fibers are not separated from the sur-rounding cells by a distinct extracellular matrix. Thefibers are comparatively small and contain only a fewgroups of myofilaments, and may be categorized assmooth fibers (Fig. 3C,D). Thick and thin myofila-ments, as well as z-elements, are distinguishable. Allfilaments follow the longitudinal axis of the append-ages. Nuclei and mitochondria are situated outsidethe myofilament-containing part (Fig. 3D). Veryprobably, these cells are not included in the epider-mal cell layer and do not reach the cuticle. In addi-tion, transverse filaments have been found in certainepithelial cells, which might be myofilaments as well,because they are grouped in thin and thick filaments(Fig. 3E). However, these were are only detectableunder cLSM at high magnifications and appear asinterrupted transverse bands just underneath thesurface.

The thick muscle fibers visible in the bases of allthree antennae are interpreted as branches from thedorsal longitudinal strands, which approach the base

186 Filippova, Purschke, Tzetlin, & Muller


of the median antenna (Figs. 1C, 2A). The fibers inthe lateral antennae are twice as long as those in themedian antenna, forming a strand of almost equaldiameter, while those of the median antenna widen

distally, forming a distinct terminal triangle. Themuscles in the bases of all other appendages belongto the respective parapodial complex. The palpsof M. prolifera are fleshy, stout, and distally fused.Together with the posterior lips, they surroundthe mouth opening. Each palp is penetrated bynumerous F-actin fibers that spread out in afan-like bow toward the midventral edge of theappendage. Shortly before ending, the musclestrands ramify again, producing two opposing cagesof fibers (Figs. 1C, 2B). Apart from muscles project-ing toward the appendages, the prostomiumwas bareof F-actin.Sphaerodoropsis sp. The prostomium bears termi-

nally two small dorsal antennae and two ventralpalps (see Rouse & Pleijel 2001); the peristomiumcarries one pair of short and indistinct tentacular cirri(Fig. 1B). The transition between prostomium andperistomium is indiscernible. Compared with the ap-parently simple morphology of the anterior end ofSphaerodoropsis sp., its musculature is very complex(Figs. 1D, 5A–C, 6): three pairs of more-or-less lon-gitudinally oriented, two pairs of chiasmata-formingand more-or-less obliquely oriented, and three trans-verse muscles can be differentiated. The first pairof longitudinal muscles stretches along both sides,from the level of the mouth opening to the tip of theantennae (Fig. 6). This muscle is an elongation of theventral longitudinal muscle band of the body wall.The second longitudinally oriented pair reaches froma lateral position in front of the mouth to the outermargin of the same appendages. Each muscle de-scribes a sigmoid curve, but together they have theoutline of a ‘‘lyre.’’ Therefore, the term ‘‘lyrate mus-cles’’ is introduced here. The third pair of longitudi-nal muscles extends from the anterior edge of themouth to the medial base of each antenna. In thesecond group of muscles, a distinct chiasma is formedby the first pair of prostomial cross-muscles (Figs.1D, 6). Each of these thick fiber bundles stretchesfrom the lateral side toward the inner bases of theantenna of the contralateral side (Fig. 6). The second,less distinct, chiasma is formed by muscle fibers orig-inating from the ventral longitudinal muscle band ofthe body wall, which form a posterior loop followingthe foregut and subsequently project to the base ofthe antenna of the contralateral side (Figs. 1D, 6).Dorsally to all of these described muscles, two par-allel transverse muscle bands (Fig. 6) follow theanterior body margin, describing an anterior convexcurve. A single transverse muscle lies beneath allother prostomial muscles, describing an anterior con-cave bow (Fig. 6). The two anteriormost muscles runinto the base of the palps.

Fig. 1. A. Myrianida prolifera. Sketch of anterior end with

median (ma) and lateral (la) antennae, dorsal (dtc) and

ventral (vtc) tentacular cirrus at the peristomium, and

dorsal cirrus of segment II (dc II). B. Ventrolateral view

of anterior end in Sphaerodoropsis sp. with antenna (a),

palp (pa), and tentacular cirrus (tc). C,D. Slightly

schematic drawings of musculature of the anterior end.

C. Myrianida prolifera. The median and lateral antennae,

the dorsal tentacular cirri, and the dorsal cirri each possess

proximal longitudinal (ma/pl, la/pl) as well as distal

longitudinal (ma/dl, la/dl, dtc/dl, dc/dl) muscles. The

longitudinal muscles of the palp (pa/l) ramify distally.

