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. 2022 Mar 24;240(6):1005–1019. doi: 10.1111/joa.13659

Osteohistology of the hyperelongate hemispinous processes of Amargasaurus cazaui (Dinosauria: Sauropoda): Implications for soft tissue reconstruction and functional significance

Ignacio A Cerda 1,2,3,✉, Fernando E Novas 1,4, José Luis Carballido 1,5, Leonardo Salgado 1,2
PMCID: PMC9119615  PMID: 35332552

Abstract

Dicraeosaurid sauropods are iconically characterized by the presence of elongate hemispinous processes in presacral vertebrae. These hemispinous processes can show an extreme degree of elongation, such as in the Argentinean forms Amargasaurus cazaui, Pilmatueia faundezi and Bajadasaurus pronuspinax. These hyperelongated hemispinous processes have been variably interpreted as a support structure for a padded crest/sail as a display, a bison‐like hump or as the internal osseous cores of cervical horns. With the purpose to test these hypotheses, here we analyze, for the first time, the external morphology, internal microanatomy and bone microstructure of the hemispinous processes from the holotype of Amargasaurus, in addition to a second dicraeosaurid indet. (also from the La Amarga Formatin; Lower Cretaceous, Argentina). Transverse thin‐sections sampled from the proximal, mid and distal portions of both cervical and dorsal hemispinous processes reveal that the cortical bone is formed by highly vascularized fibrolamellar bone interrupted with cyclical growth marks. Obliquely oriented Sharpey's fibres are mostly located in the medial and lateral portions of the cortex. Secondary remodelling is evidenced by the presence of abundant secondary osteons irregularly distributed within the cortex. Both anatomical and histological evidence does not support the presence of a keratinized sheath (i.e. horn) covering the hyperelongated hemispinous processes of Amargasaurus, and either, using a parsimonious criterium, in other dicraeosaurids with similar vertebral morphology. The spatial distribution and relative orientation of the Sharpey's fibres suggest the presence of an important system of interspinous ligaments that possibly connect successive hemispinous processes in Amargasaurus. These ligaments were distributed along the entirety of the hemispinous processes. The differential distribution of secondary osteons indicates that the cervical hemispinous processes of Amargasaurus were subjected to mechanical forces that generated higher compression strain on the anterior side of the elements. Current data support the hypothesis for the presence of a ‘cervical sail’ in Amargasaurus and other dicraeosaurids.

Keywords: bone histology, keratinous horn sheath; dicraeosauridae, neural arch morphology; interspinous ligaments; sauropoda

Life restoration of Amargasaurus cazaui. Current data support the hypothesis for the presence of a ‘cervical sail’ in Amargasaurus and other dicraeosaurids. Illustration made by Gabriel Lio.

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1. INTRODUCTION

Besides their colossal body size, Sauropoda also exhibited a characteristic bauplan, which was maintained for more than one hundred million years of evolution (Wilson & Curry Rogers, 2005). Despite this apparent simplicity in their body plan (i.e. quadrupedal stance with small heads, long necks and long tails), sauropods showed a highly morphological variation in the skeleton, particularly in the vertebrae, which exhibit a strong degree of anatomical disparity amongst the different lineages (Salgado & Powell, 2010; Wilson, 1999). Perhaps one of the most extreme examples of vertebral disparity is found in Dicraeosauridae, a clade of diplodocoid sauropods, whose fossil record comes from Argentina, Tanzania, United States and China (Bonaparte, 1986; Bonaparte, 1996; Coria et al., 2019; Gallina, 2016; Gallina et al., 2019; Harris & Dodson, 2004; Janensch, 1929; Rauhut et al., 2005; Salgado & Bonaparte, 1991; Upchurch et al., 2004; Windholz et al., 2021; Xu et al., 2018). Presacral vertebrae of dicraeosaurid sauropods are characterized by the presence of elongate bifid neural spines, which are therefore composed by two hemispinous processes (following Harris [2006] and referring to one half of a bifurcated spine. These hemispinous processes show an extreme degree of dorsoventral elongation; some of them representing four times the length of the respective vertebral centrum (Figure 1a), as is the case for Amargasaurus cazaui, Pilmatueia faundezi and Bajadasaurus pronuspinax (Coria et al., 2019; Gallina et al., 2019; Salgado & Bonaparte, 1991).

FIGURE 1.

FIGURE 1

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(a) Skeletal silhouette of Amargasaurus cazaui (preserved elements in white colour). (b) C3 in left lateral view. (c) C5 in left lateral view. (d) C10 in left lateral view. (e) D3 in right lateral view. (f) D4 in right lateral view. (g) MOZ‐Pv 626–1, second? Dorsal vertebra in anterior view. Asterisks indicate the positions from the sections were obtained. Scale bars: 10 cm. Figure 1.F. courtesy of G. Windholz

The functional significance of the hyperelongated hemispinous processes in the neck of dicraeosaurids has been discussed by different authors, which interpreted these processes either as a support structure for a padded crest/sail for intra‐interspecific display, a bison‐like hump made by muscles and other soft tissues or as internal osseous cores of keratinous sheaths (Bailey, 1997; Brusatte, 2012; Gallina et al., 2019; Schwarz et al., 2007). In this regard, based on the gross morphology, Bailey (1997) considered that Amargasaurus exhibited a short sail in the cervical region, followed behind by a thick hump of fat, reminiscent of that of a Bison. Whereas the cervical sail could be used for intra−/interspecific display, the bison‐like hump was possibly adaptive for energy storage, maintenance of gigantothermy, and heat‐shielding in unshaded habitats, amongst others (Bailey, 1997). Alternatively, Schwarz et al. (2007) proposed that the hyperelongated hemispinous processes of Amargasaurus were partially (the distal two thirds) covered by a keratinized sheath (i.e. a horn). This interpretation was founded on the purported presence of external striations in the dorsal two‐thirds of the hemispinous processes of the cervical vertebrae which were found similar to that found at the surface of the bony cores of bovid horns (Schwarz et al., 2007). A detailed view of the alleged striations was unfortunately not figured. The presence of a keratinized sheath has also been reconstructed in the recently published description of Bajadasaurus pronuspinax by Gallina et al. (2019) which exhibits distinct anteriorly pointed and curved hemispinous processes. Although the latter authors did not find such longitudinally oriented striations mentioned by Schwarz et al. (2007) for Amargasaurus, they considered that the external horn sheaths could also be ascribed to Bajadasurus due to the extreme elongation of the hemispinous processes. Accepting that a keratinized sheath was covering the hemispinous processes, Gallina et al. (2019) interpreted such coverings as an adaptive device to avoid fractures of the fragile hemispinous processes when used as passive defensive structures in these sauropod dinosaurs.

