evolution of high-performance swimming in sharks ... · within elasmobranchs (sharks and rays),...

Evolution of High-Performance Swimming in Sharks:Transformations of the Musculotendinous System FromSubcarangiform to Thunniform SwimmersSven Gemballa,1* Peter Konstantinidis,1 Jeanine M. Donley,2 Chugey Sepulveda,3 andRobert E. Shadwick4

1Evolution, Bio-Geosphere Dynamics Program (EBID), Department of Zoology, D-72076 Tubingen, Germany2Biology Department, MiraCosta College, Oceanside, California 920563Pfleger Institute of Environmental Research, Oceanside, California 920544Department of Zoology, University of British Columbia, Vancouver, B.C. V6T 2A9, Canada

ABSTRACT In contrast to all other sharks, lamnidsharks perform a specialized fast and continuous “thun-niform” type of locomotion, more similar to that of tunasthan to any other known shark or bony fish. Withinsharks, it has evolved from a subcarangiform mode. Ex-perimental data show that the two swimming modes insharks differ remarkably in kinematic patterns as well asin muscle activation patterns, but the morphology of theunderlying musculotendinous system (red muscles andmyosepta) that drives continuous locomotion remainslargely unknown. The goal of this study was to identifydifferences in the musculotendinous system of the twoswimming types and to evaluate these differences in anevolutionary context. Three subcarangiform sharks (thevelvet belly lantern shark, Etmopterus spinax, the small-spotted catshark, Scyliorhinus canicula, and the black-mouth catshark, Galeus melanostomus) from the two ma-jor clades (two galeans, one squalean) and one lamnidshark, the shortfin mako, Isurus oxyrhinchus, were com-pared with respect to 1) the 3D shape of myomeres andmyosepta of different body positions; 2) the tendinousarchitecture (collagenous fiber pathways) of myoseptafrom different body positions; and 3) the association of redmuscles with myoseptal tendons. Results show that thethree subcarangiform sharks are morphologically similarbut differ remarkably from the lamnid condition. More-over, the “subcarangiform” morphology is similar to thecondition known from teleostomes. Thus, major featuresof the “subcarangiform” condition in sharks have evolvedearly in gnathostome history: Myosepta have one mainanterior-pointing cone and two posterior-pointing conesthat project into the musculature. Within a single myo-septum cones are connected by longitudinally orientedtendons (the hypaxial and epaxial lateral and myorhab-doid tendons). Mediolaterally oriented tendons (epineuraland epipleural tendons; mediolateral fibers) connect ver-tebral axis and skin. An individual lateral tendon spansonly a short distance along the body (a fraction between0.05 and 0.075 of total length, L, of the shark). This spanis similar in all tendons along the body. Red musclesinsert into the midregion of the lateral tendons. The short-fin mako differs substantially from this condition in sev-eral respects: Red muscles are internalized and separatedfrom white muscles by a sheath of lubricative connectivetissue. They insert into the anterior part of the hypaxiallateral tendon. Rostrocaudally, this tendon becomes very

distinct and its span increases threefold (0.06L anteriorlyto 0.19L posteriorly). Mediolateral fibers do not form dis-tinct epineural/epipleural tendons in the mako. Since ourmorphological findings are in good accordance with exper-imental data it seems likely that the thunniform swim-ming mode has evolved along with the described morpho-logical specializations. J. Morphol. 267:477–493, 2006.© 2006 Wiley-Liss, Inc.

KEY WORDS: Lamnidae; Squalidae; Scyliorhinidae; redmuscles; myoseptal tendons; swimming mechanics

The field of fish locomotion has been dominatedprimarily by studies on bony fishes, while only re-cently have sharks received substantial attention.Recent experimental studies focused on hydrody-namics, kinematics, energetics, and muscle activa-tion patterns in swimming sharks (Ferry andLauder, 1996; Bernal et al., 2001a,b, 2003a,b; Don-ley and Shadwick, 2003; Wilga and Lauder, 2004;Donley et al., 2004, 2005). One issue addressed bythese studies is the mode of axial undulatory loco-motion performed by sharks. Four categories aregenerally used to describe the kinematics of axialundulations in fishes. These modes, the anguilli-form, subcarangiform, carangiform, and thunniform(e.g., Webb, 1975; Lindsey, 1978; Videler, 1993),form a continuum of types of axial undulatory loco-

Contract grant sponsors: Deutsche Forschungsgemeinschaft (DFG)(travel grant to S.G.); Reinhold und Maria Teufel-Stiftung, Tuttlingen(to P.K.); Scripps Graduate Department (to J.D.); National ScienceFoundation (NSF) (mako collection funds to R.S.).

*Correspondence to: Sven Gemballa, Evolution, Bio-GeosphereDynamics Program (EBID), Department of Zoology, University ofTuebingen, Auf der Morgenstelle 28, D-72076 Tubingen, Germany.E-mail: [email protected]

Published online 20 January 2006 inWiley InterScience (www.interscience.wiley.com)DOI: 10.1002/jmor.10412

JOURNAL OF MORPHOLOGY 267:477–493 (2006)

© 2006 WILEY-LISS, INC.

motion with the anguilliform and thunniform modebeing extreme cases. Classification of a species intoone of these categories occurs mainly due to thedegree of lateral displacement as a function of bodyposition. For the sharks being investigated in detailit has been demonstrated that they either fall intothe subcarangiform (Webb and Keyes, 1982; Donleyand Shadwick, 2003) or the thunniform mode (Don-ley et al., 2004, 2005).

Previous works have shown that the subcarangi-form mode is widely distributed among sharks. Al-though initial gross observations classified the usualmode of shark locomotion as anguilliform (e.g., Lind-sey, 1978), detailed studies in six species have dem-onstrated that kinematic parameters match the sub-carangiform mode (Webb and Keyes, 1982; Donleyand Shadwick, 2003). This mode is intermediatebetween the anguilliform (e.g., eels) and carangi-form mode (e.g., mackerels) and is characterized bylateral displacement amplitudes that rapidly in-crease over the posterior half of the body (e.g., Webb,1975). For example, in the leopard shark Triakissemifasciata, lateral displacement increases morethan threefold between (total length, L) 0.5L (mid-body) to 1.0L (tip of caudal fin; Donley and Shad-wick, 2003). Moreover, measured amplitudes at 0.5Lare only about half of that measured in eels anddouble that measured in mackerels (sharks: Donleyand Shadwick, 2003; eel, mackerel: Altringham andShadwick, 2001), clearly justifying the classificationas subcarangiform.

In contrast, among sharks the thunniform modehas only been described for members of the familyLamnidae (five species including the white sharkCarcharodon carcharias). In this mode lateral move-ments are largely confined to the caudal region. Inone lamnid, the shortfin mako Isurus oxyrhinchus, itwas demonstrated that the amplitude of lateral mo-tion increases substantially only beyond 0.8L wherethe trunk tapers to the narrow caudal peduncle. Inthis respect, the lamnid shark resembles tunasmuch more than any other group of sharks or sub-carangiform teleosts (Donley et al., 2004).