The mouth opening is posteriorly surrounded by a

circumbuccal complex (cbm). In the trunk, dorsal (bw/dl)

and ventral (bw/vl) longitudinal strands and transverse

fibers (bw/dt) are prominent. The longitudinal muscles

find their termination in the basal muscles of the head

appendages. D. Sphaerodoropsis sp. The musculature of

the trunk comprises laterodorsal (bw/ll1), lateroventral

(bw/ll2) and ventral (bw/vl) longitudinal muscles as well

as transverse muscles (bw/slt) beneath them. a, antenna;

bw, body wall; bw/dl, dorsal longitudinal muscle of bw;

bw/dt, dorsal transverse muscle of bw; bw/dv, dorsoventral

muscle of bw; bw/ll1, bw/ll2, lateral longitudinal muscle of

bw 1 and 2; bw/o, oblique muscle of bw; bw/slt,

sublongitudnal transverse muscle of bw; bw/vl, ventral

longitudinal muscle of bw; cbm, circumbuccal

musculature; dc/dl, distal longitudinal muscle of dorsal

cirrus; dcII, dorsal cirrus of segment II; dtc, dorsal

tentacular cirrus; dtc/dl, distal longitudinal muscle of dtc;

la, lateral antenna; la/dl, distal longitudinal muscle of la; la/

pl, proximal longitudinal muscle of la; ma, median

antenna; ma/dl, distal longitudinal muscle of ma; ma/pl,

proximal mucle of ma; pa, palp; pa/l, longitudinal muscle

of pa; ppc, parapodial muscle complex; tc, tentacular

cirrus; vtc, ventral tc.



Pharynx

Myrianida prolifera. The areas beside and behindthe mouth opening are characterized by a so-called

circumbuccal complex, consisting of an outer layer oftransverse and an inner layer of longitudinal muscles(Figs. 1C, 2B). Together with the musculature of thepalps described above, they form the anterior mus-



cles of the mouth and foregut. The prominent pro-ventricle is primarily made up of densely packed per-pendicular rings of radial muscle fibers (Fig. 2C).Within each ring, the individual fibers are recogniz-able and each is characterized by core devoid of actinand an actin-containing periphery surrounding thiscore. Other fiber types are invisible. Anteriorly, theproventricle continues in the pharyngeal tube linedby a dense sheath of longitudinal fibers (Fig. 2C).Posteriorly, retractors attach to the proventricle.Sphaerodoropsis sp. The mouth opening is sur-

rounded by circular muscles (Figs. 1D, 5A, 6). Thin

fibers radiate anteriorly from the stomodaeum to theperiphery in a fan-like pattern (Fig. 6). Two mouthretractor muscles extend for a short distance posteri-orly; between them, a chiasma is formed by a second,somewhat thicker muscle fiber pair (Figs. 1D, 5A, 6).Due to its dense layers of outer circular and innerlongitudinal muscles, the pharynx occurs as a brightfluorescing structure in parapodial segment II or III(Fig. 5C). The musculature is exclusively made up ofcircular and longitudinal fibers (Fig. 5C); radial fiberswere not found in the pharynx. Furthermore, an an-terior pharyngeal tube with a thin muscular wall isalso lacking in Sphaerodoropsis sp.

Trunk

In both investigated species, the muscular archi-tecture of the trunk is dominated by longitudinalmuscle strands.Myrianida prolifera. In anterior segments, two dor-

sal longitudinal strands can be distinguished (Figs.1C, 2A), while in more posterior segments the singlebundles are so closely apposed in the median thatthey form a continuous dorsal layer, reaching later-ally down to the parapodia (Figs. 2D,H, 7B). In thefirst stages of stolon formation at the beginning ofreproduction, the dorsal strands are still continuousat the transition between stock and stolon, respec-tively (Fig. 2D). Ventrally, two thick ventral and onesmaller ventromedian longitudinal muscle strands ex-tend from the peristomium to the pygidium withoutchanges in location (Figs. 2B,E–G, 7B). Posteriorly,the main strands are interconnected by transverse fi-bers (Fig. 2G). Terminally, the ventrolateral musclesform a loop and join each other (Fig. 2G). Additionaltransverse fibers surround the dorsally located anus(Fig. 2I). The more dorsally placed ventromedianstrand measures only 20% of the diameter of themain strands and terminates in front of the pygidium.At this point some longitudinal fibers of the anal cirriare attached to this muscle, whereas others are at-tached to the circular muscle surrounding the anus(Fig. 2G,I).

