Phylogenetic inference with q2-phylogeny ¶

Warning

This site has been replaced by the new QIIME 2 “amplicon distribution” documentation, as of the 2025.4 release of QIIME 2. You can still access the content from the “old docs” here for the QIIME 2 2024.10 and earlier releases, but we recommend that you transition to the new documentation at https://amplicon-docs.qiime2.org. Content on this site is no longer updated and may be out of date.

Are you looking for:

the QIIME 2 homepage? That’s https://qiime2.org.
learning resources for microbiome marker gene (i.e., amplicon) analysis? See the QIIME 2 amplicon distribution documentation.
learning resources for microbiome metagenome analysis? See the MOSHPIT documentation.
installation instructions, plugins, books, videos, workshops, or resources? See the QIIME 2 Library.
general help? See the QIIME 2 Forum.

Old content beyond this point… 👴👵

Phylogenetic inference with q2-phylogeny

Phylogenetic inference with q2-phylogeny

Note

This tutorial assumes, you’ve read through the QIIME 2 Overview documentation and have at least worked through some of the other Tutorials.

Inferring phylogenies ¶

Several downstream diversity metrics, available within QIIME 2, require that a phylogenetic tree be constructed using the Operational Taxonomic Units (OTUs) or Amplicon Sequence Variants (ASVs) being investigated.

But how do we proceed to construct a phylogeny from our sequence data?

Well, there are two phylogeny-based approaches we can use. Deciding upon which to use is largely dependent on your study questions:

1. A reference-based fragment insertion approach. Which, is likely the ideal choice. Especially, if your reference phylogeny (and associated representative sequences) encompass neighboring relatives of which your sequences can be reliably inserted. Any sequences that do not match well enough to the reference are not inserted. For example, this approach may not work well if your data contain sequences that are not well represented within your reference phylogeny (e.g. missing clades, etc.). For more information, check out these great fragment insertion examples.

2. A de novo approach. Marker genes that can be globally aligned across divergent taxa, are usually amenable to sequence alignment and phylogenetic investigation through this approach. Be mindful of the length of your sequences when constructing a de novo phylogeny, short reads many not have enough phylogenetic information to capture a meaningful phylogeny. This community tutorial will focus on the de novo approaches.

Here, you will learn how to make use of de novo phylogenetic approaches to:

generate a sequence alignment within QIIME 2
mask the alignment if needed
construct a phylogenetic tree
root the phylogenetic tree

If you would like to substitute any of the steps outlined here by making use of tools external to QIIME 2, please see the import, export, and filtering documentation where appropriate.

Sequence Alignment ¶

Prior to constructing a phylogeny we must generate a multiple sequence alignment (MSA). When constructing a MSA we are making a statement about the putative homology of the aligned residues (columns of the MSA) by virtue of their sequence similarity.

The number of algorithms to construct a MSA are legion. We will make use of MAFFT (Multiple Alignment using Fast Fourier Transform)) via the q2-alignment plugin. For more information checkout the MAFFT paper.

Let’s start by creating a directory to work in:

mkdir qiime2-phylogeny-tutorial
cd qiime2-phylogeny-tutorial

Next, download the data:

Please select a download option that is most appropriate for your environment:

Download URL: https://data.qiime2.org/2024.10/tutorials/phylogeny/rep-seqs.qza

Save as: rep-seqs.qza

            wget \
  -O "rep-seqs.qza" \
  "https://data.qiime2.org/2024.10/tutorials/phylogeny/rep-seqs.qza"
          

            curl -sL \
  "https://data.qiime2.org/2024.10/tutorials/phylogeny/rep-seqs.qza" > \
  "rep-seqs.qza"
          

Run MAFFT

qiime alignment mafft \
  --i-sequences rep-seqs.qza \
  --o-alignment aligned-rep-seqs.qza

Output artifacts:

rep-seqs.qza: view | download
aligned-rep-seqs.qza: view | download

Reducing alignment ambiguity: masking and reference alignments ¶

Why mask an alignment?

Masking helps to eliminate alignment columns that are phylogenetically uninformative or misleading before phylogenetic analysis. Much of the time alignment errors can introduce noise and confound phylogenetic inference. It is common practice to mask (remove) these ambiguously aligned regions prior to performing phylogenetic inference. In particular, David Lane’s (1991) chapter 16S/23S rRNA sequencing proposed masking SSU data prior to phylogenetic analysis. However, knowing how to deal with ambiguously aligned regions and when to apply masks largely depends on the marker genes being analyzed and the question being asked of the data.

Note

Keep in mind that this is still an active area of discussion, as highlighted by the following non-exhaustive list of articles: Wu et al. 2012, Ashkenazy et al. 2018, Schloss 2010, Tan et al. 2015, Rajan 2015.

How to mask alignment.