Within elasmobranchs (sharks and rays), lamnidsnest deep within a subgroup of sharks, the Galea(e.g., Maisey et al., 2004). The unique occurrence ofthunniform swimming in this derived group ofsharks and the widespread occurrence of the subcar-angiform swimming mode in the other shark speciesthat have been studied supports the hypothesis thatthe thunniform mode has evolved from a subcaran-giform mode on the transition to lamnid sharks.Moreover, the thunniform mode is linked to otherderived features of the locomotory system that sep-arate lamnids from other sharks. They have a spe-cific fusiform body shape with a lunate caudal finand internalized red muscles combined with a heatconservation mechanism (endothermy) (Carey et al.,1971, 1985; Reif and Weishampel, 1986; Bernal etal., 2001a). These features are adaptations of the

locomotory system for a life as pelagic predatorsexploiting the open ocean. This existence requires ahighly efficient locomotory system specialized forfast and continuous swimming including long mi-grations (Magnuson, 1978; Boustany et al., 2002).

Continuous swimming is powered by red myomeremuscles. Interestingly, these muscles work in a dif-ferent way in thunniform lamnids than in subcar-angiform sharks. In subcarangiform sharks redmuscles at a given body position shorten in phasewith local body bending during swimming (beam-like swimming; Donley and Shadwick, 2003). Inlamnids, red muscles at a given body positionshorten out of phase with local bending but shortenin phase with bending at a more posterior positionsuggesting force transmission over some distancealong the trunk (nonbeam-like swimming; Donley etal., 2004, 2005). This difference seems to be a moregeneral phenomenon because it was also found inbony fishes (thunniform tunas vs. subcarangiformfishes; e.g., Knower et al., 1999; Shadwick et al.,1999; Altringham and Shadwick, 2001; Katz et al.,2001).

The underlying musculotendinous system (i.e.,myomeres with collagenous myosepta, horizontalsepta and dermis) that is responsible for these dif-ferences is poorly understood. Forces generated byeither red or white muscles have to be transferred toaxial structures, such as the vertebral column, togenerate lateral bending moments along the trunkduring swimming. The collagenous dermis, the hor-izontal septum, and the myosepta are likely candi-dates for this force transmission (Wainwright et al.,1978; Westneat and Wainwright, 2001; Gemballaand Vogel, 2002; Gemballa and Treiber, 2003). Dataon teleosts demonstrate that both the myosepta andthe horizontal septum are quite uniform in all majorgroups but have undergone considerable evolution-ary changes in tunas (Westneat and Wainwright,2001; Gemballa et al., 2003a,b). Few data from apreliminary study on the mako shark suggest thatmyosepta in the posterior trunk might also be mor-phologically specialized (Donley et al., 2004). How-ever, we do not yet have a detailed understanding ofthe three-dimensional organization of the musculo-tendinous system of any lamnid, nor of the basalcondition in any subcarangiform shark.

In this study we investigated the musculotendi-nous system that powers continuous swimming (my-osepta, horizontal septum, and their associationwith red muscles) in three subcarangiform sharks(velvet belly lantern shark, Etmopterus spinax,smallspotted catshark, Scyliorhinus canicula, andthe blackmouth catshark, Galeus melanostomus)and one lamnid shark. The goal was twofold: first,detailed morphological information might facilitateunderstanding of the experimental data on the twodifferent locomotory modes described for sharks.Second, comparative morphological data on bothswimming types are necessary in developing hy-

478 S. GEMBALLA ET AL.

Journal of Morphology DOI 10.1002/jmor

potheses regarding the evolutionary changes thattook place from subcarangiform to thunniformsharks. Since the plesiomorphic condition of many ofthe characters of the musculotendinous system re-main unknown in elasmobranchs (see Gemballa andHagen, 2004), the polarity of characters has to beestablished by comparison with existing data onelasmobranch outgroups (teleosts: Gemballa et al.,2003a,b; Gemballa and Roder, 2004; one chondrich-thyan, the ratfish Chimaera monstrosa: Gemballaand Hagen, 2004).

MATERIALS AND METHODSInvestigation of the 3D Morphology ofMyosepta

After several weeks of fixation in 6% formalde-hyde, specimens were skinned and then cleared andstained (Alcian Blue 8GX for cartilage only) accord-ing to the procedure of Dingerkus and Uhler (1977).Visualization of the connective tissue system wasbest after stepwise transfer of the cleared andstained specimens to pure ethanol. Microdissectionsof myosepta were carried out using fine iris springscissors (Vannas, Tuebingen, Germany). Dissectionsstarted anteriorly and proceeded in a posterior di-rection by removing myoseptum by myoseptum. In-dividual myosepta were excised close to their inser-tion line to axial structures (vertebral column,vertical septum, and peritoneal membrane). Excisedmyosepta were kept separately for further micro-scopic investigations (see below). Three myosepta(an anterior, a midbody, a posterior one) were re-tained in their position in each specimen. They wereused to prepare camera lucida drawings of the 3Dshape (lateral views) and to measure the distancespanned by specific myoseptal tendons (see below).3D artwork was generated from these drawings us-ing the airbrush function of Adobe Photoshop (SanJose, CA). An additional 3D graphic representationwas prepared for the posterior body region of theshortfin mako. In this specimen, a 3D reconstructionwas obtained from histological sections. Major land-marks (vertebrae, neural arches, vertical septum,abdominal cavity, tip of main anterior, secondaryanterior and ventral posterior cone, sections of ten-dons, position of red muscle) were digitized andaligned using SurfDriver 3.5.3. Maxon Cinema 4Dwas used for choosing an adequate perspective andrendering. The obtained 3D view was edited inAdobe Photoshop (final shading, adding of myosep-tal shape).

Investigation of Myoseptal Tendons andTheir Association With Red Muscles

Major collagen fiber tracts in the myosepta andhorizontal septum were detected during microdis-sections and were integrated into the camera lucidadrawings. For better visualization of these fiber

tracts excised myosepta were spread out on glassesand analyzed under polarized light (Zeiss Stemi2000C with Zeiss Polarizer S and Analyzer A53).With this technique, collagen fibers appear as whitestrands on black background due to their birefrin-gent properties. Collagen fiber pathways were pho-tographed using a digital camera (Fujix DC HC-300Z; 1000 � 1500 pixel) mounted onto thestereomicroscope. This technique clearly confirmedthe pathways of the major collagen fiber bundlesthat had been obtained from the microdissections.

Quantitative data were obtained for one specificmyoseptal tendon, the lateral tendon. Because thistendon connects an anterior- and posterior-pointingcone of a myoseptum, its rostrocaudal span could bemeasured between the tips of the myoseptal cones.Measurements were taken in the three myosepta(anterior, midbody, posterior) that were retained intheir position during microdissections in each of thefour species.

We obtained information on the association of redmuscles with specific myoseptal tendons from trans-verse and sagittal histological sections, and addi-tionally from transverse sections of fresh specimens(Isurus oxyrinchus only). For histological investiga-tions we used pieces of the anterior and posteriortrunk in each of the four species. Sections wereobtained from paraffin blocks (20 �m), and werestained by the Azan–Domagk procedure (Romeis,1986) to distinguish between muscle and connectivetissue. Micrographs were taken as described above.

Material Examined

We examined three nonlamnid sharks and onelamnid shark. One squalean species, the velvet bellylantern shark Etmopterus spinax (L., 1758) (345 and281 mm total length) and two galean species, thesmallspotted catshark Scyliorhinus canicula (L.,1758) (315 and 489 mm total length), and the black-mouth catshark Galeus melanostomus Rafinesque,1810 (508 and 548 mm total length, L) were caughtby bottom trawls off the Catalan coast (Palamos,Mediterranean Sea) at depths of 300–500 m. Onespecimen of each species was used for clearing andstaining, the other for histological studies. For thelatter we used a piece of the anterior trunk (0.48–0.52L) and a piece of the posterior trunk (0.68–0.72L) of each species.

