Permutation tests for phylogenetic comparative analyses of high‐dimensional shape data: What you shuffle matters

Evaluating statistical trends in high‐dimensional phenotypes poses challenges for comparative biologists, because the high‐dimensionality of the trait data relative to the number of species can prohibit parametric tests from being computed. Recently, two comparative methods were proposed to circumvent this difficulty. One obtains phylogenetic independent contrasts for all variables, and statistically evaluates the linear model by permuting the phylogenetically independent contrasts (PICs) of the response data. The other uses a distance‐based approach to obtain coefficients for generalized least squares models (D‐PGLS), and subsequently permutes the original data to evaluate the model effects. Here, we show that permuting PICs is not equivalent to permuting the data prior to the analyses as in D‐PGLS. We further explain why PICs are not the correct exchangeable units under the null hypothesis, and demonstrate that this misspecification of permutable units leads to inflated type I error rates of statistical tests. We then show that simply shuffling the original data and recalculating the independent contrasts with each iteration yields significance levels that correspond to those found using D‐PGLS. Thus, while summary statistics from methods based on PICs and PGLS are the same, permuting PICs can lead to strikingly different inferential outcomes with respect to statistical and biological inferences.     

Ecological and phylogenetic influence on mandible shape variation of South American caviomorph rodents (Rodentia: Hystricomorpha)

We analyzed mandible shape variation of 17 genera belonging to three superfamilies (Cavioidea, Chinchilloidea, and Octodontoidea) of South American caviomorph rodents using geometric morphometrics. The relative influence of phylogeny and ecology on this variation was assessed using phylogenetic comparative methods. Most morphological variation was concentrated in condylar, coronoid, and angular processes, as well as the diastema. Features potentially advantageous for digging (i.e. high coronoid and condylar processes, relatively short angular process, and diastema) were present only in octodontoids; cavioids showed opposing trends, which could represent a structural constraint for fossorial habits. Chinchilloids showed intermediate features. Genera were distributed in the morphospace according to their classification into superfamilial clades. The phylogenetic signal for shape components was significant along phylogeny, whereas the relationship between mandibular shape and ecology was nonsignificant when phylogenetic structure was taken into account. An early evolutionary divergence in the mandible shape among major caviomorph clades would explain the observed strong phylogenetic influence on the variation of this structure. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 102 , 828–837.     

The evolutionary role of modularity and integration in the hominoid cranium

Patterns of morphological integration and modularity among shape features emerge from genetic and developmental factors with varying pleiotropic effects. Factors or processes affecting morphology only locally may respond to selection more easily than common factors that may lead to deleterious side effects and hence are expected to be more conserved. We briefly review evidence for such global factors in primate cranial development as well as for local factors constrained to either the face or the neurocranium. In a sample comprising 157 crania of Homo sapiens, Pan troglodytes, and Gorilla gorilla, we statistically estimated common and local factors of shape variation from Procrustes coordinates of 347 landmarks and semilandmarks. Common factors with pleiotropic effects on both the face and the neurocranium account for a large amount of shape variation, but mainly by extension or truncation of otherwise conserved developmental pathways. Local factors (modular shape characteristics) have more degrees of freedom for evolutionary change than mere ontogenetic scaling. Cranial shape is similarly integrated during development in all three species, but human evolution involves dissociation among several characteristics. The dissociation has probably been achieved by evolutionary alterations and by the novel emergence of local factors affecting characteristics that are controlled at the same time by the common factors.     

Early evolutionary differentiation of morphological variation in the mandible of South American caviomorph rodents (Rodentia, Caviomorpha)

Caviomorphs are a clade of South American rodents recorded at least since the early Oligocene (> 31.5 Ma) that exhibit ample eco-morphological variation. It has been proposed that phylogenetic structure is more important than ecological factors for understanding mandibular shape variation in this clade. This was interpreted as a result of the long-standing evolutionary history of caviomorphs and the early divergence of major lineages. In this work, we test this hypothesis through the analysis of morphological variation in the mandible of living and extinct species and compare this information with that obtained through comparative phylogenetic analyses. Our results support the hypothesis of early origin of mandibular variation; moreover, they suggest the conservation of early differentiated morphologies, which could indicate the existence of constrained evolutionary diversification.     

