Evolution rate nonuniformity in <Emphasis Type="Italic">Drosophila virilis</Emphasis> species group. II. Group of Drosophilas: Application of Tajima’s test

The steadiness of the molecular clock was estimated in 11 Drosophila species of the virilis group by sequences of five genes by applying Tajima’s Simple Method. The main characteristic of this method is the independence of phylogenetic constructions. The obtained results have completely confirmed the conclusions drawn relying on the application of the two-cluster test and the branch-length test. In addition, irregularity of the molecular clock was found in some linlages of the D. virilis species group.     

Discordance of species trees with their most likely gene trees: the case of five taxa

Under a coalescent model for within-species evolution, gene trees may differ from species trees to such an extent that the gene tree topology most likely to evolve along the branches of a species tree can disagree with the species tree topology. Gene tree topologies that are more likely to be produced than the topology that matches that of the species tree are termed anomalous, and the region of branch-length space that gives rise to anomalous gene trees (AGTs) is the anomaly zone. We examine the occurrence of anomalous gene trees for the case of five taxa, the smallest number of taxa for which every species tree topology has a nonempty anomaly zone. Considering all sets of branch lengths that give rise to anomalous gene trees, the largest value possible for the smallest branch length in the species tree is greater in the five-taxon case (0.1934 coalescent time units) than in the previously studied case of four taxa (0.1568). The five-taxon case demonstrates the existence of three phenomena that do not occur in the four-taxon case. First, anomalous gene trees can have the same unlabeled topology as the species tree. Second, the anomaly zone does not necessarily enclose a ball centered at the origin in branch-length space, in which all branches are short. Third, as a branch length increases, it is possible for the number of AGTs to increase rather than decrease or remain constant. These results, which help to describe how the properties of anomalous gene trees increase in complexity as the number of taxa increases, will be useful in formulating strategies for evading the problem of anomalous gene trees during species tree inference from multilocus data.     

Maximum likelihood molecular clock comb: analytic solutions.

Maximum likelihood (ML) is increasingly used as an optimality criterion for selecting evolutionary trees, but finding the global optimum is a hard computational task. Because no general analytic solution is known, numeric techniques such as hill climbing or expectation maximization (EM), are used in order to find optimal parameters for a given tree. So far, analytic solutions were derived only for the simplest model--three taxa, two state characters, under a molecular clock. Four taxa rooted trees have two topologies--the fork (two subtrees with two leaves each) and the comb (one subtree with three leaves, the other with a single leaf). In a previous work, we devised a closed form analytic solution for the ML molecular clock fork. In this work, we extend the state of the art in the area of analytic solutions ML trees to the family of all four taxa trees under the molecular clock assumption. The change from the fork topology to the comb incurs a major increase in the complexity of the underlying algebraic system and requires novel techniques and approaches. We combine the ultrametric properties of molecular clock trees with the Hadamard conjugation to derive a number of topology dependent identities. Employing these identities, we substantially simplify the system of polynomial equations. We finally use tools from algebraic geometry (e.g., Gr?bner bases, ideal saturation, resultants) and employ symbolic algebra software to obtain analytic solutions for the comb. We show that in contrast to the fork, the comb has no closed form solutions (expressed by radicals in the input data). In general, four taxa trees can have multiple ML points. In contrast, we can now prove that under the molecular clock assumption, the comb has a unique (local and global) ML point. (Such uniqueness was previously shown for the fork.).     

Outgroup misplacement and phylogenetic inaccuracy under a molecular clock--a simulation study

We conducted a simulation study of the phylogenetic methods UPGMA, neighbor joining, maximum parsimony, and maximum likelihood for a five-taxon tree under a molecular clock. The parameter space included a small region where maximum parsimony is inconsistent, so we tested inconsistency correction for parsimony and distance correction for neighbor joining. As expected, corrected parsimony was consistent. For these data, maximum likelihood with the clock assumption outperformed each of the other methods tested. The distance-based methods performed marginally better than did maximum parsimony and maximum likelihood without the clock assumption. Data correction was generally detrimental to accuracy, especially for short sequence lengths. We identified another region of the parameter space where, although consistent for a given method, some incorrect trees were each selected with up to twice the frequency of the correct (generating) tree for sequences of bounded length. These incorrect trees are those where the outgroup has been incorrectly placed. In addition to this problem, the placement of the outgroup sequence can have a confounding effect on the ingroup tree, whereby the ingroup is correct when using the ingroup sequences alone, but with the inclusion of the outgroup the ingroup tree becomes incorrect.     

