Evolution of Limb Proportions

Whereas the Triassic Triadobatrachus has limbs with intermediate proportions between anurans and salamanders (Lires et al. Reference Lires, Soto and Gómez2016), our data show that from the Jurassic on, limbs of fossil salientians are within the range documented in extant frogs (Fig. 3). Only over PC 3 do some fossil taxa lie slightly outside the range of living anurans, with relatively long femora and short proximal tarsals and metatarsals that partially resemble the ancestral condition (i.e., inferred from salamanders and Triadobatrachus). The group of species located in this peripheral position over PC 3 includes the Jurassic Notobatrachus degiustoi and Vieraella herbsti, the only stem salientians (Fig. 1) for which the full set of variables can be measured. This position in the morphospace indicates that their proportions might represent an intermediate grade between the ancestral configurations and modern anuran proportions. However, because the salientian Jurassic record is still very sparse and the available data indicate that homoplasy is widespread in the group, the possibility that they evolved from salientian forms with shorter femora and longer proximal tarsals not yet represented in the fossil record cannot be completely ruled out. In this regard, similar proportions have evolved convergently in pipimorphs and pelobatids ranging from the Jurassic to the Oligocene, which indicates that proportional long femora and short proximal tarsals and metatarsals are, at least in these latter cases, a derived state.

Limb proportions were already diversified by the Jurassic, pointing toward an earlier origin of the salientian Bauplan. This evidence is consistent with recent calibrated molecular phylogenies that recover the origin of Anura in the Late Triassic (Feng et al. Reference Feng, Blackburn, Liang, Hillis, Wake, Cannatella and Zhang2017; Hime et al. Reference Hime, Lemmon, Lemmon, Prendini, Brown, Thomson, Kratovil, Noonan, Pyron and Peloso2021) and with the presence of ilia that might be closely related to the crown-group in the Late Triassic Chinle Formation (Stocker et al. Reference Stocker, Nesbitt, Kligman, Paluh, Marsh, Blackburn and Parker2019).

According to our sample of Jurassic taxa (i.e., stem-anurans and the xenoanuran Rhadinosteus parvus; Supplementary Table S1), the documented early limb diversification was mainly related to variation in the relative lengths between fore- and hindlimbs (i.e., PC 1; Fig. 4). By the Early Cretaceous, limb proportions were further diversified in relation to the autopod (i.e., PC 2 and PC 3; Fig. 4, Supplementary Fig. S2), linked to a phylogenetically more diverse fossil record encompassing a number of pipimorphs, a clade with characteristically long metapodia (Trueb Reference Trueb1996; Wuttke and Poschmann Reference Wuttke and Poschmann2010; Gómez Reference Gómez2016).

It is worth noting that the fossil diversity shown in this study predominantly encompasses “archeobatrachians” (Fig. 4; Supplementary Table S1), whereas neobatrachians are poorly represented in the sample due to their scant record of articulated specimens with well-preserved limbs. Molecular time estimates (e.g., Pyron Reference Pyron2014) and the anurofauna from the Aptian Crato Formation of Brazil (Báez et al. Reference Báez, Moura and Gómez2009) show that the neobatrachian diversification was well underway during the Early Cretaceous (e.g., Pyron Reference Pyron2014; Feng et al. Reference Feng, Blackburn, Liang, Hillis, Wake, Cannatella and Zhang2017; Jetz and Pyron Reference Jetz and Pyron2018; Hime et al. Reference Hime, Lemmon, Lemmon, Prendini, Brown, Thomson, Kratovil, Noonan, Pyron and Peloso2021). Hence, it is expected that at least from the Early Cretaceous on, limb diversity was probably greater than what we have documented in our fossil sample. This bias is particularly accentuated after the Cretaceous/Paleogene boundary, the time when the species-rich Hyloidea, Microhylidae, and Natatanura, the three Neobatrachian clades comprising about 88% of extant anurans, underwent a rapid diversification (Feng et al. Reference Feng, Blackburn, Liang, Hillis, Wake, Cannatella and Zhang2017; Jetz and Pyron Reference Jetz and Pyron2018; Hime et al. Reference Hime, Lemmon, Lemmon, Prendini, Brown, Thomson, Kratovil, Noonan, Pyron and Peloso2021).

