Virtually all mt genes and introns tested hybridized well across the full spectrum of angiosperms examined, and some even hybridized well across additional blots containing the full diversity of land plants, a roughly 450-million-year time span (see, e.g., Qiu et al., 1998). The success of these hybridizations, carried out at moderate stringencies [washes at 60°C in 2x standard saline citrate (SSC)/0.1% SDS], across such large timespans testifies to the very low substitution rates of the great majority of plant mtDNAs. A probe sequence was inferred to be absent from the mt genome of a particular filter-bound preparation of total DNA if there was no detectable hybridization on an overexposed autoradiograph against two layers of controls: good hybridization to the DNA in question using other mt probes and good hybridization to other DNAs with the probe in question.

Fig. 1 shows examples of the three general categories of results obtained with the various mt probes used. Many probes, such as rRNA probes and the cox1 exon probe used in the middle panel of Fig. 1, hybridized strongly to essentially all DNAs tested; i.e., the lane-to-lane variations in hybridization intensity were reproducible across all probes in this category. We interpret these variations as primarily reflecting differences in amount of mtDNA loaded per lane, and conclude that each mtDNA probably contains an intact copy of the sequence probed (see the penultimate section for an explanation of the weak cox1 hybridization in lane 4 of the middle panel). Many other probes, such as the rps7 gene probe used in the top panel of Fig. 1, while hybridizing strongly to many lanes

FIGURE 1. Southern blot survey illustrating three distinct presence/absence patterns of mitochondrial genes and introns. BamHI-cut total DNAs from 51 of 281 angiosperms surveyed were arranged according to presumptive phylogenetic relationships, were electrophoresed, and were blotted and hybridized with probes internal to the rps7 gene (Upper; gene mostly present, with several losses evident), the cox1 coding sequence (Middle; gene universally present), and the cox1 intron (Lower; intron rarely present, each presence thought to reflect an independent acquisition by horizontal transfer).