Phylogenetic Comparative Approach Research Articles

Adaptation and innovation in darter fish cranial musculature (Etheostomatinae: Percidae): insights from diceCT

Abstract Fish skulls are often highly kinetic, with multiple linkage and lever systems powered by a diverse suite of muscles. Comparative analysis of the evolution of soft-tissue structures in the fish skull is often limited under traditional approaches, while new imaging techniques like diceCT (diffusible iodine-based contrast-enhanced computed tomography) allow for high-resolution imaging of muscles in situ. Darters (Percidae: Etheostomatinae) are a diminutive and species-rich clade of lotic freshwater fishes, which show diverse head shapes believed to be associated with different foraging strategies. We used diceCT to sample all major cranial adductors and abductors responsible for movement of the jaw, hyoid, operculum, and suspensorium from 29 species. We applied comparative phylogenetic approaches to analyse the evolutionary trends in muscle size across the clade. We found two major patterns: (i) darter cranial muscles show fundamental trade-offs relating to investment in musculature, as well as buccal expansion vs. biting attributes; early divergence in muscle size appears to be associated with shifts in habitat use and foraging; (ii) darter adductor mandibulae show high variation in architecture (fibre orientation, divisions). This study highlights how new imaging techniques can provide novel insights into the anatomy of even well-sampled/represented clades.

Biophysics of protist behaviour

Protists, an umbrella term first coined by Ernst Haeckel in 1866, are a vast collection of (primarily unicellular) eukaryotes that are "neither animals nor plants". This basic definition by exclusion has been exercised for centuries, even though recent advances have led to more rigorous taxonomic assignment of various protist groups. Pioneering comparative phylogenetic approaches have been applied to these organisms to reconstruct the deep branches of the eukaryotic tree, revealing essential clues about early eukaryotic evolution. Protists, including amoebae, flagellates, ciliates, and algae, are also vital constituents of global ecosystems, where they appear at the base of food chains, control the relative abundance of other microbes, and participate in global biogeochemical recycling. Due to their typically small size and lack of nervous systems, protists are often associated with the unfortunate label 'primitive'. Yet they exhibit remarkable behavioural sophistication and are able to feed, predate, navigate and interact with their surroundings. Unlike macroscopic animals, many protists reside in a non-intuitive physical regime where viscous forces dominate over inertia, and where they use diverse propulsion and navigation strategies. Interdisciplinary research into these cell-scale phenomena, characterised by a complex interplay of physical forces and mechanical constraints, has significantly advanced the emerging fields of active matter, microhydrodynamics, and non-equilibrium statistical physics. This primer discusses the biophysics of protist behaviour, with a focus on locomotion and feeding. I will highlight the most extensively studied principles and describe some more esoteric behaviours that have not yet been systematically explored.

Genome-wide patterns of selection-drift variation strongly associate with organismal traits across the green plant lineage.

There are many gaps in our knowledge of how life cycle variation and organismal body architecture associate with molecular evolution. Using the diverse range of green algal body architectures and life cycle types as a test case, we hypothesize that increases in cytomorphological complexity are likely to be associated with a decrease in the effective population size, because larger-bodied organisms typically have smaller populations, resulting in increased drift. For life cycles, we expect haploid-dominant lineages to evolve under stronger selection intensity relative to diploid-dominant life cycles owing to masking of deleterious alleles in heterozygotes. We use a genome-scale data set spanning the phylogenetic diversity of green algae and phylogenetic comparative approaches to measure the relative selection intensity across different trait categories. We show stronger signatures of drift in lineages with more complex body architectures compared with unicellular lineages, which we consider to be a consequence of smaller effective population sizes of the more complex algae. Significantly higher rates of synonymous as well as nonsynonymous substitutions relative to other algal body architectures highlight that siphonous and siphonocladous body architectures, characteristic of many green seaweeds, form an interesting test case to study the potential impacts of genome redundancy on molecular evolution. Contrary to expectations, we show that levels of selection efficacy do not show a strong association with life cycle types in green algae. Taken together, our results underline the prominent impact of body architecture on the molecular evolution of green algal genomes.

Spider silk tensile performance does not correlate with web use.