Rabbits, if anything, are likely Glires

Rodentia (e.g., mice, rats, dormice, squirrels, and guinea pigs) and Lagomorpha (e.g., rabbits, hares, and pikas) are usually grouped into the Glires. Status of this controversial superorder has been evaluated using morphology, paleontology, and mitochondrial plus nuclear DNA sequences. This growing corpus of data has been favoring the monophyly of Glires. Recently, Misawa and Janke [Mol. Phylogenet. Evol. 28 (2003) 320] analyzed the 6441 amino acids of 20 nuclear proteins for six placental mammals (rat, mouse, rabbit, human, cattle, and dog) and two outgroups (chicken and xenopus), and observed a basal position of the two murine rodents among the former. They concluded that "the Glires hypothesis was rejected." We here reanalyzed [loc. cit.] data set under maximum likelihood and Bayesian tree-building approaches, using phylogenetic models that take into account among-site variation in evolutionary rates and branch-length variation among proteins. Our observations support both the association of rodents and lagomorphs and the monophyly of Euarchontoglires (=Supraprimates) as the most likely explanation of the protein alignments. We conducted simulation studies to evaluate the appropriateness of lissamphibian and avian outgroups to root the placental tree. When the outgroup-to-ingroup evolutionary distance increases, maximum parsimony roots the topology along the long Mus-Rattus branch. Maximum likelihood, in contrast, roots the topology along different branches as a function of their length. Maximum likelihood appears less sensitive to the "long-branch attraction artifact" than is parsimony. Our phylogenetic conclusions were confirmed by the analysis of a different protein data set using a similar sample of species but different outgroups. We also tested the effect of the addition of afrotherian and xenarthran taxa. Using the linearized tree method, [loc. cit.] estimated that mice and rats diverged about 35 million years ago. Molecular dating based on the Bayesian relaxed molecular clock method suggests that the 95% credibility interval for the split between mice and rats is 7-17 Mya. We here emphasize the need for appropriate models of sequence evolution (matrices of amino acid replacement, taking into account among-site rate variation, and independent parameters across independent protein partitions) and for a taxonomically broad sample, and conclude on the likelihood that rodents and lagomorphs together constitute a monophyletic group (Glires).     

Bayesian Phylogenetic Inference using Relaxed-clocks and the Multispecies Coalescent

The multispecies coalescent (MSC) model accommodates both species divergences and within-species coalescent and provides a natural framework for phylogenetic analysis of genomic data when the gene trees vary across the genome. The MSC model implemented in the program bpp assumes a molecular clock and the Jukes–Cantor model, and is suitable for analyzing genomic data from closely related species. Here we extend our implementation to more general substitution models and relaxed clocks to allow the rate to vary among species. The MSC-with-relaxed-clock model allows the estimation of species divergence times and ancestral population sizes using genomic sequences sampled from contemporary species when the strict clock assumption is violated, and provides a simulation framework for evaluating species tree estimation methods. We conducted simulations and analyzed two real datasets to evaluate the utility of the new models. We confirm that the clock-JC model is adequate for inference of shallow trees with closely related species, but it is important to account for clock violation for distant species. Our simulation suggests that there is valuable phylogenetic information in the gene-tree branch lengths even if the molecular clock assumption is seriously violated, and the relaxed-clock models implemented in bpp are able to extract such information. Our Markov chain Monte Carlo algorithms suffer from mixing problems when used for species tree estimation under the relaxed clock and we discuss possible improvements. We conclude that the new models are currently most effective for estimating population parameters such as species divergence times when the species tree is fixed.     

Success of maximum likelihood phylogeny inference in the four-taxon case

We used simulated data to investigate a number of properties of maximum- likelihood (ML) phylogenetic tree estimation for the case of four taxa. Simulated data were generated under a broad range of conditions, including wide variation in branch lengths, differences in the ratio of transition and transversion substitutions, and the absence of presence of gamma-distributed site-to-site rate variation. Data were analyzed in the ML framework with two different substitution models, and we compared the ability of the two models to reconstruct the correct topology. Although both models were inconsistent for some branch-length combinations in the presence of site-to-site variation, the models were efficient predictors of topology under most simulation conditions. We also examined the performance of the likelihood ratio (LR) test for significant positive interior branch length. This test was found to be misleading under many simulation conditions, rejecting too often under some simulation conditions. Under the null hypothesis of zero length internal branch, LR statistics are assumed to be asymptotically distributed chi 2(1); with limited data, the distribution of LR statistics under the null hypothesis varies from chi 2(1).      

