The position of gnetales among seed plants: overcoming pitfalls of chloroplast phylogenomics

The phylogenetic position of Gnetales is one of the most contentious issues in the seed plant systematics. To elucidate the Gnetales position, an improved amino acid substitution matrix was estimated based on 64 chloroplast (cp) genomes and was applied to cp genome data including all three lineages of Gnetales in maximum likelihood analyses of proteins. Although the initial analysis strongly supported the sister relation of Gnetales with Cryptomeria (Cupressophyta or non-Pinaceae conifers) (the "Gnecup" hypothesis), the support seems to be caused by a long-branch attraction (LBA) artifact. Indeed, by removing fastest evolving proteins that are most likely associated with the LBA, the support drastically declined. Furthermore, another analysis of partial genome data with dense taxon sampling of conifers showed that, in psbC, rpl2, and rps7 proteins, there are many parallel amino acid substitutions between the lineages leading to Gnetales and to Cryptomeria, and by further excluding these three genes, the sister relation of Gnetales with Pinaceae (the "Gnepine" hypothesis) became supported. Overall, our analyses indicate that the LBA and parallel substitutions cause a seriously biased inference of phylogenetic position of Gnetales with the cp genome data.