Spider silk is amongst the toughest materials produced by living systems, but its tensile performance varies considerably between species. Despite the extensive sampling of the material properties and composition of dragline silk, the understanding why some silks perform better than others is still limited. Here, I adopted a phylogenetic comparative approach to re-analyse structural and mechanical data from the Silkome database and the literature across 164 species to (a) provide an extended model of silk property evolution, (b) test for correlations between structural and mechanical properties, and (c) to test if silk tensile performance differs between web-building and non-web-building species. Unlike the common notion that orb-weavers have evolved the best performing silks, outstanding tensile properties were found both in and outside the araneoid clade. Phylogenetic linear models indicated that the mechanical and structural properties of spider draglines poorly correlate, but silk strength and toughness correlated better with birefringence (an indicator of the material anisotropy) than crystallinity. Furthermore, in contrast to previous ideas, silk tensile performance did not differ between ecological guilds. These findings indicate multiple unknown pathways towards the evolution of spider silk tensile super-performance, calling for a better integration of non-orb-weaving spiders in spider silk studies.

The evolution of cultural diversity in Pama-Nyungan Australia

Explaining the processes that produce cultural diversity has long been a focus of anthropological study. Whilst linguistic diversity has frequently been shown to be associated with population splitting during migrations, much less is known about the influence of migration patterns on other forms of culture. Here we compare variation between different cultural types to understand whether their diversity might also be a consequence of historic population change. For our case study, we developed a dataset of 90 cultural traits recorded across around 100 societies in Pama-Nyungan Australia focusing on three cultural forms important in Australian life: adolescent initiation, mortuary practice and rock art motifs. Trait diversity was analysed using phylogenetic methods and compared with language diversity since previous studies have suggested an association between Pama-Nyungan language evolution and population dispersal. We were able to demonstrate a strong association between the phylogenetic history of language and initiation ritual but not between language and mortuary ritual or rock motifs. The finding suggests population migration may have had a significant influence on initiation ritual diversity. It also lends support to the idea, as suggested by some authors, that the development of complex initiation practices may have played an active role in the dispersal of Pama-Nyungan speakers throughout the Australian continent. Our study results suggest that the type of phylogenetic comparative approach applied here can make an important contribution to the reconstruction of deep cultural history over long timescales.

Convergent evolution of giant size in eurypterids.

Eurypterids-Palaeozoic marine and freshwater arthropods commonly known as sea scorpions-repeatedly evolved to remarkable sizes (over 0.5 m in length) and colonized continental aquatic habitats multiple times. We compiled data on the majority of eurypterid species and explored several previously proposed explanations for the evolution of giant size in the group, including the potential role of habitat, sea surface temperature and dissolved sea surface oxygen levels, using a phylogenetic comparative approach with a new tip-dated tree. There is no compelling evidence that the evolution of giant size was driven by temperature or oxygen levels, nor that it was coupled with the invasion of continental aquatic environments, latitude or local faunal diversity. Eurypterid body size evolution is best characterized by rapid bursts of change that occurred independently of habitat or environmental conditions. Intrinsic factors played a major role in determining the convergent origin of gigantism in eurypterids.

Integrating phylogenies into single-cell RNA sequencing analysis allows comparisons across species, genes, and cells.

Comparisons of single-cell RNA sequencing (scRNA-seq) data across species can reveal links between cellular gene expression and the evolution of cell functions, features, and phenotypes. These comparisons evoke evolutionary histories, as depicted by phylogenetic trees, that define relationships between species, genes, and cells. This Essay considers each of these in turn, laying out challenges and solutions derived from a phylogenetic comparative approach and relating these solutions to previously proposed methods for the pairwise alignment of cellular dimensional maps. This Essay contends that species trees, gene trees, cell phylogenies, and cell lineages can all be reconciled as descriptions of the same concept-the tree of cellular life. By integrating phylogenetic approaches into scRNA-seq analyses, challenges for building informed comparisons across species can be overcome, and hypotheses about gene and cell evolution can be robustly tested.

Palms of the past: can morphometric phytolith analysis inform deep time evolution and palaeoecology of Arecaceae?

