Resolution of the laurasiatherian phylogeny: evidence from genomic data

Despite great progress over the past decade, some portions of the mammalian tree of life remain unresolved. In particular, relationships among the different orders included within the supraordinal group Laurasiatheria have been proven difficult to determine, and have received poor support in the vast majority of phylogenomic studies of mammalian systematics. We estimated interordinal relationships within Laurasiatheria using sequence data from 3733 protein-coding genes. Our study included data from from 11 placental mammals, corresponding to five of the six orders of Laurasiatheria, plus five outgroup species. Ingroup and outgroup species were chosen to maximize the number single-copy ortholog genes for which sequence data was available for all species in our study. Phylogenetic analyses of the concatenated dataset using maximum likelihood and Bayesian methods resulted on an identical and well supported topology in all alignment strategies compared. Our analyses provide high support for the sister relationship between Chiroptera and Cetartiodactyla and also provide support for placing Perissodactyla as sister to Carnivora. We obtained maximal estimates of bootstrap support (100%) and posterior probability (1.00) for all nodes within Laurasiatheria. Our study provides a further demonstration of the utility of very large and conserved genomic dataset to clarify our understanding of the evolutionary relationships among mammals.     

A new phylogenetic marker,apolipoprotein B,provides compelling evidence for eutherian relationships

Higher-level relationships within, and the root of Placentalia, remain contentious issues. Resolution of the placental tree is important to the choice of mammalian genome projects and model organisms, as well as for understanding the biogeography of the eutherian radiation. We present phylogenetic analyses of 63 species representing all extant eutherian mammal orders for a new molecular phylogenetic marker, a 1.3kb portion of exon 26 of the apolipoprotein B (APOB) gene. In addition, we analyzed a multigene concatenation that included APOB sequences and a previously published data set (Murphy et al., 2001b) of three mitochondrial and 19 nuclear genes, resulting in an alignment of over 17kb for 42 placentals and two marsupials. Due to computational difficulties, previous maximum likelihood analyses of large, multigene concatenations for placental mammals have used quartet puzzling, less complex models of sequence evolution, or phylogenetic constraints to approximate a full maximum likelihood bootstrap. Here, we utilize a Unix load sharing facility to perform maximum likelihood bootstrap analyses for both the APOB and concatenated data sets with a GTR+Gamma+I model of sequence evolution, tree-bisection and reconnection branch-swapping, and no phylogenetic constraints. Maximum likelihood and Bayesian analyses of both data sets provide support for the superordinal clades Boreoeutheria, Euarchontoglires, Laurasiatheria, Xenarthra, Afrotheria, and Ostentoria (pangolins+carnivores), as well as for the monophyly of the orders Eulipotyphla, Primates, and Rodentia, all of which have recently been questioned. Both data sets recovered an association of Hippopotamidae and Cetacea within Cetartiodactyla, as well as hedgehog and shrew within Eulipotyphla. APOB showed strong support for an association of tarsier and Anthropoidea within Primates. Parsimony, maximum likelihood and Bayesian analyses with both data sets placed Afrotheria at the base of the placental radiation. Statistical tests that employed APOB to examine a priori hypotheses for the root of the placental tree rejected rooting on myomorphs and hedgehog, but did not discriminate between rooting at the base of Afrotheria, at the base of Xenarthra, or between Atlantogenata (Xenarthra+Afrotheria) and Boreoeutheria. An orthologous deletion of 363bp in the aligned APOB sequences proved phylogenetically informative for the grouping of the order Carnivora with the order Pholidota into the superordinal clade Ostentoria. A smaller deletion of 237-246bp was diagnostic of the superordinal clade Afrotheria.     

