Limb Evolution Research Articles

A Combination of Activation and Repression by a Colinear Hox Code Controls Forelimb-Restricted Expression of Tbx5 and Reveals Hox Protein Specificity

Tight control over gene expression is essential for precision in embryonic development and acquisition of the regulatory elements responsible is the predominant driver for evolution of new structures. Tbx5 and Tbx4, two genes expressed in forelimb and hindlimb-forming regions respectively, play crucial roles in the initiation of limb outgrowth. Evolution of regulatory elements that activate Tbx5 in rostral LPM was essential for the acquisition of forelimbs in vertebrates. We identified such a regulatory element for Tbx5 and demonstrated Hox genes are essential, direct regulators. While the importance of Hox genes in regulating embryonic development is clear, Hox targets and the ways in which each protein executes its specific function are not known. We reveal how nested Hox expression along the rostro-caudal axis restricts Tbx5 expression to forelimb. We demonstrate that Hoxc9, which is expressed in caudal LPM where Tbx5 is not expressed, can form a repressive complex on the Tbx5 forelimb regulatory element. This repressive capacity is limited to Hox proteins expressed in caudal LPM and carried out by two separate protein domains in Hoxc9. Forelimb-restricted expression of Tbx5 and ultimately forelimb formation is therefore achieved through co-option of two characteristics of Hox genes; their colinear expression along the body axis and the functional specificity of different paralogs. Active complexes can be formed by Hox PG proteins present throughout the rostral-caudal LPM while restriction of Tbx5 expression is achieved by superimposing a dominant repressive (Hoxc9) complex that determines the caudal boundary of Tbx5 expression. Our results reveal the regulatory mechanism that ensures emergence of the forelimbs at the correct position along the body. Acquisition of this regulatory element would have been critical for the evolution of limbs in vertebrates and modulation of the factors we have identified can be molecular drivers of the diversity in limb morphology.

Expression of arthropod distal limb-patterning genes in the onychophoran Euperipatoides kanangrensis

A current hypothesis states that the ancestral limb of arthropods is composed of only two segments. The proximal segment represents the main part of the modern leg, and the distal segment represents the tarsus and claw of the modern leg. If the distal part of the limb is an ancestral feature, one would expect conserved regulatory gene networks acting in distal limb development in all arthropods and possibly even their sister group, the onychophorans. We investigated the expression patterns of six genes known to function during insect distal limb development in the onychophoran Euperipatoides kanangrensis, i.e., clawless (cll), aristaless (al), spineless (ss), zinc finger homeodomain 2 (zfh2), rotund (rn), and Lim1. We find that all investigated genes are expressed in at least some of the onychophoran limbs. The expression patterns of most of these genes, however, display crucial differences to the known insect patterns. The results of this study question the hypothesis of conserved distal limb evolution in arthropods and highlight the need for further studies on arthropod limb development.

A Footnote to the Evolution of Digits

A Footnote to the Evolution of Digits

Conservation and Divergence of Regulatory Strategies at Hox Loci and the Origin of Tetrapod Digits

Author SummaryOur upper limbs differ from fish fins, notably by their subdivision into arm and hand regions, which are separated by a complex articulation, the wrist. The development of this anatomy is associated with two distinct waves of expression of the Hoxa and Hoxd genes during development. Would such a shared expression pattern be sufficient to infer homology between fish fins and mouse limbs? We investigated this question here, looking at whether the two phases of Hox gene transcription that are observed during tetrapod limb development also occur during zebrafish fin development. We find the answer is “not quite.” For although the mechanisms that regulate the expression of Hoxa and Hoxd are comparable between zebrafish fins and mouse limbs, when the genomic regions that regulate Hox gene expression in fish fins are introduced into transgenic mice, they trigger Hox gene expression in only the proximal limb segment (the segment nearest the body) and not in the presumptive digits. We conclude that although fish have the Hox regulatory toolkit to produce digits, this potential is not utilized as it is in tetrapods, and as a result we propose that fin radials—the bony elements of fins—are not homologous to tetrapod digits.

