The Phylogenetic Informativeness of Nucleotide and Amino Acid Sequences for Reconstructing the Vertebrate Tree

To aid in future efforts to accurately reconstruct the vertebrate tree, a quantitative measure of phylogenetic informativeness was applied to nucleotide and amino acid sequences for a set of 11 genes. We identified orthologues and assembled published fossil-calibrated divergence times between taxa that had been sequenced for each gene. Rates of molecular evolution for each site were estimated to characterize the molecular evolutionary pattern of genes and to calculate the phylogenetic informativeness. The fast-evolving gene albumin yielded the highest informativeness over the period from 60 million years ago to 500 million years ago. In contrast, calmodulin yielded the lowest informativeness, presumably because functional constraint minimized substitutions in the amino acid sequence. The gene c-myc showed an intermediate level of informativeness. The nucleotide sequence of cytochrome b showed extremely high utility for recent epochs, but low utility for times before 100 million years ago. We ranked nine other genes for their utility during the epochs of the divergence of the muroid rodents, early placental mammals, early vertebrates, and early metazoa, yielding results consistent with, but more precise than, previous studies. Interestingly, DNA sequence always exceeded amino acid sequence in informativeness over all time scales, yet support values were at best moderately higher. For epochs not subject to strong phylogenetic conflict due to convergence, we advocate gleaning the additional power of the threefold increase in number of characters that is present for DNA sequences over resorting to the less noisy but less informative amino acid sequences.     

RECONSTRUCTING CHARACTER EVOLUTION ON POLYTOMOUS CLADOGRAMS

Abstract— Algorithms to reconstruct character evolution on polytomous cladograms or phylogenetic trees have to date interpreted each polytomy literally, as if it were an event of multiple speciation, with multiple daughter species descending independently from a mother species, thus requiring any similarities shared by only some of these daughters to be accounted for by convergence. These algorithms are not appropriate when the polytomy is interpreted in the usual way, namely as representing uncertainty in the cladogram's resolution. New algorithms for both ordered and unordered characters are presented to reconstruct character evolution under the uncertain-resolution interpretation of polytomies. These algorithms allow the cladogram to resolve itself so as to be favourable for the character whose evolution is being reconstructed. Because different characters may have different favourable resolutions, it is not possible in general to use these algorithms to determine the total parsimony of a polytomous cladogram (the number of evolutionary steps required over all characters by the cladogram), for which the only adequate approach is to find a most parsimonious dichotomous resolution of the cladogram.     

Tests of turtle phylogeny: molecular, morphological, and paleontological approaches

We present phylogenetic analyses of both molecular and morphological data for the 23 major lineages of living turtles and seven key fossil taxa. Nearly 1 kilobase of cytochrome b sequence, 325 base pairs of 12S ribosomal DNA, and 115 morphological characters contained similar phylogenetic information, although each provided unique information on different nodes of chelonian history. A character-based combinability test (implemented in PAUP*) and a non-parametric test of taxonomic congruence indicated no strong evidence for heterogeneity among data sets, and we used a combined approach to estimate a final phylogeny of the major lineages of living turtles. This approach resulted in a very well-resolved tree, with only a few of the deep branches within the Cryptodira left as an unresolved polytomy. The addition of six relatively complete fossils chosen to help resolve this basal polytomy provided little added resolution to the tree and resulted in a sharp decline in bootstrap proportions for nodes near the fossils. Branch-length analysis and independent dates from the fossil record suggest that these unresolved nodes may represent a rapid radiation of the major cryptodiran lineages 90-120 million years ago.     

Phylogenetic signal and noise: predicting the power of a data set to resolve phylogeny

A principal objective for phylogenetic experimental design is to predict the power of a data set to resolve nodes in a phylogenetic tree. However, proactively assessing the potential for phylogenetic noise compared with signal in a candidate data set has been a formidable challenge. Understanding the impact of collection of additional sequence data to resolve recalcitrant internodes at diverse historical times will facilitate increasingly accurate and cost-effective phylogenetic research. Here, we derive theory based on the fundamental unit of the phylogenetic tree, the quartet, that applies estimates of the state space and the rates of evolution of characters in a data set to predict phylogenetic signal and phylogenetic noise and therefore to predict the power to resolve internodes. We develop and implement a Monte Carlo approach to estimating power to resolve as well as deriving a nearly equivalent faster deterministic calculation. These approaches are applied to describe the distribution of potential signal, polytomy, or noise for two example data sets, one recent (cytochrome c oxidase I and 28S ribosomal rRNA sequences from Diplazontinae parasitoid wasps) and one deep (eight nuclear genes and a phylogenomic sequence for diverse microbial eukaryotes including Stramenopiles, Alveolata, and Rhizaria). The predicted power of resolution for the loci analyzed is consistent with the historic use of the genes in phylogenetics.     

