Our analysis reveals that the PELA and PECA of Latimeria and Ambystoma and the PELA of Neoceratodus share a very similar, complex configuration of homologous (between PECAs) and topologically corresponding (between PECAs and PELAs) muscles (Tables S5–S7; Figs 1, 2 and 3). Among several striking similarities, the two limbs (PELA and PECA of Ambystoma) and three fins (PECA and PELA of Latimeria and PELA of Neoceratodus) share dorsal and ventral superficial muscle masses that extend from the girdles to the distal regions of the fins/limbs, a series of similar dorsal and ventral deep muscles (supinators and pronators and their derivatives), and pre- and postaxial muscles that often span more than one joint. Based on this evidence and on the strong evidence that the very simplified PECA muscle anatomy of Neoceratodus is a derived characteristic of dipnoans (see SI for details; see also in ref. 35), we propose that the characteristic muscle configuration of the tetrapod limbs arose through a series of stepwise changes from the last common ancestor (LCA) of extant osteichthyans to the LCA of tetrapods. The LCA of extant gnathostomes most likely had five muscles in each paired fin: ventrally, the abductor superficialis, abductor profundus and a preaxial muscle pterygialis cranialis; dorsally, the adductor superficialis and adductor profundus36 (Tables S5–S6). The LCA of extant bony fishes probably had the same five muscles plus a postaxial muscle (pterygialis caudalis) in both the PELA and PECA, because the plesiomorphic extant osteichthyan Polypterus (see in ref. 21 and our observations) and Latimeria share the presence of this muscle in each appendage (Tables S5–6, Figs 1 and 3). This postaxial muscle (“zonopropterygialis” in Polypterus PECA sensu Wilhelm et al.21; present in both its PECA and PELA according to our observations) and the preaxial muscle pterygialis cranialis (“coracometapterygialis I and II” in Polypterus PECA sensu Wilhelm et al.21; present in both its PECA and PELA according to our observations) are thought to be derived from the dorsal (adductor/‘levator’) and ventral (abductor/‘depressor’) fin musculature, respectively36. However, in some fishes ventral muscles may be differentiated postaxially, and dorsal muscles preaxially34.

Figure 1 Muscle maps. Right pectoral (A–D) and pelvic (E–H) appendages of Neoceratodus (A,B,E,F) and Latimeria (C,D,G,H) in dorsal (A,C,E,G) and ventral (B,D,F,H) views. Note that the use of similar colors in the pectoral and pelvic muscles does not indicate ancestral serial homology between the structures of these paired appendages, but instead the result of derived similarity (see text). Full size image

Figure 2 Hypotheses of hindlimb muscle homology; similar colors indicate homologous muscles. Latimeria (A,B), Neoceratodus (C,D), and Ambystoma (E,F). Dorsal views (A,C,E) and ventral views (B,D,F). Abbreviations: abductor dorsolateralis (abd. dorsolat.), abductor profundus (abd. prof.), abductor superficialis (abd. sup.), abductor digiti minimi (AbD5), abductor et extensor digiti I (AbED1), adductor femoris (ADD), adductor profundus (add. prof.), adductor superficialis (add. sup.), contrahentium caput longum (CCL), caudofemoralis (CDF), caudalipuboischiotibialis (CPIT), extensor cruris tibialis (ECT), extensor cruris et tarsi fibularis (ECTF), extensores digitorum breves (EDB), extensor digitorum longus (EDL), elevator lateralis (elev. lat.), extensor tarsi tibialis (ETT), extensor iliotibialis (EXILT), flexor accessorius lateralis (FAL), flexor accessorius medialis (FAM), flexores breves superficiales (FBS), flexor digitorum communis (FDC), femorofibularis (FMFB), gracilis (GRA), iliocaudalis (ILC), intermetatarsales (IMT), interosseus cruris (IOC), ischiocaudalis (ISC), puboischiofemoralis externus (PIFE), puboischiofemoralis internus (PIFI), puboischiotibialis (PIT), pubotibialis (PTB), pronator profundus (PP), pubotibialis (PTB), pterygialis caudalis (pteryg. caud.), pterygialis cranialis (pteryg. cran.), segmented muscle (seg. m.), tenuissimus (T). Full size image

