Cranial integration in the fire salamander, Salamandra salamandra (Caudata: Salamandridae)

Phenotypic integration and modularity are concepts that represent the pattern of connectivity of morphological structures within an organism. Integration describes the coordinated variation of traits, and analyses of these relationships among traits often reveals the presence of modules, sets of traits that are highly integrated but relatively independent of other traits. Phenotypic integration and modularity have been studied at both the evolutionary and static level across a variety of clades, although most studies thus far are focused on amniotes, and especially mammals. Using a high-dimensional geometric morphometric approach, we investigated the pattern of cranial integration and modularity of the Italian fire salamander (Salamandra salamandra giglioli). We recovered a highly modular pattern, but this pattern did not support either entirely developmental or functional hypotheses of cranial organisation, possibly reflecting complex interactions amongst multiple influencing factors. We found that size had no significant effect on cranial shape, and that morphological variance of individual modules had no significant relationship with degree of within-module integration. The pattern of cranial integration in the fire salamander is similar to that previously recovered for caecilians, with highly integrated jaw suspensorium and occipital regions, suggesting possible conservation of patterns across lissamphibians

Size, microhabitat, and loss of larval feeding drive cranial diversification in frogs

Habitat is one of the most important factors shaping organismal morphology, but it may vary across life history stages. Ontogenetic shifts in ecology may introduce antagonistic selection that constrains adult phenotype, particularly with ecologically distinct developmental phases such as the free-living, feeding larval stage of many frogs (Lissamphibia: Anura). We test the relative influences of developmental and ecological factors on the diversification of adult skull morphology with a detailed analysis of 15 individual cranial regions across 173 anuran species, representing every extant family. Skull size, adult microhabitat, larval feeding, and ossification timing are all significant factors shaping aspects of cranial evolution in frogs, with late-ossifying elements showing the greatest disparity and fastest evolutionary rates. Size and microhabitat show the strongest effects on cranial shape, and we identify a “large size-wide skull” pattern of anuran, and possibly amphibian, evolutionary allometry. Fossorial and aquatic microhabitats occupy distinct regions of morphospace and display fast evolution and high disparity. Taxa with and without feeding larvae do not notably differ in cranial morphology. However, loss of an actively feeding larval stage is associated with higher evolutionary rates and disparity, suggesting that functional pressures experienced earlier in ontogeny significantly impact adult morphological evolution

A practical guide to sliding and surface semilandmarks in morphometric analyses

Advances in imaging technologies, such as computed tomography (CT) and surface scanning, have facilitated the rapid generation of large datasets of high-resolution 3D specimen reconstructions in recent years. The wealth of phenotypic information available from these datasets has the potential to inform our understanding of morphological variation and evolution. However, the ever-increasing ease of compiling 3D datasets has created an urgent need for sophisticated methods of capturing high-density shape data that reflect the biological complexity in form. Landmarks often do not take full advantage of the rich shape information available from high-resolution 3D specimen reconstructions, as they are typically restricted to sutures or processes that can be reliably identified across specimens and exclude most of the surficial morphology. The development of sliding and surface semilandmark techniques has greatly enhanced the quantification of shape, but their application to diverse datasets can be challenging, especially when dealing with the variable absence of some regions within a structure. Using comprehensive 3D datasets of crania that span the entire clades of birds, squamates and caecilians, we demonstrate methods for capturing morphology across incredibly diverse shapes. We detail many of the difficulties associated with applying semilandmarks to comparable regions across highly disparate structures, and provide solutions to some of these challenges, while considering the consequences of decisions one makes in applying these approaches. Finally, we analyse the benefits of high-density sliding semilandmark approaches over landmark-only studies for capturing shape across diverse organisms and discuss the promise of these approaches for the study of organismal form

High-density three-dimensional morphometric analyses support conserved static (intraspecific) modularity in caecilian (Amphibia: Gymnophiona) crania

Analyses of phenotypic integration and modularity seek to quantify levels of covariation among traits to identify their shared functional, developmental and genetic underpinnings (‘integration’), which may delineate semi-independent subsets of highly integrated traits (‘modules’). Existing studies have focused mainly on mammals or model organisms, limiting our understanding of factors that shape patterns of integration and modularity and their importance for morphological evolution. We present the first study of static (intraspecific) integration and modularity in caecilian crania, using dense surface sliding semi-landmarks to quantify cranial morphology in Boulengerula boulengeri and Idiocranium russeli. Eleven hypotheses of modular organization were compared with a likelihood approach and best-fitting models confirmed with covariance ratio analysis. Allometric corrections and subsampling analyses demonstrated the robustness of results. Allometry had a substantially larger influence on the results of landmark- only analyses relative to analyses incorporating semi-landmarks. Idiocranium russeli displayed significantly higher variation than B. boulengeri, but they had similar 12- and 13-module patterns, respectively, suggesting that caecilian crania are highly modular and that the modularity pattern is largely conserved across these species, despite their divergent morphologies and > 175 Myr of independent evolution. As in previous mammalian studies, our caecilian study suggests that cranial modularity patterns are conserved within major vertebrate clades.Ashleigh F. Marshall, Carla Bardua, David J. Gower, Mark Wilkinson, Emma Sherratt and Anjali Goswam

