Limb Ossification in the Paleozoic Branchiosaurid Apateon (Temnospondyli) and the Early Evolution of Preaxial Dominance in Tetrapod Limb Development

EVOLUTION & DEVELOPMENT 9:1, 69 –75 (2007)

Limb ossification in the Paleozoic branchiosaurid Apateon (Temnospondyli) and the early evolution of preaxial dominance in tetrapod limb development

Nadia B. Fro¨bisch,a,Ã Robert L. Carroll,a and Rainer R. Schochb aRedpath Museum, McGill University, 859 Sherbrooke Street West, Montreal H3A 2K6, Canada bStaatliches Museum fu¨r Naturkunde Stuttgart, Rosenstein 1, 70191 Stuttgart, Germany ÃAuthor for correspondence (email: [email protected])

SUMMARY Despite the wide range of shapes and sizes that divergent evolution of these two pathways and its causes are accompany a vast variety of functions, the development of still not understood. Based on an extensive ontogenetic series tetrapod limbs follows a conservative pattern of de novo we investigated the pattern of limb development of the 300 Ma condensation, branching, and segmentation. Development of old branchiosaurid amphibian Apateon. This revealed a the zeugopodium and digital arch typically occurs in a posterior preaxial dominance in limb development that was previously to anterior sequence, referred to as postaxial dominance, with believed to be unique and derived for modern salamanders. a digital sequence of 4–3–5–2–1. The only exception to this The Branchiosauridae are favored as close relatives of pattern in all of living Tetrapoda can be found in salamanders, extant salamanders in most phylogenetic hypotheses of the which display a preaxial dominance in limb development, a de highly controversial origins and relationships of extant novo condensation of a basale commune (distal carpal/tarsal amphibians. The findings provide new insights into the 112) and a precoccial development of digits I and II. These evolution of developmental pathways in tetrapod limb divergent patterns have puzzled researchers for over a development, the relationships of modern amphibians with century leading to various explanatory hypotheses. Despite possible Paleozoic antecedents, and their initial timing of many advances in research on tetrapod limb development, the divergence.

INTRODUCTION ossification (Shubin and Alberch 1986; Rieppel 1992, 1993a, b), revealing a highly conserved order in all tetrapods. The Branchiosauridae are a clade of small-bodied, gilled tem- Initially, the stylopodium forms as de novo condensation, nospondyl amphibians from the Upper Carboniferous and which gives rise subsequently to the two zeugopodial elements Lower Permian of Central Europe. They display an overall through bifurcation, thereby establishing a preaxial and a salamander-like appearance and share similarities with urode- postaxial column (Fig. 2). The postaxial column generally les in their ecology and life history (Schoch and Fro¨ bisch leads in development before the preaxial element of the 2006) (Fig. 1). Branchiosaurids display an extraordinary fossil zeugopodium. The preaxial column never undergoes a bifur- record due to exceptional preservation resulting from anoxic cation and radiale/tibiale are segmented of the radius/tibia, as bottom conditions of the lakes in which they lived (Boy and are subsequent elements of the preaxial column. The postaxial Sues 2000). Hundreds of skeletons comprising a wide range of column undergoes a bifurcation distally to the ulna/fibula and ontogenetic stages as well as soft tissue preservation of gills, establishes the ulnare and intermedium in the forelimb and stomach contents, and ‘‘skin shadows’’ offer a rich source of fibulare (calcaneum) and intermedium (astragalus) in the ontogenetic data rarely available in the fossil record (Boy hindlimb, respectively. Later, the intermedium produces either 1978; Schoch 2002; Schoch and Carroll 2003) (Fig. 1). This a single centrale through segmentation or two centralia allowed for a detailed investigation of patterns of limb devel- through bifurcation. The postaxial element, that is, ulnare/ opment in the branchiosaurid Apateon and a direct compari- fibulare, gives rise to a new, sharply angled axis of develop- son with the patterns found in extant tetrapods. ment, extending from postaxial to preaxial across the distal In recent years, much research has focused on limb devel- carpals/tarsals: the digital arch. The fourth carpal/tarsal forms opment of extant tetrapods at the level of tissue condensation through segmentation of the ulnare/fibulare and subsequent (Hinchliffe 2002; Tickle 2002), as well as chondrification and bifurcation gives rise to the fourth metacarpal and third

