Molecular phylogeny of the Siphonocladales (Chlorophyta: Cladophorophyceae)

The Siphonocladales are tropical to warm-temperate, marine green macro-algae characterized by a wide variety of thallus morphologies, ranging from branched filaments to pseudo-parenchymatous plants. Phylogenetic analyses of partial large subunit (LSU) rDNA sequences sampled from 166 isolates revealed nine well-supported siphonocladalean clades. Analyses of a concatenated dataset of small subunit (SSU) and partial LSU rDNA sequences greatly clarified the phylogeny of the Siphonocladales. However, the position of the root of the Siphonocladales could not be determined unambiguously, as outgroup rooting and molecular clock rooting resulted in a different root placement. Different phylogenetic methods (likelihood, parsimony and distance) yielded similar tree topologies with comparable internal node resolution. Likewise, analyses under more realistic models of sequence evolution, taking into account differences in evolution between stem and loop regions of rRNA, did not differ markedly from analyses using standard four-state models. The molecular phylogeny revealed that all siphonocladalean architectures may be derived from a single Cladophora-like ancestor. Parallel and convergent evolution of various morphological characters (including those traditionally employed to circumscribe the families and genera) have occurred in the Siphonocladales. Consequently, incongruence with traditional classifications, including non-monophyly in all families and most genera, was shown.     

An empirical test of the midpoint rooting method

The outgroup method is widely used to root phylogenetic trees. An accurate root indication, however, strongly depends on the availability of a proper outgroup. An alternate rooting method is the midpoint rooting (MPR). In this case, the root is set at the midpoint between the two most divergent operational taxonomic units. Although the midpoint rooting algorithm has been extensively used, the efficiency of this method in retrieving the correct root remains untested. In the present study, we empirically tested the success rate of the MPR in obtaining the outgroup root for a given phylogenetic tree. This was carried out by eliminating outgroups in 50 selected data sets from 33 papers and rooting the trees with the midpoint method. We were thus able to compare the root position retrieved by each method. Data sets were separated into three categories with different root consistencies: data sets with a single outgroup taxon (54% success rate for MPR), data sets with multiple outgroup taxa that showed inconsistency in root position (82% success rate), and data sets with multiple outgroup taxa in which root position was consistent (94% success rate). Interestingly, the more consistent the outgroup root is, the more successful MPR appears to be. This is a strong indication that the MPR method is valuable, particularly for cases where a proper outgroup is unavailable.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 92 , 669–674.     

Alveolate phylogeny inferred using concatenated ribosomal proteins

Dinoflagellates and apicomplexans are a strongly supported monophyletic group in rDNA phylogenies, although this phylogeny is not without controversy, particularly between the two groups. Here we use concatenated protein-coding genes from expressed sequence tags or genomic data to construct phylogenies including "typical" dinophycean dinoflagellates, a parasitic syndinian dinoflagellate, Amoebophrya sp., and two related species, Oxyrrhis marina, and Perkinsus marinus. Seventeen genes encoding proteins associated with the ribosome were selected for phylogenetic analysis. The dataset was limited for the most part by data availability from the dinoflagellates. Forty-five taxa from four major lineages were used: the heterokont outgroup, ciliates, dinoflagellates, and apicomplexans. Amoebophrya sp. was included in this phylogeny as a sole representative of the enigmatic marine alveolate or syndinian lineage. The atypical dinoflagellate O. marina, usually excluded from rDNA analyses due to long branches, was also included. The resulting phylogenies were well supported in concatenated analyses with only a few unstable or weakly supported branches; most features were consistent when different lineages were pruned from the tree or different genes were concatenated. The least stable branches involved the placement of Cryptosporidium spp. within the Apicomplexa and the relationships between P. marinus, Amoebophrya sp., and O. marina. Both bootstrap and approximately unbiased test results confirmed that P. marinus, Amoebophrya sp., O. marina, and the remaining dinoflagellates form a monophyletic lineage to the exclusion of Apicomplexa.     