Palm fossils are often used as evidence for warm and wet palaeoenvironments, reflecting the affinities of most modern palms. However, several extant palm lineages tolerate cool and/or arid climates, making a clear understanding of the taxonomic composition of ancient palm communities important for reliable palaeoenvironmental inference. However, taxonomically identifiable palm fossils are rare and often confined to specific facies. Although the resolution of taxonomic information they provide remains unclear, phytoliths (microscopic silica bodies) provide a possible solution because of their high preservation potential under conditions where other plant fossils are scarce. We thus evaluate the taxonomic and palaeoenvironmental utility of palm phytoliths. We quantified phytolith morphology of 97 modern palm and other monocot species. Using this dataset, we tested the ability of five common discriminant methods to identify nine major palm clades. We then compiled a dataset of species' climate preferences and tested if they were correlated with phytolith morphology using a phylogenetic comparative approach. Finally, we reconstructed palm communities and palaeoenvironmental conditions at six fossil sites. Best-performing models correctly identified phytoliths to their clade of origin only 59 % of the time. Although palms were generally distinguished from non-palms, few palm clades were highly distinct, and phytolith morphology was weakly correlated with species' environmental preferences. Reconstructions at all fossil sites suggested that palm communities were dominated by Trachycarpeae and Areceae, with warm, equable climates and high, potentially seasonal rainfall. However, fossil site reconstructions had high uncertainty and often conflicted with other climate proxies. While phytolith morphology provides some distinction among palm clades, caution is warranted. Unlike prior spatially restricted studies, our geographically and phylogenetically broad study indicates phytolith morphology may not reliably differentiate most palm taxa in deep time. Nevertheless, it reveals distinct clades, including some likely to be palaeoenvironmentally informative.

Ecological Predictors of Organelle Genome Evolution: Phylogenetic Correlations with Taxonomically Broad, Sparse, Unsystematized Data.

Comparative analysis of variables across phylogenetically linked observations can reveal mechanisms and insights in evolutionary biology. As the taxonomic breadth of the sample of interest increases, challenges of data sparsity, poor phylogenetic resolution, and complicated evolutionary dynamics emerge. Here, we investigate a cross-eukaryotic question where all these problems exist: which organismal ecology features are correlated with gene retention in mitochondrial and chloroplast DNA (organelle DNA or oDNA). Through a wide palette of synthetic control studies, we first characterize the specificity and sensitivity of a collection of parametric and non-parametric phylogenetic comparative approaches to identify relationships in the face of such sparse and awkward datasets. This analysis is not directly focused on oDNA, and so provides generalizable insights into comparative approaches with challenging data. We then combine and curate ecological data coupled to oDNA genome information across eukaryotes, including a new semi-automated approach for gathering data on organismal traits from less systematized open-access resources including encyclopedia articles on species and taxa. The curation process also involved resolving several issues with existing datasets, including enforcing the clade-specificity of several ecological features and fixing incorrect annotations. Combining this unique dataset with our benchmarked comparative approaches, we confirm support for several known links between organismal ecology and organelle gene retention, identify several previously unidentified relationships constituting possible ecological contributors to oDNA genome evolution, and provide support for a recently hypothesized link between environmental demand and oDNA retention. We, with caution, discuss the implications of these findings for organelle evolution and of this pipeline for broad comparative analyses in other fields.

Accurate Detection of Convergent Mutations in Large Protein Alignments With ConDor.

Evolutionary convergences are observed at all levels, from phenotype to DNA and protein sequences, and changes at these different levels tend to be correlated. Notably, convergent mutations can lead to convergent changes in phenotype, such as changes in metabolism, drug resistance, and other adaptations to changing environments. We propose a two-component approach to detect mutations subject to convergent evolution in protein alignments. The "Emergence" component selects mutations that emerge more often than expected, while the "Correlation" component selects mutations that correlate with the convergent phenotype under study. With regard to Emergence, a phylogeny deduced from the alignment is provided by the user and is used to simulate the evolution of each alignment position. These simulations allow us to estimate the expected number of mutations in a neutral model, which is compared to the observed number of mutations in the data studied. In Correlation, a comparative phylogenetic approach, is used to measure whether the presence of each of the observed mutations is correlated with the convergent phenotype. Each component can be used on its own, for example Emergence when no phenotype is available. Our method is implemented in a standalone workflow and a webserver, called ConDor. We evaluate the properties of ConDor using simulated data, and we apply it to three real datasets: sedge PEPC proteins, HIV reverse transcriptase, and fish rhodopsin. The results show that the two components of ConDor complement each other, with an overall accuracy that compares favorably to other available tools, especially on large datasets.