& 2007 The Author(s) 69 Journal compilation & 2007 Blackwell Publishing Ltd. 70 EVOLUTION & DEVELOPMENT Vol. 9, No. 1, January^February 2007

Fig. 1. Different ontogenetic stages of the branchiosaurid Apateon Fig. 2. Patterns of limb formation in extant tetrapods. Limb for- from the Upper Carboniferous of Germany. The smallest larva mation proceeds through a pattern of de novo condensation, (top) has no ossifications in the skeleton and only the ‘‘skin branching, and segmentation. Frogs and amniotes display postax- shadow’’ is preserved. Later stages show successively more ossifi- ial dominance in limb development, salamanders preaxial domi- cations, starting in the skull and proceeding in the postcranium. nance. bc, basale commune; c, centrale; H/F, humerus/femur; i, Scale bar equals 2 mm. intermedium; r/t, radius/tibia; ra/ti, radiale/tibiale; u/f, ulna/fibula; ul/fi, ulnare/fibulare. In dependence on Shubin and Alberch (1986). carpal. Further bifurcations establish the remaining preaxial distal carpals/tarsals and their associated metacarpal/tarsals in Condensation and chondrification are subsequently fol- the same manner. The fifth digit arises either after the fourth lowed by the ossification of the individual elements of the or third digit somewhat independent from the remaining limbs. Inevitably, ossification must form the basis for com- digital arch. The resulting digital sequence is 4–(5 or 3)–2–1. parison with fossil taxa, where only skeletal elements are The phalangeal elements of the digits form through segmen- available. The assumption that ossification recapitulates chon- tation from their respective metacarpals/tarsals to establish drification has long been accepted and was only recently the specific phalangeal formula (Fig. 2). challenged when it was shown that these processes are not The above pattern is established in anurans and all am- always tightly coupled (Rieppel 1992, 1993a, b). Examples niotes with a distinct postaxial dominance and the digital arch from amniotes demonstrate that ossification sequences may extending from postaxial to preaxial (Fig. 2A). Salamanders be modified to some extent in accordance with functional however, show a different pattern from that found in all other needs. Despite this, it can be inferred from the same studies of tetrapods in that they display a preaxial dominance in devel- chondrification and ossification sequences in amniotes (Riep- opment, where the digital arch develops in an anterior to pel 1992, 1993a, b), but particularly investigations of frogs posterior direction with the digital sequence 2–1–3–4–(5) (Fig. (Erdmann 1933; Wiens 1989; Trueb and Hanken 1992; Hall 2B). Interestingly, despite the reversed pattern of limb devel- and Larsen 1998; Haas 1999) and salamanders (Erdmann opment in urodeles, they also show a temporal dominance of 1933; Keller 1946; Blanco and Alberch 1992; Nye et al. 2003), the penultimate digit in the formation of the digital arch that the common direction, that is, preaxial or postaxial (Shubin and Alberch 1986). Moreover, salamanders show a dominance and the general order of digit formation, are ba- precoccial development of the basale commune (fused carpal/ sically similar for both processes. Therefore, timing of ossi- tarsal 112). These unique features of salamanders are estab- fication can serve as an information basis for establishing the lished very early in their limb development and can be ob- sequence of developmental processes in the limbs of fossil served as early as in the precartilaginous condensation of the amphibians. The excellent fossil record of branchiosaurids limb elements (Wake and Shubin 1998). allowed for the investigation of such details of their