Relationships among the major lineages of Dictyoptera: the effect of outgroup selection on dictyopteran tree topology

Abstract Dictyoptera, comprising Blattaria, Isoptera, and Mantodea, are diverse in appearance and life history, and are strongly supported as monophyletic. We downloaded COII, 16S, 18S, and 28S sequences of 39 dictyopteran species from GenBank. Ribosomal RNA sequences were aligned manually with reference to secondary structure. We included morphological data (maximum of 175 characters) for 12 of these taxa and for an additional 15 dictyopteran taxa (for which we had only morphological data). We had two datasets, a 59‐taxon dataset with five outgroup taxa, from Phasmatodea (2 taxa), Mantophasmatodea (1 taxon), Embioptera (1 taxon), and Grylloblattodea (1 taxon), and a 62‐taxon dataset with three additional outgroup taxa from Plecoptera (1 taxon), Dermaptera (1 taxon) and Orthoptera (1 taxon). We analysed the combined molecular?morphological dataset using the doublet and MK models in Mr Bayes , and using a parsimony heuristic search in paup . Within the monophyletic Mantodea, Mantoida is recovered as sister to the rest of Mantodea, followed by Chaeteessa; the monophyly of most of the more derived families as defined currently is not supported. We recovered novel phylogenetic hypotheses about the taxa within Blattodea (following Hennig, containing Isoptera). Unique to our study, one Bayesian analysis places Polyphagoidea as sister to all other Dictyoptera; other analyses and/or the addition of certain orthopteran sequences, however, place Polyphagoidea more deeply within Dictyoptera. Isoptera falls within the cockroaches, sister to the genus Cryptocercus. Separate parsimony analyses of independent gene fragments suggest that gene selection is an important factor in tree reconstruction. When we varied the ingroup taxa and/or outgroup taxa, the internal dictyopteran relationships differed in the position of several taxa of interest, including Cryptocercus, Polyphaga, Periplaneta and Supella. This provides further evidence that the choice of both outgroup and ingroup taxa greatly affects tree topology.     

Optimal outgroup analysis

We present and critically examine a statistical criterion for the selection of outgroup taxa for rooting evolutionary trees. The criterion is the amount of phylogenetic signal for the ingroup when the states of the candidate outgroup taxa are assumed to be plesiomorphic relative to the ingroup for the purpose of measuring plesiomorphy content of the outgroup taxon. A statistical measure of rooted, ingroup signal was subjected to a suite of critical tests which indicate that it provides a proxy measure of plesiomorphy content. As the evolutionary distance between the ingroup ancestral node and outgroup taxa increases, the tree-independent measure of signal decreases, tracking the decay in plesiomorphy content and the increase in convergence to the ingroup states. We show that a priori generalizations about optimal outgroup taxon sampling strategies are likely to be misleading, and that testing for the suitability of available outgroup taxon sampling in specific instances is warranted. Software for optimal outgroup analysis is available.     

Mitochondrial genomes in the Hymenoptera and their utility as phylogenetic markers

Abstract.  In this study, we assessed the ability of mitochondrial genome sequences to recover a test phylogeny of five hymenopteran taxa from which phylogenetic relationships are well accepted. Our analyses indicated that the test phylogeny was well recovered in all nucleotide Bayesian analyses when all the available holometabolan (i.e. outgroup) taxa were included, but only in Bayesian analyses excluding third codon positions when only the hymenopteran representatives and a single outgroup were included. This result suggests that taxon sampling of the outgroup might be as important as taxon sampling of the ingroup when recovering hymenopteran phylogenetic relationships using whole mitochondrial genomes. Parsimony analyses were more sensitive to both taxon sampling and the analytical model than Bayesian analyses, and analyses using the protein dataset did not recover the test phylogeny. In general, mitochondrial genomes did not resolve the position of the Hymenoptera within the Holometabola with confidence, suggesting that an increased taxon sampling, both within the Holometabola and among outgroups, is necessary.     

