Abstract

ABSTRACT With a standard set of primers directed toward conserved regions, we have used the polymerase chain reaction to amplify homologous segments of mtDNA from more than 100 animal species, including mammals, birds, amphibians, fishes, and some invertebrates. Amplification and direct sequencing were possible using unpurified mtDNA from nanogram samples of fresh specimens and microgram amounts of tissues preserved for months in alcohol or decades in the dry state. The bird and fish sequences evolve with the same strong bias toward transitions that holds for mammals. However, because the light strand of birds is deficient in thymine, thymine to cytosine transitions are less common than in other taxa. Amino acid replacement in a segment of the cytochrome b gene is faster in mammals and birds than in fishes and the pattern of replacements fits the structural hypothesis for cytochrome b. The unexpectedly wide taxonomic utility ofthese primers offers opportunities for phylogenetic and population research. During the past decade, geneticists and taxonomists have used restriction endonucleases rather than sequencing to examine variation within and between species in specific segments of DNA (1-7). Although the indirect assessment of sequence variation obtained with the restriction endonuclease method is known to have many drawbacks, § sequence data have been difficult to obtain. The construction and screening of clone libraries has been too tedious and have demanded too much expertise for routine use by those geneticists and taxonomists who must analyze many individuals. Dependence on restriction analysis has limited our understanding of the dynamics of DNA sequence evolution. The presence or absence of a restriction site reveals little about the kinds of nucleotide substitutions that have occurred. Thus, although restriction analysis of mtDNA from closely related mammals first showed that these genomes have a higher rate of evolutionary substitution than does nuclear DNA, the demonstration that this acceleration results mainly from an increase in the number of transitions relative to transversions came only from conventional cloning and sequencing (1, 3). Because most studies of animal mtDNA have used restriction analysis, it has been difficult to determine whether a high rate of evolution and a transition bias are characteristic of all animal mtDNAs (8-10). There has been a need for simple methods of sequencing mtDNA to examine the pattern of evolutionary substitution in other animal groups.