Congruent Avian Phylogenies Inferred from Mitochondrial and Nuclear DNA Sequences

Recent molecular studies addressing the phylogenetic relationships of avian orders have had conflicting results. While studies using nuclear DNA sequences tend to support traditional taxonomic views, also supported by morphological data [(paleognaths (galloanseres (all other birds)))], with songbirds forming a clade within Neoaves (all other birds), analyses with complete mtDNA genomes have resulted in topologies that place songbirds as one of the earliest-diverging avian lineages. Considering that over half of the extant bird species are songbirds, these different results have very different implications for our understanding of avian evolution. We analyzed data sets comprising nearly 4 kb of mitochondrial DNA (mtDNA) (complete 12S, ND1, ND2, and cytochrome b) plus 600 bp of the nuclear gene c-mos for 15 birds that were chosen to represent all major avian clades and to minimize potential long-branch attraction problems; we used a partition-specific maximum likelihood approach. Our results show congruence with respect to the ingroup among phylogenies obtained with mtDNA and the nuclear gene c-mos, separately or combined. The data sets support a traditional avian taxonomy, with paleognaths (ratites and tinamous) occupying a basal position and with songbirds more derived and forming a monophyletic group. We also show that, for mtDNA studies, turtles may be a better outgroup for birds than crocodilians because of their slower rate of sequence evolution.     

Basal divergences in birds and the phylogenetic utility of the nuclear RAG-1 gene.

The single-copy RAG-1 gene is found throughout higher vertebrates and consists of a single 3.1-kb exon without intervening introns. A 2.9-kb region of the RAG-1 locus was sequenced for 14 basal taxa of birds plus the crocodylian outgroups Alligator and Gavialis. Phylogenetic analysis of the sequences supported the hypothesis that the deepest evolutionary split in extant birds separates paleognaths from neognaths. A deep division among neognaths separates the chicken- and duck-like birds ("galloanserines") from a clade consisting of all other birds ("plethornithines"). The relationships of these three basal clades in Aves were supported by high bootstrap (98 to 100%) and large decay index values (above 14). Additionally, the plethornithine clade is characterized by a 15-bp (five-codon) synapomorphic deletion relative to all other birds. RAG-1 evolves slowly, with a number of properties favoring its phylogenetic utility, including rarity of indels, minimal saturation of transition changes at 3rd positions of codons, nearly constant base composition across taxa, and no asymmetry in directional patterns of reconstructed change. However, RAG-1 does not evolve in a clocklike manner, suggesting that this gene cannot easily be used for estimating ages of ancient lineages.     

Resolving the root of the avian mitogenomic tree by breaking up long branches

Incomplete taxon sampling has been a major problem in resolving the early divergences in birds. Five new mitochondrial genomes are reported here (brush-turkey, lyrebird, suboscine flycatcher, turkey vulture, and a gull) and three break up long branches that tended to attract the distant reptilian outgroup. These long branches were to galliforms, and to oscine and suboscine passeriformes. Breaking these long branches leaves the root, as inferred by maximum likelihood and Bayesian phylogenetic analyses, between paleognaths and neognaths. This means that morphological, nuclear, and mitochondrial data are now in agreement on the position of the root of the avian tree and we can, move on to other questions. An overview is then given of the deepest divisions in the mitogenomic tree inferred from complete mitochondrial genomes. The strict monophyly of both the galloanseres and the passerines is strongly supported, leaving the deep six-way split within Neoaves as the next major question for which resolution is still lacking. Incomplete taxon sampling was also a problem for Neoaves, and although some resolution is now available there are still problems because current phylogenetic methods still fail to account for real features of DNA sequence evolution.     

Interordinal relationships of birds and other reptiles based on whole mitochondrial genomes

