Evolution and development of the synarcual in early vertebrates

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1 DOI /s ORIGINAL PAPER Evolution and development of the synarcual in early vertebrates Zerina Johanson Kate Trinajstic Robert Carr Alex Ritchie Received: 14 March 2012 / Revised: 20 June 2012 / Accepted: 20 June 2012 Ó Springer-Verlag 2012 Abstract The synarcual is a structure incorporating the anterior vertebrae of the axial skeleton and occurs in vertebrate taxa such as the fossil group Placodermi and the Chondrichthyes (Holocephali, Batoidea). Although the synarcual varies morphologically in these groups, it represents the first indication, phylogenetically, of a differentiation of the vertebral column into separate regions. Among the placoderms, the synarcual of Cowralepis mclachlani Ritchie, 2005 (Arthrodira) shows substantial changes during ontogeny to produce an elongate, spoolshaped structure with a well-developed dorsal keel. Because the placoderm synarcual is covered in perichondral bone, the ontogenetic history of this Cowralepis specimen is preserved as it developed anteroposteriorly, Communicated by A. Schmidt-Rhaesa. Z. Johanson (&) Earth Sciences, Natural History Museum, Cromwell Road, London SW7 5BD, UK z.johanson@nhm.ac.uk K. Trinajstic Western Australian Organic and Isotope Geochemistry Centre, Department of Chemistry, Curtin University, Bentley, WA 6845, Australia K. Trinajstic Department of Earth and Planetary Sciences, Western Australian Museum, 49 Kew Street, Welshpool, WA 6106, Australia R. Carr Department of Natural Sciences and Geography, Concordia University Chicago, 7400 Augusta St., River Forest, IL , USA A. Ritchie Palaeontology, Australian Museum, Sydney 2010, Australia dorsally and ventrally. As well, in the placoderm Materpiscis attenboroughi Long et al., 2008 (Ptyctodontida), incomplete fusion at the posterior synarcual margin indicates that both neural and haemal arch vertebral elements are added to the synarcual. A survey of placoderm synarcuals shows that taxa such as Materpiscis and Cowralepis are particularly informative because perichondral ossification occurs prior to synarcual fusion such that individual vertebral elements can be identified. In other placoderm synarcuals (e.g. Nefudina qalibahensis Lelièvre et al., 1995; Rhenanida), cartilaginous vertebral elements fuse prior to perichondral ossification so that individual elements are more difficult to recognize. This ontogenetic development in placoderms can be compared to synarcual development in Recent chondrichthyans; the incorporation of neural and haemal elements is more similar to the holocephalans, but differs from the batoid chondrichthyans. Keywords Vertebral fusion Synarcual Placodermi Chondrichthyes Holocephali Batoidea Vertebral column Introduction In certain vertebrates, the axial skeleton is modified by fusion of anterior elements into a structure known as the synarcual. A synarcual is present in the fossil group Placodermi (Figs. 1, 2, 3, 4, 5) and in certain chondrichthyans, including the holocephalans and batoids (Figs. 6, 7). The synarcual represents, phylogenetically, the first known appearance of a differentiation of the axial skeleton into a distinct anterior region, relative to the remainder of the vertebral column. These synarcuals are non-homologous, so it could be predicted that they (and the distinct anterior

2 Fig. 1 Generalized placoderm (lateral view) showing limits of headshield and trunkshield. Axial skeleton in black, with synarcual anteriorly, bridging the gap between head and trunkshield. After Dennis and Miles (1981: fig. 2) and Miles and Westoll (1968) regions they represent) developed in different ways. We will test this hypothesis by comparing the development of the synarcual in these early vertebrates. Vertebral elements that can be incorporated into the synarcual include dorsal (basidorsal/neural, interdorsal), ventral (basiventral/haemal, interventral) and central elements (e.g. Gadow 1933, see Arratia et al. (2001) for a review of the homology of the centrum, including the arcocentra in placoderms). The dorsal, ventral and central vertebral elements are considered to be homologous across the vertebrates. Placoderms (extinct armoured fish; Fig. 1) are members of the gnathostome stem group, resolved phylogenetically to the basal nodes of the jawed vertebrate clade (Janvier 1996; Brazeau 2009). Among the placoderms, a synarcual is preserved in the Rhenanida, Ptyctodontida and Arthrodira. A synarcual is also present in Stensiöella heintzi Broili 1933 (Gross 1962, 1965). Stensiöella was described as a placoderm (Gross 1962), but this has been questioned due to the absence of placoderm characters in Stensiöella and similarity to the holocephalan Deltoptychius Morris and Roberts 1862 (Coates and Sequiera 2001). A synarcual was also reconstructed in the Bothriolepididae (Antiarchi), based on the morphology of grooves and pits on the internal surface of the trunkshield plate (Moloshnikov 2008). Dorsal parts of the synarcual were said to insert into these grooves and pits, but the synarcual itself has never been observed. Current phylogenies resolve the Placodermi as a paraphyletic group (Brazeau 2009; Davis et al. 2012), although Young (2010; also Goujet and Young 1995, 2004) describes the Placodermi as monophyletic. In either instance, the Rhenanida would be resolved as a sister taxon to the Ptyctodontida and Arthrodira. Among chondrichthyans, a synarcual is present in the crown group chondrichthyans (sensu Pradel et al. 2011; Euselachii? Holocephali), but absent in stem group taxa. In chondrichthyans, the synarcual supports the dorsal fin spine in holocephalans such as Chimera monstrosa Linnaeus, 1758 (Fig. 6a), or the pectoral fin in skates and rays (González-Isáis and Domínguez 2004). The function of the synarcual in placoderms is more difficult to establish. The pectoral fin and scapulocoracoid are associated with the lateral and anterior trunkshield plates and separated from the synarcual, which is positioned under the dorsal trunkshield and extends anteriorly to articulate with the braincase (Fig. 1). In this position, the synarcual is also separated from any dorsal fin elements (but see Miles and Young 1977: fig. 34), but acts to support the headshield with the trunkshield. We will review synarcual morphology in the Placodermi and compare this to extant chondrichthyan synarcuals, in order to compare developmental patterns in both groups and the formation of this distinct anterior region of the vertebral column. Normally, the