Above the longitudinal muscles, bracing musclesform a distinct pattern. In each segment, two pairs ofbracing muscles can be discriminated (Fig. 2H). Themore anterior fibers start shortly posterior to the in-tersegmental furrow at the level of the ventral longi-tudinal strands and extend diagonally in a posteriordirection above the dorsum to the contralateral side,spanning two segments across. The more posteriorbracing muscles follow the same course, but in ananterior direction. Thus, these muscles create a pat-tern of two types of chiasmata: the first type is

Fig. 2. F-actin staining of Myrianida prolifera, depth-

coded images. Anterior end oriented to the top, in (F, G)

to the left. A.Dorsal view of anterior end. Thick muscles in

the proximal parts of the median and lateral antennae,

dorsal tentacular and parapodial cirri (ma/pl, la/pl, dtc/pl,

dc/pl), thin fibers in the distal part of the cirrus (dtc/dl), and

transverse fibers (bw/t). Arrowheads point to transition

zone in the bases of the appendages. B. Ventral view of

the anterior end. Mouth opening lined by circumbuccal

muscles (cbm) and laterally by longitudinal fibers of the

palps (pa/l). Paired ventral (bw/vl) and unpaired

ventromedian (bw/vml) strands of longitudinal muscles.

The cross indicates a chiasma formed by oblique muscles,

which continue in the parapodial muscle complex (ppc).

C. Proventricle with radial muscles (pv/ra) and pharyngeal

tube with longitudinal fibers (pt/l). D. Continuous dorsal

longitudinal layer (bw/dl) at transition between stock and

stolon. E. Transverse fibers (bw/t) run from the dorsum to

the ventral side (arrows) and terminate ventrally in three

distinct pairs (white dots). F. Chiasmata of the oblique

muscles and fiber-free areas alternate. G. Transverse

filaments interconnect the ventral bands terminally

(arrowhead). H. Bracing (bw/b), transverse (bw/t), and

longitudinal (bw/dl) muscles on the dorsal side. I. The

anus is surrounded by circular fibers (an/c); note

longitudinal fibers of anal cirrus (ac/l). J. Cross section of

segment with longitudinal (i/l) and circular (i/c) muscles

encircling the intestine. ac/l, longitudinal muscle of anal

cirrus; an/c, circular muscle of anus; bw, body wall; bw/b,

bracing muscle of bw; bw/dl, dorsal longuitudinal muscle

of bw; bw/vml, ventromedian longitudinal muscle of bw;

bw/o, oblique muscle of bw; bw/t, transverse muscle of

bw; bw/vl, ventral longitudinal muscle of bw; cbm,

circumbuccal muscle; dc/pl, proximal longitudinal muscle

of dorsal cirrus; dtc/dl, distal longitudinal muscle of dorsal

tentacular cirrus; dtc/pl, proximal longitudinal muscle of

dorsal tentacular cirrus; i/c, circular muscle of intestine; i/l,

longitudinal muscle of intestine; la/pl, proximal

longitudinal muscle of lateral antenna; ma/pl, proximal

longitudinal muscle of median antenna; pa/l, longitudinal

muscle of palp; ppc, parapodial muscle complex; pt/l,

longitudinal fibers of pharyngeal tube; pv/r, retractor

muscle of proventricle; pv/ra, radial fibers of proventricle.



formed at each side in a dorsolateral position, thesecond one mid-dorsally in posterior regions of eachsegment (Figs. 4A, 7C). Transverse muscles abovethe bracing muscles form a third layer. Numerous fi-bers are regularly distributed and cover the entiresegments dorsally (Figs. 1C, 2A,D,H, 4A,B). In thebody sides, they run in front of and behind the pa-rapodia ventrally, and finally bend mid-ventrally. Inthe ventral wall they accumulate, forming three, bi-laterally symmetric, distinct muscle strands in eachsegment, which terminate in a paramedian position(Fig. 2B,E). This pattern is repeated in every seg-

ment. Thus, the transverse fibers form, apart fromsmall midventral gaps at the level of the median lon-gitudinal muscle and the ventral nerve cord, rings insupralongitudinal position.

At the level of the segmental furrows, thin musclesextend above the ventromedian longitudinal strandfrom one side to the other (laterolateral muscles)(Fig. 7B–D).Sphaerodoropsis sp. This species possesses four

pairs of longitudinal muscle strands, the ventralones of which are the most prominent (Figs. 5E,7A). Their diameter diminishes toward the posterior

Fig. 3. Myrianida prolifera; ultrastructure of tentacular cirri. A. Part of cross section through tentacular cirrus with central

nerve (n) accompanied by a few muscle cells (arrowheads). Arrow indicates cilia of sensory cell. B. Two small longitudinal

muscle fibers (arrowheads) between epidermal tissue (ep) and nerve (n). C. Group of numerous small longitudinal fibers

(arrowheads) in tentacular cirrus. D. Enlargement of muscle cells shown in B; note the small number of myofilaments (mf)

and absence of extracellular matrix around muscular tissue; nucleus (nu), and mitochondria (m) located peripherally. E.

