The perfect storm: Gene tree estimation error, incomplete lineage sorting, and ancient gene flow explain the most recalcitrant ancient angiosperm clade, Malpighiales

The genomic revolution offers renewed hope of resolving rapid radiations
in the Tree of Life. The development of the multispecies coalescent (MSC)
model and  improved gene tree estimation methods can better
accommodate gene tree heterogeneity caused by incomplete lineage sorting
(ILS) and gene tree estimation error stemming from the short internal
branches. However, the relative influence of these factors in species tree
inference is not well understood. Using anchored hybrid enrichment, we
generated a data set including 423 single-copy loci from 64 taxa
representing 39 families to infer the species tree of the flowering plant
order Malpighiales. This order includes nine of the top ten most unstable
nodes in angiosperms, which have been hypothesized to arise from the rapid
radiation during the Cretaceous. Here, we show that coalescent-based
methods do not resolve the backbone of Malpighiales and concatenation
methods yield inconsistent estimations, providing evidence that gene tree
heterogeneity is high in this clade. Despite high levels of ILS and gene
tree estimation error, our simulations demonstrate that these two factors
alone are insufficient to explain the lack of resolution in this order. To
explore this further, we examined triplet frequencies among empirical gene
trees and discovered some of them deviated significantly from those
attributed to ILS and estimation error, suggesting gene flow as an
additional and previously unappreciated phenomenon promoting gene tree
variation in Malpighiales. Finally, we applied a novel method to quantify
the relative contribution of these three primary sources of gene tree
heterogeneity and demonstrated that ILS, gene tree estimation error, and
gene flow contributed to 15%, 52%, and 32% of the variation, respectively.
Together, our results suggest that a perfect storm of factors likely
influence this lack of resolution, and further indicate that recalcitrant
phylogenetic relationships like the backbone of Malpighiales may be better
represented as phylogenetic networks. Thus, reducing such groups solely to
existing models that adhere strictly to bifurcating trees greatly
oversimplifies reality, and obscures our ability to more clearly discern
the process of evolution.