类群取样与系统发育分析精确度之探索

Appropriate and extensive taxon sampling is one of the most important determinants of accurate phylogenetic estimation. In addition, accuracy of inferences about evolutionary processes obtained from phylogenetic analyses is improved significantly by thorough taxon sampling efforts. Many recent efforts to improve phylogenetic estimates have focused instead on increasing sequence length or the number of overall characters in the analysis, and this often does have a beneficial effect on the accuracy of phylogenetic analyses. However, phylogenetic analyses of few taxa (but each represented by many characters) can be subject to strong systematic biases, which in turn produce high measures of repeatability (such as bootstrap proportions) in support of incorrect or misleading phylogenetic results. Thus, it is important for phylogeneticists to consider both the sampling of taxa, as well as the sampling of characters, in designing phylogenetic studies. Taxon sampling also improves estimates of evolutionary parameters derived from phylogenetic trees, and is thus important for improved applications of phylogenetic analyses. Analysis of sensitivity to taxon inclusion, the possible effects of long-branch attraction, and sensitivity of parameter estimation for model-based methods should be a part of any careful and thorough phylogenetic analysis. Furthermore, recent improvements in phylogenetic algorithms and in computational power have removed many constraints on analyzing large, thoroughly sampled data sets. Thorough taxon sampling is thus one of the most practical ways to improve the accuracy of phylogenetic estimates, as well as the accuracy of biological inferences that are based on these phylogenetic trees.     

EFFECT OF TAXON SAMPLING,CHARACTER WEIGHTING,AND COMBINED DATA ON THE INTERPRETATION OF RELATIONSHIPS AMONG THE HETEROKONT ALGAE1

Nuclear ribosomal small subunit and chloroplast rbcL sequence data for heterokont algae and potential outgroup taxa were analyzed separately and together using maximum parsimony. A series of taxon sampling and character weighting experiments was performed. Traditional classes (e.g. diatoms, Phaeophyceae, etc.) were monophyletic in most analyses of either data set and in analyses of combined data. Relationships among classes and of heterokont algae to outgroup taxa were sensitive to taxon sampling. Bootstrap (BS) values were not always predictive of stability of nodes in taxon sampling experiments or between analyses of different data sets. Reweighting sites by the rescaled consistency index artificially inflates BS values in the analysis of rbcL data. Inclusion of the third codon position from rbcL enhanced signal despite the superficial appearance of mutational saturation. Incongruence between data sets was largely due to placement of a few problematic taxa, and so data were combined. BS values for the combined analysis were much higher than for analyses of each data set alone, although combining data did not improve support for heterokont monophyly.     

More genes or more taxa? The relative contribution of gene number and taxon number to phylogenetic accuracy

The relative contribution of taxon number and gene number to accuracy in phylogenetic inference is a major issue in phylogenetics and of central importance to the choice of experimental strategies for the successful reconstruction of a broad sketch of the tree of life. Maximization of the number of taxa sampled is the strategy favored by most phylogeneticists, although its necessity remains the subject of debate. Vast increases in gene number are now possible due to advances in genomics, but large numbers of genes will be available for only modest numbers of taxa, raising the question of whether such genome-scale phylogenies will be robust to the addition of taxa. To examine the relative benefit of increasing taxon number or gene number to phylogenetic accuracy, we have developed an assay that utilizes the symmetric difference tree distance as a measure of phylogenetic accuracy. We have applied this assay to a genome-scale data matrix containing 106 genes from 14 yeast species. Our results show that increasing taxon number correlates with a slight decrease in phylogenetic accuracy. In contrast, increasing gene number has a significant positive effect on phylogenetic accuracy. Analyses of an additional taxon-rich data matrix from the same yeast clade show that taxon number does not have a significant effect on phylogenetic accuracy. The positive effect of gene number and the lack of effect of taxon number on phylogenetic accuracy are also corroborated by analyses of two data matrices from mammals and angiosperm plants, respectively. We conclude that, for typical data sets, the number of genes utilized may be a more important determinant of phylogenetic accuracy than taxon number.     