Bridging “Romer’s Gap”: Limb Mechanics of an Extant Belly-Dragging Lizard Inform Debate on Tetrapod Locomotion During the Early Carboniferous

Devonian stem tetrapods are thought to have used ‘crutching’ on land, a belly-dragging form of synchronous forelimb action-powered locomotion. During the Early Carboniferous, early tetrapods underwent rapid radiation, and the terrestrial locomotion of crown-group node tetrapods is believed to have been hindlimb-powered and ‘raised’, involving symmetrical gaits similar to those used by modern salamanders. The fossil record over this period of evolutionary transition is remarkably poor (Romer’s Gap), but we hypothesize a phase of belly-dragging sprawling locomotion combined with symmetrical gaits. Since belly-dragging sprawling locomotion has differing functional demands from ‘raised’ sprawling locomotion, we studied the limb mechanics of the extant belly-dragging blue-tongued skink. We used X-ray reconstruction of moving morphology to quantify the three-dimensional kinematic components, and simultaneously recorded single limb substrate reaction forces (SRF) in order to calculate SRF moment arms and the external moments acting on the proximal limb joints. In the hindlimbs, stylopodal long-axis rotation is more emphasized than in the forelimbs, and much greater vertical and propulsive forces are exerted. The SRF moment arm acting on the shoulder is at a local minimum at the instant of peak force. The hindlimbs display patterns that more closely resemble ‘raised’ sprawling species. External moment at the shoulder of the skink is smaller than in ‘raised’ sprawlers. We propose an evolutionary scenario in which the locomotor mechanics of belly-dragging early tetrapods were gradually modified towards hindlimb-powered, raised terrestrial locomotion with symmetrical gait. In accordance with the view that limb evolution was an exaptation for terrestrial locomotion, the kinematic pattern of the limbs for the generation of propulsion preceded, in our scenario, the evolution of permanent body weight support.

Development of fore‐ and hindlimb muscles in frogs: Morphogenesis, homeotic transformations, digit reduction, and the forelimb–hindlimb enigma

Here we provide the first detailed description, based on immunohistochemistry and dissections, of the limb muscle development in the direct developing frog Eleutherodactylus coqui. We compare E. coqui with other tetrapods and discuss our results in a broad evolutionary and developmental context to address some major questions concerning the origin, evolution, and ontogeny of the tetrapod limbs. Our observations and comparisons: (1) support the "in-out" developmental mechanism of the appendicular pectoral muscles; (2) show that the protractor pectoralis and its amniote derivatives trapezius and sternocleidomastoideus clearly develop, anatomically, from the branchial muscles; (3) corroborate that the similarity between the forearm/hand and the leg/foot muscles in tetrapods is due to derived homoplasic events that occurred during the fins-limbs transition and not due to serial homology; (4) lend some support for the hypothesis that the morphological transformation of the anuran tibiale and fibulare represents a distal shift in the zeugo-autopodial border; (5) provide evidence that the identity of the tetrapod hand and foot muscles is mainly related to the topological position of the digits to which they attach; and (6) for the first time, show that apart from a proximo-distal morphogenetic gradient there is also an ulno-radial/fibulo-tibial gradient within the development of the fore- and hindlimb muscles and a dorsoventral gradient within the ontogeny of the hindlimb (but not forelimb) muscles of the frog E. coqui; the two latter gradients are seen in the ontogeny of amniotes such as chickens but are markedly different to those seen in axolotl regeneration and ontogeny.