Introns outperform exons in analyses of basal avian phylogeny using clathrin heavy chain genes

Neoaves is the most diverse major avian clade, containing ~95% of avian species, and it underwent an ancient but rapid diversification that has made resolution of relationships at the base of the clade difficult. In fact, Neoaves has been suggested to be a "hard" polytomy that cannot be resolved with any amount of data. However, this conclusion was based on slowly evolving coding sequences and ribosomal RNAs and some recent studies using more rapidly evolving intron sequences have suggested some resolution at the base of Neoaves. To further examine the utility of introns and exons for phylogenetics, we sequenced parts of two unlinked clathrin heavy chain genes (CLTC and CLTCL1). Comparisons of phylogenetic trees based upon individual partitions (i.e. introns and exons), the combined dataset, and published phylogenies using Robinson-Foulds distances (a metric of topological differences) revealed more similarity than expected by chance, suggesting there is structure at the base of Neoaves. We found that introns provided more informative sites, were subject to less homoplasy, and provided better support for well-accepted clades, suggesting that intron evolution is better suited to determining closely-spaced branching events like the base of Neoaves. Furthermore, phylogenetic power analyses indicated that existing molecular datasets for birds are unlikely to provide sufficient phylogenetic information to resolve relationships at the base of Neoaves, especially when comprised of exon or other slowly evolving regions. Although relationships among the orders in Neoaves cannot be definitively established using available data, the base of Neoaves does not appear to represent a hard polytomy. Our analyses suggest that large intron datasets have the best potential to resolve relationships among avian orders and indicate that the utility of intron data for other phylogenetic questions should be examined.     

Rapid diversification of the cotton genus (Gossypium: Malvaceae) revealed by analysis of sixteen nuclear and chloroplast genes

Previous molecular phylogenetic studies have failed to resolve the branching order among the major cotton (Gossypium) lineages, and it has been unclear whether this reflects actual history (rapid radiation) or sampling properties of the genes evaluated. In this paper, we reconsider the phylogenetic relationships of diploid cotton genome groups using DNA sequences from 11 single-copy nuclear loci (10?293 base pairs [bp]), nuclear ribosomal DNA (695 bp), and four chloroplast loci (7370 bp). Results from individual loci and combined nuclear and chloroplast DNA partitions reveal that the cotton genome groups radiated in rapid succession following the formation of the genus. Maximum likelihood analysis of nuclear synonymous sites shows that this radiation occurred within a time span equivalent to 17% of the time since the separation of Gossypium from its nearest extant relatives in the genera Kokia and Gossypioides. Chloroplast and nuclear phylogenies differ significantly with respect to resolution of the basal divergence in the genus and to interrelationships among African cottons. This incongruence is due to limited character evolution in cpDNA and either previously unsuspected hybridization or unreliable phylogenetic performance of the cpDNA characters. This study highlights the necessity of using multiple, independent data sets for resolving phylogenetic relationships of rapidly diverged lineages.     

Application of the phylogenetic informativeness method to chloroplast markers: A test case of closely related species in tribe Hydrangeeae (Hydrangeaceae)

