Rates and patterns in the evolution of snake-like body form in squamate reptiles: evidence for repeated re-evolution of lost digits and long-term persistence of intermediate body forms

An important challenge in evolutionary biology is to understand how major changes in body form arise. The dramatic transition from a lizard-like to snake-like body form in squamate reptiles offers an exciting system for such research because this change is replicated dozens of times. Here, we use morphometric data for 258 species and a time-calibrated phylogeny to explore rates and patterns of body-form evolution across squamates. We also demonstrate how time-calibrated phylogenies may be used to make inferences about the time frame over which major morphological transitions occur. Using the morphometric data, we find that the transition from lizard-like to snake-like body form involves concerted evolution of limb reduction, digit loss, and body elongation. These correlations are similar across squamate clades, despite very different ecologies and >180 million years (My) of divergence. Using the time-calibrated phylogeny and ancestral reconstructions, we find that the dramatic transition between these body forms can occur in 20 My or less, but that seemingly intermediate morphologies can also persist for tens of millions of years. Finally, although loss of digits is common, we find statistically significant support for at least six examples of the re-evolution of lost digits in the forelimb and hind limb.     

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. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.     

Are there general laws for digit evolution in squamates? The loss and re‐evolution of digits in a clade of fossorial lizards (Brachymeles,Scincinae)

Evolutionary simplification of autopodial structures is a major theme in studies of body‐form evolution. Previous studies on amniotes have supported Morse's law, that is, that the first digit reduced is Digit I, followed by Digit V. Furthermore, the question of reversibility for evolutionary digit loss and its implications for “Dollo's law” remains controversial. Here, we provide an analysis of limb and digit evolution for the skink genus Brachymeles. Employing phylogenetic, morphological, osteological, and myological data, we (a) test the hypothesis that digits have re‐evolved, (b) describe patterns of morphological evolution, and (c) investigate whether patterns of digit loss are generalizable across taxa. We found strong statistical support for digit, but not limb re‐evolution. The feet of pentadactyl species of Brachymeles are very similar to those of outgroup species, while the hands of these lineages are modified (2‐3‐3‐3‐2) and a have a reduced set of intrinsic hand muscles. Digit number variation suggests a more labile Digit V than Digit I, contrary to Morse's law. The observed pattern of digit variation is different from that of other scincid lizards (Lerista, Hemiergis, Carlia). Our results present the first evidence of clade‐specific modes of digit reduction.     

HOW LIZARDS TURN INTO SNAKES: A PHYLOGENETIC ANALYSIS OF BODY-FORM EVOLUTION IN ANGUID LIZARDS

Abstract One of the most striking morphological transformations in vertebrate evolution is the transition from a lizardlike body form to an elongate, limbless (snakelike) body form. Despite its dramatic nature, this transition has occurred repeatedly among closely related species (especially in squamate reptiles), making it an excellent system for studying macroevolutionary transformations in body plan. In this paper, we examine the evolution of body form in the lizard family Anguidae, a clade in which multiple independent losses of limbs have occurred. We combine a molecular phylogeny for 27 species, our morphometric data, and phylogenetic comparative methods to provide the first statistical phylogenetic tests of several long‐standing hypotheses for the evolution of snakelike body form. Our results confirm the hypothesized relationships between body elongation and limb reduction and between limb reduction and digit reduction. However, we find no support for the hypothesized sequence going from body elongation to limb reduction to digit loss, and we show that a burrowing lifestyle is not a necessary correlate of limb loss. We also show that similar degrees of overall body elongation are achieved in two different ways in anguids, that these different modes of elongation are associated with different habitat preferences, and that this dichotomy in body plan and ecology is widespread in limb‐reduced squamates. Finally, a recent developmental study has proposed that the transition from lizardlike to snakelike body form involves changes in the expression domains of midbody Hox genes, changes that would link elongation and limb loss and might cause sudden transformations in body form. Our results reject this developmental model and suggest that this transition involves gradual changes occurring over relatively long time scales.     

