Phylogenetic organization in the assimilation of chemically distinct substrates by soil bacteria

Soils are among the most biodiverse habitats on earth and while the species composition of microbial communities can influence decomposition rates and pathways, the functional significance of many microbial species and phylogenetic groups remains unknown. If bacteria exhibit phylogenetic organization in their function, this could enable ecologically meaningful classification of bacterial clades. Here, we show non-random phylogenetic organization in the rates of relative carbon assimilation for both rapidly mineralized substrates (amino acids and glucose) assimilated by many microbial taxa and slowly mineralized substrates (lipids and cellulose) assimilated by relatively few microbial taxa. When mapped onto bacterial phylogeny using ancestral character estimation this phylogenetic organization enabled the identification of clades involved in the decomposition of specific soil organic matter substrates. Phylogenetic organization in substrate assimilation could provide a basis for predicting the functional attributes of uncharacterized microbial taxa and understanding the significance of microbial community composition for soil organic matter decomposition.     

Phylogenetic turnover during subtropical forest succession across environmental and phylogenetic scales

Although spatial and temporal patterns of phylogenetic community structure during succession are inherently interlinked and assembly processes vary with environmental and phylogenetic scales, successional studies of community assembly have yet to integrate spatial and temporal components of community structure, while accounting for scaling issues. To gain insight into the processes that generate biodiversity after disturbance, we combine analyses of spatial and temporal phylogenetic turnover across phylogenetic scales, accounting for covariation with environmental differences. We compared phylogenetic turnover, at the species‐ and individual‐level, within and between five successional stages, representing woody plant communities in a subtropical forest chronosequence. We decomposed turnover at different phylogenetic depths and assessed its covariation with between‐plot abiotic differences. Phylogenetic turnover between stages was low relative to species turnover and was not explained by abiotic differences. However, within the late‐successional stages, there was high presence‐/absence‐based turnover (clustering) that occurred deep in the phylogeny and covaried with environmental differentiation. Our results support a deterministic model of community assembly where (i) phylogenetic composition is constrained through successional time, but (ii) toward late succession, species sorting into preferred habitats according to niche traits that are conserved deep in phylogeny, becomes increasingly important.     

The phylogenetic diversity of metagenomes

Phylogenetic diversity--patterns of phylogenetic relatedness among organisms in ecological communities--provides important insights into the mechanisms underlying community assembly. Studies that measure phylogenetic diversity in microbial communities have primarily been limited to a single marker gene approach, using the small subunit of the rRNA gene (SSU-rRNA) to quantify phylogenetic relationships among microbial taxa. In this study, we present an approach for inferring phylogenetic relationships among microorganisms based on the random metagenomic sequencing of DNA fragments. To overcome challenges caused by the fragmentary nature of metagenomic data, we leveraged fully sequenced bacterial genomes as a scaffold to enable inference of phylogenetic relationships among metagenomic sequences from multiple phylogenetic marker gene families. The resulting metagenomic phylogeny can be used to quantify the phylogenetic diversity of microbial communities based on metagenomic data sets. We applied this method to understand patterns of microbial phylogenetic diversity and community assembly along an oceanic depth gradient, and compared our findings to previous studies of this gradient using SSU-rRNA gene and metagenomic analyses. Bacterial phylogenetic diversity was highest at intermediate depths beneath the ocean surface, whereas taxonomic diversity (diversity measured by binning sequences into taxonomically similar groups) showed no relationship with depth. Phylogenetic diversity estimates based on the SSU-rRNA gene and the multi-gene metagenomic phylogeny were broadly concordant, suggesting that our approach will be applicable to other metagenomic data sets for which corresponding SSU-rRNA gene sequences are unavailable. Our approach opens up the possibility of using metagenomic data to study microbial diversity in a phylogenetic context.     