Two main patterns are observed in extant anurans. First, even though the real past diversity is underrepresented and our sample of extant taxa is much larger and phylogenetically more diverse than the fossil one, the region of the morphospace occupied by extant species is largely restricted to that of fossils. Second, the different regions of the morphospace have been repeatedly explored by non–closely related species (Fig. 3, Supplementary Figs. S4, S6, S7). Consequently, when the distribution of taxa in the limb morphospace is analyzed over geological time, the general pattern that emerges is that after an early morphological diversification that was already underway in the Jurassic, the evolution of limbs is marked by repeated convergence over a range of proportions that had already been largely explored by the Early Cretaceous (Fig. 4). These findings are in line with those of Moen et al. (Reference Moen, Morlon and Wiens2016), who found that the evolution of the general body form in anurans is also characterized by a remarkable degree of morphological conservation, alongside widespread convergence (related to microhabitat), even among phylogenetically distant clades that diverged as far back as 150 Ma. Numerous previous works on biomechanics (e.g., Herrel et al. Reference Herrel, Moureaux, Laurin, Daghfous, Crandell, Tolley, Measey, Vanhooydonck and Boistel2016) and macroevolutionary anatomical patterns in anurans (e.g., Citadini et al. Reference Citadini, Brandt, Williams and Gomes2018; Buttimer et al. Reference Buttimer, Stepanova and Womack2020; Stepanova and Womack Reference Stepanova and Womack2020; Petrović et al. Reference Petrović, Vukov and Tomašević Kolarov2021) have shown that locomotion, together with microhabitat, has been a major driver of diversification and convergence in anuran limb evolution. Our results agree with these previous findings: (1) taxa are distributed in the limb morphospace according to their locomotor modes (Fig. 3), at least partially; and (2) cross-validation errors from pFDA are rather small (Table 2).

The question of why only a subset of the theoretically possible limb proportions has actually evolved in the last 200 Myr of evolution of salientians might be addressed from at least two complementary perspectives. On the one hand, given that adaptive pressures related to locomotor modes seem to be a key driver in the evolution of limb proportions, limb configurations associated with low locomotor efficiency in the available ecological space might have been negatively selected in a consistent manner over the evolution of Salientia. Likewise, other selective pressures on limbs might act on other biological aspects, such as feeding (e.g., prey manipulation; Gray et al. Reference Gray, O'Reilly and Nishikawa1997) or mating (e.g., amplexus; Duellman Reference Duellman1992). On the other hand, intrinsic developmental processes might have constrained limb evolution. The evolution of the unique anuran Bauplan involved profound changes in development, among which the most evident are the evolution of the tadpole and a drastic metamorphosis (Duellman and Trueb Reference Duellman and Trueb1994). The fact that the entire anuran skeleton has been remarkably conserved in spite of the ecological diversity of the group suggests that these radical developmental changes might have imposed tight constraints on the skeletal system as a whole, including the limbs. In this regard, the rampant convergence at deep temporal scales observed in both body form (Moen et al. Reference Moen, Morlon and Wiens2016) and limb proportions could be attributed to the limited ways available to respond to selection due to such constraints, which are inherent to the clade (Wake Reference Wake1991; Moen et al. Reference Moen, Morlon and Wiens2016). Other constraints underlying the conservation and convergence of the appendicular morphology might be related to plesiomorphic developmental processes in limb patterning. Specifically, recent studies have shown that the macroevolutionary diversity of limb proportions in amniotes is also limited to certain areas of the morphospace and that this distribution can be predicted by interactions between activating and inhibitory signals that would modulate the proximo-distal limb patterning sequence in tetrapods (Young Reference Young2013; Young et al. Reference Young, Winslow, Takkellapati and Kavanagh2015). Preliminary studies by C.M.P.-B. suggest that the macroevolutionary patterns of limb proportions in anurans also agree with the predictions of the developmental model tested in amniotes. Interestingly, the proximo-distal sequence in which limb elements develop might have not only biased limb proportions toward certain directions but might have also impacted the rates of limb evolution in frogs (Stepanova and Womack Reference Stepanova and Womack2020). A hypothesis originally delineated for mammals proposes that later-developing bones are subjected to reduced developmental constraints, which might confer them more freedom to evolve in response to selection pressures (Weisbecker Reference Weisbecker2011; Martín-Serra et al. Reference Martín-Serra, Figueirido, Pérez-Claros and Palmqvist2015; Stepanova and Womack Reference Stepanova and Womack2020). In agreement with this hypothesis, it has been shown that the distal limb elements (i.e., later-developing bones) of frogs show higher evolutionary rates compared with the more proximal ones (Stepanova and Womack Reference Stepanova and Womack2020).