Phylogenomic data analyses provide evidence that Xenarthra and Afrotheria are sister groups

The phylogenetic positions of the 4 clades, Euarchontoglires, Laurasiatheria, Afrotheria, and Xenarthra, have been major issues in the recent discussion of basal relationships among placental mammals. However, despite considerable efforts these relationships, crucial to the understanding of eutherian evolution and biogeography, have remained essentially unresolved. Euarchontoglires and Laurasiatheria are generally joined into a common clade (Boreoeutheria), whereas the position of Afrotheria and Xenarthra relative to Boreoeutheria has been equivocal in spite of the use of comprehensive amounts of nuclear encoded sequences or the application of genome-level characters such as retroposons. The probable reason for this uncertainty is that the divergences took place long time ago and within a narrow temporal window, leaving only short common branches. With the aim of further examining basal eutherian relationships, we have collected conserved protein-coding sequences from 11 placental mammals, a marsupial and a bird, whose nuclear genomes have been largely sequenced. The length of the alignment of homologous sequences representing each individual species is 2,168,859 nt. This number of sites, representing 2840 protein-coding genes, exceeds by a considerable margin that of any previous study. The phylogenetic analysis joined Xenarthra and Afrotheria on a common branch, Atlantogenata. This topology was found to fit the data significantly better than the alternative trees.     

Phylogenomics of life-or-death switches in multicellular animals: Bcl-2, BH3-Only, and BNip families of apoptotic regulators

In this report, we conducted a comprehensive survey of Bcl-2 family members, a divergent group of proteins that regulate programmed cell death by an evolutionarily conserved mechanism. Using comparative sequence analysis, we found novel sequences in mammals, nonmammalian vertebrates, and in a number of invertebrates. We then asked what conclusions could be drawn from phyletic distribution, intron/exon structures, sequence/structure relationships, and phylogenetic analyses within the updated Bcl-2 family. First, multidomain members having a sequence pattern consistent with the conservation of the Bcl-X(L)/Bax/Bid topology appear to be restricted to multicellular animals and may share a common ancestry. Next, BNip proteins, which were originally identified based on their ability to bind to E1B 19K/Bcl-2 proteins, form three independent monophyletic branches with different evolutionary history. Lastly, a set of Bcl-2 homology 3-only proteins with unrelated secondary structures seems to have evolved after the origin of Metazoa and exhibits diverse expansion after speciation during vertebrate evolution.     

A genomic schism in birds revealed by phylogenetic analysis of DNA strings

The molecular systematics of vertebrates has been based entirely on alignments of primary structures of macromolecules; however, higher order features of DNA sequences not used in traditional studies also contain valuable phylogenetic information. Recent molecular data sets conflict over the phylogenetic placement of flightless birds (ratites - paleognaths), but placement of this clade critically influences interpretation of character change in birds. To help resolve this issue, we applied a new bioinformatics approach to the largest molecular data set currently available. We distilled nearly one megabase (1 million base pairs) of heterogeneous avian genomic DNA from 20 birds and an alligator into genomic signatures, defined as the complete set of frequencies of short sequence motifs (strings), thereby providing a way to directly compare higher order features of nonhomologous DNA sequences. Phylogenetic analysis and principal component analysis of the signatures strongly support the traditional hypothesis of basal ratites and monophyly of the nonratite birds (neognaths) and imply that ratite genomes are linguistically primitive within birds, despite their base compositional similarity to neognath genomes. Our analyses show further that the phylogenetic signal of genomic signatures are strongest among deep splits within vertebrates. Despite clear problems with phylogenetic analysis of genomic signatures, our study raises intriguing issues about the biological and genomic differences that fundamentally differentiate paleognaths and neognaths.     

Phylogenomic analysis resolves the interordinal relationships and rapid diversification of the laurasiatherian mammals

Although great progress has been made in resolving the relationships of placental mammals, the position of several clades in Laurasiatheria remain controversial. In this study, we performed a phylogenetic analysis of 97 orthologs (46,152 bp) for 15 taxa, representing all laurasiatherian orders. Additionally, phylogenetic trees of laurasiatherian mammals with draft genome sequences were reconstructed based on 1608 exons (2,175,102 bp). Our reconstructions resolve the interordinal relationships within Laurasiatheria and corroborate the clades Scrotifera, Fereuungulata, and Cetartiodactyla. Furthermore, we tested alternative topologies within Laurasiatheria, and among alternatives for the phylogenetic position of Perissodactyla, a sister-group relationship with Cetartiodactyla receives the highest support. Thus, Pegasoferae (Perissodactyla + Carnivora + Pholidota + Chiroptera) does not appear to be a natural group. Divergence time estimates from these genes were compared with published estimates for splits within Laurasiatheria. Our estimates were similar to those of several studies and suggest that the divergences among these orders occurred within just a few million years.     

