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

Abstract

Two key factors have been implicated as major impediments to phylogenomic inference: incomplete lineage sorting (ILS)—especially in cases where clades are in the anomaly zone—and erroneous gene tree estimation—commonly manifested by long-branch attraction in the Felsenstein zone. Seldom have these factors been analyzed when they occur simultaneously as in cases involving ancient adaptive radiations. Rafflesiaceae (Malpighiales) is an iconic holoparasitic plant clade growing 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 in Malpighiales. To complicate matters, parasitic plants like Rafflesiaceae are well-known to exhibit accelerated rates of nucleotide substitution. Here, using 2,141 genes we identify for the first time that accelerated substitution rates is a genome-wide phenomenon in holoparasites. We demonstrate two orders of magnitude increase in substitution rates in Rafflesiaceae compared to its free-living relatives, rendering nearly every gene prone to long-branch attraction. Our analyses rejected the monophyletic (Rafflesiaceae, Euphorbiaceae) clade proposed in previous studies but favored an earlier divergence of Rafflesiaceae in close affinity with Euphorbiaceae, Peraceae, Putranjivaceae, and Pandaceae. Rafflesiaceae is therefore prey to a joint trap of the anomaly and Felsenstein zones. Moreover, owing to massive gene loss within Rafflesiaceae, our simulation suggests that this represents an intractable phylogenetic problem that will never achieve a single phylogenetic resolution. Our case study highlights a seemingly insurmountable confluence of factors—a danger zone—that hinders phylogenomic resolution and demonstrates the need to be flexible in cases where multiple phylogenomic placements are equally likely.