Plant chloroplast genomes (plastomes) are characterized by an inverted repeat (IR) region and two larger single copy (SC) regions. Patterns of molecular evolution in the IR and SC regions differ, most notably by a reduced rate of nucleotide substitution in the IR compared to the SC region. In addition, the organization and structure of plastomes is fluid, and rearrangements through time have repeatedly shuffled genes into and out of the IR, providing recurrent natural experiments on how chloroplast genome structure can impact rates and patterns of molecular evolution. Here we examine four loci (psbA, ycf2, rps7, and rps12 exon 2-3) that were translocated from the SC into the IR during fern evolution. We use a model-based method, within a phylogenetic context, to test for substitution rate shifts. All four loci show a significant, 2- to 3-fold deceleration in their substitution rate following translocation into the IR, a phenomenon not observed in any other, nontranslocated plastid genes. Also, we show that after translocation, the GC content of the third codon position and of the noncoding regions is significantly increased, implying that gene conversion within the IR is GC-biased. Taken together, our results suggest that the IR region not only reduces substitution rates, but also impacts nucleotide composition. This finding highlights a potential vulnerability of correlating substitution rate heterogeneity with organismal life history traits without knowledge of the underlying genome structure
