How can I improve or otherwise investigate an unreliable genome tree?

Question

Summary My genome tree doesn't agree with my gene trees and I get the feeling that my genome tree might be wrong, possibly due to long branch attraction, but I don't know how to check/fix it.

Background I have a set of genes of interest recovered from genomes and metagenomes. I also have some bins built from the metagenomes of interest.

Previous work I've built multiple gene trees, which tend to agree with each other, as well as with other phylogenetic methods (16S rRNA, initial genome trees where available, conserved orientation of the genes of interest). So at this point I though I had a pretty good idea of how my genes / species of interest are linked to the species tree.

Concern I've assembled the metagenomes and recovered bins. I've refined bins of interest with anvio, based on sequence composition / differential coverage, and kept only the bins which appeared quite high quality (>70% completion*, <10 - often <5% - redundancy). Thus contamination in the metagenome assemblies (MAGs) is unlikely. The contamination would have to happen multiple times in the same way to different samples from different environments, at the same coverage, to get the same effect.

• with the exception of a bin of particular interest which I accepted with 62%

I have de-replicated these bins, added some known genomes, and tried to build a species tree with Orthofinder (with the proteomes). There is a problem occurs with a clade of 3 bins I will call 'X'.

Trees Based on the gene-of-interest phylogenies, X looks like probably a sister-group to the rest of known genomes from order 'A'. Which is very interesting. With the 16S rRNA, it's not quite as clear (there are no 16S rRNA sequences in the bins); however, some 16S rRNA amplified from one of the original samples fit the bill as 'sister-clade to basically everything else in group A'). The first (100 or so) BLAST hits for this 16S sequence are all from group 'A', still.

Tree conflict However, in the proteome tree, X as well as Y (Y being a sequenced genome that I suspected was related to X based on 16S data) end up in order 'B' instead of 'A'. GTDBtk also labels X as belonging to clade 'B', not 'A'.

Now,I know that genes and genomes don't always have the same evolutionary trajectories. But multiple sets of data lead me to a different conclusion than the proteome tree, so I have to at least question whether the proteome tree is correct; what's likely to be the problem (I suspect long-branch attraction), and, if it is long-branch attraction (or anything else) how can I uncover the correct signal?

Thank you for your time!

Answer 1

The difficulty in answering the question is:

• The absence of "Y" in the "Gene of interest" tree
• The sampling bias in the number of taxa mostly between the "genome tree" and the "gene of interest" and "16S" tree
• The "genome" tree is unrooted so its difficult to compare it with the "16S" tree, thus its possible that if "16S" was unrooted it falls within monophyly "B"
• The trees don't have any branch or statistical support
• We don't really know the tree building method (I haven't looked it up)
• The bacteria might have significant recombination (we don't know the bacteria).

The answer is there is a lot of room for different tree artefacts here, however it could be a biologically real result. I agree "the tree" is simply a tool which uses a series of assumptions and obtaining confidence in a tree requires a lot of analysis. Vast amounts of computational time are dedicated to obtaining a robust tree.

The principle step is to compare like for like:

• same number of taxa and
• same rooting position (or unrooted).
• second step is to obtain statistical robustness for the observation.

There is a formal process for identifying conflicting trees and it needs code - I have written stuff that does this and maybe I should release it. Essentially, you would data chunk gene by gene to produce a genome of gene trees and use e.g. ETE3 or similar to plot the tree conflicts. It ain't rocket science. Thus the amount of user time is trivial. The idea is that once the "problem trees" are identified, e.g. rapid evolution.

The one observation I would say is that the "Y" branch looks a little bit weird and this could indicate part of the data is has resulted from different evolutionary rate and that is the basis for long-branch attraction.

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Scenario 1 It could be that the 16S and "genome" tree are part of the same tree (monophyly B), they are just rooted differently. Thus the conflict is just a small number of genes and this could lead to ...

Scenario 2 Keep in mind bacteria do undergo 'horizontal transmission' and if 'gene of interest' is under strong selective pressure recombination is entirely possible.

Answer 2

It seems like you've got the right approach, in trying out a few different genes and looking for consistency. As you say, different genes can have different selective pressures, which influences how much they change in relation to the whole genome background.

Certainly don't put too much trust in a single gene, and especially not 16S: it has a low mutation rate, can jump around and be repeated in the genome, and is an occasional target for horizontal gene transfer.

The answer may be that there isn't any single correct phylogeny. A primary assumption of phylogenetics is that evolutionary progression has a tree-like structure (with roots and tips), but that model is broken by horizontal gene transfer and plasmid sharing. It may be the case that you need to break the genome up into fragments, each with its own inherited history.