Form and function within a phylogenetic framework: locomotory habits of extant predators and some Miocene Sparassodonta (Metatheria)

In this study, we analysed locomotory habits in extant predators and Sparassodonta species through geometric morphometric techniques and discriminant analyses of the distal humerus in anterior view, proximal ulna in lateral view, and tibia in proximal view. We included a wide sample of extant predators, and considered the phylogenetic and allometric structure in the data sets. We also included some Sparassodonta, a group of carnivorous metatherians that inhabited South America during the Cenozoic, and inferred their locomotory habits. Results suggest the presence of a close relationship between shape and locomotory habits, even after removing the shape component explained by phylogeny in the three postcranial elements. Terrestrial habits were inferred for Arctodictis sinclairi, Borhyaena tuberata, ‘Lycopsis’ longirostrus, and Thylacosmilus atrox. Some degree of cursoriality was highlighted in B. tuberata and T. atrox, and climbing abilities in ‘L.’ longirostrus, and to a lesser degree in B. tuberata. Scansorial habits were inferred for Cladosictis patagonica, Sipalocyon gracilis, Prothylacynus patagonicus, and Pseudonotictis pusillus, and in the case of C. patagonica, some digging ability was also tentatively inferred. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 165 , 224–251.     

Ecophenotypic variation and phylogenetic inheritance in first lower molar shape of extant Italian populations of Microtus (Terricola) savii (Rodentia)

Seventeen extant populations of Microtus (Terricola) savii have been investigated for correlations of first lower molar shape to climatic variables by means of geometric morphometrics, and controlling for phylogenetic inheritance. Comparative methods revealed that climatic variables and phylogeography provide a very similar contribution to variation in first lower molars morphology, whereas tooth size does not appear to be affected by climatic conditions. Climate‐related changes have been recognized in the anteroconid portion of the tooth. This indicates that molar tooth variation is strongly influenced by climatic conditions, although in a complex way. Calabrian populations, often ascribed to Microtus (Terricola) brachycercus, form a distinct cluster, in agreement with the most recent genetic analyses. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99 , 632–647.     

Body mass and foraging ecology predict evolutionary patterns of skeletal pneumaticity in the diverse "waterbird" clade

Extensive skeletal pneumaticity (air-filled bone) is a distinguishing feature of birds. The proportion of the skeleton that is pneumatized varies considerably among the >10,000 living species, with notable patterns including increases in larger bodied forms, and reductions in birds employing underwater pursuit diving as a foraging strategy. I assess the relationship between skeletal pneumaticity and body mass and foraging ecology, using a dataset of the diverse "waterbird" clade that encompasses a broad range of trait variation. Inferred changes in pneumaticity and body mass are congruent across different estimates of phylogeny, whereas pursuit diving has evolved independently between two and five times. Phylogenetic regressions detected positive relationships between body mass and pneumaticity, and negative relationships between pursuit diving and pneumaticity, whether independent variables are considered in isolation or jointly. Results are generally consistent across different estimates of topology and branch lengths. "Predictive" analyses reveal that several pursuit divers (loons, penguins, cormorants, darters) are significantly apneumatic compared to their relatives, and provide an example of how phylogenetic information can increase the statistical power to detect taxa that depart from established trait correlations. These findings provide the strongest quantitative comparative support yet for classical hypotheses regarding the evolution of avian skeletal pneumaticity.     

