Tree rooting with outgroups when they differ in their nucleotide composition from the ingroup: The Drosophila saltans and willistoni groups, a case study

Rooting is frequently the most precarious step in any phylogenetic analysis . Outgroups can become useless for rooting if they are too distantly relate d to the ingroup. Specifically, little attention has been paid to scenarios where outgroups have evolved different nucleotide frequencies from the ing roup. We investigate one empirical example that arose seeking to determine the phylogenetic relationship between the sat tons and the willistoni group s of Drosophila (subgenus Sophophora). We have analyzed 2085 coding nucleot ides from the xanthine dehydrogenase (Xdh) gene in 14 species, 6 from the s altans group and 8 from the willistoni group. We adopt a two-step strategy: (1) we investigate the phylogeny without outgroups, rooting the network by the midpoint method; (2) we reinvestigate the rooting of this phylogeny us ing predefined outgroups in both a parsimony- and a model-based maximum-lik elihood framework. A satisfactory description of the substitution process a long the Xdh region calls for six substitution types and substitution rate variation among codon positions. When the ingroup sequences are considered alone, the phylogeny obtained using this description corroborates the known relationships derived from anatomical criteria. Inclusion of the outgroups makes the root unstable, apparently because of differences between ingroup s and outgroups in the substitution processes; these differences are better accounted for by a simplified model of evolution than by more complex, rea listic descriptions of the substitution process. (C) 2000 Academic Press.