synarcual of extant chondrichthyans would be expected to provide more developmental information than the synarcual of the fossil placoderms. However, because the placoderm synarcual is perichondrally ossified, the early ontogenetic history of the synarcual can be well preserved, while it is lost due to cartilage fusion in living chondrichthyans. Therefore, the placoderm synarcual may illuminate some of the early ontogeny of the chondrichthyan synarcual as well. For example, new specimens of the arthrodire Cowralepis mclachlani and the ptyctodont Materpiscis attenboroughi preserve a substantial amount of ontogenetic information, including early stages of development. This is due to the deposition of perichondral bone around cartilaginous elements during development. By comparison, the mineralized cartilage of recent chondrichthyan synarcuals is largely fused; synarcual development is best observed posteriorly, where vertebral elements are newly incorporated into the synarcual (e.g. Fig. 6a). One feature that will be examined in detail is the development of the ventral part of the placoderm synarcual. This appears rounded or spool-like relative to the flange or keel of the dorsal part of the synarcual and may develop from a ventral expansion of the basiventrals (as in most living skates and rays, Figs. 6, 7) or from direct incorporation of separate ventral elements (as in the chimera, Fig. 6a). For example, the synarcual of Materpiscis attenboroughi indicates that placoderms are more

3 Fig. 2 a 1, a 2 Placoderm axial skeleton. NHMUK PV P Incisoscutum ritchiei, showing neural arches/spines and haemal arches/spines. Anteriorly, haemal spines are notably reduced. a 1 Inset, closeup of neural spines showing zygapophyses. b f Placoderm synarcuals. b 1, b 2 WAM , Compagopiscis croucheri (Eubrachythoracida; Arthrodira). b 1 inset shows region of the synarcual indicated by asterisk in main Figure (b 2 ), rotated counter clockwise 90 degrees. c, d WAM , Campbellodus decipiens (Ptyctodontida). c Synarcual and more posterior axial skeleton, in lateral view. Smaller white arrow indicates rounded base of neural arch. Black box in c indicates area highlighted in d. d Closeup of synarcual in Fig. 2c and more posterior vertebral elements. Smaller white arrow indicates expanded dorsal flange. e NHMUK PV P.57665, Austroptyctodus gardineri (Ptyctodontida), small white arrows indicate spool-shaped areas of the ventral synarcual, including haemal elements. f, g WAM , Materpiscis attenboroughi (Ptyctodontida). f Lateral view. Smaller white arrow indicates top of spool-shaped part of the synarcual, composed of haemal arch elements. g Medial view. White arrow indicates top of spoolshaped part of the synarcual. 3 5 indicate the individually recognizable haemal arches being added to the ventral synarcual. Abbreviations: haem. haemal arch/spine, kl median dorsal keel, na/n.sp neural arch, neural spine, n.sp neural spine, syn synarcual, zyg,zygapophyses.scale bars = a 2, b 2, e = 1cm;f = 0.5 cm. Larger white arrows indicate anterior in all figures

4 Fig. 3 Placoderm synarcual a c, CMC VP8544, Dunkleosteus sp., Devonian, Morocco. a Anterior view of articular condyle (articulating with occipital region of braincase). b Lateral view. c Ventral view. comparable to the Holocephali in that separate ventral arch elements are directly incorporated into the spool-shaped part of the synarcual, with no ventral expansion. Materials and methods Fossil placoderm synarcuals were examined from the groups Arthrodira [Incisoscutum ritchiei Dennis and Miles, 1981 (Dennis and Miles 1981), Compagopiscis croucheri Gardiner and Miles, 1994 (Gardiner and Miles 1994), Dunkleosteus sp., Cowralepis mclachlani (Ritchie 2005)], Ptyctodontida [Campbellodus decipiens Miles and Young, 1977 (Miles and Young 1977), Austroptyctodus gardineri Abbreviations: art.cond articular condyles, na neural arch, nc notochord, sp.n spino-occipital nerve foramina Fig. 4 Placoderm synarcuals. a d Cowralepis mclachlani (Devo-nian, Merriganowry Quarry, New South Wales). a, b 1, b 2 AMF129164, smaller individual. a Ventral view showing internal surface of head and trunkshield, occipital region of braincase and synarcual, larger white arrow indicates anterior. b 1, b 2, closeup of occipital and synarcual, stereopair. Black arrows indicate circular growth rings of the neural bases in both the occipital and synarcual. White arrows indicate location of spino-occipital nerves. c d New synarcual uniquely preserved in lateral and medial views. c 1, c 2 AMF , right lateral view (c 1, larger white arrow indicates anterior). d 1, d 2 AMF , medial view (d 1 larger white arrow indicates anterior). d 1, black arrows indicate circular growth lines of individualized neural arch base. Arrowheads in d 2 indicate transition from individualized neural arch bases to fusion of bases and deposition of perichondral bone along this margin. Abbreviations: vert. 1 4 vertebral elements 1 4. a, b From Johanson et al. (2010), reproduced with permission from the Int J Dev Biol. Scale bars a, c, d = 1cm

5 Miles and Young, 1977 (Miles and Young 1977; Long 1997), Materpiscis attenboroughi (Long et al. 2008; Trinajstic et al. 2012)], and the Rhenanida [Nefudina qalibahensis (Lelièvre and Carr 2009), Jagorina pandora Jaekel, 1921 (Stensiö 1963; Johanson et al. 2010)]. All ptyctodont and most arthrodire specimens are from the Gogo Formation (Devonian, Western Australia) and were prepared in weak acetic acid to remove the surrounding

6 Fig. 5 Placoderm synarcuals. a c Mb.f 510.2, Jagorina pandora. a Dorsal view of counterpart. White arrow indicates position of synarcual. b, c View of part and counterpart specimen, respectively. c white arrow indicates synarcual. Larger white arrows indicate anterior direction. Scale bars = 1 cm. Abbreviations: pect.gird pectoral girdle. b, c From Johanson et al. 2010, reproduced with permission from the Int J Dev Biol

7 Fig. 6 Recent chondrichthyan embryos, cleared and stained. a AMNH 55040, Chimera monstrosa (Holocephali), lateral view. Black arrows indicate neural arch elements being added to the synarcual. b, c Closeup of synarcual and first free vertebrae, b AMNH 4128, Torpedo torpedo. c AMNH 8193, Rhinobatos lentiginosus (Garman, 1880), ventral view. d AMNH 4128, Torpedo torpedo (Linnaeus, 1758) (Batoidea; Torpediformes). e AMNH 8193, Rhinobatos lentiginosus (Batoidea; Rhinobatiformes). f AMNH 16350, Raja texana Chandler, 1921 (Batoidea; Rajiformes), ventral view. Black arrows indicate position of first free vertebral centrum limestone nodule, with three ptyctodont specimens embedded in resin to preserve the association of skeletal elements (see Long et al for a more complete methodology). Rubber latexes of the part and counterpart moulds of Cowralepis mclachlani were taken. Recent chondrichthyan embryos from the AMNH Zoology