The lamnid specimen, a shortfin mako Isurusoxyrinchus Rafinesque, 1810 (650 mm total length),was caught by hook and line off the coast of South-ern California. The right side of this specimen wasused in part for histological investigations (trans-verse sections at 0.34L and 0.55L). The remainingparts were cleared and stained to examine the my-osepta and horizontal septum. Another specimen ofI. oxyrinchus (approx. 900 mm L) was frozen and cutinto �3-cm-thick transverse section to record the

479MUSCULOTENDINOUS SYSTEM IN SHARKS


relationship between red muscles and myoseptalcones.

RESULTS3D Morphology of Myosepta

In lateral views of skinned sharks a myoseptumappears as a simple zigzag line extending betweendorsal and ventral midline with at least one forwardand two backward flexures (Figs. 1, 2). This zigzagline represents the lateral line of attachment of amyoseptum to the skin. Medially, a myoseptum isattached to the median plane (axial skeleton, verti-cal septum, and peritoneal membrane). A myosep-tum is spanned between these two lines of attach-ment forming a complex 3D collagenous sheet.Specifically, at the level of each forward and back-ward flexure of the lateral zigzag line a myoseptumforms pointed cones that project into the myomericmusculature. The cones of the forward flexures aretermed anterior cones, those of the backward flexureare termed posterior cones (Fig. 1).

Myosepta of all four sharks investigated exhibittwo posterior-pointing cones, the dorsal and ventralposterior cones (DPC, VPC). Between these two

cones one prominent anterior-pointing cone, themain anterior cone (MAC), is present. Additionalanterior-pointing cones, the epaxial secondary ante-rior cones (eSACs) form the dorsalmost part of allmyosepta. In contrast, the hypaxial SACs (hSACs),which form the ventralmost part of myosepta, areonly developed in postanal myosepta (Fig. 1). Thesepostanal myosepta are bisected by the mid-horizontal line into epaxial and hypaxial moieties.Preanal myosepta lack this dorsoventral symmetrydue to the lack of hSACs. In this region, a collage-nous horizontal septum is present in the mid-horizontal line (Fig. 1).

Following the terminology introduced by Gem-balla et al. (2003a), we term the myoseptal sheetextending between posterior cones and the mainanterior cone as sloping parts (epaxial and hypaxialsloping part; ESP, HSP) and the myoseptal sheetextending between posterior cones and secondaryanterior cones as flanking parts (epaxial and hypax-ial flanking part; EFP, HFP). The myoseptal sheetsextending between secondary anterior cones anddorsal or ventral midline are here referred to assecondary flanking parts (SFPs) (Fig. 1).

Medially, the sloping and flanking parts are at-tached to axial structures along an almost horizon-tal attachment line that traverses across severalvertebral segments. Due to this almost horizontalarrangement subsequent flanking and sloping partsget very close to each other and form a common lineof attachment to axial structures. It is almost im-possible to separate myosepta by microdissectionsalong this line. We term these multiple structures offlanking or sloping parts “multilayers” (HSP-multilayer shown in Fig. 1).

The principle arrangement of a myoseptum con-sisting of main anterior, dorsal, and ventral poste-rior and secondary anterior cones is kept along thewhole trunk. Nevertheless, within a rostrocaudalseries of myosepta their proportions and some oftheir features change considerably depending on thespecies (see below; Fig. 2).

Collagenous Fiber Architecture of Myoseptaand Horizontal Septum

The collagenous fiber architecture of the myoseptais similar in the three subcarangiform sharks inves-tigated here. Although there are some gradualchanges from preanal to postanal myosepta in thesespecies, myosepta of the whole series in these spe-cies turned out to be relatively homogenous whencompared to the thunniform mako shark. For ease ofdescription we first address the collagenous fiberarchitecture and rostrocaudal changes in the sub-carangiform species and then describe similaritiesand differences with the thunniform mako.

Myoseptal series in subcarangiform sharks.The myoseptal series in the three subcarangiformsharks consist of �60 myosepta (Etmopterus spinax:

Fig. 1. Morphology of shark myosepta based on the four spe-cies investigated. Lateral view, anterior to left. The double blackline indicates the lateral attachment line where the myoseptum isattached to the skin. Mid-horizontally, a myoseptum is dividedinto an upper epaxial and a lower hypaxial part. Terminology ofmyoseptal structures is based on Gemballa et al. (2003a). Eachmyoseptum bears anterior cones (MAC, main anterior cone;eSAC, hSAC, epaxial and hypaxial secondary anterior cones), andposterior cones (DPC, VPC, dorsal and ventral posterior cones).Central myoseptal parts between MAC and DPC, VPC are termedepaxial and hypaxial sloping parts (ESP, HSP); those betweenDPC, VPC, and SACs are termed epaxial and hypaxial flankingparts (EFP, HFP). The upper- and lowermost parts are termedsecondary flanking parts (SFP). A: Myoseptum from the anteriorbody. Notice the presence of a horizontal septum (HS) and theabsence of a hSAC. AVPC is formed by a simple backward flexure.B: Myosepta from posterior body (i.e., postanal). Notice the ab-sence of an HS and the symmetry of epaxial and hypaxial parts.Because of the almost horizontal orientation, medial parts of themyoseptal sheet converge to form multilayer structures (shownfor HSP only).



Fig. 2. Outline of a subcarangiform shark (Etmopterus spinax, A) and the thunniform mako shark (Isurus oxyrinchus, B). For eachspecies one anterior and one posterior myoseptum is depicted in its original position in the trunk and separately at highermagnification. Pale red area in myosepta marks the area where red muscles insert into the myoseptal sheet. Collagen fiber tracts areshown schematically. ENF, epineural or epipleural fibers; LT, lateral tendon; MT, myorhabdoid tendon; SMT, secondary myorhabdoidtendon. Notice the distinct and elongated hypaxial lateral tendon in the posterior myoseptum of the mako (bright red).

57; Scyliorhinus canicula: 63; Galeus melanostomus:65). Almost all myoseptal parts within a series aredominated by longitudinally oriented collagenous fi-ber tracts that connect anterior and posterior coneswithin single myosepta. The collagen fibers in thesetracts are densely packed and form conspicuoustendon-like structures. They are structurally similarto what was referred to as myoseptal tendons inother fishes (e.g., Gemballa et al., 2003a) and thusare referred to as “tendons” in the following descrip-tion.

The epaxial sloping part (ESP) of a myoseptumexhibits a prominent longitudinally oriented tendon,the epaxial lateral tendon (eLT; Figs. 2A, 3) thatconnects the main anterior cone (MAC) and the dor-sal posterior cone (DPC). Its anterior and posteriorends lie at the tip of the myoseptal cones deep in themyomeric musculature. In contrast, the middle partof this tendon is situated laterally, right under theskin (Figs. 2, 3; see also Fig. 6A,B). Besides thelateral tendon the ESP region does not bear anyfurther tendon-like structures. However, the medialpart of the ESP is formed by two sets of collagenfibers (Figs. 2B, 3A). One set of fibers, the obliquefibers, originates from the epaxial lateral tendonand runs into the vertical septum. These fibers arecrossed by mediolaterally oriented fibers that insertinto the cartilaginous vertebral column proximallyand to the skin distally. We term this set of fibersepineural fibers because we consider them homologsof the epineural tendon in other gnathostomes(Gemballa et al., 2003a). Medial ESP of subsequentmyosepta merge to form the ESP-multilayer struc-tures. Hence, these multiple layers are reinforced bysets of epineural and oblique fibers of subsequentmyosepta. However, this reinforcement is onlypresent in preanal myosepta. Medial ESP regions ofpostanal myosepta lack the system of epineural andoblique fibers. Here, only some collagen fibers ofalmost mediolateral orientation form remnants ofthe epineural fibers (Figs. 2, 3B).