A Bayesian extension of phylogenetic generalized least squares: Incorporating uncertainty in the comparative study of trait relationships and evolutionary rates

Phylogenetic comparative methods use tree topology, branch lengths, and models of phenotypic change to take into account nonindependence in statistical analysis. However, these methods normally assume that trees and models are known without error. Approaches relying on evolutionary regimes also assume specific distributions of character states across a tree, which often result from ancestral state reconstructions that are subject to uncertainty. Several methods have been proposed to deal with some of these sources of uncertainty, but approaches accounting for all of them are less common. Here, we show how Bayesian statistics facilitates this task while relaxing the homogeneous rate assumption of the well-known phylogenetic generalized least squares (PGLS) framework. This Bayesian formulation allows uncertainty about phylogeny, evolutionary regimes, or other statistical parameters to be taken into account for studies as simple as testing for coevolution in two traits or as complex as testing whether bursts of phenotypic change are associated with evolutionary shifts in intertrait correlations. A mixture of validation approaches indicates that the approach has good inferential properties and predictive performance. We provide suggestions for implementation and show its usefulness by exploring the coevolution of ankle posture and forefoot proportions in Carnivora.     

Evaluating evolutionary pressures and phylogenetic signal in earthworms: a case study – the number of typhlosole lamellae in Hormogastridae (Annelida,Oligochaeta)

Rarely have phylogenetic comparative methods been used to study the correlation between phenotypic traits and environmental variables in invertebrates. With the widespread convergence and conservativeness of the morphological characters used in earthworms, these comparative methods could be useful to improve our understanding of their evolution and systematics. One of the most prominent morphological characters in the family Hormogastridae, endemic to Mediterranean areas, is their multilamellar typhlosole, traditionally thought to be an adaptation to soils poor in nutrients. We tested the correlation of body size and soil characteristics with the number of typhlosole lamellae through a phylogenetic generalized least squares (PGLS) analysis. An ultrametric phylogenetic hypothesis was built with a 2580‐bp DNA sequence from 90 populations, used in combination with three morphological and 11 soil variables. The best‐supported model, based on the Akaike information criterion, was obtained by optimizing the parameters lambda (λ), kappa (κ), and delta (δ). The phylogenetic signal was strong for the number of typhlosole lamellae and average body weight, and was lower for soil variables. Increasing body weight appeared to be the main evolutionary pressure behind the increase in the number of typhlosole lamellae, with soil texture and soil richness having a weaker but significant effect. Information on the evolutionary rate of the number of typhlosole lamellae suggested that the early evolution of this character could have strongly shaped its variability, as is found in an adaptive radiation. This work highlights the importance of implementing the phylogenetic comparative method to test evolutionary hypotheses in invertebrate taxa.     

Diversification of body shape in Sebastes rockfishes of the north‐east Pacific

Body shape variation is integrally related to many aspects of fish ecology, including locomotion and foraging, and can indicate the functional diversity of fish assemblages. Few studies have thoroughly characterized body shape in a diverse marine fish clade, or investigated both temporal and spatial patterns of variation in body shape disparity. Here, I use digital photographs to measure geometric body shape in 66 species of north‐east Pacific rockfish (Sebastes spp.), including a correction for error introduced by arching of specimens. Different components of interspecific shape variation show associations with fish size, depth habitat, trophic niche and phylogenetic relationships. Overall, the accumulation of body shape disparity appears to have been near‐constant over time, and shows little variation across the latitudinal range of rockfish.     