8 Fig. 7 Recent chondrichthyan embryos, cleared and stained. a, b AMNH 30607, Dasyatis americana (Batoidea; Myliobatiformes). a Lateral view. b Ventral view. Black arrow indicates distinct segmentation posterior to the developing synarcual. White arrowhead collections were cleared and stained using standard protocols (e.g. Dingerkus and Uhler 1977). All specimens are illustrated using macrophotography. Institutional abbreviations: AMF, Australian Museum, Sydney; AMNH, American Museum of Natural History, New York; CMC, Cincinnati Museum Center, Cincinnati; Mb.f, Museum fur Naturkunde, Berlin. NHMUK PV P, Natural History Museum, London; WAM, Western Australian Museum, Perth. Abbreviations: art.con, articular condyles; gr.r, growth rings; haem, haemal arch/spine; keel, keel (or flange) of the dorsal synarcual; keel, dorsal synarcual keel; kl, keel on internal surface of trunkshield; na/n.sp, neural arch/neural spine; nc, neural canal; occ, occipital; pect.gird, pectoral girdle; sp.n, foramina for spino-occipital nerves; syn, synarcual; vert.1-4, vertebrae 1-4; v.syn, ventral synarcual; zyg, zygapophysis. Results Eubrachythoracida; Arthrodira Incisoscutum ritchiei (NHMUK PV P.50934; WAM ) Acid-prepared specimens of Gogo placoderms such as I. ritchiei (NHMUK PV P ) preserve substantial morphological detail, including the vertebral column. Posterior to the bony trunkshield plates, the neural and haemal arches are well-developed, positioned dorsal and ventral to the notochord, with long spines (Fig. 2a 1, 2 ). Fusion is absent, with contact limited to zygapophyses on indicates loss of this segmentation anteriorly. White asterisk indicates ongoing incorporation of elements into synarcual, with loss of basiventral separation on one side of the free vertebra, but not the other the neural arches (Fig. 2a 1 ). Haemal arches are used here in the sense of Miles and Westoll (1968) to refer to ventral elements along the vertebral column, not necessarily restricted to the caudal region. In other specimens where the synarcual is present anteriorly (WAM ), two fused neural elements are visible immediately posterior to the ventral keel of the trunkshield median dorsal plate. A single neurapophysis (lamina of the vertebral arch lacking zygapophyses) is located directly behind the fused elements. Its morphology suggests it is a third element, which has broken away from the posterior margin of the synarcual. These are displaced but appear identical to the anterior elements of the synarcual in Compagopiscis, described below. Compagopiscis croucheri (WAM ) In Compagopiscis croucheri, at least six neural arches have fused to form the incompletely preserved synarcual. The right half of the synarcual preserves four fused neural arches. The left half of the synarcual also preserves four neural arches, however, the posterior two neural arches have a dorsolaterally expanded neural spine (Fig. 2b 2, black asterisk and in inset, Fig. 2b 1 ), although the height of this is reduced when compared to the neural spines on more posterior vertebrae. Directly posterior to this is a separated element also with an extended spine, which may have been fused to the other element in life. As in ptyctodonts (see below) the synarcual rested on the notochord and did not surround it. Although there is no expanded dorsal keel or flange present in Compagopiscis, the two posterior neural spines are incompletely fused, having an opening between

9 the two neural elements. Two small foramina are preserved, one at the base of the neural arch and a second on the anterior margin of the expanded neural spine. There is a lateral process along the dorsal margin of the neural spine and a reduced prezygapophysis on the more posterior element. There are no haemal elements preserved below the synarcual. Posterior to the right half of the synarcual, there are three isolated neural arches with neural spines preserved directly behind the fused elements. At the anterior margin of the median dorsal trunkshield keel, there is a row of separate neural elements with reduced spines. The haemal elements are separate, and there is no haemal spine development. Within the eubrachythoracid arthrodires from the Gogo Formation, neural and haemal spines only fully develop posterior to the median dorsal plate (Fig. 2a 2, b 2, compare neural arches labelled na/n.sp with those more anterior). The neural and haemal elements do not otherwise fuse at any point along the vertebral column. Dunkleosteus sp. (CMC VP8544) The synarcual of Dunkleosteus Lehman, 1956 shows the highest degree of fusion among the placoderms described here (Fig. 3), except for the rhenanid Nefudina. The dorsal synarcual is broken, so it is uncertain whether the synarcual had an expanded flange or keel as in other placoderm taxa. Seven spino-occipital foramina are preserved, indicating that at least eight neural elements have been fused into the synarcual. Just behind the last foramen is a groove, which may represent an incompletely fused neural element, or it may be part of a crack, which extends dorsoventrally in this region (Fig. 3b). The posteriormost part of the synarcual also appears to be incomplete. Anteriorly, the synarcual is modified into two articular condyles (art.con, Fig. 3a), which would have articulated with the posterior occipital region of the braincase. Openings for the neural arch and canal are also visible in anterior view. In ventral view, a faint seam can be seen extending anteroposteriorly (Fig. 3c), comparable to that described in batoid chondricthyans and representing the fusion of the basiventrals (Fig. 7a, b; Claeson 2011). Although synarcual fusion has occurred before perichondral bone deposition, addition of haemal elements to the rounded ventral portion of the synarcual in Materpiscis (see below) suggests that haemal elements also comprise the ventral synarcual in Dunkleosteus sp. Placoderm synarcuals: Arthrodira Cowralepis mclachlani (AMF129164, AMF137328, AMF137329) The synarcual of Cowralepis was described previously (Ritchie 2005; Johanson et al. 2010) and is commonly preserved in ventral view, showing significant changes through ontogeny. In the earliest known stage, the synarcual is rectangular and short (syn, Fig. 4a, b), becoming elongate and spool-like with