Last update: Jun 29, 2024, Contributors: Heiko Schmidt, Jana Trifinopoulos, Minh Bui, Rob Lanfear

Frequently asked questions

For common questions and answers.

How do I get help?

If you have questions please follow the steps below:

1. Continue to read the FAQ below, which may answer your questions already.
2. If not, read the documentation http://www.iqtree.org/doc.
3. If you still could not find the answer, search the IQ-TREE Google group. There is a “Search for topics” box at the top of the Google group web page.
4. Finally, if no answer is found, post a question to the IQ-TREE group. The average response time is one to two working days.

For other feedback and feature requests, please post a topic to the IQ-TREE Google group. We welcome all suggestions to further improve IQ-TREE! For feature request, please also explain why you think such a new feature would be useful or how can it help for your work.

How do I report a bug?

For bug report, please send the following information to the IQ-TREE Google group:

1. A description of the behaviour, which you think might be unexpected or caused by a bug.
2. The first 10 lines and last 10 lines of the .log file.

3. (If possible) the assertion message printed on the screen, which may look like this:

    iqtree: ....cpp:140: ...: Assertion '...' failed.
   

The development team will get back to you and may ask for the full .log file and input data files for debugging purpose, if necessary. In such case please only send your data files directly to the developers for confidential reason! Keep in mind that everyone can see all emails sent to the group!

How do I interpret ultrafast bootstrap (UFBoot) support values?

The ultrafast bootstrap (UFBoot) feature (-bb option) was published in (Minh et al., 2013). One conclusions from the analysis of many gene trees is that UFBoot support values are more unbiased: 95% support correspond roughly to a probability of 95% that a clade is true. So this has a different meaning than the normal (more conservative) bootstrap supports. For UFBoot, you should only start to rely on a branch if its support is >= 95%. Thus, the interpretations are different and you should not compare BS% with UFBoot% directly.

Moreover, it is recommended to also perform the SH-aLRT test (Guindon et al., 2010), e.g., by adding -alrt 1000 into the IQ-TREE command line. Each branch will then be assigned with SH-aLRT and UFBoot supports. One would be more confident if a clade has its SH-aLRT >= 80% and UFboot >= 95%.

NOTE: These recommendations only apply to single gene trees. If you reconstruct a “concatenation” tree from many genes in a phylogenomic analysis, they do not hold anymore. In fact, UFBoot supports and even the more conservative Felsenstein’s bootstrap supports will tend to be 100% and there has been plenty of literature about this issue. You are recommended to compute concordance factors for any phylogenomic analysis.

How does IQ-TREE treat gap/missing/ambiguous characters?

Gaps (-) and missing characters (? or N for DNA alignments) are treated in the same way as unknown characters, which represent no information. The same treatment holds for many other ML software (e.g., RAxML, PhyML). More explicitly, for a site (column) of an alignment containing AC-AG-A (i.e. A for sequence 1, C for sequence 2, - for sequence 3, and so on), the site-likelihood of a tree T is equal to the site-likelihood of the subtree of T restricted to those sequences containing non-gap characters (ACAGA).

Ambiguous characters that represent more than one character are also supported: each represented character will have equal likelihood. For DNA the following ambigous nucleotides are supported according to IUPAC nomenclature:

For protein sequences the following ambiguous amino-acids are supported:

The letters * and ! may found in alignments of protein and/or coding DNA sequences. Stop codon is typically translated to *. Some alignment programs also mark frameshift mutations (cf. Ranwez et al., 2011), that means since frameshift mutations in a codon alignment cause incomplete codons that cannot be unambiguously translated the resulting position in the translated protein sequence and padding positions in the respective codon are marked using !.

Can I mix DNA and protein data in a partitioned analysis?

Yes! You can specify this via a NEXUS partition file. In fact, you can mix any data types supported in IQ-TREE, including also codon, binary and morphological data. To do so, each data type should be stored in a separate alignment file (see also Partitioned analysis with mixed data). As an example, assuming dna.phy is a DNA alignment and and prot.phy is a protein alignment. Then a partition file mixing two types of data can be specified as follows:

#nexus
begin sets;
    charset part1 = dna.phy: 1-100 201-300;
    charset part2 = dna.phy: 101-200;
    charset part3 = prot.phy: 1-150;
    charset part4 = prot.phy: 151-400;
    charpartition mine = HKY:part1, GTR+G:part2, WAG+I+G:part3, LG+G:part4;
end;

NOTE: The site count for each alignment should start from 1, and not continue from the last position of a previous alignment (e.g., see part3 and part4 declared above).

