Cervical vertebrae. The processus ventralis corporis appears from C2 to C4 in Aptenodytes forsteri (Fig. 1A), Pygoscelis papua, Spheniscus magellanicus, and from C2 to C3 in Pygoscelis adeliae. Subsequently, this process manifests again from C10 to C13 in A. forsteri, P. adeliae, Pygoscelis antarcticus, S. magellanicus and Eudyptes chrysocome. In P. papua and a specimen of P. adeliae (MLP-ORN 15475), it is observed between C9 and C13. The processus carotici develop from C5 to C9 in A. forsteri (Fig. 1B), and S. magellanicus, from C4 to C8 or C9 in P. adeliae, from C5 to C8 in P. papua and from C7 to C9 in E. chrysocome. The processus costales exhibit a consistent short and spine-like structure across all specimens (Fig. 1C). The processus transversi are slightly developed up to C11 and extend laterodorsally further from C11 to C13 (Fig. 1D) in A. forsteri, P. adeliae, P. papua, S. magellanicus, and E. chrysocome. In the C13 of P. antarcticus, these processus are uniquely dorsally curved. Lastly, the processus spinosus is observed from C2 to C6 and from C12 to C13 in all specimens (Fig. 1D).

Cervicothoracic vertebrae. The vertebral body exhibits subtle variations, being slightly rectangular in A. forsteri and quadrangular in the remaining species (Fig. 1E–I). In CT1, the processus ventralis corporis is simple with two well-defined alae cristae ventrales. However, in CT2 it is bifurcated in all species. Both vertebrae have a deep fovea cranioventralis and a well-developed processus transversi, with a rounded and small eminentiae costolaterales. The processus spinosus assumes a quadrangular, flattened structure, covering the entire length of the vertebral arch in all species. Notably, in CT1 of A. forsteri, it presents a shorter length and a midline position on the vertebral arch.

Thoracic vertebrae. All vertebrae are opisthocoelus, but the cranial articular facet of T1 acquires a saddle-shape in all species. The processus ventralis corporis decreases cranio-caudally in length (Fig. 1J). It is bifurcated (Fig. 1K) from T1 to T3 transitioning to a simple form from T4 to T6 in A. forsteri, P. adeliae, P. antarcticus, E. chrysocome, and some specimens of S. magellanicus. In contrast, other specimens of S. magellanicus and P. papua, exhibit bifurcated processus ventralis corporis in T1 and T2 and simple processes from T3 to T6. The eminentiae costolaterales, rounded in shape, are prominently developed in A. forsteri, but smaller in the remaining species. The processus transversi are quadrangular, dorsoventrally flattened and laterally expanded. The processus spinosus is well-developed, quadrangular, compressed, and covers the entire length of the vertebral arch. Noteworthy variations occur when T5 is the last thoracic, resulting in a reduced and simple processus ventralis corporis. Conversely, when the T7 is present (Fig. 1L–O), this process is absent, while the dorsally curved and well-developed processus transversi make contact with the alae praeacetabulares ilii of the pelvic girdle.

Synsacrum. The synsacral body, robust and slightly curved, is divided into the classical five segments (Fig. 1J): TS, TLS, LS, SS, and CS. Between S1 and S4, the synsacral body is strongly compressed, a feature consistently observed across most species, with P. papua displaying a slightly less pronounced compression. From S4 to the caudal end, the synsacrum exhibits dorso-ventral depression, widening between S4 and S10, and subsequently decreasing in size from S11 to the caudal end. The processus transversi exhibit variability, being long in S1 and S2 and gradually decreasing in length until the CS. In the CS, these processes acquire a rectangular shape and extend laterally.

The sulcus ventralis synsacri varies in depth and width, being deep and broad in E. chrysocome, narrow in P. adeliae, P. antarcticus, P. papua, and S. magellanicus, and absent in A. forsteri. The processus costales are developed between S4–S6 in S. magellanicus, P. adeliae, A. forsteri, P. papua, and P. antarcticus, S5–S6 in E. chrysocome, and then in S8–S9 in E. chrysocome, S9– S10 in S. magellanicus, and P. adeliae, and S10–S11 in A. forsteri, P. papua, and P. antarcticus. The crista spinosa synsacri, compressed at the cranial end, decreases in height craniocaudally toward the last synsacral vertebra.