The devil in the details: interactions between the branch-length prior and likelihood model affect node support and branch lengths in the phylogeny of the Psoraceae

In popular use of Bayesian phylogenetics, a default branch-length prior is almost universally applied without knowing how a different prior would have affected the outcome. We performed Bayesian and maximum likelihood (ML) inference of phylogeny based on empirical nucleotide sequence data from a family of lichenized ascomycetes, the Psoraceae, the morphological delimitation of which has been controversial. We specifically assessed the influence of the combination of Bayesian branch-length prior and likelihood model on the properties of the Markov chain Monte Carlo tree sample, including node support, branch lengths, and taxon stability. Data included two regions of the mitochondrial ribosomal RNA gene, the internal transcribed spacer region of the nuclear ribosomal RNA gene, and the protein-coding largest subunit of RNA polymerase II. Data partitioning was performed using Bayes' factors, whereas the best-fitting model of each partition was selected using the Bayesian information criterion (BIC). Given the data and model, short Bayesian branch-length priors generate higher numbers of strongly supported nodes as well as short and topologically similar trees sampled from parts of tree space that are largely unexplored by the ML bootstrap. Long branch-length priors generate fewer strongly supported nodes and longer and more dissimilar trees that are sampled mostly from inside the range of tree space sampled by the ML bootstrap. Priors near the ML distribution of branch lengths generate the best marginal likelihood and the highest frequency of "rogue" (unstable) taxa. The branch-length prior was shown to interact with the likelihood model. Trees inferred under complex partitioned models are more affected by the stretching effect of the branch-length prior. Fewer nodes are strongly supported under a complex model given the same branch-length prior. Irrespective of model, internal branches make up a larger proportion of total tree length under the shortest branch-length priors compared with longer priors. Relative effects on branch lengths caused by the branch-length prior can be problematic to downstream phylogenetic comparative methods making use of the branch lengths. Furthermore, given the same branch-length prior, trees are on average more dissimilar under a simple unpartitioned model compared with a more complex partitioned models. The distribution of ML branch lengths was shown to better fit a gamma or Pareto distribution than an exponential one. Model adequacy tests indicate that the best-fitting model selected by the BIC is insufficient for describing data patterns in 5 of 8 partitions. More general substitution models are required to explain the data in three of these partitions, one of which also requires nonstationarity. The two mitochondrial ribosomal RNA gene partitions need heterotachous models. We found no significant correlations between, on the one hand, the amount of ambiguous data or the smallest branch-length distance to another taxon and, on the other hand, the topological stability of individual taxa. Integrating over several exponentially distributed means under the best-fitting model, node support for the family Psoraceae, including Psora, Protoblastenia, and the Micarea sylvicola group, is approximately 0.96. Support for the genus Psora is distinctly lower, but we found no evidence to contradict the current classification.     

When does the incongruence length difference test fail?

This paper examines the efficiency of the incongruence length difference test (ILD) proposed by Farris et al. (1994) for assessing the incongruence between sets of characters. DNA sequences were simulated under various evolutionary conditions: (1) following symmetric or asymmetric trees, (2) with various mutation rates, (3) with constant or variable evolutionary rates along the branches, and (4) with different among-site substitution rates. We first compared two sets of sequences generated along the same tree and under the same evolutionary conditions. The probability of a Type-I error (wrongly rejecting the true hypothesis of congruence) was substantially below the standard 5% level of significance given by the ILD test; this finding indicates that the choice of the 5% level is rather conservative in this case. We then compared two data sets, still generated along the same tree, but under different evolutionary conditions (constant vs. variable evolutionary rate, homogeneity vs. heterogeneity rate of substitution). Under these conditions, the probability of rejecting the true hypothesis of congruence was greater than the 5% given by the ILD test and increased with the number of sites and the degree to which the tree was asymmetric. Finally, the comparison of the two data sets, simulated under contrasting tree structures (symmetric vs. asymmetric) but under the same evolutionary conditions, led us to reject the hypothesis of congruence, albeit weakly, particularly when the number of informative sites was low and among-site substitution rate heterogeneous. We conclude that the ILD test has only limited power to detect incongruence caused by differences in the evolutionary conditions or in the tree topology, except when numerous characters are present and the substitution rate is homogeneous from site to site.     