Evidence for the reversibility of digit loss: a phylogenetic study of limb evolution in Bachia (Gymnophthalmidae: Squamata)

Reevolution of lost characters constitutes evidence that the capacity for producing specific phenotypes may remain latent after a trait is lost and be transmitted over many generations without visible effect. Although some evolutionary changes are easily reversible, it can be argued that the reappearance of complex characters would be nearly impossible. This idea is based on the assumption that, after a structure is lost, the genes related to its development will degenerate. In the present paper we test this idea with respect to digit loss in the gymnophthalmid genus Bachia. We present a molecular phylogeny of the genus Bachia and investigate the evolution of digit number in this taxon. Most members of this South American genus have undergone major reduction in hind limbs without ever losing all the digits in the forelimbs. We apply three statistical methods to test the hypothesis that trait loss is irreversible (Dollo's law). These are tree tests, parsimony-cost curves, and likelihood-ratio tests. Data is also analyzed under a simple probability model. All analyses provided strong evidence for reevolution of digit number in derived Bachia species. The evidence is stronger in toes (hind limb) than in fingers (forelimb). Other published examples of reevolution of complex traits are discussed in the light of the statistical approaches used in this paper. We conclude that there are a limited number of cases with strong evidence for the reevolution of lost morphological structures, raising questions about the mechanisms that retain the genetic information for a latent character.     

LOSS AND RE‐EVOLUTION OF COMPLEX LIFE CYCLES IN MARSUPIAL FROGS: DOES ANCESTRAL TRAIT RECONSTRUCTION MISLEAD?

Using phylogeny-based methods to identify evolutionary transitions has become an integral part of evolutionary biology. Here, we demonstrate the potential for these methods to give statistically well-supported but misleading inferences about character evolution. We also show how inferences of character evolution can be informed using GIS-based methods to reconstruct ancestral environmental regimes. We reconstruct a phylogeny for marsupial frogs (Hemiphractidae) using nuclear and mitochondrial DNA sequences and estimate patterns of life-history evolution across the resulting tree. We find that Gastrotheca species with complex life cycles (i.e., egg, tadpole, and adult stages) are phylogenetically nested among species and genera with direct development (i.e., egg and adult stages only). Assuming a single rate for gains and losses in likelihood reconstructions, there is strong statistical support for the hypothesis that the tadpole stage was lost early in the phylogeny but reappeared within Gastrotheca. Assuming different rates of gain and loss, the model with significantly higher statistical support, the tadpole stage seems to have been lost multiple times but never regained. Given that both hypotheses cannot be correct, at least one reconstruction model must be giving well-supported but misleading results. Several lines of evidence (including GIS-based reconstructions of the ancestral climatic regime) suggest that the former hypothesis is correct, and that the tadpole stage has evolved from direct development within Gastrotheca, the only known case of such a reversal in frogs.     

Cave-adapted evolution in the North American amblyopsid fishes inferred using phylogenomics and geometric morphometrics

Cave adaptation has evolved repeatedly across the Tree of Life, famously leading to pigmentation and eye degeneration and loss, yet its macroevolutionary implications remain poorly understood. We use the North American amblyopsid fishes, a family spanning a wide degree of cave adaptation, to examine the impact of cave specialization on the modes and tempo of evolution. We reconstruct evolutionary relationships using ultraconserved element loci, estimate the ancestral histories of eye-state, and examine the impact of cave adaptation on body shape evolution. Our phylogenomic analyses provide a well-supported hypothesis for amblyopsid evolutionary relationships. The obligate blind cavefishes form a clade and the cave-facultative eyed spring cavefishes are nested within the obligate cavefishes. Using ancestral state reconstruction, we find support for at least two independent subterranean colonization events within the Amblyopsidae. Eyed and blind fishes have different body shapes, but not different rates of body shape evolution. North American amblyopsids highlight the complex nature of cave-adaptive evolution and the necessity to include multiple lines of evidence to uncover the underlying processes involved in the loss of complex traits.     

