Rootstrap

IQ-TREE provides an option to calculate the rootstrap support values (Naser-Khdour et al 2021) for rooted and unrooted trees.

1.Using non-reversible models:

If you are using partitioned analysis, you first find the best partitioning scheme using the reversible model:

iqtree2 –s example.phy –p example.nex --prefix UNROOTED

This will infer the ML unrooted tree and print the result into UNROOTED.treefile and the best partitioning scheme and print the reult intoUNROOTED.best_scheme.nex. . If you used the UFBoot option too, then the bootstrap trees will be printed into UNROOTED.ufboot file

You then can use the best partitioning scheme to find the ML rooted tree with the non-reversible model

For DNA, use the UNREST model:

iqtree2 –s example.phy –p UNROOTED.best_scheme.nex –t UNROOTED.treefile -m UNREST -B 1000 --prefix ROOTED

For amino acid, use the NONREV model:

iqtree2 –s example.phy –p UNROOTED.best_scheme.nex –t UNROOTED.treefile -m NONREV -B 1000 --prefix ROOTED

This will print the ML rooted tree with the rootstrap support values into the ROOTED.rootstrap.nex file. The tree file in Nexus format will look like this:

#NEXUS [ This file is best viewed in FigTree. ] begin trees; tree tree_1 = ((BOS_MUT:0.0035185508[&id="2",rootstrap="0"],BOS_TAU:0.0060681062[&id="3",rootstrap="0"]):0.0306288620[&id="1",rootstrap="23.8"],((CAPRA_HIR:0.0092827982[&id="6",rootstrap="0"],OVIS_ARI:0.0104285307[&id="7",rootstrap="0"]):0.0052665552[&id="5",rootstrap="33.5"],PANTH_HOD:0.0113457232[&id="8",rootstrap="42.7"]):0.0000022608[&id="4",rootstrap="23.8"]):0.0000000000[&id="0",rootstrap="23.8"]; end;

Note: If you are not using a partitioned analysis remove the -p UNROOTED.best_scheme.nex –t UNROOTED.treefile option.

Note: It is recommended to compare AIC/BIC values of the reversible and the non-reversible models before making any conclusions about the root placement.

2.Using reversible models with outgroup:

First find the unrooted ML tree and the unrooted bootstrap trees and print them to files UNROOTED.treefile and UNROOTED.ufboot respectively:

iqtree2 –s example.phy –p example.nex –wbt -B 1000 --prefix UNROOTED

You then can use the -o option to root the ML tree and the bootstrap trees with outgroup taxons (e.g. taxon1,taxon2,taxon3) and use --rootstrap option to calculate the rootstrap support values

iqtree2 –t UNROOTED.treefile –-rootstrap UNROOTED.ufboot -o taxon1,taxon2,taxon3 --prefix ROOTED

This will print the ML rooted tree with the rootstrap support values into the ROOTED.rootstrap.nex file. and will print the topology test on rooting positions across all branches to the ROOTED.roottest.csv.

3.Perform topology tests on rooting positions across all branches

To perform topology tests on rooting positions across all branches use --root-test –zb 1000 -au option. This option will re-root the ML tree on all branches (including tips) and will print the topology test table into ROOTED.roottest.csv that looks like this:

ID,logL,deltaL,bp-RELL,p-KH,p-SH,c-ELW,p-AU 1,-1412910.023,0,0.251,0.695,1,0.3333548982,0.7135619126 8,-1412910.023,3.927876242e-05,0.338,0.305,0.837,0.3333420861,0.4428443979 5,-1412910.023,0.0001593173947,0.411,0.111,0.811,0.3333030158,0.1048951504 7,-1413575.029,665.0064102,0,0,0,3.129908119e-223,2.688384524e-44 6,-1413575.029,665.0064236,0,0,0,3.130318178e-223,2.809697961e-44 3,-1417753.19,4843.167287,0,0,0,0,0.0002968435628 2,-1417753.191,4843.167847,0,0,0,0,0.000296849965

The "id" columns in the ROOTED.roottest.csv are identical to the "id" labels in the ROOTED.rootstrap.nex file.

Note: This option can be combined with any of the options above to calculate the rootstrap support values and the AU test (and other topology tests) in one analysis.