In the epaxial flanking part (EFP) a myorhabdoidtendon runs from the tip of the dorsal posteriorcones to the tip of the epaxial secondary anteriorcones (eSACs). As with the lateral tendon, the my-orhabdoid tendon runs in the lateral region of themyoseptal part. The medial part of the EFP consistsof collagen fibers of two directions. One set origi-nates from the myorhabdoid tendon and runs cau-domedially towards the vertical septum. Another setforms a band of caudolaterally oriented fibers thatoriginates at the vertical septum (Figs. 2B,C, 3A,B).Both directions of fibers reinforce the EFP-multilayer in the preanal region. The caudomedialfibers are preserved towards the postanal region,whereas the caudolateral fibers are not (Fig. 3B).The dorsalmost secondary flanking part (eSFP) ex-hibits a third longitudinally oriented tendon, thesecondary myorhabdoid tendon that is present in allbody regions (Figs. 2B,C, 3).

In the postanal region hypaxial and epaxial myo-septa form moieties bisected by the mid horizontalline. Both moieties are not only similar in their 3Dshape but also in their tendinous architecture (Figs.2C, 3B). Hence, the longitudinally oriented myosep-tal tendons in the hypaxial parts are termed hypax-ial lateral tendon (hLT; in hypaxial sloping parts),myorhabdoid tendon (in hypaxial flanking parts),and secondary myorhabdoid tendon (in secondaryflanking parts). As with postanal epaxial myosepta,the mediolateral fibers are weakly developed in thehypaxial myosepta. Few of these fibers are presentin the hypaxial sloping parts. They correspond to theepineural fibers of the epaxial sloping parts butwould be called epipleural fibers in the hypaxial part(for terminology, see Gemballa and Britz, 1998).

In contrast, hypaxial myosepta of the preanal re-gion differ considerably from their correspondingepaxial parts in terms of their 3D shape and tendi-nous architecture. They lack ventral posterior andhypaxial secondary anterior cones but consist of rel-atively plain sheets of oblique orientation (Fig. 2A).Moreover, they lack conspicuous myoseptal tendonsbut consist of a system of more or less mediolaterallyoriented collagen fibers. The gradual change fromthe preanal to the postanal condition of hypaxialmyosepta occurs when the abdominal cavity tapersoff. The increasing space between abdominal cavityand skin is occupied by myosepta that graduallydevelop cones and the longitudinally oriented myo-septal tendons until in the postanal region the hy-paxial and epaxial part are symmetrical in shapeand architecture (Figs. 2A, 3B).

This transition in hypaxial myosepta is the mostconspicuous one in the myoseptal system of the in-vestigated subcarangiform sharks. In the epaxialseries all major features (presence of cones and ten-dons) are kept along the whole trunk. Although theepineural tendons appear to weaken rostrocaudally,the longitudinal tendons (lateral and myorhabdoidtendons) are prominent in all body regions. Propor-tions of epaxial myosepta change rostrocaudally dueto the tapering of the trunk. In all species the height(extension in dorsoventral direction) of a myoseptumdecreases remarkably (Fig. 2). However, the rostro-caudal extension (i.e., the span) of myoseptal partsdoes not vary with body position. For example, thespan of the epaxial lateral tendons (i.e., the distancebetween MAC and DPC in longitudinal direction) isalmost constant from anterior to posterior myosepta(Fig. 4). Moreover, values are even similar betweenthe three subcarangiform sharks investigated (Et-mopterus spinax: 0.071–0.074L; Scyliorhinus can-icula: 0.05–0.056L; Galeus melanostomus:0.07–0.076L; see Fig. 4). This is in sharp contrast tothe conditions found in the mako (see below).

Anterior myosepta of the mako. In the makothe myoseptal series consists of �80 myosepta. My-osepta of the anterior part of this series resemblethat of the subcarangiform sharks in bearing an



Fig. 3. Collagen fiber architecture of myosepta (MS) of Etmoperus spinax. MS were spread out under polarized light for visual-ization of collagen fiber tracts. The lateral parts of all myoseptal parts are characterized by longitudinal tendons, the lateral tendon,myorhabdoid tendon, and secondary myorhabdoid tendon. Medial parts have fewer collagen fibers that do not form distinct tendons(fiber directions indicated by dashed lines; ENF, epineural fibers; EPF, epipleural fibers; OF, oblique fibers). A: Epaxial part of anteriorMS (MS 19 out of �57) extending between 0.35L and 0.42L. B: Epaxial and hypaxial part of posterior MS (MS 43 out of �57) extendingbetween 0.68L and 0.75L. Close-up view shows detail of the hypaxial lateral tendon and some of the epipleural fibers. See Figure 1 forterminology and abbreviations of myoseptal parts.



epaxial secondary anterior cone and a main anteriorcone and in lacking corresponding cones in the hy-paxial part (Fig. 2B). Moreover, the system of cross-ing mediolateral collagenous fibers of the plain hy-paxial part in the mako (Fig. 2B) is similar to that ofthe anterior hypaxial myosepta in the subcarangi-form sharks (Fig. 2A).

As in the subcarangiform sharks, all epaxial partsof the mako exhibit longitudinal tendons that con-nect the myoseptal cones (i.e., secondary myorhab-doid tendon, myorhabdoid tendon, and epaxial lat-eral tendon). Moreover, all epaxial parts havemediolaterally oriented fibers. Those of the epaxialsloping part reinforce the ESP-multilayers, as de-scribed for the subcarangiform sharks. However, inthe latter these fibers are more conspicuous than inthe mako. At least the epineural fibers of the sub-carangiform sharks are confined to a certain regionof the ESP and appear to be similar to what istermed epineural tendons in bony fishes, whereasepineural fibers in the mako are indistinct.

Posterior myosepta of the mako and rostro-caudal changes in myoseptal morphology. To-wards the postanal region epaxial and hypaxial my-osepta in the mako gradually become symmetrical inshape and in most aspects of their collagenous ar-chitecture (Figs. 2, 5). In the hypaxial part a ventralposterior cone and hypaxial secondary anterior coneis developed. Tips of cones are connected by promi-nent longitudinal tendons, the hypaxial lateral, my-orhabdoid, and secondary myorhabdoid cone. Thistransition in the hypaxial part is similar to thatobserved in the subcarangiform sharks.

However, in contrast to the subcarangiformsharks, in the mako conspicuous changes occursalong the myoseptal series in the mako. Thesechanges include a threefold elongation of the span ofthe lateral tendons (0.06–0.19L; Fig. 4) and achange in the distinctness of the hypaxial lateraltendon (Fig. 5A,B, insert). Being broad and indis-tinct preanally (Fig. 5A) this tendon becomes aprominent and distinct tendon postanally. This

change is most obvious in the anterior part of thetendon (see also Fig. 7A,B). Adjacent collagen fibersrun into this distinct part of the tendon (Fig. 5B).Furthermore, the hypaxial lateral tendon hasshifted medially into deeper regions of the muscula-ture (Fig. 5A,B) compared to its lateral position inthe subcarangiform sharks (Fig. 3B).

Besides the conspicuous elongation of the mainanterior cones, secondary anterior cones are alsoelongated towards the posterior body (Fig. 2B). As inthe subcarangiform sharks, the mediolateral fibersof the epaxial sloping parts (epineural fibers) arepresent but less conspicuous in the mako (Figs. 3B,5B).