limb Fr˛bisch et al. Limb development in branchiosaurids 71 ossification including the sequence and direction of digit for- order is documented by differences in the degree of ossifi- mation. The results have implications for the controversial cation of different bones within individuals, as well as se- relationships of salamanders with Paleozoic amphibian taxa quentially older individuals. and the time at which preaxial dominance in limb develop- Furthermore, the terminal phalanges of Apateon ossify ment evolved in tetrapods. very early, a pattern also found in basal salamanders (Voro- byeva and Hinchliffe 1996; Vorobyeva et al. 1997) (Fig. 3) as well as some derived salamander taxa (Nye et al. 2003). The MATERIALS AND METHODS early ossification of the distal phalanges has previously been interpreted as a larval adaptation of salamanders (Holmgren The specimens investigated for this study are housed in the col- 1933), but this pattern is actually also reported for a wide lection of the Geological–Paleontological Institute of the University of Mainz, Germany (611 specimens, GPIM-N 1230-1840), as well variety of extant amniotes (Strong 1925; Petri 1935; Hodges as the Staatliches Museum fu¨ r Naturkunde Stuttgart, Germany 1953; O’Rahilly and Gardner 1972; Adams 1992; Rieppel (SMNS 51208, 51212, 51249, 51255, 51269, 51349). 1993b). The reasons for this deviation from an otherwise Ontogenetic stages were determined using the staging charac- proximal to distal direction of phalangeal ossification in this teristics established by Schoch (2004). Specimens were investigated wide range of taxonomic groups are unknown. The medio- with a Wild dissection scope (Typ 256575) and a Dolan–Jenner distal phalanges were the last elements of the manus and pes high-intensity illuminator Mi-150 (Dolan-Jenner Industries, to ossify in Apateon. Carpals and tarsals remained unossified Lawrence, MA, USA). Furthermore, the specimens were careful- even in the latest known stages of this genus (Fig. 1). Ossi- ly cleaned with acetone and a thin layer of diluted acetone was fication of carpals and tarsals is also slow in modern sala- applied with a soft brush during investigation and photography. manders or may never occur. In amniotes, ossification of The application of acetone as well as the high-intensity lightning the mesopodial elements also takes place later than in the proved to be important for the visibility of details and coloration gradients in skeletal elements. remaining autopodial elements, showing an example for a decoupling of chondro- and osteogenesis present in all tetrapods (e.g., Rieppel 1992, 1993b). RESULTS The clearly established preaxial dominance and early formation of digit II are distinct features of salamander limb The branchiosaurid Apateon represents a rare case where data development (Shubin and Alberch 1986; Shubin and Wake on a developmental process are directly available from the 2003) that are well established in the branchiosaurid Apateon. fossil record. The majority of the more than 600 specimens The differences in the sequence between frogs and amniotes that were investigated for this study come from one locality on the one hand and salamanders and Apateon on the other and horizon, the Upper Carboniferous ‘‘Kusel locality’’ near hand become particularly apparent when the ossification se- Erdesbach in the Palatinate of western Germany. Details of quence of the limb is considered relative to ontogenetic rank the ossification sequence of the branchiosaurid Apateon are (Fig. 4). The ossification of the preaxial zeugopodial elements not only exceptionally informative due to the large sample size before the postaxial ones and the digital sequence of II–(I– and wide span of ontogenetic stages available, but also be- III)–IV–V are uniquely shared features of salamanders and cause of the unique preservation of the bones themselves. Apateon that are not reported for any other tetrapod taxon. Different degrees of ossification between and within individ- Neither frogs nor any amniotes show preaxial dominance in ual skeletal elements are reflected in a different coloration of limb development. Frogs clearly display postaxial