In evolutionary biology appropriate marker selection for the reconstruction of solid phylogenetic hypotheses is fundamental. One of the most challenging tasks addresses the appropriate choice of genomic regions in studies of closely related species. Robust phylogenetic frameworks are central to studies dealing with questions ranging from evolutionary and conservation biology, biogeography to plant breeding. Phylogenetic informativeness profiles provide a quantitative measure of the phylogenetic signal in markers and therefore a method for locus prioritization. The present work profiles phylogenetic informativeness of mostly non-coding chloroplast regions in an angiosperm lineage of closely related species: the popular ornamental tribe Hydrangeeae (Hydrangeaceae, Cornales, Asterids). A recent phylogenetic study denoted a case of resolution contrast between the two strongly supported clades within tribe Hydrangeeae. We evaluate the phylogenetic signal of 13 highly variable plastid markers for estimating relationships within and among the currently recognized monophyletic groups of this tribe. A selection of combined loci based on their phylogenetic informativeness retrieved more robust phylogenetic hypotheses than simply combining individual markers performing best with respect to resolution, nodal support and accuracy or those presenting the highest number of parsimony informative characters. We propose the rpl32–ndhF intergenic spacer (IGS), trnV–ndhC IGS, trnL–rpl32 IGS, psbT–petB region and ndhA intron as the best candidates for future phylogenetic studies in Hydrangeeae and potentially in other Asterids. We also contrasted the phylogenetic informativeness of coded indels against substitutions concluding that, despite their low phylogenetic informativeness, coded indels provide additional phylogenetic signal that is nearly free of noise. Phylogenetic relationships obtained from our total combined analyses showed improved resolution and nodal support with respect to recently published results.     

Phylogenetic implications of the morphology of the braincase of caecilian amphibians (Gymnophiona)

Caecilian morphology is strongly modified in association with their fossorial mode of life. Currently phylogenetic analyses of characters drawn from the morphology of caecilians lack resolution, as well as complementarity, with results of phylogenetic analyses that employ molecular data. Stemming from the hypothesis derived from the mammal literature that the braincase has the greatest potential (in comparison to other cranial units) to yield phylogenetic information, the braincase and intimately associated stapes of 27 species (23 genera) of extant caecilians were examined using images assembled via microcomputed tomography. Thirty‐four new morphological characters pertaining to the braincase and stapes were identified and tested for congruence with previously recognized morphological characters. The results reveal that when added to previous character matrices, characters of the braincase and stapes resolve generic‐level relationships in a way that is largely congruent with the results of molecular analyses. Analysis of a combined data set of molecular and morphological data provides a framework for conducting ancestral character state reconstructions, which resulted in the identification of 95 new synapomorphies for various clades and taxa, 27 of which appear to be unique for the taxa that possess them. Together these data demonstrate the utility of the application of characters of the braincase and stapes for resolving phylogenetic relationships for a group whose morphology is largely confounded by functional modifications. In addition this study provides evidence of the utility of the braincase in resolving problematic morphology‐based phylogeny outside of Amniota, in an amphibian group. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166 , 160–201.     

Multi-locus phylogeny of dolphins in the subfamily Lissodelphininae: character synergy improves phylogenetic resolution

Background  

Dolphins of the genus Lagenorhynchus are anti-tropically distributed in temperate to cool waters. Phylogenetic analyses of cytochrome b sequences have suggested that the genus is polyphyletic; however, many relationships were poorly resolved. In this study, we present a combined-analysis phylogenetic hypothesis for Lagenorhynchus and members of the subfamily Lissodelphininae, which is derived from two nuclear and two mitochondrial data sets and the addition of 34 individuals representing 9 species. In addition, we characterize with parsimony and Bayesian analyses the phylogenetic utility and interaction of characters with statistical measures, including the utility of highly consistent (non-homoplasious) characters as a conservative measure of phylogenetic robustness. We also explore the effects of removing sources of character conflict on phylogenetic resolution.     

Transformation Series as an Ideographic Character Concept

An ideographic concept of character is indispensable to phylogenetic inference. Hennig proposed that characters be conceptualized as “transformation series”, a proposal that is firmly grounded in evolutionary theory and consistent with the method of inferring transformation events as evidence of phylogenetic propinquity. Nevertheless, that concept is usually overlooked or rejected in favor of others based on similarity. Here we explicate Hennig's definition of character as an ideographic concept in the science of phylogenetic systematics. As transformation series, characters are historical individuals akin to species and clades. As such, the related concept of homology refers to a historical identity relation and is not equivalent to or synonymous with synapomorphy. The distinction between primary and secondary homology is dismissed on the grounds that it conflates the concept of homology with the discovery operations used to detect instances of that concept. Although concern for character dependence is generally valid, it is often misplaced, focusing on functional or developmental correlation (both of which are irrelevant in phylogenetic systematics but may be valid in other fields) instead of the historical/transformational independence relevant to phylogenetic inference. As an ideographic science concerned with concrete objects and events (i.e. individuals), intensionally and extensionally defined properties are inconsistent with the individuation of characters for phylogenetic analysis, the utility of properties being limited to communicating results and facilitating future rounds of testing.     

