Most species are extinct; those that are not are often unknown. Sequenced and sampled species are often a minority of known ones. Past evolutionary events involving horizontal gene flow, such as horizontal gene transfer, hybridization, introgression and admixture, are therefore likely to involve “ghosts”, i.e. extinct, unknown or unsampled lineages. The existence of these ghost lineages is widely acknowledged, but their possible impact on the detection of gene flow and on the identification of the species involved is largely overlooked. It is generally considered as a possible source of error that, with reasonable approximation, can be ignored. We explore the possible influence of absent species on an evolutionary study by quantifying the effect of ghost lineages on introgression as detected by the popular D-statistic method. We show from simulated data that under certain frequently encountered conditions, the donors and recipients of horizontal gene flow can be wrongly identified if ghost lineages are not taken into account. In particular, having a distant outgroup, which is usually recommended, leads to an increase in the error probability and to false interpretations in most cases. We conclude that introgression from ghost lineages should be systematically considered as an alternative possible, even probable, scenario.

Introgression, endosymbiosis, and gene transfer, i.e., horizontal gene flow (HGF), are primordial sources of innovation in all domains of life. Our knowledge on HGF relies on detection methods that exploit some of its signatures left on extant genomes. One of them is the effect of HGF on branch lengths of constructed phylogenies. This signature has been formalized in statistical tests for HGF detection and used for example to detect massive adaptive gene flows in malaria vectors or to order evolutionary events involved in eukaryogenesis. However, these studies rely on the assumption that ghost lineages (all unsampled extant and extinct taxa) have little influence. We demonstrate here with simulations and data reanalysis that when considering the more realistic condition that unsampled taxa are legion compared to sampled ones, the conclusion of these studies become unfounded or even reversed. This illustrates the necessity to recognize the existence of ghosts in evolutionary studies.