Myoepithelial cell with circular myofilaments beneath apical plasma membrane; inset: enlargement of bundle of thick

myofilaments (double arrowhead) interconnected by thin filaments (arrowhead). cu, cuticle; ep, epidermis; gl, gland cell; m,

mitochondrion; mf, myofilaments; n, nerve; nu, nucleus; va, vacuole; za, zonula adhaerens.



end, where they are interconnected by transverse fil-aments (Fig. 5G). The pair of comparatively thindorsal longitudinal strands lies in a paramedian po-sition (Fig. 5D,F). Anteriorly, they project into theprostomium and continue as prostomial longitudinalmuscles (Fig. 5A,B); posteriorly they extend into thepygidium. Laterally, three bundles of longitudinal fi-bers are visible: one thick bundle more dorsally lo-cated and two more laterally located bundles withfewer fibers (Figs. 5B,D,F, 7F).

Below the longitudinal muscles transverse musclesencircle the body (Fig. 7B). They are evenly distrib-uted; segmental patterns are not visible. These trans-verse fibers in sublongitudinal position form almostcomplete rings, leaving only a narrow gap in the mid-ventral line (Fig. 5E).

Two pairs of bracing muscles, located between thelongitudinal and sublongitudinal transverse fibers,are present in each segment. The longer fibers extendfrom the dorsal longitudinal muscle strands and runeither in an anterior or in a posterior direction to-ward the ventral longitudinal strands on the contra-lateral side. By crossing each other, they formchiasmata mid-dorsally (Figs. 5D, 7F). Shorter fibersrun diagonally between the two lateral longitudinalstrands at each side, forming lateral rows of chias-mata (Fig. 7F).

Septal, stomatogastric, and parapodial musculature

Starting from the third segment onward, myo-epithelial septa separate adjacent segments. Theystretch across the entire diameter, containing horizon-

tally arranged myofilaments (Fig. 7E). The septa em-bed all structures that reach through all segments fromthe anterior to the posterior end, e.g., the nervous sys-tem and the longitudinal muscle strands. Due to theresolution of the microscope and the minute dimen-sions of these septa, the extracellular matrix betweenadjacent myoepithelia could not be observed.

The intestines ofM. prolifera and of Sphaerodorop-sis sp. each contain a regular grid made up of evenlydistributed and distinctly separated longitudinal andcircular fibers (Figs. 2J, 4C, 5D, 7F).

In M. prolifera, the notopodia are represented bydorsal cirri. Those belonging to the two anteriormostsegments are the longest, and are directed anteriorly.The bases of these distinct dorsal cirri are equippedwith thick muscle fibers, like those of the antennae andthe tentacular cirri. Moreover, similar thin fibers arepresent in these appendages as well (Fig. 2A). In livinganimals, these appendages make slow sweeping move-ments to explore the surroundings of the animals.Each individual appendage is in slightly curved mo-tion. The cirri of the remaining segments are shorter,directed backwards and lack noticeable muscle fibers.In living animals they appear more or less stiff.

In Sphaerodoropsis sp., large capsules are present atpositions where dorsal cirri occur in other polychaetes.TEM investigations demonstrated that all of these ap-pendages, besides having epithelial supporting cells,possess mono- and multiciliated sensory cells, partlyin the form of collar receptors, but lackmuscle fibers aswell as myofilament-containing epithelial cells.

In both species, the neuropodia contain several mus-cles, e.g., chaetal pro- and retractors. Oblique muscles

Fig. 4. Myrianida prolifera; F-actin staining of the trunk, optical sections of the dorsum at different levels through one

complete chaetiger and adjacent segments. A. Peripheral section with bracing (bw/b, arrows), transverse (bw/t), and

longitudinal (bw/dl) muscles. B. Slightly deeper region, most bracing muscles are cut off. C. Dorsal layer of the intestine

with circular (i/c, arrows) and longitudinal (i/l, arrows) muscles. Septa (dissepiments: s) visible as intensely stained

transverse bands of F-actin filaments; adjacent myoepithelial layers of septa not discernible due to the resolution limits of

the cLSM. bw, body wall; bw/b, bracing muscle of bw; bw/dl, dorsal longitudinal muscle of bw; bw/t, transverse muscle of

bw; i, intestine; i/c, circular muscle of i; i/l, longitudinal muscle of i; s, septum.



Fig. 5. Sphaerodoropsis sp., F-actin staining, depth-coded images. A. Ventral view of anterior end including the first two

chaetigers. Ventral longitudinal muscles (bw/vl) continue as prostomial longitudinal (pr/l) and antennal longitudinal

muscle (a/l), palp longitudinal muscles connected by prostomial ventral transverse muscle (pr/vt), lyrate (pr/ly), cross

muscles one and two (pr/cr1, pr/cr2), longitudinal muscles in the antenna (a/l), and circular muscles around the mouth

(m/c), constitute the prostomial musculature. Note transverse muscle (bw/slt) passing the longitudinal musculature (bw/

vl) in sublongitudinal position (arrowheads). Position of pharynx (asterisk). B.Dorsolateral view of anterior end, ventral

to left, showing laterodorsal (bw/ll1), lateroventral (bw/ll2), and ventral (bw/vl) longitudinal and transverse muscles in

sublongitudinal position (bw/slt). Circular muscles of the pharynx (ph/c) are superimposed on the other structures.