A significant interspecific variation is observed in the fusion of the synsacrum to the pelvic girdle. Aptenodytes forsteri, S. magellanicus, and E. chrysocome do not exhibit fusion. On the contrary, in P. adeliae fusion occurs to varying degrees, involving the last two vertebrae of the TLS, all vertebrae of the LS and SS, and the first two (e.g., MLP-ORN 15476) or three (MACN-Or 68826) vertebrae of the CS. Pygoscelis antarcticus presents full fusion, with the processus transversi and the processus costales of all synsacral vertebrae fused to the pelvic girdle. In P. papua, fusion affects the last three vertebrae of the TLS, all vertebrae of the LS and SS, and the first (MACN-Or 68596) or second (MLP-ORN 14767) vertebrae of the CS.

Free caudal vertebrae. They exhibit a cylindrical vertebral body (Fig. 1P, Q) characterized by slightly concave or flat articular facets and a triangular vertebral arch. A bifid processus spinosus is observed in varying segments across different penguin species. In E. chrysocome it occurs from Ca1 to Ca3, in some specimens (e.g. MLP-ORN 15138) of P. adeliae and in P. papua from Ca1 to Ca4. Notably, in A. forsteri, P. antarcticus, and S. magellanicus, as well as some specimens (MACN-Or 68558) of P. adeliae, the bifid processus spinosus is observed from Ca1 to Ca5. Conversely, in the remaining free caudal vertebrae, the processus spinosus assumes a simple form. The processus haemalis appear consistently from Ca4 to the last free caudal vertebra. Robust and quadrangular, the processus transversi exhibit a consistent morphology across all free caudal vertebrae.

Pygostyle. It results from the ankylosis of five caudal vertebrae, progressively decreasing craniocaudally in size, forming a triangular single structure (Fig. 1Q, R). In subadults, the initial vertebra of the pygostyle remains unfused, displaying a cylindrical vertebral body with a well-developed processus haemalis, a poorly developed processus spinosus, and weakly (or absent) processus transversi (Fig. 1S).

The present work examined the complete spine of 66 specimens (Table 2), offering new and updated insights into six species inhabiting Southern South America and Antarctica. The findings represent the first data for Pygoscelis antarcticus, and include updated observations that expand upon previous knowledge for Eudyptes chrysocome, Pygoscelis papua, Pygoscelis adeliae, Aptenodytes forsteri, and Spheniscus magellanicus (Watson 1883; Shufeldt 1901; Verheyen 1958a; Stephan 1979; Guinard and Marchand 2010; Guinard et al. 2010; Jadwiszczak 2014; Pereira 2015; Sosa and Acosta Hospitaleche 2022). As a result, the general vertebral formula for the six studied Sphenisciformes species can be established as C₁₃ + CT₂ + T₆ + S_13/14 + Ca_6/7 + P₅₍₁₎ = 41/42.

Our findings reveal a total number of 42 vertebrae for E. chrysocome, P. papua, and A. forsteri, consistent with previous reports (Watson 1883; Shufeldt 1901; Sosa and Acosta Hospitaleche 2022). Similarly, P. adeliae and P. antarcticus, not examined by previous authors, also present this same count. Spheniscus magellanicus shows a total of 41 vertebrae, contrasting with the 43 elements reported by previous studies (e.g., Watson 1883). This mismatch was also pointed out by Pereira (2015), who counted 42 or 41 vertebrae in different specimens.

The cervical region consistently comprises 13 vertebrae across all analyzed species, aligning with observations by Gervais and Alix (1877), Watson (1883), Verheyen (1958a), Stephan (1979), Guinard and Marchand (2010), Guinard et al. (2010), and Sosa and Acosta Hospitaleche (2022). Morphologically, the cervical vertebrae of the studied penguin species are similar and almost indistinguishable, except for the sturdier vertebrae of the large-bodied species A. forsteri. The main differences occurred in the presence/absence of specific structures when comparing different species. For instance, in A. forsteri, the processus spinosus is present from C2 to C7 and in C13, whereas in other species, it appears from C2 to C6 and from C12 to C13. Similarly, the processus ventralis corporis is observed from C2 to C4 in A. forsteri and P. papua, from C2 to C3 in P. adeliae, from C9 to C13 in P. papua and P. adeliae, and C10 to C13 in P. adeliae (again) and A .forsteri.