Relationships within Cornales and circumscription of Cornaceae-matK and rbcL sequence data and effects of outgroups and long branches

Phylogenetic relationships in Cornales were assessed using sequences rbcL and matK. Various combinations of outgroups were assessed for their suitability and the effects of long branches and outgroups on tree topology were examined using RASA 2.4 prior to conducting phylogenetic analyses. RASA identified several potentially problematic taxa having long branches in individual data sets that may have obscured phylogenetic signal, but when data sets were combined RASA no longer detected long branch problems. t(RASA) provides a more conservative measurement for phylogenetic signal than the PTP and skewness tests. The separate matK and rbcL sequence data sets were measured as not containing phylogenetic signal by RASA, but PTP and skewness tests suggested the reverse [corrected]. Nonetheless, the matK and rbcL sequence data sets suggested relationships within Cornales largely congruent with those suggested by the combined matK-rbcL sequence data set that contains significant phylogenetic signal as measured by t(RASA), PTP, and skewness tests. Our analyses also showed that a taxon having a long branch on the tree may not be identified as a "long-branched" taxon by RASA. The long branches identified by RASA had little effect on the arrangement of other taxa in the tree, but the placements of the long-branched taxa themselves were often problematic. Removing the long-branched taxa from analyses generally increased bootstrap support, often substantially. Use of non-optimal outgroups (as identified by RASA) decreased phylogenetic resolution in parsimony analyses and suggested different relationships in maximum likelihood analyses, although usually weakly supported clades (less than 50% support) were impacted. Our results do not recommend using t(RASA) as a sole criterion to discard data or taxa in phylogenetic analyses, but t(RASA) and the taxon variance ratio obtained from RASA may be useful as a guide for improved phylogenetic analyses. Results of parsimony and ML analyses of the sequence data using optimal outgroups suggested by RASA revealed four major clades within Cornales: (1) Curtisia-Grubbia, (2) Cornus-Alangium, (3) Nyssa-Camptotheca-Davidia-Mastixia-Diplopanax, and (4) Hydrangeaceae-Loasaceae, with clades (2) and (3) forming a monophyletic group sister to clade (4) and clade (1) sister to the remainder of Cornales. However, there was not strong bootstrap support for relationships among the major clades. The placement of Hydrostachys could not be reliably determined, although most analyses place the genus within Hydrangeaceae; ML analyses, for example, placed the genus as the sister of Hydrangeeae. Our results supported a Cornales including the systematically problematic Hydrostachys, a Cornaceae consisting of Cornus and Alangium, a Nyssaceae consisting of Nyssa and Camptotheca, a monogeneric Davidiaceae, a Mastixiaceae consisting of Mastixia and Diplopanax, and an expanded Grubbiaceae consisting of Grubbia and Curtisia, and two larger families, Hydrangeaceae and Loasaceae.     

Dynamically heterogenous partitions and phylogenetic inference: an evaluation of analytical strategies with cytochrome b and ND6 gene sequences in cranes