Nuclear mitochondrial pseudogenes as molecular outgroups for phylogenetically isolated taxa: a case study in Sphenodon

'Living fossil' taxa, by definition, have no close relatives, and therefore no outgroup to provide a root to phylogenetic trees. We identify and use a molecular outgroup in the sole extant lineage of sphenodontid reptiles, which separated from other reptiles 230 million years ago. We isolated and sequenced a partial nuclear copy of the mitochondrial cytochrome b gene. We confirm the copy is indeed not mitochondrial, is older than all extant mitochondrial copies in Sphenodon (tuatara), and is therefore useful as a molecular outgroup. Under phylogenetic analysis, the nuclear copy places the root of the tuatara mitochondrial gene tree between the northern and the southern (Cook Strait) groups of islands of New Zealand that are the last refugia for Sphenodon. This analysis supports a previous mid-point rooted mitochondrial gene tree. The mitochondrial DNA tree conflicts with allozyme analyses which place a Cook Strait population equidistant to all northern and other Cook Strait populations. This population on North Brother Island is the only natural population of extant S. guntheri; thus, we suggest that the current species designations of tuatara require further investigation.     

Elongation factor-1 alpha occurs as two copies in bees: implications for phylogenetic analysis of EF-1 alpha sequences in insects

We report the complete sequence of a paralogous copy of elongation factor-1 alpha (EF-1 alpha) in the honeybee, Apis mellifera (Hymenoptera: Apidae). This copy differs from a previously described copy in the positions of five introns and in 25% of the nucleotide sites in the coding regions. The existence of two paralogous copies of EF-1 alpha in Drosophila and Apis suggests that two copies of EF-1 alpha may be widespread in the holometabolous insect orders. To distinguish between a single, ancient gene duplication and parallel, independent fly and bee gene duplications, we performed a phylogenetic analysis of hexapod EF-1 alpha sequences. Unweighted parsimony analysis of nucleotide sequences suggests an ancient gene duplication event, whereas weighted parsimony analysis of nucleotides and unweighted parsimony analysis of amino acids suggests the contrary: that EF-1 alpha underwent parallel gene duplications in the Diptera and the Hymenoptera. The hypothesis of parallel gene duplication is supported both by congruence among nucleotide and amino acid data sets and by topology-dependent permutation tail probability (T-PTP) tests. The resulting tree topologies are also congruent with current views on the relationships among the holometabolous orders included in this study (Diptera, Hymenoptera, and Lepidoptera). More sequences, from diverse orders of holometabolous insects, will be needed to more accurately assess the historical patterns of gene duplication in EF-1 alpha.      

Hypothetical Ancestors and Rooting in Cladistic Analysis

Most hypothetical ancestors that are used to root trees in cladistic analyses summarize character-state information in one or more outgroup taxa. Nonetheless, hypothetical ancestors also provide a means of rooting trees using the ontogenetic and paleontological methods of polarizing character transformations, and for incorporating the inferences of more than one of these methods into a single analysis. However, the use of one hypothetical ancestor that combines inferences based on outgroup comparison with those based on other methods of polarizing character transformations to root a cladogram is invalid. Inferences regarding plesiomorphic character states based on outgroup comparison apply to the outgroup node, whereas inferences based on either the ontogenetic or paleontological method apply to the ingroup node. These inferences cannot be combined into a single hypothetical construct. A hypothetical ancestor based on outgroup information is included in the data matrix and used to root the resulting network; however, because this ancestor places potentially problematic constraints on the analysis, the use of actual outgroup taxa is preferable in most instances. Correct use of a hypothetical ancestor inferred with the ontogenetic and paleontological methods involves the Lundberg method in which the shortest ingroup network is rooted at the internode to which the hypothetical ancestor attaches most parsimoniously. Because inferences of polarity based on outgroup comparison cannot be combined directly with those based on other polarization methods, the synthesis of information from all three methods in a single tree must involve taxonomic congruence.     

