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.

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.

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.

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.

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.

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.

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.

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.

Inflammasomes are activated in response to SARS-CoV-2 infection and are associated with COVID-19 severity in patients.

Severe cases of COVID-19 are characterized by a strong inflammatory process that may ultimately lead to organ failure and patient death. The NLRP3 inflammasome is a molecular platform that promotes inflammation via cleavage and activation of key inflammatory molecules including active caspase-1 (Casp1p20), IL-1ß, and IL-18. Although participation of the inflammasome in COVID-19 has been highly speculated, the inflammasome activation and participation in the outcome of the disease are unknown. Here we demonstrate that the NLRP3 inflammasome is activated in response to SARS-CoV-2 infection and is active in COVID-19 patients. Studying moderate and severe COVID-19 patients, we found active NLRP3 inflammasome in PBMCs and tissues of postmortem patients upon autopsy. Inflammasome-derived products such as Casp1p20 and IL-18 in the sera correlated with the markers of COVID-19 severity, including IL-6 and LDH. Moreover, higher levels of IL-18 and Casp1p20 are associated with disease severity and poor clinical outcome. Our results suggest that inflammasomes participate in the pathophysiology of the disease, indicating that these platforms might be a marker of disease severity and a potential therapeutic target for COVID-19.

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.

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.

Diversification rates are more strongly related to microhabitat than climate in squamate reptiles (lizards and snakes).

Patterns of species richness among clades can be directly explained by the ages of clades or their rates of diversification. The factors that most strongly influence diversification rates remain highly uncertain, since most studies typically consider only a single predictor variable. Here, we explore the relative impacts of macroclimate (i.e., occurring in tropical vs. temperate regions) and microhabitat use (i.e., terrestrial, fossorial, arboreal, aquatic) on diversification rates of squamate reptile clades (lizards and snakes). We obtained data on microhabitat, macroclimatic distribution, and phylogeny for >4000 species. We estimated diversification rates of squamate clades (mostly families) from a time-calibrated tree, and used phylogenetic methods to test relationships between diversification rates and microhabitat and macroclimate. Across 72 squamate clades, the best-fitting model included microhabitat but not climatic distribution. Microhabitat explained ∼37% of the variation in diversification rates among clades, with a generally positive impact of arboreal microhabitat use on diversification, and negative impacts of fossorial and aquatic microhabitat use. Overall, our results show that the impacts of microhabitat on diversification rates can be more important than those of climate, despite much greater emphasis on climate in previous studies.

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.

Ecological associations of autopodial osteology in Neotropical geckos.

Coevolution of form and function inspires investigation of associations between morphological variation and the exploitation of specific ecological settings. Such relationships, based mostly on traits of external morphology, have been extensively described for vertebrates, and especially so for squamates. External features are, however, composed by both soft tissues and bones, and these likely play different biomechanical roles during locomotion, such as in the autopodia. Therefore, ecological trends identified on the basis of external morphological measurements may not be directly correlated with equivalent variation in osteology. Here, we investigate how refined parameters of autopodial osteology relate to ecology, by contrasting climbing and nonclimbing geckos. Our first step consisted of inferring how external and osteological morphometric traits coevolved in the group. Our results corroborate the hypothesis of coevolution between external and osteological elements in the autopodia of geckos, and provides evidence for associations between specific osteological traits and preferred locomotor habit. Specifically, nonclimbers exhibit longer ultimate and penultimate phalanges of Digit V in the manus and pes and also a longer fifth metatarsal in comparison with climbers, a pattern discussed here in the context of the differential demands made upon locomotion in specific ecological contexts. Our study highlights the relevance of osteological information for discussing the evolution of ecological associations of the tetrapod autopodium. J. Morphol. 278:290-299, 2017. © 2017 Wiley Periodicals, Inc.