Phylogenetic relationships of the Acanthocephala inferred from 18S ribosomal DNA sequences

Phylogenetic relationships within the Acanthocephala have remained unresolved. Past systematic efforts have focused on creating classifications with little consideration of phylogenetic methods. The Acanthocephala are currently divided into three major taxonomic groups: Archiacanthocephala, Palaeacanthocephala, and Eoacanthocephala. These groups are characterized by structural features in addition to the taxonomy and habitat of hosts parasitized. In this study the phylogenetic relationships of 11 acanthocephalan species are examined with 18S rDNA sequences. Maximum parsimony, minimum evolution, and maximum likelihood methods are used to estimate phylogenetic relationships. Within the context of sampled taxa, all phylogenetic analyses are consistent with monophyly of the major taxonomic groups of the Acanthocephala, suggesting that the current higher order classification is natural. The molecular phylogeny is used to examine patterns of character evolution for various structural and ecological characteristics of the Acanthocephala. Arthropod intermediate host distributions, when mapped on the phylogeny, are consistent with monophyletic groups of acanthocephalans. Vertebrate definitive host distributions among the Acanthocephala display independent radiations into similar hosts. Levels of uncorrected sequence divergence among acanthocephalans are high; however, relative-rate tests indicate significant departure from rate uniformity among acanthocephalans, arthropods, and vertebrates. This precludes comparison of 18S divergence levels to assess the relative age of the Acanthocephala. However, other evidence suggests an ancient origin of the acanthocephalan-arthropod parasitic association.     

Phylogenetic systematics of the colorful, cyanide-producing millipedes of Appalachia (Polydesmida, Xystodesmidae, Apheloriini) using a total evidence Bayesian approach

Here, we provide an exemplar-approach phylogeny of the xystodesmid millipede tribe Apheloriini with a focus on genus-group relationships-particularly of the genus Brachoria. Exemplars for the phylogenetic analysis were chosen to represent the maximum breadth of morphological diversity within all nominal genera in the tribe Apheloriini, and to broadly sample the genus Brachoria. In addition, three closely related tribes were used (Rhysodesmini, Nannariini, and Pachydesmini). Morphological and DNA sequence data were scored for Bayesian inference of phylogeny. Phylogenetic analysis resulted in polyphyletic genera Brachoria and Sigmoria, a monophyletic Apheloriini, and a "southern clade" that contains most of the tribal species diversity. We used this phylogeny to track morphological character histories and reconstruct ancestral states using stochastic character mapping. Based on the findings from the character mapping study, the diagnostic feature of the genus Brachoria, the cingulum, evolved independently in two lineages. We compared our phylogeny against prior classifications using Bayes factor hypothesis-testing and found that our phylogenetic hypothesis is inconsistent with the previous hypotheses underlying the most recent classification. With our preferred total-evidence phylogeny as a framework for taxonomic modifications, we describe a new genus, Appalachioria; supply phylogenetic diagnoses of monophyletic taxa; and provide a phylogeny-based classification for the tribe Apheloriini.     

Phylogenetic relationships of Steinernema Travassos, 1927 (Nematoda: Cephalobina: Steinernematidae) based on nuclear, mitochondrial and morphological data

Entomopathogenic nematodes of the genus Steinernema are lethal parasites of insects that are used as biological control agents of several lepidopteran, dipteran and coleopteran pests. Phylogenetic relationships among 25 Steinernema species were estimated using nucleotide sequences from three genes and 22 morphological characters. Parsimony analysis of 28S (LSU) sequences yielded a well-resolved phylogenetic hypothesis with reliable bootstrap support for 13 clades. Parsimony analysis of mitochondrial DNA sequences (12S rDNA and cox 1 genes) yielded phylogenetic trees with a lower consistency index than for LSU sequences, and with fewer reliably supported clades. Combined phylogenetic analysis of the 3-gene dataset by parsimony and Bayesian methods yielded well-resolved and highly similar trees. Bayesian posterior probabilities were high for most clades; bootstrap (parsimony) support was reliable for approximately half of the internal nodes. Parsimony analysis of the morphological dataset yielded a poorly resolved tree, whereas total evidence analysis (molecular plus morphological data) yielded a phylogenetic hypothesis consistent with, but less resolved than trees inferred from combined molecular data. Parsimony mapping of morphological characters on the 3-gene trees showed that most structural features of steinernematids are highly homoplastic. The distribution of nematode foraging strategies on these trees predicts that S. hermaphroditum, S. diaprepesi and S. longicaudum (US isolate) have cruise forager behaviours.     

