Last update: Jul 24, 2018, Contributors: M Bui
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The iqtree executable should already be installed on the MBL cluster. If you, however, want to install IQ-TREE to your computer, please first download and install the binary for your platform. For the next steps, the folder containing your iqtree executable should be added to your PATH enviroment variable so that IQ-TREE can be invoked by simply entering iqtree at the command-line. Alternatively, you can also copy iqtree binary into your system search.
TIP: For quick overview of all supported options in IQ-TREE, run the command
iqtree -h.
IQ-TREE takes as input a multiple sequence alignment and will reconstruct an evolutionary tree that is best explained by the alignment. The input alignment can be in various common formats: Phylip format, Fasta, Nexus, ClustalW. IQ-TREE will automatically detect the input file format.
Download an example alignment called example.phy in PHYLIP format. This example contains parts of the mitochondrial DNA sequences of several animals (Source: Phylogenetic Handbook).
You can now start to reconstruct a maximum-likelihood tree from this alignment using -s command line option (assuming that you are now in the same folder with example.phy):
iqtree -s example.phy
With this simple command IQ-TREE will first perform ModelFinder (see choosing the right substitution model below) to find the best-fit substitution model (i.e. no need to run jModelTest or ProtTest) and then infer a phylogenetic tree using the selected model.
At the end of the run IQ-TREE will write several output files including:
example.phy.iqtree: the main report file that is self-readable. You should look at this file to see the computational results. It also contains a textual representation of the final tree (see below).example.phy.treefile: the ML tree in NEWICK format, which can be visualized by any supported tree viewer programs like FigTree or iTOL.example.phy.log: log file of the entire run (also printed on the screen). To report bugs, please send this log file and the original alignment file to the authors.For this example data the resulting maximum-likelihood tree may look like this (extracted from .iqtree file):
NOTE: Tree is UNROOTED although outgroup taxon 'LngfishAu' is drawn at root
+-------------LngfishAu
|
| +--------------LngfishSA
+-------|
| +------------LngfishAf
|
| +--------------------Frog
+------|
| +-----------------Turtle
| +--|
| | | +------------------------Crocodile
| | +----|
| | +------------------Bird
| +--|
| | +---------------------------Sphenodon
| +-----|
| | +-------------------------------Lizard
+---------|
| +--------------Human
| +--|
| | | +------Seal
| | +--|
| | | +-----Cow
| | +--|
| | +-------Whale
| +----|
| | | +---Mouse
| | +---------|
| | +------Rat
+-----------|
| +--------------Platypus
+--|
+-----------Opossum
Here the tree is drawn at the outgroup Lungfish which is more accient than other species in this example. However, please note that IQ-TREE always produces an unrooted tree as it knows nothing about this biological background; IQ-TREE simply draws the tree this way as LngfishAu is the first sequence occuring in the alignment.
QUESTIONS:
- Does this tree make sense to you?
During the example run above, IQ-TREE periodically wrote to disk a checkpoint file example.phy.ckp.gz (gzip-compressed to save space). This checkpoint file is used to resume an interrupted run, which is handy if you have a very large data sets or time limit on a cluster system. If the run did not finish, invoking IQ-TREE again with the very same command line will recover the analysis from the last stopped point, thus saving all computation time done before.
If the run successfully completed, running again will issue an error message:
ERROR: Checkpoint (example.phy.ckp.gz) indicates that a previous run successfully finished
Use `-redo` option if you really want to redo the analysis and overwrite all output files.
This prevents lost of data if you accidentally re-run IQ-TREE. However, if you really want to re-run the analysis and overwrite all previous output files, use -redo option:
iqtree -s example.phy -redo
Finally, the default prefix of all output files is the alignment file name. You can
change the prefix using the -pre option:
iqtree -s example.phy -pre myprefix
This prevents output files being overwritten when you perform multiple analyses on the same alignment within the same folder.
IQ-TREE supports a wide range of substitution models for DNA, protein, codon, binary and morphological alignments. The previous run already determined the best-fit model that minimizes the Bayesian Information Criterion (BIC) score.
QUESTIONS:
- What is the name of the best-fit model?
- What do
+Iand+Gmean?- What is the best model according to Akaike Information Criterion (AIC) and the corrected AIC (AICc)?
