Vertebral architecture in the earliest stem tetrapods

X-ray synchrotron microtomography has revealed the three-dimensional vertebral architecture of Ichthyostega, and other crucial and celebrated early tetrapods; a surprising feature is the relationship between the vertebral elements, with the pleurocentra unexpectedly attached to the succeeding intercentrum, suggesting a ‘reverse’ rhachitomous design. A backbone of interlocking vertebrae is a prerequisite for a tetrapod — a vertebrate living on land. Skeletal details in fossils of the very earliest tetrapods, however, are very hard to make out, as they are often swathed in rocky matrix. This inconvenience has now been overcome by the application of the latest synchrotron microtomography technology to the three best-known early tetrapods — Ichthyostega, Acanthostega and Pederpes. Early tetrapods were presumed to have had 'rhachitomous' vertebrae, with a neural arch and spine above, a single intercentrum front and ventral, and paired pleurocentra to the rear and ventral. Surprisingly, the new scans reveal a 'reverse' rhachitomous design, effectively rewriting the 'textbook' view of vertebral column evolution in the first limbed vertebrates. The construction of the vertebral column has been used as a key anatomical character in defining and diagnosing early tetrapod groups1. Rhachitomous vertebrae2—in which there is a dorsally placed neural arch and spine, an anteroventrally placed intercentrum and paired, posterodorsally placed pleurocentra—have long been considered the ancestral morphology for tetrapods1,3,4,5,6. Nonetheless, very little is known about vertebral anatomy in the earliest stem tetrapods, because most specimens remain trapped in surrounding matrix, obscuring important anatomical features7,8,9. Here we describe the three-dimensional vertebral architecture of the Late Devonian stem tetrapod Ichthyostega using propagation phase-contrast X-ray synchrotron microtomography. Our scans reveal a diverse array of new morphological, and associated developmental and functional, characteristics, including a possible posterior-to-anterior vertebral ossification sequence and the first evolutionary appearance of ossified sternal elements. One of the most intriguing features relates to the positional relationships between the vertebral elements, with the pleurocentra being unexpectedly sutured or fused to the intercentra that directly succeed them, indicating a ‘reverse’ rhachitomous design10. Comparison of Ichthyostega with two other stem tetrapods, Acanthostega7 and Pederpes8, shows that reverse rhachitomous vertebrae may be the ancestral condition for limbed vertebrates. This study fundamentally revises our current understanding11 of vertebral column evolution in the earliest tetrapods and raises questions about the presumed vertebral architecture of tetrapodomorph fish12,13 and later, more crownward, tetrapods.

The construction of the vertebral column has been used as a key anatomical character in defining and diagnosing early tetrapod groups 1 . Rhachitomous vertebrae 2 -in which there is a dorsally placed neural arch and spine, an anteroventrally placed intercentrum and paired, posterodorsally placed pleurocentra-have long been considered the ancestral morphology for tetrapods 1,3-6 . Nonetheless, very little is known about vertebral anatomy in the earliest stem tetrapods, because most specimens remain trapped in surrounding matrix, obscuring important anatomical features [7][8][9] .