Horizontal septum. In all four species of sharksa horizontal septum is only present in the anteriorbody (Fig. 6C). In this region it divides the myomericmusculature into an epaxial and hypaxial part, withthe hypaxial ones being restricted to narrow stripesdue to the outbulging abdominal cavity. The hori-zontal septum diminishes towards the postanal re-gion where epaxial and hypaxial parts form moi-eties. Neither microdissections nor histologicalinvestigations revealed any remnants of this septum(Fig. 6D,E).

Two orientations of collagen fibers are present inthe horizontal septum of all four shark species. Oneset of fibers, the epicentral fibers, runs caudolater-ally from its attachment to the vertebral columntowards the skin. These fibers are crossed by an-other set, the posterior oblique fibers, that runcraniolaterally from the vertebral column to theskin. Neither set of fibers form tendons or tendon-like structures but consist of almost evenly distrib-uted fibers.

Association of Red Muscles and MyoseptalTendons

Red muscles in the subcarangiform sharks.In the subcarangiform sharks examined here redmuscle fibers are easy to detect because of theirslender appearance when compared to the deeperwhite muscle fibers (Fig. 6A,B,D,E). Moreover, redmuscle fibers are oriented longitudinally, whilewhite muscle fibers have different orientations andare almost exclusively out of a horizontal and sagit-tal plane (Fig. 6D,E).

Red muscles form a lateral and superficial bandalong the trunk that is placed around the mid-horizontal line. In transverse sections of the anteriorand posterior body this lateral band is of a uniformshape. The thickest layer of red muscle fibers ispresent around the mid-horizontal line. Here the redmuscle area makes up �20–30% of the thickness ofthe lateral musculature (i.e., red and white muscle).The red muscle fibers extend far dorsally and ven-trally from the mid-horizontal line to the level of theposterior myoseptal cones. However, in these dorsal-and ventralmost areas the thickness of the red mus-

Fig. 4. Rostrocaudal span of three selected myosepta in thefour investigated sharks as a function of location along the bodylength, L. Span is given in fraction of body length on the gray bars(light gray, subcarangiform sharks; dark gray, thunniform makoshark). The span was measured between the tip of the mainanterior and posterior cone and thus reflects the length of thelateral tendons.



cle band decreases to �5% of the thickness of thelateral musculature. This specific red muscle areaintersects with the lateral parts of the epaxial andhypaxial sloping parts of the myosepta (pale redareas in Fig. 2A). Thus, red muscle fibers insert intothe lateral tendons, as do the deeper white fibers(Figs. 2A, 6D,E). Hence, forces generated by redmuscle fibers might be delivered along these lateraltendons toward the posterior cones. Furthermore,

red muscle fibers insert into the distal ends of theepineural fibers of a myoseptum. However, as de-scribed above, these fibers are hardly developed inthe postanal region (Fig. 3).

Red muscles in the mako shark. In the mako,red muscle fibers nest deep within the white musclefibers at a more medial position (Fig. 7A,B). Thedeep band of red muscles that forms along the trunkintersects with the main anterior cone (MAC) in all

Fig. 5. Collagen fiber architecture of myosepta (MS) of Isurus oxyrhinchus. MS were spread out under polarized light forvisualization of collagen fiber tracts. All myoseptal parts bear longitudinal tendons or collagen fiber tracts (lateral tendon, myorhab-doid tendon, and secondary myorhabdoid tendon) in their lateral parts. The medial parts of the myosepta consist of mediolaterallyoriented fibers that do not form distinct tendons but are distributed more or less homogenously across the myoseptal sheet. A: Epaxialand hypaxial sloping part of anterior MS extending between 0.4L and 0.52L. Notice that the hypaxial sloping part lacks a distinctlateral tendon but only bears hypaxial lateral fibers. These are situated at a more medial position when compared to the epaxial lateraltendons. B: Epaxial and hypaxial part of posterior MS extending between 0.55L and 0.74L. Notice the hypaxial lateral tendon that issituated at a more medial position in the deeper trunk musculature. Close-up view shows detail of the distinct hypaxial lateral tendon.See Figure 1 for terminology and abbreviations of myoseptal parts.



body regions. In the anterior body the area of inter-section lies in the ventral part of the MAC (Fig. 2B,pale red area in anterior myoseptum). Toward thepostanal region this area gradually shifts and redmuscle fibers intersect with the tip of the MAC (Fig.7C, see also pale red area in posterior myoseptum ofFig. 2B).

Transverse sections and histological sectionsclearly demonstrate that red muscles attach to thedistinct and prominent hypaxial lateral tendons of

postanal myosepta (Fig. 7A,B). Sections of 12 hypax-ial lateral tendons are visible within the band of redmuscles in a single transverse section (labeled 1–12in Fig. 7B). These sections of the tendons lie close tothe tip of the main anterior cone and thus representthe anterior parts of the tendons. The posterior endsof the tendons are visible within the white muscles(labeled 13–24 in Fig. 7B). In contrast to the anteriorparts, the tendons are less distinct and broadened(see also Fig. 5). The 24 sections of lateral tendons

Fig. 6. Histological sections of Galeus melanostomus illustrating the distribution of red and white muscle fibers and the associationof red muscle fibers with myoseptal tendons. A: Transverse section of left body side at 0.68L through main anterior cone (MAC),hypaxial sloping part (HSP), and ventral posterior cone (VPC). An area of thin red muscle fibers is visible laterally and can bedistinguished from the medial area of thicker white muscle fibers. The red muscle area is broadest at the level of the MAC and HSPand narrows to a thin stripe further ventrally. B: Enlarged view of A depicting the hypaxial lateral tendon (LT) that is present in thelateralmost part of the HSP. Laterally, the LT is embedded in red musculature (RM), medially in white musculature (WM). C–E:Sagittal sections, anterior to left. C: Anterior body at 0.48–0.51L. Below the MAC, a horizontal septum (HS) subdivides the lateralmusculature into an epaxial and hypaxial part. D,E: posterior body at 0.69–0.72L. D: Superficial section showing the region betweenMAC and VPC. Enlarged view depicts slender and longitudinally oriented red muscle fibers that insert into hypaxial lateral tendons(LT). E: Deeper section showing the region between MAC and VPC. Enlarged view depicts thick and obliquely oriented white musclefibers that insert into hypaxial lateral tendons (LT). Scale bars � 2 mm.



that are present in a single transverse section be-long to 24 subsequent nesting myoseptal cones. Inother words, that means that a single hypaxial lat-eral tendon spans the distance of 24 segments(equaling 0.19L, see Fig. 4). Only the anterior part ofthis tendon is embedded into the red musculature.Figure 7C depicts the elongated main anterior coneof the mako myosepta, its intersection area with thered muscles, and how a transverse section is com-posed of sections of subsequent hypaxial lateral ten-dons.

Another conspicuous feature that is evident intransverse histological sections is the sheath of looseconnective tissue that surrounds the red muscles inits medial, dorsal, and ventral part (Fig. 7B). Thecharacteristics of this sheath are particularly evi-dent when freshly killed specimens are cut trans-versely. In these sections the band of internalizedred muscles pops out of the sections because it is notfirmly anchored within the surrounding white mus-cles. Laterally, red and white muscles are not sepa-rated by connective tissue.