maturity, particularly anteriorly (Johanson et al. 2010: fig. 2F). This anterior edge matches and articulates with a comparably rounded margin of the occipital region of the braincase (occ; Fig. 4a, b). In certain specimens, the two halves of the synarcual have become separated, allowing a partial view of the internal surface of the dorsal portion of the synarcual (Johanson et al. 2010: fig. 2B, F). Here, several rounded elements can be seen, arranged in an anteroposterior sequence, with multiple circular growth rings visible in each (Fig. 4b 1, black arrows). These represent the periodical growth of the cartilaginous neural arch bases and associated perichondral bone deposition (Ritchie 2005: fig. 18C), showing no loss of identity (via fusion) within the synarcual. Small tubes can also be seen, representing the course of the spinooccipital nerves between the neural arches (Fig. 4b 2, white arrows). The two halves of the synarcual were also separated in a larger (and so presumed older) specimen, showing the distinctive rounded neural arch bases (Johanson et al. 2010: fig. 2I). In these older specimens, the anterior synarcual was modified into a thick, rounded articular surface. A new specimen of Cowralepis (AMF , AMF ; Fig. 4c, d) preserves the synarcual in lateral view for the first time, allowing for a more complete description of synarcual development. The ventral portion of the synarcual (v.syn) extends anteroposteriorly, and, as noted, is thick and rounded anteriorly (cranio-vertebral articulation) where it meets the occipital. However, the anteriormost face of the synarcual is not visible, so the presence of separate articular condyles cannot be determined. The ventral synarcual is surmounted by a thin dorsal keel comprising the neural arch bases (Fig. 4c 1, keel), identified by the multiple, circular growth rings as described above (gr.r; Ritchie 2005). The keel is lower anteriorly, gradually increasing in height posteriorly. The anterior keel is made up of three to four neural arch bases, although this is difficult to distinguish anteriorly, particularly in external view (Fig. 4c). What appear to be single foramina for the spinooccipital nerves are present (sp.n), but not associated with the first two or three vertebrae (Fig. 4c 2,d 1, area between the arrowheads and arrows). Laterally, the growth rings associated with these first three vertebrae appear less rounded and more flattened when compared with more posterior neural arch bases (Fig. 4c 2 ). This suggests that at this point in development, the cartilaginous neural arch bases have begun to fuse and lose their identity, growing more as a unit, with corresponding appositional deposition of perichondral bone along the edges of this unit (black arrowheads, Fig. 4c 2 ). However, in medial (internal) view,

10 the neural arch bases have remained distinct, particularly ventrally, where the first three to four bases can be distinguished (Fig. 4d 1, region between the black arrows). However, along with being less distinct externally, these arches are partially covered anteriorly by the rounded ventral cranio-vertebral articulation (Fig. 4c, d). The synarcual keel increases in height posteriorly, and the growth rings are more rounded than they are anteriorly (more similar to their appearance in the more posterior neural arch bases of the vertebral column), and they are also stretched. This could be related to the growth of the synarcual, but the Cowralepis fauna in the Merriganowry Quarry has experienced postmortem deformation (Ritchie 2005: fig. 8), which could have resulted in this stretching. Nevertheless, these posterior neural arch bases are more distinct than those anteriorly, although they have fused together to produce spino-occipital nerve foramina. As well, the ventral part of the synarcual appears to bend or kink just posterior to the rounded articular area (Fig. 4c, d). Posterior to this bend, the neural arch bases appear to have retained more individuality than anteriorly. Development and composition of the ventral portion of the Cowralepis synarcual is more difficult to determine, as it is smooth and rounded, with no indication of the addition of individual elements, including in medial view (Fig. 4d). In living taxa such as the holocephalan Chimera monstrosa, the ventral elements are clearly fusing to the body of the synarcual (Fig. 6a). By comparison, in the Batoidea, it appears that the ventral parts of the synarcual develop from ventral extensions of the more dorsally positioned basiventrals growing around the vertebral centra, which remain distinct from the surrounding synarcual (Figs. 6, 7; Claeson 2010, 2011). The ventral part of the Cowralepis synarcual is distinct from the dorsal (neural arch-derived) part even in early ontogenetic stages (e.g. Fig. 4a, b), where it comprises a smooth, thin flap of perichondral bone underlying the more distinct dorsal neural arches, lacking the rounded growth rings of the neural arch bases. However, rounded growth rings also characterize the morphology of the ossified haemal arch bases (Ritchie 2005), so if these were contributing to the ventral portion of the synarcual, they must have fused completely as cartilaginous units, prior to ossification. Clearer evidence for the contribution of haemal arches to the synarcual, and more specifically to the rounded, spool-like ventral synarcual, is provided by the ptyctodont placoderm Materpiscis attenboroughi, described below. In early ontogenetic stages of Cowralepis (Fig. 4a, b), the neural arch bases are rounded and maintain an identity that can also be identified in larger, older specimens (Fig. 4c, d). The four neural arch bases seen in the smallest specimen (Fig. 4a, b) can be compared to the first four neural arches in the larger specimen (Fig. 4d 1, between the black arrows). In both, these are individual arches that show growth rings of perichondral bone. As the synarcual develops in the larger specimen, these arches merge or are fused, with further layers of cartilage and perichondral bone deposited along the edge of the fused cartilage (Fig. 4c, d 2, arrowheads). This fusion, which does not characterize the more posterior arches of the synarcual keel, may be related to the function of the synarcual, with increased fusion anteriorly increasing rigidity and strength closer to the articulation with the braincase. Although the Cowralepis neural arch keel may have been affected by postmortem distortion, it is a vertically oriented structure compared to the ventral, rounded synarcual. Of interest is the morphology