Modelling heterotachy in phylogenetic inference by reversible-jump Markov chain Monte Carlo

The rate at which a given site in a gene sequence alignment evolves over time may vary. This phenomenon--known as heterotachy--can bias or distort phylogenetic trees inferred from models of sequence evolution that assume rates of evolution are constant. Here, we describe a phylogenetic mixture model designed to accommodate heterotachy. The method sums the likelihood of the data at each site over more than one set of branch lengths on the same tree topology. A branch-length set that is best for one site may differ from the branch-length set that is best for some other site, thereby allowing different sites to have different rates of change throughout the tree. Because rate variation may not be present in all branches, we use a reversible-jump Markov chain Monte Carlo algorithm to identify those branches in which reliable amounts of heterotachy occur. We implement the method in combination with our 'pattern-heterogeneity' mixture model, applying it to simulated data and five published datasets. We find that complex evolutionary signals of heterotachy are routinely present over and above variation in the rate or pattern of evolution across sites, that the reversible-jump method requires far fewer parameters than conventional mixture models to describe it, and serves to identify the regions of the tree in which heterotachy is most pronounced. The reversible-jump procedure also removes the need for a posteriori tests of 'significance' such as the Akaike or Bayesian information criterion tests, or Bayes factors. Heterotachy has important consequences for the correct reconstruction of phylogenies as well as for tests of hypotheses that rely on accurate branch-length information. These include molecular clocks, analyses of tempo and mode of evolution, comparative studies and ancestral state reconstruction. The model is available from the authors' website, and can be used for the analysis of both nucleotide and morphological data.     

Efficient biased estimation of evolutionary distances when substitution rates vary across sites

This paper deals with phylogenetic inference when the variability of substitution rates across sites (VRAS) is modeled by a gamma distribution. We show that underestimating VRAS, which results in underestimates for the evolutionary distances between sequences, usually improves the topological accuracy of phylogenetic tree inference by distance-based methods, especially when the molecular clock holds. We propose a method to estimate the gamma shape parameter value which is most suited for tree topology inference, given the sequences at hand. This method is based on the pairwise evolutionary distances between sequences and allows one to reconstruct the phylogeny of a high number of taxa (>1,000). Simulation results show that the topological accuracy is highly improved when using the gamma shape parameter value given by our method, compared with the true (unknown) value which was used to generate the data. Furthermore, when VRAS is high, the topological accuracy of our distance-based method is better than that of a maximum likelihood approach. Finally, a data set of Maoricicada species sequences is analyzed, which confirms the advantage of our method.     

Disk-covering, a fast-converging method for phylogenetic tree reconstruction.

The evolutionary history of a set of species is represented by a phylogenetic tree, which is a rooted, leaf-labeled tree, where internal nodes represent ancestral species and the leaves represent modern day species. Accurate (or even boundedly inaccurate) topology reconstructions of large and divergent trees from realistic length sequences have long been considered one of the major challenges in systematic biology. In this paper, we present a simple method, the Disk-Covering Method (DCM), which boosts the performance of base phylogenetic methods under various Markov models of evolution. We analyze the performance of DCM-boosted distance methods under the Jukes-Cantor Markov model of biomolecular sequence evolution, and prove that for almost all trees, polylogarithmic length sequences suffice for complete accuracy with high probability, while polynomial length sequences always suffice. We also provide an experimental study based upon simulating sequence evolution on model trees. This study confirms substantial reductions in error rates at realistic sequence lengths.     

Molecular clock fork phylogenies: closed form analytic maximum likelihood solutions

Maximum likelihood (ML) is increasingly used as an optimality criterion for selecting evolutionary trees, but finding the global optimum is a hard computational task. Because no general analytic solution is known, numeric techniques such as hill climbing or expectation maximization (EM) are used in order to find optimal parameters for a given tree. So far, analytic solutions were derived only for the simplest model-three-taxa, two-state characters, under a molecular clock. Quoting Ziheng Yang, who initiated the analytic approach,"this seems to be the simplest case, but has many of the conceptual and statistical complexities involved in phylogenetic estimation."In this work, we give general analytic solutions for a family of trees with four-taxa, two-state characters, under a molecular clock. The change from three to four taxa incurs a major increase in the complexity of the underlying algebraic system, and requires novel techniques and approaches. We start by presenting the general maximum likelihood problem on phylogenetic trees as a constrained optimization problem, and the resulting system of polynomial equations. In full generality, it is infeasible to solve this system, therefore specialized tools for the molecular clock case are developed. Four-taxa rooted trees have two topologies-the fork (two subtrees with two leaves each) and the comb (one subtree with three leaves, the other with a single leaf). We combine the ultrametric properties of molecular clock fork trees with the Hadamard conjugation to derive a number of topology dependent identities. Employing these identities, we substantially simplify the system of polynomial equations for the fork. We finally employ symbolic algebra software to obtain closed formanalytic solutions (expressed parametrically in the input data). In general, four-taxa trees can have multiple ML points. In contrast, we can now prove that each fork topology has a unique(local and global) ML point.     

