Fins to limbs: what the fossils say

A broad phylogenetic review of fins, limbs, and girdles throughout the stem and base of the crown group is needed to get a comprehensive idea of transformations unique to the assembly of the tetrapod limb ground plan. In the lower part of the tetrapod stem, character state changes at the pectoral level dominate; comparable pelvic level data are limited. In more crownward taxa, pelvic level changes dominate and repeatedly precede similar changes at pectoral level. Concerted change at both levels appears to be the exception rather than the rule. These patterns of change are explored by using afternative treatments of data in phylogenetic analyses. Results highlight a large data gap in the stem group preceding the first appearance of limbs with digits. It is also noted that the record of morphological diversity among stem tetrapods is somewhat worse than that of basal crown group tetrapods. The pre-limbed evolution of stem tetrapod paired fins is marked by a gradual reduction in axial segment numbers (mesomeres); pectoral fins of the sister group to limbed tetrapods include only three. This reduction in segment number is accompanied by increased regional specialization, and these changes are discussed with reference to the phylogenetic distribution of characteristics of the stylopod, zeugopod, and autopod.     

Reconstructing pectoral appendicular muscle anatomy in fossil fish and tetrapods over the fins‐to‐limbs transition

The question of how tetrapod limbs evolved from fins is one of the great puzzles of evolutionary biology. While palaeontologists, developmental biologists, and geneticists have made great strides in explaining the origin and early evolution of limb skeletal structures, that of the muscles remains largely unknown. The main reason is the lack of consensus about appendicular muscle homology between the closest living relatives of early tetrapods: lobe‐finned fish and crown tetrapods. In the light of a recent study of these homologies, we re‐examined osteological correlates of muscle attachment in the pectoral girdle, humerus, radius, and ulna of early tetrapods and their close relatives. Twenty‐nine extinct and six extant sarcopterygians were included in a meta‐analysis using information from the literature and from original specimens, when possible. We analysed these osteological correlates using parsimony‐based character optimization in order to reconstruct muscle anatomy in ancestral lobe‐finned fish, tetrapodomorph fish, stem tetrapods, and crown tetrapods. Our synthesis revealed that many tetrapod shoulder muscles probably were already present in tetrapodomorph fish, while most of the more‐distal appendicular muscles either arose later from largely undifferentiated dorsal and ventral muscle masses or did not leave clear correlates of attachment in these taxa. Based on this review and meta‐analysis, we postulate a stepwise sequence of specific appendicular muscle acquisitions, splits, and fusions that led from the ancestral sarcopterygian pectoral fin to the ancestral tetrapod forelimb. This sequence largely agrees with previous hypotheses based on palaeontological and comparative work, but it is much more comprehensive in terms of both muscles and taxa. Combined with existing information about the skeletal system, our new synthesis helps to illuminate the genetic, developmental, morphological, functional, and ecological changes that were key components of the fins‐to‐limbs transition.     

Anatomical network analysis of the musculoskeletal system reveals integration loss and parcellation boost during the fins‐to‐limbs transition

Tetrapods evolved from within the lobe‐finned fishes around 370 Ma. The evolution of limbs from lobe‐fins entailed a major reorganization of the skeletal and muscular anatomy of appendages in early tetrapods. Concurrently, a degree of similarity between pectoral and pelvic appendages also evolved. Here, we compared the anatomy of appendages in extant lobe‐finned fishes (Latimeria and Neoceratodus) and anatomically plesiomorphic amphibians (Ambystoma, Salamandra) and amniotes (Sphenodon) to trace and reconstruct the musculoskeletal changes that took place during the fins‐to‐limbs transition. We quantified the anatomy of appendages using network analysis. First, we built network models—in which nodes represent bones and muscles, and links represent their anatomical connections—and then we measured network parameters related to their anatomical integration, heterogeneity, and modularity. Our results reveal an evolutionary transition toward less integrated, more modular appendages. We interpret this transition as a diversification of muscle functions in tetrapods compared to lobe‐finned fishes. Limbs and lobe‐fins show also a greater similarity between their pectoral and pelvic appendages than ray‐fins do. These findings on extant species provide a basis for future quantitative and comprehensive reconstructions of the anatomy of limbs in early tetrapod fossils, and a way to better understand the fins‐to‐limbs transition.     