Rates of dinosaur limb evolution provide evidence for exceptional radiation in Mesozoic birds

Birds are the most diverse living tetrapod group and are a model of large-scale adaptive radiation. Neontological studies suggest a radiation within the avian crown group, long after the origin of flight. However, deep time patterns of bird evolution remain obscure because only limited fossil data have been considered. We analyse cladogenesis and limb evolution on the entire tree of Mesozoic theropods, documenting the dinosaur-bird transition and immediate origins of powered flight. Mesozoic birds inherited constraints on forelimb evolution from non-flying ancestors, and species diversification rates did not accelerate in the earliest flying taxa. However, Early Cretaceous short-tailed birds exhibit both phenotypic release of the hindlimb and increased diversification rates, unparalleled in magnitude at any other time in the first 155 Myr of theropod evolution. Thus, a Cretaceous adaptive radiation of stem-group birds was enabled by restructuring of the terrestrial locomotor module, which represents a key innovation. Our results suggest two phases of radiation in Avialae: with the Cretaceous diversification overwritten by extinctions of stem-group birds at the Cretaceous-Palaeogene boundary, and subsequent diversification of the crown group. Our findings illustrate the importance of fossil data for understanding the macroevolutionary processes generating modern biodiversity.

The Deep Homology of the Autopod: Insights from Hox Gene Regulation

The evolution of tetrapod limbs from fish fins was a significant functional and morphological shift, but how significant was it in terms of the underlying genetic mechanisms? The fossil record provides insight into the morphological changes. However, to understand the underlying mechanisms, we must peer into the gene regulatory networks of living vertebrates. Analysis of HoxA/D expression in a basal actinopterygian, the North American paddlefish, Polyodon spathula, reveals patterns of expression long considered to be a unique developmental signature of the autopod (hands/feet, digits) and shown in tetrapods to be controlled by a "digit enhancer" regulatory landscape. These data, along with recent interspecific transgenic experiments, expression results from chondrichthyans, and data from fossils support the notion that the autopod shares a deep homology with the distal endoskeleton of the fin (distal radials) of other gnathostomes.

The development of the nervous system in Laevicaudata (Crustacea, Branchiopoda): insights into the evolution and homologies of branchiopod limbs and ‘frontal organs’

We investigated the development of the external morphology and of the nervous system in Lynceus biformis and Lynceus brachyurus (Laevicaudata, Branchiopoda), by using immunohistochemical methods in combination with a confocal laser scanning analysis. In both Lynceus species, a free-swimming nauplius larva, equipped with three appendages, hatches from resting eggs. Despite their close phylogenetic relationship to each other, considerable differences are present in their external morphology. Hatching L. brachyurus larvae are equipped with a large and flattened labrum, where in contrast, the L. biformis larvae possess a smaller labrum with four conspicuous posteriorly directed spines at its margin. Despite these differences, the development of the nervous system is quite similar in both species. The hatching larvae are equipped with a naupliar nervous system, and only in the more advanced stages, the development of the ventral nerve cord starts. Furthermore, our investigation into the nervous system provided insights into architecture and evolution of protocerebral sensory organs, the dorsal setae field and the dorsal frontal organ, only present in Laevicaudata. The identification of frontal filaments with an associated frontal filament organ in Lynceus revealed—after a comprehensive comparison with other branchiopods—that these organs exist throughout Branchiopoda and are comparable to those in other crustaceans. Additionally, our results of the peripheral nervous system analysis showed that the innervation pattern of the naupliar appendages (antenna and mandible) and the trunk appendages could be serially homologized, despite much difference in gross morphology of these. Based on the innervation pattern of limbs, we suggest that the larval uniramous mandibular palp, found in the larvae of all ‘large’ branchiopods, is largely exopodal of nature (contrary to most earlier statements) and that the endopodite of the trunk limbs consists of only one distal endite-like segment (confirming some earlier statements) and not of three as proposed by others.

Silurian horseshoe crab illuminates the evolution of arthropod limbs.

The basic arrangement of limbs in euarthropods consists of a uniramous head appendage followed by a series of biramous appendages. The body is divided into functional units or tagmata which are usually distinguished by further differentiation of the limbs. The living horseshoe crabs are remnants of a much larger diversity of aquatic chelicerates. The limbs of the anterior and posterior divisions of the body of living horseshoe crabs differ in the loss of the outer and inner ramus, respectively, of an ancestral biramous limb. Here we report a new fossil horseshoe crab from the mid-Silurian Lagerstätte in Herefordshire, United Kingdom (approximately 425 Myr B.P.), a site that has yielded a remarkably preserved assemblage of soft-bodied fossils. The limbs of the new form can be homologized with those of living Limulus, but retain an ancestral biramous morphology. Remarkably, however, the two limb branches originate separately, providing fossil evidence to suggest that repression or loss of gene expression might have given rise to the appendage morphology of Limulus. Both branches of the prosomal limbs of this new fossil are robust and segmented in contrast to their morphology in Cambrian arthropods, revealing that a true biramous limb was once present in chelicerates as well as in the mandibulates.