Here we describe the three-dimensional vertebral architecture of the Late Devonian stem tetrapod Ichthyostega using propagation phase-contrast X-ray synchrotron microtomography. Our scans reveal a diverse array of new morphological, and associated developmental and functional, characteristics, including a possible posterior-to-anterior vertebral ossification sequence and the first evolutionary appearance of ossified sternal elements. One of the most intriguing features relates to the positional relationships between the vertebral elements, with the pleurocentra being unexpectedly sutured or fused to the intercentra that directly succeed them, indicating a 'reverse' rhachitomous design 10 . Comparison of Ichthyostega with two other stem tetrapods, Acanthostega 7 and Pederpes 8 , shows that reverse rhachitomous vertebrae may be the ancestral condition for limbed vertebrates. This study fundamentally revises our current understanding 11 of vertebral column evolution in the earliest tetrapods and raises questions about the presumed vertebral architecture of tetrapodomorph fish 12,13 and later, more crownward, tetrapods. Jarvik 3,14,15 reassembled the vertebral column of the Devonian stem tetrapod Ichthyostega from 'imperfectly' preserved material. Each vertebral unit was thought to consist of a relatively straight neural spine; paired, round pleurocentra situated underneath the neural arch in front and spanning the intervertebral joint; and an anteroventrally placed crescent-shaped intercentrum (Fig. 1a, b). The morphology and positional arrangement of the vertebral elements in Ichthyostega was primarily fashioned after Jarvik's restoration of the vertebrae in Eusthenopteron 3 -a tetrapodomorph fish considered to possess tetrapod-like rhachitomous vertebrae 1 . Extensive re-examination of the Ichthyostega fossil material revealed pronounced regionalization of the neural arch/spine morphology, and resulted in a reconstruction of the vertebral column differing radically from Jarvik's interpretation 9 . However, little information could be gleaned about the remaining vertebral elements as they were concealed by other anatomical features (for example, ribs) or remained embedded in matrix.
Recently, the three-dimensional skeletal anatomy of Ichthyostega was recreated from microcomputed tomography (mCT) scans 16 . The mCT data exposed all elements within the vertebral column and indicated the presence of new anatomical features, as well as an unexpected vertebral configuration. In light of this, two key specimens were subjected to phase-contrast X-ray synchrotron microtomography (PPC-SRmCT) using a specially developed scanning protocol 17 (see Methods) to obtain the highest possible resolution and sensitivity to low-contrast structures. The specimens consist of an almost complete thoracic region with associated ribs (MGUH VP 6115) and a 'lumbar' region preserving most elements except for the more posterior neural arches/spines (MGUH VP 29017a). For comparison, a section of the vertebral column from the Devonian tetrapod Acanthostega gunnari (MGUH f.n. 1227) was also scanned using PPC-SRmCT, and the skeletal remains of the more crownward Tournaisian tetrapod Pederpes finneyae (GLAHMS 100815) were imaged using mCT. For visualization of the segmented scan data see Supplementary Videos 1-4.
As has been previously noted 9 , most of the thoracic neural spines of Ichthyostega are caudally inclined (Figs 1c and 2); however, our PPC-SRmCT data have revealed that the anterior thoracic neural spines, flanked by the cleithra, are vertical in orientation (Fig. 2c, d). In addition, we have discovered further regionalization in transverse process morphology (Figs 1c, d, 2 and 3). The anterior thoracic and lumbar vertebral arches have ventrally projecting transverse processes, whereas those in the posterior thoracic region are horizontally flat, and square-shaped, with small triangular-shaped anterior extensions (see further below). Rib heads in the thoracic region are not bicipital (or incipiently bicipital; Fig. 1e, f, g), but rather anteroposteriorly elongated-articulating with the entire length of the transverse processes (Figs 1c and 2c, e). Comparing vertebral arch morphology with that of Acanthostega 7 (Fig. 1e, f) and Pederpes 8 (Fig. 1g), we suggest that vertically oriented neural spines and ventrally projecting transverse processes are the general condition for the tetrapod stem group. This indicates that the posterior thorax, rather than the lumbar region 9 , is uniquely constructed in Ichthyostega. Considering that vertebral processes tend to align themselves perpendicular to the predominant forces acting on them 18,19 , the morphology of the posterior thoracic vertebrae in Ichthyostega signifies a departure from the primary role of the epaxial musculature.