Phylogenetic diversity and its conservation in the presence of phylogenetic uncertainty: a case study of cladoceran communities in urban waterbodies

The need to protect and preserve biodiversity is a pressing issue and requires that conservation projects be based on solid foundations. Knowledge of species evolutionary history can serve as a tool to help guide conservation projects on the basis of evolutionary heritage. We used communities of Cladocera (Crustacea, Branchiopoda) in urban waterbodies to identify which sites should be prioritized for phylogenetic diversity conservation. Phylogenetic trees were inferred using DNA sequences from two mitochondrial genes. Furthermore, we also evaluated the consequences of phylogenetic uncertainty for identifying sites for conservation priority. Using results from Bayesian analyses, we considered the effect of uncertainty in the phylogenetic tree on phylogenetic diversity (PD) estimation. When phylogenetic uncertainty was taken into account, the conservation value of individual sites became uncertain and several potential comparisons between sites could not be supported. Consequently prioritization of one site over the other could not be defended in biodiversity conservation projects. Our study highlights the fact that accounting for phylogenetic uncertainty can alter the relative conservation priority of sites, as assessed by their phylogenetic diversity. Therefore, variability in the phylogenetic estimates should be consistently considered and integrated into estimates of phylogenetic diversity and conservation decisions to avoid making suboptimal choices.     

Escaping from the Felsenstein Zone by Detecting Long Branches in Phylogenetic Data

Long branches in a true phylogeny tend to disrupt hierarchical character covariation (phylogenetic signal) in the distribution of traits among organisms. The distortion of hierarchical structure in character-state matrices can lead to errors in the estimation of phylogenetic relationships and inconsistency of methods of phylogenetic inference. Examination of trees distorted by long-branch attraction will not reveal the identities of problematic taxa, in part because the distortion can mask long branches by reducing inferred branch lengths and through errors in branching order. Here we present a simple method for the detection of taxa whose placement in evolutionary trees is made difficult by the effects of long-branch attraction. The method is an extension of a tree-independent conceptual framework of phylogenetic data exploration (RASA). Taxa that are likely to attract are revealed because long branches leave distinct footprints in the distribution of character states among taxa, and these traces can be directly observed in the error structure of the RASA regression. Problematic taxa are identified using a new diagnostic plot called the taxon variance plot, in which the apparent cladistic and phenetic variances contributed by individual taxa are compared. The procedure for identifying long edges employs algorithms solved in polynomial time and can be applied to morphological, molecular, and mixed characters. The efficacy of the method is demonstrated using simulated evolution and empirical evidence of long branches in a set of recently published sequences. We show that the accuracy of evolutionary trees can be improved by detecting and combating the potentially misleading influences of long-branch taxa.     

Phylogenetic trait conservation in the partner choice of a group of ectomycorrhizal trees

Ecological interactions are frequently conserved across evolutionary time. In the case of mutualisms, these conserved interactions may play a large role in structuring mutualist communities. We hypothesized that phylogenetic trait conservation could play a key role in determining patterns of association in the ectomycorrhizal symbiosis, a globally important trophic mutualism. We used the association between members of the pantropical plant tribe Pisonieae and its fungal mutualist partners as a model system to test the prediction that Pisonieae‐associating ectomycorrhizal fungi will be more closely related than expected by chance, reflecting a conserved trait. We tested this prediction using previously published and newly generated sequences in a Bayesian framework incorporating phylogenetic uncertainty. We report that phylogenetic trait conservation does exist in this association. We generated a five‐marker phylogeny of members of the Pisonieae and used this phylogeny in a Bayesian relaxed molecular clock analysis. We established that the most recent common ancestors of Pisonieae species and Pisonieae‐associating fungi sharing phylogenetic conservation of their patterns of ectomycorrhizal association occurred no more recently than 14.2 Ma. We therefore suggest that phylogenetic trait conservation in the Pisonieae ectomycorrhizal mutualism association represents an inherited syndrome which has existed for at least 14 Myr.     

