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may actually be far smaller than it appears, representing as few as four maternal lineages (one <br />from each pond and one from each individual collected in the main channel). <br />Isolation and analysis. of mtDNA.- MtDNA has been a useful marker for studies of <br />population structure (reviewed by Avise et al., 1987; Avise, 1992) and estimates of genetic <br />diversity (Avise et al., 1988, 1989). It is maternally inherited (Dawid and Blackler, 1972, and <br />reviewed in Avise and Lansman, 1983; but see Satta et al., 1988; Kondo et al., 1990; Hoeh et <br />al., 1991; and Gyllensten et al., 1991 for exceptions) and evolves rapidly in many vertebrates <br />(reviewed in Moritz et al., 1987; but see Avise et al., 1992), making it a highly polymorphic <br />character for tracing maternal lineages. <br />Tissue samples were typically removed by non-lethal methods (mostly ova/milt stripped <br />from gravid individuals, razely muscle plugs); however, occasionally other tissues (muscle, <br />heart, liver, and/or gonad) were occasionally available from accidental moralities. MtDNA <br />was isolated from tissues of each individual either by phenol-chloroform extraction (Chapman <br />and Powers, 1984) or equilibrium-density ultracentrifugation (Dowling et al., 1990). The <br />former method was used initially on a small number of egg samples, while the latter was used <br />to purify intact, circular mtDNA from the remainder. <br />Genetic variation was characterized by cleaving each sample with the following <br />restriction endonucleases: BanI (GGPyPuCC), BstEII (GGTNACC), HinfI (GANTC), HinPI <br />(GCGC), MboI (GATC), NheI (GCTAGC), ScrFI (CCNGG), and TagI (TCAG}. Resulting <br />cleavage fragments were end-labelled, electrophoretically separated on 1.0-1.5% agarose and <br />4.0% polyacrylamide gels, and visualized by autoradiography (Dowling et al., 1940), <br />Distinctive restriction fragment patterns for a specific enzyme were identified by alphabetical <br />code. Letters were assigned by order of discovery and not similarity of patterns. The <br />composite haglotype for each individual is identifed by an eight-letter code describing the <br />4 <br />