Morphological integration and modularity in the hyperkinetic feeding system of aquatic‐foraging snakes

The kinetic skull is a key innovation that allowed snakes to capture, manipulate, and swallow prey exclusively using their heads using the coordinated movement of eight bones. Despite these unique feeding behaviors, patterns of evolutionary integration and modularity within the feeding bones of snakes in a phylogenetic framework have yet to be addressed. Here, we use a dataset of 60 μCT‐scanned skulls and high‐density geometric morphometric methods to address the origin and patterns of variation and integration in the feeding bones of aquatic‐foraging snakes. By comparing alternate superimposition protocols allowing us to analyze the entire kinetic feeding system simultaneously, we find that the feeding bones are highly integrated, driven predominantly by functional selective pressures. The most supported pattern of modularity contains four modules, each associated with distinct functional roles: the mandible, the palatopterygoid arch, the maxilla, and the suspensorium. Further, the morphological disparity of each bone is not linked to its magnitude of integration, indicating that integration within the feeding system does not strongly constrain morphological evolution, and that adequate biomechanical solutions to a wide range of feeding ecologies and behaviors are readily evolvable within the constraint due to integration in the snake feeding system.     

Evolution of breeding plumages in birds: A multiple‐step pathway to seasonal dichromatism in New World warblers (Aves: Parulidae)

Many species of birds show distinctive seasonal breeding and nonbreeding plumages. A number of hypotheses have been proposed for the evolution of this seasonal dichromatism, specifically related to the idea that birds may experience variable levels of sexual selection relative to natural selection throughout the year. However, these hypotheses have not addressed the selective forces that have shaped molt, the underlying mechanism of plumage change. Here, we examined relationships between life‐history variation, the evolution of a seasonal molt, and seasonal plumage dichromatism in the New World warblers (Aves: Parulidae), a family with a remarkable diversity of plumage, molt, and life‐history strategies. We used phylogenetic comparative methods and path analysis to understand how and why distinctive breeding and nonbreeding plumages evolve in this family. We found that color change alone poorly explains the evolution of patterns of biannual molt evolution in warblers. Instead, molt evolution is better explained by a combination of other life‐history factors, especially migration distance and foraging stratum. We found that the evolution of biannual molt and seasonal dichromatism is decoupled, with a biannual molt appearing earlier on the tree, more dispersed across taxa and body regions, and correlating with separate life‐history factors than seasonal dichromatism. This result helps explain the apparent paradox of birds that molt biannually but show breeding plumages that are identical to the nonbreeding plumage. We find support for a two‐step process for the evolution of distinctive breeding and nonbreeding plumages: That prealternate molt evolves primarily under selection for feather renewal, with seasonal color change sometimes following later. These results reveal how life‐history strategies and a birds' environment act upon multiple and separate feather functions to drive the evolution of feather replacement patterns and bird coloration.     

Tempo and mode of evolutionary divergence in modern and Holocene Vancouver Island marmots (Marmota vancouverensis) (Mammalia, Rodentia)

Marmota vancouverensis is the only insular species among the 14 species of marmots. The evolutionary history of this species is unresolved. Although M. vancouverensis is strongly differentiated in osteological and other morphological characters, its low genetic divergence suggests recent evolution from an ancestral continental species. We used geometric morphometric techniques to assess the morphology of hemimandibles from 239 modern M. vancouverensis , Marmota caligata , Marmota flaviventris , Marmota olympus and 30 Holocene (9435–735 cal. yr bp) subfossil M. vancouverensis . Our results confirm that the mandible of M. vancouverensis is strongly differentiated in shape from continental marmot species, but similar in size to its mainland sister species M. caligata . Temporal variation in size and shape over the past 2500 years among allochronic samples of M. vancouverensis was minimal suggesting that the morphological divergence of this species occurred in a period of rapid change following its isolation from mainland populations in the late Pleistocene. Selection pressures associated with environmental changes and founder effects and genetic drift resulting from population bottlenecks created by population declines and habitat fragmentation are hypothesized as factors contributing to the morphological divergence of this species.     