The convergent evolution of snake‐like forms by divergent evolutionary pathways in squamate reptiles*

Convergent evolution of phenotypes is considered evidence that evolution is deterministic. Establishing if such convergent phenotypes arose through convergent evolutionary pathways is a stronger test of determinism. We studied the evolution of snake‐like body shapes in six clades of lizards, each containing species ranging from short‐bodied and pentadactyl to long‐bodied and limbless. We tested whether body shapes that evolved in each clade were convergent, and whether clades evolved snake‐like body shapes following convergent evolutionary pathways. Our analyses showed that indeed species with the same numbers of digits in each clade evolved convergent body shapes. We then compared evolutionary pathways among clades by considering patterns of evolutionary integration and shape of relationship among body parts, patterns of vertebral evolution, and models of digit evolution. We found that all clades elongated their bodies through the addition, not elongation, of vertebrae, and had similar patterns of integration. However, patterns of integration, the body parts that were related by a linear or a threshold model, and patterns of digit evolution differed among clades. These results showed that clades followed different evolutionary pathways. This suggests an important role of historical contingency as opposed to determinism in the convergent evolution of snake‐like body shapes.     

REPTILIAN VIVIPARITY AND DOLLO'S LAW

It has been suggested repeatedly that the evolutionary transition from oviparity (egg-laying) to viviparity (live-bearing) in reptiles is irreversible. However, these adaptive arguments have yet to be tested by detailed examination of the phylogenetic distribution of oviparity and viviparity across a broad range of taxa. Using available data on reproductive modes and phylogenetic relationships within reptiles, we here quantify the numbers and directions of evolutionary transitions between oviparity and viviparity. Phylogenetic relationships among three diverse squamate groups (scincid lizards, colubrid snakes, elapid snakes) are currently inadequately known for inclusion in this study Among the remaining reptiles, oviparity has given rise to viviparity at least 35 times. Five possible instances of reversals (from viviparity to oviparity) are identified, but closer examination indicates that all have weak empirical support (i.e., they could be “unreversed” with little loss in parsimony, and/or are based on poorly substantiated phylogenetic hypotheses). Viviparity is clearly more frequently (and presumably easily) gained than lost in several disparate groups so far examined (reptiles, fishes, polychaete worms); this evolutionary bias should be considered when reproductive mode is optimized on a phylogeny or employed in phylogenetic reconstruction.     

A single origin of Batesian mimicry among hybridizing populations of admiral butterflies (Limenitis arthemis) rejects an evolutionary reversion to the ancestral phenotype

Batesian mimicry is a fundamental example of adaptive phenotypic evolution driven by strong natural selection. Given the potentially dramatic impacts of selection on individual fitness, it is important to understand the conditions under which mimicry is maintained versus lost. Although much empirical and theoretical work has been devoted to the maintenance of Batesian mimicry, there are no conclusive examples of its loss in natural populations. Recently, it has been proposed that non-mimetic populations of the polytypic Limenitis arthemis species complex represent an evolutionary loss of Batesian mimicry, and a reversion to the ancestral phenotype. Here, we evaluate this conclusion using segregating amplified fragment length polymorphism markers to investigate the history and fate of mimicry among forms of the L. arthemis complex and closely related Nearctic Limenitis species. In contrast to the previous finding, our results support a single origin of mimicry within the L. arthemis complex and the retention of the ancestral white-banded form in non-mimetic populations. Our finding is based on a genome-wide sampling approach to phylogeny reconstruction that highlights the challenges associated with inferring the evolutionary relationships among recently diverged species or populations (i.e. incomplete lineage sorting, introgressive hybridization and/or selection).     

Evolutionary change of the numbers of homeobox genes in bilateral animals

It has been known that the conservation or diversity of homeobox genes is responsible for the similarity and variability of some of the morphological or physiological characters among different organisms. To gain some insights into the evolutionary pattern of homeobox genes in bilateral animals, we studied the change of the numbers of these genes during the evolution of bilateral animals. We analyzed 2,031 homeodomain sequences compiled from 11 species of bilateral animals ranging from Caenorhabditis elegans to humans. Our phylogenetic analysis using a modified reconciled-tree method suggested that there were at least about 88 homeobox genes in the common ancestor of bilateral animals. About 50-60 genes of them have left at least one descendant gene in each of the 11 species studied, suggesting that about 30-40 genes were lost in a lineage-specific manner. Although similar numbers of ancestral genes have survived in each species, vertebrate lineages gained many more genes by duplication than invertebrate lineages, resulting in more than 200 homeobox genes in vertebrates and about 100 in invertebrates. After these gene duplications, a substantial number of old duplicate genes have also been lost in each lineage. Because many old duplicate genes were lost, it is likely that lost genes had already been differentiated from other groups of genes at the time of gene loss. We conclude that both gain and loss of homeobox genes were important for the evolutionary change of phenotypic characters in bilateral animals.     