Re-examination of the phylogeny of Rhacophoridae (Anura) based on mitochondrial and nuclear DNA

The phylogenetic relationships among rhacophorid frogs are under dispute. We use partial sequences of three mitochondrial (12S rRNA, 16S rRNA, and cytochrome b) and three nuclear protein-coding (Rag-1, rhodopsin exon 1, and tyrosinase exon 1) genes from 57 ingroup taxa and eight outgroup taxa to propose a hypothesis for phylogenetic relationships within Rhacophoridae. Our results support recognition of the genus Feihyla, and Chiromantis is the sister taxon to the clade formed by Feihyla, Polypedates and Rhacophorus. We place Aquixalus odontotarsus within Kurixalus, and the remaining species of Aquixalus and Philautus jinxiuensis into the genus Gracixalus. We give Philautus (Kirtixalus) the rank of genus and place Philautus menglaensis within it. The division of species groups among Chinese Rhacophorus needs revision, and a cryptic species is revealed within Rhacophorus nigropunctatus. Rhacophorus pingbianensis is considered a synonym of Rhacophorus omeimontis. The validity of Rhacophorus hui is confirmed by present molecular evidence.     

A global phylogeny of apple snails: Gondwanan origin, generic relationships, and the influence of outgroup choice (Caenogastropoda: Ampullariidae)

Apple snails (Ampullariidae) are a diverse family of pantropical freshwater snails and an important evolutionary link to the common ancestor of the largest group of living gastropods, the Caenogastropoda. A clear understanding of relationships within the Ampullariidae, and identification of their sister taxon, is therefore important for interpreting gastropod evolution in general. Unfortunately, the overall pattern has been clouded by confused systematics within the family and equivocal results regarding the family's sister group relationships. To clarify the relationships among ampullariid genera and to evaluate the influence of including or excluding possible sister taxa, we used data from five genes, three nuclear and two mitochondrial, from representatives of all nine extant ampullariid genera, and species of Viviparidae, Cyclophoridae, and Campanilidae, to reconstruct the phylogeny of apple snails, and determine their affinities to these possible sister groups. The results obtained indicate that the Old and New World ampullariids are reciprocally monophyletic with probable Gondwanan origins. All four Old World genera, Afropomus, Saulea, Pila, and Lanistes, were recovered as monophyletic, but only Asolene, Felipponea, and Pomella were monophyletic among the five New World genera, with Marisa paraphyletic and Pomacea polyphyletic. Estimates of divergence times among New World taxa suggest that diversification began shortly after the separation of Africa and South America and has probably been influenced by hydrogeological events over the last 90 Myr. The sister group of the Ampullariidae remains unresolved, but analyses omitting certain outgroup taxa suggest the need for dense taxonomic sampling to increase phylogenetic accuracy within the ingroup. The results obtained also indicate that defining the sister group of the Ampullariidae and clarifying relationships among basal caenogastropods will require increased taxon sampling within these four families, and synthesis of both morphological and molecular data. © 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 98 , 61–76.     

Two issues in archaeological phylogenetics: taxon construction and outgroup selection

Cladistics is widely used in biology and paleobiology to construct phylogenetic hypotheses, but rarely has it been applied outside those disciplines. There is, however, no reason to suppose that cladistics is not applicable to anything that evolves by cladogenesis and produces a nested hierarchy of taxa. This includes cultural phenomena such as languages and tools recovered from archaeological contexts. Two methodological issues assume primacy in attempts to extend cladistics to archaeological materials: the construction of analytical taxa and the selection of appropriate outgroups. In biology the species is the primary taxonomic unit used, irrespective of the debates that have arisen in phylogenetic theory over the nature of species. Also in biology the phylogenetic history of a group of taxa usually is well enough known that an appropriate taxon can be selected as an outgroup. No analytical unit parallel to the species exists in archaeology, and thus taxa have to be constructed specifically for phylogenetic analysis. One method of constructing taxa is paradigmatic classification, which defines classes (taxa) on the basis of co-occurring, unweighted character states. Once classes have been created, a form of occurrence seriation-an archaeological method based on the theory of cultural transmission and heritability-offers an objective basis for selecting an outgroup.     

Phylogenetic evidence for the herbaceous origin of angiosperms

The ancestral angiosperm is commonly interpreted as an arborescent to shrubby magnolialean with large, multiparted, complex flowers. We examined this hypothesis using a phylogenetic analysis of new and reevaluated characters polarizabled with outgroup comparison. Our cladistic analysis of basal angiosperms placed the nonmagnolialeanChloranthaceae andPiperaceae at the bottom of the tree. We further inferred the probable ancestral states of characters not polarizable with outgroup comparison by examining their distribution among taxa at the base of our cladogram. The sum of ancestral character states suggests that the protoangiosperm was a diminutive, rhizomatous to scrambling perennial herb, with small, simple flowers.     