Potential phylogenetic utility of the low-copy nuclear gene pistillata in dicotyledonous plants: comparison to nrDNA ITS and trnL intron in Sphaerocardamum and other Brassicaceae.

We report the potential phylogenetic utility of DNA sequence data from the last 700 bp of a ca. 1-kb intron of the MADS-box gene pistillata from a sampling of Sphaerocardamum species and other Brassicaceae. These results are compared with nrDNA ITS and the chloroplast trnL intron for the same taxa to demonstrate the potential phylogenetic utility of this pistillata intron and to identify potential historically independent sequences for an ongoing study of relationships within Sphaerocardamum. Analyses of the DNA sequence data for Brassicaceae indicated that pairwise divergences and potentially informative characters were higher in the pistillata intron (0.6-30.8%, 284 characters) and ITS (0-24%, 94 characters) than in the chloroplast trnL intron (0-4.2%, 17 characters). A comparison of Sphaerocardamum sequences identified low divergences and numbers of informative characters for trnL intron (0-2.4%, 1 character) and nrDNA ITS (0-2.5%, 2 characters) and substantially more variation among the pistillata sequences (0.15-3.7%, 19 characters). Phylogenetic analyses of these pistillata sequences fully resolve ingroup relationships without character conflict. Results of pistillata PCR amplifications from a broader dicot sample showed that some primers may be useful in amplifying orthologous pistillata sequences. Ultimately this pistillata intron may be a valuable source of phylogenetic characters at lower taxonomic levels.     

Efficiently resolving the basal clades of a phylogenetic tree using Bayesian and parsimony approaches: a case study using mitogenomic data from 100 higher teleost fishes

Many phylogenetic analyses that include numerous terminals but few genes show high resolution and branch support for relatively recently diverged clades, but lack of resolution and/or support for "basal" clades of the tree. The various benefits of increased taxon and character sampling have been widely discussed in the literature, albeit primarily based on simulations rather than empirical data. In this study, we used a well-sampled gene-tree analysis (based on 100 mitochondrial genomes of higher teleost fishes) to test empirically the efficiency of different methods of data sampling and phylogenetic inference to "correctly" resolve the basal clades of a tree (based on congruence with the reference tree constructed using all 100 taxa and 7990 characters). By itself, increased character sampling was an inefficient method by which to decrease the likelihood of "incorrect" resolution (i.e., incongruence with the reference tree) for parsimony analyses. Although increased taxon sampling was a powerful approach to alleviate "incorrect" resolution for parsimony analyses, it had the general effect of increasing the number of, and support for, "incorrectly" resolved clades in the Bayesian analyses. For both the parsimony and Bayesian analyses, increased taxon sampling, by itself, was insufficient to help resolve the basal clades, making this sampling strategy ineffective for that purpose. For this empirical study, the most efficient of the six approaches considered to resolve the basal clades when adding nucleotides to a dataset that consists of a single gene sampled for a small, but representative, number of taxa, is to increase character sampling and analyze the characters using the Bayesian method.     

Lower level relationships in the mushroom genus Cortinarius (Basidiomycota, Agaricales): a comparison of RPB1, RPB2, and ITS phylogenies

We sampled and analyzed approximately 2900bp across the three loci from 54 taxa belonging to a taxonomically difficult group of Cortinarius subgenus Phlegmacium. The combined analyses of ITS and variable regions of RPB1 and RPB2 greatly increase the resolution and nodal support for phylogenies of these closely related species belonging to clades that until now have proven very difficult to resolve with the ribosomal markers, nLSU and ITS. We present the first study of the utility of variable regions of the genes encoding the two largest subunits of RNA polymerase II (RPB1 and RPB2) for inferring the phylogeny of mushroom-forming fungi in combination with and compared to the widely used ribosomal marker ITS. The studied region of RPB1 contains an intron of the size and variability of ITS along with many variable positions in coding regions. Though almost entirely coding, the studied region of RPB2 is more variable than ITS. Both RNA polymerase II genes were alignable across all taxa. Our results indicate that several sections of Cortinarius need redefinition, and that several taxa treated at subspecific and varietal level should be treated at specific level. We suggest a new section for the two species, C. caesiocortinatus and C. prasinocyaneus, which constitute a well-supported separate lineage. We speculate that sequence information from RNA polymerase II genes have the potential for resolving phylogenetic problems at several levels of the diverse and taxonomically very challenging genus Cortinarius.     