C. Enlargement of pharynx with circular (ph/c) and longitudinal (ph/l) muscles.D.Dorsal view of the trunk; four bundles

of longitudinal (bw/ll1, bw/dl) and bracing muscles (bw/b, yellow arrowheads) and in deeper layers circular (i/c) as well as

longitudinal (i/l) muscles of the intestine are visible. E. Ventral view of trunk with oblique muscles (bw/o) forming

‘‘double-inverted-V’’ (arrows) and transverse muscles running (bw/slt) almost to the midventral line, leaving a small gap

at their attachment zone (yellow arrowhead). F. Dorsolateral view with dorsal, laterodorsal, and lateroventral

longitudinal bundles (bw/dl, bw/ll1, bw/ll2), and bracing muscles (bw/b, yellow arrowheads) G. Ventral view of the

posterior end; arrowheads point to ventral longitudinal musculature extending into the very posterior end. a/l,

longitudinal muscle of antenna; bw, body wall; bw/b, bracing muscle of bw; bw/dl, dorsal longitudinal muscle of bw;

bw/ll1, dorsolateral longitudinal muscle of bw; bw/ll2, lateral longitudinal muscle of bw; bw/o, oblique muscle of bw;

bw/slt, sublongitudinal transverse muscle of bw; bw/vl, ventral longitudinal muscle of bw; i/c, circular muscle of intestine;

i/l, longitudinal muscle of intestine; p/l, longitudinal muscle of palp; ph, pharynx; ph/c, circular muscle of ph; ph/l,

longitudinal muscle of ph; ppc, parapodial muscle complex; pr, prostomium; pr/cl, cross muscle 1 of pr; pr/cr2, cross

muscle 2 of pr; pr/l, longitudinal muscle of pr; pr/ly, lyrate muscle of pr; pr/vt, ventral transverse muscle of pr.



extending from the midventral line toward the para-podia form distinct patterns. InM. prolifera they form‘‘dorsoventrally flattened, X-shaped’’ patterns in theposterior third of each segment (Figs. 2B, 7C,D), sep-arated by large fiber-free areas. The anterior branchesof the ‘‘X’’ belong to the corresponding segment, theposterior branches to the following segment. At firstglance, the pattern looks different in Sphaerodoropsissp., but the only difference is that the node of the ‘‘X’’is shifted anteriorly, resulting in an ‘‘inverse double-V’’pattern (Fig. 5E). Furthermore, this pattern is super-imposed on the numerous sublongitudinal transversemuscles, almost reaching the midline.

Discussion

While it is beyond discussion and generally ac-cepted that Sphaerodoridae belong to Phyllodocida,

their position within this taxon remains uncertain(Rouse & Fauchald 1997; Pleijel & Dahlgren 1998;Rouse & Pleijel 2001). In some of their cladistic an-alyses, Rouse & Fauchald (1997: 160) found a sister-relationship of Syllidae and Sphaerodoridae. Com-mon characters like complex chaetae, reducednotopodia, and presence of an axial pharynx (pro-ventricle) inspire such a close grouping (Ushakov1972). Due to their densely papillar covering, theshort-bodied Sphaerodoridae resemble Syllidae ofthe genus Sphaerosyllis. In other trees, however, Syll-idae turn out as a more basal taxon, and the Sphaer-odoridae as sister taxon to Pilargidae (Rouse &Fauchald 1997: 163). In more recent morphologicalcladistic analyses, Sphaerodoropsis anae AGUADO &ROUSE, 2006 as representative of Sphaerodoridae ap-pears as sister to monophyletic Syllidae, in an anal-ysis with a reduced data set, while in a completeanalysis it falls in a more distant position, with Syn-elmis albini (LANGERHANS, 1881) (Pilargidae) beingthe sister group of Syllidae (Aguado & San Martin2009). In the only molecular study available, a sister-group relationship of Syllidae and Sphaerodoridaewas also not found (Aguado et al. 2007). The sameholds true for a combined analysis using molecularandmorphological data carried out byWorsaae et al.

