J Mol Evol (1991) 33:525-536 Journal of Molecular Evolution ~) Springer-Verlag New York Inc. 1991 Molecular Phylogenetic Analysis of Actin Genie Regions from Achlya bisexualis (Oomycota) and Costaria costata (Chromophyta) Debashish Bhattacharya, Shawn K. Stickel, and Mitchell L. Sogin Center for Molecular Evolution, Marine BiologicalLaboratory, Woods Hole, MA 02543, USA Summary. Actin genie regions were isolated and characterized from the heterokont-flagellated pro- tists, Achlya bisexualis (Oomycota) and Costaria costata (Chromophyta). Restriction enzyme and cloning experiments suggested that the genes are present in a single copy and sequence determina- tions revealed the existence of two introns in the C. costata actin genie region. Phylogenetic analyses of actin genie regions using distance matrix and max- imum parsimony methods confirmed the close evo- lutionary relationship ofA. bisexuafis and C. costata suggested by ribosomal DNA (rDNA) sequence comparisons and reproductive ceil ultrastructure. The higher fungi, green plants, and animals were seen as monophyletic groups; however, a precise order of branching for these assemblages could not be determined. Phylogenetic frameworks inferred from comparisons of rRNAs were used to assess rates of evolution in actin genie regions of diverse eukaryotes. Actin genie regions had nonuniform rates of nucleotide substitution in different lineages. Comparison of rates of actin and rDNA sequence divergence indicated that actin genie regions evolve 2.0 and 5.3 times faster in higher fungi and flowering plants, respectively, than their rDNA sequences. Conversely, animal actins evolve at approximately one-fifth the rate of their rDNA sequences. Key words: Actin -- Chromophytes -- Phyloge- netic analysis --Small subunit RNA -- Monophyly -- Molecular evolution Introduction Evolutionarily conserved actins and/or their genes have been isolated from many eukaryotic phyla in- Offprint requests to: M.L. Sogin eluding plants, fungi, animals, and divergent protist groups (Hightower and Meagher 1986). In most taxa studied, actin mRNAs are transcribed from multi- gene families, though Saccharomyces cerevisiae (Ng and Abelson 1980), Aspergilhts nidulans (Fidel et al. 1988), the oomycete Phytophthora megasperma (Dudler 1990), Tetrahymena sp. (Cupples and Pearlman 1986; Hirono et al. 1987), and Volvox carteri (Cresnar et al., unpublished) are defined by single-copy genes. Despite a high degree of primary structure conservation, these homologous proteins serve diverse functional roles in cytoskeletal archi- tecture and ceil motility. For example, animals have structurally distinct cytoplasmic and muscle-specif- ic actins (Vandekerckhove and Weber 1978, 1979, 1984; Hightower and Meagher 1986) with mammals expressing at least six different actins including a-skeletal, a-cardiac, and/3- and ~-nonmuscle actins (Alonso 1987). Recent studies show that the higher green plant, Glycine max, contains three similar ac- tin families of divergent function (McLean et al. 1990). Understanding the evolution of single-copy and multiple gene families leading to divergent function is frequently biased by the selection of organisms and complications of unequal rates of amino acid substitution. Most studies do not include species that represent the extreme phylogenetic depths ex- hibited by divergent protistan lineages, and as- sumptions implicit in the molecular clock hypoth- esis (Wilson et al. 1977) are frequently inconsistent with observed rates of change in different evolu- tionary lines of descent. In contrast, small subunit (16S-like) rRNA sequences have been determined for a broad spectrum ofeukaryotes (Sogin and Gun- derson 1987; Sogin et al. 1989). All 16S-like rRNA genic regions are mosaics of genetic elements that evolve at different rates, yet, these "multi-handed