Figure 3 Hypotheses of forelimb muscle homology; similar colors indicate homologous muscles. Latimeria (A,B), Neoceratodus (C,D), and Ambystoma (E,F). Dorsal views (A,C,E) and ventral views (B,D,F). Colors indicate homologous muscles. Abbreviations: abductor digiti minimi (AbD4), abductor et extensor digit 1 (AbED1), coracobrachialis (CB), contrahentium caput longum (CCL), contrahentes digitorum (CD), deltoideus scapularis (DS), extensor antebrachii et carpi ulnaris (EACU), extensor carpi radialis + supinator (ECR + S), extensor digitorum (ED), extensores digitorum breves (EDB), flexor antebrachii et carpi radialis (FACR), flexor antebrachii et carpi ulnaris (FACU), flexor accessorius lateralis (FAL), flexor accessorius medialis (FAM), flexores breves profundi (FBP), flexor digitorum communis (FDC), humeroantebrachialis (HAB), intermetacarpales (IMC), latissimus dorsi (LD), levator scapulae (LS), pectoralis (P), procoracohumeralis (PCH), palmaris profundus 1 (PP1), serratus anterior (SA), supracoracoideus (SC), triceps coracoideus (TC), triceps humeralis lateralis (THL), triceps humeralis medialis (THM), triceps scapularis medialis (TSM), coracoradialis (CR), subcoracoscapularis (SCS), retractor lateralis ventralis pectoralis (ret. lat. vent. pect.), adductor superficialis (add. sup.), retractor lateralis ventralis pectoralis (ret. lat. vent. pect.), adductor profundus (add. prof.), segmented muscle (seg. m.), abductor superficialis (abd. sup.), abductor profundus (abd. prof.), pterygialis cranialis (pteryg. cran.), pterygialis caudalis (pteryg. caud.). Full size image

A striking implication of our synthesis is that the LCA of extant sarcopterygians probably already had the basic tetrapod limb phenotype in both PECA and PELA, with the exception of the characteristic tetrapod autopod (hand/foot) (Tables S5–S7; Fig. 4C). Specifically, this LCA probably had at least two layers of adductor and abductor muscles that were partially segmented proximo-distally at the level of each joint. That is, the dramatic changes between the LCA of extant bony fishes and the LCA of extant sarcopterygians affected in a markedly similar way the ventral and dorsal sides of both the PECA and PELA. In particular, the deep musculature (adductor profundus dorsally; abductor profundus ventrally) gave rise to a series of smaller muscles (pronators dorsally; supinators ventrally) (Figs 1, 2, 3 and 4; Tables S5–S7). An illustrative example of the pronounced overall PECA-PELA similarity of sarcopterygians is the almost identical configuration of the Latimeria PELA and PECA, which is in turn strikingly similar to that of the Neoceratodus PELA (Tables S5–S6; Figs 2 and 3). Because of this marked PECA-PELA similarity, most hypotheses of homology shown in Tables S5–S6 are straightforward. An extensive account of the rationale and evidence behind each of the hypotheses shown in these tables is provided in the SI.

Figure 4 Evolutionary and developmental transitions leading to the modern adult tetrapod limb. Evolutionary transitions in adult morphology exemplified by ventral musculature, based on the present paper (A): LCA of extant osteichthyans; (B): stem sarcopterygians; (C): LCA of extant sarcopterygians; (D): LCA of extant tetrapods: see cladogram on top left), and developmental transitions from early stages to adult morphology in tetrapods (E–H), exemplified by ontogeny of ventral musculature in chicken hindlimb (based on Kardon26). All images show dorsal views with dorsal muscles (therefore including the dorsal, postaxial muscle pterygialis caudalis) removed. Full size image

