Postnatal ontogeny of the spine of the Emperor Penguin Aptenodytes forsteri (Aves, Sphenisciformes) and modularity of the neck

Abstract

The Emperor Penguin (Aptenodytes forsteri), the largest extant penguin, inhabits exclusively in Antarctica. Little is known about the spine of this species; this work presents a description of the vertebral column of an adult of A. forsteri, compared with specimens from different ontogenetic stages (chick, fledgling, and juvenile). We also analyze the regionalization of the spine with a principal component analysis (PCA), and the modularity with an elliptical Fourier analysis, performing a PCA, followed by a linear discriminant analysis on the PC scores. Our results show that the vertebrae are completely ossified in adults, while the suture lines between the different elements are visible in the previous stages and some structures remain cartilaginous. A total of 42/43 vertebrae were counted (13 cervicals, 2 cervicothoracics, 6/7 thoracics, 13 synsacrals, and 8 caudals), and an extra vertebra appears in the thoracic region of one specimen. These regions are already differentiated in fledglings, but not in chicks. Six modules associated with the S-shape of the adult neck were recognized and are represented by different numbers of vertebrae: module 1 (C1), module 2 (C2), module 3 (C3–5), module 4 (C6), module 5 (C7–9) and module 6 (C10–13). Differences in the vertebral configuration in modules 5 and 6 of previous stages were found, where cervical 10 is part of module 5 due to the absence of the processus ventralis corporis.

Introduction

Penguins (Aves, Sphenisciformes) are highly specialized birds for marine life. They present a body with a hydrodynamic shape, short neck, paddle-like forelimbs, and short and caudally positioned hindlimbs (Winkler et al. 2020). The Antarctic Aptenodytes forsteri Gray 1844, that reaches 115 cm height and 46 kg weight (Martínez et al. 2020), is the largest species among the living forms.

The Emperor Penguin A. forsteri has been the subject of numerous studies (Prévost 1961; Isenmann and Jouventin 1970; Jouventin 1971; Klages 1989; Wienecke and Robertson 1997; Cherel and Kooyman 1998), but only few of them focus on its osteological anatomy (Stephan 1979; Guinard and Marchand 2010; Sosa and Acosta Hospitaleche 2018). In the present contribution, we analyze the spine of A. forsteri, taking into account the following aspects: general morphology, postnatal ontogenetic variation, regionalization, and modularity of the cervical portion.

The spine is the part of the axial skeleton that protects the spinal cord, provides support and stability to the entire body, and serves as the origin and insertion of muscles. The spine is divided into regions (Galbusera 2018; Galbusera and Bassani 2019) that, in birds, are named as cervical, thoracic, synsacral, caudal, and pygostyle. The cervical vertebrae have fused or absent ribs (Zusi 1962; Baumel and Witmer 1993). The thoracic ones carry complete ribs, articulating directly or indirectly with the sternum (Baumel and Witmer 1993). Some vertebrae called “transitional” (Fürbringer 1888) or “cervicothoracic” (Newton 1896; Pycraft 1898; Virchow 1931; Zusi 1962; Baumel and Witmer 1993; Jadwiszczak 2014a) carry free ribs (not contacting with the sternum) and exhibit a transitional morphology between the cervical and thoracic vertebrae (Baumel and Witmer 1993). The synsacral region corresponds to the synsacrum, a rigid structure of ankylosed vertebrae in adults, that incorporates some thoracic, lumbar, sacral, and the most proximal caudal vertebrae (Baumel and Witmer 1993). Finally, the caudal region is subdivided into a portion of free vertebrae and the pygostyle, an element formed by postnatal ankylosis of the last five–nine caudal vertebrae (Baumel 1988; Baumel and Witmer 1993).

The number of vertebrae in penguins differs from 42 vertebrae in Eudyptes chrysocome, Eudyptes chrysolophus, Eudyptula minor, Pygoscelis papua, and Aptenodytes patagonicus (Watson 1883; Shufeldt 1901; Stephan 1979) to 43 in Spheniscus demersus, Spheniscus magellanicus, and Spheniscus mendiculus (Watson 1883). The cervical region comprises 13 (Watson 1883; Stephan 1979; Guinard and Marchand 2010; Guinard et al. 2010) or 15 vertebrae (sensu Shufeldt 1901 and including the 2 cervicothoracics) depending on the author. Recent approaches consider the cervicothoracic vertebrae as a distinct region (Jadwiszczak 2014a). On the other hand, the thoracic region includes six (Shufeldt 1901) or eight–nine vertebrae (Stephan 1979 and Watson 1883 respectively, due to the inclusion of the cervicothoracics).

The constitution of the synsacrum varies from 12 (Watson 1883, considering the first synsacral vertebra as thoracic) to 14 (Stephan 1979). Depending on the species and the ontogenetic stage, the synsacrum includes from one to three caudal vertebrae (Pycraft 1898). In penguins, it is divided in five segments: thoracic, thoracolumbar, lumbar, sacral, and caudal (Stephan 1979). The thoracic segment comprises a single vertebra with ribs; the thoracolumbar has five vertebrae with wide processi transversi (and the last three vertebrae with processi costale). The lumbar segment comprises two–three vertebrae without processi costale, whereas the sacral has two vertebrae with strong processi costale. The caudal segment presents two–three vertebrae morphologically similar to the free caudals (see below).

The caudal region comprises six–seven free vertebrae with well-developed processi transversi (Watson 1883; Shufeldt 1901; Stephan 1979) and the pygostyle. The pygostyle is constituted by the fusion of the six–seven last caudal vertebrae (Stephan 1979).

It is worth mentioning here that postnatal variation of the penguin spine was only occasionally evaluated. A complex analysis of the cervical region was made with specimens of different ages (Guinard and Marchand 2010; Guinard et al. 2010), and more recently, the synsacra of juveniles of Aptenodytes and Pygoscelis were examined with comparative purposes (Jadwiszczak 2014b).

Analyses of modularity in the cervical region of penguins were made by Guinard et al. (2010) following the definition of modules provided by Klingenberg (2002). In this context, a module is considered as a group of elements morphologically similar and identified by a biomechanical function. The number of vertebrae included in each module varies interspecifically. According to that, the cervical region was divided into six portions reflecting the morphologic variations during growth and the neck flexibility in adults (Guinard and Marchand 2010; Guinard et al. 2010).

The first three modules (Mo1, Mo2, Mo3) connect the skull with the rest of the skeleton and contribute to the formation of the most proximal neck curvature. These segments help bring the head back, and rotate it. The fourth module (Mo4) is a transitional zone toward the fifth module (Mo5). It is identified as “mechanical curvature” because the folding of this zone is induced by the preceding and the posterior modules. The sixth module (Mo6) comprises the second cervical curvature and keeps the base of the neck backwards (Guinard et al. 2010).

These modules vary along postnatal ontogeny. In juveniles, the neck does not have a strongly folded S-shape mainly because the processi articulare craniale are not completely developed, particularly in the middle zone of the neck (Guinard et al. 2010). The modular configuration changes along ontogenetic growth, probably related to the ecological requirements of each stage. While chicks and juveniles are more terrestrial, adults acquire an eminently marine mode of life. For that reason, the folding of the neck must be more efficient in adults to bring the head closer to the body and thus achieve a hydrodynamic shape (Guinard et al. 2010).

Starting from these premises as the only background in this topic, we characterized each vertebra and the spine regions of A. forsteri, highlighting the individual and postnatal ontogenetic variations. The cervical modules in fledgling and adult specimens were also determined, omitting chicks that were too small or un-ossified for this kind of analysis.

Materials and methods

Materials

Skeletons analyzed here are deposited in the collections of the Vertebrate Zoology Department (adult MLP-O-15192, juvenile MLP-O-15035) and the Vertebrate Paleontology Department (adult MLP-478, fledgling MLP-1014, chicks MLP-1010, MLP-1011, MLP-1012, MLP-1013) of the Museo de La Plata, and the Ornithology Section of the Museo Argentino de Ciencias Naturales (adult MACN-OR-8462), Ciudad de Buenos Aires, Argentina. Four specimens were collected on Snow Hill Island (Antarctica) immediately after natural death during the austral summer field trip of 2014 by members of the Instituto Antártico Argentino. Ontogenetic stages concur with Stonehouse (1953).

