Deep evolutionary origin of limb and fin regeneration.

Salamanders and lungfishes are the only sarcopterygians (lobe-finned vertebrates) capable of paired appendage regeneration, regardless of the amputation level. Among actinopterygians (ray-finned fishes), regeneration after amputation at the fin endoskeleton has only been demonstrated in polypterid fishes (Cladistia). Whether this ability evolved independently in sarcopterygians and actinopterygians or has a common origin remains unknown. Here we combine fin regeneration assays and comparative RNA-sequencing (RNA-seq) analysis of Polypterus and axolotl blastemas to provide support for a common origin of paired appendage regeneration in Osteichthyes (bony vertebrates). We show that, in addition to polypterids, regeneration after fin endoskeleton amputation occurs in extant representatives of 2 other nonteleost actinopterygians: the American paddlefish (Chondrostei) and the spotted gar (Holostei). Furthermore, we assessed regeneration in 4 teleost species and show that, with the exception of the blue gourami (Anabantidae), 3 species were capable of regenerating fins after endoskeleton amputation: the white convict and the oscar (Cichlidae), and the goldfish (Cyprinidae). Our comparative RNA-seq analysis of regenerating blastemas of axolotl and Polypterus reveals the activation of common genetic pathways and expression profiles, consistent with a shared genetic program of appendage regeneration. Comparison of RNA-seq data from early Polypterus blastema to single-cell RNA-seq data from axolotl limb bud and limb regeneration stages shows that Polypterus and axolotl share a regeneration-specific genetic program. Collectively, our findings support a deep evolutionary origin of paired appendage regeneration in Osteichthyes and provide an evolutionary framework for studies on the genetic basis of appendage regeneration.

A histological study of normal and pathological limb regeneration in the Mexican axolotl Ambystoma mexicanum.

Salamanders show unparalleled capacities of tissue regeneration amongst tetrapods (four-legged vertebrates), being able to repair and renew lost or damage body parts, such as tails, jaws, and limbs in a seemingly perfect fashion. Despite countless studies on axolotl (Ambystoma mexicanum) regeneration, only a few studies have thus far compared gross morphological and histological features of the original and regenerated limb skeleton. Therein, most studies have focused on nerves or muscles, while even fewer have provided detailed information about bones and cartilage. This study compares skeletal tissue structures of original and regenerated limbs with respect to tissue level histology. Histological serial sections of 55 axolotl larvae were generated, including 29 limbs that were severed by conspecifics, and 26 that were subject to targeted amputations. Amputations were executed in several larval stages (48, 52, and 53) and at different limb positions (humeral midshaft, above the mesopod). In addition, 3D reconstructions were prepared based on X-ray microtomography scans. The results demonstrate that regenerated forelimbs show a diversity of limb and digit abnormalities as a result of imperfect regeneration. Furthermore, abnormalities were more severe and more frequent in regenerated forelimbs caused by natural bites as compared with regenerated forelimbs after amputation. The results indicate that abnormalities occur frequently after regeneration in larval axolotls contradicting the notion of regeneration generally resulting in perfect limbs.

Deep-time evolution of regeneration and preaxial polarity in tetrapod limb development.

Among extant tetrapods, salamanders are unique in showing a reversed preaxial polarity in patterning of the skeletal elements of the limbs, and in displaying the highest capacity for regeneration, including full limb and tail regeneration. These features are particularly striking as tetrapod limb development has otherwise been shown to be a highly conserved process. It remains elusive whether the capacity to regenerate limbs in salamanders is mechanistically and evolutionarily linked to the aberrant pattern of limb development; both are features classically regarded as unique to urodeles. New molecular data suggest that salamander-specific orphan genes play a central role in limb regeneration and may also be involved in the preaxial patterning during limb development. Here we show that preaxial polarity in limb development was present in various groups of temnospondyl amphibians of the Carboniferous and Permian periods, including the dissorophoids Apateon and Micromelerpeton, as well as the stereospondylomorph Sclerocephalus. Limb regeneration has also been reported in Micromelerpeton, demonstrating that both features were already present together in antecedents of modern salamanders 290 million years ago. Furthermore, data from lepospondyl 'microsaurs' on the amniote stem indicate that these taxa may have shown some capacity for limb regeneration and were capable of tail regeneration, including re-patterning of the caudal vertebral column that is otherwise only seen in salamander tail regeneration. The data from fossils suggest that salamander-like regeneration is an ancient feature of tetrapods that was subsequently lost at least once in the lineage leading to amniotes. Salamanders are the only modern tetrapods that retained regenerative capacities as well as preaxial polarity in limb development.