Locomotor Modes over Geological Time

The results of the pFDA and the distribution of species in the morphospace show that limb proportions represent valuable evidence to discuss locomotor modes in extinct salientians. Even though locomotor groups partially overlap in the morphospace (Fig. 3) and differences in proportions between jumpers and swimmers are not statistically significant (Supplementary Table S3), the cross-validation error rates obtained for the training sample (i.e., extant anurans) in the pFDA are rather small, even between swimmers and jumpers (Table 2, Supplementary Table S8). Swimming is the most challenging locomotor mode when trying to infer locomotion from limb morphology, because swimmers have intermediate proportions between walker-hoppers and jumpers. In contrast, walker-hoppers and jumpers with more extreme limb configurations tend to be correctly classified. Importantly, the inference of locomotor modes from limb proportions is applicable to the fossil taxa sampled herein because they are largely within the range documented in extant anurans. Moreover, there is no evidence that the functional relationship between limb morphology and locomotor behavior was different in fossils with respect to living species, because the postcranial skeleton of extinct taxa is very similar to that of living anurans.

The three locomotor modes might have already been present in the Jurassic. Notobatrachus is recovered in an area of the morphospace PC 1–PC 2 only shared with walker-hopper extant anurans, far from the overlap zone with the Sw and J modes (Fig. 3). This is in turn reflected in a high pFDA posterior probability (i.e., p _WH = 0.999; Table 1). Furthermore, Reilly and Jorgensen (Reference Reilly and Jorgensen2011), in their comprehensive study on the pelvic system morphology, have shown that the ilio-sacral configuration of Notobatrachus is consistent with walking-hopping locomotion. In contrast, the classification of Prosalirus and Vieraella as swimmers is not as well supported. Importantly, Prosalirus lacks preserved metatarsi, which are a main feature in the morphological diversification of swimmers (i.e., over PC 2 of the full set of variables; Fig. 3, Supplementary Table S2). Furthermore, in contrast to our results, Prosalirus was interpreted as a jumper in its original description (Shubin and Jenkins Reference Shubin and Jenkins1995; Jenkins and Shubin Reference Jenkins and Shubin1998) and was later recovered as a walker-hopper according to its ilio-sacral morphology (Reilly and Jorgensen Reference Reilly and Jorgensen2011). Vieraella is recovered as Sw with a rather low posterior probability (p _Sw = 0.54), which is consistent with its intermediate position over PC 1 and PC 2 (Fig. 3). In addition, it presents the leiopelmatoid-like type of sacral diapophyses (Báez and Basso Reference Báez and Basso1996) that Reilly and Jorgensen (Reference Reilly and Jorgensen2011) linked to walker-hoppers, although this species was not considered in their study. The xenoanuran Rhadinosteus, the only crown-anuran sampled for the Jurassic, is classified as J (p _J = 0.94; Table 1). Its postcranial anatomy has not been previously described in relation to jumping, but it has been noted that it lacks the burrowing and swimming specializations typical of extant xenoanurans (i.e., Rhinophrynus and pipids, respectively; Henrici Reference Henrici1998). Regarding the pelvic morphology, the ilium resembles that of Prosalirus (R.O.G., personal observation), but the poor preservation of the sacrum makes it difficult to classify the species in the terms of Reilly and Jorgensen (Reference Reilly and Jorgensen2011).