DISCUSSION

This study presents the first comparative dataseton the musculotendinous system of the locomotorysystem in sharks (i.e., myoseptal architecture andassociation of myoseptal tendons with red muscles).It reveals both similarities and differences betweenone thunniform and three subcarangiform species.In this section we compare the morphological fea-tures of these distantly related sharks to thoseknown from other gnathostomes. The goal of thiscomparison is to analyze the evolutionary transfor-mations of the musculotendinous system in sharks.On the one hand we demonstrate that some featuresof this system are evolutionarily conserved, i.e., theyhave evolved in the last common ancestor of gnatho-stomes or chondrichthyans or elasmobranchs (mod-ern sharks and rays) and remained relatively un-changed during evolution. On the other hand, wedemonstrate that some features were transformedwithin elasmobranchs and represent derived fea-tures of certain elasmobranch groups. Moreover, we

Fig. 7. Association of red muscles and myosepta in Isurus oxyrinchus. A,B: Transverse sections through main anterior cone andadjacent hypaxial musculature, lateral to the left. A: Fresh specimen illustrating the deep position of red muscles within the whitemuscles. A series of sections through hypaxial lateral tendons (white) is clearly visible along the ventral margin of the red muscles.B: Histological section at 0.54L. Twenty-four hypaxial lateral tendons are visible (1–12 within red muscles; 13–24 within whitemuscles). Dorso- and ventromedially the red muscles are separated from the white muscles by a sheath of connective tissue. Theenlarged view illustrates the connective tissue sheath and tendons 1–9. C: 3D reconstruction of a posterior myoseptum. Only theelongated main anterior cone and hypaxial part are shown. The anterior transverse plane of the block is indicated by the transparentpale red front plane. The intersection of red muscles within this plane is indicated by the darker red area. Notice the sections ofhypaxial lateral tendons (white) within the red muscle and its correspondence with the sections shown in A,B. The pale red triangulararea highlighted on the main anterior cone represents the intersection of red muscles and myoseptum. The hypaxial lateral tendon(bright red) of this main anterior cone is embedded into the red muscles with its anterior half and into the white muscles in its posteriorhalf. The anterior tip of this tendon is cut at its intersection with the transverse plane in front.



analyze whether any of these derived features arelinked to either the subcarangiform or the thunni-form mode of locomotion and whether these morpho-logical findings are in accordance with experimentaldata on kinematics and muscle activity patternsobtained from subcarangiform and thunniformsharks.

Analysis of Evolutionary Transformations ofthe Musculotendinous System

Horizontal septum and the series of collage-nous myosepta. Gnathostomes fall into two sistergroups, the cartilaginous fishes (Chondrichthyes)and the Teleostomi, including the ray-finned fishes(Actinopterygii) and the lobe-finned fishes (Sarcop-terygii; see Fig. 8; Janvier, 1996; Liem et al., 2001;Venkatesh et al., 2001). Past studies consideringmore than 70 species from all major groups demon-strated that the myoseptal system is remarkablyconservative within the Teleostomi. The set of myo-septal tendons consisting of epineural and epipleu-ral tendons, hypaxial and epaxial lateral tendons,and hypaxial and epaxial myorhabdoid tendons ispresent in basal actinopterygians (Gemballa et al.,2003a; Gemballa and Roder, 2004; Gemballa, 2004),in derived actinopterygians (Gemballa and Britz,1998; Gemballa et al., 2003a; Gemballa and Treiber,2003), and in basal sarcopteryginas (Gemballa andEbmeyer, 2003). In contrast, we have only limiteddata on the myoseptal system in chondrichthyans.The only study so far (Gemballa and Hagen, 2004)investigated ratfish (Chimaera monstrosa), a repre-sentative of the Holocephali that forms the sistergroup of all remaining chondrichthyans, the elasmo-branchs (� sharks � rays; Fig. 8). It demonstratedthat most of the myoseptal features known fromteleostomes are present in holocephalans as well.Thus, suggesting that these myoseptal featuresmust have evolved at the latest in the gnathostomeancestor. In addition, a dataset on the fiber archi-tecture of the horizontal septum based on 35 speciesfrom all major gnathostome clades (Gemballa et al.,

2003b) provides a basis for the analysis of evolution-ary transformations of this structure.

These studies have provided the framework forinterpretation of the data on the myoseptal systemand horizontal septum obtained for the four sharkspecies in this study. In the light of the currentphylogenetic hypothesis, any similarities between

Fig. 8. Evolutionary transformations of the musculotendinoussystem discussed in this article. Characters are mapped onto aphylogenetic tree of gnathostomes. Distribution of characterstates among gnathostome groups is indicated on terminal verti-cal lines by Roman and Arabic numbers for lampreys (outgroup),Holocephali (chimaeras), Elasmobranchii (sharks and rays), Ac-tinopterygii (ray-finned fishes), and Sarcopterygii (lobe-finnedfishes). Gray boxes in lower part indicate evolution of the ances-tral character state in gnathostomes as concluded from the dis-tribution of character states. Evolution of characters I and IVremains equivocal; arrows indicate alternative interpretations.See Table 1 and text for description of character states. Note thatall ancestral states of characters I–IX evolved early in gnathos-tome evolution and were retained within sharks in its plesiomor-phic condition. The only exceptions to this were detected in thethunniform mako (characters V [newly evolved character state 2],VII [3], VIII [2], IX [2]).



certain groups of sharks and teleostomes or holo-cephalans are likely to represent features of thegnathostome groundplan, and thus plesiomorphicfeatures within elasmobranchs. Any features thatare shared by the four sharks but are absent inteleostomes or holocephalans are likely to representevolutionary novelties of all elasmobranchs or atleast a larger group of elasmobranchs. With thisinformation at hand, differences among any of thefour sharks investigated here can be polarized aseither plesiomorphic features among sharks or de-rived features that have only evolved within a sub-group of sharks. In this study, we reveal some ofthese derived features and demonstrate that allthese features are related to the thunniform mode oflocomotion of lamnids. All features discussed in thefollowing section are summarized in Table 1 andFigure 8.

Horizontal septum. Since a horizontal septum isabsent in all nongnathostome vertebrates (characterI, state [0] in Fig. 8, Table 1) but is present in themajor gnathostome groups except for sarcoptery-gians, it has obviously only evolved within gnathos-tomes. All four sharks investigated bear a horizontalseptum with an array of crossing collagen fibers(epicentral and posterior oblique fibers; see Gem-balla et al., 2003b) in the anterior body that dimin-ishes posteriorly. This condition (character I [1] inFig. 8, Table 1) matches the condition found in twoother chondrichthyans, (Squalus acanthias [Alex-ander, 1969], Chimaera monstrosa [Gemballa andHagen, 2004]), suggesting that it represents agroundplan feature of the Chondrichthyes. Thesame condition I [1] is present in one group of basalactinopterygians, the sturgeons, while other basalactinopterygians either lack the horizontal septum(lepisosteids; I [0]) or have a fully developed one (I

[2]; polypterids, Amia and teleosts; Gemballa et al.,2003b). Moreover, sarcopterygians lack a horizontalseptum (Gemballa et al., 2003b). Given this charac-ter distribution it is impossible to decide at whichnode of the tree the horizontal septum of condition I[1] evolved (Fig. 8; Table 1). It was either evolvedonce at the gnathostome level or at least twice in thechondrichthyan ancestor and within the acti-nopterygians. However, the particular structure ofthe horizontal septum was conserved within sharksregardless of their locomotory specializations, indi-cating that this is not a relevant structure in under-standing the evolution of thunniform swimming insharks. In contrast, in tunas the horizontal septumis assumed to play a significant role in force trans-mission during thunniform swimming (Westneatand Wainwright, 2001).