A challenge to categories: “What, if anything, is a mosasaur?”

Abstract The concept of “mosasaur” is explored from the perspective of its historical origins, and tested empirically and phylogenetically in order to examine the concept in its modern application. Historical analysis of the origins of the concept of “mosasaur” makes it clear that the term bears significant historical burden (comparative anatomic, empirical, phylogenetic, paleontological, etc.). In order to address the flaws in the concept of mosasaur properly, this treatise critically assesses Camp’s [1923] diagnostic characters for Anguimorpha, Platynota, Varanoidea, and Mosasauroidea, concluding that Camp’s data permit mosasaurs to be viewed only as anguimorphans, not platynotans nor varanoids. A similar critical assessment is given for the characters used to diagnose anguimorphans and varanoids in Estes et al. [1988], concluding here that not a single character out of twenty-two is shared between varanoids and mosasaurs. The character concept developed by Romer [1956] for the “posteriorly retracted nares” of varanoids, and then later mosasaurs, is critically examined and found to be insufficient as a test of similarity of the intended primary homologs. The recent work of Rieppel et al. [2007], Conrad [2008] and Conrad et al. [2010] is critically reviewed as these authors revive the use, and subdivision, of the “posteriorly retracted nares” as a character in anguimorph phylogenetic analysis. Based on these criticisms, it is concluded here that there is no character-based evidence to support phylogenetic hypotheses that mosasaurs are derived aquatic varanoid lizards. A key recommendation of this treatise is that the hypothesis conceiving of mosasaurs as derived aquatic varanoids be abandoned. The final critical review presented in this treatise examines the taxonomic implications, relating to the concept of “mosasaur”, arising from the hypothesis of convergent paddle-like limb evolution in mosasaurs as presented by Bell and Polcyn [2005]. In conclusion, it is recognized that the concept and term “mosasaur” has ceased to exist in any biologically meaningful way, and that the future requires the construction of a new suite of terms and concepts to convey what we now think we know about these animals.

Developmental Pattern and Morphology of Salamandrella keyserlingii Limbs (Amphibia, Hynobiidae) Including Some Evolutionary Aspects

In this paper, we consider the pattern of skeletal development and morphology in the limbs of the hynobiid species, Salamandrella keyserlingii , with the overall aim of analyzing tetrapod limb evolution in context of the phylogeny. In comparison with anurans the urodeles demonstrate caenogenetic adaptation in distal structures of the limbs. As the most primitive family of Caudata hynobiids are of special interest since a study of early stages is likely to display some plesiomorphic features. In common with other larval salamanders Salamandrella has early development of digits 1 + 2. However, in contrast to them this species displays a number of distinctive characters, including larval adaptations and features which can be considered as phylogenetically primitive for caudates and sometimes for uniseriate sarcopterygians in general. The main peculiarities of Salamandrella can been summarized as follows: 1) early formation of the mesenchyme mass between the zeugopod bifurcation as the base for the proximodistal differentiation of preaxial, median and postaxial columns; 2) initial connection of the median column with postaxial one in the region of intermedium condensation; 3) early formation of the intermedium condensation in close contact with ulnar/fibular condensation; 4) dominance of the posterior or postaxial branch (ulnare/fibulare) in the early limb development; 5) comparatively late formation of the basale commune which arises primarily as a small distal 2 condensation at the base of the digit 2 and later fuses with distal 1 condensation; 6) fusion of the skeletal elements in longitudinal rather than in transverse direction; usually amalgamation of the median elements with postaxial than with preaxial ones; 7) frequent (in 75%) amalgamation of the intermedium with ulnare; 8) presence of two central elements in the standard morphology; 9) presence of a well developed, long temporary epidermal fin between first and second digits used in balance and locomotion of the larvae. This is caenogenetic adaptation. The comparison of Salamandrella with other caudates and some fossil sarcopterygians led us to conclusion that many developmental pattern of its limb (1 – 4) can been considered as an ancestral features. It may be supposed that such characters of urodeles as the precocious appearance of anterior digits and basale commune, the gap between them and proximal (zeugopod/stylopod) portion, distal-to-proximal sequence in development of carpal and tarsal elements have a secondary character, probably correlated with their caenogenetic adaptations, and do not support the hypothesis of the polyphyly of tetrapods.