Phylogenetic relationships of Steinernema Travassos, 1927 (Nematoda: Cephalobina: Steinernematidae) based on nuclear, mitochondrial and morphological data

Entomopathogenic nematodes of the genus Steinernema are lethal parasites of insects that are used as biological control agents of several lepidopteran, dipteran and coleopteran pests. Phylogenetic relationships among 25 Steinernema species were estimated using nucleotide sequences from three genes and 22 morphological characters. Parsimony analysis of 28S (LSU) sequences yielded a well-resolved phylogenetic hypothesis with reliable bootstrap support for 13 clades. Parsimony analysis of mitochondrial DNA sequences (12S rDNA and cox 1 genes) yielded phylogenetic trees with a lower consistency index than for LSU sequences, and with fewer reliably supported clades. Combined phylogenetic analysis of the 3-gene dataset by parsimony and Bayesian methods yielded well-resolved and highly similar trees. Bayesian posterior probabilities were high for most clades; bootstrap (parsimony) support was reliable for approximately half of the internal nodes. Parsimony analysis of the morphological dataset yielded a poorly resolved tree, whereas total evidence analysis (molecular plus morphological data) yielded a phylogenetic hypothesis consistent with, but less resolved than trees inferred from combined molecular data. Parsimony mapping of morphological characters on the 3-gene trees showed that most structural features of steinernematids are highly homoplastic. The distribution of nematode foraging strategies on these trees predicts that S. hermaphroditum, S. diaprepesi and S. longicaudum (US isolate) have cruise forager behaviours.     

Evolution of habitat preference in Clitellata (Annelida)

Clitellata (earthworms, leeches, and allies) is a clade of segmented annelid worms that comprise more than 5000 species found worldwide in many aquatic and terrestrial habitats. According to current views, the first clitellates were either aquatic (marine or freshwater) or terrestrial. To address this question further, we assessed the phylogenetic relationships among clitellates using parsimony, maximum likelihood and Bayesian analyses of 175 annelid 18S ribosomal DNA sequences. We then defined two ecological characters (Habitat and Aquatic‐environment preferences) and mapped those characters on the trees from the three analyses, using parsimony character‐state reconstruction (i.e. Fitch optimization). We accommodated phylogenetic uncertainty in the character mapping by reconstructing character evolution on all the trees resulting from parsimony and maximum likelihood bootstrap analyses and, in the Bayesian inference, on the trees sampled using the Markov chain Monte Carlo algorithm. Our analyses revealed that an ‘aquatic’ ancestral state for clitellates is a robust result. By using alterations of coding characters and constrained analyses, we also demonstrated that the hypothesis for a terrestrial origin of clitellates is not supported. Our analyses also suggest that the most recent ancestor of clitellates originated from a freshwater environment. However, we stress the importance of adding sequences of some rare marine taxa to more rigorously assess the freshwater origin of Clitellata. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95 , 447–464.     

Perils of paralogy: using HSP70 genes for inferring organismal phylogenies

Conserved genes have found their way into the mainstream of molecular systematics. Many of these genes are members of multigene families. A difficulty with using single genes of multigene families for phylogenetic inference is that genes from one species may be paralogous to those from another taxon. We focus attention on this problem using heat shock 70 (HSP70) genes. Using polymerase chain reaction techniques with genomic DNA, we isolated and sequenced 123 distinct sequences from 12 species of sharks. Phylogenetic analysis indicated that the sequences cluster with constituitively expressed cytoplasmic heat shock-like genes. Three highly divergent gene clades were sampled. A number of similar sequences were sampled from each species within each distinct gene clade. Comparison of published species trees with an HSP70 gene tree inferred using Bayesian phylogenetic analysis revealed several cases of gene duplication and differential sorting of gene lineages within this group of sharks. Gene tree parsimony based on the objective criteria of duplication and losses showed that previously published hypotheses of species relationships and two novel hypothesis based on Bayesian phylogenetics were concordant with the history of HSP70 gene duplication and loss. By contrast, two published hypotheses based on morphological data were not significantly different from the null hypothesis of a random association between species relatedness and the HSP70 gene tree. These results suggest that gene tree parsimony using data from multigene families can be used for inferring species relationships or testing published alternative hypotheses. More importantly, the results suggest that systematic studies relying on phylogenetic inferences from HSP70 genes may by plagued by unrecognized paralogy of sampled genes. Our results underscore the distinction between gene and species trees and highlight an underappreciated source of discordance between gene trees and organismal phylogeny, i.e., unrecognized paralogy of sampled genes.     