What is the interpretation of branch lengths when mixing codon and DNA data?

When mixing codon and DNA data in a partitioned analysis, the branch lengths are interpreted as the number of nucleotide substitutions per nucleotide site! This is different from having only codon data, where branch lengths are the number of nucleotide substitutions per codon site (thus typically 3 times longer than under DNA models).

Note that if you mix codon, DNA and protein data, the branch lengths are then the number of character substitutions per site, where character is either nucleotide or amino-acid.

What is the purpose of composition test?

At the beginning of each run, IQ-TREE performs a composition chi-square test for every sequence in the alignment. The purpose is to test for homogeneity of character composition (e.g., nucleotide for DNA, amino-acid for protein sequences). A sequence is denoted failed if its character composition significantly deviates from the average composition of the alignment.

More specifically, for each sequence, compute:

chi2 = \sum_{i=1}^k (O_i - E_i)^2 / E_i

where k is the size of the alphabet (e.g. 4 for DNA, 20 for amino acids) and the values 1 to k correspond uniquely to one of the characters. O_i is the character frequency in the sequence tested. E_i is the overall character frequency from the entire alignment.

Whether the character composition deviates significantly from the overall composition is done by testing the chi2 value using the chi2-distribution with k-1 degrees of freedom (df=3 for DNA or df=19 for amino acids). By and large it is a normal Chi^2 test.

This test should be regarded as an explorative tool which might help to nail down problems in a dataset. One would typically not remove failing sequences by default. But if the tree shows unexpected topology the test might point in direction of the origin of the problem.

Furthermore, please keep in mind, this test is performed at the very beginning, where IQ-TREE does not know anything about the models yet. That means:

• If you have partitioned (multi-gene) data, it might be more reasonable to test this separately for each partition in a partition analysis. Here, one might want to be able to decide whether some partitions should better be discarded if it is hard to find a composition representing the sequences in the partition. Or on the other hand if a sequence fails for many partitions and show very unexpected phylogenetic topologies, try without it.
• If you have (phylogenomic) protein data, you can also try several protein mixture models, which account for different amino-acid compositions along the sequences, for example, the C10 to C60 profile mixture models.
• Finally, it is recommended to always check the alignment (something one should always do anyway), especially if they have been collected and produced automatically.

What is the good number of CPU cores to use?

Starting with version 1.5.1, you can use option -nt AUTO to automatically determine the best number of threads for your current data and computer.

If you want to know more details: IQ-TREE can utilize multicore machines to speed up the analysis via -nt option. However, it does not mean that using more cores will always result in less running time: if your alignment is short, using too many cores may even slow down the analysis. This is because IQ-TREE parallelizes the likelihood computation along the alignment. Thus, the parallel efficiency is only increased with longer alignments.

If you want to restrict the number of CPU cores allocated by -nt AUTO use -ntmax to specify the maximal number of CPU cores allowed (DEFAULT: #CPU cores on the current machine).

How do I save time for standard bootstrap?

The standard bootstrap is rather slow and may take weeks/months for large data sets. One way to speed up is to use the multicore version. However, this only works well for long alignments (see What is the good number of CPU cores to use?). Another way is to use many machines or a computing cluster and split the computation among the machines. To illustrate, you want to perform 100 bootstrap replicates and have 5 PCs, each has 4 CPU cores. Then you can:

1. Perform 5 independent bootstrap runs (each with 20 replicates) on the 5 machines with 5 prefix outputs (such that output files are not overwritten). For example:

    # For old IQ-TREE versions <= 1.5.X, change iqtree to iqtree-omp
    iqtree -nt 4 -s input_alignment -bo 20 ... -pre boot1
    iqtree -nt 4 -s input_alignment -bo 20 ... -pre boot2
    iqtree -nt 4 -s input_alignment -bo 20 ... -pre boot3
    iqtree -nt 4 -s input_alignment -bo 20 ... -pre boot4
    iqtree -nt 4 -s input_alignment -bo 20 ... -pre boot5
   

   Note that if you have access to a computing cluster, you may want to submit these jobs onto the cluster queue in parallel and with even more fined grained parallelization (e.g. one replicate per job).

2. Once all 5 runs finished, combine the 5 .boottrees file into one file (e.g. by cat command under Linux):

    cat boot*.boottrees > alltrees
   

3. Construct a consensus tree from the combined bootstrap trees:

    iqtree -con -t alltrees
   

   The consensus tree is then written to .contree file.