Exploring the relationship between environment and brain morphology in anurans: a comparative phylogenetic approach

Abstract The correlation of ecological and behavioural characteristics with morphological features of brain structures has been recurrently corroborated for different vertebrate taxa, such as mammals, birds, and fishes. Here, we asked whether such correlations can also be detected in amphibians, an animal group of great interest due to its great behavioural and ecological diversity. We analysed the shape of two brain regions, cerebellum and optic tectum, of 67 species from 32 families of the order Anura (frogs and toads), applying 2D geometric morphometrics in a phylogenetic context. We found species that share similar environments and daily patterns have a similarly shaped cerebellum and optic tectum, with diurnal and arboreal species with more varied brain shapes. We did not find a correlation between activity mode or lifestyle with shape for both brain regions; however, we found that shared ancestry had an influence in their evolution. Future studies including a higher number of species from each ecological group, and from more families, would help to better clarify whether the patterns found here are a general rule for anurans.

The evolution of positive allometry and exaggerated traits in a diverse beetle clade

Abstract Understanding variation in trait allometry and the drivers of variation in sexually selected traits is a major theme in evolutionary biology. The static allometries of sexually selected traits are often positive due to disproportional investment by large individuals into trait size. Comparative studies of weapon allometry are rare, and typically focus on variation along a single dimension (e.g. length), despite weapons often diverging dramatically in shape. Furthermore, studies predominantly focus on groups where weapons have evolved as novel structures (e.g. horns), despite most weapons arising from modifications of existing structures. We test the hypothesis that sexual selection drives variation in static allometry using a phylogenetic comparative approach with 59 brentine weevil species showing remarkable variation in trait exaggeration and sexual size dimorphism (SSD). Contrasting two dimensions of head size exaggeration (a sexually selected trait) and elytron length (a nonsexual trait), positive static allometry was common across both measures of male head size, but not females, and there was positive covariance between male head static allometry and SSD across species. Positive static allometry for male elytron length was less common, but did show positive covariance with SSD, possibly due to the function of wings as compensatory structures for bearing large weapons. Our findings support the hypothesis that sexual selection drives the evolution of allometries both within and among species, and that the modification of existing structures to be used as weapons does not limit the potential for positive static allometry. Read the free Plain Language Summary for this article on the Journal blog.

Phylogeny, Ecology, and Gene Families Covariation Shaped the Olfactory Subgenome of Rodents.

Olfactory receptor (OR) genes represent the largest multigenic family in mammalian genomes and encode proteins that bind environmental odorant molecules. The OR repertoire is extremely variable among species and is subject to many gene duplications and losses, which have been linked to ecological adaptations in mammals. Although they have been studied on a broad taxonomic scale (i.e., placental), finer sampling has rarely been explored in order to better capture the mechanisms that drove the evolution of the OR repertoire. Among placental mammals, rodents are well-suited for this task, as they exhibit diverse life history traits, and genomic data are available for most major families and a diverse array of lifestyles. In this study, 53 rodent published genomes were mined for their OR subgenomes. We retrieved more than 85,000 functional and pseudogene OR sequences that were subsequently classified into phylogenetic clusters. Copy number variation among rodents is similar to that of other mammals. Using our OR counts along with comparative phylogenetic approaches, we demonstrated that ecological niches such as diet, period of activity, and a fossorial lifestyle strongly impacted the proportion of OR pseudogenes. Within the OR subgenome, phylogenetic inertia was the main factor explaining the relative variations of the 13 OR gene families. However, a striking exception was a convergent 10-fold expansion of the OR family 14 among the phylogenetically divergent subterranean mole-rat lineages belonging to Bathyergidae and Spalacidae families. This study illustrates how the diversity of the OR repertoire has evolved among rodents, both shaped by selective forces stemming from species life history traits and neutral evolution along the rodent phylogeny.