Macropredatory ichthyosaur from the Middle Triassic and the origin of modern trophic networks.

The biotic recovery from Earth's most severe extinction event at the Permian-Triassic boundary largely reestablished the preextinction structure of marine trophic networks, with marine reptiles assuming the predator roles. However, the highest trophic level of today's marine ecosystems, i.e., macropredatory tetrapods that forage on prey of similar size to their own, was thus far lacking in the Paleozoic and early Mesozoic. Here we report a top-tier tetrapod predator, a very large (>8.6 m) ichthyosaur from the early Middle Triassic (244 Ma), of Nevada. This ichthyosaur had a massive skull and large labiolingually flattened teeth with two cutting edges indicative of a macropredatory feeding style. Its presence documents the rapid evolution of modern marine ecosystems in the Triassic where the same level of complexity as observed in today's marine ecosystems is reached within 8 My after the Permian-Triassic mass extinction and within 4 My of the time reptiles first invaded the sea. This find also indicates that the biotic recovery in the marine realm may have occurred faster compared with terrestrial ecosystems, where the first apex predators may not have evolved before the Carnian.

Early evolution of limb regeneration in tetrapods: evidence from a 300-million-year-old amphibian.

Salamanders are the only tetrapods capable of fully regenerating their limbs throughout their entire lives. Much data on the underlying molecular mechanisms of limb regeneration have been gathered in recent years allowing for new comparative studies between salamanders and other tetrapods that lack this unique regenerative potential. By contrast, the evolution of animal regeneration just recently shifted back into focus, despite being highly relevant for research designs aiming to unravel the factors allowing for limb regeneration. We show that the 300-million-year-old temnospondyl amphibian Micromelerpeton, a distant relative of modern amphibians, was already capable of regenerating its limbs. A number of exceptionally well-preserved specimens from fossil deposits show a unique pattern and combination of abnormalities in their limbs that is distinctive of irregular regenerative activity in modern salamanders and does not occur as variants of normal limb development. This demonstrates that the capacity to regenerate limbs is not a derived feature of modern salamanders, but may be an ancient feature of non-amniote tetrapods and possibly even shared by all bony fish. The finding provides a new framework for understanding the evolution of regenerative capacity of paired appendages in vertebrates in the search for conserved versus derived molecular mechanisms of limb regeneration.

A stem batrachian from the Early Permian of Texas and the origin of frogs and salamanders.

The origin of extant amphibians (Lissamphibia: frogs, salamanders and caecilians) is one of the most controversial questions in vertebrate evolution, owing to large morphological and temporal gaps in the fossil record. Current discussions focus on three competing hypotheses: a monophyletic origin within either Temnospondyli or Lepospondyli, or a polyphyletic origin with frogs and salamanders arising among temnospondyls and caecilians among the lepospondyls. Recent molecular analyses are also controversial, with estimations for the batrachian (frog-salamander) divergence significantly older than the palaeontological evidence supports. Here we report the discovery of an amphibamid temnospondyl from the Early Permian of Texas that bridges the gap between other Palaeozoic amphibians and the earliest known salientians and caudatans from the Mesozoic. The presence of a mosaic of salientian and caudatan characters in this small fossil makes it a key taxon close to the batrachian (frog and salamander) divergence. Phylogenetic analysis suggests that the batrachian divergence occurred in the Middle Permian, rather than the late Carboniferous as recently estimated using molecular clocks, but the divergence with caecilians corresponds to the deep split between temnospondyls and lepospondyls, which is congruent with the molecular estimates.

A new recumbirostran 'microsaur' from the lower Permian Bromacker locality, Thuringia, Germany, and its fossorial adaptations.

Several recumbirostran 'microsaurs' are known from early Permian sites across Germany, including the Tambach Formation in Thuringia, central Germany. The only 'microsaur' thus far described from the Tambach Formation was the ostodolepid recumbirostran Tambaroter carrolli. However, there is also the documented presence of an undescribed recumbirostran 'microsaur' at the well-known Bromacker locality. The Bromacker locality is highly recognized and best known for its very diverse and extremely well-preserved terrestrial tetrapod assemblage combined with the co-occurrence of an exceptional vertebrate ichnofossil record. Here we describe a second new recumbirostran taxon from the Tambach Formation, which is also the first from the Bromacker locality itself. Phylogenetic analysis indicates that the new taxon, Bromerpeton subcolossus gen. et sp. nov., is a brachystelechid recumbirostran, a group also known elsewhere in Germany. The following features differentiate Bromerpeton from the other members of the clade: the presence of 13 maxillary teeth, narrow postorbitals that do not substantially contribute to the postorbital region of the skull, the frontal does not contribute to the orbital margin, and the presence of five manual digits. This new recumbirostran 'microsaur' further adds to the unique ecosystem that is preserved at the Bromacker locality, granting us a better understanding of what was living underfoot the larger more well-known animals at the locality. Likewise, it expands our understanding of the evolution of recumbirostran 'microsaurs', especially with regards to digit and limb reduction within the clade.

