Nadot and colleagues (1994) were first to demonstrate that nucleotide sequences of chloroplast ribosomal protein S4 (rps) were phylogenetcally informative. In spite of early promise, rps4 has been largely ignored since. Comprehensive analysis of sequences drawn from over 200 representatives of all extant lineages of embryophytes demonstrates that this neglect is unwarranted. For a plastid gene, rps4 exhibits a remarkable degree of nucleotide substitution rate heterogeneity. The gene can be divided into five regions based on nucleotide substitution rate. Slowly evolving first (5' end), last (3' end) and middle regions exhibit substitution rates similar to rbcL, and may be functionally constrained. The two regions flanking either side of the middle region evolve at a much faster rate. Furthermore, these regions are prone to insertion and deletion, with some insertions exceeding 140 bp’s. Both insertions and deletions can be phylogenetically informative, either individually or in groups and sometimes define large clades with long evolutionary histories (e.g., euphyllophytes, moniliforms (ferns + horsetails), polypodiaceous ferns, and yew-conifers). Further phylogenetic information can be culled from the spacer region immediately adjacent to the 3' end of the gene. This region is highly variable in length with different size classes characteristic of different major clades of land plants. The spacers of the three “bryophyte” clades, lycopsids and most seed plants are short (generally <50 bp), whereas those of the moniliforms are considerably longer (generally >300 bp). The spacer region evolves at a rate similar to ITS in ferns and angiosperms and is generally useful for detecting radiations occurring during the Tertiary (i.e., within “families” of tracheophytes). Although rps4 is significantly shorter (and thus provides fewer characters for analysis) than phylogenetically comparable plastid genes, such as rbcL or atpB, the phylogenetic resolving power of rps4 is often greater, providing both fuller and more well supported resolution of radiations occurring from the Devonian to the early Tertiary.

Key words: chloroplast, ferns, land plants, molecular systematics, phylogeny, rps4