Reversibility of digit loss revisited: Limb diversification in Bachia lizards (gymnophthalmidae).

Strict interpretations of the Dollo's Law lead to postulation that trait loss is irreversible and organisms never recover ancestral phenotypes. Dollo, however, admitted the possibility of reversals in trait loss when predicted differences between reversed (derived) and ancestral forms. Phenotypic signatures from reversals are expected, as the historical context of a reversal in trait loss differs from the initial setting where the trait originally evolved. This article combines morphological and molecular information for Bachia scolecoides to discuss phenotypic and genetic patterns established during processes that reversed digit loss in Gymnophthalmidae (also termed microteiid lizards). Results suggest that pathways leading to the derived tetradactyl state of B. scolecoides comprise particularities in their origin and associated processes. Autopodial bones of B. scolecoides lack digit identity, and muscle anatomy is very similar between manus and pes. Gymnophthalmidae sequence patterns in the limb-specific sonic hedgehog enhancer (ZRS) suggest that regulation of shh expression did not degenerate in Bachia, given the prediction of similar motifs despite mutations specific to Bachia. Persistence of developmental mechanisms might explain intermittent character expression leading to reversals of digit loss, as ZRS signaling pathways remain active during the development of at least one pair of appendices in Bachia, especially if some precursors persisted at early stages. Patterns of ZRS sequences suggest that irreversibility of trait loss might be lineage-specific (restricted to Gymnophthalmini) and contingent to the type of signature established. These results provide insights regarding possible mechanisms that may allow reactivation of developmental programs in specific regions of the embryo.

Evolution of territoriality in Hylinae treefrogs: Ecological and morphological correlates and lineage diversification.

Given the diverse nature of traits involved in territorial defence, they may respond to different selective pressures and then exhibit distinct patterns of evolution. These selective pressures also may cause territorial behaviour to be associated with environmental and morphological variables. Such associations, however, have mostly been studied at the intraspecific level, being phylogenetic analyses of territoriality in a broad taxonomic framework rare in the literature. We used the anuran subfamily Hylinae to test (1) whether two territorial-behaviour traits with different levels of aggression-territorial call and physical combat-are evolutionarily more labile than a morphological trait used in physical combat-the spine-shaped prepollex; (2) whether reproduction in lentic waters and phytotelmata, as well as resource scarcity, might favour the occurrence of territoriality; (3) if physical combat is more important than territorial call for the evolution of body size and sexual size dimorphism and (4) the relationships between territorial-behaviour traits and lineage diversification. We mainly used the literature to build two datasets with different levels of certainty. Territorial-behaviour traits exhibited intermediate levels of phylogenetic signal in Hylinae, whereas the phylogenetic signal for the presence of the spine-shaped prepollex was strong. We found support for the hypothesis that reproduction in lentic water favours the occurrence of territorial behaviour, because the expression of territorial-behaviour traits was more associated with reproduction in lentic than in lotic waters. Territorial-behaviour traits were not correlated with annual precipitation nor with habitat complexity. Body size and sexual size dimorphism were not correlated with the presence of territorial call nor with physical combat. We identified negative correlations between diversification rates and physical combat. Relationships of territorial call and physical combat with diversification rates suggest that these territorial behaviours influence evolutionary processes in different ways.

Repeated evolution of similar phenotypes: Integrating comparative methods with developmental pathways.

Repeated phenotypes, often referred to as 'homoplasies' in cladistic analyses, may evolve through changes in developmental processes. Genetic bases of recurrent evolution gained attention and have been studied in the past years using approaches that combine modern analytical phylogenetic tools with the stunning assemblage of new information on developmental mechanisms. In this review, we evaluated the topic under an integrated perspective, revisiting the classical definitions of convergence and parallelism and detailing comparative methods used to evaluate evolution of repeated phenotypes, which include phylogenetic inference, estimates of evolutionary rates and reconstruction of ancestral states. We provide examples to illustrate how a given methodological approach can be used to identify evolutionary patterns and evaluate developmental mechanisms associated with the intermittent expression of a given trait along the phylogeny. Finally, we address why repeated trait loss challenges strict definitions of convergence and parallelism, discussing how changes in developmental pathways might explain the high frequency of repeated trait loss in specific lineages.