Lack of evidence for coevolution between oxytocin receptor N-terminal variants and monogamy in placental mammals

Oxytocin (OXT) is a neurohypophyseal hormone that influences a wide range of affiliative behaviors, such as pair-bonding and infant care, across mammals. The effects of OXT depend significantly on an adequate interaction with its receptor, OXTR. OXTR belongs to the G-protein coupled receptor family. The extracellular N-terminal domain of OXTR interacts with the linear C-terminal tail of OXT and is required for OXT binding. Across mammalian species there is a genetic diversity in OXTR terminal sequence. Previous work on primates has shown an association between OXTR phylogeny and monogamy. However, it is not clear whether this variation coevolved with either mating system (monogamy) or infant care behaviors (such as allomaternal care). Here, we take a phylogenetic comparative and evolutionary modeling approach across a wide range of placental mammals (n = 60) to test whether OXTR N-terminal variants co-evolved with either monogamy or allomaternal care behaviors. Our results indicate that the diversity in OXTR N-terminal region is unlikely to provide the underlying genetic bases for variation in mating system and/or allomaternal behavior as we find no evidence for co-evolution between protein sequence and affiliative behaviors. Hence, the role played by OXT in influencing affiliative behaviors is unlikely to be mediated by the genetic diversity of its receptor.

Evolution End Classification of tfd Gene Clusters Mediating Bacterial Degradation of 2,4-Dichlorophenoxyacetic Acid (2,4-D).

The tfd (tfdI and tfdII) are gene clusters originally discovered in plasmid pJP4 which are involved in the bacterial degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) via the ortho-cleavage pathway of chlorinated catechols. They share this activity, with respect to substituted catechols, with clusters tcb and clc. Although great effort has been devoted over nearly forty years to exploring the structural diversity of these clusters, their evolution has been poorly resolved to date, and their classification is clearly obsolete. Employing comparative genomic and phylogenetic approaches has revealed that all tfd clusters can be classified as one of four different types. The following four-type classification and new nomenclature are proposed: tfdI, tfdII, tfdIII and tfdIV(A,B,C). Horizontal gene transfer between Burkholderiales and Sphingomonadales provides phenomenal linkage between tfdI, tfdII, tfdIII and tfdIV type clusters and their mosaic nature. It is hypothesized that the evolution of tfd gene clusters proceeded within first (tcb, clc and tfdI), second (tfdII and tfdIII) and third (tfdIV(A,B,C)) evolutionary lineages, in each of which, the genes were clustered in specific combinations. Their clustering is discussed through the prism of hot spots and driving forces of various models, theories, and hypotheses of cluster and operon formation. Two hypotheses about series of gene deletions and displacements are also proposed to explain the structural variations across members of clusters tfdII and tfdIII, respectively. Taking everything into account, these findings reconstruct the phylogeny of tfd clusters, have delineated their evolutionary trajectories, and allow the contribution of various evolutionary processes to be assessed.

Physiological cold tolerance evolves faster than climatic niches in plants.

Understanding how plants respond to thermal stress is central to predicting plant responses and community dynamics in natural ecosystems under projected scenarios of climate change. Although physiological tolerance is suggested to evolve slower than climatic niches, this comparison remains to be addressed in plants using a phylogenetic comparative approach. In this study, we compared i) the evolutionary rates of physiological tolerance to extreme temperatures with ii) the corresponding rates of climatic niche across three major vascular plant groups. We further accounted for the potential effects of hardening when examining the association between physiological and climatic niche rates. We found that physiological cold tolerance evolves faster than heat tolerance in all three groups. The coldest climatic-niche temperatures evolve faster than the warmest climatic-niche temperatures. Importantly, evolutionary rates of physiological cold tolerance were faster than rates of change in climatic niches. However, an inverse association between physiological cold tolerance and responding climatic niche for plants without hardening was detected. Our results indicated that plants may be sensitive to changes in warmer temperatures due to the slower evolutionary rates of heat tolerance. This pattern has deep implications for the framework that is being used to estimate climate-related extinctions over the upcoming century.

Ecological correlates of cranial evolution in the megaradiation of dipsadine snakes

BackgroundDipsadine snakes represent one of the most spectacular vertebrate radiations that have occurred in any continental setting, with over 800 species in South and Central America. Their species richness is paralleled by stunning ecological diversity, ranging from arboreal snail-eating and aquatic eel-eating specialists to terrestrial generalists. Despite the ecological importance of this clade, little is known about the extent to which ecological specialization shapes broader patterns of phenotypic diversity within the group. Here, we test how habitat use and diet have influenced morphological diversification in skull shape across 160 dipsadine species using micro-CT and 3-D geometric morphometrics, and we use a phylogenetic comparative approach to test the contributions of habitat use and diet composition to variation in skull shape among species.ResultsWe demonstrate that while both habitat use and diet are significant predictors of shape in many regions of the skull, habitat use significantly predicts shape in a greater number of skull regions when compared to diet. We also find that across ecological groupings, fossorial and aquatic behaviors result in the strongest deviations in morphospace for several skull regions. We use simulations to address the robustness of our results and describe statistical anomalies that can arise from the application of phylogenetic generalized least squares to complex shape data.ConclusionsBoth habitat and dietary ecology are significantly correlated with skull shape in dipsadines; the strongest relationships involved skull shape in snakes with aquatic and fossorial lifestyles. This association between skull morphology and multiple ecological axes is consistent with a classic model of adaptive radiation and suggests that ecological factors were an important component in driving morphological diversification in the dipsadine megaradiation.