In all species, two cervicothoracic vertebrae, also referred as “transitional” (Fürbringer 1888), were identified. These vertebrae exhibit an intermediate morphology between cervical and thoracic vertebrae. While mentioned and described only by Jadwiszczak (2014) and Sosa and Acosta Hospitaleche (2022) in penguins, other authors include them either within the cervical (Shufeldt 1901; Verheyen 1958a, although the latter recognized their distinct morphology) or thoracic region (Watson 1883; Stephan 1979).

The thoracic vertebrae, characterized by the presence of unfused ribs articulating with the sternum (Baumel and Witmer 1993) appear usually in a number of six. Except by the cranial articular facet of T1, all thoracics are opisthocoelus (see Baumel and Witmer 1993; Verheyen 1958a; Bellairs and Jenkin 1960; Watson 1883; Sosa and Acosta Hospitaleche 2022). Confirming Verheyen (1858a) observations, we noted that the processus ventralis corporis are bifid only in the first two or three thoracic vertebrae.

The first vertebra, ankylosed to the synsacrum and bearing ribs, used to be included in the thoracic region (Watson 1883; Takashima and Mizuma 1981; Hiraga et al. 2013), contributing to variations in vertebrae count across different author criteria (Pereira 2015). Indeed, the synsacrum, with 13–14 vertebrae, is the most variable element in terms of size, robustness, composition, and association with the pelvic girdle. In line with the classifications of Bellairs and Jenkin (1960) and Stephan (1979), the five synsacral segments were identified. Contrary to Watson (1883), in all Pygoscelis species, the synsacrum is fused to the pelvic girdle.

Most species, such as P. adeliae, P. antarcticus, P. papua, and S. magellanicus, exhibit six free caudal vertebrae. However, seven free caudal vertebrae are counted in A. forsteri and E. chrysocome. Variations in the number of caudal vertebrae have also been reported in cranes (­Gruidae) (Hiraga et al. 2013). The pygostyle resulted as the most conservative element, composed of five vertebrae ankylosed forming a triangular structure in all species described here (see also Felice and O’Connor 2014). However, Verheyen (1958a) identified four to five elements in most of the living penguin species, and Stephan (1979) described six to seven caudal vertebrae constituting the pygostyle.

Numerical changes in the vertebral series can result from meristic variations, also called numerical anomalies (Verheyen 1858a, 1858b; Verheyen 1960; Oostra et al. 2005), or homeotic transformations. Meristic variations involve alterations in the total number of segments or elements, while homeotic transformations encompass variations in the number and ordinal position of vertebrae within or between regions, or the acquisition of morphology typical of another position in the spine. Importantly, the latter does not necessarily lead to a change in the total number of vertebrae (Bateson 1894; Guinard 2012). Examples of homeotic transformations includes where some lumbar vertebrae adopt the morphology the sacral vertebrae, with modified processus transversi supporting the pelvic girdle (Bateson 1894). While numerical variations in the vertebral column have been extensively studied in humans (Decker 1915; Danforth 1930; Schultz 1947; Bornstein and Peterson 1966; de Beer Kaufman 1974; Cimen and Elden 1999; Oostra et al. 2005; Lian et al. 2018), similar occurrences have been reported in goats (Simoens et al. 1982 and literature cited there), primates (Schultz 1947), salamanders (Bumpus 1897), and rabbits (Sawin et al. 1967). In general, supernumerary vertebrae are most commonly observed in the thoraco-lumbar or lumbo-sacral transition zones (Sawin et al. 1967), although occurrences have been noted in thoracic and lumbar vertebrae (de Beer Kaufman 1974; Hiraga et al. 2013) and even in cervical regions (Hiraga et al. 2013). Various factors contribute to these numerical variations, including sexual differences, responses to different stimuli, genetic variability (Danforth 1930; Borstein and Peterson 1966; Lian et al. 2018), growth and body size due to differences in the number of genes (Sawin 1967 and literature cited there), and population differences (Borstein and Peterson 1966), but not to age differences (Bornstein and Peterson 1966).