What snakes and caecilians have in common? Molecular interaction units and the independent origins of similar morphotypes in Tetrapoda.

Developmental pathways encompass transcription factors and cis-regulatory elements that interact as transcription factor-regulatory element (TF-RE) units. Independent origins of similar phenotypes likely involve changes in different parts of these units, a hypothesis promisingly tested addressing the evolution of the rib-associated lumbar (RAL) morphotype that characterizes emblematic animals such as snakes and elephants. Previous investigation in these lineages identified a polymorphism in the Homology region 1 [H1] enhancer of the Myogenic factor-5 [Myf5], which interacts with HOX10 proteins to modulate rib development. Here we address the evolution of TF-RE units focusing on independent origins of RAL morphotypes. We compiled an extensive database for H1-Myf5 and HOX10 sequences with two goals: (i) evaluate if the enhancer polymorphism is present in amphibians exhibiting the RAL morphotype and (ii) test a hypothesis of enhanced evolutionary flexibility mediated by TF-RE units, according to which independent origins of the RAL morphotype might involve changes in either component of the interaction unit. We identified the H1-Myf5 polymorphism in lineages that diverged around 340 Ma, including Lissamphibia. Independent origins of the RAL morphotype in Tetrapoda involved sequence variation in either component of the TF-RE unit, confirming that different changes may similarly affect the phenotypic outcome of a given developmental pathway.

Development and function explain the modular evolution of phalanges in gecko lizards.

Selective regimes favouring the evolution of functional specialization probably affect covariation among phenotypic traits. Phalanges of most tetrapods develop from a conserved module that constrains their relative proportions. In geckos, however, biomechanical specializations associated with adhesive toepads involve morphological variation in the autopodium and might reorganize such modular structures. We tested two hypotheses to explain the modular architecture of hand bones in geckos, one based on developmental interactions and another incorporating functional associations related to locomotion, and compared the empirical support for each hypothetical module between padded and padless lineages. We found strong evidence for developmental modules in most species, which probably reflects embryological constraints during phalangeal formation. Although padded geckos exhibit a functional specialization involving the hyperextension of the distal phalanges that is absent in padless species, the padless species are the ones that show a distal functional module with high integration. Some ancestrally padless geckos apparently deviate from developmental predictions and present a relatively weak developmental module of phalanges and a strongly integrated distal module, which may reflect selective regimes involving incipient frictional adhesion in digit morphology. Modularity of digit elements seems dynamic along the evolutionary history of geckos, being associated with the presence/absence of adhesive toepads.

A guide to incubate eggs of Tropidurus lizards under laboratory conditions.

Studies in Evo-Devo benefit from the use of a variety of organisms, as comparative approaches provide a better understanding of Biodiversity and Evolution. Standardized protocols to incubate eggs and manipulate embryo development enable postulation of additional species as suitable biological systems for research in the field. In the past decades, vertebrate lineages such as Squamata (lizards, snakes, and amphisbaenians) emerged as crucial study systems for addressing topics as diverse as phenotypic evolution and climate change. However, protocols for maintaining gravid females and incubating eggs in the lab under experimental conditions are available to only a few squamate species. This resource article presents a simple incubation guide that standardizes conditions to maintain embryos of Tropidurus catalanensis (Squamata: Tropiduridae) under different experimental conditions, manipulating relevant environmental factors like temperature and humidity. We identified associated effects relating the egg incubation condition to developmental stage, incubation time, hatching success, and resulting morphotypes. Temperature and humidity play a key role in development and require attention when establishing the experimental design. Current literature comprises information for Tropidurus lizards that ponders how general in Squamata are the ecomorphs originally described for Anolis. Studies evaluating phenotypic effects of developmental environments suggest plasticity in some of the traits that characterize the ecomorphological associations described for this family. We expect that this incubation guide encourages future studies using Tropidurus lizards to address Evo-Devo questions.

Different developmental environments reveal multitrait plastic responses in South American Anostomidae fish.

