Time Flies, a New Molecular Time-Scale for Brachyceran Fly Evolution Without a Clock

The insect order Diptera, the true flies, contains one of the four largest Mesozoic insect radiations within its suborder Brachycera. Estimates of phylogenetic relationships and divergence dates among the major brachyceran lineages have been problematic or vague because of a lack of consistent evidence and the rarity of well-preserved fossils. Here, we combine new evidence from nucleotide sequence data, morphological reinterpretations, and fossils to improve estimates of brachyceran evolutionary relationships and ages. The 28S ribosomal DNA (rDNA) gene was sequenced for a broad diversity of taxa, and the data were combined with recently published morphological scorings for a parsimony-based phylogenetic analysis. The phylogenetic topology inferred from the combined 28S rDNA and morphology data set supports brachyceran monophyly and the monophyly of the four major brachyceran infraorders and suggests relationships largely consistent with previous classifications. Weak support was found for a basal brachyceran clade comprising the infraorders Stratiomyomorpha (soldier flies and relatives), Xylophagomorpha (xylophagid flies), and Tabanomorpha (horse flies, snipe flies, and relatives). This topology and similar alternative arrangements were used to obtain Bayesian estimates of divergence times, both with and without the assumption of a constant evolutionary rate. The estimated times were relatively robust to the choice of prior distributions. Divergence times based on the 28S rDNA and several fossil constraints indicate that the Brachycera originated in the late Triassic or earliest Mesozoic and that all major lower brachyceran fly lineages had near contemporaneous origins in the mid-Jurassic prior to the origin of flowering plants (angiosperms). This study provides increased resolution of brachyceran phylogeny, and our revised estimates of fly ages should improve the temporal context of evolutionary inferences and genomic comparisons between fly model organisms. (Bayesian analysis; Brachycera; Diptera; divergence times; molecular systematics; 28S ribosomal DNA.) In 1984, Beverley and Wilson (1984) published a sem- inal work estimating divergence times for Drosophila and several other derived flies based on the assump- tion of a constant rate of protein evolution in larval hemolymph proteins. This early molecule-based time scale has been widely used for dating comparisons be- tween Drosophila and other dipterans (e.g., Clark and Henikoff, 1992; Bonneton et al., 1997; Pitnick et al., 1999; Shaw et al., 2001; Bolshakov et al., 2002). Since that time, the existence of a molecular clock has been widely questioned and empirically challenged (e.g., Gillespie, 1986, 1991; Ayala, 2000). A number of new methods have been proposed that incorporate heterogeneity of evolutionary rates over time into divergence time es- timation (Thorne et al., 1998; Huelsenbeck et al., 2000; Kishino et al., 2001; Sanderson, 2002). The revolutionary advances of genomic and phylogenetic data and method- ology and the need to date precisely the age of diver- gences between key fly model organisms (e.g., Drosophila melanogaster, Culex pipiens, Anopheles gambiae, and Musca domestica) make it essential to revise and extend our cur- rent understanding of the time scale for dipteran lineage origins. The Diptera (true flies) are among the largest radia- tions of terrestrial eukaryotic organisms. As for other holometabolous insect orders, major diversification of fly lineages occurred in Mesozoic environments (Yeates and Wiegmann, 1999). Flies are arguably the most im- portant insect order in terms of their impact on human and animal health, being the vectors of such devastating afflictions as malaria, yellow fever, and sleeping sick-