The performance of outgroup-free rooting under evolutionary radiations.

Tree rooting implies a temporal dimension to phylogenies. Only after defining the position of the root node is that the ancestral-descendant relationship between branches can be fully deduced. Rooting has been usually carried out by employing evolutionarily close outgroup lineages, which is a drawback when these lineages are unavailable or unknown. Alternatively, outgroup-free rooting methods were proposed, which rely on the constancy of evolutionary rates to varying degrees. In this work we analyzed the performance of two of these methods, the midpoint rooting (MPR) and the minimal ancestor deviation (MAD), in rooting topologies evolved under challenging scenarios of fast evolutionary radiations derived from empirical data, characterized by short internal branches near the crown node. Considering all branch length combinations investigated, both methods exhibited average success rates below 50%, although MAD slightly outperformed MPR. Moreover, tree balance significantly impacted the relative performance of the methods. We found that, in four-taxa unrooted trees, the outcome of whether both methodologies will correctly root the tree can be roughly predicted by two simple dimensionless metrics: the coefficient of variation of the external branch lengths, and the ratio between the internal branch length to the total sum of branch lengths, which were employed to devise a general linear model that allowed calculating the probability of correct placing the root node for any four-taxa tree. We predicted that the performance of both outgroup-free rooting methods on loci representing the placental mammal radiation ranged between 50% and 75%.