Construction of a Phylogenetic Tree of Photosynthetic Prokaryotes Based on Average Similarities of Whole Genome Sequences

Phylogenetic trees have been constructed for a wide range of organisms using gene sequence information, especially through the identification of orthologous genes that have been vertically inherited. The number of available complete genome sequences is rapidly increasing, and many tools for construction of genome trees based on whole genome sequences have been proposed. However, development of a reasonable method of using complete genome sequences for construction of phylogenetic trees has not been established. We have developed a method for construction of phylogenetic trees based on the average sequence similarities of whole genome sequences. We used this method to examine the phylogeny of 115 photosynthetic prokaryotes, i.e., cyanobacteria, Chlorobi, proteobacteria, Chloroflexi, Firmicutes and nonphotosynthetic organisms including Archaea. Although the bootstrap values for the branching order of phyla were low, probably due to lateral gene transfer and saturated mutation, the obtained tree was largely consistent with the previously reported phylogenetic trees, indicating that this method is a robust alternative to traditional phylogenetic methods.     

Bayesian inference of character evolution

Much recent progress in evolutionary biology is based on the inference of ancestral states and past transformations in important traits on phylogenetic trees. These exercises often assume that the tree is known without error and that ancestral states and character change can be mapped onto it exactly. In reality, there is often considerable uncertainty about both the tree and the character mapping. Recently introduced Bayesian statistical methods enable the study of character evolution while simultaneously accounting for both phylogenetic and mapping uncertainty, adding much needed credibility to the reconstruction of evolutionary history.     

Phylogenetic investigations of Antarctic notothenioid fishes (Perciformes: Notothenioidei) using complete gene sequences of the mitochondrial encoded 16S rRNA

The Notothenioidei dominates the fish fauna of the Antarctic in both biomass and diversity. This clade exhibits adaptations related to metabolic function and freezing avoidance in the subzero Antarctic waters, and is characterized by a high degree of morphological and ecological diversity. Investigating the macroevolutionary processes that may have contributed to the radiation of notothenioid fishes requires a well-resolved phylogenetic hypothesis. To date published molecular and morphological hypotheses of notothenioids are largely congruent, however, there are some areas of significant disagreement regarding higher-level relationships. Also, there are critical areas of the notothenioid phylogeny that are unresolved in both molecular and morphological phylogenetic analyses. Previous molecular phylogenetic analyses of notothenioids using partial mtDNA 12S and 16S rRNA sequence data have resulted in limited phylogenetic resolution and relatively low node support. One particularly controversial result from these analyses is the paraphyly of the Nototheniidae, the most diverse family in the Notothenioidei. It is unclear if the phylogenetic results from the 12S and 16S partial gene sequence dataset are due to limited character sampling, or if they reflect patterns of evolutionary diversification in notothenioids. We sequenced the complete mtDNA 16S rRNA gene for 43 notothenioid species, the largest sampling to-date from all eight taxonomically recognized families. Phylogenetic analyses using both maximum parsimony and maximum likelihood resulted in well-resolved trees with most nodes supported with high bootstrap pseudoreplicate scores and significant Bayesian posterior probabilities. In all analyses the Nototheniidae was monophyletic. Shimodaira–Hasegawa tests were able to reject two hypotheses that resulted from prior morphological analyses. However, despite substantial resolution and node support in the 16S rRNA trees, several phylogenetic hypotheses among closely related species and clades were not rejected. The inability to reject particular hypotheses among species in apical clades is likely due to the lower rate of nucleotide substitution in mtDNA rRNA genes relative to protein coding regions. Nevertheless, with the most extensive notothenioid taxon sampling to date, and the much greater phylogenetic resolution offered by the complete 16S rRNA sequences over the commonly used partial 12S and 16S gene dataset, it would be advantageous for future molecular investigations of notothenioid phylogenetics to utilize at the minimum the complete gene 16S rRNA dataset.     