For our purposes, we’ll assume that we have ambiguously aligned columns in the MAFFT alignment we produced above. The default settings for the --p-min-conservation of the alignment mask approximates the Lane mask filtering of QIIME 1. Keep an eye out for updates to the alignment plugin.

qiime alignment mask \
  --i-alignment aligned-rep-seqs.qza \
  --o-masked-alignment masked-aligned-rep-seqs.qza

Output artifacts:

masked-aligned-rep-seqs.qza: view | download

Reference based alignments

There are several tools that attempt to reduce the amount of ambiguously aligned regions by using curated reference alignments. Traditional, de novo alignment methods mututally align a set of unaligned sequences to create a multiple sequence alignment (MSA) from scratch. Re-running these methods with additional sequences will create MSAs with varying numbers of columns and assignments of bases to each column. These alignments is therefore incompatible with one another and may not be joined through concatenation.

Reference based alignments, on the other hand, are meant to add sequences to an existing alignment. Alignments computed using reference based alignment tools always have widths identical to the reference alignment and maintain the meaning of each column. Therefore, these alignments may be concatenated.

QIIME 2 currently does not wrap any methods for reference-based alignments, but alignments created using these methods can be imported into QIIME 2 as FeatureData[AlignedSequence] artifacts, provided that the alignments are standard FASTA formats. Some examples of tools for reference-based alignment include PyNAST (using NAST), Infernal, and SINA. SILVA Reference alignments are particularly powerful for rRNA gene sequence data, as knowledge of secondary structure is incorporated into the curation process, thus increasing alignment quality.

Note

Alignments constructed using reference based alignment approaches can be masked too, just like the above MAFFT example. Also, the reference alignment approach we are discussing here is distinct from the reference phylogeny approach (i.e. q2-fragment-insertion) we mentioned earlier. That is, we are not inserting our data into an existing tree, but simply trying to create a more robust alignment for making a better de novo phylogeny.

Construct a phylogeny ¶

As with MSA algorithms, phylogenetic inference tools are also legion. Fortunately, there are many great resources to learn about phylogentics. Below are just a few introductory resources to get you started:

There are several methods / pipelines available through the q2-phylogeny plugin of :qiime2:. These are based on the following tools:

Methods ¶

fasttree ¶

FastTree is able to construct phylogenies from large sequence alignments quite rapidly. It does this by using the using a CAT-like rate category approximation, which is also available through RAxML (discussed below). Check out the FastTree online manual for more information.

qiime phylogeny fasttree \
  --i-alignment masked-aligned-rep-seqs.qza \
  --o-tree fasttree-tree.qza

Output artifacts:

fasttree-tree.qza: view | download

Tip

For an easy and direct way to view your tree.qza files, upload them to iTOL. Here, you can interactively view and manipulate your phylogeny. Even better, while viewing the tree topology in “Normal mode”, you can drag and drop your associated alignment.qza (the one you used to build the phylogeny) or a relevent taxonomy.qza file onto the iTOL tree visualization. This will allow you to directly view the sequence alignment or taxonomy alongside the phylogeny. 🕶️

raxml ¶

Like fasttree, raxml will perform a single phylogentic inference and return a tree. Note, the default model for raxml is --p-substitution-model GTRGAMMA. If you’d like to construct a tree using the CAT model like fasttree, simply replace GTRGAMMA with GTRCAT as shown below:

qiime phylogeny raxml \
  --i-alignment masked-aligned-rep-seqs.qza \
  --p-substitution-model GTRCAT \
  --o-tree raxml-cat-tree.qza \
  --verbose

stdout:

Warning, you specified a working directory via "-w"
Keep in mind that RAxML only accepts absolute path names, not relative ones!

RAxML can't, parse the alignment file as phylip file 
it will now try to parse it as FASTA file

Using BFGS method to optimize GTR rate parameters, to disable this specify "--no-bfgs" 

This is RAxML version 8.2.12 released by Alexandros Stamatakis on May 2018.

With greatly appreciated code contributions by:
Andre Aberer      (HITS)
Simon Berger      (HITS)
Alexey Kozlov     (HITS)
Kassian Kobert    (HITS)
David Dao         (KIT and HITS)
Sarah Lutteropp   (KIT and HITS)
Nick Pattengale   (Sandia)
Wayne Pfeiffer    (SDSC)
Akifumi S. Tanabe (NRIFS)
Charlie Taylor    (UF)

Alignment has 157 distinct alignment patterns

Proportion of gaps and completely undetermined characters in this alignment: 39.77%

RAxML rapid hill-climbing mode

Using 1 distinct models/data partitions with joint branch length optimization

Executing 1 inferences on the original alignment using 1 distinct randomized MP trees

All free model parameters will be estimated by RAxML
ML estimate of 25 per site rate categories