ki ctes over whether molecular sequence data should be partitioned for phylogenetic analysis often confound two types of heterogeneity among partitions. We distinguish historical heterogeneity (i.e., different partitions have different evolutionary relationships) from dynamic heterogeneity (i.e., different partitions show different patterns of sequence evolution) and explore the impact of the latter on phylogenetic accuracy and precision with a two-gene, mitochondrial data set for cranes. The well-established phylogeny of cranes allows us to contrast tree-based estimates of relevant parameter values with estimates based on pairwise comparisons and to ascertain the effects of incorporating different amounts of process information into phylogenetic estimates. We show that codon positions in the cytochrome b and NADH dehydrogenase subunit 6 genes are dynamically heterogenous under both Poisson and invariable-sites + gamma-rates versions of the F84 model and that heterogeneity includes variation in base composition and transition bias as well as substitution rate. Estimates of transition-bias and relative-rate parameters from pairwise sequence comparisons were comparable to those obtained as tree-based maximum likelihood estimates. Neither rate-category nor mixed-model partitioning strategies resulted in a loss of phylogenetic precision relative to unpartitioned analyses. We suggest that weighted-average distances provide a computationally feasible alternative to direct maximum likelihood estimates of phylogeny for mixed-model analyses of large, dynamically heterogenous data sets.     

Complete nucleotide sequence of Japanese flounder (Paralichthys olivaceus) mitochondrial genome: structural properties and cue for resolving teleostean relationships

We cloned and sequenced the complete mitochondrial genome of Japanese flounder (Paralichthys olivaceus). A circular 17,090 bp mitochondrial genome from the flounder contains 37 structural genes as in other vertebrates so far reported. This is the first report of the complete mitochondrial sequence from a higher teleostean fish (Acanthopterygii). The organization including gene order is quite similar to that of other teleostean fishes as well as placental mammals. The putative control region of the Japanese flounder mitochondrial genome contains a length variable region of about a 74 bp tandem repeat cluster. As a preliminary study we adopted the maximum likelihood and neighbor-joining inference methods to examine phylogenetic relationships among teleostean and related fishes. Comparisons of amino acid sequences of protein-coding genes and nucleotide sequences of tRNA genes resolved some middle to deep branches among some teleostean fishes. The flounder mitochondrial genome does not show an indication of evolutionary rate difference among teleosts leading to difficulty in phylogenetic analyses, and our data is useful for future evolutionary studies dealing with higher teleostean fishes.     

Branch lengths, support, and congruence: testing the phylogenomic approach with 20 nuclear loci in snakes

Many authors have claimed that short branches in the Tree of Life will be very difficult to resolve with strong support, even with the large multilocus data sets now made possible by genomic resources. Short branches may be especially problematic because the underlying gene trees are expected to have discordant phylogenetic histories when the time between branching events is very short. Although there are many examples of short branches that are difficult to resolve, surprisingly, no empirical studies have systematically examined the relationships between branch lengths, branch support, and congruence among genes. Here, we examine these fundamental relationships quantitatively using a data set of 20 nuclear loci for 50 species of snakes (representing most traditionally recognized families). A combined maximum likelihood analysis of the 20 loci gives strong support for 69% of the nodes, but many remain weakly supported, with bootstrap values for 20% ranging from 21% to 66%. For the combined-data tree, we find significant correlations between the length of a branch, levels of bootstrap support, and the proportion of genes that are congruent with that branch in the separate analyses of each gene. We also find that strongly supported conflicts between gene trees over the resolution of individual branches are common (roughly 35% of clades), especially for shorter branches. Overall, our results support the hypothesis that short branches may be very difficult to confidently resolve, even with large, multilocus data sets. Nevertheless, our study provides strong support for many clades, including several that were controversial or poorly resolved in previous studies of snake phylogeny.     

Incongruence between primary sequence data and the distribution of a mitochondrial atp1 group II intron among ferns and horsetails