Differential correlates of diet and phylogeny on the shape of the premaxilla and anterior tooth in sparid fishes (Perciformes: Sparidae)

We explore the correlational patterns of diet and phylogeny on the shape of the premaxilla and anterior tooth in sparid fishes (Perciformes: Sparidae) from the western Mediterranean Sea. The premaxilla is less variable, and in spite of the presence of species-specific features, a common structural pattern is easily recognizable in all species (i.e. the ascending and the articular processes are fused in a single branch, as in many percoid fishes). In contrast, tooth shape is more variable, and different structural types can be recognized (e.g. canine-like or incisive). Coupling geometric morphometric and comparative methods we found that the relationship between shape, diet and phylogeny also differs between premaxilla and tooth. Thus, the shape of the premaxilla is significantly correlated with food type, whereas the shape of the teeth is not correlated with diet, and probably reflects the species phylogenetic relationships. Two biological roles, resistance against compressive forces generated in the buccal cavity and the size of the oral gape, would explain the ecomorphological patterns of the premaxilla. The premaxilla and anterior tooth appear to evolve at different rates (mosaic evolution) and represent an example of morphological traits belonging to the same functional unit but following uncoupled evolutionary pathways.     

Evolution of molar shape in didelphid marsupials (Marsupialia: Didelphidae): analysis of the influence of ecological factors and phylogenetic legacy

The diversity of items consumed by modern didelphids, varying from mostly fruits in Caluromys Allen to mostly small vertebrates in Lutreolina O. Thomas, may cause changes in molar size and shape. We evaluated the morphometric variation of the first and third upper and lower molars of 16 genera of didelphid marsupials, with the aim of assessing the relationship between molar shape change, diet and phylogeny. We used a geometric morphometric approach to analyse how shape changes with diet. We mapped shape onto the phylogeny of the group to reconstruct ancestral states and analyse the evolution of molar shape. Finally, we statistically estimated the effect of size, diet and phylogeny on molar shape. All the analyses indicated little correlation between diet and molar shape and a strong correlation between the position of each genus on the phylogeny and molar shape. We believe that the wide ecological niche used by most of the groups (at least regarding diet) makes the evolutionary changes not strong enough to override pre‐existing differences that occur among clades, and the absence of highly diet‐specialized species (e.g. hypercarnivory or obligate folivory) causes the need for retaining a molar shape that can be useful to process different kinds of food items. © 2014 The Linnean Society of London     

The influence of feeding on the evolution of sensory signals: a comparative test of an evolutionary trade‐off between masticatory and sensory functions of skulls in southern African Horseshoe bats (Rhinolophidae)

The skulls of animals have to perform many functions. Optimization for one function may mean another function is less optimized, resulting in evolutionary trade‐offs. Here, we investigate whether a trade‐off exists between the masticatory and sensory functions of animal skulls using echolocating bats as model species. Several species of rhinolophid bats deviate from the allometric relationship between body size and echolocation frequency. Such deviation may be the result of selection for increased bite force, resulting in a decrease in snout length which could in turn lead to higher echolocation frequencies. If so, there should be a positive relationship between bite force and echolocation frequency. We investigated this relationship in several species of southern African rhinolophids using phylogenetically informed analyses of the allometry of their bite force and echolocation frequency and of the three‐dimensional shape of their skulls. As predicted, echolocation frequency was positively correlated with bite force, suggesting that its evolution is influenced by a trade‐off between the masticatory and sensory functions of the skull. In support of this, variation in skull shape was explained by both echolocation frequency (80%) and bite force (20%). Furthermore, it appears that selection has acted on the nasal capsules, which have a frequency‐specific impedance matching function during vocalization. There was a negative correlation between echolocation frequency and capsule volume across species. Optimization of the masticatory function of the skull may have been achieved through changes in the shape of the mandible and associated musculature, elements not considered in this study.     