Tri-phasic expression of posterior Hox genes during development of pectoral fins in zebrafish: implications for the evolution of vertebrate paired appendages

During development of the limbs, Hox genes belonging to the paralogous groups 9-13 are expressed in three distinct phases, which play key roles in the segmental patterning of limb skeletons. In teleost fishes, which have a very different organization in their fin skeletons, it is not clear whether a similar patterning mechanism is at work. To determine whether Hox genes are also expressed in several distinct phases during teleost paired fin development, we re-analyzed the expression patterns of hox9-13 genes during development of pectoral fins in zebrafish. We found that, similar to tetrapod Hox genes, expression of hoxa/d genes in zebrafish pectoral fins occurs in three distinct phases, in which the most distal/third phase is correlated with the development of the most distal structure of the fin, the fin blade. Like in tetrapods, hox gene expression in zebrafish pectoral fins during the distal/third phase is dependent upon sonic hedgehog signaling (hoxa and hoxd genes) and the presence of a long-range enhancer (hoxa genes), which indicates that the regulatory mechanisms underlying tri-phasic expression of Hox genes have remained relatively unchanged during evolution. Our results suggest that, although simpler in organization, teleost fins do have a distal structure that might be considered comparable to the autopod region of limbs.     

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.     

Expression of Hoxa-11 and Hoxa-13 in the pectoral fin of a basal ray-finned fish, Polyodon spathula: implications for the origin of tetrapod limbs

Summary Paleontological and anatomical evidence suggests that the autopodium (hand or foot) is a novel feature that distinguishes limbs from fins, while the upper and lower limb (stylopod and zeugopod) are homologous to parts of the sarcopterygian paired fins. In tetrapod limb development Hoxa-11 plays a key role in differentiating the lower limb and Hoxa-13 plays a key role in differentiating the autopodium. It is thus important to determine the ancestral functions of these genes in order to understand the developmental genetic changes that led to the origin of the tetrapod autopodium. In particular it is important to understand which features of gene expression are derived in tetrapods and which are ancestral in bony fishes. To address these questions we cloned and sequenced the Hoxa-11 and Hoxa-13 genes from the North American paddlefish, Polyodon spathula, a basal ray-finned fish that has a pectoral fin morphology resembling that of primitive bony fishes ancestral to the tetrapod lineage. Sequence analysis of these genes shows that they are not orthologous to the duplicated zebrafish and fugu genes. This implies that the paddlefish has not duplicated its HoxA cluster, unlike zebrafish and fugu. The expression of Hoxa-11 and Hoxa-13 in the pectoral fins shows two main phases: an early phase in which Hoxa-11 is expressed proximally and Hoxa-13 is expressed distally, and a later phase in which Hoxa-11 and Hoxa-13 broadly overlap in the distal mesenchyme of the fin bud but are absent in the proximal fin bud. Hence the distal polarity of Hoxa-13 expression seen in tetrapods is likely to be an ancestral feature of paired appendage development. The main difference in HoxA gene expression between fin and limb development is that in tetrapods (with the exception of newts) Hoxa-11 expression is suppressed by Hoxa-13 in the distal limb bud mesenchyme. There is, however, a short period of limb bud development where Hoxa-11 and Hoxa-13 overlap similarly to the late expression seen in zebrafish and paddlefish. We conclude that the early expression pattern in tetrapods is similar to that seen in late fin development and that the local exclusion by Hoxa-13 of Hoxa-11 from the distal limb bud is a derived feature of limb developmental regulation.     

Homology and a generative theory of biological form

Homology continues to be a concept of central importance in the study of phylogenetic relations, but its relation to ontogenetic processes remains problematical. A definition of homology in terms of equivalent morphogenetic processes is defined and applied to the comparative study of tetrapod limbs. This allows for a consistent treatment of relations of similarity and difference of appendage structure in vertebrates, and the distinction between fishes fins and tetrapod limbs in terms of the concept of equivalence is described. The role of genes can also be clarified in this context, in particular the influence of the Hox 4 complex in determining digit character and the homeotic transformations that arise from changes in their expression patterns. It is argued that these observations are not compatible with the notion of homology between individual digits (I, II, III, etc.) across the tetrapods, and that homology cannot be consistently identified with gene action. The relations between homology and the properties of the morphogenetic limb field are discussed.     