Single-copy nuclear genes recover cretaceous-age divergences in bees

We analyzed the higher level phylogeny of the bee family Halictidae based on the coding regions of three single-copy nuclear genes (long-wavelength [LW] opsin, wingless, and elongation factor 1-alpha [EF-1 alpha]). Our combined data set consisted of 2,234 aligned nucleotide sites (702 base pairs [bp] for LW opsin, 405 bp for wingless, and 1,127 bp for EF-1 alpha) and 779 parsimony-informative sites. We included 58 species of halictid bees from 33 genera, representing all subfamilies and tribes, and rooted the trees using seven outgroups from other bee families: Colletidae, Andrenidae, Melittidae, and Apidae. We analyzed the separate and combined data sets by a variety of methods, including equal weights parsimony, maximum likelihood, and Bayesian methods. Analysis of the combined data set produced a strong phylogenetic signal with high bootstrap and Bremer support and high posterior probability well into the base of the tree. The phylogeny recovered the monophyly of the Halictidae and of all four subfamilies and both tribes, recovered relationships among the subfamilies and tribes congruent with morphology, and provided robust support for the relationships among the numerous genera in the tribe Halictini, sensu Michener (2000). Using our combined nucleotide data set, several recently described halictid fossils from the Oligocene and Eocene, and recently developed Bayesian methods, we estimated the antiquity of major clades within the family. Our results indicate that each of the four subfamilies arose well before the Cretaceous-Tertiary boundary and suggest that the early radiation of halictid bees involved substantial African-South American interchange roughly coincident with the separation of these two continents in the late Cretaceous. This combination of single-copy nuclear genes is capable of recovering Cretaceous-age divergences in bees with high levels of support. We propose that LW opsin, wingless, and EF-1 alpha(F2 copy) may be useful in resolving relationships among bee families and other Cretaceous-age insect lineages.     

Resolving the relationships of apid bees (Hymenoptera: Apidae) through a direct optimization sensitivity analysis of molecular,morphological, and behavioural characters

Phylogenetic analyses that incorporate the most character information also provide the most explanatory power. Here I demonstrate the value of such an approach through a direct optimization sensitivity analysis of apid bee phylogeny. Whereas prior studies have relied solely on one class of data or the other, this analysis combines previously published molecular, morphological, and behavioural characters into a single supermatrix. The final dataset includes 191 ingroup and 30 outgroup taxa, and includes data from seven unaligned gene sequences (18S, 28S, wingless, EF1‐α, polII, Nak, LW rhodopsin), 209 adult and larval morphological characters, and two behavioural characters. Nine different sets of transformation cost parameters are evaluated, along with their relative degrees of character incongruence. The preferred parameter set returns a strict consensus tree somewhat similar to, but more resolved than, a previous parsimony tree based on molecules alone. I also describe the effects of including EF1‐α and LW rhodopsin intron sequences on the outcome of the direct optimization analysis. By accounting for more evidence, this study provides the most comprehensive treatment yet of apid phylogenetic relationships.     

Turning the crown upside down: gene tree parsimony roots the eukaryotic tree of life

The first analyses of gene sequence data indicated that the eukaryotic tree of life consisted of a long stem of microbial groups "topped" by a crown-containing plants, animals, and fungi and their microbial relatives. Although more recent multigene concatenated analyses have refined the relationships among the many branches of eukaryotes, the root of the eukaryotic tree of life has remained elusive. Inferring the root of extant eukaryotes is challenging because of the age of the group (～1.7-2.1 billion years old), tremendous heterogeneity in rates of evolution among lineages, and lack of obvious outgroups for many genes. Here, we reconstruct a rooted phylogeny of extant eukaryotes based on minimizing the number of duplications and losses among a collection of gene trees. This approach does not require outgroup sequences or assumptions of orthology among sequences. We also explore the impact of taxon and gene sampling and assess support for alternative hypotheses for the root. Using 20 gene trees from 84 diverse eukaryotic lineages, this approach recovers robust eukaryotic clades and reveals evidence for a eukaryotic root that lies between the Opisthokonta (animals, fungi and their microbial relatives) and all remaining eukaryotes.     

Phylogenetic utility and evidence for multiple copies of elongation factor-1alpha in the spider genus Habronattus (Araneae: Salticidae)

In the continuing quest for informative genes for use in molecular systematics, the protein-coding gene Elongation factor-1alpha (EF-1alpha) has rapidly become one of the most prevalent "single-copy" nuclear genes utilized, particularly in arthropods. This paper explores the molecular evolutionary dynamics and phylogenetic utility of EF-1alpha in the salticid spider genus Habronattus. As has been reported for other arthropod lineages, our studies indicate that multiple (two) copies of EF-1alpha exist in Habronattus. These copies differ in intron structure and thus in size, making it possible to easily separate PCR amplification products. We present data for an intronless EF-1alpha copy for three Habronattus species. The presence of nonsense mutations and generally elevated rates of amino acid change suggest that this copy is evolving under relaxed functional constraints in Habronattus. A larger taxon sample (50 species plus outgroups) is presented for an EF-1alpha copy that includes both intron and exon regions. Characteristics of both regions suggest that this is a functional, orthologous copy in the species sampled. Maximum-likelihood relative-rate comparisons show that exon third codon sites are evolving more than 100 times as fast as second codon sites in these sequences and that intron sites are evolving about twice as fast as exon third sites. In combination, the EF-1alpha data provide robust, species-level phylogenetic signal that is largely congruent with morphologically well supported areas of Habronattus phylogeny. The recovery of some novel clades, and the unexpected fragmentation of others, suggests areas requiring further phylogenetic attention.     