One of the bodies was preserved in a 70% ethyl alcohol solution for further studies, and only the cervical vertebrae were macerated. The synsacrum and pygostyle were examined through X-ray images obtained in the Radiological Area of the Diagnostic Imaging Service of the Hospital Escuela de la Facultad de Ciencias Veterinarias (Universidad Nacional de La Plata). The other four specimens were frozen for transportation and then thawed at room temperature for further maceration in the laboratories of the MLP. Additionally, we incorporated information from the 1-day hatchling of A. forsteri, available on DigiMorph (Triche, 2005).

Osteological descriptions

Osteological terms concur with Baumel and Witmer (1993), Landolt and Zweers (1984), and Livezey and Zusi (2006). Vertebrae were grouped a priori by morphological similarities and according to regions (cervical, cervicothoracic, thoracic, synsacrum, caudal and pygostyle). Then, for each region, the vertebrae were subdivided according to morphologic similarity: cervicals were grouped following the modules suggested for A. forsteri by Guinard and Marchand (2010), and the rest of the regions were grouped based on the morphological similarity observed directly on the material.

Descriptions include: the corpus vertebrae (corpus) in a general aspect and in cranial, caudal, ventral, and lateral perspectives; and arcus vertebrae (arcus) in cranial, caudal, and dorsal views. The order follows the position in the spine from cranial to caudal ends, indicating the element of the spine with Arabic numbers following the region letter (C: cervical, CT: cervicothoracic, T: thoracic, and Ca: free caudal).

Regionalization analysis

Vertebrae were allocated into regions according to our direct observations. Then, the morphological consistency of the cervical, cervicothoracic, and thoracic vertebrae was evaluated through a dataset constructed with the nine linear measurements proposed by Guinard et al. (2010) (Online Resource 1, Supplementary Fig. S1; Online Resource 2). A principal component analysis (PCA), using a correlation matrix, was performed using InfoStat Software 2020 version (Di Rienzo et al. 2020). The missing data (13.33%) were imputed using Euclidean distance. The total components retained were those that exceed 70% of the total accumulated variation.

Modularity analysis

Two independent datasets were used to evaluate the modularity of the cervical region of fledgling and adults. The first one was based on the outline of the articular facet of the right processus articularis cranialis (according to Guinard et al. 2010) of two adults (MLP-O-15192, MACN-OR-8462) and the fledgling (MLP-1014). Photographs were taken with a Panasonic Lumix LZ40 camera perpendicular to the articular facet of the right processus articularis cranialis. Each photo was converted to a solid black image with a white background and saved as jpg files at 300 dpi and stored in a single directory. The extraction of the silhouettes from the images, the morphogeometric analysis of the outlines of the shape, and the analysis of the data were carried out using the Momocs (Bonhomme et al. 2014) package from R (R Core Team, 2021) and carried out in RStudio (RStudio Team, 2021) software. The latest version is available at https://momx.github.io/Momocs/index.html.

The coordinates of the outlines were extracted using the import_jpg function. The resulting list was combined in a single vector to obtain a list to be analyzed. The classification category of each element was imported as a table from a txt file. The new coordinate list was classified as an Out object and inspected with the panel and stack methods. First, the outlines were smoothed with the coo_smooth function to remove the digitization noise, and then they were normalized using a sequence of the following functions: coo_center, coo_scale, coo_align_coo_slidedirection (“down”), and inspected with the stack method to corroborate the quality of the alignment and the homology of the first point of the outlines.

The quantification of the outlines was made with an elliptical Fourier analysis (EFA; Kuhl and Giardina, 1982), where the closed outlines are decomposed into a series of harmonics; each harmonic represented by four coefficients. The harmonics calibration was calculated with the calibrate_harmonicpower_efourier and calibrate_reconstruction_efourier functions to determine the appropriate number of harmonics needed to achieve 99.9% of their total power. Fourier analysis was carried out with the efourier function.

A PCA using the PCA function, followed by summary and plot_PCA functions to display the results, was performed. The number of components to retain was determined with the scree and scree_plot, and screeplot functions (the last one from the vegan package (Oksanen et al. 2020) that includes the broken stick model). To calculate and plot the variation of the shape along the axes, the PCcontrib function was used. Finally, a discriminant analysis on PC scores was performed using the LDA function, followed by plot_LDA and plot_CV functions to display the results.

The second dataset was built from linear measurements, indices, and angles, as proposed by Guinard et al. (2010) (Online Resource 1, Fig. S1; Online Resource 3). The PCA was carried out using a correlation matrix that was analyzed with InfoStat Software (Di Rienzo et al. 2020), imputing the missing data (6.56%) through Euclidean distance. The total components retained were those that exceed 70% of the total accumulated variation.

Results

Osteological descriptions

Cervical 1 (Atlas) has a ring shape (Fig. 1a–d)

Corpus

The kidney-shaped fossa condyloidea presents a concave surface that articulates with the condylus occipitalis of the skull. There are two projections obliquely located in the dorsal end of this fossa. The processus ventralis corporis is small and keel-shaped. It is medio-ventrally located and projects caudally. The facies articularis cranialis is slightly convex. The incisura fossae is semicircular.

Fig. 1

Adult vertebrae MLP-O-15192. a–d C1 in a cranial, b left lateral, c caudal, and d dorsal views, e–i C2 in e cranial, f left lateral, g caudal, h dorsal, and i ventral views, j–n C3 in j cranial, k left lateral, l caudal, m dorsal, and n ventral views, o–s C4 in o cranial, p left lateral, q caudal, r dorsal, and s ventral views, t–x C5 in t cranial, u left lateral, v caudal, w dorsal, and x ventral views. aat, arcus atlantis; aax, arcus axis; ac, ansa costotransversaria; av, arcus vertebrae; cat, corpus atlantis; cax, corpus axis; cl, cristae laterale; cv, corpus vertebrae; da, dens axis; faca, facies articularis caudalis; facr, facies articularis cranialis; fat, facies articularis atlantis; fax, facies articularis axialis; fc, fovea cranioventralis; fco, fossa condyloidea; ft, foramen transversarium; if, incisura fossae; lda, lamina dorsalis arcus; lla, lamina lateralis arcus; paca, processus articularis caudalis; pacr, processus articularis cranialis; pc, processus costalis; pp, processus postlateralis; ps, processus spinosus; psa, processus spinosus axis; pvc, processus ventralis corporis; ta, tuberculum ansae; td, torus dorsalis. Scale bar 10 mm

Arcus

It is oval and mainly forms the ring of the Atlas. The processi articulares caudales are dorsolaterally positioned in a caudal perspective. The processus spinosus is absent.

Cervical 2 (Axis) (Fig. 1e–i)

Corpus

It is craniocaudally elongated and its caudal half is compressed. Both facies articularis are located at the same level on the craniocaudal axis. The facies articularis atlantica is kidney-shaped, cranio-ventrally leaned, and presents a dorsal notch. The dens axis is cranially projected and presents a flattened dorsal surface. The processus ventralis corporis is highly developed and projects ventrocaudally from the caudal half of the vertebral body. The distal end of the processus ventralis corporis is widened. The facies articularis caudalis is quadrangular and saddle-shaped.

Arcus

Compared to the width of the lamina lateralis arcus, lamina dorsalis arcus is very expanded. The entire arch covers approximately two thirds of the dorsal side of the vertebral body. The processi articulare craniale are smaller than the processi articulares caudales. The processis articularis cranialis are oval and laterally positioned. The joint surfaces are dorsoventral and faced laterally, obliquely positioned with respect to the sagittal plane and approaching each other caudally. Two small ridges are positioned ventrocaudally to the processi articulare craniale and slightly protrude from the lamina lateralis arcus. The processi articulares caudales have a joint surface, which is ventrally directed. Dorsally and caudally located, two semicircular bulges constitute the tori dorsales which have a cranially projected spine. The processus spinosus axis develops a widened oval distal end.

