Chloroplast DNA systematics: a review of methods and data analysis

The field of plant molecular systematics is expanding rapidly, and with it new and refined methods are coming into use. This paper reviews recent advances in experimental methods and data analysis, as applied to the chloroplast genome. Restriction site mapping of the chloroplast genome has been used widely, but is limited in the range of taxonomic levels to which it can be applied. The upper limits (i.e., greatest divergence) of its application are being explored by mapping of the chloroplast inverted repeat region, where rates of nucleotide substitution are low. The lower limits of divergence amenable to restriction site study are being examined using restriction enzymes with 4-base recognition sites to analyze polymerase chain reaction (PCR)-amplified portions of the chloroplast genome that evolve rapidly. The comparison of DNA sequences is the area of molecular systematics in which the greatest advances are being made. PCR and methods for direct sequencing of PCR products have resulted in a mushrooming of sequence data. In theory, any degree of divergence is amenable to comparative sequencing studies. In practice, plant systematists have focused on two slowly evolving sequences (rbcL and rRNA genes). More rapidly evolving DNA sequences, including rapidly changing chloroplast genes, chloroplast introns, and intergenic spacers, and the noncoding portions of the nuclear ribosomal RNA repeat, also are being investigated for comparative purposes. The relative advantages and disadvantages of comparative restriction site mapping and DNA sequencing are reviewed. For both methods, the analysis of resulting data requires sufficient taxon and character sampling to achieve the best possible estimate of phylogenetic relationships. Parsimony analysis is particularly sensitive to the issue of taxon sampling due to the problem of long branches attracting on a tree. However, data sets with many taxa present serious computational difficulties that may result in the inability to achieve maximum parsimony or to find all shortest trees.