The problem of rooting rapid radiations

There are many examples of groups (such as birds, bees, mammals, multicellular animals, and flowering plants) that have undergone a rapid radiation. In such cases, where there is a combination of short internal and long external branches, correctly estimating and rooting phylogenetic trees is known to be a difficult problem. In this simulation study, we tested the performances of different phylogenetic methods at estimating a tree that models a rapid radiation. We found that maximum likelihood, corrected and uncorrected neighbor-joining, and corrected and uncorrected parsimony, all suffer from biases toward specific tree topologies. In addition, we found that using a single-taxon outgroup to root a tree frequently disrupts an otherwise correct ingroup phylogeny. Moreover, for uncorrected parsimony, we found cases where several individual trees (in which the outgroup was placed incorrectly) were selected more frequently than the correct tree. Even for parameter settings where the correct tree was selected most frequently when using extremely long sequences, for sequences of up to 60,000 nucleotides the incorrectly rooted trees were each selected more frequently than the correct tree. For all the cases tested here, tree estimation using a two taxon outgroup was more accurate than when using a single-taxon outgroup. However, the ingroup was most accurately recovered when no outgroup was used.     

Evaluating nuclear protein-coding genes for phylogenetic utility in beetles

Although nuclear protein-coding genes have proven broadly useful for phylogenetic inference, relatively few such genes are regularly employed in studies of Coleoptera, the most diverse insect order. We increase the number of loci available for beetle systematics by developing protocols for three genes previously unused in beetles (alpha-spectrin, RNA polymerase II and topoisomerase I) and by refining protocols for five genes already in use (arginine kinase, CAD, enolase, PEPCK and wingless). We evaluate the phylogenetic performance of each gene in a Bayesian framework against a presumably known test phylogeny. The test phylogeny covers 31 beetle specimens and two outgroup taxa of varying age, including three of the four extant beetle suborders and a denser sampling in Adephaga and in the carabid genus Bembidion. All eight genes perform well for Cenozoic divergences and accurately separate closely related species within Bembidion, but individual genes differ markedly in accuracy over the older Mesozoic and Permian divergences. The concatenated data reconstruct the test phylogeny with high support in both Bayesian and parsimony analyses, indicating that combining data from multiple nuclear loci will be a fruitful approach for assembling the beetle tree of life.     

Morphology,molecules, and the phylogenetics of cetaceans

Recent phylogenetic analyses of cetacean relationships based on DNA sequence data have challenged the traditional view that baleen whales (Mysticeti) and toothed whales (Odontoceti) are each monophyletic, arguing instead that baleen whales are the sister group of the odontocete family Physeteridae (sperm whales). We reexamined this issue in light of a morphological data set composed of 207 characters and molecular data sets of published 12S, 16S, and cytochrome b mitochondrial DNA sequences. We reach four primary conclusions: (1) Our morphological data set strongly supports the traditional view of odontocete monophyly; (2) the unrooted molecular and morphological trees are very similar, and most of the conflict results from alternative rooting positions; (3) the rooting position of the molecular tree is sensitive to choice of artiodactyls outgroup taxa and the treatment of two small but ambiguously aligned regions of the 12S and 16S sequences, whereas the morphological root is strongly supported; and (4) combined analyses of the morphological and molecular data provide a well-supported phylogenetic estimate consistent with that based on the morphological data alone (and the traditional view of toothed-whale monophyly) but with increased bootstrap support at nearly every node of the tree.     