Vertebral evolution and the diversification of squamate reptiles

Taxonomic, morphological, and functional diversity are often discordant and independent components of diversity. A fundamental and largely unanswered question in evolutionary biology is why some clades diversify primarily in some of these components and not others. Dramatic variation in trunk vertebral numbers (14 to >300) among squamate reptiles coincides with different body shapes, and snake-like body shapes have evolved numerous times. However, whether increased evolutionary rates or numbers of vertebrae underlie body shape and taxonomic diversification is unknown. Using a supertree of squamates including 1375 species, and corresponding vertebral and body shape data, we show that increased rates of evolution in vertebral numbers have coincided with increased rates and disparity in body shape evolution, but not changes in rates of taxonomic diversification. We also show that the evolution of many vertebrae has not spurred or inhibited body shape or taxonomic diversification, suggesting that increased vertebral number is not a key innovation. Our findings demonstrate that lineage attributes such as the relaxation of constraints on vertebral number can facilitate the evolution of novel body shapes, but that different factors are responsible for body shape and taxonomic diversification.     

Parsimony-based test for identifying changes in evolutionary trends for quantitative characters: implications for the origin of the amniotic egg

The origin of the amniotic egg was a major event in vertebrate evolution and is thought to have contributed to the spectacular evolutionary radiation of amniotes. We test one of the most popular scenarios proposed by Carroll in 1970 to explain the origin of the amniotic egg using a novel method based on an asymmetric version of linear parsimony (aka Wagner parsimony) for identifying the most parsimonious split of a tree into two parts between which the evolution of the character is allowed to differ. The new method evaluates the cost of splitting a phylogenetic tree at a given node as the integral, over all pairs of asymmetry parameters, of the most parsimonious costs that can be achieved by using the first parameter on the subtree pending from this node and the second parameter elsewhere. By testing all the nodes, we then obtain the most parsimonious split of a tree with regard to the character values at its tips. Among the nine trees and two characters tested, our method yields a total of 517 parsimonious trend changes in Permo-Carboniferous stegocephalians, a single one of which occurs in a part of the tree (among stem-amniotes) where Carroll's scenario predicts that there should have been distinct changes in body size evolutionary trends. This refutes the scenario because the amniote stem does not appear to have elevated rates of evolutionary trend shifts. Our nodal body size estimates offer less discriminating power, but they likewise fail to find strong support for Carroll's scenario.     

A Phylogenetic Analysis of the Evolution of the Stridulatory Apparatus in True Crickets (Orthoptera, Grylloidea)

The stridulatory apparatus (or stridulum) is currently assumed ancestral in crickets. Models of its subsequent evolution consider only one modality of evolutionary change: the stridulum would have been progressively lost in multiple cricket lineages. A phylogenetic test of this hypothesis is presented here. The morpho-functional types of stridulum have been optimized on the cladistic phylogenies of two monophyletic cricket clades, and parsimonious evolutionary scenarios of the evolution of the stridulum in these clades have been derived. The phylogenetic patterns thus obtained support the hypothesis that the stridulum has been lost several times convergently in crickets. They indicate, however, that the loss of the stridulum could be reversible, and that several modalities of evolutionary change exist for the stridulum. Phylogenetic analysis thus reveals an unsuspected complexity in the evolution of acoustic communication in crickets.     

