Testing the variability of chloroplast sequences for plant phylogeography

Phylogeography in plants is hampered by lack of DNA-sequence regions that detect sufficient variation in intra-specific lineages to reveal historical patterns. We tested 13 putatively highly variable non-coding chloroplast regions in six species complexes, from four different angiosperm families, where phylogeographic patterns have previously been identified using restriction fragment length polymorphism analysis of the chloroplast genome. All regions tested amplified in most of the species. The intergenic spacer regions trnQ–rps16, trnS–trnG, psbA–trnH, psbD–trnT and ndhC–trnV were the five most promising regions for phylogeographic analysis in terms of variability, and petB and rpl16 were variable, given the utility of being amplified in a single reaction. The trnQ–rps16 and psbA–trnH intergenic spacer regions and the rpl16 D4-loop intron showed variation between known lineages in all species. The psbA–trnH intergenic spacer that has been suggested as a suitable barcoding gene for plants, generally showed a level of variation similar to that in other variable regions in the species investigated here, suggesting that some caution is required in the use of this region for barcoding applications. The present analysis identified a set of seven chloroplast regions that are a useful basis for informed selection of sequences for assessment of phylogeographic structure in plants.