Ribosomal RNA and Tetrapod Phylogeny

— Previous phylogenetic analyses of tetrapod 18S ribosomal RNA (rRNA) sequences support the grouping of birds with mammals, whereas other molecular data, and morphological and paleontological data favor the grouping of birds with crocodiles. The 18S rRNA gene has consequently been considered odd, serving as “definitive evidence of different genes providing significantly different estimates of phylogeny in higher organisms” (p. 156; Huelsenbeck et al., 1996, Trends Ecol. Evol. 11:152–158). Our research indicates that the previous discrepancy of phylogenetic results between the 18S rRNA gene and other genes is caused mainly by (1) the misalignment of the sequences, (2) the inappropriate use of the frequency parameters, and (3) poor sequence quality. When the sequences are aligned with the aide of the secondary structure of the 18S rRNA molecule and when the frequency parameters are estimated either from all sites or from the variable domains where substitutions have occurred, the 18S rRNA sequences no longer support the grouping of the avian species with the mammalian species. [alignment; 18S rRNA; RNA secondary structure; Indel; molecular phylogenetics; tetrapod phylogeny. ] One of the early controversies in the phylogenetic relationship among tetrapods is whether birds are more closely related to crocodilians (Romer, 1966; Carroll, 1988; Gauthier et al., 1988) or to mammals (Gardiner, 1982; Løvtrup, 1985). Hedges et al. (1990) collected a set of 18S ribosomal RNA (rRNA) sequences to evaluate the relationships among tetrapods and found that the bird–mammal grouping was much more strongly supported, with a bootstrap value of 88%, than the bird– crocodilian grouping. A subset of these 18S rRNA sequences was used subsequently in a statistical test, based on the minimum-evolution criterion, to evaluate relative support of these alternative phylogenetic hypotheses (Rzhetsky and Nei, 1992). The nine shortest trees, including the neighbor-joining tree, all grouped the avian and mammalian species together as a monophyletic taxon. The bird–mammal grouping contradicts both the traditional classification and the results derived from a large amount of other molecular data (Hedges, 1994; Seutin et al., 1994; Caspers et al., 1996; Janke and Arnason, 1997; Zardoya and Meyer, 1998; Ausio et al., 1999) and morphological and paleontological data (Eernisse and Kluge, 1993). For this reason, the proposal of the bird– mammal grouping based on the 18S rRNA gene has received critical examination from many different perspectives to determine what bias could have been introduced in analyzing the 18S rRNA sequences among tetrapod species. Four kinds of potential bias involving the 18S rRNA gene have been proposed. First, the genomes of homeotherms such as birds and mammals tend to be more GC rich than those of poikilotherms (Bernardi, 1993). This shift in nucleotide frequencies, i.e., the problem of nonstationarity in the substitution process, is generally not accommodated in either parsimony or maximum-likelihood phylogenetic methods. For this reason, the LogDet (Lockhart et al., 1994) distance, which is based on a substitution model that presumably should correct for the nucleotide frequency shift, was used to see whether the annoying bird–mammal grouping would disappear (Huelsenbeck and Bull, 1996; Huelsenbeck et al., 1996). It did not. Second, the substitution pattern is biased favoring U↔C transitions in rRNA genes (Marshall, 1992). This bias is expected and is mainly caused by the fact that the nucleotide G can pair with either U or C in maintaining the secondary structure of the rRNA molecule. For example, in pairing with a G, a C can be replaced by a U with little effect on the secondary structure. This ease of substitution is partly the reason for the model (Tamura and Nei, 1993) that uses one parameter for the T↔C transition and another for the A↔G transition. However, the substitution pattern is not unique in the 18S rRNA gene and therefore cannot explain the difference in phylogenetic outcome between the 18S rRNA and the rest of rRNA molecules. The use of the most general distance such as the LogDet still does not break up the bird–mammal grouping when applied to the 18S rRNA sequences (Huelsenbeck and Bull, 1996; Huelsenbeck et al., 1996). The combination of a higher GC content in homeotherms and the biased substitution pattern favoring U↔C transitions may jointly increase the problems associated with long-branch attraction (Marshall, 1992; Huelsenbeck et al., 1996). If U↔C transition is the predominant substitution type and if birds and mammals have experienced an increase in C, then convergent U→C transitions may occur independently in the lineages leading to birds and to mammals. However, Hedges and Maxon (1992) dismissed long-branch attraction because the 18S rRNA sequences do not seem to have experienced substitutional saturation. A weighted parsimony method (Williams and Fitch, 1990; Fitch et al., 1995) produced equivocal results (Marshall, 1992). When the paleontological tree grouping birds with crocodilians was used as the starting tree, the method ended up supporting this starting tree. However, when the tree grouping birds with mammals was used as the starting tree, the method ended up supporting this new starting tree. The weighted parsimony method is