A Bayesian perspective on a non-parsimonious parsimony model

Several stochastic models of character change, when implemented in a maximum likelihood framework, are known to give a correspondence between the maximum parsimony method and the method of maximum likelihood. One such model has an independently estimated branch-length parameter for each site and each branch of the phylogenetic tree. This model--the no-common-mechanism model--has many parameters, and, in fact, the number of parameters increases as fast as the alignment is extended. We take a Bayesian approach to the no-common-mechanism model and place independent gamma prior probability distributions on the branch-length parameters. We are able to analytically integrate over the branch lengths, and this allowed us to implement an efficient Markov chain Monte Carlo method for exploring the space of phylogenetic trees. We were able to reliably estimate the posterior probabilities of clades for phylogenetic trees of up to 500 sequences. However, the Bayesian approach to the problem, at least as implemented here with an independent prior on the length of each branch, does not tame the behavior of the branch-length parameters. The integrated likelihood appears to be a simple rescaling of the parsimony score for a tree, and the marginal posterior probability distribution of the length of a branch is dependent upon how the maximum parsimony method reconstructs the characters at the interior nodes of the tree. The method we describe, however, is of potential importance in the analysis of morphological character data and also for improving the behavior of Markov chain Monte Carlo methods implemented for models in which sites share a common branch-length parameter.     

Consequences of recombination on traditional phylogenetic analysis

We investigate the shape of a phylogenetic tree reconstructed from sequences evolving under the coalescent with recombination. The motivation is that evolutionary inferences are often made from phylogenetic trees reconstructed from population data even though recombination may well occur (mtDNA or viral sequences) or does occur (nuclear sequences). We investigate the size and direction of biases when a single tree is reconstructed ignoring recombination. Standard software (PHYLIP) was used to construct the best phylogenetic tree from sequences simulated under the coalescent with recombination. With recombination present, the length of terminal branches and the total branch length are larger, and the time to the most recent common ancestor smaller, than for a tree reconstructed from sequences evolving with no recombination. The effects are pronounced even for small levels of recombination that may not be immediately detectable in a data set. The phylogenies when recombination is present superficially resemble phylogenies for sequences from an exponentially growing population. However, exponential growth has a different effect on statistics such as Tajima's D. Furthermore, ignoring recombination leads to a large overestimation of the substitution rate heterogeneity and the loss of the molecular clock. These results are discussed in relation to viral and mtDNA data sets.     

Phylogeny and divergence of the pinnipeds (Carnivora: Mammalia) assessed using a multigene dataset

Background  

Phylogenetic comparative methods are often improved by complete phylogenies with meaningful branch lengths (e.g., divergence dates). This study presents a dated molecular supertree for all 34 world pinniped species derived from a weighted matrix representation with parsimony (MRP) supertree analysis of 50 gene trees, each determined under a maximum likelihood (ML) framework. Divergence times were determined by mapping the same sequence data (plus two additional genes) on to the supertree topology and calibrating the ML branch lengths against a range of fossil calibrations. We assessed the sensitivity of our supertree topology in two ways: 1) a second supertree with all mtDNA genes combined into a single source tree, and 2) likelihood-based supermatrix analyses. Divergence dates were also calculated using a Bayesian relaxed molecular clock with rate autocorrelation to test the sensitivity of our supertree results further.     

When is it safe to use an oversimplified substitution model in tree- making?

