Rooting phylogenetic trees in R using Ape's root function: Trees don't seem to root.

Question

[spiral01]

Hi, I have a dataset of roughly 7000 trees in newick format that I have loaded into R. Each tree has 5 tips, each representing a species. As the trees are not rooted, I am trying to root the trees using one of the species (AGAP) as the outgroup. However, when I run the root function like so:

x <- root(nh.tree.list[[1]], outgroup = "AGAP")

The tree does indeed shift so that the node below the tip AGAP is at the base of the tree, but the tree remains unrooted (which I can confirm using the is.rooted command). This tree in question in newick format is:

(AMEM:0.06608854676,AARA:0.05958550382((AGAP:0.08235019885,AQUA:0.05921768275):0.0120129681,AMEC:0.06541441488):0.007929218113);

I need to root the trees as the analysis I intend to do requires rooted trees. In case it helps, these trees were created from multiple sequence alignment fasta files, using neighbour joining methods (njs function). I cannot fathom why the trees aren't being rooted. Can anyone shed any light on this? Thanks.

[fanli.gcb]

That tree shows up as rooted for me:

> tree = read.tree()
1: (AMEM:0.06608854676,AARA:0.05958550382((AGAP:0.08235019885,AQUA:0.05921768275):0.0120129681,AMEC:0.06541441488):0.007929218113);
2: 
> tree

Phylogenetic tree with 4 tips and 3 internal nodes.

Tip labels:
[1] "AMEM" "AARA" "AGAP" ""    
Node labels:
[1] ""     "AMEC" "AQUA"

Rooted; includes branch lengths.

[moranr]

I got the same as Fanli.gcb when i ran on Ape too. Plot the tree, you will see if its rooted or not. Maybe there is a problem with function to check rooting or something.

[spiral01]

This is very strange. I am still getting:

Phylogenetic tree with 5 tips and 3 internal nodes.

Tip labels:
[1] "AMEM" "AARA" "AGAP" "AQUA" "AMEC"

Unrooted; includes branch lengths.

When I plotted it is still not rooted. I tried to read in the tree independently from the same information I have given you and I still got the above message, using no arguments other than file for read.tree. The fact that two of you got the same means there's something wrong my end, though I am not sure what it might be.

[spiral01]

Ok I think I know what my issue is. I was using read.tree(file = "filename") which seems to result in an unrooted tree. You guys did read.tree(), and then entered tree details on the next line, as fanli.gcb already showed. However, how can I do this if I need to read in 7000 files? To be clear, I am talking about the difference between this:

tree <- read.tree(file = "species_tree_align_0.fasta.nh")

Which produces an unrooted tree, and this:

> tree3 = read.tree()
1:(AMEM:0.06608854676,AARA:0.05958550382((AGAP:0.08235019885,AQUA:0.05921768275):0.0120129681,AMEC:0.06541441488):0.007929218113);
2:

Which produces the rooted tree. What I need to know is how to achieve the latter tree when reading in multiple files?

Answer 1

[spiral01]

Apologies for the multiple posting, but have resolved this issue by using the root function, and the resolve.root argument set to true as per the below:

x <- root(nh.tree.list[[1]], outgroup = "AGAP", resolve.root = TRUE)

Thanks for all the help!

[fanli.gcb]

Glad to see you figured it out, but I'm still a bit worried that your read.tree seems to give an unrooted tree. I don't think it's due to differences in reading from file versus command line:

> tree=read.tree(file="tree.nh")
> tree

Phylogenetic tree with 4 tips and 3 internal nodes.

Tip labels: [1] "AMEM" "AARA" "AGAP" ""     Node labels: [1] ""    
"AMEC" "AQUA"

Rooted; includes branch lengths.

What version of ape are you using?

> sessionInfo()
R version 3.2.3 Patched (2015-12-22 r69809)
Platform: x86_64-suse-linux-gnu (64-bit)
Running under: openSUSE 13.1 (Bottle) (x86_64)

locale:
 [1] LC_CTYPE=en_US.UTF-8       LC_NUMERIC=C              
 [3] LC_TIME=en_US.UTF-8        LC_COLLATE=en_US.UTF-8    
 [5] LC_MONETARY=en_US.UTF-8    LC_MESSAGES=en_US.UTF-8   
 [7] LC_PAPER=en_US.UTF-8       LC_NAME=C                 
 [9] LC_ADDRESS=C               LC_TELEPHONE=C            
[11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C       

attached base packages:
[1] stats     graphics  grDevices utils     datasets  methods   base     

other attached packages:
[1] ape_3.4

loaded via a namespace (and not attached):
[1] nlme_3.1-126    grid_3.2.3      lattice_0.20-33