Was the ANITA rooting of the angiosperm phylogeny affected by long-branch attraction?

Five groups of basal angiosperms, Amborella, Nymphaeales, Illiciales, Trimeniaceae, and Austrobaileya (ANITA), were identified in several recent studies as representing a series of the earliest-diverging lineages of the angiosperm phylogeny. All of these studies except one employed a multigene analysis approach and used gymnosperms as the outgroup to determine the ingroup topology. The high level of divergence between gymnosperms and angiosperms, however, has long been implicated in the difficulty of reconstructing relationships at the base of angiosperm phylogeny using DNA sequences, for fear of long-branch attraction (LBA). In this study, we replaced the gymnosperm sequences from the five-gene matrix (mitochondrial atpI and matR, plastid atpB and rbcL. and nuclear 18S rDNA) used in our earlier study with four categories of divergent sequences-random sequences with equal base frequencies or equally AT- and GC-rich contents, homopolymers and heteropolymers, misaligned gymnosperm sequences, and aligned lycopod and bryophyte sequences-to evaluate whether the gymnosperms were an appropriate outgroup to angiosperms in our earlier study that identified the ANITA rooting. All 24 analyses performed rooted the angiosperm phylogeny at either Acorus or Alisma (or Alisma-Triglochin-Potamogeton in one case due to use of a slightly different alignment) and placed the monocots as a basal grade, producing genuine LBA results. These analyses demonstrate that the identification of ANITA as the basalmost extant angiosperms was based on historical signals preserved in the gymnosperm sequences and that the gymnosperms were an appropriate outgroup with which to root the angiosperm phylogeny in the multigene sequence analysis. This strategy of evaluating the appropriateness of an outgroup using artificial sequences and a series of outgroups with increments of divergence levels can be applied to investigations of phylogenetic patterns at the bases of other major clades, such as land plants, animals, and eukaryotes.