Several different groups of birds have been proposed as being the oldest or earliest diverging extant lineage within the avian phylogenetic tree, particularly ratites (Struthioniformes), waterfowl (Anseriformes), and shorebirds (Charadriiformes). Difficulty in resolving this issue stems from several factors, including the relatively rapid radiation of primary (ordinal) bird lineages and the lack of characters from an extant outgroup for birds that is closely related to them by measure of time. To help resolve this question, we have sequenced entire mitochondrial genomes for five birds (a rhea, a duck, a falcon, and two perching birds), one crocodilian, and one turtle. Maximum parsimony and maximum likelihood analyses of these new sequences together with published sequences (18 taxa total) yield the same optimal tree topology, in which a perching bird (Passeriformes) is sister to all the other bird taxa. A basal position for waterfowl among the bird study taxa is rejected by maximum likelihood analyses. However, neither the conventional view, in which ratites (including rhea) are basal to other birds, nor tree topologies with falcon or chicken basal among birds could be rejected in the same manner. In likelihood analyses of a subset of seven birds, alligator, and turtle (9 taxa total), we find that increasing the number of parameters in the model shifts the optimal topology from one with a perching bird basal among birds to the conventional view with ratites diverging basally; moreover, likelihood scores for the two trees are not significantly different. Thus, although our largest set of taxa and characters supports a tree with perching birds diverging basally among birds, the position of this earliest divergence among birds appears unstable. Our analyses indicate a sister relationship between a waterfowl/chicken clade and ratites, relative to perching birds and falcon. We find support for a sister relationship between turtles and a bird/crocodilian clade, and for rejecting both the Haemothermia hypothesis (birds and mammals as sister taxa) and the placement of turtles as basal within the phylogenetic tree for amniote animals.     

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

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

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

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

The early history of modern birds inferred from DNA sequences of nuclear and mitochondrial ribosomal genes

The traditional view of avian evolution places ratites and tinamous at the base of the phylogenetic tree of modern birds (Neornithes). In contrast, most recent molecular studies suggest that neognathous perching birds (Passeriformes) compose the oldest lineage of modern birds. Here, we report significant molecular support for the traditional view of neognath monophyly based on sequence analyses of nuclear and mitochondrial DNA (4.4 kb) from every modern avian order. Phylogenetic analyses further show that the ducks and gallinaceous birds are each other's closest relatives and together form the basal lineage of neognathous birds. To investigate why other molecular studies sampling fewer orders have reached different conclusions regarding neognath monophyly, we performed jackknife analyses on our mitochondrial data. Those analyses indicated taxon-sampling effects when basal galloanserine birds were included in combination with sparse taxon sampling. Our phylogenetic results suggest that the earliest neornithines were heavy-bodied, ground-dwelling, nonmarine birds. This inference, coupled with a fossil bias toward marine environments, provides a possible explanation for the large gap in the early fossil record of birds.     

Phylogeny and sex chromosome evolution of Palaeognathae

Many paleognaths (ratites and tinamous) have a pair of homomorphic ZW sex chromosomes in contrast to the highly differentiated sex chromosomes of most other birds. To understand the evolutionary causes for the different tempos of sex chromosome evolution, we produced female genomes of 12 paleognathous species and reconstructed the phylogeny and the evolutionary history of paleognathous sex chromosomes. We uncovered that Palaeognathae sex chromosomes had undergone stepwise recombination suppression and formed a pattern of “evolutionary strata”. Nine of the 15 studied species' sex chromosomes have maintained homologous recombination in their long pseudoautosomal regions extending more than half of the entire chromosome length. We found that in the older strata, the W chromosome suffered more serious functional gene loss. Their homologous Z-linked regions, compared with other genomic regions, have produced an excess of species-specific autosomal duplicated genes that evolved female-specific expression, in contrast to their broadly expressed progenitors. We speculate such “defeminization” of Z chromosome with underrepresentation of female-biased genes and slow divergence of sex chromosomes of paleognaths might be related to their distinctive mode of sexual selection targeting females rather than males, which evolved in their common ancestors.     

Complete mitochondrial DNA sequences of the green turtle and blue-tailed mole skink: statistical evidence for archosaurian affinity of turtles.

Turtles have highly specialized morphological characteristics, and their phylogenetic position has been under intensive debate. Previous molecular studies have not established a consistent and statistically well supported conclusion on this issue. In order to address this, complete mitochondrial DNA sequences were determined for the green turtle and the blue-tailed mole skink. These genomes possess an organization of genes which is typical of most other vertebrates, such as placental mammals, a frog, and bony fishes, but distinct from organizations of alligators and snakes. Molecular evolutionary rates of mitochondrial protein sequences appear to vary considerably among major reptilian lineages, with relatively rapid rates for snake and crocodilian lineages but slow rates for turtle and lizard lineages. In spite of this rate heterogeneity, phylogenetic analyses using amino acid sequences of 12 mitochondrial proteins reliably established the Archosauria (birds and crocodilians) and Lepidosauria (lizards and snakes) clades postulated from previous morphological studies. The phylogenetic analyses further suggested that turtles are a sister group of the archosaurs, and this untraditional relationship was provided with strong statistical evidence by both the bootstrap and the Kishino-Hasegawa tests. This is the first statistically significant molecular phylogeny on the placement of turtles relative to the archosaurs and lepidosaurs. It is therefore likely that turtles originated from a Permian-Triassic archosauromorph ancestor with two pairs of temporal fenestrae behind the skull orbit that were subsequently lost. The traditional classification of turtles in the Anapsida may thus need to be reconsidered.     