of the dorsal surface of the occipital. Most of the Cowralepis occipitals are visible in ventral view, and in the earliest ontogenetic stage preserved, have the beginnings of the spool-like morphology, while the synarcual is rectangular in shape (Fig. 4a). One specimen preserved the dorsal surface of the occipital, with opposing, but broken ridges running anteroposteriorly (Johanson et al. 2010: fig. 2H, H ). The presence of these vertical ridges was confusing, given the flattened headshield that was thought to characterize Cowralepis (Ritchie 2005: fig. 20A). However, these can now be reinterpreted in light of the morphology of the synarcual, as representing a vertical keel on the occipital, derived from articulating neural arch bases. This corresponds to observations reviewed above, of growth rings (representing the dorsal occipital surface) when the occipital was preserved in ventral view. These were originally interpreted as vertebral elements added to the rear of the occipital region (Johanson et al. 2010), to which can be added the presence of a vertical keel on the occipital, formed by the neural arches/ spines, and the maintenance of the identity of these arches (i.e. growth circles not lost, as in the synarcual keel). Comparable occipital and synarcual regions were already noted by Johanson et al. (2010), who suggested that similarity of these structures in Cowralepis may have been due to Hox gene misexpression. Placoderm synarcuals: Ptyctodontida Campbellodus decipiens (WAM ) Although broken dorsally and ventrally, the synarcual in Campbellodus decipiens (Fig. 2c, d) is complete anteriorly and posteriorly, comprising three fused neural elements. Incomplete fusion creates open spaces between the neural spines for the spino-occipital nerves. However, the spaces between these elements are approximately equal in size, indicating that fusion was regular through the synarcual. The most anterior neural spine is the widest. Dorsally, the neural spines have expanded and fused into a hollow flange

11 or keel (Fig. 2d, smaller white arrow). Four neural elements from the main vertebral column are preserved posterior to the synarcual (Fig. 2c, d) and show the rounded neural arch base (Fig. 2c, smaller white arrow) that also characterizes Cowralepis (Arthrodira). There is no indication that these posterior elements are being fused and incorporated into the synarcual. A slight protrusion on the neural elements directly behind the synarcual may represent the zygapophysis (zyg, Fig. 2d), but there is no evidence of zygapophyses on the more posterior vertebral elements (Fig. 2c), or fusion medially between the left and right neurapophysis or spines. In a second specimen of Campbellodus (WAM ), the synarcual shows better preservation. It comprises three to four fused neural arches, with incomplete fusion between three of the neural spines resulting in two open spaces between them for the spino-occipital nerves, allowing the original number of neural spines to be determined. The most anterior neural spine is the widest, although the posterior one is broken and the synarcual is also broken ventrally. Dorsally, the neural spines have expanded and fused into a hollow flange. Austroptyctodus gardineri (NHMUK PV P.57665) The synarcual is short, comprising four neural elements (Miles and Young 1977; fig. 2E). In the ventral portion of the synarcual, distinct spool-shaped regions can be seen (Fig. 2e, small white arrows), believed to represent ventral haemal elements that have become incorporated into the synarcual (see Materpiscis attenboroughi, below). Anteriorly, the ventral synarcual is modified into the rounded, concave articulation with the occipital. As in Campbellodus and Materpiscis, the dorsal flange of the synarcual is large and expanded. The most posterior arch is less completely fused, although this is seen in the flange (neural spine, n.sp), rather than the spool-shaped part of the synarcual. The incomplete fusion results in an elongate opening between the last neural element, and the rest of the synarcual. Zygapophyses are absent. Materpiscis attenboroughi (WAM ) A synarcual is present, but incomplete, with only the right side being preserved (Fig. 2f, g). In Materpiscis, the synarcual includes five neural arches/spines, which differ morphologically from more posterior elements of the column in that zygapophyses appear to be absent. In lateral view (Fig. 2f), the last three arches are discrete and less completely fused into the synarcual, with a more complete fusion of the anterior two arches. The anterior margin of the synarcual is expanded into a rounded anterior contact with the occipital. A large dorsal flange extends posteriorly, possibly resulting from an expansion and fusion of the neural spines of the first two arches. More posteriorly, neural spines are distinct, although the spine associated with the third arch is noticeably shorter and smaller than the more posterior two arches. The spine of the fourth arch runs along the posterolateral margin of the flange, while the spine of the fifth is positioned along the posterior flange (Fig. 2f). The fifth neural arch is the most distinct arch of the synarcual, but is associated with the spool-like morphology of the base, and is considered part of the synarcual. Internally, the relative contributions of neural and haemal elements to the basal part of the synarcual can be determined. The two most posterior elements added to the synarcual (Figs. 2f, g, 4, 5) were described above as being distinct in lateral view. The neural arch base of the last element added to the synarcual is also discrete and visible in internal or medial view (na, Fig. 2g), possessing a squared base, but more importantly, a rounded depression in this base, comparable to the base of more posterior neural arches in the axial skeleton (e.g. Fig. 2c, smaller white arrow). In the last element, a second square base with a rounded depression can be seen ventral to the neural arch base. This is identified as a haemal arch base (Fig. 2g, haem, compare to Fig. 2c, haem). More anteriorly in the synarcual, the haemal arch base of the fourth element is also present, with the more dorsal neural arch base less clearly visible (4, Fig. 2g). A haemal arch element may also be present associated with the third element of the synarcual (3, Fig. 2g), as indicated by its position in the synarcual relative to the haemal arches associated with the fourth and fifth elements, and the presence of a depression in the haemal base. The bases of the first two arches are clearly