Czech L, Huerta-Cepas J, Stamatakis A, 2017. A critical review on the use of support values in tree viewers and bioinformatics toolkits. Molecular Biology and Evolution. DOI: 10.1093/molbev/msx055The authors found something that, in retrospect, seems glaringly obvious. Phylogenetic trees are nearly always saved in the Newick format of nested brackets, for example as follows:
(A:2,(B:1,C:2)99:1);In this case we are dealing with a rooted tree of only three taxa. A is sister to a clade of B and C. The numbers after the colons indicate branch lengths, and the 99 directly after the brackets is a support value, most likely bootstrap, for the sister group relationship (B,C).
The problem explored by Czech et al. is ultimately that under the Newick format branch support values or other branch annotations are not actually attached to branches; they are attached to nodes. In this case, for example, the 99 is attached to the node that is the hypothetical common ancestor of B and C. Logically, because the tree is rooted we can assume that the support value is meant for the branch leading down from the ancestor of B and C towards the root.
But what if we reroot a tree with node annotations that are really meant to be branch annotations a posterioiri? (My post on the various options for rooting phylogenies can be found here.) Czech et al. found that the behaviour of the these values is undefined. For some software they were able to demonstrate that the branch annotation ended up on the wrong branch after rerooting.
How serious an issue is that? I guess it depends on what one's practice is. The problem should be pretty much limited to analyses producing unrooted trees (e.g. in RAxML, PAUP or MrBayes) under the assumption of reversibility, where the user then uses outgroup rooting to polarise the tree a posteriori. Any analysis using a clock model would avoid it, as would asymmetric step-matrices or, crucially, those analyses specifying the outgroup before the start of the analysis.
In addition, it seems as if the problem would be limited to a few branches between the pseudo-root used to save unrooted trees and the new root after rerooting, so that most relationships should be fine. I may look at one or two of my published phylogenies to see if I ever had that problem, but I am not worried; in the most recent case where support values were a critical part of my argumentation, for example, they are fairly deep inside the tree, because we sampled widely around the ingroup, and I also used Templeton tests and suchlike to demonstrate the non-monophyly of certain taxa.
Apparently Czech et al. have already achieved some success at getting software providers to make changes that will help solve the confusion around where the branch annotations end up. But nonetheless my main take-home from this is to be less blasé about a posteriori rooting. In the future I will make sure to always define an outgroup already when I set up a PAUP or RAxML run, so that the need to reroot does not arise.