CHLOROPLAST DNA EVOLUTION IN Atriplex

Analysis of chloroplast DNA varia­ tion in species of Lycopersicon (Solan­ aceae) has demonstrated the utility of the techniques for assessing phyloge­ netic relationships among species (Pal­ mer and Zamir, 1982). A similar but more extensive survey has been initi­ ated in the genus Atriplex (Chenopo­ diaceae) using the same techniques. Atriplex is a genus of over 400 de­ scribed species and has representatives on all continents except Antarctica. The centers of greatest concentration are in western North America (59 species), the Mediterranean and Near East (42 spe­ cies), Asia (49 species), South America (79 species), and Australia (59 species). The genus is of additional interest in that it contains species having either C3 or C4 pathways of photosynthesis. Preliminary work has been carried out on 60 species: 22 from western America, 33 from Australia, and 5 from Eurasia. Only 6 of these are C3 species; the rest possess C4 photosynthesis. We have treated chloroplast DNA from these species with ten different restriction endonucleases-enzymes which recog­ nize and cleave the DNA duplex at spe­ cific six-base-pair sequences. The characteristic set of fragments gener­ ated by each enzyme is resolved by aga­ rose gel electrophoresis and visualized by UV-illumination of the ethidium bromide-stained gel. Species-specific changes in the frag­ ment patterns produced by a given restriction enzyme are usually inter­ pretable as the consequence of either the loss or gain of a specific restriction cleavage site and are generally caused by a single base-pair substitution. The phylogenetic utility of this type of chlo­ roplast DNA mutational analysis lies in the fact that many restriction site changes are shared by two or more spe­ cies and can be used to infer shared ev­ olutionary histories. Many different mutations have been found which lead to various affinity groups among the 60 Atriplex species examined. Summation of these group­ ings according to principles of parsi­ mony analysis (Ferris et al., 1981) leads to a composite phylogenetic tree. Con­ sideration of the tree allows (1) deter­ mination of relative branching orders among the species, (2) determination of the ancestral chloroplast genome for the group under study, (3) assignment ofan evolutionary direction to most muta­ tions, i.e., determination of which group has the ancestral fragment pattern and which has the derived pattern, (4) de­ termination ofthe nature and incidence of parallelisms, and (5) calculation of relative divergence distances between the different chloroplast DNA lineages. Although the present investigation is far from complete, both in terms of number of restriction enzymes and number of species to be surveyed, sev­ eral tentative but important conclu­ sions have emerged. First, there is a remarkable homogeneity among most Atriplex chloroplast genomes. Sequence divergence estimates for the great ma­ jority ofC4 species studied are less than 1%. Only in comparisons involving the most primitive C3 species (see below) do we observe divergence values of even a few percent. Thus, the A trip lex chlo­ roplast genome appears to be evolving significantly more slowly than single­ copy nuclear DNA sequences from the same species (Belford and Thompson, 1981). Moreover, the chloroplast gen­ ome appears to be a much more sensi­ tive molecular yardstick than the nuclear genome for measuring evolu­ tionary distances within Atriplex. The range ofdivergence values encountered in an equivalent group of species is at least tenfold for the chloroplast gen­ ome, while no more than twofold for the nuclear genome (Belford and Thomp­ son, 1981). A central issue in A triplex phylogeny