The postcranial skeleton of the Devonian tetrapod Tulerpeton curtum Lebedev

Postcranial remains of the Russian Late Devonian tetrapod Tulerpeton include the hexadactylous fore limb, hind limb, anocleithral pectoral girdle, squamation, and associated disarticulated postcranial bones. A cladistic analysis indicates that Tulerpeton is a reptiliomorph stem-group amniote and the earliest known crown-group tetrapod: Acanthostega and Ichthyostega are successively more derived plesion stem-group tetrapods and do not consititute a monophyletic ichthyostegalian radiation. Previous analyses suggesting a profound split in tetrapod phylogeny are thereby corroborated, and likewise the interpretation of Westlothiana as a stem-group amniote. The divergence of reptiliomorphs from batrachomorphs occurred before the Devonian-Carboniferous boundary. Tulerpeton originates from an entirely aquatic environment with a diverse fish fauna. The morphologies of its limbs and those of Devonian stem-tetrapods suggest that dactyly predates the elaboration of the carpus and tarsus, and that Polydactyly persisted after the evolutionary divergence of the principal lineages of living tetrapods. The apparent absence of a branchial lamina and gill skeleton suggests that Tulerpeton was primarily air-breathing, whereas contemporary stem-group tetrapods and more recent batrachomorphs retained greater emphasis on gill-breathing.     

Elimination of a long-range cis-regulatory module causes complete loss of limb-specific Shh expression and truncation of the mouse limb

Mutations in a conserved non-coding region in intron 5 of the Lmbr1 locus, which is 1 Mb away from the sonic hedgehog (Shh) coding sequence, are responsible for mouse and human preaxial polydactyly with mirror-image digit duplications. In the mouse mutants, ectopic Shh expression is observed in the anterior mesenchyme of limb buds. Furthermore, a transgenic reporter gene flanked with this conserved non-coding region shows normal polarized expression in mouse limb buds. This conserved sequence has therefore been proposed to act as a long-range, cis-acting regulator of limb-specific Shh expression. Previous phylogenetic studies have also shown that this sequence is highly conserved among tetrapods, and even in teleost fishes. Paired fins of teleost fishes and tetrapod limbs have evolved from common ancestral appendages, and polarized Shh expression is commonly observed in fins. In this study, we first show that this conserved sequence motif is also physically linked to the Shh coding sequence in a teleost fish, the medaka, by homology search of a newly available genomic sequence database. Next, we show that deletion of this conserved intronic sequence by targeted mutation in the mouse results in a complete loss of Shh expression in the limb bud and degeneration of skeletal elements distal to the stylopod/zygopod junction. This sequence contains a major limb-specific Shh enhancer that is necessary for distal limb development. These results suggest that the conserved intronic sequence evolved in a common ancestor of fishes and tetrapods to control fin and limb development.     

The evolution of paired appendages in vertebrates: T-box genes in the zebrafish

The presence of two sets of paired appendages is one of the defining features of jawed vertebrates. We are interested in identifying genetic systems that could have been responsible for the origin of the first set of such appendages, for their subsequent duplication at a different axial level, and/or for the generation of their distinct identities. It has been hypothesized that four genes of the T-box gene family (Tbx2–Tbx5) played important roles in the course of vertebrate limb evolution. To test this idea, we characterized the orthologs of tetrapod limb-expressed T-box genes from a teleost, Danio rerio. Here we report isolation of three of these genes, tbx2, tbx4, and tbx5. We found that their expression patterns are remarkably similar to those of their tetrapod counterparts. In particular, expression of tbx5 and tbx4 is restricted to pectoral and pelvic fin buds, respectively, while tbx2 can be detected at the anterior and posterior margins of the outgrowing fin buds. This, in combination with conserved expression patterns in other tissues, suggests that the last common ancestor of teleosts and tetrapods possessed all four of these limb-expressed T-box genes (Tbx2–Tbx5), and that these genes had already acquired, and have subsequently maintained, their gene-specific functions. Furthermore, this evidence provides molecular support for the notion that teleost pectoral and pelvic fins and tetrapod fore- and hindlimbs, respectively, are homologous structures, as suggested by comparative morphological analyses. Received: 14 July 1999 / Accepted: 4 September 1999     

