Direct evidence for the descent of a group of species from a single common ancestor is rarely possible in the absence of fossils. Consequently, phylogenetic taxonomy relies on comparative analysis of morphological, chromosomal, and biochemical characters for evidence of relationship. However, even when many characters are concordant in their variation patterns, phylogenetic inferences can be confused by character convergence, functional correlation, unequal rates of evolution in different lineages, and ignorance of primitive and derived character states. In principle, a monophyletic relationship for a large number of diploid species could be inferred if they possessed in common a character that had a single and unique origin. Such a character appears to have been identified in the annual plant genus Clarkia (Onagraceae). It is shared by more than a dozen diploid. species which are morphologically and karyotypically diverse, and assigned to four sections in the current taxonomy (Lewis and Lewis, 1955). The character is an apparent duplication of the structural gene which codes the subunits of the cytoplasmic isozyme of phosphoglucoisomerase (PGI) (EC 5.3. 1.9). PGIs are dimeric enzymes that catalyze the reversible isomerization of glucose-6phosphate and fructose-6-phosphate. The duplication was discovered during the course of electrophoretic studies of enzyme variation in Clarkia (Gottlieb, 1973, 1974, 1977a). Its electrophoretic variability in several species, mode of inheritance, linkage, and several biochemical properties were recently described (Gottlieb, 1977b). The duplication is considered unique because its presumed mode of origin (Gottlieb, 1977b) required the sequential occurrence of a number of independent and rare events: (1) two reciprocal translocations that involve the same two pairs of nonhomologous chromosomes; (2) each translocation must overlap the chromosomal segment that bears the ancestral PGI locus; (3) a chromosomal heterozygote for the translocations must be formed presumably from a cross between the single translocations; and (4) the duplicated progeny class must become homozygous and be viable and fertile. These steps have been demonstrated experimentally in maize (Burnham, 1962). The major genetic prediction of origin by translocation is that the ancestral and duplicate loci not be linked. This appears to be the case in Clarkia since the two loci assort independently in C. xantiana (Gottlieb, 1977b), and we report here similar results in C. biloba, C. dudleyana, and C. unguiculata. This paper describes the electrophoretic variability of PGI and the number of coding gene loci in 30 species of Clarkia (about 75% of those currently recognized). In addition, we provide both genetic and biochemical evidence of overlaps on starch gels of the electrophoretic mobilities of isozymes coded by the ancestral and duplicate genes. The taxonomic distribution of the duplication in Clarkia requires a new phylogenetic alignment of the sections of the genus.