Bridging morphological transitions to the metazoa

Our inability to answer many questions regarding the developmentof metazoan complexity may be due in part to the prevailingidea that most eukaryote "phyla" originated within a short periodof geologic time from simple unicellular ancestors. This view,however, is contradicted by evidence that larger groups of eukaryotesshare characters, suggesting that these assemblages inheritedcharacters from a common ancestor. Because molecular analyseshave had limited success in resolving the relationships of highereukaryote taxa, we have undertaken a phylogenetic analysis basedprimarily on morphological characters. The analysis emphasizescharacters considered to have a high probability of having evolvedonly once. Transitions between taxa are evaluated for the likelihoodof character-state transformations. The analysis indicates thatthe evolutionary history of the clade containing the Metazoahas been complex, encompassing the gain and loss of a secondaryand perhaps a primary photosynthetic endosymbiont with accompanyingchanges in trophic level. The history also appears to have includeda hetero-autotrophic ancestor that possessed a "conoid" feedingapparatus and may have involved a transformation from a flagellateto an amoeboid body form, a trend toward increased intracellularcompartmentation, and the development of complex social behavior.Such changes could have been critical for establishing the underlyingcomplexity required for a rapid diversification of cell andtissue types in the early stages of metazoan evolution.     

Re-evolution of lost mandibular teeth in frogs after more than 200 million years, and re-evaluating Dollo's law

Dollo's law states that structures that are evolutionarily lost will not be regained. Recent phylogenetic studies have revealed several potential examples in which Dollo's law seems to be violated, where lost structures appear to have been regained over evolutionary time. However, these examples have recently been questioned and suggested to be methodological artifacts. In this article, I document a striking and incontrovertible phylogenetic example of the re-evolution of a lost, complex structure: mandibular teeth in the frog genus Gastrotheca. I use a time-calibrated phylogeny for 170 amphibian species to show that mandibular teeth were lost in the ancestor of modern frogs at least 230 million years ago (Mya) and have been regained in the last ～ 5-17 My. I review recent studies on trait re-evolution and show that this long period of trait absence prior to re-acquisition is largely unprecedented. I also argue that there are several methodological issues that may cause trait re-evolution to be hardest to detect under those conditions when it is most likely to occur, leading to erroneous failures to reject Dollo's law. Finally, I discuss a mechanism that may facilitate trait re-evolution, and the evolution of mandibular teeth in frogs as an example of developmental constraint.     

Myxosporea (Myxozoa,Cnidaria) Lack DNA Cytosine Methylation

DNA cytosine methylation is central to many biological processes, including regulation of gene expression, cellular differentiation, and development. This DNA modification is conserved across animals, having been found in representatives of sponges, ctenophores, cnidarians, and bilaterians, and with very few known instances of secondary loss in animals. Myxozoans are a group of microscopic, obligate endoparasitic cnidarians that have lost many genes over the course of their evolution from free-living ancestors. Here, we investigated the evolution of the key enzymes involved in DNA cytosine methylation in 29 cnidarians and found that these enzymes were lost in an ancestor of Myxosporea (the most speciose class of Myxozoa). Additionally, using whole-genome bisulfite sequencing, we confirmed that the genomes of two distant species of myxosporeans, Ceratonova shasta and Henneguya salminicola, completely lack DNA cytosine methylation. Our results add a notable and novel taxonomic group, the Myxosporea, to the very short list of animal taxa lacking DNA cytosine methylation, further illuminating the complex evolutionary history of this epigenetic regulatory mechanism.     

Understanding the evolutionary processes of fungal fruiting bodies: correlated evolution and divergence times in the Psathyrellaceae

Fruiting body evolution is one of the central topics in fungal evolutionary biology. A number of hypotheses have been developed to explain the contemporary diversity of fruiting body forms, but their evaluation has been hampered by the lack of well-sampled data sets and suitable statistical methods. Phylogenetic evidence of the physiological changes that accompany switches in fruiting body type is lacking, and very little is known about the age of major events of fruiting body evolution. Based on a new multigene phylogeny, by using Bayesian methods, we demonstrate the existence of correlation between a number of morphological features and switches from nondeliquescent to deliquescent (autodigesting) fruiting bodies in the mushroom family Psathyrellaceae. Our results show that switches in the anatomy of two types of spacer cells (cystidia and pseudoparaphyses) and basidia (bimorphic or monomorphic) as well as the structure of the mushroom cap follow the evolution of deliquescent fruiting bodies, which suggests strong functional linkage between these traits. We performed Bayes factor-based tests, referred hereafter to as evolutionary pathway test (EPT), to decide which of the correlated characters were gained first during evolution. The EPTs strongly suggest that deliquescence was gained first, followed after short waiting times by the other morphological features. Bayesian relaxed molecular clock analyses suggest that the various events of switching between fruiting body types occurred independently at various ages during the history of the family. The utility of two mushroom fossils (Archaemarasmius and Protomycena), the only ones with unambiguous taxonomic positions, for the calibration of agaric trees were also examined. Based on our results, we suggest that the evolutionary benefit of deliquescence may be prevention against desiccation via accelerated ontogeny of the fruiting body. Hypotheses regarding the functional significance of the correlated evolution are presented and discussed. Further, we argue that the changes in fruiting body types in mushrooms in general can be attributed to independent events (e.g., dispersal and adaptation) and not to particular geologic ages.     