Increased taxon sampling greatly reduces phylogenetic error

Several authors have argued recently that extensive taxon sampling has a positive and important effect on the accuracy of phylogenetic estimates. However, other authors have argued that there is little benefit of extensive taxon sampling, and so phylogenetic problems can or should be reduced to a few exemplar taxa as a means of reducing the computational complexity of the phylogenetic analysis. In this paper we examined five aspects of study design that may have led to these different perspectives. First, we considered the measurement of phylogenetic error across a wide range of taxon sample sizes, and conclude that the expected error based on randomly selecting trees (which varies by taxon sample size) must be considered in evaluating error in studies of the effects of taxon sampling. Second, we addressed the scope of the phylogenetic problems defined by different samples of taxa, and argue that phylogenetic scope needs to be considered in evaluating the importance of taxon-sampling strategies. Third, we examined the claim that fast and simple tree searches are as effective as more thorough searches at finding near-optimal trees that minimize error. We show that a more complete search of tree space reduces phylogenetic error, especially as the taxon sample size increases. Fourth, we examined the effects of simple versus complex simulation models on taxonomic sampling studies. Although benefits of taxon sampling are apparent for all models, data generated under more complex models of evolution produce higher overall levels of error and show greater positive effects of increased taxon sampling. Fifth, we asked if different phylogenetic optimality criteria show different effects of taxon sampling. Although we found strong differences in effectiveness of different optimality criteria as a function of taxon sample size, increased taxon sampling improved the results from all the common optimality criteria. Nonetheless, the method that showed the lowest overall performance (minimum evolution) also showed the least improvement from increased taxon sampling. Taking each of these results into account re-enforces the conclusion that increased sampling of taxa is one of the most important ways to increase overall phylogenetic accuracy.     

Taxon sampling and seed plant phylogeny

We investigated the effects of taxon sampling on phylogenetic inference by exchanging terminals in two sizes of rbcL matrices for seed plants, applying parsimony and bayesian analyses to ten 38‐taxon matrices and ten 80‐taxon matrices. In comparing tree topologies we concentrated on the position of the Gnetales, an important group whose placement has long been disputed. With either method, trees obtained from different taxon samples could be mutually contradictory and even disagree on groups that seemed strongly supported. Adding terminals improved the consistency of results for unweighted parsimony, but not for parsimony with third positions excluded and not for bayesian analysis, particularly when the general time‐reversible model was employed. This suggests that attempting to resolve deep relationships using only a few taxa can lead to spurious conclusions, groupings unlikely to be repeatable with different taxon samplings or larger data sets. The effect of taxon sampling has not generally been recognized, and phylogenetic studies of seed plants have often been based on few taxa. Such insufficient sampling may help explain the variety of phylogenetic hypotheses for seed plants proposed in recent years. We recommend that restricted data sets such as single‐gene subsets of multigene studies should be reanalyzed with alternative selections of terminals to assess topological consistency.     

Phylogenetic analysis of RAD‐seq data: examining the influence of gene genealogy conflict on analysis of concatenated data

One of the major issues in phylogenetic analysis is that gene genealogies from different gene regions may not reflect the true species tree or history of speciation. This has led to considerable debate about whether concatenation of loci is the best approach for phylogenetic analysis. The application of Next‐generation sequencing techniques such as RAD‐seq generates thousands of relatively short sequence reads from across the genomes of the sampled taxa. These data sets are typically concatenated for phylogenetic analysis leading to data sets that contain millions of base pairs per taxon. The influence of gene region conflict among so many loci in determining the phylogenetic relationships among taxa is unclear. We simulated RAD‐seq data by sampling 100 and 500 base pairs from alignments of over 6000 coding regions that each produce one of three highly supported alternative phylogenies of seven species of Drosophila. We conducted phylogenetic analyses on different sets of these regions to vary the sampling of loci with alternative gene trees to examine the effect on detecting the species tree. Irrespective of sequence length sampled per region and which subset of regions was used, phylogenetic analyses of the concatenated data always recovered the species tree. The results suggest that concatenated alignments of Next‐generation data that consist of many short sequences are robust to gene tree/species tree conflict when the goal is to determine the phylogenetic relationships among taxa.     