Except for a possible pair of small pleurocentra sitting between the third and fourth neural arches ( Fig. 2d-f), there is a complete lack of ossified centrum elements in the anterior part of the thoracic column (Figs 2 and 3). Fully developed intercentrum/pleurocentra units first appear around the fifth preserved neural arch. The articulated condition of the specimen (Supplementary Fig. 1 and Supplementary Video 1), including the skull preserved in anatomically correct position 9,15 , suggests that this pattern is not a preservational artefact and may indicate an unusual posterior-to-anterior ossification sequence. Although vertebral development most commonly occurs in an anteriorto-posterior direction 20,21 (possibly linked to somitogenesis 22 and Hox gene expression patterns 23 ), some fish have been shown to exhibit the reverse pattern (for example, Eusthenopteron and various actinopterygians) 21 . As these fish tend to be phylogenetically disparate, it has been suggested that a posterior-to-anterior ossification sequence may be related to functional constraints, such as the locomotor behaviours of hatchlings and juveniles 21 . Such a hypothesis is consistent with Ichthyostega being primarily aquatic in habit as a juvenile 24 and using its paddle-like hindlimbs and tail for swimming 16 , although alternative explanations may exist.
The centra of Ichthyostega display a remarkable array of morphological features. In the thoracic region, the intercentra are long anteroposteriorly and widely spaced (Figs 1c and 2d-f); however, they become noticeably shorter and more closely packed in the lumbar region (Figs 1d and 3a-c). As the dimensions of vertebral bodies have an effect on passive flexibility at intervertebral joints 25 , shortening of the lumbar intercentra (in combination with changes in neural arch morphology) indicate a functional shift in this region. In particular, short, closely packed centra indicate increased intervertebral joint stiffness in the lumbar region due to a minimal degree of angular deflection before adjacent vertebrae obstruct each other 18 . Furthermore, all the left and right intercentra halves appear fused along the length of the vertebral column, except for the first two which are distinctly paired (Figs 2d-f and 3 and Supplementary Fig. 1). The delayed fusion of the anterior intercentra lends further support to a posterior-to-anterior ossification sequence, as intercentra are thought to ossify by two condensations lateral to the notochord before fusing along the midline 26 .
Directly below the first five preserved thoracic intercentra is a series of mid-ventral circular discs. These elements are evenly spaced, one for vertebral elements only (f). The ribcage has been mediolaterally compressed, entombing the vertebral column. On the basis of intervertebral spacing, the first preserved centrum has probably been displaced posteriorly and should correspond to the preceding neural arch. In turn, the second preserved centrum and associated stenebral element are disarticulated and sitting inside the succeeding intercentrum. The boundary between fused pleurocentra/intercentra is approximated. Anterior towards left. Scale bar, 10 mm.

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each centrum unit (in blue, Fig. 2b, d-f). The PPC-SRmCT data show that the micro-anatomy of these elements is very similar to the other endochondral bones in the specimen (Supplementary Fig. 2). The consistent shape, segmental arrangement, and probable endochondral origin indicate that these mid-ventral elements may represent ossifications (sternebrae) within a cartilaginous sternum, which has been displaced into the dorsal part of the body cavity along with an ulna after death (Supplementary Fig. 3). Whether or not the ribs articulated with the sternebrae cannot be determined, as the distal ends of the anterior ribs are missing. However, the edges of the sternebrae show no noticeable pitting for reception of the rib ends. In living tetrapods, the sternum (or sternebrae) are thought to strengthen the body wall and aid in locomotion and, in some amniotes, ventilation 27 .
Conceivably, the presence of sternebrae in the anterior thorax of Ichthyostega may have helped to reinforce the ribcage during synchronous forelimb 'crutching' on land 16 , allowing the animal to balance its body weight on its chest during the swing phase of forelimb movement.