Complex phenotypes result from developmental processes integrating genetic, epigenetic, and environmental information. Although changing environments combine several signals that may induce multitrait plastic responses, literature often decodes developmental plasticity into single trait variation as a function of isolated environmental signals. To address the multivariate nature of developmental plasticity, we evaluated how different combinations of environmental signals influence the development of morphological and behavioral traits. We raised Megaleporinus macrocephalus (Anostomidae) in four different developmental environments, and found that foraging position and structural complexity during development induced different morphotypes, which overlapped with behavioral patterns. Foraging position induced distinct patterns of mouth and fin positioning and overall body shape, which were accentuated by structural complexity. Moreover, fish most often chose conditions similar to their developmental environments. Combined signals during development, therefore, revealed environment-specific phenotypic patterns associating morphology and behavior. Such results endorse the ability of developmental processes to influence the variation present in natural populations. Implications of addressing the multivariate essence of developmental plasticity transcend the evolutionary theory and inspire applications in several fields.

Shifts in space and time: ecological transitions affect the evolution of resting metabolic rates in microteiid lizards.

Ecological diversification often encompasses exposure to new thermal regimes given by the use of specific spatial (microhabitat) and temporal (activity periods) niches. Empirical evidence provides links between temperature and physiology (e.g. rates of oxygen consumption), fostering predictions of evolutionary changes in metabolic rates coupled with ecological shifts. One example of such correspondence is the evolution of fossoriality and nocturnality in vertebrate ectotherms, where changes in metabolic rates coupled with niche transitions are expected. Because most studies address single transitions (fossoriality or nocturnality), metabolic changes associated with concomitant shifts in spatial and temporal components of habitat usage are underestimated, and it remains unclear which transition plays a major role for metabolic evolution. Integrating multiple ecological aspects that affect the evolution of thermosensitive traits is essential for a proper understanding of physiological correlates in niche transitions. Here, we provide the first phylogenetic multidimensional description of effects from ecological niche transitions both in space (origin of fossorial lineages) and in time (origin of nocturnal lineages) on the evolution of microteiid lizard (Gymnophthalmidae) metabolic rates. We found that evolution of resting metabolic rates was affected by both niche transitions, but with opposite trends. Evolution of fossoriality in endemic diurnal microteiids is coupled with a less thermally sensitive metabolism and higher metabolic rates. In contrast, a reduction in metabolic rates was detected in the endemic fossorial-nocturnal lineage, although metabolic thermal sensitivity remained as high as that observed in epigeal species, a pattern that likely reduces locomotion costs at lower temperatures and also favors thermoregulation in subsuperficial sand layers.

Phenotypic integration mediated by hormones: associations among digit ratios, body size and testosterone during tadpole development.

BACKGROUND: Developmental associations often explain phenotypic integration. The intersected hormonal regulation of ontogenetic processes fosters predictions of steroid-mediated phenotypic integration among sexually dimorphic traits, a statement defied by associations between classical dimorphism predictors (e.g. body size) and traits that apparently lack sex-specific functions (e.g. ratios between the lengths of Digits II and IV - 2D:4D). Developmental bases of female-biased 2D:4D have been identified, but these remain unclear for taxa presenting male-biased 2D:4D (e.g. anura). Here we propose two alternative hypotheses to investigate evolution of male-biased 2D:4D associated with sexually dimorphic body size using Leptodactylus frogs: I)'hypothesis of sex-specific digit responses' - Digit IV would be reactive to testosterone but exhibit responses in the opposite direction of those observed in female-biased 2D:4D lineages, so that Digit IV turns shorter in males; II) 'hypothesis of identity of the dimorphic digit'- Digit II would be the dimorphic digit. RESULTS: We compiled the following databases using Leptodactylus frogs: 1) adults of two species from natural populations and 2) testosterone-treated L. fuscus at post-metamorphic stage. Studied traits seem monomorphic in L. fuscus; L. podicipinus exhibits male-biased 2D:4D. When present, 2D:4D dimorphism was male-biased and associated with dimorphic body size; sex differences resided on Digit II instead of IV, corroborating our 'hypothesis of identity of the dimorphic digit'. Developmental steroid roles were validated: testosterone-treated L. fuscus frogs were smaller and exhibited masculinized 2D:4D, and Digit II was the digit that responded to testosterone. CONCLUSION: We propose a model where evolution of sexual dimorphism in 2D:4D first originates from the advent, in a given digit, of increased tissue sensitivity to steroids. Phenotypic integration with other sexually dimorphic traits would then occur through multi-trait hormonal effects during development. Such process of phenotypic integration seems fitness-independent in its origin and might explain several cases of steroid-mediated integration among sexually dimorphic traits.

