Analysing fungal ITS data - questions about the pipeline - phylogenetic analysis

I have always been under the impression, and read in several places including in the QIIME and QIIME2 ITS tutorials, that it is not advisable to use phylogenetic methods with ITS data. When analysing my own data I have avoided phylogenetic methods.
I have recently come across somebody who had used a bacterial 16S pipeline for their fungal ITS data, including phylogenetic analyses. I suggested they reanalyse their data using a fungal ITS pipeline and using alternatives to the phylogenetic analyses.
I was told that, whilst not often seen, the ITS1 region does produce a reasonable tree and is used frequently in published analyses including gold standard taxonomy papers from eg CBS.
I would argue that a taxonomy paper is a far cry from a mycobiome study, but I was hoping somebody could point me in the direction of some good publications on this topic. Whilst I have read that ITS is not advised, I can’t put my finger on anything to support this and have been going through my reference databases all morning!


Hi @Cat ,

This is sort of true — ITS alignments are sometimes used for examining phylogeny of closely related fungal species. Thus, fungal taxonomy papers (which are generally concerned with parsing closely related species/groups) will sometimes use this for comparison of isolated strains.

Indeed it is. In a mycobiome study you are almost inevitably comparing fungi from highly divergent groups. ITS will not generate a reliable alignment/phylogeny for such divergent groups.

As a non-coding region, it is highly variable, which is a two-edged sword: on the one hand, this means that it can be used to differentiate even many closely related species (taxonomically and phylogenetically) but on the other hand the mutation rate is so high that it loses the “molecular clock” aspects of conserved genes (such as rRNA genes) that are used for molecular phylogeny of highly divergent groups.

This looks like a relevant paper on the topic:

I hope that helps!


@Nicholas_Bokulich Thank you. It is reassuring that my own perceptions are the same, but you have worded it better so I may steal some of your words for my own response.
Moreover, that paper looks great, thank you.

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