Effect of taxon sampling on recovering the phylogeny of squamate reptiles based on complete mitochondrial genome and nuclear gene sequence data

The complete nucleotide sequences of the mitochondrial (mt) genomes of three species of squamate lizards: Blanus cinereus (Amphisbaenidae), Anguis fragilis (Anguidae), and Tarentola mauritanica (Geckkonidae) were determined anew. The deduced amino acid sequences of all 13 mt protein-coding genes were combined into a single data set and phylogenetic relationships among main squamate lineages were analyzed under maximum likelihood (ML) and Bayesian Inference (BI). Within Squamata, the monophyly of Iguanidae, Anguimorpha, Amphisbaenia, Gekkota, Serpentes, and Acrodonta received high statistical support with both methods. It is particularly striking that this is the first molecular analysis that recovers the monophyly of Scincomorpha (including Scincidae, Xantusiidae, Cordylidae, and Lacertidae), although it is only supported in the Bayesian analysis, and it is sensitive to changes in the outgroup (see below). Phylogenetic relationships among the main squamate lineages could not be resolved with ML but received strong support with BI (above 95%). The newly reconstructed phylogeny of squamates does not support the Iguania-Scleroglossa split. Acrodonta and Serpentes form a clade, which is the sister group of the remaining squamate lineages. Within these, Gekkota were the first branching out, followed by Amphisbaenia, and a clade including Anguimorpha as sister group of Scincomorpha + Iguanidae. The recovered topology differed substantially from previously reported hypotheses on squamate relationships, and the relative effect of using different outgroups, genes, and taxon samplings were explored. The sister group relationship of Serpentes + Acrodonta, and their relative basal position within Squamata could be due to a long-branch attraction artifact. Phylogenetic relationships among Scincomorpha, Amphisbaenia, and Anguimorpha were found to be rather unresolved. Future improving of squamate phylogenetic relationships would rely on finding snake and acrodont species with slower mt evolutionary rates, ensuring thorough taxon coverage of squamate diversity, and incorporating more nuclear genes with appropriate evolutionary rates.     

Musculoskeletal anatomical changes that accompany limb reduction in lizards

Muscles, bones, and tendons in the adult tetrapod limb are intimately integrated, both spatially and functionally. However, muscle and bone evolution do not always occur hand in hand. We asked, how does the loss of limb bones affect limb muscle anatomy, and do these effects vary among different lineages? To answer these questions, we compared limb muscular and skeletal anatomy among gymnophthalmid lizards, which exhibit a remarkable variation in limb morphology and different grades of digit and limb reduction. We mapped the characters onto a phylogeny of the group to assess the likelihood that they were acquired independently. Our results reveal patterns of reduction of muscle and bone elements that did not always coincide and examples of both, convergent and lineage‐specific non‐pentadactyl musculoskeletal morphologies. Among lineages in which non‐pentadactyly evolved independently, the degree of convergence seems to depend on the number of digits still present. Most tetradactyl and tridactyl limbs exhibited profound differences in pattern and degree of muscle loss/reduction, and recognizable morphological convergence occurred only in extremely reduced morphologies (e.g., spike‐like appendix). We also found examples of muscles that persisted although the bones to which they plesiomorphically attach had been lost, and examples of muscles that had been lost although their normal bony attachments persisted. Our results demonstrate that muscle anatomy in reduced limbs cannot be predicted from bone anatomy alone, meaning that filling the gap between osteological and myological data is an important step toward understanding this recurrent phenomenon in the evolution of tetrapods. J. Morphol. 276:1290–1310, 2015. © 2015 Wiley Periodicals, Inc.