Bayesian estimation of ancestral character states on phylogenies

Biologists frequently attempt to infer the character states at ancestral nodes of a phylogeny from the distribution of traits observed in contemporary organisms. Because phylogenies are normally inferences from data, it is desirable to account for the uncertainty in estimates of the tree and its branch lengths when making inferences about ancestral states or other comparative parameters. Here we present a general Bayesian approach for testing comparative hypotheses across statistically justified samples of phylogenies, focusing on the specific issue of reconstructing ancestral states. The method uses Markov chain Monte Carlo techniques for sampling phylogenetic trees and for investigating the parameters of a statistical model of trait evolution. We describe how to combine information about the uncertainty of the phylogeny with uncertainty in the estimate of the ancestral state. Our approach does not constrain the sample of trees only to those that contain the ancestral node or nodes of interest, and we show how to reconstruct ancestral states of uncertain nodes using a most-recent-common-ancestor approach. We illustrate the methods with data on ribonuclease evolution in the Artiodactyla. Software implementing the methods (BayesMultiState) is available from the authors.     

Geographic origin of human mitochondrial DNA: accommodating phylogenetic uncertainty and model comparison

Many biogeographic problems are tested on phylogenetic trees. Typically, the uncertainty in the phylogeny is not accommodated when investigating the biogeography of the organisms. Here we present a method that accommodates uncertainty in the phylogenetic trees. Moreover, we describe a simple method for examining the support for competing biogeographic scenarios. We illustrate the method using mitochondrial DNA sequences sampled from modern humans. The geographic origin of modern human mtDNA is inferred to be in Africa, although support for this hypothesis was ambiguous for data from an early paper.     

Phylogenetic diversity of macromycetes and woody plants along an elevational gradient in Eastern Mexico

Phylogenetic information provides insight into the ecological and evolutionary processes that organize species assemblages. We compared patterns of phylogenetic diversity among macromycete and woody plant communities along a steep elevational gradient in eastern Mexico to better understand the evolutionary processes that structure their communities. Macrofungi and trees were counted and identified in eight sites from 100 to 3500 m asl, and sequence data retrieved from GenBank for the same or closely related species were used to reconstruct their phylogenies. Patterns of species richness and phylogenetic diversity were similar for both macrofungi and trees, but macromycete richness and diversity peaked at mid‐elevations, whereas woody plant richness and diversity did not show significant trends with elevation. Phylogenetic similarity among sites was low for both groups and decreased as elevational distance between sites increased. Macromycete communities displayed phylogenetic overdispersion at low elevations and phylogenetic clustering at high elevations; the latter is consistent with environmental filtering at high elevation sites. Woody plants generally exhibited phylogenetic clustering, consistent with the potential importance of environmental filtering throughout the elevational gradient.     

Body Size and Geographic Range Do Not Explain Long Term Variation in Fish Populations: A Bayesian Phylogenetic Approach to Testing Assembly Processes in Stream Fish Assemblages

We combine evolutionary biology and community ecology to test whether two species traits, body size and geographic range, explain long term variation in local scale freshwater stream fish assemblages. Body size and geographic range are expected to influence several aspects of fish ecology, via relationships with niche breadth, dispersal, and abundance. These traits are expected to scale inversely with niche breadth or current abundance, and to scale directly with dispersal potential. However, their utility to explain long term temporal patterns in local scale abundance is not known. Comparative methods employing an existing molecular phylogeny were used to incorporate evolutionary relatedness in a test for covariation of body size and geographic range with long term (1983 – 2010) local scale population variation of fishes in West Fork White River (Indiana, USA). The Bayesian model incorporating phylogenetic uncertainty and correlated predictors indicated that neither body size nor geographic range explained significant variation in population fluctuations over a 28 year period. Phylogenetic signal data indicated that body size and geographic range were less similar among taxa than expected if trait evolution followed a purely random walk. We interpret this as evidence that local scale population variation may be influenced less by species-level traits such as body size or geographic range, and instead may be influenced more strongly by a taxon’s local scale habitat and biotic assemblages.     

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.