Considerations for Reconstructing Metazoan History: Signal, Resolution, and Hypothesis Testing

SYNOPSIS. Certain issues of metazoan phytogeny remain difficultto resolve even with advances in phylogenetic theory and moleculartools. This lack of resolution might be due to limited phylogeneticsignal in regions of the metazoan tree. For example, 18S rDNAdata are consistent with a rapid radiation early in the historyof bilaterian animals. Despite this lack of resolution, ourunderstanding of metazoan evolutionary history can be furtheredby extracting phylogenetic signal from data with more appropriateevolutionary models and by explicitly testing alternative hypotheses.I examined 18S rDNA data across a diverse number of phyla toidentify potential problems with using genetic data to reconstructmetazoan history. Although the 18S performs well at the mostgeneral level of metazoan phytogeny, it has shortcomings particularlyamong the non-arthropod protostomes. Examples of parametricbootstrap simulation are given to illustrate how we may beginto address methodological issues when there is limited phylogeneticsignal. These simulations suggest that we are more likely tounderstand the bilaterian radiation event by increasing thelength of nucleotide sequences collected than by employing fastergenes.     

Female genitalia are moderately informative for phylogenetic inference and not concerted with male genitalia in Saprininae beetles (Coleoptera: Histeridae)

Our study – the first of its kind within Histeridae (Coleoptera) – deals with the female genitalia in the subfamily Saprininae, with respect to their variation and utility for phylogenetic reconstruction. Morphology of female genitalia (chiefly characters of the spermatheca, and variation of the gonocoxites, articulating sclerites and gonostyli) is herein described and depicted. We perform parsimony‐based morphological phylogenetic analyses of Saprininae using combined datasets that comprise somatic, male and female genitalia characters. We assess phylogenetic utility of female genital characters and test for their correlation with male genital characters. We found that female genitalia are generally moderately informative for phylogenetic reconstruction. The somatic and male genitalia characters (on average) possess higher phylogenetic signal; female genitalia provide better support and diagnoses for particular clades (emphasizing their importance for systematics), in which the evolution of female genitalia can be linked to environmental factors imposed by the mode of life. Regardless of the degree of informativeness, structures of female genitalia represent an additional interesting and important source of information for systematics and taxonomy. We did not find any evidence of correlated evolution between male and female genitalia.     

Relative apparent synapomorphy analysis (RASA). I: The statistical measurement of phylogenetic signal

We have developed a new approach to the measurement of phylogenetic signal in character state matrices called relative apparent synapomorphy analysis (RASA). RASA provides a deterministic, statistical measure of natural cladistic hierarchy (phylogenetic signal) in character state matrices. The method works by determining whether a measure of the rate of increase of cladistic similarity among pairs of taxa as a function of phenetic similarity is greater than a null equiprobable rate of increase. Our investigation of the utility and limitations of RASA using simulated and bacteriophage T7 data sets indicates that the method has numerous advantages over existing measures of signal. A first advantage is computational efficiency. A second advantage is that RASA employs known methods of statistical inference, providing measurable sensitivity and power. The performance of RASA is examined under various conditions of branching evolution as the number of characters, character states per character, and mutations per branch length are varied. RASA appears to provide an unbiased and reliable measure of phylogenetic signal, and the general approach promises to be useful in the development of new techniques that should increase the rigor and reliability of phylogenetic estimates.      

Integrating genomic and phenotypic data to evaluate alternative phylogenetic and species delimitation hypotheses in a recent evolutionary radiation of grasshoppers