Some hints:
-m MF.-mset to restrict the testing procedure to a subset of base models instead of testing the entire set of all available models. For example, -mset GTR to only test GTR+... models for DNA data, or -mset WAG,LG will test only models like WAG+... or LG+... for protein data.For more details about ModelFinder see:
S. Kalyaanamoorthy, B.Q. Minh, T.K.F. Wong, A. von Haeseler, and L.S. Jermiin (2017) ModelFinder: fast model selection for accurate phylogenetic estimates. Nat. Methods, 14:587–589. DOI: 10.1038/nmeth.4285
To overcome the computational burden required by the nonparametric bootstrap, IQ-TREE introduces an ultrafast bootstrap approximation (UFBoot) (Minh et al., 2013; Hoang et al., 2018) that is orders of magnitude faster than the standard procedure and provides relatively unbiased branch support values. IQ-TREE also provides an implementation of the SH-like approximate likelihood ratio test (SH-aLRT) (Guindon et al., 2010). We can do both UFBoot and SH-aLRT by a single command line:
iqtree -s example.phy -m TIM2+I+G -bb 1000 -alrt 1000 -pre example.boot
Options explained:
-m specifies the model to avoid ModelFinder, as it was already found to be the best-fit model.-bb specifies the number of bootstrap replicates where 1000 is the minimum number recommended.-alrt specifies the number of bootstrap replicates for SH-aLRT where 1000 is the minimum number recommended.-pre to specify output file prefix so that they do not overwrite the previous analysis.The section MAXIMUM LIKELIHOOD TREE in example.boot.iqtree shows a textual representation of the maximum likelihood tree with branch support values in percentage. The NEWICK format of the tree is printed to the file example.boot.treefile. In addition, IQ-TREE writes the following files:
example.boot.contree: the consensus tree with assigned branch supports where branch lengths are optimized on the original alignment.example.boot.splits.nex: support values in percentage for all splits (bipartitions), computed as the occurence frequencies in the bootstrap trees. This file is in NEXUS format, which can be viewed with the program SplitsTree to explore the conflicting signals in the data. So it is more informative than consensus tree, e.g. you can see how highly supported the second best conflicting split is, which had no chance to enter the consensus tree.NOTE: UFBoot support values have a different interpretation to the standard bootstrap. Refer to FAQ: UFBoot support values interpretation for more information.
You can now look at the textual tree figure in .iqtree file:
NOTE: Tree is UNROOTED although outgroup taxon 'LngfishAu' is drawn at root
Numbers in parentheses are SH-aLRT support (%) / ultrafast bootstrap support (%)
+-------------LngfishAu
|
| +--------------LngfishSA
+-------| (100/100)
| +------------LngfishAf
|
| +--------------------Frog
+------| (99.8/100)
| +-----------------Turtle
| +--| (85/72)
| | | +------------------------Crocodile
| | +----| (96.5/97)
| | +------------------Bird
| +--| (39/51)
| | +---------------------------Sphenodon
| +-----| (98.2/99)
| | +-------------------------------Lizard
+---------| (100/100)
| +--------------Human
| +--| (92.3/93)
| | | +------Seal
| | +--| (68.3/75)
| | | +-----Cow
| | +--| (99.7/100)
| | +-------Whale
| +----| (99.1/100)
| | | +---Mouse
| | +---------| (100/100)
| | +------Rat
+-----------| (100/100)
| +--------------Platypus
+--| (93/98)
+-----------Opossum
QUESTIONS:
- Discuss the branch supports. What are the unstable branches of the tree?
We won’t do the standard nonparametric bootstrap because it’ll be too slow. For your information, it is invoked by -b 100 option to perform 100 bootstrap replicates.
For more details see D.T. Hoang, O. Chernomor, A. von Haeseler, B.Q. Minh, and L.S. Vinh (2018) UFBoot2: Improving the ultrafast bootstrap approximation. Mol. Biol. Evol., 35:518–522. https://doi.org/10.1093/molbev/msx281
IQ-TREE can utilize multiple CPU cores to speed up the analysis. A complement option -nt allows specifying the number of CPU cores to use. For example:
iqtree -s example.phy -m TIM2+I+G -nt 2
Here, IQ-TREE will use 2 CPU cores to perform the analysis.
Note that the parallel efficiency is only good for long alignments. A good practice is to use -nt AUTO to determine the best number of cores:
iqtree -s example.phy -m TIM2+I+G -nt AUTO
Then while running IQ-TREE on a computer with 4 physical CPU cores, it may print something like this on to the screen:
Measuring multi-threading efficiency up to 8 CPU cores
Threads: 1 / Time: 8.001 sec / Speedup: 1.000 / Efficiency: 100% / LogL: -22217
Threads: 2 / Time: 4.346 sec / Speedup: 1.841 / Efficiency: 92% / LogL: -22217
Threads: 3 / Time: 3.381 sec / Speedup: 2.367 / Efficiency: 79% / LogL: -22217
Threads: 4 / Time: 4.385 sec / Speedup: 1.825 / Efficiency: 46% / LogL: -22217
BEST NUMBER OF THREADS: 3
Therefore, I would only use 3 cores for this example data. For later analysis with your same data set, you can stick to the determined number.