High-density morphometric analysis of shape and integration: the good, the bad, and the not-really-a-problem

The field of comparative morphology has entered a new phase with the rapid generation of high-resolution three-dimensional data. With freely available 3D data of thousands of species, methods for quantifying morphology that harness this rich phenotypic information are quickly emerging. Among these techniques, high-density geometric morphometric approaches provide a powerful and versatile framework to robustly characterize shape and phenotypic integration, the covariances among morphological traits. These methods are particularly useful for analyses of complex structures and across disparate taxa, which may share few landmarks of unambiguous homology. However, high-density geometric morphometrics also brings challenges, for example with statistical, but not biological, covariances imposed by placement and sliding of semilandmarks and registration methods such as Procrustes superimposition. Here, we present simulations and case studies of high-density datasets for squamates, birds, and caecilians that exemplify the promise and challenges of high-dimensional analyses of phenotypic integration and modularity. We assess: (1) the relative merits of "big" high-density geometric morphometrics data over traditional shape data; (2) the impact of Procrustes superimposition on analyses of integration and modularity; and (3) differences in patterns of integration between analyses using high-density geometric morphometrics and those using discrete landmarks. We demonstrate that for many skull regions 20-30 landmarks and/or semilandmarks are needed to accurately characterize their shape variation, and landmark-only analyses do a particularly poor job of capturing shape variation in vault and rostrum bones. Procrustes superimposition can mask modularity, especially when the number of landmarks is low and they covary in parallel directions, but this effect decreases with increasing landmark number or more biologically complex covariance patterns. Landmark-only and landmark-plus-sliding-semilandmark analyses of integration are generally congruent in overall pattern of integration, but landmark-only analyses tend to show higher integration between adjacent bones, especially when landmarks placed on the sutures between bones introduces a boundary bias. Allometry may be a stronger influence on patterns of integration in landmark-only analyses, which show stronger integration prior to removal of allometric effects compared to analyses including semilandmarks. High-density geometric morphometrics has its challenges and drawbacks, but our analyses of simulated and empirical datasets demonstrate that these potential issues are unlikely to obscure genuine biological signal. Rather, high-density geometric morphometric data exceeds traditional landmark-based methods in characterization of morphology and allow more nuanced comparisons across disparate taxa. Combined with the rapid increases in 3D data availability, high-density morphometric approaches have immense potential to propel a new class of studies of comparative morphology and phenotypic integration

High-density three-dimensional morphometric analyses support conserved static (intraspecific) modularity in caecilian (Amphibia: Gymnophiona) crania

Analyses of phenotypic integration and modularity seek to quantify levels of covariation among traits to identify their shared functional, developmental and genetic underpinnings (‘integration’), which may delineate semi-independent subsets of highly integrated traits (‘modules’). Existing studies have focused mainly on mammals or model organisms, limiting our understanding of factors that shape patterns of integration and modularity and their importance for morphological evolution. We present the first study of static (intraspecific) integration and modularity in caecilian crania, using dense surface sliding semi-landmarks to quantify cranial morphology in Boulengerula boulengeri and Idiocranium russeli. Eleven hypotheses of modular organization were compared with a likelihood approach and best-fitting models confirmed with covariance ratio analysis. Allometric corrections and subsampling analyses demonstrated the robustness of results. Allometry had a substantially larger influence on the results of landmarkonly analyses relative to analyses incorporating semi-landmarks. Idiocranium russeli displayed significantly higher variation than B. boulengeri, but they had similar 12- and 13-module patterns, respectively, suggesting that caecilian crania are highly modular and that the modularity pattern is largely conserved across these species, despite their divergent morphologies and > 175 Myr of independent evolution. As in previous mammalian studies, our caecilian study suggests that cranial modularity patterns are conserved within major vertebrate clades