to preaxial the bone. These extend from shades of yellow for weakly formation of the digital arch with an early condensation and ossified bones, to tones of red and brown and finally black in chondrification of digit IV, which is followed by the identical fully ossified elements (Schoch 1992) (Fig. 3). sequence of ossification (Erdmann 1933; Wiens 1989; Trueb The results of the study reveal that the sequence of limb and Hanken 1992; Hall and Larsen 1998; Haas 1999). These ossification of the branchiosaurid Apateon is remarkably observations show that the branchiosaurid Apateon shares similar to that of modern salamanders. Both branchiosaurids fundamental features of limb development with extant sala- and salamanders have four digits in the hand and five in the manders that were previously considered unique, derived foot. The most striking similarity of limb development in the characters of urodeles (Shubin and Wake 2003). branchiosaurid Apateon with salamanders is a clearly established preaxial dominance in ossification. This is reflected in DISCUSSION the advanced ossification of the preaxial zeugopodial elements before the postaxial ones (Fig. 3A). Digit II leads in the Explanatory hypotheses for the divergent pattern digital sequence of ossification followed in order by digits III, in salamander limb development I, IV, and in the case of the pes V. This is well established in For over a century the divergence of salamander limb devel- the metapodials as well as the phalangeal elements. The opment from the general tetrapod pattern has puzzled 72 EVOLUTION & DEVELOPMENT Vol. 9, No. 1, January^February 2007

Fig. 4. Graph depicting ossification sequences of limb elements in representative tetrapod taxa with events relative to ontogenetic rank (sequence order); methodology following Smith (2001). The sequential ossification of stylo-, zeugo-, and metapodial elements is represented as events. More distal phalangeal elements were excluded from the analysis, because they can differ in having a strictly proximal to distal direction in ossification or starting phalangeal ossification with the terminal phalanges. This variation is widely distributed within and among different tetrapod clades without Fig. 3. Ossification patterns and degrees of ossification in the representing a clear functional or phylogenetic signal. It, however, manus (A) and pes (B) of Apateon (left) and a differentially cleared adds noise to the sequence order that is irrelevant for the repre- and stained larva of the basal salamander Cryptobranchus (right; sentation of axial dominance in limb development and was for this red stain depicts ossification, blue stain cartilaginous elements). F, reason excluded. Note that the sequence is identical in Apateon and femur; f, fibula; mc, metacarpal; mt, metatarsal; p, phalanx; r, ra- the salamanders Ambystoma and Triturus. Ossification sequence dius; t, tibia; tp, terminal phalanx; u, ulna. Note the differences in data is based on personal observations and literature (Erdmann the bone coloration in Apateon, which reflect different degrees of 1933; Rieppel 1992; Vorobyeva and Mednikov 2004). ossification. Scale bar equals 2 mm. researchers and a number of explanatory hypotheses have sion limit at the distal end of the zeugopodium in other tetra- been formulated. Early investigators considered the differen- pods. In contrast, Hinchliffe later noted that the well- ces between salamanders and other tetrapods so striking that differentiated digital arch in axolotls provides evidence for they proposed a diphyletic origin of tetrapods (Holmgren digital identity with all other tetrapods (Hinchliffe 2002). 