Complete mitochondrial DNA genome sequences of extinct birds: ratite phylogenetics and the vicariance biogeography hypothesis

The ratites have stimulated much debate as to how such large flightless birds came to be distributed across the southern continents, and whether they are a monophyletic group or are composed of unrelated lineages that independently lost the power of flight. Hypotheses regarding the relationships among taxa differ for morphological and molecular data sets, thus hindering attempts to test whether plate tectonic events can explain ratite biogeography. Here, we present the complete mitochondrial DNA genomes of two extinct moas from New Zealand, along with those of five extant ratites (the lesser rhea, the ostrich, the great spotted kiwi, the emu and the southern cassowary and two tinamous from different genera. The non-stationary base composition in these sequences violates the assumptions of most tree-building methods. When this bias is corrected using neighbour-joining with log-determinant distances and non-homogeneous maximum likelihood, the ratites are found to be monophlyletic, with moas basal, as in morphological trees. The avian sequences also violate a molecular clock, so we applied a non-parametric rate smoothing algorithm, which minimizes ancestor-descendant local rate changes, to date nodes in the tree. Using this method, most of the major ratite lineages fit the vicariance biogeography hypothesis, the exceptions being the ostrich and the kiwi, which require dispersal to explain their present distribution.     

Evolutionary shifts in the melanin‐based color system of birds

Melanin pigments contained in organelles (melanosomes) impart earthy colors to feathers. Such melanin‐based colors are distributed across birds and thought to be the ancestral color‐producing mechanism in birds. However, we have had limited data on melanin‐based color and melanosome diversity in Palaeognathae, which includes the flighted tinamous and large‐bodied, flightless ratites and is the sister taxon to all other extant birds. Here, we use scanning electron microscopy and spectrophotometry to assess melanosome morphology and quantify reflected color for 19 species within this clade. We find that brown colors in ratites are uniquely associated with elongated melanosomes nearly identical in shape to those associated with black colors. Melanosome and color diversity in large‐bodied ratites is limited relative to other birds (including flightless penguins) and smaller bodied basal maniraptoran dinosaur outgroups of Aves, whereas tinamous show a wider range of melanosome forms similar to neognaths. The repeated occurrence of novel melanosome forms in the nonmonophyletic ratites suggests that melanin‐based color tracks changes in body size, physiology, or other life history traits associated with flight loss, but not feather morphology. We further anticipate these findings will be useful for future color reconstructions in extinct species, as variation in melanosome shape may potentially be linked to a more nuanced palette of melanin‐based colors.     

Phylogeny and biogeography of ratite birds inferred from DNA sequences of the mitochondrial ribosomal genes

The origin of the flightless ratite birds of the southern continents has been debated for over a century. Whether dispersal or vicariance (continental breakup) best explains their origin depends largely on their phylogenetic relationships. No consensus has been reached on this issue despite many morphological and molecular studies. To address this question further we sequenced a 2.8-kb region of mitochondrial DNA containing the ribosomal genes in representative ratites and a tinamou. Phylogenetic analyses indicate that Struthio (Africa) is basal and Rhea (South America) clusters with living Australasian ratites. This phylogeny agrees with transferrin and DNA hybridization studies but not with sequence analyses of some protein-coding genes. These results also require reevaluation of the phylogenetic position of the extinct moas of New Zealand. We propose a new hypothesis for the origin of ratites that combines elements of dispersal and vicariance.      

Complete Mitochondrial Genome Sequences of the South American and the Australian Lungfish: Testing of the Phylogenetic Performance of Mitochondrial Data Sets for Phylogenetic Problems in Tetrapod Relationships

