Assessing the Cretaceous Superordinal Divergence Times within Birds and Placental Mammals by Using Whole Mitochondrial Protein Sequences and an Extended Statistical Framework

— Using the set of all vertebrate mtDNA protein sequences published as of May 1998, plus unpublished examples for elephant and birds, we examined divergence times in Placentalia and Aves. Using a parsimony-based test, we identiŽed a subset of slower evolutionary rate placental sequences that do not appear to violate the clock assumption. Analyzing just these sequences decreases support for Marsupionta and the carnivore + perissodactyl group but increases support for armadillo diverging earlier than rabbit (which may represent the whole Glires group). A major theme of the paper is to use more comprehensive estimates of divergence time standard error (SE). From the well-studied horse/rhino split, estimated to be 55 million years before present (mybp), the splitting time within carnivores is conŽdently shown to be older than 50 million years. Some of our estimates of divergence times within placentals are relatively old, at up to 169 million years, but are within 2 SE of other published estimates. The whale/cow split at 65 mybp may be older than commonly assumed. All the sampled splits between the main groups of fereuungulates (the clade of carnivores, cetartiodactyls, perissodactyls, and pholidotes) seem to be distinctly before the Cretaceous/Tertiary boundary. Analyses suggest a close relationship between elephants (representing Afrotheria) and armadillos (Xenarthra), and our timing of this splitting is coincident with the opening of the South Atlantic, a major vicariant event. Recalibrating with this event (at 100 mybp), we obtain younger estimates for the earliest splits among placentals . Divergence times within birds are also assessed by using previously unpublished sequences. We fail to reject a clock for all bird taxa available. Unfortunately, available deep calibration points for birds are questionable, so a new calibration based on the age of the Anseriform stem lineage is estimated. The divergence time of rhea and ostrich may be much more recent than commonly assumed, while that of passerines may be older. Our major concern is the rooting point of the bird subtree, as the nearest outgroup (alligator) is very distant. [bird phylogeny; mammal order phylogeny; mitochondrial genomes; molecular divergence times; sequencing errors.] It is becoming increasingly popular to infer divergence time estimates based on molecular sequences. An important statistical principle is that a single point estimate without an associated standard error (SE) is near useless for critical assessment. In this paper, we pay particular attention to giving more realistic estimates of errors on these divergence times by incorporating and integrating alternative sources of error. This is necessary because if molecular biologists are to have a meaningful discussion with each other, or with paleontologists, on the issue of molecular divergence times, then they must be prepared to calculate comprehensive SE estimates (Waddell and Penny, 1996). Here our approach is to check if a clockconstrained maximum-likelihood (ML) tree is, or is not, rejected under the best-Žtting model we have. A gamma ( G ) model is typically more appropriate for a coding sequence than is the assumption that all sites evolve at the same rate (see Swofford et al., 1996, for a review of such models). The model should allow better estimation of edge lengths, enabling more accurate inference of divergence times (Waddell and Penny, 1996). The edge lengths of the ML tree give arguably the most reliable estimates of relative divergence times because of the statistical efŽciency of ML estimation (Hasegawa et al., 1985, 1987, 1989; Kishino and Hasegawa, 1990). The next step is to pick the best calibration point(s) based on fossil or other information. Lastly, modelbased divergence timeestimates ought to be 3Present address: Institute of Molecular Biosciences, Massey University, Palmerston North, New Zealand, Email: waddell@onyx.si.edu. 120 SYSTEMATIC BIOLOGY VOL. 48 accompanied by SE values (Hasegawa et al., 1989), preferably those that attempt to take into account all the major sources of error that can be quantiŽed (Waddell, 1995:473– 476; Waddell and Penny, 1996). Following the techniques of the last two references, we integrate three major sources of error (fossil calibration error, Žnite sequence length error, and ancestral polymorphism) and quantify a fourth, sequencing error. We hope this approach will let people more clearly see the beneŽts and pitfalls of calibration of molecular divergence times just as Felsenstein (1985) was able to highlight the need for good statistical support in analysis of molecular evolutionary trees. Here, we estimate when major lineages within mammals and birds originated. It has long been recognized that these divergences may have begun well back into the Cretaceous (e.g., Gregory, 1910; Novacek, 1993; Simpson, 1945). Recently, molecular data are conŽrming this view (e.g., Hedges et al., 1996; Cooper and Penny, 1997; Springer, 1997; Kumar and Hedges, 1998), but with much uncertainty remaining as to exactly when and which clades formed in the Cretaceous. This is changing. For example, evidence has been found for an elephant (representing Afrotheria) + armadillo (Xenarthra) clade (Stanhope et al., 1998; Waddell et al., 1999). Results below suggest a link between this group (named Atlantogenata) and the opening of the South Atlantic Ocean. MATERIALS AND METHODS