The presence of both jumpers and swimmers is more certain in the Early Cretaceous. In particular, Wealdenbatrachus, classified herein as a jumper with a high posterior probability (p _J = 0.98; Supplementary Table S4) consistent with the results of Gómez and Lires (Reference Gómez and Lires2019), has previously been proposed to be an efficient long-distance jumper based not only on its long hindlimbs, but also on its iliac anatomy (Báez and Gómez Reference Báez and Gómez2019). Likewise, some early Cretaceous taxa were almost undoubtedly swimmers: pipimorphs such as Cratopipa and Cordicephalus are classified as swimmers (p _Sw = 0.96 and 0.84, respectively; Supplementary Table S4) and present postcranial features of extant pipids that have been classically linked to their aquatic lifestyle (e.g., robust hindlimbs, proportionally long fingers in hands and feet, expanded sacral diapophyses; Cannatella and Trueb Reference Cannatella and Trueb1988; Trueb Reference Trueb1996; Báez et al. Reference Báez, Gómez and Taglioretti2012; Cannatella Reference Cannatella2015; Gómez and Pérez-Ben Reference Gómez and Pérez-Ben2019; Turazzini and Gómez Reference Turazzini and Gómez2023).

In summary, the Mesozoic phylogenetic and morphological diversification of salientians correlates with the evolution of locomotor diversity during this period. During the Jurassic, even though the distinction between swimmers and jumpers is not clear, both walker-hopper (i.e., Notobatrachus) and non-walker-hopper modes might have already been present. It is very likely that swimming and jumping behaviors had already evolved by the Early Cretaceous linked to the limb diversification toward other dimensions of the morphospace documented for that time (i.e., PC 2).

Evolution of the Salientian Bauplan in Relation to Jumping Locomotion

In recent years, the classical view that the anuran Bauplan evolved in relation to jumping has been challenged by novel evidence and a renewed discussion of the issue from an updated phylogenetic perspective (see “Introduction”). In line with these studies, our findings cast further doubts on the jumping hypothesis, because: (1) none of the stem-salientians are recovered as a jumper specialist; (2) the last common ancestor of all frog-like salientians is recovered in an area of the morphospace where the locomotor modes overlap (Fig. 3); and (3) accordingly, jumping is not unequivocally inferred as the ancestral state for this clade (Fig. 5). More importantly, independent of the specific locomotor abilities inferred herein, the patterns observed in the morphospace together with the phylogenetic distribution of locomotor modes in extant taxa (Supplementary Figs. S6–S9) reveal fundamental shortcomings in testing this hypothesis not only from limb proportions, but probably from any other evidence.

The most evident hurdle is obtaining confident inferences of locomotor modes in fossil taxa, even under the (reasonable) assumption that the correlation between skeletal morphology and function observed in living anurans can be extrapolated to forms that diverged 200 Ma. When such inferences are made from limb proportions, the main problems, as discussed earlier, are that locomotor modes overlap to some extent in the limb morphospace and that the resulting classifications are not necessarily consistent with evidence from other parts of the skeleton (e.g., the pelvic system morphology). However, it remains possible that other new or still underexplored approaches, such as bone microanatomy, may provide more reliable insights into the locomotor modes of fossil frogs in the future.