Cervicals 3–5 (Fig. 1j–x)

Corpus

It is small, with the craniocaudal axis ventrally inclined, positioning both facies articularis in different levels. The facies articularis cranialis is rectangular and dorsoventrally flattened. The foramen transversarium is small and oval, with its major axis dorsoventrally located and progressively increasing in size in the successive vertebrae. The foramen is bounded by a thin ansa costotransversaria that carry two slightly marked cristae laterale in the three vertebrae. A tuberculum ansae dorsal to the crests appears only in C4 and C5. Ventrally, the processi costale are reduced in C3 and C4. It is thorn-shaped and caudally projected in C5. The fovea cranioventralis is deep and well marked, located on the cranial end (in the articulation areas with the ventral lip of the facies articularis caudalis of the preceding vertebra during the ventral flexion of the neck). In the caudal half, the processus ventralis corporis projects ventrocaudally and develops an oval and expanded distal end in C3, smaller than those of C1–2 The processus ventralis corporis is rounded and develops a slight caudal expansion in C4, or it is absent and two little crests cranially projected corresponding to the processi postlaterales are developed instead in C5. The facies articularis caudalis is quadrangular, saddle-shaped, and dorsoventrally elongated.

Arcus

The lamina dorsalis arcus is laterally expanded and surpasses the lateral limits of the vertebral body. It makes the vertebra acquire a quadrangular shape in dorsal view. On the lateral limits of the lamina lateralis arcus there is a small and slightly marked semicircular notch located on the caudal end of C3. This notch is slightly larger in C4, and is located in the half of the lamina dorsalis arcus in C5. On the lateral edges of the lamina dorsalis there is another semicircular small and slightly marked notch in C3. The lamina lateralis arcus, less developed than the lamina dorsalis arcus, constitutes the laterals of the vertebral arch. The cranially inclined processi articulare craniale are semicircular in C3, oval in C4, and triangular in C5. The processi articulares caudales are faced ventrally. They are oval in C3, kidney-shaped in C4 and triangular in C5. Dorsal to these processes, the tori dorsales are strongly projected dorsolaterally. On the mediodorsal line of the lamina dorsalis arcus, the laterally compressed processus spinosus presents a rounded outline; it is slightly caudally inclined in C3 and C4, and dorsally projected in C5.

Cervical 6 (Fig. 2a–e)

Corpus

It is elongated and slightly larger than C5. The facies articularis cranialis is rectangular, dorsoventrally compressed, and saddle-shaped. The foramen transversarium is large and rounded, laterally bounded by the ansa costotransversaria, with a dorsoventrally expanded tuberculum processi transversi, and a spine-shaped ventral processi costale, which are flattened and ventrally projected. Cranially and ventral to the facies articularis cranialis, the paired processi carotici enclose the canalis caroticus cervicalis. On the caudal end and extending cranially for about a third of the ventrolateral surface of the corpus, two small and flattened wing-like structures correspond to the processi postlaterales. The facies articularis caudalis is rectangular and saddle-shaped.

Fig. 2

Adult vertebrae MLP-O-15192. a–e C6 in a cranial, b left lateral, c caudal, d dorsal, and e ventral views, f–j C7 in f cranial, g left lateral, h caudal, i dorsal, and j ventral views, k–o C8 in k cranial, l left lateral, m caudal, n dorsal, and o ventral views, p–t C9 in p cranial, q left lateral, r caudal, s dorsal, and t ventral views, u–y C10 in u cranial, v left lateral, w caudal, x dorsal, and y ventral views. ac, ansa costotransversaria; av, arcus vertebrae; cv, corpus vertebrae; faca, facies articularis caudalis; facr, facies articularis cranialis; ft, foramen transversarium; lda, lamina dorsalis arcus; lla, lamina lateralis arcus; paca, processus articularis caudalis; pacr, processus articularis cranialis; pc, processus costalis; pca, processus caroticus; pp, processus postlateralis; ps, processus spinosus; pvc, processus ventralis corporis; td, torus dorsalis. Scale bar 10 mm

Arcus

The lamina dorsalis arcus slightly exceeds the lateral limit of the corpus and carries a tuberculum on its caudal end. The oval processi articulare craniale face medially and are cranio-ventrally inclined, although the caudal most part of these processes are dorsally and barely medially faced. The oval processi articulares caudales face ventrolaterally, and are smaller than the processi articulare craniale. Dorsally, and caudally located on the lamina dorsalis arcus, the tori dorsales are distinctly flattened, tip-shaped, and cranially projected. The processus spinosus is thinner than those of the preceding vertebrae, with a quadrangular outline, and slightly caudally inclined. Two weak crests project from the tori dorsales to the processus spinosus.

Cervicals 7–9 (Fig. 2f–t)

Corpus

It is elongated with its craniocaudal axis aligned to the spine. The facies articularis cranialis is trapezoidal, dorsoventrally compressed, saddle-shaped, and slightly larger than that of C6. The foramen transversarium is large and rounded. Laterally, the ansa costotransversaria has a strong dorsoventrally expanded tuberculum processus transversi that projects laterally beyond the limit of the processus articularis cranialis. Ventrally, two cristae laterale are developed. On the ventral side of the ansa costotransversaria, the processi costale are flattened, spine-shaped, and ventrocaudally projected, decreasing caudally in length. The processi carotici are similar to those of C6. On the caudal end, the processi postlaterales project cranially on a third of the ventrolateral surface of the corpus in C7, whereas they are slightly developed in C8, and absent in C9. In all these vertebrae, the facies articularis caudalis is quadrangular, saddle-shaped, and similar in size to that of C6.

Arcus

The lamina dorsalis arcus slightly surpasses laterally the corpus; a longitudinal crest runs along the lamina lateralis arcus in C7 and C8; these crests are absent in C9. The processi articulare craniale are drop-shaped with the sharpest end pointing cranially and the articular surface slightly inclined toward the midline. The processi articulares caudales are oval and smaller than the processi articulare craniale; and the joint surface is ventrolaterally positioned. The tori dorsales are located on the caudal half of the lamina dorsalis arcus and cranial to the processi articulares caudales. The tori dorsales are flattened and tip-shaped, cranially projected in C7, oval and craniocaudally extended in C8, oval and midline-located on of the processi articulare caudali, and dorsally projected in C9. The processus spinosus is significantly smaller than that of the preceding vertebrae in C7, extremely reduced in C8, and absent in C9. Two weak crests project from the tori dorsales to the processus spinosus.

Cervicals 10–13 (Figs. 2u–y, 3a–o)

Corpus

It is elongated with a horizontal and slightly inclined craniocaudalin C12. The facies articularis cranialis is trapezoidal and saddle-shaped, dorsoventrally compressed, and more laterally expanded than the previous ones. The foramina transversaria is oval and their major axis is craniocaudally oriented. These foramina are larger in C12–13. In C10–12 the tuberculi processi transversi are dorsoventrally expanded and do not surpass the lateral limits of the processi articularis craniale. In C11, however, they slightly surpass it. Two cristae laterale (the dorsal is stronger), are developed caudoventral to these tubercles. The processi costale are spine-shaped and flattened. In C10, they project caudally with a length equivalent to half the length of the previous vertebra. In C11, the processi costale project twice the length of C11. In C12, they are short, rounded and robust; and in C13 they are blunt and weakly developed. Only in C10, the processi carotici delimit a small canalis caroticus cervicalis. The processus ventralis corporis is developed from these vertebrae. It is smaller and rounded in C10; rounded, laterally compressed, and cranially inclined in C11; quadrangular and cranially inclined in C12; and smaller and ventrally projected in C13. The processus ventralis corporis covers two thirds of the total length of the vertebral body in C11 and C12, but only a third in C10 and C13. The fovea cranioventralis appears from the C11 where it is barely marked. It is also barely marked in C12, and well developed in C13. The facies articularis caudalis is saddle-shaped and similar in size in all the vertebrae, although in C13 it is slightly larger than in the previous ones.