1933; Jarvik 1965). This is in conflict with the vast majority of Moreover, other evidence showed that the anterior border of morphological and molecular data available today that expression of Hoxd-11 in all tetrapods occurs between pro- strongly support the monophyly of Tetrapoda. spective digits I and II, supporting their homology among More recently, Wagner et al. (1999) proposed that the salamanders, frogs, and amniotes (Torok et al. 1998). In ad- unique sequence of digit development in salamanders is a dition, this hypothesis seems unlikely in the light of the here result of the loss of their original digits I and II early in their presented data, because it would imply that the digit reduc- evolutionary history. Support for this hypothesis was based tion took place at least once in a temnospondyl clade pre- on sirenids, proteids, and amphiumids as modern examples of ceding the Upper Carboniferous branchiosaurids, since the salamanders that have undergone digit reduction and that latter already display preaxial dominance. There is no evi- were at the time considered to have a basal position in the dence in the fossil record that branchiosaurids or related salamander phylogeny (but see Wiens 2005 for a recent per- groups went through a phase of digit reduction or show a spective). Subsequently, the postaxial digits were reinvented tendency for the latter. implicating that salamander digits I and II are homologous to An alternative hypothesis is that the salamander autopo- digits III and IV in all other tetrapods. With the loss of the dium is homologous to that of other tetrapods, but has simply original digits I and II, the more posterior digits III and IV in evolved a different ontogenetic sequence (Shubin and Wake the forelimb, and V in the hind limb would assume an an- 2003) (see also Fig. 4). Such changes of the developmental terior position, but would be expressed before the appearance sequence in the clade leading to extant salamanders are at- of more lateral digits that had evolved de novo. Additional tributed to larval adaptations, in which the development of support for this hypothesis was based on the exceptional ex- preaxial digits is required at an early stage for climbing and pression pattern of Hoxa-11 in the salamander autopodium, adherence to water vegetation (Holmgren 1933; Shubin et al. where it is first expressed in digit III, but has a sharp expres- 1995; Hinchliffe 2002; Shubin and Wake 2003). Fr˛bisch et al. Limb development in branchiosaurids 73

Phylogenetic implications The interrelationships of the three modern amphibian groups, that is, frogs, salamanders, and caecilians, and their relationships to possible Paleozoic antecedents are highly controversial and represent one of the most puzzling problems in vertebrate evolution and systematics (Carroll 2001). Despite the long research history surrounding this problem (Haeckel 1866) and paleontological, morphological and molecular ap- proaches to it, no consensus has been reached. Three different competing hypotheses of basal tetrapod relationships are currently discussed (see Schoch and Milner 2004 for a review). On the one hand monophyly of lissamphibians with either a temnospondyl (Trueb and Clouthier 1991; Milner 1993; Ruta et al. 2003) or a lepospondyl origin (Laurin and Reisz 1997; Laurin 1998; Vallin and Laurin 2004) is proposed; on the other hand, a polyphyletic origin of the three modern amphibian groups with caecilians as a sister taxon to lepospondyl microsaurs and salamanders and frogs nested within temnospondyl dissorophoids is suggested (Anderson in press; Car- roll 2001) (Fig. 5). Interweaved with the problematic phylogenetic relationships is the timing of the split of the lineages leading to the three modern groups and the taxonomic level at which a potential monophyly of the lissamphibians is considered. Whatever the selective advantages of preaxial dominance in limb development, profound changes in the control of development were presumably necessary to reverse the polarity of digit formation. The complexity of a change from postaxial to preaxial limb development suggests a single evolutionary origin of preaxial dominance. The discovery of preaxial dominance of limb development in the branchiosaurid Apateon has far reaching consequences for the evolution of limb development when considered in the phylogenetic framework of basal tetrapods. Unfortunately, the excellent fossil record of branchiosaurids is not repeated for other Paleozoic amphibian taxa and for this as well as for analytical reasons it is very difﬁcult to include the data into a comprehensive phylogenetic analysis (Anderson in press). Therefore the evolution of preaxial dominance in limb development is here discussed in the light of existing phylogenetic hypotheses.