3D morphology of myosepta. The absence oftwo (dorsal and ventral) posterior cones in nongna-thostome outgroups (character II, state [0]) and thepresence of such cones in all major gnathostomeclades (character II [1]; Gemballa et al., 2003a; thisstudy for sharks) clearly indicate that they evolvedwithin the gnathostome lineage (Fig. 8, Table 1).These cones point posteriorly into the trunk muscu-lature and appear as concentric rings in transversesections. This shape is retained in the four sharksinvestigated here, and was also reported for anotherspecies (Squalus acanthias; Alexander, 1969). Thus,these cones represent plesiomorphic features withinelasmobranchs.

An anterior-pointing cone (the main anterior cone,MAC) is also absent in nongnathostomes (characterIII [0] in Fig. 8, Table 1), but present in all majorgnathostome groups (III [1]; see Gemballa et al.,2003a), including the four sharks investigated here(e.g., see concentric ring in Fig. 6A). The shape of the

TABLE 1. Character states of characters I–IX of the musculotendinous system in sharks and related gnathostome clades

Character Description of character states

I: Horizontal septum with epicentraland posterior oblique fibers

I [0]: absent I [1]: present anteriorly I [2]: present alongwhole trunk

II: Posterior myoseptal cones II [0]: absent II [1]: present —III: Main anterior myoseptal cones III [0]: no cone; simple

forward flexureIII [1]: undivided cone III [2]: bifid cone

IV: Secondary anterior cones IV [0]: absent IV [1]: present —V: Lateral tendons V [0]: absent V [1]: present —VI: Myorhabdoid tendons VI [0]: absent VI [1]: present —VII: Epineural/epipleural tendons VII [0]: absent VII [1]: present as fiber

setVII [2]: present as

distinct tendonproximally

VIII: Increase in rostrocaudal spanof lateral tendons along body

VIII [0]: tendonabsent

VIII [1]: slight increaseof span, max. span0.1L

—

IX: Position of red muscles andassociation with lateral tendon

IX [0]: RM distributedhomogenously; nospecific association

IX [1]: lateral mid-horizontal RMassociated withmiddle part of lateraltendon

—

See Figure 8 for evolutionary tranformations between character states. For derived character states in thunniform sharks, see text andFigure 8.



main anterior cone, however, differs between gna-thostome groups. In derived actinopterygians, theteleosts, this cone is generally subdivided at themidhorizontal plane into a dorsal and a ventral an-terior cone (character III [2]; Nursall, 1956; Alex-ander, 1969; Vronskiy and Nikolaichuk, 1989; Gem-balla and Britz, 1998; Westneat and Wainwright,2001; Gemballa and Treiber, 2003; Gemballa et al.,2003a). The same has been demonstrated for thepostanal myosepta of one group of basal actinoptery-gians (polypterids: Gemballa, 2004). However, theundivided main anterior cone (III [1]) is present insharks (this study), chimaeras (Gemballa and Ha-gen, 2004), basal sarcopterygians (Gemballa and Eb-meyer, 2003), and basal actinopterygians (except forpolypterids; Gemballa, 2004; Gemballa and Roder,2004). The sister group of gnathostomes, the lam-preys, bears a simple undivided forward flexure atthe mid-horizontal line (Vogel and Gemballa, 2000).This distribution indicates that an undivided MACrepresents a derived groundplan feature of gnatho-stomes (character III [1] in Fig. 8, Table 1) that wasretained in sharks and other basal groups but wasindependently transformed into bifid cones (i.e., dor-sal and ventral anterior cones) in polypterids andteleosts.

Two additional myoseptal cones, the secondaryanterior cones (SACs), are present in all sharks in-vestigated here (character IV [1] in Fig. 8, Table 1)but are absent in the basal chondrichthyan Chi-maera monstrosa and nongnathostome outgroups(character IV [0]; Vogel and Gemballa, 2000; Gem-balla and Hagen, 2004). Moreover, they are absentin most major actinopterygian and sarcopterygianclades, but have been found in the postanal regionsof two basal groups of actinopterygians (polypteridsand lepisosteids; Gemballa, 2004) and in one basalgroup of sarcopterygians, the Dipnoi (lungfishes;Gemballa and Ebmeyer, 2003). The presence ofthese cones in representatives of the major groups ofsharks (Galea and Squalea) suggests that SACs area common feature in elasmobranchs. However, fromthe distribution outside the elasmobranchs it re-mains unclear whether these cones have evolvedearly in the gnathostome ancestor and were reducedseveral times or whether they evolved indepen-dently in elasmobranchs and several teleostomegroups (character IV [1] in Fig. 8, Table 1).

Myoseptal tendons. All four sharks investigatedhere bear two longitudinally oriented myoseptal ten-dons in the central myoseptal parts, namely, thehypaxial and epaxial lateral. These tendons weredemonstrated to be present in the basal chondrich-thyan Chimaera monstrosa, in actinopterygians,and basal sarcopterygians (character V, state [1] inFig. 8, Table 1; Gemballa and Ebmeyer, 2003; Gem-balla et al., 2003a; Gemballa and Hagen, 2004; Gem-balla and Roder, 2004). Since they are absent innongnathostome outgroups (character V [0]; Vogeland Gemballa, 2000), they must have evolved in the

gnathostome lineage, and were retained as plesi-omorphic features within elasmobranchs (see char-acter V [1] in Fig. 8).

The lateral tendons in all gnathostomes investi-gated so far are uniform: They are broad and indis-tinct, most condensed in their middle part, withslightly diverging collagen fibers towards their an-terior and posterior ends at the myoseptal cones.Their middle parts connect to the skin (Gemballa etal., 2003a). We found this condition to be completelyconserved in the three subcarangiform sharks, butonly partially conserved in the mako. In this species,only the epaxial lateral tendon matches this condi-tion, whereas the hypaxial lateral tendon has be-come more distinct and has shifted medially. Thisfinding suggests that this is a derived feature of themusculotendinous system of the mako (character V[state 2] in Fig. 8).

Another set of longitudinally oriented myoseptaltendons, the myorhabdoid tendons, have now beenidentified in all gnathostome clades (sharks: thisstudy; actinopterygians: Gemballa et al., 2003a;Gemballa and Roder, 2004; tetrapods: Gemballa andEbmeyer, 2003) as well as in basal vertebrates, thelampreys (Vogel and Gemballa, 2000). Also, thebasal chondrichthyan Chimaera monstrosa bearsthese tendons (Gemballa and Hagen, 2004). Clearly,these tendons represent gnathostome groundplanfeatures that were retained as plesiomorphic fea-tures within elasmobranchs (character VI, state [1]in Fig. 8; Table 1).

The tendinous architecture of secondary anteriorcones has never been addressed so far. In this re-gion, the four sharks investigated here bear second-ary myorhabdoid tendons (SMTs). This suggeststhat SMTs are, most probably, a common featureamong sharks and might represent an elasmo-branch groundplan feature. Any further discussionsof this feature will only be possible after additionaldata on the presence and tendinous architecture ofsecondary anterior cones have been gathered frombasal teleostomes.

The mediolaterally oriented epineural and epi-pleural fibers detected in the three subcarangiformsharks are homologs of the epineural and epipleuraltendons known from all major groups of gnathos-tomes including the chondrichthyan Chimaera(Gemballa et al., 2003a). In teleosts and the basalsarcopterygian Latimeria, these tendons are gener-ally distinct in their proximal parts (character VI [2]in Fig. 8, Table 1), whereas in more basal gnathos-tomes, as well as in the sharks investigated here,these tendons appear to be much less distinct (char-acter VI [1] in Fig. 8, Table 1). Since these tendonsare absent in nongnathostomes (character VI [0];Vogel and Gemballa, 2000), we interpret conditionVI [1] as an apomorphic gnathostome groundplanfeature that was retained as a plesiomorphic condi-tion in sharks.



