The danger zone: the joint trap of incomplete lineage sorting and long-branch attraction in placing Rafflesiaceae

Two key factors have been implicated as major impediments to phylogenomic inference: incomplete lineage sorting (ILS), especially in cases where clades are in the so-called anomaly zone, and erroneous gene tree estimation, commonly manifested by long-branch attraction in the Felsenstein zone. Rafflesiaceae (Malpighiales) is an iconic parasitic plant clade whose species occur west of Wallace's line in tropical Southeast Asia. This clade has been notoriously difficult to place phylogenetically and is nested within an explosive ancient radiation, rendering the Malpighiales one of the thorniest nodes across the angiosperm Tree of Life. The parasitic family Apodanthaceae has recently been positioned as sister to Rafflesiaceae, offering the hope to stabilize their placements. Here, using a dataset of 2,135 genes and complex species tree inference methods, the monophyletic Rafflesiaceae+Apodanthaceae clade is variously placed with Euphorbiaceae, Peraceae, Putranjivaceae, and Pandaceae. Such unstable placements appear to be the result of excessive levels of rate heterogeneity and ILS, which contribute to a phylogenetic danger zone where simulation suggests that current methods and genomic data will never provide a tidy phylogenetic resolution for species trees alike. Despite the topological uncertainty, however, our divergence time estimation identifies a mid-Cretaceous origin of stem group Rafflesiaceae and Apodanthaceae, not only making them the oldest parasitic plant lineage reported to date, but also suggests a likely Gondwana vicariance scenario to explain their disjunct distribution in South America, Africa, Australia, and Northern India.