Fig. 6. Sphaerodoropsis sp.; musculature of the anterior

end. A. Ventral view of the anterior end with prostomial

muscles, depth-coding image. B. Schematic drawing of A

with color-coded prostomial muscles. Seven main pairs of

muscle fibers contribute to the prostomial musculature:

peripheral longitudinal muscles (pr/l, brown) which

originate from the ventral longitudinal muscle of the

body wall (bw/vl), lyriform (pr/ly, red), central

longitudinal (pr/cl, pink) muscles, a larger pair of

chiasma-forming (A, arrowhead) cross muscles (pr/cr1,

dark blue) and a smaller pair (pr/cr2, light blue)

originating from bw/vl, and three pairs of transverse

muscles: one ventral (pr/vt, orange) and two dorsal (pr/

dt1, pr/dt2, light orange). The foregut is surrounded by

circular muscles (m/c, light green), anterior protractor

fibers spread out in a fan-like pattern (m/pr, yellow), and

retractor muscles of the stomodaeum (m/r, dark green). a,

antenna; a/l, antennal longitudinal muscle; bw/vl, ventral

longitudinal muscle of body wall; m/c, circular muscles of

mouth region; m/pr, protractor muscles of mouth; m/r,

retractor muscles of mouth; p, palp; p/l, longitudinal

muscle of palp; ppc, parapodial muscle complex; pr,

prostomium; pr/c1, central longitudinal muscle of pr; pr/

crl, pr/cr2, cross muscle 1, 2 of pr; pr/dt1, pr/dt2, dorsal

transverse muscle 1, 2 of pr; pr/l, longitudinal muscle of pr;

pr/ly, lyriform muscle of pr; pr/vt, ventral transverse

muscle of pr.



(2005). In the following, we compare the musculararrangement in the Syllidae and Sphaerodoridae, inorder to evaluate the questionable close relationship.

Phylogenetically important characters

Syllidae and Sphaerodoridae, but also Pontodor-idae and Nautiliniellidae, possess a structure in theirforegut commonly called ‘‘proventricle,’’ but its ex-istence may be uncertain and requires further inves-tigation (see Fauchald & Rouse 1997; Rouse & Pleijel2001). Such a proventricle is regarded as a derived

structure of the axial muscular pharynx generallypresent in Phyllodocida (see Purschke 1988; Tzetlin& Purschke 2005). However, to date it is uncertainwhether these structures, summarized as proventri-cles, are in fact the result of a single evolutionaryevent leading to all of the taxa mentioned above(Rouse & Pleijel 2001). To date, ultrastructural dataare only available for Syllidae (see Purschke 1988;Tzetlin & Purschke 2005). Although the musculatureof the proventricle in Syllidae is considered to consistof circular, longitudinal, and radial fibers by certainauthors (see Aguado & San Martin 2009), it includes

Fig. 7. Schematic drawings of the muscular architecture of the trunk.A. Sphaerodoropsis sp. B–F.Myrianida prolifera. B.

Trunk. C. Bracing muscles. D. Oblique muscles. E. Oblique and bracing muscles. C–E, dorsal view. F. Segmental border

with septum and mesentery, intestine, longitudinal muscles of body wall and parapodium. Dorsal (bw/dl), laterodorsal

(bw/ll1), lateroventral (bw/ll2), ventral (bw/vl), ventromedian (bw/vml) longitudinal, transverse supralongitudinal (bw/t),

transverse sublongitudinal (bw/slt), bracing (bw/b), and oblique muscles (bw/o) of the body wall; dorsal cirrus (dc),

neuropod (nep), and muscle complex (ppc) of the parapodia; muscle filaments within the mesenterium (me) and septum

(s); longitudinal (i/l) and circular (i/c) fibers of the intestine; arrowhead indicates ‘‘Querbinder’’5 laterolateral muscle;

stars in C–E indicate position of parapodia.



only the predominant radial fibers arranged in rows,and the thin outer and inner layers of so-called an-nular (circular) fibers (Malaquin 1893; Haswell 1921;Wissocq 1974). The present data corroborate a gen-eral structure composed of mainly radial fibers witha peculiar ultrastructure including the presence of aso-called hirudinean type of fiber with central sarco-plasm and peripheral cylinder of the contractilefilaments (Wissocq 1974; Purschke 1988). Thearrangement of muscle fibers in the proventricle inSyllidae is regarded as unique for this taxon and con-sequently as its autapomorphy (Fauchald & Rouse1997; Rouse & Fauchald 1997). Moreover, the pro-ventricle is preceded by a pharyngeal tube with adense layer of longitudinal fibers. These two compo-nents form a functional unit perfectly adapted to var-ious modes of nutritional uptake by sucking.