Amphibian development in the fossil record.

Ontogenetic series of extinct taxa are extremely rare and when preserved often incomplete and difficult to interpret. However, the fossil record of amphibians includes a number of well-preserved ontogenetic sequences for temnospondyl and lepospondyl taxa, which have provided valuable information about the development of these extinct groups. Here we summarize the current knowledge on fossil ontogenies of amphibians, their potential and limitations for relationship assessments, and discuss the insights they have provided for our understanding of the anatomy, life history, and ecology of extinct amphibians.

Salamander limb development: integrating genes, morphology, and fossils.

The development of the tetrapod limb during skeletogenesis follows a highly conservative pattern characterized by a general proximo-distal progression in the establishment of skeletal elements and a postaxial polarity in digit development. Salamanders represent the only exception to this pattern and display an early establishment of distal autopodial structures, specifically the basale commune, an amalgamation of distal carpal and tarsal 1 and 2, and a distinct preaxial polarity in digit development. This deviance from the conserved tetrapod pattern has resulted in a number of hypotheses to explain its developmental basis and evolutionary history. Here we summarize the current knowledge of salamander limb development under consideration of the fossil record to provide a deep time perspective of this evolutionary pathway and highlight what data will be needed in the future to gain a better understanding of salamander limb development specifically and tetrapod limb development and evolution more broadly.

Regionalization, constraints, and the ancestral ossification patterns in the vertebral column of amniotes.

The development of the vertebral column has been studied extensively in modern amniotes, yet many aspects of its evolutionary history remain enigmatic. Here we expand the existing data on four major vertebral developmental patterns in amniotes based on exceptionally well-preserved specimens of the early Permian mesosaurid reptile Mesosaurus tenuidens: (i) centrum ossification, (ii) neural arch ossification, (iii) neural arch fusion, and (iv) neurocentral fusion. We retrace the evolutionary history of each pattern and reconstruct the ancestral condition in amniotes. Despite 300 million years of evolutionary history, vertebral development patterns show a surprisingly stability in amniotes since their common ancestor. We propose that this stability may be linked to conservatism in the constraints posed by underlying developmental processes across amniotes. We also point out that birds, mammals, and squamates each show specific trends deviating from the ancestral condition in amniotes, and that they remain rather unchanged within these lineages. The stability of their unique patterns demonstrates a certain homogeneity of vertebral developmental constraints within these lineages, which we suggest might be linked to their specific modes of regionalization. Our research provides a framework for the evolution of axial development in amniotes and a foundation for future studies.

Testing the impact of miniaturization on phylogeny: Paleozoic dissorophoid amphibians.

Among the diverse clade of Paleozoic dissorophoid amphibians, the small, terrestrial amphibamids and the neotenic branchiosaurids have frequently been suggested as possible antecedents of either all or some of the modern amphibian clades. Classically, amphibamids and branchiosaurids have been considered to represent distinct, but closely related clades within dissorophoids, but despite their importance for the controversial lissamphibian origins, a comprehensive phylogenetic analysis of small dissorophoids has thus far not been attempted. On the basis of an integrated data set, the relationships of amphibamids and branchiosaurids were analyzed using parsimony and Bayesian approaches. Both groups represent miniaturized forms and it was tested whether similar developmental pathways, associated with miniaturization, lead to an artificial close relationship of branchiosaurids and amphibamids. Moreover, the fit of the resulting tree topologies to the distribution of fossil taxa in the stratigraphic rock record was assessed as an additional source of information. The results show that characters associated with a miniaturized morphology are not responsible for the close clustering of branchiosaurids and amphibamids. Instead, all analyses invariably demonstrate a monophyletic clade of branchiosaurids highly nested within derived amphibamids, indicating that branchiosaurids represent a group of secondarily neotenic amphibamid dissorophoids. This understanding of the phylogenetic relationships of small dissorophoid amphibians provides a new framework for the discussion of their evolutionary history and the evolution of characters shared by branchiosaurids and/or amphibamids with modern amphibian taxa.