It is worth noting that the aforementioned hypotheses regarding the soft tissue association and functional significance of the hyperelongated hemispinous processes, at least for the Argentinean dicraeosaurids, has not been tested using other information than that of the external surface texture, as described by Schwarz et al. (2007). One possible way to further test this could be the histology. Paleohistology has been demonstrated to be highly useful in discerning diverse aspects of skeletal structures, including: the identification of unusual bony elements (e.g. Bellardini & Cerda, 2017), histogenesis (e.g. Cerda et al., 2015a, 2015b; Scheyer & Sander, 2004), function (e.g. de Buffrénil et al., 1986; Main et al., 2005) and relationships with soft tissues (e.g. Hieronymus et al., 2009; Lambertz et al., 2018; Pereyra et al., 2019; Woodruff et al., 2016). Strictly taking into account hyperelongated structures of the vertebrate neural arches, paleohistological studies have been conducted on Edaphosauridae and Sphenacodontidae (e.g. Huttenlocker et al., 2010, 2011; Ricqlès, 1974). These studies have provided insights about the relationships of hyperelongated neural spines with soft tissues (i.e. epaxial musculature), and also provide evidence to test some hypotheses about the functional significance of these elements (Huttenlocker et al., 2010, 2011).

In the present study, we analyze the bone histology of the hyperelongate hemispinous processes of Amargasaurus cazaui and an indeterminate dicreosaurid, both from the lower Cretaceous of Argentina. Our primary goal was to obtain evidence to test previous hypotheses regarding the tissues that covered these structures in dicraeosaurid sauropods. In a broader sense, this paleohistological approach may provide novel data about the possible functional significance of the hyperelongate hemispinous processes.

Institutional abbreviations. MACN, Museo Argentino de Ciencias Naturales ‘Bernardino Rivadavia’, Buenos Aires, Buenos Aires Province, Argentina; MOZ, Museo Olsacher, Zapala, Neuquén Province, Argentina; MPCA‐Pv, Paleovertebrate collection of the Museo Provincial ‘Carlos Ameghino’, Cipolletti, Río Negro, Argentina. PVL: Instituto Miguel Lillo, San Miguel de Tucumán, Tucumán, Argentina.

2. MATERIALS AND METHODS

Hemispinous processes from presacral vertebrae of two dicraeosaurids, the holotype of Amargasaurus cazaui (MACN PV N1), plus a fragmentary dicreaosaurid specimen (MOZ‐Pv 6126‐1) were sampled for histological analysis. Both individuals come from Puesto Antigual Member outcrops, corresponding to the lowermost part of the La Amarga Formation, Barremian‐lower Aptian (Lower Cretaceous), which are exposed close to La Amarga stream, north of China Muerta Hill, Neuquén Province, Argentina (Apesteguía, 2007; Leanza et al., 2004; Salgado & Bonaparte, 1991; Windholz et al., 2021). The holotype of A. cazaui includes several cranial and postcranial elements originally described by Salgado and Bonaparte (1991). The left hemispinous process of three cervical (C3, C5 and C10) and two dorsal (D3 and D4) vertebrae from this individual were sampled (Figure 1b‐f). The sections were obtained in the proximal (C3, C5, C10, D3), mid (C3, C5, C10, D3) and distal (C5, C10, D3, D4) portions of the hemispinous processes. In the case of MOZ‐Pv 6126‐1, the same corresponds with a fragmentary element (i.e. anterior dorsal vertebra) recently assigned to Dicraeosauridae indet. by Windholz et al. (2021). A single section from this specimen was obtained from the mid‐portion of the spine (Figure 1g). Prior to sampling, detailed pictures of the vertebrae were obtained. In the case of MACN PV N15, complete sections of around 20 mm thickness were obtained using a Dremel® rotary tool equipped with a thin diamond‐edged saw. To avoid loss of anatomical information, mould and casts of the extracted samples were made and replaced in the original hemispinous processes (Cerda et al., 2020). For comparative purpose, histological sections from extant mammals were also sampled and studied. Regarding extant samples, cross‐sections were obtained from a horn core of Capra sp. (PVL A06) and an ungueal phalanx of Equus ferus (MPCA un‐numbered specimen). In both cases, these elements were entirely or partially covered in life by a keratinized sheath.

Histological thin sections were performed in the Museo Paleontológico Egidio Feruglio (Trelew, Argentina) and the Paleohistological Laboratory of the Museo Provincial Carlos Ameghino (Cipolletti, Río Negro Province, Argentina) following standard procedures (e.g. Cerda et al., 2020; Chinsamy & Raath, 1992). The thin sections were studied using petrographic polarizing microscopes (BestScope and Leica DM 750P). Nomenclature and definitions of structures used in this study are derived from Francillon‐Vieillot et al. (1990) and Currey (2002). In the case of the term ‘intrinsic fibres’, we follow the definition of Ricqlès et al. (1991), for which this term refers to the collagenous fibres that are deposited by osteoblasts during the ossification process and form the matrix of the bone tissue. In identifying lines of arrested growth (LAGs) and annuli, we opted to use the term ‘cyclical growth marks’ (CGM) (sensu Woodward et al., 2013). Nomenclature of neural laminae and fossae follows Wilson (1999) and Wilson et al. (2011) respectively.