Limb specialization in living marsupial and eutherian mammals: constraints on mammalian limb evolution

Evolution of the marsupial forelimb complex is argued to be constrained by the functional requirements of the newborn's crawl to the teat. This constraint is often used to explain why marsupials are limited in their diversity relative to eutherians. However, despite the constraint's importance to mammalian evolution, one of its fundamental corollaries, that marsupials exhibit less forelimb specialization than eutherians, has not been tested explicitly. We used morphometric analyses of mammalian limbs to test 2 hypotheses concerning their specialization: marsupials have less-specialized forelimbs than eutherians, and marsupials tend to specialize their hind limbs rather than forelimbs and eutherians their forelimbs rather than hind limbs. In support of the 1st, marsupial forelimbs are more similar among functional groups, display smaller morphospaces, and have lower morphologic divergence from the average mammalian state than eutherian forelimbs. In support of the 2nd, marsupial hind limbs are more dissimilar than forelimbs and display higher morphological divergence, but the opposite is true for eutherians. Results suggest fundamental differences in the mechanistic underpinnings of limb specialization in marsupials and eutherians and support a constraint on the marsupial forelimb complex, and possibly the mammalian hind limb.

Evolution of vertebrate appendicular structures: Insight from genetic and palaeontological data

The new body of evidence from fossils and comparative-developmental analysis of subset of appendicular patterning genes has revealed that limb elements seen in tetrapods are assembled in fish fin over evolutionary time. However, despite of deep homology in basic structure and underlying developmental system, there remains a large morphological gap between distal elements of tetrapod limb and distal fin skeleton of tetrapodomorph fish. Understanding the genetic basis of major transformations in distal-limb morphology is the next challenge for evolutionary developmental biologists. Here by integrating data from fossils, comparative-developmental and genetic studies, models are proposed describing the evolution of cis-regulatory elements as a basis for diversification of appendicular architecture. Instead of emphasizing the subset of developmental genes, for instance Hoxd genes, the focus here is on the significance of elucidating cis-regulatory elements for multiple other key molecular players of limb/fin development and genetic/molecular interactions among them, for a better understanding of the developmental and genetic basis of limb evolution.

Evolution of the axial system in craniates: morphology and function of the perivertebral musculature

The axial musculoskeletal system represents the plesiomorphic locomotor engine of the vertebrate body, playing a central role in locomotion. In craniates, the evolution of the postcranial skeleton is characterized by two major transformations. First, the axial skeleton became increasingly functionally and morphologically regionalized. Second, the axial-based locomotion plesiomorphic for craniates became progressively appendage-based with the evolution of extremities in tetrapods. These changes, together with the transition to land, caused increased complexity in the planes in which axial movements occur and moments act on the body and were accompanied by profound changes in axial muscle function. To increase our understanding of the evolutionary transformations of the structure and function of the perivertebral musculature, this review integrates recent anatomical and physiological data (e.g., muscle fiber types, activation patterns) with gross-anatomical and kinematic findings for pivotal craniate taxa. This information is mapped onto a phylogenetic hypothesis to infer the putative character set of the last common ancestor of the respective taxa and to conjecture patterns of locomotor and muscular evolution. The increasing anatomical and functional complexity in the muscular arrangement during craniate evolution is associated with changes in fiber angulation and fiber-type distribution, i.e., increasing obliqueness in fiber orientation and segregation of fatigue-resistant fibers in deeper muscle regions. The loss of superficial fatigue-resistant fibers may be related to the profound gross anatomical reorganization of the axial musculature during the tetrapod evolution. The plesiomorphic function of the axial musculature -mobilization- is retained in all craniates. Along with the evolution of limbs and the subsequent transition to land, axial muscles additionally function to globally stabilize the trunk against inertial and extrinsic limb muscle forces as well as gravitational forces. Associated with the evolution of sagittal mobility and a parasagittal limb posture, axial muscles in mammals also stabilize the trunk against sagittal components of extrinsic limb muscle action as well as the inertia of the body's center of mass. Thus, the axial system is central to the static and dynamic control of the body posture in all craniates and, in gnathostomes, additionally provides the foundation for the mechanical work of the appendicular system.