fused, although their dorsal margins remain somewhat distinct (Fig. 2g, smaller white arrow). Also internally, the large posterodorsal flange has been broken and is hollow. The posterior part of the flange extends ventromedially to form a canal for the spinal cord. Foramina for the spinal nerves appear to be absent. Materpiscis is similar to other ptyctodonts in that the synarcual rests upon the notochord, rather than enclosing it, as occurs in the most other placoderms and chondrichthyans (Miles and Young 1977). Like Campbellodus and Austroptyctodus, Materpiscis has a large posterodorsally oriented flange, but one that appears to result from the fusion and expansion of only the first two neural spines, while the other remain distinct. In Campbellodus, all neural spines appear to be involved in forming the flange, while in Austroptyctodus, the last, and most distinct neural arch appears to only contribute to the posterior margin of the dorsal flange. As well, the dorsal flange in Materpiscis shows a distinct ventromedial extension internally. All synarcuals lack the zygapophyses that can be present on the neural elements of more posterior vertebrae. Materpiscis

12 also provides important information, along with Cowralepis mclachlani, as to the vertebral elements contributing to the development of the synarcual. The last vertebral element added to the Materpiscis synarcual shows a more dorsal neural arch base (preserved throughout the Cowralepis synarcual) and a ventral haemal arch base. Haemal arch bases can also be recognized more anteriorly, and externally, these preserve the rounded morphology that characterizes the ventral synarcual of most of the placoderms described here. For example, haemal arch bases were not visible in the Cowralepis synarcual, but Materpiscis shows how these can be fully incorporated into the ventral synarcual. This is relevant to the development of the recent chondrichthyan synarcual, which shows differences in the composition of the ventral part of the synarcual between holocephalans and the batoids. Placoderm synarcuals: Rhenanida Nefudina qalibahensis The synarcual of Nefudina (Lelièvre and Carr 2009: fig. 3) is well fused with the original vertebral elements indicated only by the foramina for the spino-occipital nerves. Anteroventrally, the synarcual forms paired articulations with the occipital region that consist of thick and solid lateral expansions (forming a basal element). A reduced notochordal space separates the two lateral articular surfaces. However, the axis for the lateral thickenings is oblique to the anterior posterior axis of the neural canal, extending ventrally and tapering posteriorly. The basal element may represent cranial zygapophyses that are structurally reinforced for the functional craniovertebral joint. The presence of five grooves on the dorsal surface of the basal element suggests the fusion of six vertebrae in its formation. A low neural arch extends anteriorly over the basal element. It increases in depth as the basal element angles ventrally. The arch consists of internal and external perichondral laminae. The external lamina of the arch is fully fused. Posterior to the basal element, only the neural arch is present. The ventral edge of the arch is at the level of, or just above, the spino-occipital foramina so that no estimate can be made for the number of fused vertebrae. However, based on the spacing of foramina over the basal element, the fused neural arches in this posterior region may represent an additional six vertebral elements. Jagorina pandora (Mb.f 510.2) The synarcual of Jagorina is short and was reconstructed as being formed from three vertebral elements (Stensiö 1963: fig. 7). The holotype of Jagorina pandora is preserved as part and counterpart, with the synarcual being more complete in the counterpart (Fig. 5c, smaller white arrow), although only preserved as an impression. In Fig. 5b, the most anterior spino-occipital foramen is broken, while in Fig. 5c, the foramen is complete (as part of an elongate opening). The two spino-occipital foramina present indicate the presence of three neural spines in the dorsal synarcual keel. A concave indentation is present dorsally along the posterior margin of the synarcual, representing one half of the spino-occipital foramen, which would have been matched by a comparable indentation on the next, independent vertebral element. The ventral part of the synarcual, below the dorsal keel, is similar to the ventral synarcuals described above in Cowralepis, as well as Dunkleosteus sp. and the ptyctodonts. This region appears rounded, with a well-developed anterior portion articulating to the occipital region of the braincase (craniovertebral articulation). The concave anteriormost margin of the articulation surface is preserved, followed by a bend or kink in the ventral synarcual, with the dorsal keel of the synarcual increasing in height posterior to this point. Posterior to the synarcual, the neural arches/spines are distinct (Fig. 5b), with elongate spines and rounded, spool-shaped elements ventrally (Fig. 5c, possibly arcocentra sensu Arratia et al. 2001). There is no indication that additional neural arch elements are in the process of being fused to the rear of the synarcual, nor of the elongate haemal arch/ spines that characterize other placoderms (e.g. Fig. 2a 2 ), although the Jagorina specimen is incomplete posteriorly. In Cowralepis, the concentric growth lines of the individual neural arch bases were clearly visible, particularly posteriorly. This suggested that the keel was distinct from the ventral part of the synarcual, and that the latter developed from haemal elements. Direct addition of haemal arches was described in Materpiscis. However, in Jagorina, the neural arch bases are less clearly individualized, placoderm-type haemal arches are not preserved, and vertebral centra or arcocentra may be present just posteriorly in the vertebral column. It is possible that the rounded ventral part of the synarcual developed from expansion of the neural, rather than haemal, arch bases with some incorporation of vertebral centra (as in skates and rays, described below). Chondrichthyan synarcuals Holocephali In Chimera monstrosa, separate dorsal elements are added to the posterior margin of the developing synarcual, with the elongate space between these developing into the dorsal and ventral spino-occipital nerve foramina that are present more anteriorly in the mineralized synarcual (Fig. 6a, black arrows; Garman 1913; Didier 1995; Johanson et al. 2010).