Given the similarity regarding anatomical and histological features that were observed between different parts of a single hemispinous process and between different hemispinous processes, instead of describing each section/spine separately, we provide a general description of the main anatomical and histological features of the element. Our descriptions are focused on: spine external texture; spine gross morphology in cross‐section; bone microanatomy (i.e. presence and relative amount of compact and cancellous bone); degree of secondary remodelling, organization of the intrinsic fibres of the primary bone tissue, degree of vascularization, orientation of vascular canals, presence, density and orientation of Sharpey's fibres; presence, number and distribution of cyclical growth marks and evidence of modelling. For practical purposes, since the samples obtained from MACN PV N15 correspond with five well‐identified vertebrae (three cervicals and two dorsals), we refer to them based only on their position within the vertebral column and along the spine (e.g. distal portion of C3). In the case of the only section obtained from MOZ‐Pv 6126‐1, we refer to it using the collection number.

3. DESCRIPTION

3.1. External texture

The external surface of the hemispinous processes has been partially eroded and/or covered with plaster, which obscures a proper characterization of the external texture of the bone surface in several areas. The best‐preserved areas reveal that the hemispinous processes usually exhibit a rather smooth external surface (Figure 2a‐d). However, variations of this pattern are present along several portions of the hemispinous processes. For example, some portions of the surface exhibit an irregular texture (Figure 2b). Also, a series of thin and shallow, longitudinally oriented ridges and furrows (Figure 2e‐h), are observed along some vertebral elements (e.g. C9 and C11). However, in many cases, these ridges and furrows are attributable to postmortem breakage of the subperiosteal surface of the bone. A series of wide ridges with low relief is observed in mid and distal portions of D3 and D5, and unlike the previous features, clearly do not correspond to postmortem breakage of the subperiosteal surface (Figure 2i,j). Additionally, variations in the surface texture are observed in the distal portion of D8 (Figure 2k,l), whereas the anterior and the posteromedial areas of the spine are smooth, the posterolateral surface exhibits a naturally roughened texture (i.e. abrupt disruptions of the subperiosteal bone surface are absent). Finally, a pattern of diminutive furrows and ridges are present along the lateral surface of the distal portion of D3. This pattern, which does not seem to correspond, nor is consistent with postmortem alteration, is clear and distinguishable in cross‐section (Figure 3).

FIGURE 2.

FIGURE 2

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External texture of Amargasaurus cazaui (MACN PV N1A) cervical (a‐h) and dorsal (i‐l) hyperelongate hemispinous processes. (a) C4. (b) C5. (c) C6. (d) C12. (e) C3. (f) C9. (g) C10. (h) D11. (i) D3. (j) D4. (k‐l) D8 in posterior (k) and anterior (l) views. In the cervical vertebrae, the best‐preserved surfaces exhibit a smooth or gently rugose texture. The longitudinally oriented ridges and furrows observed E‐H correspond in with postmortem alterations. Shallow but wider ridges and grooves, which do not correspond with alterations, are observed in the mid and distal portions of the dorsals (i, j). Note the distinct change of the external texture in the posterior side of the eighth dorsal. Scale bars: 10 mm

FIGURE 3.

FIGURE 3

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Amargasaurus cazaui (MACN PV N1A), detail of the outer cortex of hemispinous process of the D4 (distal portion). Note the undulate appearance of the subperiosteal cortex. Abbreviations: Ce: Cementing line; its: Intertrabecular space; sm: Sedimentary matrix. Scale bars: 0.2 mm (a), 0.1 mm (b)

3.2. Cross‐section morphology

The hemispinous processes are usually longer than wide (i.e. more anteroposteriorly elongate than laterally wide) in the transverse section (Figure 4a‐f), but their exacting morphology exhibits minor degrees of variation. For example, a roughly triangular cross‐sectional morphology is observed in C5 (mid‐portion) and C10 (proximal and mid‐portion) and the mid‐portion of the D3 (Fig. 4B2, C1, C2, D2). In all these cases, one of the ‘triangle vertices' is always located along the anterior edge of the hemispinous process. The hemispinous processes exhibit a distinct elliptical morphology in the proximal and mid area of C3, as well as in the distal portion of C5 and D3 (Figure 4a1,a2,c3,d3). This elliptical morphology is also observed in the single section sampled from MOZ‐Pv 6126‐1 (Figure 4f). Roughly rhomboidal to circular cross‐sectional shapes are respectively observed in the proximal and distal portions of C5 (Figure 4b1,b3). These more complex cross‐sectional morphologies are observed in the proximal and distal portions of D3 and D4, respectively. In D3, the cross‐section has a distinct ‘hook’ shape, with a deep concavity that posteriorly divides the spine in two distinct processes, one short (lateral) and one very long (medial) (Figure 4d1). The medial process is approximately three times the length of the shorter one. Whereas the concavity corresponds to the location of the postzygapophyseal spinodiapophyseal fossa (posdf), the short and long processes correspond to the spinodiapophyseal (spdl) and spinopostzygapophyseal (spol) laminae, respectively. The spol, whilst less developed, is still distinct in the mid‐portion of the spine, where the lamina is observed as a protuberance along the posterior side. In the case of the distal portion of D4, the spine is lateromedially compressed (more than two times longer than wide), slightly concave in lateral surface and slightly convex in the medial surface (Figure 4e).

FIGURE 4.