Fig. 3

Adult vertebrae MLP-O-15192. a–e C11 in a cranial, b left lateral, c caudal, d dorsal, and e ventral views, f–j C12 in f cranial, g left lateral, h caudal, i dorsal, and j ventral views, k–o C13 in k cranial, l left lateral, m caudal, n dorsal, and o ventral views, p–t CT1 in p cranial, q left lateral, r caudal, s dorsal, and t ventral views, u–y CT2 in u cranial, v left lateral, w caudal, x dorsal, and y ventral views, z–d′ T1 in z cranial, a′ left lateral, b′ caudal, c′ dorsal, and d′ ventral views. ac, ansa costotransversaria; acv, alae cristae ventralis; av, arcus vertebrae; cv, corpus vertebrae; ec, eminentia costolateralis; faca, facies articularis caudalis; facr, facies articularis cranialis; fc, fovea cranioventralis; fcc, fovea costalis capituli; fct, fovea costalis tuberculi; lda, lamina dorsalis arcus; lla, lamina lateralis arcus; paca, processus articularis caudalis; pacr, processus articularis cranialis; pc, processus costalis; ps, processus spinosus; pt, processus transversus; pvc, processus ventralis corporis; pvl, processus ventrolateralis; td, torus dorsalis. Scale bar 10 mm

Arcus

The lamina dorsalis arcus extends laterally as much as the corpus. Only in C10, a weak longitudinal ridge is on the lamina lateralis arcus. The processi articulare craniale are drop-shaped in C10, while they are oval in the rest of the vertebrae. In a dorsal perspective, the joint surface of these processes is medially leaning in C10 and C11, and inclined toward the midline and cranially curved in C12 and C13. The processi articulares caudales are similar in size to the processi articulare craniale and their joint surface are lateroventrally leaning in C10 and C11, while in C12 and C13 they are smaller than the processi articulare craniale, and their joint surface are rounded and concave. The tori dorsales present a variable location. On C10 they are located on the medial edge of the dorsal side of the processi articulares caudales and caudo-laterally projected. On C11, they are expanded on the dorsal side of the processi articulares caudales, and extended from the caudal end to the midline. On C12, they are rounded and flattened, and expand from the caudal end to the lateral limit of these processes. On C13 they are rounded and expand over the entire dorsal surface of the processi articulares caudales. The processus spinosus is absent in C10 and C11, barely developed in C12, and well developed in C13. In the later, the processus spinosus is compressed and presents a heart-shaped dorsal expansion that occupies two thirds of the total length of the lamina dorsalis arcus. Two weak crests extend from the cranial end of the tori dorsales to the midline of the arcus. In C13, the area ligamentum elasticum is represented by a depression located in the midline, caudal to the processus spinosus and the caudal edge of the lamina dorsalis arcus.

Cervicothoracic 1 (Fig. 3p–t)

Corpus

Rectangular and similar in size to C13. Its craniocaudal axis is aligned with the spine (not inclined). The facies articularis cranialis is trapezoidal, laterally elongated and saddle-shaped, larger than that of C13. Ventrally, the fovea cranioventralis is cranial to the small and rounded processus ventralis corporis that is ventrally projected. The processus ventrolateralis extends ventrolaterally from the lateral end of the facies articularis cranialis to the midline of the body. The facies articularis caudalis is trapezoidal and saddle-shaped, with the major axis dorsoventrally positioned. At the latero-cranial end of the vertebral body, the barely developed eminentia costolateralis presents a rounded fovea costalis capituli.

Arcus

The lamina dorsalis arcus and the lamina lateralis arcus are well developed, but they do not surpass the limit of the corpus. The processi articulare craniale are oval and their main axis are craniocaudally positioned. The joint surfaces of these processes are slightly convex and inclined with the minor axis converging in the midline. The base of the processi transversi extends from the cranial end of the processi articulare craniale to the center of the vertebra. These processes spread latero-cranially, decreasing in width and bending dorsally toward the ends. The fovea costalis tuberculi is barely developed. The processi articulares caudales and craniale are similar in size; their joint surfaces are rounded and slightly concave. The quadrangular and laterally compressed processus spinosus is almost entirely covering the lamina dorsalis arcus. It develops a dorsal, flattened and drop-shaped expansion which is cranially oriented. Caudally, a depressed area with a triangular shape corresponds to the area ligamentum elasticum. The tori dorsales are rounded and barely developed on the dorsal surface.

Cervicothoracic 2 (Fig. 3u–y)

Corpus

It is laterally compressed. The facies articularis cranialis is trapezoidal and saddle-shaped, with the dorsoventral axis more developed than in CT1. The fovea cranioventralis is slightly developed. Caudal to this fovea are the compressed and ventrolateral directed alae cristae ventralis. The facies articularis caudalis is concave and rectangular, and its major axis is dorsoventrally positioned. The eminentia costolaterales are slightly more developed than those of the preceding vertebra, and bear the rounded fovea costalis capituli.

Arcus

The processi articulare craniale and the processi articulares caudales are oval and smaller than those of the preceding vertebrae. The processi transversi are quadrangular, wider but less laterally expanded than in CT1. Ventrally, the fovea costalis tuberculi is represented by a rounded protuberance. The processus spinosus is quadrangular and occupies the whole dorsal surface of the lamina dorsalis arcus.

Thoracics 1–3 (Figs. 3z–d′, 4a–j)

Corpus

It is laterally compressed. The facies articularis cranialis is saddle-shaped in T1, and rounded and concave in T2–3. In ventral view, the processus ventralis corporis bears a pair of ventrolaterally projected alae cristae ventrales that decrease in size from T1 to T3. The alae cristae ventrales originate on the proximal end in T1, in the center of the process in T2, and on the distal end in T3. The facies articularis caudalis is rounded and concave. The eminentia costolaterales, on the latero-cranial end of the body, bears a concave and rounded fovea costalis capituli.

Fig. 4

Adult vertebrae MLP-O-15192. a–e T2 in a cranial, b left lateral, c caudal, d dorsal, and e ventral views, f–j T3 in f cranial, g left lateral, h caudal, i dorsal, and j ventral views, k–o T4 in k cranial, l left lateral, m caudal, n dorsal, and o ventral views, p–t T5 in p cranial, q left lateral, r caudal, s dorsal, and t ventral views, u–y T6 in u cranial, v left lateral, w caudal, x dorsal, and y ventral views. acv, alae cristae ventralis; ale, area ligamentum elasticum; av, arcus vertebrae; cv, corpus vertebrae; ec, eminentia costolateralis; faca, facies articularis caudalis; facr, facies articularis cranialis; fcc, fovea costalis capituli; fct, fovea costalis tuberculi; lda, lamina dorsalis arcus; lla, lamina lateralis arcus; paca, processus articularis caudalis; pacr, processus articularis cranialis; ps, processus spinosus; pt, processus transversus; pvc, processus ventralis corporis; td, torus dorsalis. Scale bar 10 mm

Arcus

The lamina dorsalis arcus and the lamina lateralis arcus do not exceed the lateral limits of the corpus. The processi articulare craniale are rounded and the joint surface is medially inclined in a dorsal perspective. The processi transversi are laterocaudally directed with the fovea articularis tuberculi developed on their ventral surface. In T3, the aponeurosis ossificans projects cranially on the cranial margin of the processi transversi. The processi articulares caudales are rounded and equal in size to the processi articulare craniale. Dorsally, the slightly developed and elongated tori dorsales occupy the whole lateral margin of the processi articulares caudales in T1–2, and are slightly developed in T3. The well-developed processus spinosus is laterally compressed and covers all the extension of the vertebral arch. This process acquires a quadrangular shape, with a dorsoventrally flattened expansion at its dorsal end.

Thoracics 4–6 (Fig. 4k–y)

Corpus

It is laterally compressed. The oval facies articularis cranialis presents a convex joint surface. The processus ventralis corporis occupies approximately two thirds of the length of the vertebral body in the three vertebrae, and its size progressively decreases from T4 to T6. This process is laterally compressed and, unlike the previous vertebra, the alae cristae ventralis are absent. The facies articularis caudalis are rounded and concave. Laterocranially, the eminentia costolateralis bears the concave and rounded fovea costalis capituli.