The Morphological Radiation of Setae in the Cladocera (Crustacea) and Their Potential for Morphogenesis

AbstractThe present review seeks to demonstrate the presence of an inherent biological tendency for the modification of form in cladoceran setae. We present a comparative description of the setal equipment of the cladoceran antenna I, antenna II, maxilla I, maxilla II, each thoracic limb, the carapace valves, and the postabdomen. We revealed significant differences between planktonic and benthic species of the Cladocera. Also our analysis stresses the great differences between the “daphniid” line (Daphniidae + Moinidae) and the “chydorid” line (all other families) of the order Anomopoda.Comments on cladoceran limb evolution are made, and some general conclusions concerning the major tendencies of setal evolutionary transformations are drawn. The morphological range of homologous structures in different species is generally wide, from an undifferentiated seta to either its disappearance or to enlargement, heavy chitinization, and specialization. Special issues, such as parallelisms, oligomerization, asymmetry, abnormalities, and conjugated morphogenesis are discussed. It is assumed that, having been originated, if not eliminated, these modifications either find some adaptive use in the process of radiation at the next level – adaptive radiation – or restrict the niches of specialized groups, or just remain ecologically neutral and then give rise to a series of similar species. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Rate Heterogeneity, Ancestral Character State Reconstruction, and the Evolution of Limb Morphology in Lerista (Scincidae, Squamata)

Rates of phenotypic evolution derive from numerous interrelated processes acting at varying spatial and temporal scales and frequently differ substantially among lineages. Although current models employed in reconstructing ancestral character states permit independent rates for distinct types of transition (forward and reverse transitions and transitions between different states), these rates are typically assumed to be identical for all branches in a phylogeny. In this paper, I present a general model of character evolution enabling rate heterogeneity among branches. This model is employed in assessing the extent to which the assumption of uniform transition rates affects reconstructions of ancestral limb morphology in the scincid lizard clade Lerista and, accordingly, the potential for rate variability to mislead inferences of evolutionary patterns. Permitting rate variation among branches significantly improves model fit for both the manus and the pes. A constrained model in which the rate of digit acquisition is assumed to be effectively zero is strongly supported in each case; when compared with a model assuming unconstrained transition rates, this model provides a substantially better fit for the manus and a nearly identical fit for the pes. Ancestral states reconstructed assuming the constrained model imply patterns of limb evolution differing significantly from those implied by reconstructions for uniform-rate models, particularly for the pes; whereas ancestral states for the uniform-rate models consistently entail the reacquisition of pedal digits, those for the model incorporating among-lineage rate heterogeneity imply repeated, unreversed digit loss. These results indicate that the assumption of identical transition rates for all branches in a phylogeny may be inappropriate in modeling the evolution of phenotypic traits and emphasize the need for careful evaluation of phylogenetic tests of Dollo's law.