Our results confirm the architecture of the syllidproventricle and, furthermore, demonstrate that theso-called proventricle in Sphaerodoropsis sp. containstransverse and longitudinal but, most importantly,no radial fibers of the type present in Syllidae. Thus,the similarities in the foregut are at most superficialand do not indicate a closer relationship of these twotaxa. On the basis of the present observations thepharynx of Spaerodoridae more or less resembles thetypical axial pharynx generally present in members ofPhyllodocida. However, an ultrastructural investiga-tion of the foregut in Sphaerodoridae is desirable, toelucidate with a higher degree of probability whetherthis structure in fact represents an axial pharynxcomparable to that of other taxa of Phyllodocida(see Rouse & Pleijel 2001; Tzetlin & Purschke 2005).

Evidence for monophyly of Sphaerodoridae is pro-vided by the fact that members of all species possessinflated capsules (dorsal cirri) and tubercles presentin two or more rows on the dorsum (Fauchald &Rouse 1997). Rudermann (1911) and Reimers (1933)postulated homology of the capsules with dorsal cirriof other polychaetes and thus termed them alike.Doubting such homology, Fauchald (1974) calledthe capsules ‘‘macrotubercles’’; Rouse & Fauchald(1997) cautioned that morphological similarities ordifferences must be explored to test the homologystatement. The present investigation demonstratesthat the muscular equipment of the dorsal cirri inMyrianida prolifera depends on their function for,and position within, the animal. The dorsal cirri ofthe two anteriormost segments resemble antennaeand tentacular cirri, and one can assume that they,like the former, palpate the environment for varioussensory inputs, e.g., food. Presence of longitudinalfilaments ensures a degree of motility, and move-ments are mainly restricted to shortening and spiral

bending. More discriminatory locomotion would re-quire additional musculature, such as occurs in thepalps of Spionidae (Filippova et al. 2005). Further-more, the similar muscular architecture within theanterior parapodial cirri and the tentacular cirri inM. prolifera can be interpreted as support for the hy-pothesis of serial homology for these appendages aspostulated by Glasby (1993). All other dorsal cirrilack musculature as do the inflated capsules inSphaerodoropsis sp. This negative character cannotprove the supposed homology of capsules with dorsalcirri.

Equipment of the distal parts of the antennae ortentacular cirri with muscle fibers has seldom beenaddressed in phyllodocidan polychaetes. Whereasthe occurrence of muscle fibers in appendages (pri-marily in the palps) of so-called sedentary poly-chaetes is well documented (e.g., Gardiner 1988), itis seldom reported for errant forms and, for exam-ple, has been shown to occur in nerillids, where eachsection of the articulated antennae is provided witha thick bundle of F-actin (Muller & Worsaae 2006).The observation of a distinct gap between distal andproximal fibers in M. prolifera, as well as the ab-sence of muscle elements in the appendages of me-iofaunal members of the Hesionidae as shown byTEM (G. Purschke, unpubl. data), shed doubt oncategorization of the phalloidin-labeled structuresas muscle fibers and initiated their investigation byTEM. These observations indisputability revealedthat the longitudinal fibers present in these append-ages are in fact muscle fibers. Hence, the gap is dueto a comparatively thick attachment site as observedvia ECM, where the two sets of fibers adjoin eachother. In contrast, the groups of circular fibers pres-ent in some epithelial cells were not detected withphalloidin, but are organized in groups of thick andthin filaments, indicating a presence of sarcomeresrather than something different such as bundles ofintermediate filaments.

Similarities between taxa

Members of M. prolifera and Sphaerodoropsis sp.possess bracing muscles that span the dorsal and lat-eral sides of the body, forming, especially in the for-mer species, a distinct pattern of dorsomedian anddorsolateral chiasmata. They might enable twistingmovements of the body. While these muscles arepositioned outside the longitudinal muscles inM. prolifera, as in other polychaetes, they lie betweenthe longitudinal and sublongitudinal transverse fibersin Sphaerodoropsis sp. Members of Syllidae andSphaerodoridae have, like other annelids, a regular



grid of longitudinal and circular intestinal muscles,enabling waves of contractions to transport the nour-ishment-pulp posteriorly.

Differences between taxa

The only character uniting the Sphaerodoridae–Syllidae clade found in the analysis by Rouse &Fauchald (1997: 160 [character 28], 176) is: ‘‘four orfive longitudinal muscle bands.’’ From our results itis obvious that this only applies to the syllid repre-sentative, M. prolifera: it possesses four or five lon-gitudinal muscle strands, depending on whether thedorsal layer is counted as one or two strands. Bycontrast, Sphaerodoropsis sp. possesses eight distinctlongitudinal muscle bands. Regarding the longitudi-nal musculature, the major differences between thetaxa are: (1) a complete dorsal layer in M. proliferaversus two separate bands in Sphaerodoropsis sp.; (2)two lateral pairs in Sphaerodoropsis sp. versus nosuch muscles in M. prolifera; (3) median musclestrand in M. prolifera versus no such band inSphaerodoropsis sp. The ventromedian strand in asupraneural position has been found in various poly-chaete species (e.g., Purschke & Muller 2006). It isobvious that oblique muscles reach from the level ofthe median band toward the parapodia. Whether thelongitudinal muscles are needed to anchor the ob-lique ones has yet to be determined. The same appliesto the assumption that presence and diameter of themedian longitudinal muscle strand are correlatedwith the degree to which parapodia are involved inmovement (Tzetlin & Filippova 2005). Presence of sixseparate strands in contrast to one dorsal layer makesthe body of Sphaerodoropsis sp. more flexible, whichmight be helpful for digging in the muddy substratewhere members of this species live.