Arcus

The lamina dorsalis arcus and the lamina lateralis arcus do not exceed the lateral limits of the body. The processi articulare craniale are oval and the joint surface is medially inclined in a dorsal perspective. Unlike in the previous vertebrae, the processi transversi have a rectangular outline and are laterocaudally directed. On the cranial edge these processes present a cranial projection corresponding to the aponeurosis ossificans that are thinner than those of the previous vertebra. Ventrally, on the distal end of the processus transversus, the fovea articularis tuberculi is developed. The processi articulares caudales are rounded and their joint surfaces are laterally inclined. Both the processi articulare craniale and caudale are equal in size. Dorsally, on these processes, the tori dorsales develop an elongated shape. On the midline of the lamina dorsalis arcus, the processus spinosus is strongly developed, covering all the extension of the vertebral arch. It is quadrangular, laterally compressed, and presents a dorsoventrally flattened expansion at its dorsal end.

The specimen MLP-478 presents an extra thoracic vertebra (Fig. 5a, b) morphologically similar to T6, but distinguishable from it by the absence of the processus ventralis corporis. Furthermore, the processi transversi develop a caudal projection dorsally curved which contacts with the ventral side of the ala preacetabularis ilii of the pelvic girdle.

Fig. 5

Adult vertebrae MLP-478. a–b T7 in a left lateral, and b dorsal views, c synsacrum, ventral view. Adult vertebrae MLP-O-15192. d–h Synsacrum in d cranial, e caudal, f left lateral, g ventral, and h dorsal views, i–m Ca1 in i cranial, j left lateral, k caudal, l dorsal, and m ventral views, n–r Ca2 in n cranial, o left lateral, p caudal, q dorsal, and r ventral views, s–w Ca3 in s cranial, t left lateral, u caudal, v dorsal, and w ventral views, x–b′ Ca4 in x cranial, y left lateral, z caudal, a′ dorsal, and b′ ventral views, c′–g′ Ca5 in c′ cranial, d′ left lateral, e′ caudal, f′ dorsal, and g′ ventral views, h′–l′ Ca6 in h′ cranial, i′ left lateral, j′ caudal, k′ dorsal, and l′ ventral views, m′–q′ Ca7 in m′ cranial, n′ left lateral, o′ caudal, p′ dorsal, and q′ ventral views, r′–v′ pygostyle in r′ cranial, s′ left lateral, t′ caudal, u′ dorsal, and v′ ventral views. Synsacral segments: CS, caudal segment; LS, lumbar segment; SS, sacral segment; TLS, thoracolumbar segment; TS, thoracal segment. av, arcus vertebrae; bp, basis pygostyli; cs, corpus synsacri; css, crista spinose synsacri; cv, corpus vertebrae; ec, eminentia costolaterales; faca, facies articularis caudalis; facr, facies articularis cranialis; fcc, fovea costalis capituli; fct, fovea costalis tuberculi; fi, foramen intertransversarium; lp, lamina pygostyli; paca, processus articularis caudalis; pacr, processus articularis cranialis; pc, processus costalis; ph, processus haemalis; ps, processus spinosus; pt, processus transversus. Scale bar 10 mm

Synsacrum

It consists of 13 ankylosed vertebrae (Fig. 5c–h). The corpus synsacri is compressed at the cranial half and cylindrical at the caudal half, decreasing caudally in size. The facies articularis cranialis is rounded and convex. The sulcus ventralis corporis is absent or slightly marked, and the facies articularis caudalis is rounded and flattened. Each vertebra is distinguished by the opening on both sides of a rounded and small foramina intervertebralia. Dorsally, the crista spinosa synsacri extends from the cranial end of the first vertebra to the cranial end of the last synsacral one, and corresponds to the ankylosis of all the processi spinosi. The crista spinosa synsacri is compressed, dorsally thickened and progressively lower toward the caudal end. Only in some specimens, an oval foramen opens between the processus spinosus of the first and second vertebra. The synsacrum is divided into the 1: thoracal (TS), 2: thoracolumbar (TLS), 3: lumbar (LS), 4: sacral (SS), and 5: caudal segments (CS).

1-Thoracal segment

It comprises a single vertebra corresponding to the last thoracic fused to the synsacrum during the ontogeny. The corpus is compressed and the facies articularis cranialis is typically convex. Dorsally, the eminentia costolaterales are rounded. The arcus presents a similar configuration to that of T6, but the distal end of the processi transversi are dorsally curved and contact with the ventral side of the ala preacetabularis illi. Distally, the rounded fovea articularis tuberculi is developed. This vertebra is completely different in the specimen MLP-478, where the characters described above cannot be recognized and, instead, a vertebra similar to the first thoracolumbar is developed.

2-Thoracolumbar segment

It includes five vertebrae, from which the first two are characterized by a compressed body and a well-developed processi transversi (which is longer in the first vertebra). Each process has cranial and caudal extensions contacting with the preceding and the following processi transversi, constituting a “bridge” for the contact with the ventral surface of the ala preacetabularis ilii. The following three vertebrae are more robust and their bodies are more cylindrical than the ones of the preceding vertebrae. The processi transversi are reduced and distinguishable only by the reduced foramina intertransversaria. The processi costale, turn caudally into a more ventral position. The crista spinosa is continuous along this segment, becoming caudally lower. It develops a dorsal thickening.

3-Lumbar segment

It comprises three vertebrae characterized by a cylindrical and robust corpus without processi costale. The processi transversi present a lateral and curved expansion. The foramina intervertebralia has the same size as the foramina in the thoracolumbar segment. The crista spinosa decrease caudally in height.

4-Sacral segment

It comprises two vertebrae from which the first one has the same lateral extension as the thoracal segment. The corpus vertebrae are depressed and wide. The processi transversi are short and small, and the highly reduced foramina intertransversaria bear large and rectangular processi costale which are laterally projected. The foramina intervertebralia are larger than those of the preceding segments. The crista spinosa decrease caudally.

5-Caudal segment

It comprises two vertebrae that present the typical morphology of the caudal vertebrae (see below). The corpus are cylindrical, the processi transversi are rectangular and laterocaudally expanded. The crista spinosa has a bifid end on the last vertebra. The facies articularis caudalis is rounded and flattened.

Caudals 1–7 (Fig. 5i–q′)

Corpus

It is cylindrical and both, the facies articularis cranialis and the facies articularis caudalis, are flat. The first three vertebrae lack any structure, but the processus haemalis appears in Ca4. This process is simple, or bifid and short in Ca4; and bifid and cranially projected in Ca5–7, ventrally reaching the previous vertebra.

Arcus

It is dorsoventrally extended with an eight-shaped foramen vertebrale. The processus spinosus is bifid in Ca1–5 and not bifid and slightly caudally inclined in Ca6–7. The processi transversi are rectangular and short, dorsoventrally flattened, and expand along the vertebral body.

Pygostyle

It is formed by the fusion of the last five caudal vertebrae (Fig. 5r′–v′), compressed and triangular in lateral view, and triangular in cross section, decreasing caudally in height. The facies articularis cranialis is rounded and concave. The vertebral bodies are fused forming the basis pygostyli, although each vertebra is still distinguishable. The arcus are fused constituting the lamina pygostyli and are only differentiated in the first two vertebrae. In ventral view, the processus haemalis converge in a single structure at the level of the third vertebrae. This process is cranially projected beyond the ventral side of the preceding vertebra.

Ontogenetic variations

Juvenile

The cervical vertebrae (Fig. 6a–e) are completely ossified, except for the distal end of all the processes (processus ventralis corporis, processi transversi, and processus spinosus). The processi costale, only proximally ossified, are not totally fused to the rest of the vertebra; the suture line between the body and the ansa costotransversaria is still visible. In C10 (Fig. 6d), the processus ventralis corporis is absent, whereas the processi carotici are well developed. The elements of the synsacrum (Fig. 6f, g) are partially fused, including the bodies of the last three vertebrae of the thoracolumbar segment, the complete lumbar and sacral segments, and the first vertebra of the caudal segment. On the contrary, all the processi spinose are free. In the pygostyle (Fig. 6h, i), the sutures between the elements are still visible. The last two vertebrae are fused, and the rest are free.