However, the mako appears to be derived in thisrespect, since it does not bear tendon-like epineuralstructures (Fig. 8; character VII [state 3]). Particu-larly in the posterior region, fibers of the mediolat-eral orientation seem almost absent. So far this con-siderable modification of the epineural/epipleuralfiber system has only been recorded in a carangiformteleost, the mackerel (Gemballa and Treiber, 2003).Experimental data that might provide an explana-tion for this modification are lacking.

Rostrocaudal changes in the series of myo-septa. So far, only length changes of the lateraltendon have been considered in studies on myosep-tal series (in each case measured as the rostrocaudalspan between anterior and posterior cones). In mostspecies, the rostrocaudal span is between 0.05 and0.075L in anterior lateral tendons. Posteriorly, amoderate increase of 11–13% of the initial tendonlength is observed (Gemballa and Treiber, 2003;Gemballa and Roder, 2004; for review, see Shadwickand Gemballa, 2006). Although not quantified, asimilar increase in length was demonstrated in chi-maeras (Gemballa and Hagen, 2004). Likewise, wefound a slight increase in length between anteriorand posterior lateral tendons in the three subcaran-giform sharks investigated here (increase of 10–12%of initial length; Fig. 4). In all species, the rostrocau-dal span of posterior lateral tendons does not exceed0.1L. The wide distribution of this limited span oflateral tendons in sharks and actinopterygian fishessuggests that it represents a gnathostome ground-plan feature (character VIII, state [1] in Fig. 8, Table1).

Few exceptions from this have been recorded sofar. Two carangiform (the mackerel Scomber scom-brus and the carangid Trachurus mediterraneus)and one thunniform teleost (the little tunny Euthyn-nus alletteratus) have been reported to bear poste-rior lateral tendons with spans between 0.16 and0.25L (Shadwick and Gemballa, 2006). A similarelongation of posterior lateral tendons was demon-strated in this study for the thunniform mako shark(threefold increase in length from 0.06 to 0.19L). Atpresent, we regard these extreme elongations asexceptions that are confined to specialized carangi-form and thunniform swimmers, and we hypothe-size that they evolved independently in several gna-thostome groups. Within sharks this is a derivedcondition, and so far has only been reported for themako shark (Fig. 8; character VIII [state 2]).

Position of Red Muscles and AssociationWith Myoseptal Tendons

It has long been noticed that the lateral position ofred muscles above and below the horizontal septumis present in almost all gnathostomes (e.g., Videler,1993), suggesting that this represents a groundplanfeature of this clade. This arrangement was alteredconvergently within a few groups (lamnid sharks,

thresher sharks and tunas; e.g., Bernal et al., 2003a;Graham and Dickson, 2004) where the red musclesare placed medially within the core of white mus-cles. The important thing to add here is the associ-ation of lateral (superficial) and internalized (me-dial) red muscles with myoseptal tendons.

In the subcarangiform sharks the lateral red mus-cles insert into the middle part of the lateral tendons(see Fig. 6D), suggesting that this lateral red muscle– lateral tendon association represents an elasom-branch groundplan feature. The same arrangementhas been demonstrated for Scomber (Gemballa andTreiber, 2003), and is easily observed during grossdissections of other teleosts (e.g., salmon, eel, car-angids; S.G., pers. obs.). These findings might sug-gest that the lateral red muscle – lateral tendonassociation represents a gnathostome groundplanfeature (character IX, state [1] in Fig. 8, Table 1).However, further comparative studies that show thepresence or absence of this arrangement are lackingand thus this interpretation needs further confirma-tion.

In contrast, the internalized red muscles of themako insert into the anterior part of the distinct andelongated hypaxial lateral tendon (Fig. 8; characterIX [2]). Interestingly, the same principal arrange-ment was found in one tuna where both epaxial andhypaxial lateral tendons are associated with the redmuscles (Shadwick and Gemballa, 2006), but isnever present in nonthunniform swimmers. Thiscorrespondence of swimming type and derived anat-omy of the musculotendinous system in two dis-tantly related groups of thunniform swimmers sug-gests that it is part of a specific internal design ofthunniform swimmers that can be added to the well-known external design features of thunniform swim-mers (e.g., stiff hydrofoil-like caudal fin, teardropshape, highly tapering body with narrow caudal pe-duncle; e.g., Bernal et al., 2001a). Moreover, thesefindings fit well into experimental data on swim-ming kinematics and muscle dynamics of thunni-form swimmers. Thus, they are part of a new andintegrative biomechanical understanding of thunni-form swimming.

Function of the Musculotendinous System:Integration of Morphological andExperimental Data

Studies of kinematics and muscle dynamics of twodistantly related thunniform swimmers, the makoshark and tunas, revealed strong similarities be-tween both groups (Shadwick et al., 1999; Katz etal., 2001; Donley et al., 2004, 2005). In both groupsthe contraction of the deep red muscle fibers at agiven axial position is uncoupled from bending atthis position, but rather is in phase with bending ata more posterior location. This is in contrast to whatis known from nonthunniform swimmers where redmuscles are in phase with local bending (Coughlin et



al., 1996; Katz et al., 1999; Donley and Shadwick,2003). This finding has at least two important con-sequences.

First, tunas and mako sharks should possess aunique muscle-tendon architecture that allows mid-body red muscles to cause lateral motion in theposterior region rather than local bending. Our mor-phological findings suggest that the extreme elonga-tion of the hypaxial lateral tendon and its associa-tion with red muscles in the mako facilitates thislong-reaching force transmission. A similar condi-tion was convergently evolved in tunas (Shadwickand Gemballa, 2006), indicating that selection pres-sures for fast and continuous thunniform swimminghave not only led to a specific external morphologybut also to a specific biomechanical design that in-cludes changes in the musculotendinous system andmuscle dynamics. Second, because red muscles areout of phase with local bending while white musclesare in phase with local bending, a certain degree ofshearing between red muscle fibers and surroundingwhite muscle fibers must occur during thunniformswimming, particularly in the posterior portion ofthe body. In the mako this shearing might be facil-itated by the loose connective tissue that wrapsaround the red muscles (e.g., Fig 7B).

So far, physical uncoupling of bending and redmuscle contraction has not been demonstrated forany nonthunniform swimmer. In these species redmuscle contractions cause local bending at the sameaxial position. As shown in this study for three sub-carangifom sharks, red muscle fibers attach to themiddle part of relatively short (span between 0.05Land 0.076L) lateral tendons. Thus, red muscle forceswill only travel around 0.025–0.038L posteriorlywhen transmitted along these tendons. Of course,this view oversimplifies the mechanical functions ofmyomeres but it shows that morphological findingsare principally in accordance with experimentaldata in both thunniform and nonthunniform swim-mers. Thus, comparative anatomy is one helpful tooltowards a thorough biomechanical understanding ofthe lateral musculature in swimming fishes.

ACKNOWLEDGMENTS

Many of the time-consuming histological proce-dures were carried out by Monika Meinert andChristina Nitzsche. Any technical problems withthe 3D-reconstruction were solved by ThomasSchmelzle (Tubingen).

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evolution of high-performance swimming in sharks ... · within elasmobranchs (sharks and rays),...

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