Christopher J. Huddleston

A phylogenomic study of birds reveals their evolutionary history.

Deep avian evolutionary relationships have been difficult to resolve as a result of a putative explosive radiation. Our study examined ∼32 kilobases of aligned nuclear DNA sequences from 19 independent loci for 169 species, representing all major extant groups, and recovered a robust phylogeny from a genome-wide signal supported by multiple analytical methods. We documented well-supported, previously unrecognized interordinal relationships (such as a sister relationship between passerines and parrots) and corroborated previously contentious groupings (such as flamingos and grebes). Our conclusions challenge current classifications and alter our understanding of trait evolution; for example, some diurnal birds evolved from nocturnal ancestors. Our results provide a valuable resource for phylogenetic and comparative studies in birds.

CHAPTER 8 – Phylogenetic Relationships among and within Select Avian Orders Based on Mitochondrial DNA

Among the many challenges facing avian systematists is the need for additional discrete character data sets and knowledge of the constraints influencing the character change over time. The objective of this chapter is to present phylogenetic analyses of new molecular sequence data for select avian lineages, to place these analyses in the context of existing phylogenetic hypotheses, and to discuss pertinent issues regarding methods of phylogenetic inference. Phylogenetic analyses presented in this chapter, based on mitochondrial DNA characters, address a number of controversial issues. Anseriformes and Galliformes are supported as sister taxa that are more closely related to a paleognath ( Rhea ) than to a set of neognaths. Placement of the root is critical in this latter determination, and addition of sequences from more taxa within the avian ingroup and within the crocodilian outgroup clade (including alligatorids, crocodilids, and gavialids) may help reduce the potential attraction among long branches. It is doubtful, however, that greater sampling of extant forms will ever eliminate the problem entirely. In analyses of two different data sets, Passeriformes are indicated as basal among five lineages representing the oldest divergences among extant birds and among a set of neognaths.

Phylogenomic evidence for multiple losses of flight in ratite birds

Ratites (ostriches, emus, rheas, cassowaries, and kiwis) are large, flightless birds that have long fascinated biologists. Their current distribution on isolated southern land masses is believed to reflect the breakup of the paleocontinent of Gondwana. The prevailing view is that ratites are monophyletic, with the flighted tinamous as their sister group, suggesting a single loss of flight in the common ancestry of ratites. However, phylogenetic analyses of 20 unlinked nuclear genes reveal a genome-wide signal that unequivocally places tinamous within ratites, making ratites polyphyletic and suggesting multiple losses of flight. Phenomena that can mislead phylogenetic analyses, including long branch attraction, base compositional bias, discordance between gene trees and species trees, and sequence alignment errors, have been eliminated as explanations for this result. The most plausible hypothesis requires at least three losses of flight and explains the many morphological and behavioral similarities among ratites by parallel or convergent evolution. Finally, this phylogeny demands fundamental reconsideration of proposals that relate ratite evolution to continental drift.

True and False Gharials: A Nuclear Gene Phylogeny of Crocodylia

The phylogeny of Crocodylia offers an unusual twist on the usual molecules versus morphology story. The true gharial (Gavialis gangeticus) and the false gharial (Tomistoma schlegelii), as their common names imply, have appeared in all cladistic morphological analyses as distantly related species, convergent upon a similar morphology. In contrast, all previous molecular studies have shown them to be sister taxa. We present the first phylogenetic study of Crocodylia using a nuclear gene. We cloned and sequenced the c-myc proto-oncogene from Alligator mississippiensis to facilitate primer design and then sequenced an 1,100-base pair fragment that includes both coding and noncoding regions and informative indels for one species in each extant crocodylian genus and six avian outgroups. Phylogenetic analyses using parsimony, maximum likelihood, and Bayesian inference all strongly agreed on the same tree, which is identical to the tree found in previous molecular analyses: Gavialis and Tomistoma are sister taxa and together are the sister group of Crocodylidae. Kishino-Hasegawa tests rejected the morphological tree in favor of the molecular tree. We excluded long-branch attraction and variation in base composition among taxa as explanations for this topology. To explore the causes of discrepancy between molecular and morphological estimates of crocodylian phylogeny, we examined puzzling features of the morphological data using a priori partitions of the data based on anatomical regions and investigated the effects of different coding schemes for two obvious morphological similarities of the two gharials.

Parsimony and Model-Based Analyses of Indels in Avian Nuclear Genes Reveal Congruent and Incongruent Phylogenetic Signals

Insertion/deletion (indel) mutations, which are represented by gaps in multiple sequence alignments, have been used to examine phylogenetic hypotheses for some time. However, most analyses combine gap data with the nucleotide sequences in which they are embedded, probably because most phylogenetic datasets include few gap characters. Here, we report analyses of 12,030 gap characters from an alignment of avian nuclear genes using maximum parsimony (MP) and a simple maximum likelihood (ML) framework. Both trees were similar, and they exhibited almost all of the strongly supported relationships in the nucleotide tree, although neither gap tree supported many relationships that have proven difficult to recover in previous studies. Moreover, independent lines of evidence typically corroborated the nucleotide topology instead of the gap topology when they disagreed, although the number of conflicting nodes with high bootstrap support was limited. Filtering to remove short indels did not substantially reduce homoplasy or reduce conflict. Combined analyses of nucleotides and gaps resulted in the nucleotide topology, but with increased support, suggesting that gap data may prove most useful when analyzed in combination with nucleotide substitutions.