Fig. 6

Juvenile vertebrae MLP-O-15035. a C7, cranial view, b C8, dorsal view, c C9, caudal view, d C10, left lateral view, e C11, ventral view, f X-ray of the caudal zone, right lateral view, g scheme of the synsacrum, right lateral view, h X-ray of the caudal zone, ventral view, i scheme of the pygostyle, ventral view, j C1, cranial view, k C 2, left lateral view, l C6, cranial view, m CT1, caudal view, n T3, left lateral view, o synsacrum, left lateral view, p Ca3, caudal view, and q pygostyle, cranial view; r pygostyle, left lateral view. Synsacral segments: CS, caudal segment; LS, lumbar segment; SS, sacral segment; TLS, thoracolumbar segment; TS, thoracal segment. ac, ansa costotransversaria; av, arcus vertebrae; cat, corpus atlantis; cv, corpus vertebrae; da, dens axis; faca, facies articularis caudalis; facr, facies articularis cranialis; fc, fovea cranioventralis; fcc, fovea costalis capituli; fct, fovea costalis tuberculi; ft, foramen transversarium; if, incisura fossae; lda, lamina dorsalis arcus; lla, lamina lateralis arcus; paca, processus articularis caudalis; pacr, processus articularis cranialis; pc, processus costalis; ph, processus haemalis; ps, processus spinosus; pt, processus transversus; pvc, processus ventralis corporis; td, torus dorsalis. Scale bars 10 mm

Fig. 7

Cervical 10 at different ontogenetic stages, showing the processi carotici (pc) and the processus ventralis corporis (pvc): a chick MLP-1010, cranial view, b fledgling MLP-1014, cranial view, c juvenile MLP-O-15035, cranial view, d adult MLP-O-15192, cranial view, e chick MLP-1010, lateral view, f fledgling MLP-1014, lateral view, g juvenile MLP-O-15035, lateral view, and h adult MLP-O-15192, cranial view. Scale bars 10 mm

Fledgling

The processus ventralis corporis in C1 (Fig. 6j) is proximally ossified, whereas the arcus atlantis is cartilaginous. In C2 (Fig. 6k), the dens axis is not fused to the corpus axis and the distal end of the processes are free. In C3–13 (Fig. 6l, m), the corpus and the arcus are ossified, although the distal end of the processus ventralis corporis, processi carotici, processi transversi, and processus spinosus are rounded and still cartilaginous. When present, the fovea cranioventralis is deep and well marked. The facies articularis cranialis and facies articularis caudalis are rounded due to lack of ossification, and the ansa costotransversaria and the processi costale are absent.

In CT1–2 (Fig. 6n), the corpus and arcus are ossified. On the contrary, the eminentia costolateralis and the distal end of the processus ventralis corporis, processi transversi and processus spinosus are still cartilaginous. In T1–6 (Fig. 6o), the corpus and the arcus are completely ossified. The processus ventralis corporis, the distal end of the processi transversi, and the processus spinosus are rounded and still cartilaginous.

Within the synsacral vertebrae (Fig. 6p), the corpus of the three vertebrae of the LS are fused, with their sutures still visible. The body of the last two vertebrae of the TCS and the two vertebrae of the SS are partially fused to the LS. The processus spinosus is always independent. The rest of the vertebrae of the TCS, the TS, and the two vertebrae of the CS are unfused and more ovoidal in comparison with the free caudals. The sulcus ventralis runs along the ventral side of the LS and SS. The distal end of the processi costale, processi transversus and processus spinosus are still cartilaginous.

Ca1–6 (Fig. 6q) are similar to those in adults, except for the distal end of the processi transversi, the processus spinosus, and the complete processus haemalis that are not yet ossified. In the pygostyle (Fig. 6r), the last two vertebrae are fused and the suture is still visible. The rest of the vertebrae are still free. The corpus are cylindrical and each processi spinose are small, projected cranially, and contacting the previous vertebra.

Newborn and smallest chicks

The cervical vertebrae are differentiated from the rest due to their butterfly shape (in a dorsal perspective) given by the lamina dorsalis arcus. Only the center of the corpus and the proximal end of the processi costale of C5–7 are ossified. Both parts of the lamina dorsalis arcus are unfused. The processi articulare craniale and the processi articulares caudales are the only slightly ossified processes.

The cervicothoracic vertebrae have short ribs. The corpus have only the center ossified, whereas all the processes are still cartilaginous. Both parts of the arcus are independent and the area where the processus spinosus will be placed remains cartilaginous. The processi transversi are only proximally ossified.

The thoracic vertebrae are ossified only on the center. In the arcus, both parts are still independent and all the processes remain cartilaginous. Only the processi transversi are proximally ossified.

All the synsacral vertebrae are slightly ossified and the segments are not differentiated. Each vertebral body is free and cylindrical. The arcus are poorly ossified.

The caudal vertebrae are poorly ossified and all the structures are very small and barely developed. The elements of the pygostyle are not differentiated from the free caudal ones.

Regionalization analysis for C–CT–T vertebrae (Online Resource 1, Supplementary Tables 1, 2)

Adults

PC1 (66.1%) and PC2 (18.5%) represent the 84.6% of the explained variation (Online Resource 1, Fig. S2). The PC1 separates the cervical vertebrae which are located in the upper and lower right quadrants, from the cervicothoracics and thoracics ones, which are located in the upper and lower left quadrants. The cervicothoracics are separated from the thoracics by the PC2. The variables that affect the ordering of these vertebrae are K–J (the length between the cranial end and the caudal end of the corpus), E–F (the width between the most caudal ends of the processi articulares caudales), B–F (the length between the cranial end of the processi articulare craniale, and the caudal end of the processi articulares caudales), and I–J (the total length of the arcus) on the PC1; and L–M (the length between the fovea cranioventralis and the caudal end of the corpus), and G–H (the width of the cranial ends of the) on the PC2.

Fledgling

The 84.1% of the observed variation is explained when the PC1 (54.9%) and PC2 (29.2%) are retained (Online Resource 1, Supplementary Fig. S3). Cervicals are located in the right quadrants and separated from the cervicothoracics and thoracics ordered in the left quadrants. The variables that most contributed to the ordering of the vertebrae according to regions are G–H (the width between the tori dorsales), E–F (the width between the most caudal ends of the processi articulares caudales), I–J (the total length of the arcus), and K–J (the length between the cranial end of the corpus and the caudal end of the arcus) on PC1; and K–N (the length between the facies articularis cranialis and the facies articularis caudalis), and L–M (the length between the fovea cranioventralis and the caudal end of the corpus) on PC2.

Modularity analyses

Elliptical Fourier analysis

Adult (MLP-O-15192)

The harmonic power calibration revealed that 15 harmonics for each normalized outlines were sufficient to achieve 99.9% of the total power. The representativeness of this range was confirmed visually with the reconstructed shapes, and therefore used to do the EFA. The PCA carried out with the matrix of harmonic coefficients obtained with the EFA revealed that the cumulative proportion of the variance explained with the first three components was 92.9%. The first PC explains the shape of the articular facet placed along their horizontal axis, ranging from drop-shapes with angular ends to laterally elongated shapes. The second PC explains the roundness of the shapes. The third PC explains the lateral variations of the shapes, varying from concave to convex to straight sides.

Even though the broken stick model limited the number of PCs to three, an LDA was carried out revealing that, under the highest precision for the discrimination of the groups, the number of PC to retain was six. This value was used to carry out the discriminant analysis, which allowed the ordering of the groups. The first two LD axes captured 93.1% of the variance between groups. The first LD corresponds to the axis that clearly separates the groups that correspond to Mo2–5–6 from the groups that correspond to Mo3–4. The second LD corresponds to the axis that slightly discriminates the Mo2–4 from Mo5, and from the groups of Mo3–6. The leave-one-out cross-validation, had a classification accuracy of only 20%, classifying correctly only two out of ten elements, whereas its precision was also low (see Online Resource 1, Supplementary Figs. S4, S5, S6, S7, S8, S9, S10, S11, S12, S13, S14).

Adult (MACN-OR-8462)

In this analysis, the harmonic power calibration revealed that 22 harmonics for each normalized outline were sufficient to achieve 99.9% of the total power. The results of the PCA revealed a 79.1% of the cumulative proportion of the variance explained with the first two components. The first PC captured the variation of the shape along a horizontal axis of the articular facet, meaning that it explains the width of said facets. The second PC captured the variation of the shape along the longitudinal axis of the facet, explaining the length of the articular facets, and the roundness or angularity of its ends.