A phylogenetic hypothesis for Asilidae based on a total evidence analysis of morphological and DNA sequence data (Insecta: Diptera: Brachycera: Asiloidea)

An hypothesis of phylogenetic relationships of Asilidae and its constituent taxa is presented, combining morphological and DNA sequence data in a total evidence framework. It is based on 77 robber fly species, 11 Asiloidea outgroup species, 211 morphological characters of the adult fly, and approximately 7300 bp of nuclear DNA from five genes (18S and 28S rDNA, AATS, CAD, and EF-1α protein-encoding DNA). The equally weighted, simultaneous parsimony analysis under dynamic homology in POY resulted in a single most parsimonious cladogram with a cost of 27,582 (iterative pass optimization; 27,703 under regular direct optimization). Six of the 12 included subfamily taxa are recovered as monophyletic. Trigonomiminae, previously always considered as monophyletic based on morphology, is shown to be non-monophyletic. Two of the three Trigonomiminae genera, Holcocephala Jaennicke, 1867 and Rhipidocephala Hermann, 1926, group unexpectedly as the sister taxon to all other Asilidae. Laphriinae, previously seen in the latter position, is the sister group of the remaining Asilidae. Five other subfamily taxa, i.e. Brachyrhopalinae, Dasypogoninae, Stenopogoninae, Tillobromatinae, and Willistonininae, are also shown to be non-monophyletic. The phylogenetic relationships among the higher-level taxa are partly at odds with findings of a recently published morphological study based on more extensive taxon sampling. The total evidence hypothesis is considered as the most informative one, but the respective topologies from the total-evidence, morphology-only, and molecular-only analyses are compared and contrasted in order to discuss the signals from morphological versus molecular data, and to analyze whether the molecular data outcompete the fewer morphological characters. A clade Apioceridae+Mydidae is corroborated as the sister taxon to Asilidae.     

1 Taxon sampling and inferences about diatom phylogeny

Proper taxon sampling is one of the greatest challenges to understanding phylogenetic relationships, perhaps as important as choice of optimality criterion or data type. This has been demonstrated in diatoms where centric diatoms may either be strongly supported as monophyletic or paraphyletic when analyzing SSU rDNA sequences using the same optimality criterion. The effect of ingroup and outgroup taxon sampling on relationships of diatoms is explored for diatoms as a whole and for the order Thalassiosirales. In the latter case, SSU rDNA and rbcL sequence data result in phylogenetic relationships that appear to be strongly incongruent with morphology and broadly incongruent with the fossil record. For example, Cyclotella stelligera Cleve & Grunow behaves like a rogue taxon, jumping from place to place throughout the tree. Morphological data place C. stelligera near the base of the freshwater group as sister to the extinct genus Mesodictyon Theriot and Bradbury, suggesting that it is an old, long branch that might be expected to “misbehave” in poorly sampled trees. Cyclotella stelligera and C. bodanica Grunow delimit the diameter of morphological diversity in Cyclotella, so increased sampling of intermediate taxa will be critical to resolving this part of the tree. Morphology is sampled for a much greater number of taxa and many transitional states of putative synapomorphies seem to suggest a robust morphological hypothesis. The Thalassiosirales are unstable with regards to taxon sampling in the genetic data, suggesting that perhaps the morphological hypothesis is (for now) preferable.     

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.     

Phylogenetic relationships of the Santalales and relatives

Summary Determining relationships among parasitic angiosperms has often been difficult owing to frequent morphological reductions in floral and vegetative features. We report 18S (small-subunit) rRNA sequences for representative genera of three families within the Santalales (Olacaceae, Santalaceae, and Viscaceae) and six outgroup dicot families (Celastraceae, Cornaceae, Nyssaceae, Buxaceae, Apiaceae, and Araliaceae). Using Wagner parsimony analysis, one most parsimonius tree resulted that shows the Santalales to be a holophyletic taxon most closely related toEuronymus (Celastraceae). The santalalean taxa showed approximately 13% more transitional mutations than the group of seven other dicot species. This suggests a higher fixation rate for mutations in these organisms, possibly owing to a relaxation of selection pressures at the molecular level in parasitic vs nonparasitic plants. Outgroup relationships are generally in accord with current taxonomic classifications, such as the grouping of Nyssaceae and Cornaceae together (Cornales) and the grouping of Araliaceae with Apiaceae (Apiales). These data provide the first nucleotide sequences for any parasitic flowering plant and support the contention that rRNA sequence analysis can result in robust phylogenetic comparisons at the family level and above.     