Using DNA sequence data from multiple genes (often from more than one genome compartment) to reconstruct phylogenetic relationships has become routine. Augmenting this approach with genomic structural characters (e.g., intron gain and loss, changes in gene order) as these data become available from comparative studies already has provided critical insight into some long-standing questions about the evolution of land plants. Here we report on the presence of a group II intron located in the mitochondrial atp1 gene of leptosporangiate and marattioid ferns. Primary sequence data for the atp1 gene are newly reported for 27 taxa, and results are presented from maximum likelihood-based phylogenetic analyses using Bayesian inference for 34 land plants in three data sets: (1) single-gene mitochondrial atp1 (exon+intron sequences); (2) five combined genes (mitochondrial atp1 [exon only]; plastid rbcL, atpB, rps4; nuclear SSU rDNA); and (3) same five combined genes plus morphology. All our phylogenetic analyses corroborate results from previous fern studies that used plastid and nuclear sequence data: the monophyly of euphyllophytes, as well as of monilophytes; whisk ferns (Psilotidae) sister to ophioglossoid ferns (Ophioglossidae); horsetails (Equisetopsida) sister to marattioid ferns (Marattiidae), which together are sister to the monophyletic leptosporangiate ferns. In contrast to the results from the primary sequence data, the genomic structural data (atp1 intron distribution pattern) would seem to suggest that leptosporangiate and marattioid ferns are monophyletic, and together they are the sister group to horsetails--a topology that is rarely reconstructed using primary sequence data.     

Four new mitochondrial genomes and the increased stability of evolutionary trees of mammals from improved taxon sampling

We have sequenced four new mitochondrial genomes to improve the stability of the tree for placental mammals; they are two insectivores (a gymnure, Echinosorex gymnurus and Formosan shrew Soriculus fumidus); a Formosan lesser horseshoe bat (Rhinolophus monoceros); and the New Zealand fur seal (Arctocephalus forsteri). A revision to the hedgehog sequence (Erinaceus europaeus) is also reported. All five are from the Laurasiatheria grouping of eutherian mammals. On this new data set there is a strong tendency for the hedgehog and its relative, the gymnure, to join with the other Laurasiatherian insectivores (mole and shrews). To quantify the stability of trees from this data we define, based on nuclear sequences, a major four-way split in Laurasiatherians. This ([Xenarthra, Afrotheria], [Laurasiatheria, Supraprimates]) split is also found from mitochondrial genomes using either protein-coding or RNA (rRNA and tRNA) data sets. The high similarity of the mitochondrial and nuclear-derived trees allows a quantitative estimate of the stability of trees from independent data sets, as detected from a triplet Markov analysis. There are significant changes in the mutational processes within placental mammals that are ignored by current tree programs. On the basis of our quantitative results, we expect the evolutionary tree for mammals to be resolved quickly, and this will allow other problems to be solved.     

Phylogenetic analysis of 1.5 Mbp and platypus EST data refute the Marsupionta hypothesis and unequivocally support Monotremata as sister group to Marsupialia/Placentalia

The extant mammalian groups Monotremata, Marsupialia and Placentalia are, according to the 'Theria' hypothesis, traditionally classified into two subclasses. The subclass Prototheria includes the monotremes and subclass Theria marsupials and placental mammals. Based on some morphological and molecular data, an alternative proposition, the Marsupionta hypothesis, favours a sister group relationship between monotremes and marsupials to the exclusion of placental mammals. Phylogenetic analyses of single genes and even multiple gene alignments have not yet been able to conclusively resolve this basal mammalian divergence. We have examined this problem using one data set composed of expressed sequence tags (EST) and another containing 1 510 509 nucleotide (nt) sites from 1358 inferred cDNA genomic sequences. All analyses of the concatenated sequences unambiguously supported the Theria hypothesis. The Marsupionta hypothesis was rejected with high statistical confidence from both data sets. In spite of the strong support for Theria, a non-negligible number of single genes supported either of the two alternative hypotheses. The divergence between monotremes and therian mammals was estimated to have taken place 168–178 Mya, a dating compatible with the fossil record. Considering the long common evolutionary branch of therians, it is surprising that sequence data from many thousand amino acid sites were needed to conclusively resolve their relationship to monotremes. This finding draws attention to other mammalian divergences that have been taken as unequivocally settled based on much smaller alignments. EST data provide a comprehensive random sample of protein coding sequences and an economic way to produce large amounts of data for phylogenetic analysis of species for which genomic sequences are not yet available.     