Fig. 5. The three currently discussed hypotheses of lissamphibian relationships to basal tetrapods. A, polyphyly hypothesis (following Carroll 2001); B, temnospondyl hypothesis (following Trueb & Clouthier 1991; Milner 1993; Ruta et al. 2003); C, lepospondyl hypothesis (following Laurin & Reisz 1997; Laurin 1998; Vallin & Laurin 2004). Bars indicate where preaxial dominance in limb development (PD) is known to be present. Note that different character optimizations are possible for the evolution of PD: the most parsimonious solution is a synapomorphy of PD for salamanders and Apateon (A); other optimizations require two or more steps (B, C). 74 EVOLUTION & DEVELOPMENT Vol. 9, No. 1, January^February 2007

The most parsimonious scenario for the evolution of pre- urodeles, but had already evolved in branchiosaurids by the axial dominance in limb development is its single origin as a Upper Carboniferous, 300 Ma ago. The developmental infor- shared derived character of salamanders and branchiosaurids. mation from the branchiosaurid Apateon provides new data This would support a close relationship of these groups to the relevant for the controversial phylogenetic relationships of exclusion of frogs (Carroll 2001) (Fig. 5A). This also has modern amphibians and the timing of the spilt of their lin- wider phylogenetic consequences. The fact that frogs share eages. postaxial dominance with all other tetrapods indicates an early divergence of the lineage leading to branchiosaurids and Acknowledgments subsequently to salamanders, prior to the evolution of pre- We would like to thank Ju¨ rgen Boy for access to the material, as well axial dominance in branchiosaurids. A divergence of the Bat- as Jo¨ rg Fro¨ bisch for many constructive discussions. We are grateful to Hans C. E. Larsson for discussions on limb development; all rachia (frogs1salamanders) lineage in the Late Carboniferous members of the Palaeontology Laboratory at McGill University for (approximately 308 Ma) has previously been suggested in discussions and comments on this work. The project was funded by molecular analyses (e.g., Zhang et al. 2005), but was thus far the Natural Sciences and Engineering Research Council of Canada not supported by data from the fossil record. and McGill University Graduate Student Fellowships. A single origin of all lissamphibians from within either temnospondyls (Trueb and Clouthier 1991; Milner 1993; Ru- ta et al. 2003) or lepospondyls (Laurin and Reisz 1997; Laurin REFERENCES 1998; Vallin and Laurin 2004) represent less parsimonious Adams, R. A. 1992. Stages of development and sequence of bone formation scenarios with respect to the evolution of preaxial dominance in the little brown bat, Myotis lucifugus. J. Mammal. 73: 160–167. in limb development, if a single Paleozoic taxon is taken as Anderson, J. S. in press. Incorporating ontogeny into the matrix: a phy- immediate sister taxon to the Lissamphibia (Fig. 5, A and B). logenetic evaluation of developmental evidence for the origin of modern amphibians. In J. S. Anderson and H.-D. Sues (eds.). Major Transitions This, however, does not include the possibility that the three in Vertebrate Evolution. Indiana University Press. modern clades all derive from either temnospondyls or lepo- Blanco, M. J., and Alberch, P. 1992. Caenogenesis, developmental vari- spondyls and may be monophyletic on a higher taxonomic ability, and evolution in the carpus and tarsus of the marbled newt Triturus marmoratus. Evolution 46: 677–687. level. Boy, J. A. 1978. Die Tetrapodenfauna (Amphibia, Reptilia) des saarpfa¨ lzis- The monophyly of lissamphibians is frequently favored on chen Rotliegenden (Unter-Perm; SW-Deutschland). 1.Branchiosaurus. the basis of other morphological as well as molecular data Mainzer geowiss. Mitt. 7: 27–76. Boy, J. A., and Sues, H.