13 Ventral to the notochord, individual rectangular elements are also added, and incorporated into, the posterior margin of the synarcual. Didier (1995) identified these dorsal and ventral elements as homologous to the basidorsals and basiventrals comprising the chondrichthyan vertebral column, which is followed here (and see above). However, in Chimera, the ventral elements are located along the ventral margin of the notochord, while most batoid basidorsals and basiventrals are dorsal and dorsolateral to the notochord, respectively (e.g. Fig. 7b; Miyake 1988; Claeson 2010). As well, in elasmobranchs such as Dasyatis americana Hildebrand and Schroeder, 1928 (Fig. 7a, Myliobatiformes), spinal nerve foramina pierce the basidorsals and basiventrals, but as noted above, only the neural arch elements of Chimera contribute to the spinal nerve foramina, not the more ventral elements. The posterior margin of the Chimera synarcual has a scalloped appearance, as the development of this margin lags behind the addition of the dorsal and ventral elements. The dorsal synarcual has become highly modified in shape and supports the first dorsal fin spine. Elasmobranchii; Batoidea The Batoidea includes the Torpediniformes, Pristiformes, Myliobatiformes, Rhinobatiformes and Rajiformes. The batoid synarcual is complex, with the morphology of the synarcual differing among these groups (Garman 1913; Miyake 1988; Figs. 6b e, 7). Development of the batoid synarcual was recently described by Claeson (2011; also Miyake 1988), who noted that the synarcual was formed from the fusion of vertebral centres, and in early ontogenetic stages of Raja asterias Delaroche, 1809, a synarcual composed of uncalcified cartilage and a surficial layer of tesselated cartilage surrounded the first free vertebrae. The free vertebrae comprise the vertebral centra, which in chondrichthyans are composed of areolar cartilage. The presence of free vertebrae characterizes the batoid axial skeleton (Figs. 6b f, 7). In Raja asterias, the first free vertebrae is positioned near the midregion of the gill arches, but this position shifts posteriorly in older specimens (Claeson 2011; Figs. 6b e, 7). Neural elements are incorporated into the synarcual from anterior to posterior (Claeson 2011). In the myliobatid Dasyatis americana (Fig. 7), the vertebral elements being added to the posterior margin of the synarcual include the neural arch (basidorsal, neural spine) and basiventral, both carrying the spinal foramina; this also is the case in the other batoid groups (Garman 1913; Miyake 1988). An usual feature of the vertebral column (except the Torpediniformes) is that the basiventrals are positioned dorsolaterally relative to the notochord, rather than ventrally (Fig. 7b, Miyake 1988; Claeson 2010). In Dasyatis, as in other batoids (Claeson 2011), the basiventrals grow ventrally and meet medially, creating a seam along the ventral surface of the synarcual (Figs. 6c e, 7b). It appears that the basiventrals fail to meet around the free centra, with the first visible free centra in Figs. 6, 7 located approximately halfway along the synarcual, near the lateral pectoral fin articulations. Claeson (2011) noted that the free vertebrae were anterior in early ontogenetic stages and shifted posteriorly to this position near the pectoral fin. As an alternative explanation to this shift, the first free vertebrae visible in Fig. 6b, c are small and/or poorly mineralized. These vertebrae also lack the distinctive spool-shape of more posterior free vertebrae, indicating their reduced development. Comparable vertebrae may have been present even more anteriorly, but resorbed and incorporated into the synarcual during development, rather than shifted posteriorly. In Dasyatis, the basiventrals develop ventrally towards the free vertebra, and in ventral view, the basiventral appears to be associated with the vertebrae, with spaces anteriorly and posteriorly between basiventrals (Fig. 7b). Just posterior to the developing synarcual, a more overt segmentation occurs, associated with the basiventral and free vertebra (Fig. 7b, black arrow). In ventral view, this segmentation is unequal, occurring on the right side (to the bottom of the Figure), but not yet on the left. Segmentation is lost anteriorly, presumably as the basiventral of the neural arch is incorporated into the synarcual (Fig. 7b, white arrowhead). Incorporation into the synarcual is also indicated by the loss of one of the spaces between the basiventrals ventrally, but the retention, for the moment, of the space on the other side of the free vertebra (Fig. 7b, white asterisk). Segmentation is absent posteriorly, representing the formation of the second synarcual characteristic of Dasyatis and the Family Myliobatidae (Compagno 1977). Discussion The anterior vertebrae form a synarcual in placoderms (Rhenanida, Ptyctodontida, Arthrodira) and some chondrichthyans (Holocephali, Batoidea). The synarcuals in these groups are not homologous, suggesting that development of the synarcual in these groups may differ. Synarcual morphology varies among these groups, and although comparable vertebral elements (neural, haemal, centra) are added, differing numbers of these elements are incorporated into the synarcual, and the way these are added to the developing synarcual differs substantially in the batoids. The synarcuals of placoderms and holocephalans are the simplest, while those of the batoids are more complex. For example, the batoid synarcual possesses protrusions called lateral stays, and posterior to this, lateral