Selection on different genes with equivalent functions: the convergence story told by Hox genes along the evolution of aquatic mammalian lineages.

BACKGROUND: Convergent evolution has been a challenging topic for decades, being cetaceans, pinnipeds and sirenians textbook examples of three independent origins of equivalent phenotypes. These mammalian lineages acquired similar anatomical features correlated to an aquatic life, and remarkably differ from their terrestrial counterparts. Whether their molecular evolutionary history also involved similar genetic mechanisms underlying such morphological convergence nevertheless remained unknown. To test for the existence of convergent molecular signatures, we studied the molecular evolution of Hox genes in these three aquatic mammalian lineages, comparing their patterns to terrestrial mammals. Hox genes are transcription factors that play a pivotal role in specifying embryonic regional identity of nearly any bilateral animal, and are recognized major agents for diversification of body plans. RESULTS: We detected few signatures of positive selection on Hox genes across the three aquatic mammalian lineages and verified that purifying selection prevails in these sequences, as expected for pleiotropic genes. Genes found as being positively selected differ across the aquatic mammalian lineages, but we identified a substantial overlap of their developmental functions. Such pattern likely resides on the duplication history of Hox genes, which probably provided different possible evolutionary routes for achieving the same phenotypic solution. CONCLUSIONS: Our results indicate that convergence occurred at a functional level of Hox genes along three independent origins of aquatic mammals. This conclusion reinforces the idea that different changes in developmental genes may lead to similar phenotypes, probably due to the redundancy provided by the participation of Hox paralogous genes in several developmental functions.

Beyond body size: muscle biochemistry and body shape explain ontogenetic variation of anti-predatory behaviour in the lizard Salvator merianae.

Anti-predatory behaviour evolves under the strong action of natural selection because the success of individuals avoiding predation essentially defines their fitness. Choice of anti-predatory strategies is defined by prey characteristics as well as environmental temperature. An additional dimension often relegated in this multilevel equation is the ontogenetic component. In the tegu Salvator merianae, adults run away from predators at high temperatures but prefer fighting when it is cold, whereas juveniles exhibit the same flight strategy within a wide thermal range. Here, we integrate physiology and morphology to understand ontogenetic variation in the temperature-dependent shift of anti-predatory behaviour in these lizards. We compiled data for body shape and size, and quantified enzyme activity in hindlimb and head muscles, testing the hypothesis that morphophysiological models explain ontogenetic variation in behavioural associations. Our prediction is that juveniles exhibit body shape and muscle biochemistry that enhance flight strategies. We identified biochemical differences between muscles mainly in the LDH:CS ratio, whereby hindlimb muscles were more glycolytic than the jaw musculature. Juveniles, which often use evasive strategies to avoid predation, have more glycolytic hindlimb muscles and are much smaller when compared with adults 1-2 years old. Ontogenetic differences in body shape were identified but marginally contributed to behavioural variation between juvenile and adult tegus, and variation in anti-predatory behaviour in these lizards resides mainly in associations between body size and muscle biochemistry. Our results are discussed in the ecological context of predator avoidance by individuals differing in body size living at temperature-variable environments, where restrictions imposed by the cold could be compensated by specific phenotypes.

Lungs of the first amniotes: why simple if they can be complex?

We show-in contrast to the traditional textbook contention-that the first amniote lungs were complex, multichambered organs and that the single-chambered lungs of lizards and snakes represent a secondarily simplified rather than the plesiomorphic condition. We combine comparative anatomical and embryological data and show that shared structural principles of multichamberedness are recognizable in amniotes including all lepidosaurian taxa. Sequential intrapulmonary branching observed during early organogenesis becomes obscured during subsequent growth, resulting in a secondarily simplified, functionally single-chambered lung in lepidosaurian adults. Simplification of pulmonary structure maximized the size of the smallest air spaces and eliminated biophysically compelling surface tension problems that were associated with miniaturization evident among stem lepidosaurmorphs. The remaining amniotes, however, retained the multichambered lungs, which allowed both large surface area and high pulmonary compliance, thus initially providing a strong selective advantage for efficient respiration in terrestrial environments. Branched, multichambered lungs instead of simple, sac-like organs were part and parcel of the respiratory apparatus of the first amniotes and pivotal for their success on dry land, with the sky literally as the limit.