Examining the relationship between sexual dimorphism in skin anatomy and body size in the white-lipped treefrog, Litoria infrafrenata (Anura: Hylidae).

Amphibians transport water, oxygen, carbon dioxide and various ions (e.g. sodium and potassium) across their skin. This cutaneous permeability is thought to affect their ability to respond to environmental change and to play a role in global population declines. Sexual dimorphism of skin anatomy has been accepted in some species, but rejected in others. The species in which such dimorphism has been detected have all been sexually dimorphic in body size, with males that are smaller and have thinner skin. It is unclear whether this difference in skin thickness manifests a functional difference or if it is related to body size alone. Skin thickness (epidermis, spongy dermis, compact dermis and total thickness) was examined in males and females of the white-lipped treefrog (Litoria infrafrenata). Although the skin of males is absolutely thinner than that of females, this difference is explained by body size differences between the sexes. Overall, we conclude that skin thickness in male and female L. infrafrenata correlates with body size dimorphism and suggest that future studies on amphibian skin anatomy include measures of body size, test the ecological significance of sexually dimorphic skin anatomy and better document the prevalence of sexually dimorphic amphibian skin anatomy.

Intercentrum versus pleurocentrum growth in early tetrapods: A paleohistological approach.

A variety of vertebral centrum morphologies have evolved within early tetrapods which range from multipartite centra consisting of intercentra and pleurocentra in stem-tetrapods, temnospondyls, seymouriamorphs, and anthracosaurs up to monospondylous centra in lepospondyls. With the present study, we aim to determine the formation of both intercentrum and pleurocentrum and asked whether these can be homologized based on their bone histology. Both intercentra and pleurocentra ossified endochondrally and periosteal bone was subsequently deposited on the outer surface of the centra. Our observations indicate low histological variation between intercentrum and pleurocentrum in microstructural organization and growth which inhibits the determination of homologies. However, intercentrum and pleurocentrum development differs during ontogeny. As previously assumed, the intercentrum arises from ventrally located and initially paired ossification centers that fuse ventromedially to form the typical, crescentic, rhachitomous intercentrum. In contrast, presacral pleurocentra may be ancestrally represented by four ossification centers: a ventral and a dorsal pair. Subsequently, two divergent developmental patterns are observed: In stem-tetrapods and temnospondyls, the pleurocentrum evolves from the two dorsally located ossification centers which may occasionally fuse to form a dorsal crescent. In some dvinosaurian temnospondyls, the pleurocentrum may even ossify to full rings. In comparison, the pleurocentrum of stem-amniotes (anthracosaurs, chroniosuchids, seymouriamorphs, and lepospondyls) arises from the two ventrally located ossification centers whereby the ossification pattern is almost identical to that of temnospondyls but mirror-inverted. Thus, the ring-shaped pleurocentrum of Discosauriscus ossifies from ventral to dorsal. We also propose that the ossified portions of the intercentrum and pleurocentrum continued as cartilaginous rings or discs that surrounded the notochord in the living animals.

Metamorphosis and neoteny: alternative pathways in an extinct amphibian clade.

The Branchiosauridae was a clade of small amphibians from the Permo-Carboniferous with an overall salamander-like appearance. The clade is distinguished by an extraordinary fossil record that comprises hundreds of well-preserved specimens, representing a wide range of ontogenetic stages. Branchiosaurids had external gills and weakly ossified skeletons, and due to this larval appearance their status as neotenic (perennibranchiate) forms has long been accepted. Despite their extensive fossil record large specimens with an adult morphology appeared to be lacking altogether, but recently two adult specimens were identified in a rich sample of Apateon gracilis collected in the 19th century from a locality near Dresden, Saxony. These specimens are unique among branchiosaurids in showing a high level of ossification, including bones that have never been reported in a branchiosaur. These highlight the successive formation of features believed to indicate terrestrial locomotion, as well as feeding on larger prey items. Moreover, these transformations occurred in a small time window (whereas the degree of size increase is used as a proxy of time) and the degree of concentration of developmental events in branchiosaurids is unique among tetrapods outside the lissamphibians. These specimens are compared with large adults of the neotenic branchiosaurid Apateon caducus from the Saar-Nahe Basin, which despite their larger body size lack the features found in the adult A. gracilis specimens. These specimens give new insight into patterns of metamorphosis (morphological transformation) in branchiosaurids that are believed to be correlated to a change of habitat, and clearly show that different life-history pathways comparable to those of modern salamanders were already established in this Paleozoic clade.