In the partition model, you can specify a substitution model for each gene/character set. IQ-TREE accepts partition file in RAxML-style and NEXUS format. IQ-TREE will then estimate the model parameters separately for every partition. Moreover, IQ-TREE provides edge-linked or edge-unlinked branch lengths between partitions:
-q partition_file: all partitions share the same set of branch lengths (like -q option of RAxML).-spp partition_file: like above but allowing each partition to have its own evolution rate.-sp partition_file: each partition has its own set of branch lengths (like combination of -q and -M options in RAxML) to account for, e.g. heterotachy (Lopez et al., 2002).NOTE:
-sppis recommended for typical analysis.-qis unrealistic and-spis very parameter-rich. One can also perform all three analyses and compare e.g. the BIC scores to determine the best-fit partition model.
Please now download a DNA alignment originally analysed to study the phylogenetic position of Turtles (caretta, chelonoidis_nigra, emys_orbicularis, phrynops) with Crocodiles (alligator & caiman) and Birds (Gallus & Taeniopygia) (Chiari et al., 2012). This question was highly debatable some 6 years ago.
First, we will perform an analysis with single model (no partitions) where branch supports are assessed with SH-aLRT and UFBoot:
iqtree -s turtle_nt.phy -alrt 1000 -bb 1000 -m GTR+R5
Here we use -m GTR+R5 to set the GTR+R5 model and skip ModelFinder because it might take too long.
Hint: The above command will only use 1 thread, so that users on the cluster at the same time do not interfere with each other. If you however run this on your own laptop, then you might want to add -nt AUTO to further speedup the analysis.
QUESTIONS:
- What is the best-fit model and its AIC/BIC scores?
- Use a tree viewer program (e.g. FigTree) to visualize the resulting tree and root it at the outgroup taxon
protopterus. Where is Turtle position in the tree? Does it agree with the analysis onexample.phydone above?
Now download a partition NEXUS file containing 248 genes for this Turtle data set, which looks like this:
#nexus
begin sets;
charset ENSGALG00000000041.macse_DNA_gb = 1-498;
charset ENSGALG00000000169.macse_DNA_gb = 499-1047;
...
charset ENSGALG00000020605.macse_DNA_gb = 186499-187026;
end;
This NEXUS file contains a SETS block with CharSet commands to specify individual partitions.
Perform an edge-linked partitioned analysis:
iqtree -s turtle_nt.phy -alrt 1000 -bb 1000 -spp turtle_nt.nex
The output files will have now the prefix turtle_nt.nex.*.
QUESTIONS:
- Is the partition model better than the single model in terms of AIC/BIC scores?
- Visualize the tree. What is the difference in tree topology compared with the previous tree?
- Which tree agrees with Chiari et al., 2012?
Further reading on our approach to speedup partitioned analysis:
O. Chernomor, A. von Haeseler, and B.Q. Minh (2016) Terrace aware data structure for phylogenomic inference from supermatrices. Syst. Biol., 65:997-1008. https://doi.org/10.1093/sysbio/syw037
NOTE: This section is optional.
When there are “short” partitions, it is a good practice to perform PartitionFinder (Lanfear et al., 2012), which tries to merge partitions to reduce the number of parameters and improve model fit. When you have many partitions, you can reduce the computational burden with the relaxed hierarchical clustering algorithm (Lanfear et al., 2014) using -rcluster option.
All these techniques are already implemented in ModelFinder. However, we won’t however perform this analysis here due to excessive computations. Nevertheless here are a few useful options for such analysis:
-m MFP+MERGE is to perform PartitionFinder algorithm followed by tree reconstruction.-rcluster 5 is to only examine the top 5% partitioning schemes (similar to the --rcluster-percent 10 option in PartitionFinder).-mset GTR to restrict the set of testing models to just GTR. This also helps to save computations.QUESTIONS (if you performed this analysis):
- How many partitions does not best partitioning scheme have now?
- What are the AIC/BIC scores?
- Is there any change in the tree topology?
For partitioned analysis, IQ-TREE will by default resample the sites within partitions (i.e., the bootstrap replicates are generated per partition separately and then concatenated together). However, it is recommended to resample partitions (Nei et al., 2001). This can be done with -bsam GENE option. Moreover, IQ-TREE allows an even more complicated strategy: resampling partitions and then sites within resampled partitions (Gadagkar et al., 2005). This may help to reduce false positives (i.e. wrong branch receiving 100% support). This can be done with -bsam GENESITE.