The pleurocentra of Ichthyostega are positioned posteroventrally to their respective neural arch throughout the column (Figs 1c, d, 2d and 3); however, in the thoracic region they are clasped laterally by triangular extensions of the following transverse processes (Figs 1c and  2d). This anatomical arrangement essentially forms an intervertebral locking mechanism in the thorax, restricting the range of joint mobility. A more evolutionarily important feature of the vertebral column is that the pleurocentra are either sutured (anteriorly) or fused (posteriorly) to the anterodorsal aspect of the intercentrum that directly succeed them (Figs 1c, d, 2d and 3 and Supplementary Videos 5 and 6). This indicates a 'reverse' rhachitomous condition 10 , with one vertebra encompassing a dorsally placed neural arch above and a posteroventrally placed pleurocentra-intercentrum unit (Fig. 1c, d). On the basis of this discovery, our re-evaluation of the articulated vertebrae in Acanthostega 7 (Fig. 1e and Supplementary Video 3) shows that this stem tetrapod also possesses reverse rhachitomous vertebrae, and that Pederpes 8 can be interpreted in this way ( Fig. 1g and Supplementary Fig. 4). However, rather than being fused to the dorsal aspect as in Ichthyostega, the pleurocentra of these taxa articulate at a 45u angle with the anterior border of the intercentrum (Supplementary Fig. 5).
A similar anterior pleurocentra association (or 'anteropleural') has, in the past, also been proposed for an assortment of 'osteolepids' and even some temnospondyls 10 , although the traditional arrangement is usually attributed to the latter. The potentially widespread distribution of the reverse rhachitomous condition may indicate either a misinterpretation of the fossil record or that the multipartite vertebral structure was developmentally plastic in stem tetrapods. In living tetrapods, vertebrae form by a lineage-restricted process called resegmentation, in which the posterior and anterior sclerotomal halves of consecutive somites fuse 28 . Conversely, the vertebrae of fish develop through notochordal signalling 29 and leaky resegmentation 30 , such that cells cross anterior-posterior domains. This more variable vertebral patterning mechanism may explain why both normal and reverse rhachitomous vertebrae appear in the fossil record, and in some cases, why both conditions have been recognized in closely related species 10 . Perhaps the plasticity of tetrapod vertebral morphology stabilized over the course of evolution, with modern forms acquiring a conserved, unipartite vertebral structure.
The vertebral architecture of Ichthyostega described here is markedly different from Jarvik's textbook reconstruction 3,14,15 (Fig. 1a, b). It displays even more regionalization than previously proposed 9 , an apparent posterior-to-anterior ossification sequence, and various functional innovations-including the development of sternebrae. Furthermore, the individual vertebral units reveal an unambiguous reverse rhachitomous design, with pleurocentra fused to each succeeding intercentrum (Figs 1c, d, 2 and 3). This centrum arrangement is also present in Acanthostega and possibly Pederpes (Fig. 1f, g), suggesting that the reverse rhachitomous arrangement could be the ancestral tetrapod condition. Overall, the findings described here demonstrate that the traditional rhachitomous model 1,2 has been misguidedly projected onto the earliest limbed vertebrates 3,7,8 , producing inaccurate reconstructions that have been replicated in the literature for more than half a century [4][5][6]11,14,15,27 . This study raises questions about the interpretation of vertebral anatomy in tetrapodomorph fish, such as Eusthenopteron 12 and Panderichthys 13 , and more crownward tetrapods, and has broad ramifications for our future understanding of early tetrapod skeletal evolution.

METHODS SUMMARY
PPC-SRmCT scans were performed at the European Synchrotron Radiation Facility (ID19, France). They were scanned using a white beam, filtered with 3-9 mm of copper and 60 mm of aluminium 17 , and the W150 wiggler (gap: 38-80 mm), thereby leading to energy with a narrow bandwidth (83.7-133.2 6 15 keV full-width at halfmaximum). Samples were imaged through a single crystal LuAG:Ce scintillator of 750 mm thick, coupled with a CCD FReLoN camera placed at 4-9 m from the sample (voxel size: 29.4 mm) (see Methods for details). The scan data were scaled Most neural spines, right transverse processes and pleurocentra are not preserved; centrum elements are tipped anteriorly; and rib ends are displaced dorsally. The first preserved transverse processes seem to be transitional between the horizontally flat transverse processes of the thoracic region and the smaller and more vertically oriented ones in the remainder of the lumbar region. The boundary between the fused pleurocentra/intercentra is approximated. Anterior towards left. Scale bar, 10 mm.