We determined the complete nucleotide sequences (16403 and 16572 base pairs, respectively) of the mitochondrial genomes of the South American lungfish, Lepidosiren paradoxa, and the Australian lungfish, Neoceratodus forsteri (Sarcopterygii, Dipnoi). The mitochondrial DNA sequences were established in an effort to resolve the debated evolutionary positions of the lungfish and the coelacanth relative to land vertebrates. Previous molecular phylogenetic studies based on complete mtDNA sequences, including only the African lungfish, Protopterus dolloi, sequence were able to strongly reject the traditional textbook hypothesis that coelacanths are the closest relatives of land vertebrates. However, these studies were unable to statistically significantly distinguish between the two remaining scenarios: lungfish as the closest relatives to land vertebrates and lungfish and coelacanths jointly as their sister group (Cao et al. 1998; Zardoya et al. 1998; Zardoya and Meyer 1997a). Lungfish, coelacanths, and the fish ancestors of the tetrapod lineage all originated within a short time window of about 20 million years, back in the early Devonian (about 380 to 400 million years ago). This short divergence time makes the determination of the phylogenetic relationships among these three lineages difficult. In this study, we attempted to break the long evolutionary branch of lungfish, in an effort to better resolve the phylogenetic relationships among the three extant sarcopterygian lineages. The gene order of the mitochondrial genomes of the South American and Australian lungfish conforms to the consensus gene order among gnathostome vertebrates. The phylogenetic analyses of the complete set of mitochondrial proteins (without ND6) suggest that the lungfish are the closest relatives of the tetrapods, although the support in favor of this scenario is not statistically significant. The two other smaller data sets (tRNA and rRNA genes) give inconsistent results depending on the different reconstruction methods applied and cannot significantly rule out any of the three alternative hypotheses. Nuclear protein-coding genes, which might be better phylogenetic markers for this question, support the lungfish–tetrapod sister-group relationship (Brinkmann et al. 2004).This article contains online supplementary material.Reviewing Editor: Dr. Rafael Zardoya     

Analyses of mitochondrial DNA nest ratite birds within the Neognathae: supporting a neotenous origin of ratite morphological characters

It is commonly acknowledged that the basal divergence among extant birds is between Palaeognathae and Neognathae. However, recent analyses of mitochondrial sequences have challenged that notion. In order to investigate this fundamental phylogenetic question, the complete mitochondrial DNA (mtDNA) molecule of the rook Corvus frugilegus (Passeriformes) was sequenced and included in phylogenetic analyses with the previously reported complete mtDNAs of the chicken Gallus gallus (Galliformes) and two ratite species, the ostrich Struthio camelus and the rhea Rhea americana (Struthioniformes). The analyses reconstructed a split between Passeriformes and a branch including Galliformes and Struthioniformes. Thus, the result is inconsistent with the traditional understanding of a basal avian divergence between Palaeognathae and Neognathae. The findings suggest that the morphological characteristics of the ratites are secondarily acquired, probably through neoteny and that the ratites are descendants of flying, neognathous ancestors.     

Discovery and characterization of mammalian endogenous parvoviruses

Public databases of nucleotide sequences contain exponentially increasing amounts of sequence data from mammalian genomes. Through the use of large-scale bioinformatic screening for sequences homologous to exogenous mammalian viruses, we found several sequences related to human and animal parvoviruses (PVs) in the Parvovirus and Dependovirus genera within genomes of several mammals, including rats, wallabies, opossums, guinea pigs, hedgehogs, African elephants, and European rabbits. However, phylogenetic analysis of these endogenous parvovirus (EnPV) sequences demonstrated substantial genetic divergence from exogenous mammalian PVs characterized to date. Entire nonstructural and capsid gene sequences of a novel EnPV were amplified and genetically characterized from rat (Rattus norvegicus) genomic DNA. Rat EnPV sequences were most closely related to members of the genus Parvovirus, with >70% and 65% amino acid identities to nonstructural and capsid proteins of canine parvovirus, respectively. Integration of EnPV into chromosome 5 of rats was confirmed by PCR cloning and sequence analysis of the viral and chromosomal junctions. Using inverse PCR, we determined that the rat genome contains a single copy of rat EnPV. Considering mammalian phylogeny, we estimate that EnPV integrated into the rat genome less than 30 million years ago. Comparative phylogenetic analysis done using all known representative exogenous parvovirus (ExPV) and EnPV sequences showed two major genetic groups of EnPVs, one genetically more similar to genus Parvovirus and the other genetically more similar to the genus Dependovirus. The full extent of the genetic diversity of parvoviruses that have undergone endogenization during evolution of mammals and other vertebrates will be recognized only once complete genomic sequences from a wider range of classes, orders, and species of animals become available.     