Please now perform ultrafast bootstrap with partition resamplings.
QUESTIONS:
- Is there any change in tree topology?
- Do the bootstrap support values get smaller or larger? Why?
We now want to know whether the trees inferred for the Turtle data set have significantly different log-likelihoods or not. This can be conducted with Shimodaira-Hasegawa test (Shimodaira and Hasegawa, 1999), or expected likelihood weights (Strimmer and Rambaut, 2002).
First, concatenate the trees constructed by single and partition models into one file:
cat turtle_nt.phy.treefile turtle_nt.nex.treefile >turtle_nt.trees
Now pass this file into IQ-TREE via -z option:
iqtree -s turtle_nt.phy -m MODEL_NAME -z turtle_nt.trees -pre turtle_nt.phy.treetest -n 0 -zb 1000
Options explained:
MODEL_NAME to the best-fit model found in the single model run.-pre is to specify a prefix for output files, so that they do not overwrite previous analysis.-zb is to specify the number of boostrap replicates for the resampling estimated log-likelihood method (RELL) (Kishino et al., 1990).-n 0 is to avoid tree search and estimate model parameters based on an initial parsimony tree.Now have a look at turtle_nt.phy.treetest.iqtree. The results of the tests will be printed to a section called USER TREES.
QUESTIONS:
- Do the two trees have significantly different log-likelihoods?
- How do you do tree tests with partition model? How do the results look like?
HINTS:
The KH and SH tests return p-values, thus a tree is rejected if its p-value < 0.05 (marked with a - sign).
bp-RELL and c-ELW return posterior weights which are not p-value. The weights sum up to 1 across the trees tested.
The KH test (Kishino and Hasegawa, 1989) was designed to test 2 trees and thus has no correction for multiple testing. The SH test (Shimodaira and Hasegawa, 1999) fixes this problem.
NOTE: This section is optional if you still have time.
Now we want to investigate the cause for such topological difference between trees inferred by single and partition model. One way is to identify genes contributing most phylogenetic signal towards one tree but not the other.
How can one do this? Well, we can look at the gene-wise log-likelihood differences between the two trees, called T1 and T2. Those genes having the largest lnL(T1)-lnL(T2) will be in favor of T1. Whereas genes showing the largest lnL(T2)-lnL(T1) are favoring T2.
For this purpose, we will do tree tests with partition model and utilize -wpl option for writing partition log-likelihoods:
iqtree -s turtle_nt.phy -spp turtle_nt.nex.best_scheme.nex -z turtle_nt.trees -pre turtle_nt.nex.treetest -n 0 -zb 1000 -wpl
Here we input the best partitioning scheme (turtle_nt.nex.best_scheme.nex) found previously to avoid model selection again.
The partition-wise log-likelihoods will be printed to turtle_nt.nex.treetest.partlh.
QUESTIONS:
- What are the two genes that most favor the tree inferred by single model? HINT: Use Excel or some R script to process
.partlhfile.- Have a look at the paper by (Brown and Thomson, 2016). Compare the two genes you found with those from this paper. What is special about these two genes?
Previous sections only dealt with DNA sequences. We now switch to an interesting protein data set used to examine the position of Microsporidia, a Fungus. Please download the alignment file here, which is a subset (10 genes) of the full data set (Brinkmann et al., 2005). This data set contains some Archaea as outgroup to the remaining Eukaryotes:
First perform a single model analysis:
iqtree -s microspo.fa -m LG+F+G -nt AUTO -bb 1000
QUESTIONS:
- On which branch do you think to root the tree?
- Where is the position of Microsporidia, the taxon named
Encephalit_10G?- Does this make sense to you?
We will now use the CAT-like protein mixture model called C10 (Le et al., 2008a) to analyze the same data set. Moreover, to speed up the analysis we will use the PMSF approximation (Wang et al., 2018):
iqtree -s microspo.fa -nt AUTO -m LG+C10+F+G -bb 1000 -ft microspo.fa.treefile -pre microspo.C10
Options explained:
-m LG+C10+F+G is to specify C10+F mixture models (with 11 classes).-ft is to specify the guide tree for PMSF approximation. Here we just used the tree constructed from single model as it is a reasonable enough tree. Without -ft, IQ-TREE will use a full mixture model, which may take a lot of time to finish.QUESTIONS:
- Where is the position of
Encephalit_10Gnow?- Does it make sense?
If you still have time, you can also perform tree tests for this protein data set.
See Command Reference for a complete list of all options available in IQ-TREE.
Tutorial for Workshop on Molecular Evolution