We found numerical variabilities in the thoracic, synsacral, and caudal regions among species (Fig. 3). Aptenodytes forsteri, P. adeliae, P. papua, and S. magellanicus show numerical anomalies in the thoracic (+1, or –1) or in the caudal (+2, +1, or –1) regions. For instance, one specimen of of A. forsteri (MLP-ORN 1586) exhibited an additional vertebra (T7), consistent with findings reported by Sosa and Acosta Hospitaleche (2022) and verified in this study. In the caudal region, additional vertebrae were found in four species: a seventh free caudal in P. adeliae (MLP-ORN 15038 and MLP-ORN 14680), P. papua (MLP-ORN 14921), and S. magellanicus (MLP-ORN 949 and MLP-ORN 950), and an eighth free caudal in one specimen of S. magellanicus (MLP-ORN 920). Furthermore, instances of missing vertebrae concerning the generalized vertebral formula were observed, such as in one specimen of S. magellanicus (MLP-ORN 1408) with only five thoracic vertebrae, and in P. papua, where one vertebra was absent in the TLS of the synsacrum (MLP-ORN 15410), or with only five free caudal vertebrae (MLP-ORN 14669 and MLP-ORN 14610).

The numerical anomalies identified in Sphenisciformes were attributed to ontogenetic causes by Verheyen (1958a). However, it is plausible that homeotic transformations account for the variations observed in the synsacrum among certain penguin species examined in this study. Specifically, in some specimens of P. adeliae and S. magellanicus, a vertebra from the TLS acquires the morphology of another vertebra from the TS, maintaining the total number of vertebrae for the species. Other examples include cases of P. adeliae (MLP-ORN 15137 and MACN-Or 68557) with seven free caudal vertebrae and a missing element in the CS (MLP-ORN 15038 and MLP-ORN 14680 in the two former specimens). Despite this variations, they retain a total of 42 vertebrae, with 13 vertebrae in the synsacrum and one fewer in the CS (MLP-ORN 15137 and MACN-Or 68557), a pattern also observed in the latter two specimens. Remarkably, these two last specimens also develop seven thoracic vertebrae and six free caudals. In the case of S. magellanicus, one specimen (MACN-Or 71167) presented a T7 (also mentioned by Pereira (2014) in the 19% of her sample) and a missing free caudal vertebra, but maintain the count of 41 vertebrae, which is typical for the species. All these specimens exhibit the thoracic vertebra corresponding to the TS ankylosed to the synsacrum, eliminating the possibility of confusion arising from the absence of fusion or incomplete ontogenetic development.

Other cases of numerical variation probably caused by homeotic transformations are observed within the synsacrum, characterized by counts of either 13 or 14 vertebrae. These variations predominantly occur in the TLS and CS, although occurrences were also noted in the TS. In one specimen of P. papua (MLP-ORN 14921) the TLS displayed an unusual development: the first vertebra exhibited eminentiae costolaterales (like those of the TS), while the last two vertebrae possessed processus costales, a feature typically seen in three vertebrae among the majority of specimens of this species. This specimen presented six thoracic vertebrae and a total of 14 synsacral vertebrae, which aligns with the general count for the species. Another specimen of P. papua (MLP-ORN 15410) presented only 13 vertebrae in the synsacrum, with the missing vertebra corresponding to the TLS, where the first two vertebrae displayed processus transversi, while the last two presented processus costales. Despite this variation, this specimen exhibited six thoracic and six caudal vertebrae, representing the most common count for this species.

The most significant variability in the number and configuration of synsacral vertebrae (ranging from 13 to 14 vertebrae) among penguins was observed in S. magellanicus. Specimens with 13 synsacral vertebrae exhibit one missing element in the CS, maintaining the typical total count for the species. However, the specimen MACN-Or 73286 has one vertebra less in the TLS.