and D. miranda (Olvera et al., 1979) and because it pairs more fully with the miranda homolog of the pseudoobscura third chromosome in interspecies hybrids than does either SC or ST (Wallace, 1966; Morrow, 1970; Wallace, 1988). The only informative molecular data on this topic come from a recent analysis of restriction site polymorphism (RSP) within these arrangements (Aquadro et al., 1991) that produced two important results. First, the phylogeny based on RSP data corroborates the cytogenetic phylogeny. Second, it was estimated that the TL branch diverged from the SC-ST group about 1.7 million years ago. The greater depth of the TL branch and its early splitting have been used to support the ancestral status of the TL arrangement, which represents the consensus view today (Aquadro et al., 1991; Powell, 1992).
We decided to approach the question of the ancestral arrangement in the following way. First, an independent assessment of the phylogenetic relationships among the central gene arrangements was made on the basis of nucleotide sequences flanking the amylase 1 (Amy1) gene, which is located within the inversions. Second, a framework of competing hypotheses regarding the ancestral type was devised on the basis of the known relationships among central arrangements, and each of these hypotheses was compared with the empirically derived phylogenies. Third, several additional chromosomes were added to our earlier RSP data set (Aquadro et al., 1991) and analyzed by several different methods to determine whether this more extensive phylogenetic analysis still supported an ancestral status for TL.
Lines of D. pseudoobscura stocks homozygous for the third chromosome were constructed using balancer stocks (Pavlovsky and Dobzhansky, 1966). Salivary glands were dissected from third-instar larvae, 30 per line, and gene arrangements were diagnosed from squash preparations of the polytene chromosomes. The six strains used for determining DNA sequences flanking Amy1 are as follows: ST, Ayala reference strain, from northern California; SC, strain BAJA 859#3, from Baja California, Mexico; Chiricahua (CH), strain AH 87#2, from northern California; TL, strain AH 73#2, from northern California; Estes Park (EP), strain BC p430#4, from British Columbia, Canada; Olympic (OL), strain s14AR-D; from British Columbia, Canada. Three SC strains from Michoacan, Mexico, were added to the original RSP data set: strain MEX z67w, strain MEX z53y, and strain MEX z13w. Restriction mapping of these strains was carried out as described previously (Aquadro et al., 1991). In addition to the inversions already identified, the RSP data set includes Arrowhead (AR).