Limb development in the gekkonid lizard gonatodes albogularis: A reconsideration of homology in the lizard carpus and tarsus

Despite the attention squamate lizards have received in the study of digit and limb loss, little is known about limb morphogenesis in pentadactyl lizards. Recent developmental studies have provided a basis for understanding lizard autopodial element homology based on developmental and comparative anatomy. In addition, the composition and identity of some carpal and tarsal elements of lizard limbs, and reptiles in general, have been the theme of discussions about their homology compared to non-squamate Lepidosauromorpha and basal Amniota. The study of additional embryonic material from different lizard families may improve our understanding of squamate limb evolution. Here, we analyze limb morphogenesis in the gekkonid lizard Gonatodes albogularis describing patterns of chondrogenesis and ossification from early stages of embryonic development to hatchlings. Our results are in general agreement with previous developmental studies, but we also show that limb development in squamates probably involves more chondrogenic elements for carpal and tarsal morphogenesis, as previously recognized on the grounds of comparative anatomy. We provide evidence for the transitory presence of distal carpale 1 and intermedium in the carpus and tibiale, intermedium, distal centralia, and distal tarsale 2 in the tarsus. Hence, we demonstrate that some elements that were believed to be lost in squamate evolution are conserved as transitory elements during limb development. However, these elements do not represent just phylogenetic burden but may be important for the morphogenesis of the lizard autopodium.

Plausibility of inferred ancestral phenotypes and the evaluation of alternative models of limb evolution in scincid lizards

Phylogenetic approaches to inferring ancestral character states are becoming increasingly sophisticated; however, the potential remains for available methods to yield strongly supported but inaccurate ancestral state estimates. The consistency of ancestral states inferred for two or more characters affords a useful criterion for evaluating ancestral trait reconstructions. Ancestral state estimates for multiple characters that entail plausible phenotypes when considered together may reasonably be assumed to be reliable. However, the accuracy of inferred ancestral states for one or more characters may be questionable where combined reconstructions imply implausible phenotypes for a proportion of internal nodes. This criterion for assessing reconstructed ancestral states is applied here in evaluating inferences of ancestral limb morphology in the scincid lizard clade Lerista. Ancestral numbers of digits for the manus and pes inferred assuming the models that best fit the data entail ancestral digit configurations for many nodes that differ fundamentally from configurations observed among known species. However, when an alternative model is assumed for the pes, inferred ancestral digit configurations are invariably represented among observed phenotypes. This indicates that a suboptimal model for the pes (and not the model providing the best fit to the data) yields accurate ancestral state estimates.

Early crustacean evolution and the appearance of epipodites and gills

Epipodites are structures on the outer edges of crustacean appendages serving as gills or for osmoregulation. Their evolutionary origin has been debated for a long time. Three major issues are of relevance: 1) the function of epipodites, 2) their development, and 3) the fossil record. While it has long been a problem to distinguish the gill and osmoregulatory functions of epipodites histologically, this has recently become possible based on ultrastructure. A respiratory function has particularly been claimed for the limbs or parts of limbs of early arthropod fossils. Not only rami and cuticular structures, but also entire appendages, have been referred to as “gills”. Among living taxa, the opisthosomal limbs of limulids are called gills or gill limbs, although the numerous leaf-like gill structures occur only on the posterior side of the exopods. It has long been known that crustacean exopods do not serve a respiratory function, which is restricted to structures along the outer proximal edge of the limbs. Three-dimensionally preserved fossil crustaceans from the ‘Orsten’ have contributed much to our understanding of the evolution and phylogeny of Crustacea, in particular limb evolution. The recently discovered Yicaris dianensis from the Lower Cambrian demonstrates not only the presence but also the morphogenesis of three epipodites on all post-maxillulary appendages. Yicaris dianensis may therefore be a valuable model for understanding the evolutionary origin of epipodites, not least since comparative morphology demonstrates that epipodites show a large plasticity among living eucrustacean taxa and may not even be homologous. Epipodites are discussed here in the light of 1) other putative respiratory and osmoregulatory structures in other ‘Orsten’ taxa, 2) morphological and functional variations of epipodites in living eucrustaceans, and 3) the discovery of two new species of ‘Orsten’ eucrustaceans, also showing evidence of three epipodites. This contribution aims to provide a guide for further investigations on the evolution of crustacean epipodites.