FIGURE 4

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Transversal sections of hyperelongate hemispinous processes of Amargasaurus cazaui MACN PV N1A (a‐e) and Dicreosauridae indet. MOZ‐Pv 6126–1 (f). Each image of the complete section is accompanied by a schematic drawing showing the main histological features. For simplification, the position and orientation of Sharpey's fibres are indicated outside the section scheme. Asterisks indicate the position where bone modelling is evident (e.g. presence of reversal lines). (a) Proximal (a.1) and mid (a.2) portions of C3. (b) Proximal (b.1), mid (b.2) and distal (b.3) portions of C5. (c) Proximal (c.1), mid (c.2) and distal (c.3) portions of C10. (d) Proximal (d.1), mid (d.2) and distal (d.3) portions of D3. (e) Distal portion of D4. (f) Mid‐portion of the second? Dorsal. Abbreviations: Posdf: Postzygapophyseal spinodiapophyseal fossa; spdl: Spinodiapophyseal lamina; spol: Spinopostzygapophyseal lamina. Scale bars: 10 mm

3.3. Microanatomy

The hemispinous processes primarily consist of compact bone tissue (Figure 4a‐f). Whereas cancellous bone is absent in the proximal and mid‐portions of C3 (i.e. the sections are entirely compact medullary structures), the same is usually restricted to a small area in the medullary region of most of the other samples. An exception of this pattern is observed in the distal portions of C5 and D4, which exhibits a thin cortex of compact bone surrounding a large area filled with cancellous bone (Figure 4b3,e). Cancellous bone consists of thin trabeculae formed by lamellar bone, which has been secondarily deposited during different episodes of remodelling (Figure 5a).

FIGURE 5.

FIGURE 5

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Bone histology of hyperelongate hemispinous processes of Amargasaurus cazaui MACN PV N1A. In all the images the outer cortex is oriented upward. White arrowheads indicate CGM. Hemi braces signal semicircular patches of secondary osteons. (a) Cancellous bone in the distal portion of D3. (b, c) general view (b) and detail (c) of the fibrolamellar bone tissue in the proximal portion of C10. (d) Outer cortex of the proximal portion of C10. Primary bone tissue is formed by poorly vascularized parallel fibered bone. (e) Proximal portion of D3. Vascular canals are mostly longitudinally oriented. (f, g) general view (f) and detail (g) of mid‐portion of C3 showing longitudinally and obliquely oriented vascular canals. Note the several anastomoses. Oblique anastomoses tend to form a reticular pattern in some areas. (h) Mid‐portion of C5. Note the predominance of radial and longitudinal vascular canals. (i) General view of the cortical bone of C3 (mid‐portion). Note the decreasing of the vascular canals density toward the outer portion of the cortex. a‐d: Cross polarized light with lambda filter; e, g: Normal transmitted light; f, h, i: Plane polarized light. Abbreviations: Its: Intertrabecualr spaces; lb: Lamellar bone; po: Primary osteon; rc: Resorption cavity; so: Secondary osteon; scale bars: 0.5 mm (a, b); 0.3 mm (c‐e, g); 1 mm (f, h, i)

3.4. Primary bone

Although the cortical bone of the sampled elements has been profusely remodeled, several portions of the compacta preserve primary bone, which is mostly formed by fibrolamellar bone tissue (Figure 5b,c). The intrinsic fibres of the primary bone matrix tend to be more spatially organized (i.e. from woven‐fibered to parallel‐fibered bone) toward the outer cortex in the proximal portion of C10 (Figure 5d). Fibrolamellar‐ and parallel‐fibered bone are also observed in the dorsal vertebrae hemispinous processes; however, compared to the cervical samples, parallel‐fibered tissue appears to be proportionally more abundant. This relative abundance of parallel‐fibered bone is more pronounced in the proximal area of D3.

3.5. Vascularization

The primary bone tissue is well vascularized with primary osteons which exhibit variable canal orientations (i.e. longitudinal, radial, circumferential and oblique). Interestingly, the arrangement of the vascular canal orientation varies amongst the hemispinous processes of different vertebrae, different areas (i.e. proximal, mid and distal) of the same hemispinous process and even within a single section. Longitudinally oriented vascular canals predominate in dorsal vertebrae, in the mid and distal portions of C10 and the distal portion of C5 (Figure 5e). Besides the predominating longitudinal‐oriented canals, the proximal and mid‐portion of C5, and the mid‐portion of C10 also exhibit numerous obliquely oriented canals, which sometimes anastomose and form patches of reticular bone (Figure 5b,e,f). No particular predominance of one vascular canal orientation type is evident in C3, in which longitudinal, radial, circumferential and oblique canal orientations are uniformly observed; however, reticular bone is observed in several areas. As previously mentioned, the vascularization pattern varies even within a single section. For example, in the section obtained from the mid‐portion of the C3 hemispinous process, radial canals tend to be more abundant along the lateral margins of the outer cortex. Regarding vascular canal density, this tends to decrease toward the outer cortex of the hemispinous processes.

3.6. Cyclical growth marks (CGMs)

Except for the distal section taken from the hemispinous process of D4, CGMs are recorded in all the sections. Single or double lines of arrested growth (LAGs), sometimes accompanied by annuli, are recorded in the primary bone tissue (Figures 5e‐i, 6a‐c). A maximum number of 14 and 10 CGMs were observed in MACN PV N15 and MOZ‐Pv 6126‐1, respectively. The number of CGMs was always higher in sections taken in the proximal portion of the hemispinous processes. Although the spacing between successive CGMs is irregular, they largely tend to decrease toward the outer cortex.

FIGURE 6.