Vertebral Development in Paleozoic and Mesozoic Tetrapods Revealed by Paleohistological Data.

Basal tetrapods display a wide spectrum of vertebral centrum morphologies that can be used to distinguish different tetrapod groups. The vertebral types range from multipartite centra in stem-tetrapods, temnospondyls, and seymouriamorphs up to monospondylous centra in lepospondyls and have been drawn upon for reconstructing major evolutionary trends in tetrapods that are now considered textbook knowledge. Two modes of vertebral formation have been postulated: the multipartite vertebrae formed first as cartilaginous elements with subsequent ossification. The monospondylous centrum, in contrast, was formed by direct ossification without a cartilaginous precursor. This study describes centrum morphogenesis in basal tetrapods for the first time, based on bone histology. Our results show that the intercentra of the investigated stem-tetrapods consist of a small band of periosteal bone and a dense network of endochondral bone. In stereospondyl temnospondyls, high amounts of calcified cartilage are preserved in the endochondral trabeculae. Notably, the periosteal region is thickened and highly vascularized in the plagiosaurid stereospondyls. Among "microsaur" lepospondyls, the thickened periosteal region is composed of compact bone and the notochordal canal is surrounded by large cell lacunae. In nectridean lepospondyls, the periosteal region has a spongy structure with large intertrabecular spaces, whereas the endochondral region has a highly cancellous structure. Our observations indicate that regardless of whether multipartite or monospondylous, the centra of basal tetrapods display first endochondral and subsequently periosteal ossification. A high interspecific variability is observed in growth rate, organization, and initiation of periosteal ossification. Moreover, vertebral development and structure reflect different lifestyles. The bottom-dwelling Plagiosauridae increase their skeletal mass by hyperplasy of the periosteal region. In nectrideans, the skeletal mass decreases, as the microstructure is spongy and lightly built. Additionally, we observed that vertebral structure is influenced by miniaturization in some groups. The phylogenetic information that can be drawn from vertebral development, however, is limited.

Ossification patterns in the tetrapod limb--conservation and divergence from morphogenetic events.

Two different patterns of the condensation and chondrification of the limbs of tetrapods are known from extensive studies on their early skeletal development. These are on the one hand postaxial dominance in the sequential formation of skeletal elements in amniotes and anurans, and on the other, preaxial dominance in urodeles. The present study investigates the relative sequence of ossification in the fore- and hindlimbs of selected tetrapod taxa based on a literature survey in comparison to the patterns of early skeletal development, i.e. mesenchymal condensation and chondrification, representing essential steps in the late stages of tetrapod limb development. This reveals the degree of conservation and divergence of the ossification sequence from early morphogenetic events in the tetrapod limb skeleton. A step-by-step recapitulation of condensation and chondrification during the ossification of limbs can clearly be refuted. However, some of the deeper aspects of early skeletal patterning in the limbs, i.e. the general direction of development and sequence of digit formation are conserved, particularly in anamniotes. Amniotes show a weaker coupling of the ossification sequence in the limb skeleton with earlier condensation and chondrification events. The stronger correlation between the sequence of condensation/chondrification and ossification in the limbs of anamniotes may represent a plesiomorphic trait of tetrapods. The pattern of limb ossification across tetrapods also shows that some trends in the sequence of ossification of their limb skeleton are shared by major clades possibly representing phylogenetic signals. This review furthermore concerns the ossification sequence of the limbs of the Palaeozoic temnospondyl amphibian Apateon sp. For the first time this is described in detail and its patterns are compared with those observed in extant taxa. Apateon sp. shares preaxial dominance in limb development with extant salamanders and the specific order of ossification events in the fore- and hindlimb of this fossil dissorophoid is almost identical to that of some modern urodeles.

Variability and conservation in late chondrichthyan development: ontogeny of the winter skate (Leucoraja ocellata).

The ontogeny of the northwestern Atlantic batoid, Leucoraja ocellata, is described with special focus on the development of skate specific morphologies and the development of the fins. The results show that the sequence of events involving the early outgrowth of the paired fins and the initial development of the pharyngeal region is remarkably constant in skates, holocephalians, and sharks. However, differences exist in timing of the reshaping of the mandibular arch region, development of branchial filaments, median fins, denticles, and the order of branchial cleft formation. Despite the similarities of early events related to development of the branchial region and initial outgrowth of the fins, later stages are increasingly characterized by taxon-specific morphologies making a universal staging table for chondrichthyans less applicable. The staging table presented in this study represents an important resource for future studies on batoid embryology.