Dealing with incongruence in the quest for the species tree: A case study from the orchid genus Satyrium

A species tree was reconstructed for the mainly African terrestrial orchid genus Satyrium. Separate phylogenetic analysis of both plastid and ribosomal nuclear DNA sequences for 63 species, revealed extensive topological conflict. Here we describe a detailed protocol to deal with incongruence involving three steps: identifying incongruence and testing its significance, assessing the cause of incongruence, and reconstructing the species tree. The Incongruence Length Difference test revealed that many cases of incongruence were non-significant. For the remaining significant cases, results from taxon jack-knifing experiments and parametric bootstrap suggested that non-biological artefacts such as sparse taxon sampling and long-branch attraction could be excluded as causes for the observed incongruence. In order to evaluate biological causes, such as orthology/paralogy conflation, lineage sorting, and hybridization, the number of events was counted that needs to be invoked a-posteriori to explain the observed pattern. In most cases where incongruence was significant, this resulted in a similar number of events for each of these different causes. Only for the three species from south east Asia, that form a monophyletic clade, hybridization was favoured over the alternative causes. This conclusion is based on the large number of events that needs to be invoked, in order for either orthology/paralogy conflation or lineage sorting to have been the cause of the incongruence + morphological evidence. The final species tree presented here is the product of the combined analysis of plastid and ITS sequences for all non-incongruent species and a-posteriori grafting of the incongruent clades or accessions onto the tree.