-D. 2000. Branchiosaurs: larvae, metamorphosis (Trueb and Clouthier 1991; Milner 1993; Laurin and Reisz and heterochrony in temnospondyles and seymouriamorphs. In H. 1997; Laurin 1998; Ruta et al. 2003; Vallin & Laurin 2004; Heatwole and R. L. Carroll (eds.). Amphibian Biology. Surrey Beatty & Zhang et al. 2005) (Fig. 5, B, and C). When plotted on these Sons PTY limited, Chipping Norton, pp. 1150–1197. Carroll, R. L. 2001. The origin and early radiation of terrestrial tetrapods. previously proposed phylogenetic hypotheses, it requires two J. Paleont. 75: 1202–1213. steps to account for a convergent evolution of preaxial dom- Erdmann, K. 1933. Zur Entwicklung des kno¨ chernen Skelets von Triton inance in limb development in Apateon and salamanders in und Rana unter besonderer Beru¨ cksichtigung der Zeitfolge der Ossifika- tionen. Zeitschr. ges. Anat., I. Abt. 101: 566–649. either the temnospondyl hypothesis (Fig. 5B) or the lepo- Haas, A. 1999. Larval and metamorphic skeletal development in the fast- spondyl hypothesis (Fig. 5C). Likewise, two steps are required developing frog Pyxicephalus adspersus (Anura, Ranidae). Zoomorph- if a reversal to postaxial dominance in the limb development ology 119: 23–35. Haeckel, E. 1866. Generelle Morphologie der Organismen. Georg Reimer, of frogs is proposed (Fig. 5B). Two steps would also be ne- Berlin. cessary if preaxial dominance in limb development had been Hall, J. A., and Larsen, J. H. J. 1998. Postembryonic ontogeny of the the plesiomorphic state in tetrapods, with subsequent, con- spadefoot toad, Scaphiopus intermontanus (Anura: Pelobatidae): skeletal morphology. J. Morphol. 238: 179–244. vergent evolution of postaxial dominance occurring in the Hinchliffe, R. J. 2002. Developmental basis of limb evolution. Int. J. Biol. lineages leading to frogs and amniotes. In that case, sala- 46: 835–845. manders would be the only living tetrapod group still dis- Hodges, P. C. J. 1953. Ossification in the fetal pig. Anat. Rec. 116: 315–325. Holmgren, N. 1933. On the origin of the tetrapod limb. Acta Zool. 14: 185– playing the ancestral state of limb development. 295. Limbs adapted for terrestrial locomotion are a hallmark in Jarvik, E. 1965. On the origin of girdles and paired fins. Israel J. Zool. 14: the tetrapod body plan and the origin of and causes for the 141–172. Keller, R. 1946. Morphogenetische Untersuchungen am Skelett von Sireno- divergent pattern in the limb development of salamanders has don mexicanus Shaw mit besonderer Beruecksichtigung des Ossifikat- received considerable attention from developmental biolo- ionsmodus beim neotenen Axolotl. Rev. Suisse Zool. 53: 329–426. gists, morphologists, and paleontologists (Holmgren 1933; Laurin, M. 1998. The importance of global parsimony and historical bias in understanding tetrapod evolution. Part I. Systematics, middle ear evo- Jarvik 1965; Shubin and Alberch 1986; Vorobyeva and Hin- lution and jaw suspension. Ann. Sci. Nat. 1: 1–42. chliffe 1996; Vorobyeva et al. 1997; Wagner et al. 1999; Hin- Laurin, M., and Reisz, R. R. 1997. A new perspective on tetrapod chliffe 2002; Tickle 2002; Nye et al. 2003; Shubin and Wake phylogeny. In S. S. Sumida and K. L. Martin (eds.). Amniote Origins. Academic Press, New York, pp. 9–59. 2003). The results of this study demonstrate that preaxial Milner, A. R. 1993. The Paleozoic relatives of lissamphibians. Herp. dominance in limb development is not unique and derived for Monogr. 7: 8–27. Fr˛bisch et al. Limb development in branchiosaurids 75

Nye, H. L. D., Cameron, J. A., Chernoff, E. A. G., and Stocum, D. L. 2003. Smith, K. K. 2001. Heterochrony revisited: the evolution of developmental Extending the table of stages of normal development in the axolotl: limb sequences. Biol. J. Linn. Soc. 73: 169–186. development. Dev. Dyn. 226: 555–560. Strong, R. M. 1925. The order, time, and rate of ossification of the albino O’Rahilly, R., and Gardner, E. 1972. The intitial appearance of ossification rat (Mus norvegicus albinus) skeleton. Am. J. Anat. 36: 313–355. in staged human embryos. Am. J. Anat. 134: 291–308. Tickle, C. 2002. Vertebrate limb development and possible clues to diversity Petri, C. 1935. Die Skelettentwicklung beim Meerschwein. Vierteljahrsschr. of limb form. J. Morphol. 