Assessing Uncertainty in the Rooting of the SARS-CoV-2 Phylogeny

The rooting of the SARS-CoV-2 phylogeny is important for understanding the origin and early spread of the virus. Previously published phylogenies have used different rootings that do not always provide consistent results. We investigate several different strategies for rooting the SARS-CoV-2 tree and provide measures of statistical uncertainty for all methods. We show that methods based on the molecular clock tend to place the root in the B clade, whereas methods based on outgroup rooting tend to place the root in the A clade. The results from the two approaches are statistically incompatible, possibly as a consequence of deviations from a molecular clock or excess back-mutations. We also show that none of the methods provide strong statistical support for the placement of the root in any particular edge of the tree. These results suggest that phylogenetic evidence alone is unlikely to identify the origin of the SARS-CoV-2 virus and we caution against strong inferences regarding the early spread of the virus based solely on such evidence.     

Comparison of methods for rooting phylogenetic trees: A case study using Orcuttieae (Poaceae: Chloridoideae)

DNA sequence data (cpDNA trnL intron and nrDNA ITS1 and ITS2) were analyzed to identify relationships within Orcuttieae, a small tribe of endangered grasses endemic to vernal pools in California and Baja California. The tribe includes three genera: Orcuttia, Tuctoria, and Neostapfia. All three genera carry out C₄ photosynthesis but aquatic taxa of Orcuttia lack Kranz anatomy. The unusual habitat preference of the tribe is coupled with the atypical development of C₄ photosynthesis without Kranz anatomy. Furthermore, the tribe has no known close relatives and has been noted to be phylogenetically isolated within the subfamily Chloridoideae. In this study we examine the problem of inferring the root of the tribe in the absence of an identified outgroup, analyze the phylogenetic relationships of the constituent taxa, and evaluate the evolutionary development of C₄ photosynthesis. We compare four methods for inferring the root of the tree: (1) the outgroup method, (2) midpoint rooting, the imposition of a molecular clock for both (3) maximum likelihood (ML) and (4) Bayesian analysis. We examine the consequences of each method for the inferred phylogenetic relationships. Three of the methods (outgroup rooting and the ML and Bayesian molecular clock analyses) suggest that the root of Orcuttieae is between Neostapfia and the Tuctoria/Orcuttia lineage, while midpoint rooting gives a different root. The Bayesian method additionally provides information about probabilities associated with other possible root locations. Assuming that the true root of Orcuttieae is between Neostapfia and the Tuctoria/Orcuttia lineage, our data indicate Neostapfia and Orcuttia are both monophyletic, while Tuctoria is paraphyletic (with no synapomorphies in either dataset) and forming a grade between the other two genera and needs taxonomic revision. Our data support the hypothesis that Orcuttieae was derived from a terrestrial ancestor and evolved specializations to an aquatic environment, including C₄ photosynthesis without Kranz anatomy.     

An evaluation of elongation factor 1 alpha as a phylogenetic marker for eukaryotes

Elongation factor 1 alpha (EF-1 alpha) is a highly conserved ubiquitous protein involved in translation that has been suggested to have desirable properties for phylogenetic inference. To examine the utility of EF-1 alpha as a phylogenetic marker for eukaryotes, we studied three properties of EF-1 alpha trees: congruency with other phyogenetic markers, the impact of species sampling, and the degree of substitutional saturation occurring between taxa. Our analyses indicate that the EF-1 alpha tree is congruent with some other molecular phylogenies in identifying both the deepest branches and some recent relationships in the eukaryotic line of descent. However, the topology of the intermediate portion of the EF-1 alpha tree, occupied by most of the protist lineages, differs for different phylogenetic methods, and bootstrap values for branches are low. Most problematic in this region is the failure of all phylogenetic methods to resolve the monophyly of two higher-order protistan taxa, the Ciliophora and the Alveolata. JACKMONO analyses indicated that the impact of species sampling on bootstrap support for most internal nodes of the eukaryotic EF-1 alpha tree is extreme. Furthermore, a comparison of observed versus inferred numbers of substitutions indicates that multiple overlapping substitutions have occurred, especially on the branch separating the Eukaryota from the Archaebacteria, suggesting that the rooting of the eukaryotic tree on the diplomonad lineage should be treated with caution. Overall, these results suggest that the phylogenies obtained from EF-1 alpha are congruent with other molecular phylogenies in recovering the monophyly of groups such as the Metazoa, Fungi, Magnoliophyta, and Euglenozoa. However, the interrelationships between these and other protist lineages are not well resolved. This lack of resolution may result from the combined effects of poor taxonomic sampling, relatively few informative positions, large numbers of overlapping substitutions that obscure phylogenetic signal, and lineage-specific rate increases in the EF-1 alpha data set. It is also consistent with the nearly simultaneous diversification of major eukaryotic lineages implied by the "big-bang" hypothesis of eukaryote evolution.     