Molecular Evidence for the Major Clades of Placental Mammals

Higher-level relationships among placental mammals, as well as the historical biogeography of this group against the backdrop of continental fragmentation and reassembly, remain poorly understood. Here, we analyze two independent molecular data sets that represent all placental orders. The first data set includes six genes (A2AB, IRBP, vWF, 12S rRNA, tRNA valine, 16S rRNA; total = 5.71 kb) for 26 placental taxa and two marsupials; the second data set includes 2.95 kb of exon 11 of the BRCA1 gene for 51 placental taxa and four marsupials. We also analyzed a concatenation of these data sets (8.66 kb) for 26 placentals and one marsupial. Unrooted and rooted analyses were performed with parsimony, distance methods, maximum likelihood, and a Bayesian approach. Unrooted analyses provide convincing support for a fundamental separation of placental orders into groups with southern and northern hemispheric origins according to the current fossil record. On rooted trees, one or both of these groups are monophyletic depending on the position of the root. Maximum likelihood and Bayesian analyses with the BRCA1 and combined 8.66 kb data sets provide strong support for the monophyly of the northern hemisphere group (Boreoeutheria). Boreoeutheria is divided into Laurasiatheria (Carnivora + Cetartiodactyla + Chiroptera + Eulipotyphla + Perissodactyla + Pholidota) and Euarchonta (Dermoptera + Primates + Scandentia) + Glires (Lagomorpha + Rodentia). The southern hemisphere group is either monophyletic or paraphyletic, depending on the method of analysis used. Within this group, Afrotheria (Proboscidea + Sirenia + Hyracoidea + Tubulidentata + Macroscelidea + Afrosoricida) is monophyletic. A unique nine base-pair deletion in exon 11 of the BRCA1 gene also supports Afrotheria monophyly. Given molecular dates that suggest that the southern hemisphere group and Boreoeutheria diverged in the Early Cretaceous, a single trans-hemispheric dispersal event may have been of fundamental importance in the early history of crown-group Eutheria. Parallel adaptive radiations have subsequently occurred in the four major groups: Laurasiatheria, Euarchonta + Glires, Afrotheria, and Xenarthra.     

Hidden likelihood support in genomic data: can forty-five wrongs make a right?

Combined analysis of multiple phylogenetic data sets can reveal emergent character support that is not evident in separate analyses of individual data sets. Previous parsimony analyses have shown that this hidden support often accounts for a large percentage of the overall phylogenetic signal in cladistic studies. Here, reanalysis of a large comparative genomic data set for yeast (genus Saccharomyces) demonstrates that hidden support can be an important factor in maximum likelihood analyses of multiple data sets as well. Emergent signal in a concatenation of 106 genes was responsible for up to 64% of the likelihood support at a particular node (the difference in log likelihood scores between optimal topologies that included and excluded a supported clade). A grouping of four yeast species (S. cerevisiae, S. paradoxus, S. mikatae, and S. kudriavzevii) was robustly supported by combined analysis of all 106 genes, but separate analyses of individual genes suggested numerous conflicts. Forty-eight genes strictly contradicted S. cerevisiae + S. paradoxus + S. mikatae + S. kudriavzevii in separate analyses, but combined likelihood analyses that included up to 45 of the "wrong" data sets supported this group. Extensive hidden support also emerged in a combined likelihood analysis of 41 genes that each recovered the exact same topology in separate analyses of the individual genes. These results show that isolated analyses of individual data sets can mask congruence and distort interpretations of clade stability, even in strictly model-based phylogenetic methods. Consensus and supertree procedures that ignore hidden phylogenetic signals are, at best, incomplete.     