252: 29–37. Naturfor. Ges. Zu¨rich 80: 157–240. Torok, M. A., Gardiner, D. M., Shubin, N., and Bryant, S. V. 1998. Ex- Rieppel, O. 1993a. Studies on skeleton formation in reptiles. V. Patterns of pression of HoxD genes in developing and regenerating axolotl limbs. ossification in the skeleton of Alligator mississipiensis Daudin (Reptilia, Dev. Biol. 200: 225–233. Crocodylia). Zool. J. Linn. Soc. 109: 301–325. Trueb, L., and Clouthier, R. 1991. A phylogenetic investigation of the inter- Rieppel, O. 1993b. Studies on skeleton formation in reptilesFpatterns of and intrarelationships of the Lissamphibia (Amphibia: Temnospondyli). ossification in the skeleton of Chelydra-Serpentina (Reptilia, Testudines). In H.-P. Schultze and L. Trueb (eds.). Origins of the Higher Groups of J. Zool., London 231: 487–509. Tetrapods: Controversy and Consensus. Comstock Pub. Associates, Ith- Rieppel, O. 1992. Studies on skeletal formation in reptiles III: patterns of aca, pp. 223–313. ossification in the skeleton of Lacerta viviparia Jacquin (Reptilia, Squa- Trueb, L., and Hanken, J. 1992. Skeletal development in Xenopus laevis mata). Fieldiana (Zool.) 1437: 1–25. (Anura: Pipidae). J. Morphol. 214: 1–41. Ruta, M., Jeffrey, J. E., and Coates, M. I. 2003. A supertree of early Vallin, G., and Laurin, M. 2004. Cranial morphology and affinities of tetrapods. Proc. Royal Soc. London B 270: 2507–2516. Microbrachis, and a reappraisal of the phylogeny and lifestyle of the first Schoch, R. R. 2004. Skeleton formation in the Branchiosauridae: a Case amphibians. J. Vert. Paleo. 24: 56–72. study in comparing ontogenetic trajectories. J. Vert. Paleo. 24: 309–319. Vorobyeva, E. I., and Hinchliffe, R. J. 1996. Developmental pattern Schoch, R. R. 2002. The early formation of the skull in extant and Paleozoic and morphology of Salamandrella keyserlingii limbs (Amphibia, amphibians. Paleobiology 28: 278–296. Hynobiidae) including some evolutionary aspects. Russ. J. Herp. 3: Schoch, R. R. 1992. Comparative ontogeny of early Permian branchios- 68–81. aurid amphibians from southwestern Germany. Palaeontographica, Abt. Vorobyeva, E. I., and Mednikov, D. N. 2004. Ossifikation of the limb A 222: 43–83. skeleton of Triturus vittatus and Salamandrella keyserlingii. Dokl. Biol. Schoch, R. R., and Carroll, R. L. 2003. Ontogenetic evidence for the Sci. 394: 840–844. Paleozoic ancestry of salamanders. Evol. and Dev. 5: 314–324. Vorobyeva, E. I., Ol’shevskaya, O. P., and Hinchliffe, R. J. 1997. Specific Schoch, R. R., and Fro¨ bisch, N. B. 2006. Metamorphosis and neoteny: alter- features of development of the paired limbs in Ranodon sibiricus Kessler native pathways in an extinct amphibian clade. Evolution 60: 1467–1475. (Hynobiidae, Caudata). Russ. J. Dev. Biol. 28: 188–197. Schoch, R. R., and Milner, A. R. 2004. Structure and implications of the- Wagner, G. P., Khan, P. A., Blanco, M. J., Misof, B., and Liversgae, R. A. ories on the origins of lissamphibians. In G. Arratia and M. V. H. 1999. Evolution of Hoxa-11 expression in amphibians: is the urodele Wilson (eds.). Recent Advances in the Origin and Early Radiation of autopodium an innovation? Am. Zool. 39: 686–694. Vertebrates. Verlag Dr. Friedrich Pfeil, Munich, pp. 345–377. Wake, D. B., and Shubin, N. H. 1998. Limb development in the Pacific Shubin, N. H., and Alberch, P. 1986. A morphogenetic approach to the giant salamander, Dicamtodon (Amphibia, Caudata, Dicamptodont- origin and basic organisation of the tetrapod limb. Evol. Biol. 20: 319–387. idae). Can. J. Zool. 76: 2058–2066. Shubin, N. H., and Wake, D. B. 2003. Morphological variation, develop- Wiens, J. J. 2005. Ontogeny discombobulates phylogeny. Syst. Biol. 54: ment, and evolution of the limb skeleton of salamanders. In H. Heatwole 91–110. and M. Davies (eds.). Amphibian Biology. Surrey Beatty & Sons PTY Wiens, J. J. 1989. Ontogeny of the skeleton of Spea bombifrons (Anura: limited, Chipping Norton, pp. 1782–1808. Pleobatidae). Herpetologica 47: 21–28. Shubin, N. H., Wake, D. B., and Crawford, A. J. 1995. Morphological Zhang, P., Zhou, H., Chen, Y.-Q., Liu, Y.-F., and Qu, L.-H. 2005. Mito- variation in the limbs of Trachia granulosa (Caudata: Salamandridae): genomic perspectives on the origin and phylogeny of living amphibians. evolutionary and phylogenetic implication. Evolution 49: 874–884. Syst. Biol. 54: 391–400.