RESEARCH LETTER
isotropically to 58.8 mm for segmentation. The mCT scans were performed on an X-Tek HMX 225KeV system, at a voxel size of 90 mm using 175 kV, 127 mA and a 0.5 mm copper filter. All scan data were segmented in Mimics software (Materialise) and rendered in Autodesk 3D Studio Max. Ichthyostega and Acanthostega specimens are housed in the Geological Museum, Copenhagen (MGUH), and Pederpes in the Hunterian Museum, Glasgow (GLAHMS).
Full Methods and any associated references are available in the online version of the paper.

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METHODS PPC-SRmCT scan protocol. To maximize contrast of the structures, a 4-9-m propagation distance was used to make possible the use of propagation phasecontrast despite the large size (for example, 15 3 10 3 5 cm) of the samples and the high energy. As the samples were very dense owing to metallic oxide infilling, the specimens were scanned using the high energy white beam of the ID19 W150 wiggler at a gap of 38-80 mm filtered, with 3-9 mm of copper and 60 mm of aluminium, resulting in a beam with a relatively narrow bandwidth (83.7-133.2 6 15 keV full-width at half-maximum). To homogenize absorption of the heterogeneous samples and to improve contrast, the fossil specimens were put in a 10-cm cylinder filled with aluminium/glass balls of 2 mm, and a beam profiler was used to compensate for absorption. The addition of the balls and the beam profiler provide the 60 mm of aluminium presented above for the beam filtering. This scanning protocol, derived from one originally developed for a high-energy monochromatic beam 17 , allows working with relatively low energy (when compared with the energy necessary to reach the usual 10% of transmission through the sample) while maximizing the sensitivity and the contrast (both absorption and phase) in the recorded data. Moreover, as the quantity of material crossed by the beam is nearly the same everywhere, this approach removes most of the beam hardening effect, except on the very dense metallic oxide particles. A half-acquisition scanning geometry (off-axis centre of rotation) with 5,000 projections of 0.1 s over 360u was used to cover a lateral field of view of 100 mm with an isotropic voxel size of 29.4 mm. In addition, a single crystal LuAG:Ce scintillator of 750 mm thick was used, coupled through a lenses system with a CCD FReLoN (Fast Readout Low Noise) camera. To cover the full vertical field of view, series of scans were performed with vertical displacement between each scan in such a way that every position of the sample was scanned twice. This approach is used to reduce ring artefacts, as well as to minimize the artefacts due to differences in power and spectrum in the beam along its vertical profile. Following this, a single distance phase-retrieval algorithm 31 was used to maximize the contrast of the structures in the data. Ring artefacts were corrected both on the radiographs before reconstruction by using filtered averages of all the projections, and after the reconstruction using a ring filter 32 . Data from all the scans were then combined into a single volume with moving average protocol to go from one scan to the next one. A three-dimensional unsharp mask was applied to the reconstructed data to retrieve the smallest details 33 . The scan data were scaled isotropically to 58.8 mm using binning to reduce file size for segmentation while preserving a high level of resolution and the sensitivity of the original data set with an enhanced signal to noise ratio. mCT scan protocol. The specimen used in this study was scanned on an X-Tek HMX 225 keV system with a flat panel detector. The voxel size was set to 90 mm using 175 kV and 127 mA. Owing to the dense material, a 0.5 mm copper filter was used to filter out the low-energy X-rays and reduce beam-hardening effects. The scan data were corrected for ring artefacts and beam hardening using CT-Pro reconstruction software and the image stack was created using VGStudioMax. Segmentation and rendering. All scan data were segmented in Mimics software (Materialize) and rendered in Autodesk 3D Studio Max. Fossil elements were linked and assigned object colours for visualization and movie generation. Specimen information. Ichthyostega and Acanthostega specimens are housed in the Geological Museum, Copenhagen (MGUH), and Pederpes in the Hunterian Museum, Glasgow (GLAHMS).