Response to “On the impact of incomplete taxon sampling on the relative timing of gene transfer events”

In a previous article [1], we used simulations to show how acknowledging the existence of extinct and unsampled lineages, or "ghosts," could substantially influence conclusions regarding gene flow derived from gene tree branch lengths analyses.We mentioned that this could in particular challenge the claim by Pittis and Gabaldon [2,3] that genes of alpha-proteobacterial origin are more recently transferred than those originating from actinobacteria.Bernabeu and colleagues [4] propose a critique of our results based on 2 additional interesting analyses.The first one, summarized in their Fig 1C, is a description of the conditions under which the conclusions of Pittis and Gabaldon [2,3] can actually be reversed ("conditions to shift" in their terminology), thus acknowledging this possibility.Bernabeu and colleagues conclude that these conditions are "rather restrictive" or "highly constrained," suggesting that they are unlikely.This calls for 3 comments.One is that the list of conditions they give can be slightly relaxed.Indeed, it is not necessary that the earlier transfer (from alpha-proteobacteria) originates from a ghost lineage (as in Fig 1C in [4]).The shift is also obtained if this transfer comes directly from a non-ghost alpha-proteobacteria (α1 in their Fig 1) at an earlier time than the transfer from a ghost actinobacteria.This decreases the restrictivity of the conditions to shift.Another comment is that the conditions are not necessarily independent.Constraining the events to happen in a limited timeframe (the FECA-to-LECA period) is presented as a condition that decreases the chance that it occurs at all [4], but according to our analyses (Fig 6B in [1]) it may at the same time increase the probability that it leads to a shift.A last comment is that "rather restrictive" and "highly constrained" are vague and subjective qualifications.An opposite intuition is arguable: if we consider that most of the microbial diversity is unknown [5], and if we observe that numerous complete clades (such as the CPR, which represents a large part of the ToL presented as Fig 1A in Ref [4]) are probably missing from such an analysis, the conditions for shift seem sufficiently plausible to question the robustness of the stemlength based claims.Further work is needed to transform these intuitions into a probability and assess the likelihood that the conclusions of Pittis and Gabaldon [2,3] can be reversed (or not) when acknowledging the overwhelming presence of ghosts.Our simulations [1] were an attempt to quantify this effect.Surely they are not sufficient to give a definitive answer, because they were indeed not informed by all the knowledge we can have about the tree of life.At least, and in the light of the work of Bernabeu and colleagues [4], the question remains, in our view, largely open.