FIGURE 6

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Bone histology of hyperelongate hemispinous processes of Amargasaurus cazaui MACN PV N1A. In all the images the outer cortex is oriented upward. White arrowheads indicate cyclical growth marks. (a) Loosely grouped Sharpey's fibres in the proximal portion of C3. (b‐d) densely grouped Sharpey's fibres in the mid and distal portions of D3 (b and d respectively) and proximal portion of C5 (c). Note the variation regarding the orientation of the extrinsic fibres. (e, f) the proximal portion of C3 (e) and mid‐portion of C5 (f) showing secondary osteons with multiple Haversian canals (black arrowheads). (g‐i) evidence of bone modelling in the proximal and distal portions of C10 (g and i respectively) and the distal portion of D3 (h). In each case, the reversal line is followed by a layer of new periosteal bone tissue. a, i: Normal transmitted light; b‐f, h: Plane polarized light; g: Cross polarized light with lambda filter. Abbreviations: Pb, periosteal bone; rc, resorption cavity; so, secondary osteon; scale bars: 0.2 mm (a, b, i); 0.3 mm (c, d, g, h); 0.5 mm (e, f)

3.7. Sharpey's fibres

These extrinsic fibres are recorded in all the analyzed sections. There is, however, strong variation regarding their distribution, relative density and orientation (Figure 4, 6). Such variation is not only observed amongst different elements, but also between different portions of a single spine and even within the same section. Whereas Sharpey's fibres are commonly absent or just loosely distributed in the posterior area of the cervical vertebrae hemispinous processes (Figure 6a), they are usually abundant in the same area of the dorsal vertebrae. This is particularly distinct in the proximal portion of D3, in which abundant anteroposteriorly oriented Sharpey's fibres are observed in the posterior edge of the spinopostzygapophyseal lamina and in the surface of the postzygapophyseal spinodiapophyseal fossa (Figure 4d). Sharpey's fibres are more commonly observed in the lateral and medial portions of the cortices, where they usually also exhibit a high density (Figure 6c,d). The orientation of the Sharpey's fibres regarding the subperiosteal surface is in general terms oblique. There is, however, an important degree of variation regarding the main axes of the elements. For example, whereas the Sharpey's fibres located in the posterolateral area of a proximal portion of the hemispinous processes of C5 and C10 are posterolaterally oriented, these extrinsic fibres are anterolaterally oriented in the same portion of C3 (Figure 4a‐c). Local variations of Sharpey's fibres orientation are also evident in several samples. For example, a gradual change from posterolateral to anterolateral orientation is recorded from the posterior to the anterior areas of the lateral cortex in the distal portion of C5 (Figure 4d3).

3.8. Secondary bone

Secondary remodelling is profuse in the hemispinous processes, not only in the perimedullary region but also in the mid and even in the outer cortex (Figure 6e). In this regard, Haversian bone usually reaches the subperiosteal cortex. Haversian bone includes osteons of a second and even third generation. Some peculiarities regarding secondary osteons are observed in the sampled bones. First, mostly in the hemispinous processes of the cervical vertebrae, secondary osteons with two or three Haversian canals are observed within the cortex (Figure 6e,f). These osteons usually exhibit irregular shapes and are comparatively larger than ‘normal’ osteons (i.e. those with roughly circular in section and a single Haversian canal). Second, a particular organization of the secondary osteons is observed in the proximal portions of C3, C5 and D3; more precisely in the posterior area (posterior cortex) of the spinodiapophyseal lamina in D3. In these areas, secondary osteons are grouped at the outer cortex forming large semicircular patches (Figure 5f,h). Third, despite their relative abundance, the distribution of the secondary osteons is generally not homogenous within the cortex. In this regard, the three sampled cervicals tend to exhibit a more pronounced degree of secondary remodelling in the anterior region, which can also be profuse in the medial (C3) or lateral (C5 and 10) areas. Such a pattern is not evident in the dorsal vertebrae. Fourth, although secondary osteons are oriented mostly in parallel with the main axes of the spines, the cortical bone of D3 exhibits some ‘radially’ oriented osteons in both proximal (outer cortex of the spinopostzygapophyseal lamina) and mid (lateral) portions of the spine.

3.9. Bone modelling

Clear evidence of modelling, mostly primarily based on the presence of reversal lines that disrupt the continuity of the primary or secondary tissue, is recorded in C3, C5, C10 and D3 (Figure 4a‐d, 6g‐i). These reversal lines are commonly observed in the medial portion of the cortex. The only exception to this pattern is recorded in the distal portion of C10, in which reversal lines are observed in both lateral and medial regions (Figure 4c, 6i). Another evidence for bone modelling is in C3 and C5, where CGMs contact with the subperiosteal margin of the cortex in some areas (Figure 4a,b).

4. DISCUSSION

4.1. Histological features of hyperelongate hemispinous processes

The main histological features of the hyperelongate hemispinous processes of Amargasaurus cazaui MACN PV N15 and Dicreosauridae indet. MOZ‐Pv 6126‐1 comprise a highly vascularized fibrolamellar bone interrupted with CGMs, presence of obliquely oriented Sharpey's fibres, and abundant secondary osteons irregularly distributed within the cortex. Several of these characteristics have been recorded from previous sauropod examples, including other elements of A. cazaui MACN PV N15 recently described by Windholz and Cerda (2021), particularly within the dorsal rib of this individual. The highly vascularized fibrolamellar bone in the hemispinous processes indicates high rates of bone apposition in the cortical bone (Chinsamy, 2005; Reid, 1996). Periodically deposited CGM reveals that cyclical (possibly annual) interruptions (sometimes accompanied by an important slowdown of the apposition rate, as evidenced by the presence of annuli) occurred. It is worth noting that the maximum number of CGMs in A. cazaui MACN PV N15 is higher in the hemispinous process (14) than in the dorsal rib (10) and in the stylopodial bones (5), which suggest that the first elements are better for age estimations. Variations in the CGMs count between axial and appendicular bones of the same individual have been recorded in other sauropod dinosaurs (e.g. Waskow & Sander, 2014). Regarding the presence of extrinsic fibres, although other bones from A. cazaui MACN PV N15 exhibited Sharpey’s fibres, these are more abundant in the hemispinous processes. Other typical feature of these vertebral elements, particularly from the cervical vertebrae, corresponds with the strongly compacted appearance in cross‐section, in which cancellous bone is mostly reduced or even absent.