As shown in Fig. 4, the inferred order of phylogenetic events leading to the origin of tetrapod limbs is very similar to that of the ontogeny of the limbs of extant tetrapods. Moreover, the rotation of the paired appendages (internal rotation sensu human anatomy) that occurred over the fins-limbs transition, turning the ventrolateral abductor (‘depressor’) fin musculature towards the body to become the limb ‘flexor musculature’ in tetrapods, is also paralleled by a similar rotation during the ontogeny of tetrapods such as salamanders21. The chief exception to this developmental-phylogenetic similarity is that the preaxial and postaxial muscles pterygialis cranialis and caudalis were differentiated evolutionarily long before the appearance of clear tendinous intersections segmenting proximo-distally the main abductor/adductor fin musculature. In contrast, such intersections appear at very early stages of tetrapod limb development, before any observable antero-posterior (i.e., radio-ulnar or tibio-fibular) division of the musculature is evident (although recent studies of myobast migration in mice suggest that such an antero-posterior division of the limb musculature might actually happen earlier in development than previously thought: Sevan Hopyan, pers. comm.). However, this difference makes sense from a biomechanical perspective: segmented or divided muscles that cross only one joint are only effective when the fin skeleton is elongated and segmented proximo-distally into numerous bones connected by numerous and/or more mobile joints, as is the case in lobe-finned fishes but not in most other fishes (Fig. 4B). Accordingly, early morphogenesis of limb skeletal cartilages and joints in tetrapods is associated with early morphogenesis of proximal and intermediate tendons lying in the region of the major limb joints: the elbow/knee and wrist/ankle joints, respectively (Fig. 4F). In fact, this is probably a chief developmental constraint in extant tetrapods, as such limb tendons likely can only develop ontogenetically in the neighborhood of joints26. One interesting point is that, contrary to what is usually seen in the ontogeny of extant tetrapods, in sarcopterygian fishes such as Latimeria the intersections of the superficial layer mainly lie at the level of the major fin bones, and not between these bones, i.e. in the region of the joints connecting them (Figs 1 and 4B,C).

Also interestingly, some aspects of our evolutionary hypothesis (Fig. 4A–D) are similar to those proposed more than 120 years ago by Gadow37. He suggested that muscles running all the way from the axial skeleton/musculature and/or girdles to the distal region of the fins became proximo-distally partitioned in the region of major joints - particularly those related to the overall internal rotation of the fins - during the fins-limbs transition. This view, which is supported by the present work, contradicts the statements of more recent works, particularly paleontological ones. For example, in Bishop’s detailed reconstruction of the shoulder/arm/forearm muscles of a stem tetrapod it was assumed that ancestrally these muscles did not cross more than one joint17. However, it should be noted that a few paleontologists did propose a proximo-distal partition of muscles that originally crossed more than one joint, during the fins-limbs transition, as suggested by Gadow (see, e.g. in ref. 35).

Most authors agree that the tetrapod stylopod and zeugopod bones are homologous with the proximal bones of sarcopterygian fins, but whether the tetrapod autopodia are neomorphic structures or include structures homologous to specific fin structures remains controversial2,38,39,40,41,42,43,44. Some evidence from soft tissue development favours the neomorphic hypothesis. For example, during tetrapod development the distal tendon primordium that gives rise to most tendons of the intrinsic hand/foot muscles appears later than the primordia of the proximal and intermediate tendons associated with girdle, stylopod (arm/thigh) and zeugopod muscles (Fig. 4H). Additionally, there are significant differences between the morphogenesis of the proximal/intermediate tendons vs. the distal tendon26 (see also, e.g., more recent works from Schweitzer’s group, reviewed in Huang et al.27). While the segregation of the primordia of the former tendons depends on interactions with muscle, the distal tendons 1) develop by a two-step process in which their primordium segregates into various tendon blastemas – each associated with a digit – that in turn subdivide into individual tendons; 2) develop mainly in spatial isolation from, and likely independently of interactions with, the muscles to which they will attach; and 3) express the transcription factors six-1 and six-2 and the eph-related receptor tyrosine kinase cek-8, while proximal/intermediate tendons do not (reviewed by Kardon26). These developmental data, combined with our comparative anatomical data, support the idea that the overall musculotendinous configuration of the hand/foot constitutes a tetrapod evolutionary novelty26, probably acquired later in evolution than were most of the girdle/stylopod/zeugopod muscles (Fig. 4D).