Anchored hybrid enrichment provides new insights into the phylogeny and evolution of longhorned beetles (Cerambycidae)

Cerambycidae is a species‐rich family of mostly wood‐feeding (xylophagous) beetles containing nearly 35 000 known species. The higher‐level phylogeny of C erambycidae has never been robustly reconstructed using molecular phylogenetic data or a comprehensive sample of higher taxa, and its internal relationships and evolutionary history remain the subjects of ongoing debate. We reconstructed the higher‐level phylogeny of C erambycidae using phylogenomic data from 522 single copy nuclear genes, generated via anchored hybrid enrichment. Our taxon sample (31 C hrysomeloidea, four outgroup taxa: two C urculionoidea and two C ucujoidea) included exemplars of all families and 23 of 30 subfamilies of C hrysomeloidea (18 of 19 non‐chrysomelid C hrysomeloidea), with a focus on the large family C erambycidae. Our results reveal a monophyletic C erambycidae s.s. in all but one analysis, and a polyphyletic C erambycidae s.l. When monophyletic, C erambycidae s.s. was sister to the family D isteniidae. Relationships among the subfamilies of C erambycidae s.s. were also recovered with strong statistical support except for C erambycinae being made paraphyletic by Dorcasomus A udinet‐S erville (D orcasominae) in the nucleotide (but not amino acid) trees. Most other chrysomeloid families represented by more than one terminal taxon – C hrysomelidae, D isteniidae, V esperidae and O rsodacnidae – were monophyletic, but M egalopodidae was rendered paraphyletic by Cheloderus G ray (O xypeltidae). Our study corroborates some relationships within C hrysomeloidea that were previously inferred from morphological data, while also reporting several novel relationships. The present work thus provides a robust framework for future, more deeply taxon‐sampled, phylogenetic and evolutionary studies of the families and subfamilies of C erambycidae s.l. and other C hrysomeloidea.     

Phylogenetic analyses of Vitis (Vitaceae) based on complete chloroplast genome sequences: effects of taxon sampling and phylogenetic methods on resolving relationships among rosids

Background  

The Vitaceae (grape) is an economically important family of angiosperms whose phylogenetic placement is currently unresolved. Recent phylogenetic analyses based on one to several genes have suggested several alternative placements of this family, including sister to Caryophyllales, asterids, Saxifragales, Dilleniaceae or to rest of rosids, though support for these different results has been weak. There has been a recent interest in using complete chloroplast genome sequences for resolving phylogenetic relationships among angiosperms. These studies have clarified relationships among several major lineages but they have also emphasized the importance of taxon sampling and the effects of different phylogenetic methods for obtaining accurate phylogenies. We sequenced the complete chloroplast genome of Vitis vinifera and used these data to assess relationships among 27 angiosperms, including nine taxa of rosids.     

Heterogeneity of nucleotide frequencies among evolutionary lineages and phylogenetic inference

A major assumption of many molecular phylogenetic methods is the homogeneity of nucleotide frequencies among taxa, which refers to the equality of the nucleotide frequency bias among species. Changes in nucleotide frequency among different lineages in a data set are thought to lead to erroneous phylogenetic inference because unrelated clades may appear similar because of evolutionarily unrelated similarities in nucleotide frequencies. We tested the effects of the heterogeneity of nucleotide frequency bias on phylogenetic inference, along with the interaction between this heterogeneity and stratified taxon sampling, by means of computer simulations using evolutionary parameters derived from genomic databases. We found that the phylogenetic trees inferred from data sets simulated under realistic, observed levels of heterogeneity for mammalian genes were reconstructed with accuracy comparable to those simulated with homogeneous nucleotide frequencies; the results hold for Neighbor-Joining, minimum evolution, maximum parsimony, and maximum-likelihood methods. The LogDet distance method, specifically designed to deal with heterogeneous nucleotide frequencies, does not perform better than distance methods that assume substitution pattern homogeneity among sequences. In these specific simulation conditions, we did not find a significant interaction between phylogenetic accuracy and substitution pattern heterogeneity among lineages, even when the taxon sampling is increased.