Relationships of song structure to phylogenetic history,habitat, and morphology in the vireos,greenlets, and allies (Passeriformes: Vireonidae)

Acoustic signals show immense variation among passerines, and several hypotheses have been proposed to explain this diversity. In this study, we tested, for the first time, the relationships of song structure to phylogeny, habitat type, and morphology in the vireos and allies (Vireonidae). Every measure of song structure considered in this study had moderate and significant phylogenetic signal. Furthermore, two song-constraining morphological traits, bill shape and body mass, also exhibited significant phylogenetic signal. Song length showed the largest within-clade similarity; longer songs were highly conserved in part of the greenlet (Hylophilus) clade, whereas shorter songs characterized the remaining seven genera. We found no differences in song structure among vireonids living in different habitat types. However, vireonids with shorter, stouter bills and larger bodies sang songs with lower minimum and maximum peak frequency, compared with species with longer, thinner bills and smaller bodies. We conclude that Vireonidae song evolution is driven partially by phylogenetically conserved morphological traits. Our findings support the phylogenetic signal and morphological constraints hypotheses explaining structural diversity in avian acoustic signals.     

Three‐dimensional reconstruction of the odontophoral cartilages of Caenogastropoda (Mollusca: Gastropoda) using micro‐CT: Morphology and phylogenetic significance

Odontophoral cartilages are located in the molluscan buccal mass and support the movement of the radula during feeding. The structural diversity of odontophoral cartilages is currently known only from limited taxa, but this information is important for interpreting phylogeny and for understanding the biomechanical operation of the buccal mass. Caenogastropods exhibit a wide variety of feeding strategies, but there is little comparative information on cartilage morphology within this group. The morphology of caenogastropod odontophoral cartilages is currently known only from dissection and histology, although preliminary results suggest that they may be structurally diverse. A comparative morphological survey of 18 caenogastropods and three noncaenogastropods has been conducted, sampling most major caenogastropod superfamilies. Three‐dimensional models of the odontophoral cartilages were generated using X‐ray microscopy (micro‐CT) and reconstruction by image segmentation. Considerable morphological diversity of the odontophoral cartilages was found within Caenogastropoda, including the presence of thin cartilaginous appendages, asymmetrically overlapping cartilages, and reflexed cartilage margins. Many basal caenogastropod taxa possess previously unidentified cartilaginous support structures below the radula (subradular cartilages), which may be homologous to the dorsal cartilages of other gastropods. As subradular cartilages were absent in carnivorous caenogastropods, adaptation to trophic specialization is likely. However, incongruence with specific feeding strategies or body size suggests that the morphology of odontophoral cartilages is constrained by phylogeny, representing a new source of morphological characters to improve the phylogenetic resolution of this group. J. Morphol. 2009. © 2008 Wiley‐Liss, Inc.     

Ecogeographical patterns of morphological variation in pygmy shrews Sorex minutus (Soricomorpha: Soricinae) within a phylogeographical and continental‐and‐island framework

Ecogeographical patterns of morphological variation were studied in the Eurasian pygmy shrew Sorex minutus aiming to understand the species’ morphological diversity in a continental and island setting, and within the context of previous detailed phylogeographical studies. In total, 568 mandibles and 377 skulls of S. minutus from continental and island populations from Europe and Atlantic islands were examined using a geometric morphometrics approach, and the general relationships of mandible and skull size and shape with geographical and environmental variables were studied. Samples were then pooled into predefined geographical groups to evaluate the morphological differences among them using analyses of variance, aiming to contrast the morphological and genetic relationships based on morphological and genetic distances and ancestral state reconstructions, as well as assess the correlations of morphological, genetic, and geographical distances with Mantel tests. We found significant relationships of mandible size with geographical and environmental variables, fitting the converse Bergmann's rule; however, for skull size, this was less evident. Continental groups of S. minutus could not readily be differentiated from each other by shape. Most island groups of S. minutus were easily discriminated from the continental groups by being larger, indicative of an island effect. Moreover, morphological and genetic distances differed substantially and, again, island groups were distinctive morphologically. Morphological and geographical distances were significantly correlated, although this was not the case for morphological and genetic distances, indicating that morphological variation does not reflect genetic subdivision in S. minutus. Our analyses showed that environmental variables and insularity had important effects on the morphological differentiation of S. minutus.