The broken stick model limited the number of PCs to two, and the LDA carried out revealed that the number of PC to retain was seven. This value was used to carry out the discriminant analysis, which allowed the ordering of the groups. The first two LD axes captured 99.9% of the variance between groups. The first LD corresponds to the axis that clearly separates all the groups corresponding to all the modules. The second LD corresponds to the axis that slightly discriminates the Mo5–3 from Mo4 to 6 groups. The leave-one-out cross-validation had a classification accuracy of only 11.1%, classifying correctly only one out of nine elements, whereas the precision was also low (see Online Resource 1, Supplementary Figs. S15, S16, S17, S18, S19, S20, S21, S22, S23, S24, S25).

Fledgling (MLP-1014)

Harmonic power calibration revealed that 14 harmonics for each normalized outline were enough to achieve 99.9% of total power. The PCA using the harmonic coefficients matrix revealed a 95.7% of cumulated variance explained with the first three components. The first PC explains the variation of the shape in the upper end of the articular facets, varying from angular to rounded. The second PC explains the variability of the shape at the lower end, also varying from angular to rounded. The third PC explains the width of the articular facet, varying from rounded to oval shaped.

The broken stick model limited the number of PCs to three, and the LDA carried out revealed that the number of PC to retain was five. The discriminant analysis carried out with this value allowed the ordering of the groups. The first two LD axes captured 99.9% of the variance between groups. The first LD corresponds to the axis that clearly separates the Mo5, the Mo6 and the Mo3–4 groups. The second LD corresponds to the axis that slightly discriminates the Mo3 from the Mo4 groups. The leave-one-out cross-validation had a classification accuracy of 50%, classifying correctly five out of ten elements, whereas the precision was low (see Online Resource 1, Supplementary Figs. S26, S27, S28, S29, S30, S31, S32, S33, S34, S35, S36).

Linear measurements

The PCA based on linear measurements allowed the determination of modules according to morphological similarity (Online Resource 1, Tables 6, 7).

Adults

PC1 represents the 61.2% of the total variation, and PC2 adds the 18.5%, representing the 79.7% of the accumulated explained variation (Online Resource 1, Supplementary Fig. S37). The PC1 shows the vertebrae corresponding to Mo3 (C3–5) on the left side of the graph separated from the rest which are located on the right. The PC2 separates the vertebrae of Mo4 and Mo5 (C6–9) on the lower part of the graph, from those of Mo6 (C10–13) shown on the upper part. In turn, among the former, the Mo4 (C6) can be differentiated from the others vertebrae of the Mo5. The variables with the greatest weight were I3 (development of the ansa costotransversaria), I4 (elongation index), L–M (the length between the fovea cranioventralis and the facies articularis caudalis), Ang (angle between processi articulare craniale), and K–N (the length between the facies articularis cranialis and the facies articularis caudalis) on the PC1; and K–J (the length between the cranial end of the corpus and the caudal end of the arcus), I3 (development of the ansa costotransversaria), B–F (the length between the cranial end of the processi articulare craniale and the caudal end of the processi articulares caudales), and I–J (the length of the arcus) on the PC2.

Fledgling

PC1 gathers the 57.1% of the explained variation, and PC2 accumulates a 27.0% more, making a total of 84.1% of the variation (Online Resource 1, Supplementary Fig. S38). The PC1 divides the vertebrae of Mo3 (C3–5) in the lower left quadrant, from those of Mo4 (C6) and Mo5 (C7–9) in the right lower quadrant. The vertebrae of Mo6 (C11–13) are grouped on the upper right and left quadrants. The variables that affect the ordering of these vertebrae are K–N (the length between the facies articularis cranialis and the facies articularis caudalis), I3 (development of the ansa costotransversaria), B–F (the length between the cranial end of the processi articulare craniale and the caudal end of the processi articulares caudales), and G–H (the width between the tori dorsales) on the PC1; and I–J (the length of the arcus), I2 (development or functional part of the facies articularis caudalis), A–B (the width between the most cranial ends of the processi articulare craniale); and I3 (development of the ansa costotransversaria) on the PC2.

Discussion and conclusions

Remarks on spine morphology and regionalization

The comparative morphology and the morphometric analyses of the cervical, cervicothoracic, and thoracic vertebrae of A. forsteri allowed us to recognize the same regions previously known for other penguin species. The spine of A. forsteri presents 42/43 vertebrae divided as follows: 13 cervicals, 2 cervicothoracics, 6/7 thoracics, 13 vertebrae fused into a synsacrum, 7 free caudals, and 5 caudals fused into a pygostyle (counted here as a single vertebral element).

Our results are coherent with the morphological descriptions of the cervicals for penguins provided by Zusi (1962) and Baumel and Witmer (1993), and the number of elements established by Watson (1883), Stephan (1979), Guinard et al. (2010), and Guinard and Marchand (2010). Except for C1 (Atlas) and C2 (Axis), the remaining cervical vertebrae are characterized by a homogeneous morphology, which is reflected in the PCA analyses.

The characters described above vary according to their location on the spine. The processus ventralis corporis is projected caudally in C2–4 and cranially in C11–13. The fovea cranioventralis is only present in these same vertebrae. The processus ventralis corporis and the fovea cranioventralis are absent in the middle region of the neck (C5–10), where the processi carotici appear delimiting the sulcus caroticus. The processus spinosus appears with a variable development and inclination in the cranialmost (C2–7) and caudalmost (C13) vertebrae, and it is reduced or absent in the vertebrae of the middle region (C8–12). The processi articulare craniale varies from rounded (C2–5) to teardrop-shaped (C6–10) or oval (C11–13), and it is dorsomedially (C3–6, C12–13) or dorsocranially (C7–11) positioned along the neck. The tori dorsales varies from blunt (C2–5) to elongated (C10–12) and are located at the caudal end of the lamina dorsalis arcus (on the processi articulares caudales), or cranially displaced in the vertebrae of the middle region (C6–9). Among living species, the axis presents a peculiarity. Although the general morphology is similar to the rest of the cervicals, a distal expansion appears on the processus ventralis corporis; feature also observed in the fossil penguin Icadyptes salasi (see Ksepka et al. 2008).

The cervicothoracic vertebrae are morphologically intermediate between the cervicals and the thoracics. The similarities with the last cervicals and the first thoracics are also reflected in the PCA analysis. Like the cervicals, the cervicothoracics are heterocoelous, with a flattened vertebral body and a well-developed processus ventralis corporis, processi transversi laterally elongated with a dorsal curvature at the distal end, and oval and large projected processi articulare craniale dorsocranially. Like the thoracics, the cervicothoracics are laterally compressed and present a rectangular processus spinosus, and eminentia costolaterales for the articulation of the ribs. Precisely, the presence of non-fused but short ribs that do not contact the sternum is the distinctive feature of these vertebrae. Beyond the similarities with the cervicals and thoracics, and from a morpho-functional perspective, the cervicothoracics could be considered as a part of the neck (see Vanden Berge 1999).

In agreement with Shufeldt (1901), we counted six opisthocoelous vertebrae in the thoracic region (see also Shufeldt 1901; Baumel and Witmer 1993). These vertebrae are characterized by a high and laterally compressed corpus. The first thoracic vertebra (T1) presents heterocoelous facies articularis cranialis, although the facies articularis caudalis is concave. This heterocoelous condition for the cranial end of the corpus of T1 has already been observed in E. chrysocome by Watson (1883), and in P. papua by Jadwiszczak (2014a). The processus ventralis corporis is highly developed, especially in T1–4. From T1 to 3, this process is bifid, character related to diving adaptations (Kuroda 1954). In this region, the arcus extends laterally beyond the vertebral body. As it was observed by Watson (1883), the processus spinosus is rectangular and spanning the entire arch. It is laterally compressed, and has a flattened distal expansion in all the vertebrae of this region. Caudal to this process is an angular notch (area ligamentum elasticum) where the interlaminar and interspinous elastic ligaments are inserted (Baumel and Witmer 1993). Unlike the processi articulare of the vertebrae of the previous two regions, the processi articulare craniale and processi articulares caudales of the thoracics are small and rounded; projected dorsally and ventrally, respectively. These vertebrae also have highly developed processi transversi that are flattened and directed backwards with the eminentia costolateralis on the ventral surface of the distal ends of these processes, as it was also described by Watson (1883). These vertebrae articulate with each other constituting a straight and rigid structure.