Taking into account the degree of secondary remodelling in the hemispinous processes of A. cazaui MACN PV N15 we note that groups of secondary osteons forming large semicircular patches were also recognized in the dorsal rib of the same individual. Another important feature related to the secondary remodelling is the distribution of the Haversian tissue within the cortex of the cervical hemispinous processes, which is more profuse in the anterior region. Such a distribution of osteons within a single bone is strongly related to continuous biomechanical stresses experienced by the bones (Currey, 2002). In this sense, osteon density increases in the side of the bone subjected to compression loads and a decrease in the side where tension strains are predominant (Currey, 2002; Skedros et al., 1994; Zedda et al., 2008). Therefore, histological evidence suggests that the cervical hemispinous processes of Amargasaurus were subjected to mechanical forces that generated higher compression strain along the anterior side of the elements. Such forces could be originated by the osteological neutral pose of the neck (sensu Stevens & Parrish, 1999) proposed by Paulina Carabajal et al. (2014), in which the neck has a horizontally or slightly ventrally inclined position.

4.2. Were the hyperelongate hemispinous processes covered by keratinous sheaths?

The anatomical and histological characterization of the hyperelongate hemispinous processes of Amargasaurus cazaui holotype and MOZ‐Pv 6126‐1 allows, for the first time, a discussion regarding the nature of the tissues associated with these structures and evaluating their possible functional significance. The most recent hypothesis of these hyper‐elongated neural spines considered that the distal two were covered by a keratinized, horn‐like sheath (Apesteguía et al., 2019; Gallina et al., 2019; Schwarz et al., 2007). Such interpretation was singularly based on the presence of apparent external longitudinal striations in the distal portion of the process (Schwarz et al., 2007). Although this single feature has been also considered as evidence for the presence of a keratinized horn sheath in diverse extinct taxa (e.g. Delcourt, 2018; Goodwin et al., 2006; Main et al., 2005), the same does not appear to be applicable for Amargasaurus. In their comprehensive survey regarding the morphological and histological correlates of connective tissues structures in amniotes, Hieronymus et al. (2009) found that cornified sheaths that cover bony cores, such as the horns of bovid artiodactyls and the beaks of birds and turtles, are consistently associated with a set of distinct external features, including:

  • 1

    Prominent neurovascular grooves on bone surfaces.

  • 2

    A low profile for any rugose bone that may be present, with bone spicules directed tangentially along the bone surface.

  • 3

    Presence of neurovascular foramina that reach the bone surface at shallow, oblique angles.

  • 4

    Presence of a pronounced ‘lip’ or bony overgrowth at the transition between heavily cornified skin and adjacent soft skin.

The first, third and fourth characteristics were clearly observed in the Capra horn and the Equus ungual (Figure S1, S2). In contrast, the hemispinous processes of Amargasaurus lack the four features identified by Hieronymus et al. (2009) as related to horn sheath. Although Schwarz et al. (2007) mentioned the presence of a ‘ripple−and – striation’ pattern in the dorsal part of the cervical hemispinous processes of Amargasaurus MACN PV N15, we note that the better‐preserved areas indicate that the hemispinous processes usually exhibits a rather smooth surface. Furthermore, the presence of thin and shallow, longitudinally oriented ridges and furrows located along some areas (the ‘ripple‐and‐striation’ pattern of Schwarz et al., 2007) actually correspond with postmortem alterations of the bone surface. The only clear pattern of longitudinal ridges and grooves were observed on mid and distal portions of D3, D4 and D5. Such texturing, nevertheless, does not correspond with that commonly associated with horn cores (Hieronymus et al., 2009). In summation, the hypothesis for the presence of a keratinized horn sheath is not supported by the external features of the bone surface.

Taking into account histological features, Hieronymus et al. (2009) found that the presence of dense concentrations of mineralized extrinsic fibres (i.e. Sharpey's fibres) that meet the osseous surface at oblique angles is commonly correlated with the presence of cornified sheaths (although some degree of variation on this pattern is also evident). Additionally, Hieronymus et al. (2009) mentioned that the presence of obliquely oriented neurovascular canals opened to the surface. Our histological observations of Capra horn and Equus ungueal phalanx reveal that such extrinsic fibres were entirely absent (Capra horn; Figure S3a,b) or densely grouped forming a distinct layer at the outer cortex (Equus phalanx; Figure S3C–G). In both cases, however, obliquely oriented neurovascular canals are recorded in the outer cortex. These results imply that the sole presence and orientation of Shapey's fibres cannot be causally related to the presence of a cornified structure as a keratinous horn sheath. Furthermore, Sharpey's fibres may correspond with different soft tissues associated with the bone surface, including dermis, ligaments, tendons and muscles (Aaron, 2012; de Buffrénil & Quilhac, 2021b; Francillon Vieillot et al., 1990; Petermann & Sander, 2013; Wilson et al., 2016).

Another histological evidence against the presence of a keratinous horn sheath is based on the evidence of bone modelling in the hemispinous processes. Modelling refers to a local modification of the outer or inner morphology of a bone through a single process of apposition or resorption (de Buffrénil & Quilhac, 2021b; Martin et al., 2015; Ricqlès et al., 1991). The bone modelling allows accommodating the shape and maintaining the structural integrity of each skeletal element during growth. Histological evidence for this process involves the presence of compacted coarse cancellous bone (inter cortex) and reversal lines (outer cortex) (de Buffrénil & Quilhac, 2021a; Enlow, 1963). Bone modelling can occur because, despite being a mineralized, hard tissue, bone is an active tissue. Conversely, since a horn sheath is formed from a layer of keratinous fibres, it actually corresponds with a dead tissue that is incapable of resorption/apposition process, unlike osseous tissues (Bubenik & Bubenik, 1990; Hall, 2005). Compared to osseous tissues, keratinous horn sheath growth is basal (i.e. the older layers of keratin fibres are pushed ahead of the new layers beneath), which implies that a horn sheath, contrary to an osseous element, cannot microanatomically change its own shape during life (Hall, 2005). The evidence of bone modelling (e.g. presence of reversal lines) in the hemispinous process of Amargasaurus reveals important shape variations during the ontogeny, mostly in the medial side of the cervical processes. Such morphological variations cannot be coupled with the simultaneous growth of a keratinous sheath covering the hemispinous process, which cannot modify its shape. In addition, there is no histological evidence for bone modelling in horn cores (Hieronymus et al., 2009, Scannella & Horner, 2010; I.A.C personal observation in goat horn core).