14 pectoral processes with a varying number of pectoral condyles (Garman 1913; González-Isáis and Domínguez 2004; Claeson 2011). The pectoral process is associated with a suprascapular element in Torpediformes, Myliobatiformes and Rajiformes that can be broad in taxa such as Raja (Fig. 6e). Among the Batoidea, the Myliobatiformes are also characterized by a second, more posterior thoracolumbar synarcual (Garman 1913; Compagno 1973; de Carvahlo et al. 2004). As well, the batoid synarcuals surround free vertebrae posteriorly (de Carvahlo et al. 2004; Claeson 2011; Figs. 6, 7). In the holocephans and the placoderms described above, the synarcual is composed of the neural/basidorsal and haemal/basiventral elements, but lateral stays and pectoral processes are absent. There is no involvement of independent or free vertebral centra that characterize the batoids (sensu Claeson 2011; centra generally absent in placoderms, or represented by arcocentra, and represented by notochordal rings in holocephalans; Patterson 1965; Didier 1995). An exception to this may be the rhenanid Jagorina, with putative arcocentra just posterior to the rounded ventral part of the synarcual, suggesting the ventral synarcual may have surrounded the centra as in batoids. In placoderms and holocephalans, fusion of dorsal vertebral elements creates spino-occipital foramina between the elements. In the holocephalans (Chimera and Callorhinchus Lacepède, 1798), both neural and haemal elements contribute to the synarcual, with mineralization occurring shortly thereafter (e.g. Chimera, Fig. 6a, indicated by alizarin red staining). The relative contribution of neural and haemal elements to the placoderm synarcual can also be determined because the synarcual is surrounded by perichondral bone which effectively preserves early stages of ontogenetic development. For example, in the arthrodire Cowralepis mclachlani, the anterior synarcual preserves the first vertebral elements incorporated into the synarcual, including rounded neural arch bases and more ventral haemal elements. The anterior synarcual also preserves the change from these discrete, growing bases to a fusion of these bases and a more continuous bone deposition (Fig. 4c f). In the ptyctodont Materpiscis attenboroughi, vertebral elements added to the posterior synarcual were not completely incorporated and preserve the distinctive neural arch bases dorsally within the synarcual, and the haemal arch bases ventrally (Fig. 2f). These observations indicate that the placoderm synarcual resembles that of the Holocephali in being simpler, generally lacking batoid-like centra and incorporating ventral vertebral elements directly into the synarcual with no ventral expansion (the synarcual of the arthrodire Dunkleosteus sp. may also be an exception in this regard). It is noteworthy that such ontogenetic detail can be obtained from fossil synarcuals. Growth of the synarcual in the Holocephali effectively obscures earlier ontogenetic stages (e.g. Fig. 6a), while in placoderms, perichondral bone deposition around discrete cartilaginous elements may have inhibited further incorporation. In the synarcuals of the arthrodire Dunkleosteus sp. (Fig. 6) and the rhenanid Nefudina (Lelièvre and Carr 2009), individual vertebral elements are more difficult to recognize, such that these may have largely fused or been incorporated into the synarcual prior to perichondral bone deposition. Observations in the placoderm synarcual, and developmental similarity with respect to the Holocephali supports previous observations that in chondrichthyans, the synarcual first forms from separate vertebral centres (Miyake 1988; Claeson 2011), rather than from a failure of proper vertebral segmentation. The latter can affect the vertebrate vertebral column, being a condition recognized in many human skeletal pathologies where the anterior cervical vertebrae are also fused (e.g. Klippel-Feil syndrome; Schaffer et al. 2005). In the batoids, Claeson (2011) noted that in early ontogenetic stages, the synarcual consists of uncalcified cartilage, formed from coalescing vertebral chondrification centres, covered by a layer of prismatic cartilage. This thin mineralized layer allows the synarcual to grow and develop. At later stages, neural arches are added or incorporated into the synarcual, and the synarcual continues to develop posteriorly, with the more dorsally positioned basiventrals growing ventrally, on either side of the free vertebral centra (Fig. 7b; Miyake 1988). By comparison, in placoderms and holocephalans, haemal/basiventrals are more ventral in position and do not expand, but are added to the synarcual directly. Claeson (2011) observed that the first free vertebra was located under the gill arches in a younger specimen of Raja, which then shifted posteriorly in older specimens. This would require the addition of neural arch elements anterior to the first free vertebrae, and rostrocaudal growth of the synarcual relative to the free vertebrae. Alternatively, it was noted above that anterior free vertebrae were smaller and less mineralized (Fig. 6b, c) and could have been resorbed as the synarcual developed, rather than shifted posteriorly. However, Dean et al. (2009) noted that chondrichthyan cartilage is incapable of remodelling itself and growth is only possible via cartilage deposition. This may be true as a general condition of the avascular cartilaginous skeleton (Dean et al. 2009), but more localized resorption, in conjunction with the most anterior free vertebrae in the synarcual, may have been possible. The presence of a synarcual in early vertebrates is important because it represents the first indication of the differentiation of the vertebral column into a distinct anterior region. In other vertebrates, the transition between the anterior cervical and more posterior thoracic region in the zebrafish, as well as birds, mice and Xenopus was