The second, and in our view the most important, issue is to what extent the morphology and locomotion of both fossil and living forms are useful to reconstruct the ancestral locomotor mode of frog-like salientians and, in turn, to test whether jumping is a synapomorphy of this clade. On the one hand, the distribution of species in the (phylo)morphospace (Fig. 3, Supplementary Figs. S4, S6 S7), where closely related species can occupy very different regions (e.g., the myobatrachids Myobatrachus gouldii and Crinia georgiana with a PC 1 values of −4.6 and 2.5, respectively), and the large disparity already documented in the Jurassic (Fig. 4) reveal that limb proportions are highly evolvable and have been so since the early days of salientians. Consequently, it is not clear whether the limbs of the oldest and early divergent taxa with the full Bauplan are actually representative of the plesiomorphic state of frog-like salientians, as discussed above for Notobatrachus and Vieraella.

On the other hand, independent of the evolutionary pace of the different skeletal structures and the correlation between osteology and locomotor behavior used to make inferences in fossils, the phylogenetic distribution of locomotor modes among extant taxa (Fig. 3, Supplementary Figs. S4, S6–S9) reveals that this trait is also highly evolvable: the evolution of locomotor modes is signed by repeated convergence, with multiple transitions from WH and J to any of the other modes, even among closely related taxa (e.g., within the genus Ranoidea). Therefore, even if new early stem forms with a clear jumping morphology were discovered, and jumping was thus reconstructed as the ancestral state, the high evolvability of locomotor modes would still hamper any conclusion regarding the origin of the Bauplan in relation to jumping. Likewise, hypothetical new “intermediate” fossils (i.e., without the full set of anuran features, like Triadobatrachus) would also be uninformative due to the limitations of inferring a behavior as specialized as anuran jumping from anatomical configurations that are not represented in the modern taxa.

In contrast to these impediments and uncertainties, it is clear that salientians evolved from forms with undulatory locomotion (either temnospondyls [e.g., Anderson Reference Anderson2008; Kligman et al. Reference Kligman, Gee, Marsh, Nesbitt, Smith, Parker and Stocker2023] or lepospondyls [e.g., Marjanović and Laurin Reference Marjanović and Laurin2019]) and that, based on Triadobatrachus, at least some of the unique features of the anuran Bauplan are likely to have evolved before the origin of jumping (Lires et al. Reference Lires, Soto and Gómez2016). In addition, it is a fact that living anurans are functionally and ecologically very diverse in spite of their conserved anatomy and that within this diversity, jumping locomotion, although widespread, is not the rule (Fig. 3, Supplementary Fig. S8). Furthermore, species can show not one but a repertoire of locomotory movements, such as arboreal frogs that climb and jump (Reilly and Jorgensen Reference Reilly and Jorgensen2011) or some hylids and ranids, which are good jumpers and swimmers (Nauwelaerts et al. Reference Nauwelaerts, Ramsay and Aerts2007; Soliz et al. Reference Soliz, Tulli and Abdala2017). In this context, after weighing this evidence and the limitations of inferring the ancestral locomotor mode, we wonder how fruitful it is to keep discussing the origin of the anuran Bauplan in relation to jumping. After all, anurans are not only unique in their adult morphology, but also, and maybe more profoundly, in their development (e.g., Handrigan and Wassersug Reference Handrigan and Wassersug2007), which is marked by a biphasic life cycle with a free-living tadpole and a complex metamorphosis. Tadpoles are under very different selective pressures than post-metamorphic individuals (Roelants et al. Reference Roelants, Haas and Bossuyt2011), and it has even been suggested that the bizarre adult morphology of frogs is the default “by-product” of evolutionary processes that acted on the larval stage during the early evolution of the clade (Altig Reference Altig2006). In spite of this, it is still poorly understood to what extent adult anatomy is the result of constraints imposed by metamorphosis and the tadpole stage. Taking this into account, we think that a shift of focus from a functional to a developmental perspective would renew the debate on the origin of the Bauplan and might shed new light on the matter.