The goals of the proposed research are to study chloroplast genetics and phylogeny in two genera of conifers, Pinus and Pseudotsuga. Molecular genetics and evolution has been largely neglected in conifers, despite their ecologic, systematic, and economic importance, and despite reasons to expect them to differ from angiosperms. This study is likely to be the first comprehensive analysis of molecular genetics and evolution in a group of gymnosperms. -- Fifteen to twenty restriction endonucleases will be employed for studying variability in the nucleotide sequences of chloroplast DNA. The chloroplast DNA of Douglas-fir will be isolated and cloned, and then used as probe in filter hybridizations with the other species to be studied. Chloroplast maps will be created using restriction enzymes and both homologous and heterologous probes, allowing gene organization in the Pinaceae to be compared to that of other taxa, and aiding in the interpretation of the mutational bases of observed restriction fragment length polymorphisms. -- Intraspecific chloroplast DNA diversity is generally low in angiosperms, but may be substantial in conifers. Intraspecific variability will be studied in the widespread and economically important species Douglas-fir (pseudotsuga menziesii) and ponderosa pine (Pinus ponderosa). A rangewide sample will be examined within each species, spanning several geologically ancient races that differ in terpene chemistry, isozyme frequency, and morphology. This will allow chloroplast gene diversity to be compared to that of nuclear genes, and, if sufficient variability is found, will permit the construction of intraspecific phylogenies. -- Several long-standing phylogenetic problems are likely to be resolved by study of interspecific variability in chloroplast DNA. Utilizing a cladistic analysis of shared mutations, genus-wide phylogenies will be constructed in relation to outgroups in the Pinoid section of the Pinaceae (Pinus, Pseudotsuga, Picea, and Larix). These analyses will shed light on the origin of the hard and soft pine subgenera, the origins of Douglas-fir and Washoe pine, the relationships of Jeffrey pine to subsections Ponderosae and Sabinianae, and the proposed polyphyletic origins of bristlecone pine, subsection Oocarpae, and subsection Cembrae.