Although resolving phylogenetic relationships and establishing species limits are primary goals of systematics, these tasks remain challenging at both conceptual and analytical levels. Here, we integrated genomic and phenotypic data and employed a comprehensive suite of coalescent‐based analyses to develop and evaluate competing phylogenetic and species delimitation hypotheses in a recent evolutionary radiation of grasshoppers (Chorthippus binotatus group) composed of two species and eight putative subspecies. To resolve the evolutionary relationships within this complex, we first evaluated alternative phylogenetic hypotheses arising from multiple schemes of genomic data processing and contrasted genetic‐based inferences with different sources of phenotypic information. Second, we examined the importance of number of loci, demographic priors, number and kind of phenotypic characters and sex‐based trait variation for developing alternative species delimitation hypotheses. The best‐supported topology was largely compatible with phenotypic data and showed the presence of two clades corresponding to the nominative species groups, one including three well‐resolved lineages and the other comprising a four‐lineage polytomy and a well‐differentiated sister taxon. Integrative species delimitation analyses indicated that the number of employed loci had little impact on the obtained inferences but revealed the higher power provided by an increasing number of phenotypic characters and the usefulness of assessing their phylogenetic information content and differences between sexes in among‐taxa trait variation. Overall, our study highlights the importance of integrating multiple sources of information to test competing phylogenetic hypotheses and elucidate the evolutionary history of species complexes representing early stages of divergence where conflicting inferences are more prone to appear.     

Comparison of phylogenetic signal between male genitalia and non-genital characters in insect systematics

It is generally accepted that male genitalia evolve more rapidly and divergently relative to non-genital traits due to sexual selection, but there is little quantitative comparison of the pattern of evolution between these character sets. Moreover, despite the fact that genitalia are still among the most widely used characters in insect systematics, there is an idea that the rate of evolution is too rapid for genital characters to be useful in forming clades. Based on standard measures of fit used in cladistic analyses, we compare levels of homoplasy and synapomorphy between genital and non-genital characters of published data sets and demonstrate that phylogenetic signal between these two character sets is statistically similar. This pattern is found consistently across different insect orders at different taxonomic hierarchical levels. We argue that the fact that male genitalia are under sexual selection and thus diverge rapidly does not necessarily equate with the lack of phylogenetic signal, because characters that evolve by descent with modification make appropriate characters for a phylogenetic analysis, regardless of the rate of evolution. We conclude that male genitalia are a composite character consisting of different components diverging separately, which make them ideal characters for phylogenetic analyses, providing information for resolving varying levels of hierarchy.
© The Willi Hennig Society 2009.     

Model‐based approach to test hard polytomies in the Eulaemus clade of the most diverse South American lizard genus Liolaemus (Liolaemini,Squamata)

Lack of resolution in a phylogenetic tree is usually represented as a polytomy, and often adding more data (loci and taxa) resolves the species tree. These are the ‘soft’ polytomies, but in other cases additional data fail to resolve relationships; these are the ‘hard’ polytomies. This latter case is often interpreted as a simultaneous radiation of lineages in the history of a clade. Although hard polytomies are difficult to address, model‐based approaches provide new tools to test these hypotheses. Here, we used a clade of 144 species of the South American lizard clade Eulaemus to estimate phylogenies using a traditional concatenated matrix and three species tree methods: *BEAST, BEST, and minimizing deep coalescences (MDC). The different species tree methods recovered largely discordant results, but all resolved the same polytomy (e.g. very short internodes amongst lineages and low nodal support in Bayesian methods). We simulated data sets under eight explicit evolutionary models (including hard polytomies), tested these against empirical data (a total of 14 loci), and found support for two polytomies as the most plausible hypothesis for diversification of this clade. We discuss the performance of these methods and their limitations under the challenging scenario of hard polytomies. © 2015 The Linnean Society of London     

New nuclear evidence for the oldest divergence among neognath birds: the phylogenetic utility of ZENK (i)

To date, there is little consensus concerning the phylogenetic relationships among neognath orders, which include all extant birds except ratites and tinamous. Different data sets, both molecular and morphologic, have yielded radically different and often unresolved ordinal topologies, especially within the neoaves clade. This lack of resolution and ongoing conflict indicates a need for additional phylogenetic characters to be applied to the question of higher-level avian phylogeny. In this study, sequences of a single-copy nuclear gene, ZENK, were used to reconstruct an ordinal-level phylogeny of neognath birds. Strong support was indicated for the oldest divergence within Neognathae; the chicken- and duck-like birds formed a clade that was sister to all other modern birds. In addition, many families of traditional taxonomic orders clustered together in the ZENK tree, indicating the gene's general phylogenetic reliability. However, within the neoaves clade, there was little support for relationships among orders, which is a result similar to all other recent molecular studies of higher-level avian phylogeny. This similarity among studies suggests the possibility of a rapid radiation of the major neoaves lineages. Despite the ongoing lack of neoaves resolution, ZENK's sequence divergence and base composition patterns indicate its general utility as a new phylogenetic marker for higher-level avian systematics.