Coevolutionary maintenance of forked tails and song in hirundines (Aves: Hirundininae)

Both conspicuous plumage ornamentation and song are well‐known examples of sexually selected traits, but their interrelationship is not well known, perhaps in part because of confounding factors, including interspecific variation in ecology, habitat, morphology, and type of ornamentation. Here, using a phylogenetic comparative approach and the 69 species with available information (i.e. 96% of all 72 species), I examined the evolutionary relationship between forked tails and the presence/absence of song in hirundines (Aves: Hirundininae). Hirundines have similar ecology (e.g. aerial insectivores, social monogamy, and biparental provisioning), morphology (e.g. syrinx with nearly complete bronchial rings), and plumage ornamentation (i.e. a sexually selected forked tail), which provides a unique opportunity to examine the evolutionary associations between plumage ornamentation and song. In particular, hirundines have repeatedly lost their ornamentation, forked tails, setting up a condition to test their association with the evolutionary gain/loss of their simple song. After controlling for phylogeny and covariates, I demonstrated that song was less likely to be found in species with forkless tails than in species with forked tails. Two correlates of tail shape, sexual dimorphism in the overall plumage characteristics as a well‐known proxy of sexual selection and incubation type as a measure of extrapair mating opportunity, were not positively related to the presence or absence of song, indicating the importance of forked tails, rather than their correlates. The analysis of the correlated evolution of discrete characters further supported the correlated evolution of the two traits, in which forked tails and song are maintained together and less likely to be lost under the presence of each other. The current study provided macroevolutionary support for the integrated use of visual and acoustic courtship traits.

Functional Trait Variation Along Animal Invasion Pathways

Functional trait–based mediation of animal invasions is only now developing, yet it is already showing as much promise as the approach has for plant invasion biology. Here, we provide a theory-founded examination of functional trait–based ecology with respect to animal invasions, together with a review of the empirical research. Recent developments in the scaling of traits to ecosystems, along with the frameworks for invasion ecology, provide a powerful foundation for investigations. Despite growing empirical research in this field, interspecific comparisons incorporating comparative phylogenetic approaches remain uncommon, and those examining assemblages are even more so. Despite the importance of time since introduction, it is rarely included in studies. Broad environmental tolerances, life history traits associated with fast growth or greater reproduction, and in some cases, greater phenotypic plasticity of traits, mediate the success of animal alien species at various stages of invasion, although introduction effort remains one of the most important factors affecting success.

Brain size and shape diversification in a highly diverse South American clade of rodents (Echimyidae): a geometric morphometric and comparative phylogenetic approach

Abstract Brain morphological variation is analysed through virtual endocasts in a highly diversified clade of caviomorph rodents belonging to the family Echimyidae. Diversification in brain size and shape is explored through geometric morphometrics and comparative phylogenetic analyses. The results indicate that brain shape is largely independent of general size and reveal different trends in brain size and shape. Fossorial Euryzygomatominae, arboreal Echimyini and the semi-aquatic Myocastorini Myocastor show high encephalization; the former with a greater contribution from the olfactory bulb and petrosal lobe, and the latter two with a larger surface area of neocortex. The Euryzygomatomyinae and Myocastorini of terrestrial habits show low encephalization with a low contribution of the neocortex. Phylogenetic comparative analyses suggest that endocranial morphological evolution would have been influenced by both phylogeny and locomotor habits. The concurrence of the best fit of the Ornstein–Uhlenbeck model and the significant phylogenetic signal in the datasets suggests the involvement of constraints on morphological diversification within the major clades, as expected under phylogenetic conservatism. This could be explained by an early establishment of a particular endocranial morphology in each major clade, which would have been maintained with relatively little change.