Apart from the overall similar orientation, thetransverse fibers of the two species have nothing incommon. In M. prolifera, they are located in a su-pralongitudinal position, fuse ventrally, and formthree bundles that terminate in a paramedian posi-tion. Unexpected for Annelida, in Sphaerodoropsissp. the transverse fibers lie in a sublongitudinal posi-tion and reach ventrally to the midventral line. Thearrangement of the transverse fibers in M. proliferaresembles that found in Dorvillea spp. (T. Hinken &M.C.M. Muller, unpubl. data; Filippova et al. 2006;Purschke &Muller 2006; Bergter et al. 2008). In somedorvilleids, however, the fibers accumulate ventrallyinto seven paired bundles. Apart from the midventralgap, very likely caused by the basiepithelial ventralnerve cord (Purschke 2002; Purschke &Muller 2006),the fibers form complete rings and therefore it is jus-

tified to call them ‘‘circular muscles.’’ However, inSphaerodoropsis sp. the placement of circular andlongitudinal muscles within the body wall is inverted,compared with the annelid ground pattern: the trans-verse fibers lie beneath the longitudinal ones. Suchinversion has hitherto only been reported by Rieger(1991) for Lobatocerebridae, a taxon with otherunique characters and uncertain phylogenetic posi-tion, and for Spionidae (Filippova et al. 2005). Weneither have an explanation for the origin of this in-version nor for its functional advantages. The lowbut increasing number of animals with inverted po-sitions of longitudinal and transverse muscles mightbe used to argue that such a ‘‘circular’’ muscle layerevolved convergently in several annelid taxa.

Unique inM. prolifera are the laterolateral musclesthat interconnect the lateral sides of the body. Suchfibers have been reported by Storch (1968) for thesyllid polychaete Eusyllis inflata (MARENZELLER,1879). He called these muscles ‘‘Querbinder,’’ whichcould be translated as ‘‘transverse linker,’’ suggestingthat tension of these muscles could bilaterally com-press the body. Possibly these muscles are apomor-phic for Syllidae.

With respect to the arrangement of the muscula-ture in the anterior end, the two species have nothingin common. Members of Sphaerodoropsis sp., beingexternally more ‘‘simple,’’ have the more complex in-ternal organization. High flexibility of its anteriorend is realized by five muscle pairs and three trans-verse fibers penetrating pro- and peristomium. Theselongitudinal, transverse, and chiasmata-formingmuscles enable nearly all deformations, allowing pro-trusion into tiny holes and caves in the substrate. InM. prolifera, the palps very likely are the most flexiblestructures. The ‘‘cages’’ of F-actin filaments allowvariable movements, necessary for grasping and han-dling of food particles and transport to the mouthopening. Thus, the palps look and act like lips.

In conclusion, we found important differences be-tween the syllid and the sphaerodorid species andonly a few similarities. These similarities are notsynapomorphic for the two taxa, indicating thatthey very likely do not represent sister groups. Forinstance, the proventricle and arrangement of thelongitudinal musculature speaks against such agrouping. The pharynx in Sphaerodoropsis sp. mostlikely corresponds to the type of pharynx generallyfound in members of Phyllodocida. To allow a morebroadly founded conclusion, on the one hand anultrastructural investigation of the sphaerodoridforegut is needed, and on the other hand, the taxonsampling has to be increased to elucidate the vari-ability of the characters observed, both of which are



under way. Furthermore, with Sphaerodoropsis sp.,another species can be added to the constantly grow-ing list of polychaetes lacking true circular muscles inthe body wall.

Acknowledgments. We thank the head of the zoologydepartment, Prof. Dr. A. Paululat, Osnabruck, fordiscussions and support. We are grateful to twoanonymous reviewers and their useful comments andsuggestions for improvements. A grant from the DFGallowing A.F. to carry out the experimental work inOsnabruck is gratefully acknowledged. We thank C.Franken and D. Suschenko for assistance with the TEMsectioning. M.C.M.M. wishes to thank Prof. Dr. TillyBakker-Grunwald for mental and the ZKFG of theUniversity of Osnabruck for financial support.

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