Functional analysis of limb enhancers in the developing fin

Despite diverging ～365 million years ago, tetrapod limbs and pectoral fins express similar genes that could be regulated by shared regulatory elements. In this study, we set out to analyze the ability of enhancers to maintain tissue specificity in these two divergent structures. We tested 22 human sequences that were previously reported as mouse limb enhancers for their enhancer activity in zebrafish (Danio rerio). Using a zebrafish enhancer assay, we found that 10/22 (45 %) were positive for pectoral fin activity. Analysis of the various criteria that correlated with positive fin activity found that both spatial limb activity and evolutionary conservation are not good predictors of fin enhancer activity. These results suggest that zebrafish enhancer assays may be limited in detecting human limb enhancers, and this limitation does not improve by the use of limb spatial expression or evolutionary conservation.     

Morphogenetic approach to the formation of paired limbs in the course of tetrapodization

Transition from sarcopterygians to tetrapods is analyzed based on new paleontological, ontogenetic, and molecular data. It is shown that transformation of skeletal fin elements into the tetrapod limb followed the patterns of divergent, parallel, and mosaic development. Morphogenetic plasticity and autonomy of these processes as well as the same developmental bauplan for the limbs of Urodela and Anura are proposed. Variations observed in these processes are regarded as a result of larval adaptations and heterochronies. The latter excludes recapitulation of successive archetypical states (transformation-development of the fish fin into tetrapod limb). The idea that the digits are a novelty relative to the distal radials of the fin is supported.     

Starting from fins: parallelism in the evolution of limbs and genitalia. The fin-to-limb transition

The March/April 2002 issue of Evolution and Development focused on three presentations made at the Starting from Fins: Parallelism in the Evolution of Limbs and Genitalia symposium held as part of the 2001 Chicago meeting of the Society of Integrative and Comparative Biology. The intention of the symposium and the publication of the presentations was to extend discussion of the potential and the limits of using serial homologues to understand developmental aspects of morphological evolution. The March/April 2002 issue concentrated on unpaired fin to genitalia transitions. This issue focuses on paired fins to limbs and highlights the need for developmental data to be integrated with data from fossil materal, phylogenetic analysis, and explicitly comparative studies. Coates et al. use phylogenetic methods to explore the limb/fin characters of taxa, but their analysis departs somewhat from the usual in that the reference group for organisms includes sister group taxa not usually considered true tetrapods. They state that including finned taxa from the stem group permits an attempt to distinguish the primitive condition of the characteristics demonstrated by the crown group, that is, "limbed tetrapods." In focusing on limb characters specifically and including aspects of the appendicular girdles, Coates et al. highlight morphological details and trends within a given phylogeny. They also demonstrate the degree of relevance of limb characters during the establishment of lineages and their branching patterns by using only limb characters to generate a tree and use a direct comparison of serial versus special homologies to explore the degree of evolutionary parallelism between fore-and hindlimbs. The preliminary conclusions indicate a high level of independence between the serially homologous fore-and hindlimb. Innes et al. present outcomes from the use of cutting edge molecular genetic approaches to understand developmental aspects of limb morphology. In a manner conceptually similar to Coates et al.'s use of fossil characters, Innes et al. use the serial analysis of gene expression to sort differences from similarities in the gene expression profiles of fore-and hindlimbs of the same embryos. Although these gene expression pattems are likely to reflect the serial homology of the paired limbs, they are silent in terms of our understanding both the profound and subtle differences between fore- and hindlimbs in any given species. Innes et al. point out the volume of data generated by SAGE far exceeds our ability to interpret its biological meaning. The studies presented here and in the March/April issue are excellent examples of the need to interpret complex data in light of collective knowledge of evolutionary history. We hope the insights gained from the symposium and papers contribute to a dialogue on how to integrate different approaches and assist in moving forward the field of Evolution and Development.     