The synsacrum, formed by the fusion of 13 vertebrae in the adult, is composed by 5 segments, (Stephan, 1979). The free caudals have a distinctive morphology with respect to the other regions. These vertebrae have a cylindrical corpus and a triangular arcus. Contrarily with the observations of Watson (1883), who described a distally bifid processus spinosus in Ca1–4 only, the specimens here examined have this condition in Ca1–5 and a simple and distally curved distal end in Ca6–7. The processus transversus are rectangular and flattened as described by Watson (1883). Although the fusion of six to seven vertebrae along the ontogeny of penguins was described by Stephan (1979) and Pycraft (1898), we found only five vertebrae fused into the pygostyle of A. forsteri.

Remarks on individual variations

A numerical variation was observed in the thoracic region of the specimen MLP-478. As we described above, the thoracic region of A. forsteri is characterized by the presence of six vertebrae. However, MLP-478 has seven thoracic vertebrae. The last of these vertebrae (named here as T7, Fig. 5a, b) is morphologically indistinguishable from the rest of the thoracics. It means that the corpus is opisthocoelous, the arcus does not extend beyond the laterals of the vertebral body, the processi articulare craniale and caudale are small and rounded, the processus spinosus is quadrangular and compressed and covers the entire extension of the arch, and the eminentia costolaterales are developed. However, T7 is completely devoid of processus ventralis corporis, and the distal end of the processi transversi are dorsally curved and contact the ventral side of the ala preacetabularis ilii; morphology found in the thoracal segment of the synsacrum in the rest of the specimens. However, if T7 would correspond to the thoracal segment, the synsacrum of specimen MLP-478 would have only twelve fused vertebrae. This is not the case since the synsacrum of MLP-478 has the thirteen vertebrae described above (Fig. 5c), plus the additional T7 element. Another particular condition is that the first synsacral vertebra of MLP-478 presents a morphology according to the thoracolumbar segment. We discard that T7 could correspond to the thoracal segment due to the absence of the suture lines between the synsacral segments that characterize the sub-adult.

The presence of the T7 could represent an intra-specific condition or a frequent abnormality. These numerical variations in the spine elements were already described in humans (Decker 1915; Cimen and Elden 1999) and treated numerical anomalies (Oostra et al. 2005) in goats (Simoens et al. 1983), and cranes (Family Gruidae) within wild birds (Hiraga et al. 2014). As the case reported here, these anomalies usually occur in transitional zones.

Remarks on ontogenetic variations

As expected, differences in the ossification degree of the vertebral elements between each region appear along the postnatal ontogeny. None of the spine elements in chicks are completely developed, and the corpus are always separated from the arcus. The processi costale of the cervical vertebrae are partially ossified, but not fused to the vertebrae in C5–7. Neither the synsacrum nor the pygostyle are formed; therefore, all the constituting vertebra are free.

The ossification is greater in fledgling specimens, in which each vertebra partially acquires the adult configuration and, therefore, they can be grouped according to regions. The cervicothoracic region is not completely defined, although the particular features of these vertebrae are already present. Although the synsacrum is not completely developed (only the lumbar segment is fused), each vertebra is perfectly distinguishable based on their oval body and general proportions (laterally wide and craniocaudally short shape). The same occurs with the pygostyle, which is still unfused, but each vertebra exhibits a distinctive shape that makes them also differentiable from the free caudals. In juveniles, the vertebrae are fully formed, and differ from adults due to the size and the presence of the suture lines between the elements. The suture lines between the elements of the synsacrum are also observed in juveniles of Aptenodytes and Pygoscelis specimens (Jadwiszczak 2014b). Only in the adult stage the whole spine is completely ossified and the sutures between the elements disappear.

Remarks on modularity

Although the outline of the articular facet of the right processus articularis cranialis was a useful tool for the recognition of cervical modules in other penguins (see Guinard et al. 2010), this parameter it was not as adequate as expected for their recognition in A. forsteri. The results of the PCA in adults and fledglings were not clear in determining the different modules. Clearer results were obtained in the LDA were the vertebrae of each module were slightly grouped. The grouping of the vertebrae into modules was more notable in adults, where Mo2 (Axis) groups vertebrae with a particular oval shape and a lateral prominence; Mo3 (C3–5) is characterized by being laterally elongated, and triangular to oval in shape; Mo4 (C6) tends to be quadrangular; Mo5 (C7–9) differs by being drop-shaped; and Mo6 (C10–13) is oval and longitudinally elongated. In the fledgling, this groups were not so clear. Although the vertebral groups corresponding to the modules showed some differentiation, this was not so marked. This can be attributed to the fact that at this stage the processi articulare craniale are characterized by being mostly oval in shape, and not completely ossified.

In contrast, when using linear measurements, the same modules recognized for the cervical region of other penguin species were recognized in A. forsteri. Mo1 (C1 or atlas) and Mo2 (C2 or axis) are composed by a single element, Mo3 includes the C3, C4, and C5, elements. Mo4, called “transitional” (Guinard et al. 2010), is represented by C6. Mo5, is represented by vertebrae C7 to C9.

It is necessary to highlight a particularity with respect to Mo6. According to previous observations made in A. patagonicus and P. papua during the postnatal ontogeny, the main identifying character of Mo6 should be the sudden appearance of the processus ventralis corporis accompanied by the disappearance of the processi carotici in C10 (Guinard et al. 2010). The C10 of the adult MLP-O-15192 (Fig. 7d, h) has a barely developed processus ventralis corporis accompanied by the processi carotici. Further comparisons were not possible due to the lack of preservation of C10 in MACN-OR-8462 and MLP-478. As this transformation occurs during growth, the processus ventralis corporis in C10 could be a vestige of the pre-adult stages. However, this process is not present in any of the immature specimens studied here (Fig. 7a–c, e–g). In fledgling and juveniles, the processus ventralis corporis is ossified in vertebrae C11 to C13. This feature is relevant in terms of neck mobility because the M. longus colli ventralis inserts on the processus ventralis corporis of the most caudal vertebrae and the processus caroticus of the mid-cervical region (Baumel and Witmer 1993).

Remarks on the mobility of the neck

Given that individuals of A. forsteri begin their aquatic life once they molt (Stonehouse 1953), several skeletal and muscular structures are not completely developed while they are kept on land and fed by their parents. The spine, and particularly the neck muscles, are a clear example of that.

As excellent swimmers, penguins require a hydrodynamic shape to optimize their underwater movements. Thanks to the variated morphology of the vertebrae along the spine, the neck acquires an S-shape during diving and the head is retracted against the body (Guinard et al. 2010). However, this is not possible if the ossification degree is insufficient, situation observed in the chicks here examined.

A significant change in the ossification degree and the development of the articular surfaces that participate in the spine mobility is observed in the juveniles of A. forsteri. Although the involved structures are not fully fused yet, the characteristic S-shape observed in penguins is already marked. Equivalent ontogenetic stages examined in other penguin species were considered unsuitable for an efficient diving (Guinard et al. 2010).

In adults with the spine completely developed, and as in other penguin species, the S-shaped is automatically generated by contacting each of the cervical vertebrae (Fig. 8). The first ventral curvature of the neck is determined by Mo1, Mo2, and Mo3; Mo4 is interpreted as transitional. The following dorsal curvature (mechanical curvature sensu Guinard et al. 2010) is represented by Mo5. The second ventral curvature is restricted to Mo6 which contacts with the cervicothoracic region. Summarizing, this modular organization in the curvatures of the neck is coincident with that proposed for other adult penguins by Guinard et al. (2010).

Fig. 8

Schematic draw showing the modularity in the neck of Aptenodytes forsteri, MLP-O-15192 left lateral view. Mo indicates the modules; C1–13 indicates the number of cervical vertebrae. Scale bar 10 mm