4. Estimate branch lengths of the consensus tree using the original alignment:

    iqtree -s input_alignment -te alltrees.contree -pre alltrees.contree
   

5. You can also perform the analysis on the original alignment:

    # For old IQ-TREE versions <= 1.5.X, change iqtree to iqtree-omp
    iqtree -nt 4 -s input_alignment ...
   

   and map the support values onto the obtained ML tree:

    iqtree -sup input_alignment.treefile -t alltrees 
   

   The ML tree with assigned bootstrap supports is written to .suptree file.

Why does IQ-TREE complain about the use of +ASC model?

When using ascertainment bias correction (ASC) model, sometimes you may get an error message:

ERROR: Invaid use of +ASC because of ... invariant sites in the alignment

or when performing model testing:

Skipped since +ASC is not applicable

This is because your alignment contains invariant sites (columns), which violate the mathematical condition of the model. The invariant sites can be:

• Constant sites: containing a single character state over all sequences. For example, all sequences show an A (Adenine) at a particular site in a DNA alignment.
• Partially constant sites: containing a single character, gap or unknown character. For example, at a particular site some sequences show a G (Guanine), some sequences have - (gap) and the other have N.
• Ambiguously constant sites: For example, some sequences show a C (Cytosine), some show a Y (meaning C or T) and some show a - (gap).

All these sites must be removed from the alignment before a +ASC model can be applied.

TIP: Starting with IQ-TREE version 1.5.0, an output alignment file with suffix .varsites is written in such cases, which contain only variable sites from the input alignment. The .varsites alignment can then be used with the +ASC model.

How does IQ-TREE treat identical sequences?

Among a group of identical sequences, IQ-TREE will keep the first two and ignore the rest. If the sequence is the 2nd one, it will be “kept for subsequent analysis”. If it is the 3rd or more, it will be “ignored but added at the end”. The rationale for this is to still be able to calculate the bootstrap support for this group of identical sequences: it is not always 100%. Because by bootstrap resampling, on average only two third of the sites will be present in a bootstrap alignment (due to sampling with replacement), and suddenly another sequence not in this group may actually become identical to this group of sequences. In that case, the bootstrap value will be < 100%.

Therefore, the .uniqueseq.phy printed by IQ-TREE may still contain the identical sequences, but no more than two of each identical group.

What are the differences between alignment columns/sites and patterns?

Columns are the columns/sites in the alignment and the number of columns is the length of the alignment. In the alignment there might be the same columns. Different columns are called patterns. While (parsimony) informative sites are patterns that have at least two different characters (nucleotides or amino acids) and each character should occur in at least two species. Essentially, informative sites have information for the grouping of species. These patterns are mainly important in the context of parsimony, where no evolutionary model is used. In maximum likelihood inference all patterns containing different characters are important for the estimation of tree topology and branch lengths, while constant/invariant sites (containing only the same character: only A’s or only G’s etc) are important for the correct estimation of the branch lengths. Therefore, should not be excluded from the alignment.

Example:

            123456789
species_1   AACGTACGT
species_2   AACGATCGT
species_3   AACCGTCCT
species_4   AACCTACCT

• sites/columns 1 and 2 are identical and contain only A’s - invariant site pattern, uninformative
• sites/columns 3 and 7 are identical and contain only C’s - invariant site pattern, uninformative
• sites/columns 4 and 8 are identical and contain 2 G’c and 2 C’s - informative site pattern
• sites/columns 5, 6 and 9 occur only once, each site is a different pattern. 5th is uninformative, 6th is informative, 9th is invariant and uninformative

Summing up, the alignment has 9 columns,

6 patterns

A C G T A T
A C G A T T
A C C G T T
A C C T A T

(2 informative)

G A 
G T 
C T 
C A

Can I use IQ-TREE to concatenate alignments?

Yes! If you put all of your alignments you want to concatenate into a single folder, you can do this:

iqtree2 -p FOLDER_NAME --out-aln OUTFILE_NAME

This will produce:

• OUTFILE_NAME: the concatenated alignment (default is phylip format)
• OUTFILE_NAME.nex: the partition file in nexus format
• OUTFILE_NAME.partitions: the partition file in RAxML format

Optionally, you can add --out-format FASTA|NEXUS option to specify concatenated alignment format, e.g.

iqtree2 -p FOLDER_NAME --out-aln OUTFILE_NAME --out-format NEXUS

would output the alignment in nexus format.