Molecular evolution of HoxA13 and the multiple origins of limbless morphologies in amphibians and reptiles.

Developmental processes and their results, morphological characters, are inherited through transmission of genes regulating development. While there is ample evidence that cis-regulatory elements tend to be modular, with sequence segments dedicated to different roles, the situation for proteins is less clear, being particularly complex for transcription factors with multiple functions. Some motifs mediating protein-protein interactions may be exclusive to particular developmental roles, but it is also possible that motifs are mostly shared among different processes. Here we focus on HoxA13, a protein essential for limb development. We asked whether the HoxA13 amino acid sequence evolved similarly in three limbless clades: Gymnophiona, Amphisbaenia and Serpentes. We explored variation in ω (dN/dS) using a maximum-likelihood framework and HoxA13sequences from 47 species. Comparisons of evolutionary models provided low ω global values and no evidence that HoxA13 experienced relaxed selection in limbless clades. Branch-site models failed to detect evidence for positive selection acting on any site along branches of Amphisbaena and Gymnophiona, while three sites were identified in Serpentes. Examination of alignments did not reveal consistent sequence differences between limbed and limbless species. We conclude that HoxA13 has no modules exclusive to limb development, which may be explained by its involvement in multiple developmental processes.

Functional modularity and mechanical stress shape plastic responses during fish development.

The adaptive potential of plastic phenotypes relies on combined developmental responses. We investigated how manipulation of developmental conditions related to foraging mode in the fish Megaleporinus macrocephalus induces plastic responses at different levels: 1) functional modularity of skull bones, 2) biomechanical properties of the chondrocranium using Finite Element Models, 3) bmp4 expression levels, used as a proxy for molecular pathways involved in bone responses to mechanical load. We identified new modules in experimental groups, suggesting increased integration in specific head bone elements associated with the development of subterminal and upturned mouths, which are major features of Megaleporinus plastic morphotypes released in the lab. Plastic responses in head shape involved differences in the magnitude of mechanical stress, which seem restricted to certain chondrocranium regions. Three bones represent a 'mechanical unit' related to changes in mouth position induced by foraging mode, suggesting that functional modularity might be enhanced by the way specific regions respond to mechanical load. Differences in bmp4 expression levels between plastic morphotypes indicate associations between molecular signaling pathways and biomechanical responses to load. Our results offer a multilevel perspective of epigenetic factors involved in plastic responses, expanding our knowledge about mechanisms of developmental plasticity that originate novel complex phenotypes.

Native Lizards Living in Brazilian Cities: Effects of Developmental Environments on Thermal Sensitivity and Morpho-Functional Associations of Locomotion.

Environmental conditions often affect developmental processes and consequently influence the range of phenotypic variation expressed at population level. Expansion of urban sites poses new challenges for native species, as urbanization usually affects the intensity of solar exposure and shade availability, determining the thermal regimes organisms are exposed to. In this study, we evaluate the effects of different developmental conditions in a Tropidurus lizard commonly found in Brazilian urban sites. After incubating embryos of Tropidurus catalanensis in two different thermal regimes (Developmental Environments [DE]: cold 24°C and warm 30°C), we measured morphological traits in the neonates and quantified locomotor performance in horizontal and vertical surfaces at three temperatures [Test Temperatures (TT) = 24°C, 30°C and 36°C]. Results indicate effects of developmental temperatures on morphological features, expressing functional implications that might be decisive for the viability of T. catalanensis in urbanized areas. Lizards ran similarly on horizontal and vertical surfaces, and isolated analyses did not identify significant effects of DE or TT on the sprint speeds measured. Absolute Vmax (i.e., the maximum sprint speed reached among all TTs) positively correlated with body size (SVL), and neonates from the warm DE (30°C) were larger than those from the cold DE (24°C). Morpho-functional associations of absolute Vmax also involved pelvic girdle width and forelimb, hindlimb, trunk, and tail lengths. Emerging discussions aim to understand how animals cope with abrupt environmental shifts, a likely common challenge in urbanized sites. Our findings add a new dimension to the topic, providing evidence that temperature, an environmental parameter often affected by urbanization, influences the thermal sensitivity of locomotion and the morphological profile of T. catalanensis neonates. Thermal sensitivity of specific developmental processes may influence the ability of these lizards to remain in habitats that change fast, as those suffering rapid urbanization due to city growth.