Rooting the eukaryotic tree with mitochondrial and bacterial proteins

By exploiting the large body of genome data and the considerable progress in phylogenetic methodology, recent phylogenomic studies have provided new insights into the relationships among major eukaryotic groups. However, confident placement of the eukaryotic root remains a major challenge. This is due to the large evolutionary distance separating eukaryotes from their closest relatives, the Archaea, implying a weak phylogenetic signal and strong long-branch attraction artifacts. Here, we apply a new approach to the rooting of the eukaryotic tree by using a subset of genomic information with more recent evolutionary origin-mitochondrial sequences, whose closest relatives are α-Proteobacteria. For this, we identified and assembled a data set of 42 mitochondrial proteins (mainly encoded by the nuclear genome) and performed Bayesian and maximum likelihood analyses. Taxon sampling includes the recently sequenced Thecamonas trahens, a member of the phylogenetically elusive Apusozoa. This data set confirms the relationships of several eukaryotic supergroups seen before and places the eukaryotic root between the monophyletic "unikonts" and "bikonts." We further show that T. trahens branches sister to Opisthokonta with significant statistical support and question the bikont/excavate affiliation of Malawimonas species. The mitochondrial data set developed here (to be expanded in the future) constitutes a unique alternative means in resolving deep eukaryotic relationships.     

Multidimensional vector space representation for convergent evolution and molecular phylogeny

With growing amounts of genome data and constant improvement of models of molecular evolution, phylogenetic reconstruction became more reliable. However, our knowledge of the real process of molecular evolution is still limited. When enough large-sized data sets are analyzed, any subtle biases in statistical models can support incorrect topologies significantly because of the high signal-to-noise ratio. We propose a procedure to locate sequences in a multidimensional vector space (MVS), in which the geometry of the space is uniquely determined in such a way that the vectors of sequence evolution are orthogonal among different branches. In this paper, the MVS approach is developed to detect and remove biases in models of molecular evolution caused by unrecognized convergent evolution among lineages or unexpected patterns of substitutions. Biases in the estimated pairwise distances are identified as deviations (outliers) of sequence spatial vectors from the expected orthogonality. Modifications to the estimated distances are made by minimizing an index to quantify the deviations. In this way, it becomes possible to reconstruct the phylogenetic tree, taking account of possible biases in the model of molecular evolution. The efficacy of the modification procedure was verified by simulating evolution on various topologies with rate heterogeneity and convergent change. The phylogeny of placental mammals in previous analyses of large data sets has varied according to the genes being analyzed. Systematic deviations caused by convergent evolution were detected by our procedure in all representative data sets and were found to strongly affect the tree structure. However, the bias correction yielded a consistent topology among data sets. The existence of strong biases was validated by examining the sites of convergent evolution between the hedgehog and other species in mitochondrial data set. This convergent evolution explains why it has been difficult to determine the phylogenetic placement of the hedgehog in previous studies.     

Cladistic analysis of anuran POMC sequences

Procedures for performing cladistic analyses can provide powerful tools for understanding the evolution of neuropeptide and polypeptide hormone coding genes. These analyses can be done on either amino acid data sets or nucleotide data sets and can utilize several different algorithms that are dependent on distinct sets of operating assumptions and constraints. In some cases, the results of these analyses can be used to gauge phylogenetic relationships between taxa. Selecting the proper cladistic analysis strategy is dependent on the taxonomic level of analysis and the rate of evolution within the orthologous genes being evaluated. For example, previous studies have shown that the amino acid sequence of proopiomelanocortin (POMC), the common precursor for the melanocortins and beta-endorphin, can be used to resolve phylogenetic relationships at the class and order level. This study tested the hypothesis that POMC sequences could be used to resolve phylogenetic relationships at the family taxonomic level. Cladistic analyses were performed on amphibian POMC sequences characterized from the marine toad, Bufo marinus (family Bufonidae; this study), the spadefoot toad, Spea multiplicatus (family Pelobatidae), the African clawed frog, Xenopus laevis (family Pipidae) and the laughing frog, Rana ridibunda (family Ranidae). In these analyses the sequence of Australian lungfish POMC was used as the outgroup. The analyses were done at the amino acid level using the maximum parsimony algorithm and at the nucleotide level using the maximum likelihood algorithm. For the anuran POMC genes, analysis at the nucleotide level using the maximum likelihood algorithm generated a cladogram with higher bootstrap values than the maximum parsimony analysis of the POMC amino acid data set. For anuran POMC sequences, analysis of nucleotide sequences using the maximum likelihood algorithm would appear to be the preferred strategy for resolving phylogenetic relationships at the family taxonomic level.