Morphological evolution and modularity of the caecilian skull

Background: Caecilians (Gymnophiona) are the least speciose extant lissamphibian order, yet living forms capture approximately 250 million years of evolution since their earliest divergences. This long history is reflected in the broad range of skull morphologies exhibited by this largely fossorial, but developmentally diverse, clade. However, this diversity of form makes quantification of caecilian cranial morphology challenging, with highly variable presence or absence of many structures. Consequently, few studies have examined morphological evolution across caecilians. This extensive variation also raises the question of degree of conservation of cranial modules (semi-autonomous subsets of highly-integrated traits) within this clade, allowing us to assess the importance of modular organisation in shaping morphological evolution. We used an intensive surface geometric morphometric approach to quantify cranial morphological variation across all 32 extant caecilian genera. We defined 16 cranial regions using 53 landmarks and 687 curve and 729 surface sliding semilandmarks. With these unprecedented high-dimensional data, we analysed cranial shape and modularity across caecilians assessing phylogenetic, allometric and ecological influences on cranial evolution, as well as investigating the relationships among integration, evolutionary rate, and morphological disparity. Results: We found highest support for a ten-module model, with greater integration of the posterior skull. Phylogenetic signal was significant (Kmult = 0.87, p < 0.01), but stronger in anterior modules, while allometric influences were also significant (R2 = 0.16, p < 0.01), but stronger posteriorly. Reproductive strategy and degree of fossoriality were small but significant influences on cranial morphology (R2 = 0.03–0.05), after phylogenetic (p < 0.03) and multiple-test (p < 0.05) corrections. The quadrate-squamosal ‘cheek’ module was the fastest evolving module, perhaps due to its pivotal role in the unique dual jaw-closing mechanism of caecilians. Highly integrated modules exhibited both high and low disparities, and no relationship was evident between integration and evolutionary rate. Conclusions: Our high-dimensional approach robustly characterises caecilian cranial evolution and demonstrates that caecilian crania are highly modular and that cranial modules are shaped by differential phylogenetic, allometric, and ecological effects. More broadly, and in contrast to recent studies, this work suggests that there is no simple relationship between integration and evolutionary rate or disparity.Carla Bardua, Mark Wilkinson, David J. Gower, Emma Sherratt and Anjali Goswam

Phage capsid nanoparticles with defined ligand arrangement block influenza virus entry

Multivalent interactions at biological interfaces occur frequently in nature and mediate recognition and interactions in essential physiological processes such as cell-to-cell adhesion. Multivalency is also a key principle that allows tight binding between pathogens and host cells during the initial stages of infection. One promising approach to prevent infection is the design of synthetic or semisynthetic multivalent binders that interfere with pathogen adhesion1,2,3,4. Here, we present a multivalent binder that is based on a spatially defined arrangement of ligands for the viral spike protein haemagglutinin of the influenza A virus. Complementary experimental and theoretical approaches demonstrate that bacteriophage capsids, which carry host cell haemagglutinin ligands in an arrangement matching the geometry of binding sites of the spike protein, can bind to viruses in a defined multivalent mode. These capsids cover the entire virus envelope, thus preventing its binding to the host cell as visualized by cryo-electron tomography. As a consequence, virus infection can be inhibited in vitro, ex vivo and in vivo. Such highly functionalized capsids present an alternative to strategies that target virus entry by spike-inhibiting antibodies5 and peptides6 or that address late steps of the viral replication cycle

Tracking CNS and systemic sources of oxidative stress during the course of chronic neuroinflammation

The functional dynamics and cellular sources of oxidative stress are central to understanding MS pathogenesis but remain elusive, due to the lack of appropriate detection methods. Here we employ NAD(P)H fluorescence lifetime imaging to detect functional NADPH oxidases (NOX enzymes) in vivo to identify inflammatory monocytes, activated microglia, and astrocytes expressing NOX1 as major cellular sources of oxidative stress in the central nervous system of mice affected by experimental autoimmune encephalomyelitis (EAE). This directly affects neuronal function in vivo, indicated by sustained elevated neuronal calcium. The systemic involvement of oxidative stress is mirrored by overactivation of NOX enzymes in peripheral CD11b(+) cells in later phases of both MS and EAE. This effect is antagonized by systemic intake of the NOX inhibitor and anti-oxidant epigallocatechin-3-gallate. Together, this persistent hyper-activation of oxidative enzymes suggests an "oxidative stress memory" both in the periphery and CNS compartments, in chronic neuroinflammation

Evolutionary integration of the frog cranium

Evolutionary integration (covariation) of traits has long fascinated biologists because of its potential to elucidate factors that have shaped morphological evolution. Studies of tetrapod crania have identified patterns of evolutionary integration that reflect functional or developmental interactions among traits, but no studies to date have sampled widely across the species‐rich lissamphibian order, Anura (frogs). Frogs exhibit a vast range of cranial morphologies, life history strategies and ecologies. Here, using high‐density morphometrics we capture cranial morphology for 172 anuran species, sampling every extant family. We quantify the pattern of evolutionary modularity in the frog skull and compare patterns in taxa with different life history modes. Evolutionary changes across the anuran cranium are highly modular, with a well‐integrated ‘suspensorium’ involved in feeding. This pattern is strikingly similar to that identified for caecilian and salamander crania, suggesting replication of patterns of evolutionary integration across Lissamphibia. Surprisingly, possession of a feeding larval stage has no notable influence on cranial integration across frogs. However, late‐ossifying bones exhibit higher integration than early‐ossifying bones. Finally, anuran cranial modules show diverse morphological disparities, supporting the hypothesis that modular variation allows mosaic evolution of the cranium, but we find no consistent relationship between degree of within‐module integration and disparity