The choice of an "optimal" mathematical model for computing evolutionary distances from real sequences is not currently supported by easy-to-use software applicable to large data sets, and an investigator frequently selects one of the simplest models available. Here we study properties of the observed proportion of differences (p- distance) between sequences as an estimator of evolutionary distance for tree-making. We show that p-distances allow for consistent tree- making with any of the popular methods working with evolutionary distances if evolution of sequences obeys a "molecular clock" (more precisely, if it follows a stationary time-reversible Markov model of nucleotide substitution). Next, we show that p-distances seem to be efficient in recovering the correct tree topology under a "molecular clock," but produce "statistically supported" wrong trees when substitutions rates vary among evolutionary lineages. Finally, we outline a practical approach for selecting an "optimal" model of nucleotide substitution in a real data analysis, and obtain a crude estimate of a "prior" distribution of the expected tree branch lengths under the Jukes-Cantor model. We conclude that the use of a model that is obviously oversimplified is inadvisable unless it is justified by a preliminary analysis of the real sequences.      

Interior-branch and bootstrap tests of phylogenetic trees

We have compared statistical properties of the interior-branch and bootstrap tests of phylogenetic trees when the neighbor-joining tree- building method is used. For each interior branch of a predetermined topology, the interior-branch and bootstrap tests provide the confidence values, PC and PB, respectively, that indicate the extent of statistical support of the sequence cluster generated by the branch. In phylogenetic analysis these two values are often interpreted in the same way, and if PC and PB are high (say, > or = 0.95), the sequence cluster is regarded as reliable. We have shown that PC is in fact the complement of the P-value used in the standard statistical test, but PB is not. Actually, the bootstrap test usually underestimates the extent of statistical support of species clusters. The relationship between the confidence values obtained by the two tests varies with both the topology and expected branch lengths of the true (model) tree. The most conspicuous difference between PC and PB is observed when the true tree is starlike, and there is a tendency for the difference to increase as the number of sequences in the tree increases. The reason for this is that the bootstrap test tends to become progressively more conservative as the number of sequences in the tree increases. Unlike the bootstrap, the interior-branch test has the same statistical properties irrespective of the number of sequences used when a predetermined tree is considered. Therefore, the interior-branch test appears to be preferable to the bootstrap test as long as unbiased estimators of evolutionary distances are used. However, when the interior-branch is applied to a tree estimated from a given data set, PC may give an overestimate of statistical confidence. For this case, we developed a method for computing a modified version (P'C) of the PC value and showed that this P'C tends to give a conservative estimate of statistical confidence, though it is not as conservative as PB. In this paper we have introduced a model in which evolutionary distances between sequences follow a multivariate normal distribution. This model allowed us to study the relationships between the two tests analytically.      

Phylogenetic mixtures on a single tree can mimic a tree of another topology

Phylogenetic mixtures model the inhomogeneous molecular evolution commonly observed in data. The performance of phylogenetic reconstruction methods where the underlying data are generated by a mixture model has stimulated considerable recent debate. Much of the controversy stems from simulations of mixture model data on a given tree topology for which reconstruction algorithms output a tree of a different topology; these findings were held up to show the shortcomings of particular tree reconstruction methods. In so doing, the underlying assumption was that mixture model data on one topology can be distinguished from data evolved on an unmixed tree of another topology given enough data and the "correct" method. Here we show that this assumption can be false. For biologists, our results imply that, for example, the combined data from two genes whose phylogenetic trees differ only in terms of branch lengths can perfectly fit a tree of a different topology.     

Evolution of the salmonid mitochondrial control region.

To explore the evolutionary nature of the salmonid mitochondrial DNA (mtDNA) control region (D-loop) and its utility for inferring phylogenies, the entire region was sequenced from all eight species of anadromous Pacific salmon, genus Oncorhynchus; the Atlantic salmon, Salmo salar; and the Arctic grayling, Thymallus arcticus. A comparison of aligned sequences demonstrates that the generally conserved sequence elements that have been previously reported for other vertebrates are maintained in these primitive teleost fishes. Results reveal a significantly nonrandom distribution of nucleotide substitutions, insertions, and deletions that suggests that portions of the salmonid D-loop may be under differential selective constraints and that most of the control region of these fishes may evolve at a rate similar to that of the remainder of their mtDNA genomes. Maximum likelihood and Fitch parsimony analyses of 9 kb of aligned salmonid sequence data give evolutionary trees of identical topology. These results are consistent with previous molecular studies of a limited number of salmonid taxa and with more comprehensive, classical analyses of salmonid evolution. Predictions from these data, based on a molecular clock assumption for the mtDNA control region, are also consistent with fossil evidence that suggests that species of Oncorhynchus could be as old as the Middle Pliocene and would have thus given rise to the extant Pacific salmon prior to about 5 or 6 million years ago.