The hypothesis for the presence of keratinized sheaths covering the hyperelongate hemispinous processes of dicraeosaurids has been most recently invoked by Gallina et al. (2019) on the basis of the long and extremely gracile nature of these processes, which were considered too weak to prevent fractures and breakage without an external covering or support (Gallina et al., 2019). However, contra Gallina et al. (2019) we consider a keratinized horn sheath does not unequivocally represent structural reinforcement of the hemispinous processes. Case in point, a keratinized horn sheath could generate additional mechanical loading issues on such a structure. For example, the tension generated by a lateral force applied to a keratinous sheathed hemispinous process along its dorsal portion will possibly be concentrate forces along the keratinous‐osseous boundary; potentially making this site more damage‐prone. If the keratinous sheat is absent, the tension generated by the lateral force will be possibly distributed in the complete shaft of the process (i.e. the stress is not concentrated in a particular area of the hemispinous process), avoiding the generation of areas of weakness and thus premature breakage. However, we must note that all discussion regarding the tensile strength of the hemispinous processes must be done on the basis of quantitative analyses (e.g. finite elements analysis) focused on the mechanical properties of the hyperelongate hemispinous processes, and in consideration of all the various, and hypothesized, associated soft‐tissues (e.g. keratinous sheath, ligaments, tendons, muscles). The absence of a keratinized horn sheath covering the hemispinous processes casts doubt towards the interpretation of the hyperelongated hemispinous processes acting as a defensive structure against predation attacks (Apesteguía et al., 2019; Gallina et al., 2019; Salgado & Bonaparte, 1991). Furthermore, the hemispinous processes lack internal sinus or abundant cancellous bone typical from bovid horn cores, which increases the mechanical resistance and act as shock absorbers in these structures (Farke, 2008; Nasoori, 2020). Additionally, as noted above, a pronounced ‘lip’ or bony overgrowth probably acting as a reinforcement of this critical section, is also absent.

4.3. Evidence for pneumatic diverticula

Besides the presence of a keratinous sheath covering the hyperelongated hemispinous processes of Amargasaurus, Schwarz et al. (2007) proposed that the medial surface along the proximal third of these process was in contact with a supravertebral pneumatic diverticula in the cervical vertebrae. However, osteological correlates for this soft tissue were absent for this taxon (and other dicraeosaurids). In a recent publication, Lambertz et al. (2018) reported the presence of thin fibres associated with the pneumatic diverticula epithelium and proposed the term ‘pneumosteum’ for this tissue. The histological features described for the ‘pneumosteal tissue’ correspond with extrinsic fiber (i.e. Sharpey's fibres) anchored to the subperiosteal or subendosteal tissue. Pneumosteal tissue is described as densely packed thin parallel fibres, which are inclined at approximately 30–45° to the bone surface. Although Sharpey's fibres are clearly observed on the outer cortex of the hemispinous processes of Amargasaurus, they do not resemble those of the pneumosteal tissue described by Lambertz et al. (2018). Furthermore, a distinct pattern regarding the arrangement and density of Sharpey's fibres is not present along the medial side of the proximal portion of the hemispinous processes. On the contrary, the density and arrangement of Sharpey's fibres are high along both the medial and lateral surfaces of each hemispinous process in the cervical vertebrae. Therefore, as occurs with the gross morphology, there is no histological evidence supporting the presence of a supraspinous pneumatic diverticulum. However, it is worth noting that, with the exception of Pilmatueia faundezi (Windholz et al., 2019), unambiguous evidence of postcranial pneumaticity (i.e. presence of large cortical openings connected directly with large internal cavities within the bone; O'Connor, 2006) have not been reported for any other dicraeosaurid sauropod.