Origin and early evolution of eukaryotes inferred from the amino acid sequences of translation elongation factors 1α/Tu and 2/G

Phylogenetic placements of archaebacteria and protozoa are important in understanding the origin and early evolution of eukaryotes. These problems have been analyzed mainly by comparisons of small subunit ribosomal RNA (SrRNA) sequences. However, the SrRNA phylogeny may sometimes be unreliable, especially when base compositions are biased among species. Because it is difficult to take full account of the bias in inferring the SrRNA tree, alternative examinations using protein sequence data have been very much desired. We analyzed the phylogenetic relationship among eukaryotes, archaebacteria, and eubacteria by the ML method of protein phylogeny using amino acid sequence data of EF-1α/Tu and 2/G. The unrooted tree analyses of both the EF-1α/Tu and 2/G consistently demonstrated that the ‘eocyte’ tree, in which archaebacteria are not monophyletic but eocytes are closer to eukaryotes than to other archaebacteria, is very likely. Further analysis using a composite tree of EF-1α/Tu and 2/G suggested that archaebacteria are closer to eukaryotes than to eubacteria but are not monophyletic. These results clearly support the hypothesis that eukaryotes have evolved from the eocyte-like organism. We also analyzed a protozoan phylogeny including mitochondrion-lacking species by the ML method using EF-1α and EF-2 data sets, and demonstrated (a) that two mitochondrion-lacking species, G. plecoglossi (Microsporidians) and G. lamblia (Diplomonads) probably represent the first and the second earliest offshoots of eukaryotes, respectively; (b) that Trypanosoma is not likely to have diverged next to Giardia as suggested by the SrRNA tree, but shows high affinity with higher eukaryotes; and (c) that protein phylogeny would give a robust estimation because amino acid compositions of conservative proteins do not differ significantly among species.     

Different gymnosperm outgroups have (mostly) congruent signal regarding the root of flowering plant phylogeny

We examined multiple plastid genes from a diversity of gymnosperm lineages to explore the consistency of signal among different outgroups for rooting flowering plant phylogeny. For maximum parsimony (MP), most outgroups attach on a branch of the underlying ingroup tree that leads to Amborella. Maximum likelihood (ML) analyses either root angiosperms on a nearby branch or find split support for these neighboring root placements, depending on the outgroup. The inclusion of two species of Hydatellaceae, recently recognized as an ancient line of angiosperms, does not aid in inference of the root. Cost profiles for placing the root in suboptimal locations are highly correlated across most outgroup comparisons, even comparing MP and ML profiles. Those for Gnetales are the most deviant of all those considered. This divergent outgroup either attaches on a long eudicot branch with moderate bootstrap support in MP analyses or supports no particular root location in ML analysis. Removing the most rapidly evolving sites in rate classifications based on two divergent angiosperm root placements with Gnetales yields strongly conflicting root placements in MP analysis, despite substantial overlap in the estimated sets of conservative sites. However, the generally high consistency in rooting signal among distantly related gymnosperm clades suggests that the long branch connecting angiosperms to their extant relatives may not interfere substantially with inference of the angiosperm root.     

Two new avian mitochondrial genomes (penguin and goose) and a summary of bird and reptile mitogenomic features

We report complete mitochondrial (mt) genomes for a penguin (little blue, Eudyptula minor) and a goose (greater white-fronted, Anser albifrons). A revised annotation of avian and reptile mt genomes has been carried out, which improves consistency of labeling gene start and stop positions. In conjunction with this, a summary of mt gene features is presented and a number of conserved patterns and interesting differences identified. The protein-coding genes from the two new genomes were analysed together with those from 17 other birds plus outgroup (reptile) taxa. The unrooted amino acid tree from 19 avian genomes was locally stable with many high bootstrap values using several maximum likelihood methods. In particular, Anseriformes (goose and duck) grouped strongly with Galliformes (chicken) to form Gallianseres, while the penguin paired firmly with the stork. The position where the outgroup joined the avian tree varied with the combination of outgroup taxa used. The three best supported positions of the root were passerine, but the traditional rooting position between paleognaths and neognaths could not be excluded.