Why Do Phylogenomic Data Sets Yield Conflicting Trees? Data Type Influences the Avian Tree of Life more than Taxon Sampling

&NA; Phylogenomics, the use of large‐scale data matrices in phylogenetic analyses, has been viewed as the ultimate solution to the problem of resolving difficult nodes in the tree of life. However, it has become clear that analyses of these large genomic data sets can also result in conflicting estimates of phylogeny. Here, we use the early divergences in Neoaves, the largest clade of extant birds, as a “model system” to understand the basis for incongruence among phylogenomic trees. We were motivated by the observation that trees from two recent avian phylogenomic studies exhibit conflicts. Those studies used different strategies: 1) collecting many characters [∼ 42 mega base pairs (Mbp) of sequence data] from 48 birds, sometimes including only one taxon for each major clade; and 2) collecting fewer characters (∼ 0.4 Mbp) from 198 birds, selected to subdivide long branches. However, the studies also used different data types: the taxon‐poor data matrix comprised 68% non‐coding sequences whereas coding exons dominated the taxon‐rich data matrix. This difference raises the question of whether the primary reason for incongruence is the number of sites, the number of taxa, or the data type. To test among these alternative hypotheses we assembled a novel, large‐scale data matrix comprising 90% non‐coding sequences from 235 bird species. Although increased taxon sampling appeared to have a positive impact on phylogenetic analyses the most important variable was data type. Indeed, by analyzing different subsets of the taxa in our data matrix we found that increased taxon sampling actually resulted in increased congruence with the tree from the previous taxon‐poor study (which had a majority of non‐coding data) instead of the taxon‐rich study (which largely used coding data). We suggest that the observed differences in the estimates of topology for these studies reflect data‐type effects due to violations of the models used in phylogenetic analyses, some of which may be difficult to detect. If incongruence among trees estimated using phylogenomic methods largely reflects problems with model fit developing more “biologically‐realistic” models is likely to be critical for efforts to reconstruct the tree of life. [Birds; coding exons; GTR model; model fit; Neoaves; non‐coding DNA; phylogenomics; taxon sampling.]

Homoplastic microinversions and the avian tree of life

BackgroundMicroinversions are cytologically undetectable inversions of DNA sequences that accumulate slowly in genomes. Like many other rare genomic changes (RGCs), microinversions are thought to be virtually homoplasy-free evolutionary characters, suggesting that they may be very useful for difficult phylogenetic problems such as the avian tree of life. However, few detailed surveys of these genomic rearrangements have been conducted, making it difficult to assess this hypothesis or understand the impact of microinversions upon genome evolution.ResultsWe surveyed non-coding sequence data from a recent avian phylogenetic study and found substantially more microinversions than expected based upon prior information about vertebrate inversion rates, although this is likely due to underestimation of these rates in previous studies. Most microinversions were lineage-specific or united well-accepted groups. However, some homoplastic microinversions were evident among the informative characters. Hemiplasy, which reflects differences between gene trees and the species tree, did not explain the observed homoplasy. Two specific loci were microinversion hotspots, with high numbers of inversions that included both the homoplastic as well as some overlapping microinversions. Neither stem-loop structures nor detectable sequence motifs were associated with microinversions in the hotspots.ConclusionsMicroinversions can provide valuable phylogenetic information, although power analysis indicates that large amounts of sequence data will be necessary to identify enough inversions (and similar RGCs) to resolve short branches in the tree of life. Moreover, microinversions are not perfect characters and should be interpreted with caution, just as with any other character type. Independent of their use for phylogenetic analyses, microinversions are important because they have the potential to complicate alignment of non-coding sequences. Despite their low rate of accumulation, they have clearly contributed to genome evolution, suggesting that active identification of microinversions will prove useful in future phylogenomic studies.

A molecular phylogenetic survey of caprimulgiform nightbirds illustrates the utility of non-coding sequences

The order Caprimulgiformes comprises five bird families adapted to nocturnal activity. The order has been regarded as monophyletic, but recent evidence suggests that swifts and hummingbirds (Apodiformes) belong within it. To explore the groups phylogeny, we obtained more than 2000 bp of DNA sequence from the cytochrome b and c-myc genes for 35 taxa, representing all major lineages and outgroups. Non-coding sequences of the c-myc gene were unsaturated, readily alignable and contained numerous informative insertions and deletions (indels), signalling broad utility for higher level phylogenetics. A 12 bp insertion in c-myc links Apodiformes with owlet-nightjars, confirming paraphyly of the traditional Caprimulgiformes. However, even this rare genomic change is homoplasious when all birds are considered. Monophyly of each of the five traditional families was strongly confirmed, but relationships among families were poorly resolved. The tree structure argues against family status for Eurostopodus and Batrachostomus, which should be retained in Caprimulgidae and Podargidae, respectively. The genus Caprimulgus and both subfamilies of Caprimulgidae appear to be polyphyletic. The phylogeny elucidates the evolution of adaptive traits such as nocturnality and hypothermia, but whether nocturnality evolved once or multiple times is an open question.