4.4. Cervical sails and dorsal humps

Bailey (1997) considered that Amargasaurus was the only non‐avian dinosaur that showed convincing morphological evidence for the presence of a short sail in the cervical region, followed posteriorly by a broad hump. Cervical sails associated with hyperelongated spinous processes have been inferred for other fossil vertebrates, including Late Paleozoic synapsids in the Edaphosauridae and Sphenacodontidae families. Although histological analyses of these particular elements have been conducted (e.g. Enlow, 1969; Huttenlocker et al., 2010, 2011; Ricqlès, 1974), evidence for a causal relationship between particular histological features and the membranous sails were not provided. Furthermore, some histological features associated with particular soft tissues are differently interpreted depending on the position within the element. For example, whereas the presence of abundant Sharpey's fibres along the proximal portion of the spinous processes of Edaphosaurus was associated with the epaxial musculature, such an association was not invoked for the presence of the same extrinsic fibres along the more distal portion of the spine (Huttenlocker et al., 2011). Despite the clear difficulties of making inferences without comparative data from extant forms, the histological information here obtained for Amargasaurus can bring some insights regarding the ‘cervical sail’ hypothesis. Although the sole occurrence of Sharpey's fibres cannot be considered as unequivocal evidence for a singular soft tissue present in relation to an osseous structure (e.g. Pereyra et al., 2019; Petermann & Sander, 2013), their particular distribution and orientation along the hemispinous process of Amargasaurus and the dicreosaurid indet. MOZ‐Pv 6126–1 suggest their association with a particular soft tissue. Sharpey's fibres present along the hemispinous processes of the cervical vertebrae are more commonly observed on the lateral and medial sides of the cortex, where they usually also exhibit a high density. Such distribution does not appear to be related to the possible insertions of dorsal epaxial muscles, which were hypothesized to be only inserted along the lateral portions of the hemispinous processes of dicraeosaurid sauropods and other archosaurs (Schwarz et al., 2007, Schwarz‐Wings, 2009; Woodruff, 2017). Furthermore, whereas the dorsal epaxial musculature of Amargasaurus has been inferred to contact only the proximal third of the hemispinous processes (Schwarz et al., 2007), Sharpey's fibres are distinct along the proximal to the distal portions of these processes. Besides their spatial distribution, Sharpey's fibres exhibit a common pattern with regard to their relative orientation. In this sense, although the orientation of these fibres regarding the subperiosteal surface is, in general terms, oblique, they usually have some degree of inclination toward the anterior or posterior directions. The spatial distribution and relative orientation of the Sharpey's fibres could be related to the presence of an important system of interspinous ligaments that possibly connected with, amongst other elements, successive hemispinous processes in Amargasaurus. The continuity of this system of interspinous ligaments, which was covered by the integumentary system, resulted in the formation of a prominent cervical sail in this taxon (and other dicraeosaurids with the same feature, Figure 7). Morphological and histological evidence for interspinous ligaments has been previously reported for some groups of non‐avian dinosaurs (Bertozzo et al., 2021; Cerda et al., 2015a; Woodruff et al., 2016), including theropods (Wilson et al., 2016). The absence of distinct morphological evidence of interspinous ligaments in Amargasaurus can be explained in the function of the hyperelongate condition of the hemispinous processes in this taxon. In this regard, important tensile forces produced by a ligament over a determinate osseous surface could induce the formation of metaplastic bone tissue, which results in the formation of a distinct rugosity or projection in the bone (Wilson et al., 2016). The extremely long hemispinous processes of Amargasaurus possibly dissipated the stress generated by tensile forces from the interspinous ligaments, precluding the formation of ligament scars on the bone surface. A similar explanation has been invoked to explain the variation regarding the presence of morphological correlates of interspinous ligaments amongst spinosaurid theropods, where taxa with short neural spines (i.e. Baryonyx) exhibits distinct ‘metaplastic projections’, which are absent in taxa with long neural spines (i.e. Spinosaurus) (Wilson et al., 2016). A system of interspinous ligaments could have provided an important mechanical reinforcement to the hyperelongated hemispinous processes. In this regard, the elastic fibres, which constitute an important component of the interspinous ligaments (Heylings, 1980; Barros et al., 2002; Scapinelli et al., 2006), have the property to absorb strain (Aaron, 2012).

FIGURE 7.

FIGURE 7

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Life restoration of Amargasaurus cazaui. Illustration made by Gabriel Lio

Current data precludes asserting on the possible functional meaning of a cervical sail in Amargasaurus and other dicraeosaurid sauropods, although they possibly could be used as a display device, a rather broad term that includes ‘intraspecific agonistic, deterrent, or sexual display structures’ or ‘interspecific deterrent display structures’ (Main et al., 2005). The dicraeosaurid record is still too scarce to determine the existence of sexual dimorphism in this clade.

4.5. Concluding remarks and future perspectives

  • Both anatomical and histological evidence does not support the presence of a keratinized sheath covering the hyperelongated hemispinous processes of Amargasaurus and Dicraeosauridae indet. MOZ‐Pv 6126‐1. It is possible that the same occur in other dicraeosaurids with similar structures (i.e. Bajadasaurus, Pilmatueia). Future studies are required to test such a possibility.

  • Unambiguous evidence for the presence of a supravertebral pneumatic diverticula contacting the medial side of the proximal third of the hemispinous processes of Amargasaurus is not supported by histological evidence.

  • The spatial distribution and relative orientation of the Sharpey's fibres suggest the presence of a complex system of interspinous ligaments that possibly connect successive hemispinous processes in Amargasaurus. These ligaments were distributed along the entire length of the hemispinous processes.

  • Histological evidence indicates that the cervical hemispinous processes of Amargasaurus were subjected to mechanical forces that generate higher compression strain in the anterior side of the elements.

  • Although the current data support the hypothesis for the presence of a ‘cervical sail’ in Amargasaurus and other dicraeosaurids, the same must be re‐evaluated using more morphological and histological data based on living forms. Furthermore, the functional significance of these bizarre structures must be analysed using different approaches (e.g. finite elements analyses).

Supporting information

Data S1

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Fig S1

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Fig S2

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Fig S3

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Fig S4

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Fig S5

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Fig S6

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Fig S7

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Fig S8

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Fig S9

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Fig S10

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Fig S11

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Fig S12

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Fig S13

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Fig S15

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Fig S16

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ACKNOWLEDGMENTS

We thank all the curators that allowed us access to collections, as well as for their assistance and hospitality during this study. Alejandro Kramarz, Stella Álvarez and Martín Ezcurra (Museo Argentino Ciencias Naturales), Pablo Ortiz (Instituto Miguel Lillo) and Alberto Garrido (Museo Olsacher de Ciencias Naturales). The extraction and restoration of all the samples could be done, thanks to the invaluable collaboration of Marcelo Miñana, Marcelo Isasi and Carlos Alsina. Several of the histological sections were prepared by Mariano Caffa. Gillermo Windholz generously provides a picture for Figure 1g. The constructive comments of Holly Woodward and Cary Woodruff have greatly improved this manuscript. Sci‐hub library and Wikipaleo group shared valuable publications for this research. Part of this project was developed with the financial support of Agencia Nacional de Promoción Científica y Tecnológica (PICT‐2011‐1181 to I.A.C.).

Cerda, I.A. , Novas, F.E. , Carballido, J.L. & Salgado, L. (2022) Osteohistology of the hyperelongate hemispinous processes of Amargasaurus cazaui (Dinosauria: Sauropoda): Implications for soft tissue reconstruction and functional significance. Journal of Anatomy, 240, 1005–1019. Available from: 10.1111/joa.13659

DATA AVAILABILITY STATEMENT

Data sharing is not applicable to this article as no new data were created or analyzed in this study.

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Data Availability Statement

Data sharing is not applicable to this article as no new data were created or analyzed in this study.

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