Fish fingers: digit homologues in sarcopterygian fish fins

A defining feature of tetrapod evolutionary origins is the transition from fish fins to tetrapod limbs. A major change during this transition is the appearance of the autopod (hands, feet), which comprises two distinct regions, the wrist/ankle and the digits. When the autopod first appeared in Late Devonian fossil tetrapods, it was incomplete: digits evolved before the full complement of wrist/ankle bones. Early tetrapod wrists/ankles, including those with a full complement of bones, also show a sharp pattern discontinuity between proximal elements and distal elements. This suggests the presence of a discontinuity in the proximal-distal sequence of development. Such a discontinuity occurs in living urodeles, where digits form before completion of the wrist/ankle, implying developmental independence of the digits from wrist/ankle elements. We have observed comparable independent development of pectoral fin radials in the lungfish Neoceratodus (Osteichthyes: Sarcopterygii), relative to homologues of the tetrapod limb and proximal wrist elements in the main fin axis. Moreover, in the Neoceratodus fin, expression of Hoxd13 closely matches late expression patterns observed in the tetrapod autopod. This evidence suggests that Neoceratodus fin radials and tetrapod digits may be patterned by shared mechanisms distinct from those patterning the proximal fin/limb elements, and in that sense are homologous. The presence of independently developing radials in the distal part of the pectoral (and pelvic) fin may be a general feature of the Sarcopterygii.     

The Fish–Tetrapod Transition: New Fossils and Interpretations

Our information on the transition between fish with fins and tetrapods with limbs and digits has increased manyfold in the last 15–20 years and especially in the last 5 or 10 years, with some spectacular finds of new material. Some of these include new tetrapod-like fish and very primitive tetrapods that help to resolve questions of the sequence of acquisition of tetrapod characters, the approximate timing of the events, the likely geographic location, and the circumstances under which it happened. Forelimbs and skulls became modified in advance of hind limbs, adapted for supporting the head and front of the body out of the water, probably in connection with air breathing. The likely time of origin for limbed tetrapods is between 385 and 380 million years ago, probably in the northern continent of Laurussia. The origin of limbed tetrapods did not coincide with the acquisition of full terrestriality, an outcome that probably arose in the Early Carboniferous. This later part of the story is documented by few fossils, though two in particular give key information. Studies of modern vertebrates, especially the evolutionary developmental genetics of Hox genes, are beginning to provide clues to the origin of digits.     

First discovery of a primitive coelacanth fin fills a major gap in the evolution of lobed fins and limbs

The fossil record provides unique clues about the primitive pattern of lobed fins, the precursors of digit-bearing limbs. Such information is vital for understanding the evolutionary transition from fish fins to tetrapod limbs, and it guides the choice of model systems for investigating the developmental changes underpinning this event. However, the evolutionary preconditions for tetrapod limbs remain unclear. This uncertainty arises from an outstanding gap in our knowledge of early lobed fins: there are no fossil data that record primitive pectoral fin conditions in coelacanths, one of the three major groups of sarcopterygian (lobe-finned) fishes. A new fossil from the Middle-Late Devonian of Wyoming preserves the first and only example of a primitive coelacanth pectoral fin endoskeleton. The strongly asymmetrical skeleton of this fin corroborates the hypothesis that this is the primitive sarcopterygian pattern, and that this pattern persisted in the closest fish-like relatives of land vertebrates. The new material reveals the specializations of paired fins in the modern coelacanth, as well as in living lungfishes. Consequently, the context in which these might be used to investigate evolutionary and developmental relationships between vertebrate fins and limbs is changed. Our data suggest that primitive actinopterygians, rather than living sarcopterygian fishes and their derived appendages, are the most informative comparators for developmental studies seeking to understand the origin of tetrapod limbs.     

A near-tetrapod from the Baltic Middle Devonian

The tetrapodomorph sarcopterygian Livoniana multidentata gen. et sp. nov. is described on the basis of lower jaw fragments from the Middle Devonian(late Givetian) of Latvia and Estonia. It possesses a suite of derived characters previously only known from tetrapods, which first appear in the late Devonian (late Frasnian), and a phylogenetic analysis places it on the internode between Panderichthys and the base of the Tetrapoda. The analysis also reveals that the 'Elpistostegalia' are paraphyletic to Tetrapoda, with Elpistostege closer to tetrapods than is Panderichthys. Owing to incompleteness of the material, there is almost no overlap between the data sets for Elpistostege Livoniana ; the analysis places the two genera in an unresolved trichotomy. In addition to the tetrapod features, Livoniana has a strikingly autapomorphic dentary dentition comprising multiple tooth rows. It thus provides evidence both for the unexpectedly early evolution of tetrapod characteristics and for morphological radiation around the fish-tetrapod transition.