A recent compilation of comparative anatomical, paleontological and developmental data strongly suggests that the PECA and PELA were markedly different from each other anatomically in the earliest fishes that had both, and that their most proximal regions (i.e., pelvic vs. pectoral girdles) have remained anatomically, developmentally and genetically quite different45,46,47. In contrast, the co-option of various similar genes in the development of the more distal, and phylogenetically more recent, stylopod/zeugopod and particularly autopod regions of the PECA and PELA of tetrapods led to a marked derived anatomical and developmental similarity between these structures in both appendages (i.e., a ‘similarity bottleneck’ sensu Diogo et al.46, Diogo and Molnar48, and sensu the present work). These more distal limb regions, principally the autopodia, display developmental patterns that are quite different from those of the fins of plesiomorphic gnathostomes41, and of more proximal limb regions in tetrapods. This information agrees with the notes in the previous paragraph regarding the distal vs. proximal/intermediate tendons (Fig. 4B) and with data on the development and genetic networks of tetrapod limbs47. However, it remained an open question whether such a co-option and/or other (e.g., functional/topological) factors leading to the PECA-PELA similarity bottlenecks might have occurred even before the rise of tetrapods. Our results suggest that there was in fact a second, much earlier major similarity bottleneck between the muscles of the PECA and PELA: during the transition from the LCA of extant bony fishes to the LCA of extant sarcopterygians. This latter LCA probably already displayed striking muscular similarities not only between the dorsal and ventral sides of each fin, but also between the two PECA and two PELA, thus essentially having eight copies of the same highly complex configuration. This condition is exemplified by Latimeria, in which 14 PECA muscles have clear, straightforward one-to-one topological correspondences with PELA muscles, and the dorsal muscles of each fin have clear one-to-one correspondences with ventral muscles on the same fin (Figs 2 and 3; Tables S5–S7). In contrast, the similarity between six of the muscles of each paired appendage of plesiomorphic actinopterygians and osteichthyans, such as Polypterus, is mainly due to the fact that these fins display a very simple, basic condition that was acquired much earlier in gnathostome evolution: the presence of poorly differentiated deep and superficial abductor/adductor masses36 (Fig. 4; Tables S5–S6).

Our study therefore allows us, for the first time, to propose a detailed scheme of topological correspondences between all PECA vs. PELA muscles, including girdle/stylopod muscles, based on the same empirical comparative, evolutionary, and developmental data used for the homology hypotheses (Table S7). Such schemes have previously been attempted, mostly in the 19th/early 20th centuries, but they were strongly biased by the old Romantic ‘archetypal’, idealistic view of evolution46. As seen in Table S7, the topological correspondences inferred here between the girdle/stylopod muscles of the PECA and PELA are, in both salamanders and humans, mainly between groups of muscles, without clear one-to-one equivalences, while those between the zeugopod/autopod muscles are mainly one-to-one. Therefore, our results reinforce the idea that muscles associated with the pectoral and pelvic girdles have remained more different from each other since the appearance of these appendages in basal gnathostome fishes in comparison to the more distal muscles, which were affected by similarity bottlenecks during the transitions leading to sarcopterygians and then to tetrapods.

In summary, the fins-limbs transition was a long, stepwise process, and the characteristic tetrapod musculoskeletal limb configuration was very likely present in the Silurian LCA of extant sarcopterygians, more than 400 MYA. In addition to the fact that proximal bones and numerous muscles of the paired appendages of Latimeria and Neoceratodus have clear homologues in tetrapods, the absolute numbers of muscles in each appendage suggest that the muscle configuration of extant sarcopterygian fishes is, in fact, more similar to that of tetrapods than to that of any other extant fishes. Chondrichthyans such as sharks have five muscles in each paired appendage (Total = 10) and plesiomorphic osteichthyans such as Polypterus have six pectoral and six pelvic (T = 12), while Latimeria has 20 and 15 (T = 35), Neoceratodus five and 25 (T = 30) and anatomically plesiomorphic tetrapods such as Ambystoma have 48 and 59 (T = 107), respectively (Tables S5–S6; see cladogram of Fig. 4). If we exclude intrinsic hand/foot muscles, which do not seem to be directly homologous to any specific fish muscles, Ambystoma has 28 and 27 (T = 55), only 20 more than the number found in Latimeria, so the difference between Polypterus and Latimeria (35–12 = 23) is, strikingly, larger than that between Latimeria and Ambystoma. Moreover, the data provided here point out that the major transitions that led to the characteristic phenotype of tetrapod limbs (one leading to sarcopterygians and the other to tetrapods) corresponded to the two major similarity bottlenecks that led to the striking derived myological similarity between the PECA and PELA. Finally, by providing one-to-one homology hypotheses for each muscle of the paired appendages of all these taxa, this work lays the foundation for the use of Extant Phylogenetic Bracketing in musculoskeletal reconstructions in paleontological studies on the origin/early evolution of limbs.