Ratite nonmonophyly: independent evidence from 40 novel Loci.

Large-scale multilocus studies have become common in molecular phylogenetics, but the best way to interpret these studies when their results strongly conflict with prior information about phylogeny remains unclear. An example of such a conflict is provided by the ratites (the large flightless birds of southern land masses, including ostriches, emus, and rheas). Ratite monophyly is strongly supported by both morphological data and many earlier molecular studies and is used as a textbook example of vicariance biogeography. However, recent studies have indicated that ratites are not monophyletic; instead, the volant tinamous nest inside the ratites rather than forming their sister group within the avian superorder Palaeognathae. Large-scale studies can exhibit biases that reflect a number of factors, including limitations in the fit of the evolutionary models used for analyses and problems with sequence alignment, so the unexpected conclusion that ratites are not monophyletic needs to be rigorously evaluated. A rigorous approach to testing novel hypotheses generated by large-scale studies is to collect independent evidence (i.e., excluding the loci and/or traits used to generate the hypotheses). We used 40 nuclear loci not used in previous studies that investigated the relationship among ratites and tinamous. Our results strongly support the recent molecular studies, revealing that the deepest branch within Palaeognathae separates the ostrich from other members of the clade, rather than the traditional hypothesis that separates the tinamous from the ratites. To ensure these results reflected evolutionary history, we examined potential biases in types of loci used, heterotachy, alignment biases, and discordance between gene trees and the species tree. All analyses consistently supported nonmonophyly of the ratites and no confounding biases were identified. This confirmation that ratites are not monophyletic using independent evidence will hopefully stimulate further comparative research on paleognath development and genetics that might reveal the basis of the morphological convergence in these large, flightless birds.