Environmental temperature predicts resting metabolic rates in tropidurinae lizards.

Interspecific variation in metabolic rates may be associated with climate, habitat structure, and resource availability. Despite a strong link between ecology and physiology, there is a dearth in the understanding of how the costs of body maintenance change during ecological transitions. We focused on an ecologically diverse group of neotropical lizards (Tropidurinae) to investigate whether and how resting metabolic rate (RMR) evolved under divergent micro- and macrohabitat conditions. Using a phylogenetic framework, we tested whether species from hot and dry habitats had lower RMRs in relation to those from cooler and mesic habitats, and investigated whether microhabitat usage had an effect over body mass-adjusted RMRs. Our results suggest that RMRs are not phylogenetically structured in Tropidurinae. We found no correlation between metabolism, precipitation, and microhabitat usage. Species from warmer habitats had lower RMR compared to those from cooler habitats, supporting a mechanism of negative compensation in metabolic responses to temperature. Ectotherms from warmer habitats can limit energetic demand and expenditure through reduced RMR, whereas those from cooler habitats may sustain activity despite thermal constraints via increased RMR. Our work highlights the role of temperature in shaping metabolic responses in lizards, giving additional support to the notion that physiology and ecological contexts are intertwined.

Developmental plasticity reveals hidden fish phenotypes and enables morphospace diversification.

The establishment of a given phenotype is only one expression from a range of hidden developmental possibilities. Developmental plasticity at hidden reaction norms might elicit phenotypic diversification under new developmental environments. Current discussion benefits from empirical analyses that integrate multiple environmental stimuli to evaluate how plastic responses may shape phenotypic variation. We raised Megaleporinus macrocephalus fish in different environmental settings to address contributions of developmental plasticity for emergence of new phenotypes and subsequent morphospace diversification. Plastic morphotypes were evaluated at two complementary scales, the M. macrocephalus morphospace and the higher taxonomic level of Anostomidae family. Morphospace analyses demonstrated that developmental plasticity quickly releases distinct head morphotypes that were hidden in the parental monomorphic population. Plastic morphotypes occupied discrete and previously unfilled morphospace regions, a result obtained from comparisons with a control population and in analyses including several Anostomidae species. Plastic responses involved adjustments in shape and relative position of head bonesets, and fish raised under specific environmental combinations rescued phenotypic patterns described for different genera. Therefore, developmental plasticity possibly contributes to adaptive radiation in Anostomidae. Results illustrate how plastic responses enable morphospace diversification and contribute to evolution.

Peculiar relationships among morphology, burrowing performance and sand type in two fossorial microteiid lizards.

Associations among ecology, morphology and locomotor performance have been intensively investigated in several vertebrate lineages. Knowledge on how phenotypes evolve in natural environments likely benefits from identification of circumstances that might expand current ecomorphological equations. In this study, we used two species of Calyptommatus lizards from Brazilian Caatingas to evaluate if specific soil properties favor burrowing performance. As a derived prediction, we expected that functional associations would be easily detectable at the sand condition that favors low-resistance burrowing. We collected two endemic lizards and soil samples in their respective localities, obtained morphological data and recorded performance of both species in different sand types. As a result, the two species burrowed faster at the fine and homogeneous sand, the only condition where we detected functional associations between morphology and locomotion. In this sand type, lizards from both Calyptommatus species that have higher trunks and more concave heads were the ones that burrowed faster, and these phenotypic traits did not morphologically discriminate the two Calyptommatus populations studied. We discuss that integrative approaches comprising manipulation of environmental conditions clearly contribute to elucidate processes underlying phenotypic evolution in fossorial lineages.