A mitogenomic timescale for birds detects variable phylogenetic rates of molecular evolution and refutes the standard molecular clock

Current understanding of the diversification of birds is hindered by their incomplete fossil record and uncertainty in phylogenetic relationships and phylogenetic rates of molecular evolution. Here we performed the first comprehensive analysis of mitogenomic data of 48 vertebrates, including 35 birds, to derive a Bayesian timescale for avian evolution and to estimate rates of DNA evolution. Our approach used multiple fossil time constraints scattered throughout the phylogenetic tree and accounts for uncertainties in time constraints, branch lengths, and heterogeneity of rates of DNA evolution. We estimated that the major vertebrate lineages originated in the Permian; the 95% credible intervals of our estimated ages of the origin of archosaurs (258 MYA), the amniote-amphibian split (356 MYA), and the archosaur-lizard divergence (278 MYA) bracket estimates from the fossil record. The origin of modern orders of birds was estimated to have occurred throughout the Cretaceous beginning about 139 MYA, arguing against a cataclysmic extinction of lineages at the Cretaceous/Tertiary boundary. We identified fossils that are useful as time constraints within vertebrates. Our timescale reveals that rates of molecular evolution vary across genes and among taxa through time, thereby refuting the widely used mitogenomic or cytochrome b molecular clock in birds. Moreover, the 5-Myr divergence time assumed between 2 genera of geese (Branta and Anser) to originally calibrate the standard mitochondrial clock rate of 0.01 substitutions per site per lineage per Myr (s/s/l/Myr) in birds was shown to be underestimated by about 9.5 Myr. Phylogenetic rates in birds vary between 0.0009 and 0.012 s/s/l/Myr, indicating that many phylogenetic splits among avian taxa also have been underestimated and need to be revised. We found no support for the hypothesis that the molecular clock in birds "ticks" according to a constant rate of substitution per unit of mass-specific metabolic energy rather than per unit of time, as recently suggested. Our analysis advances knowledge of rates of DNA evolution across birds and other vertebrates and will, therefore, aid comparative biology studies that seek to infer the origin and timing of major adaptive shifts in vertebrates.     

Quality and quantity of data recovered from massively parallel sequencing: Examples in Asparagales and Poaceae

? Premise of the study: Genome survey sequences (GSS) from massively parallel sequencing have potential to provide large, cost-effective data sets for phylogenetic inference, replace single gene or spacer regions as DNA barcodes, and provide a plethora of data for other comparative molecular evolution studies. Here we report on the application of this method to estimating the molecular phylogeny of core Asparagales, investigating plastid gene losses, assembling complete plastid genomes, and determining the type and quality of assembled genomic data attainable from Illumina 80-120-bp reads. ? Methods: We sequenced total genomic DNA from samples in two lineages of monocotyledonous plants, Poaceae and Asparagales, on the Illumina platform in a multiplex arrangement. We compared reference-based assemblies to de novo contigs, evaluated consistency of assemblies resulting from use of various references sequences, and assessed our methods to obtain sequence assemblies in nonmodel taxa. ? Key results: Our method returned reliable, robust organellar and nrDNA sequences in a variety of plant lineages. High quality assemblies are not dependent on genome size, amount of plastid present in the total genomic DNA template, or relatedness of available reference sequences for assembly. Phylogenetic results revealed familial and subfamilial relationships within Asparagales with high bootstrap support, although placement of the monotypic genus Aphyllanthes was placed with moderate confidence. ? Conclusions: The well-supported molecular phylogeny provides evidence for delineation of subfamilies within core Asparagales. With advances in technology and bioinformatics tools, the use of massively parallel sequencing will continue to become easier and more affordable for phylogenomic and molecular evolutionary biology investigations.     

G4-quadruplexes and genome instability

In silico comparative analysis of nuclear and mitochondrial genomes of vertebrates and yeast revealed the distribution of nucleotide sequences that are predisposed to the formation of noncanonical DNA structures, G-quadruplexes, which are closely related to the regulation of expression of a number of genes and are abundant within the hot-spots of double-strand DNA breaks. The obtained data indicate the preferred localization of potential quadruplexes in the noncoding DNA sequences, their evolutionary conservation, and the existence of homology between them in the mitochondrial and nuclear genomes. The relationship between quadruplexes, Pif1 helicase, and genomic instability is discussed.     

More than 1000 ultraconserved elements provide evidence that turtles are the sister group of archosaurs

We present the first genomic-scale analysis addressing the phylogenetic position of turtles, using over 1000 loci from representatives of all major reptile lineages including tuatara. Previously, studies of morphological traits positioned turtles either at the base of the reptile tree or with lizards, snakes and tuatara (lepidosaurs), whereas molecular analyses typically allied turtles with crocodiles and birds (archosaurs). A recent analysis of shared microRNA families found that turtles are more closely related to lepidosaurs. To test this hypothesis with data from many single-copy nuclear loci dispersed throughout the genome, we used sequence capture, high-throughput sequencing and published genomes to obtain sequences from 1145 ultraconserved elements (UCEs) and their variable flanking DNA. The resulting phylogeny provides overwhelming support for the hypothesis that turtles evolved from a common ancestor of birds and crocodilians, rejecting the hypothesized relationship between turtles and lepidosaurs.