Likelihood of final tree will be evaluated and optimized under GAMMA

GAMMA Model parameters will be estimated up to an accuracy of 0.1000000000 Log Likelihood units

Partition: 0
Alignment Patterns: 157
Name: No Name Provided
DataType: DNA
Substitution Matrix: GTR

RAxML was called as follows:

raxmlHPC -m GTRCAT -p 2467 -N 1 -s /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/qiime2/jgc/data/d34420d9-336e-4d9e-aee8-99e64b3315f0/data/aligned-dna-sequences.fasta -w /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpki5avn1s -n q2 

Partition: 0 with name: No Name Provided
Base frequencies: 0.243 0.182 0.319 0.256 

Inference[0]: Time 0.319252 CAT-based likelihood -1243.089529, best rearrangement setting 5

Conducting final model optimizations on all 1 trees under GAMMA-based models ....

Inference[0] final GAMMA-based Likelihood: -1387.920037 tree written to file /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpki5avn1s/RAxML_result.q2

Starting final GAMMA-based thorough Optimization on tree 0 likelihood -1387.920037 .... 

Final GAMMA-based Score of best tree -1387.235674

Program execution info written to /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpki5avn1s/RAxML_info.q2
Best-scoring ML tree written to: /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpki5avn1s/RAxML_bestTree.q2

Overall execution time: 0.638849 secs or 0.000177 hours or 0.000007 days

Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command: raxmlHPC -m GTRCAT -p 2467 -N 1 -s /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/qiime2/jgc/data/d34420d9-336e-4d9e-aee8-99e64b3315f0/data/aligned-dna-sequences.fasta -w /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpki5avn1s -n q2

Saved Phylogeny[Unrooted] to: raxml-cat-tree.qza

Output artifacts:

raxml-cat-tree.qza: view | download

Perform multiple searches using raxml ¶

If you’d like to perform a more thorough search of “tree space” you can instruct raxml to perform multiple independent searches on the full alignment by using --p-n-searches 5. Once these 5 independent searches are completed, only the single best scoring tree will be returned. Note, we are not bootstrapping here, we’ll do that in a later example. Let’s set --p-substitution-model GTRCAT. Finally, let’s also manually set a seed via --p-seed. By setting our seed, we allow other users the ability to reproduce our phylogeny. That is, anyone using the same sequence alignment and substitution model, will generate the same tree as long as they set the same seed value. Although, --p-seed is not a required argument, it is generally a good idea to set this value.

qiime phylogeny raxml \
  --i-alignment masked-aligned-rep-seqs.qza \
  --p-substitution-model GTRCAT \
  --p-seed 1723 \
  --p-n-searches 5 \
  --o-tree raxml-cat-searches-tree.qza \
  --verbose

stdout:

Warning, you specified a working directory via "-w"
Keep in mind that RAxML only accepts absolute path names, not relative ones!

RAxML can't, parse the alignment file as phylip file 
it will now try to parse it as FASTA file



Using BFGS method to optimize GTR rate parameters, to disable this specify "--no-bfgs" 



This is RAxML version 8.2.12 released by Alexandros Stamatakis on May 2018.

With greatly appreciated code contributions by:
Andre Aberer      (HITS)
Simon Berger      (HITS)
Alexey Kozlov     (HITS)
Kassian Kobert    (HITS)
David Dao         (KIT and HITS)
Sarah Lutteropp   (KIT and HITS)
Nick Pattengale   (Sandia)
Wayne Pfeiffer    (SDSC)
Akifumi S. Tanabe (NRIFS)
Charlie Taylor    (UF)


Alignment has 157 distinct alignment patterns

Proportion of gaps and completely undetermined characters in this alignment: 39.77%

RAxML rapid hill-climbing mode

Using 1 distinct models/data partitions with joint branch length optimization


Executing 5 inferences on the original alignment using 5 distinct randomized MP trees

All free model parameters will be estimated by RAxML
ML estimate of 25 per site rate categories

Likelihood of final tree will be evaluated and optimized under GAMMA

GAMMA Model parameters will be estimated up to an accuracy of 0.1000000000 Log Likelihood units

Partition: 0
Alignment Patterns: 157
Name: No Name Provided
DataType: DNA
Substitution Matrix: GTR




RAxML was called as follows:

raxmlHPC -m GTRCAT -p 1723 -N 5 -s /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/qiime2/jgc/data/d34420d9-336e-4d9e-aee8-99e64b3315f0/data/aligned-dna-sequences.fasta -w /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpsanvl1cp -n q2 


Partition: 0 with name: No Name Provided
Base frequencies: 0.243 0.182 0.319 0.256 

Inference[0]: Time 0.251948 CAT-based likelihood -1238.242991, best rearrangement setting 5
Inference[1]: Time 0.213775 CAT-based likelihood -1249.502284, best rearrangement setting 5
Inference[2]: Time 0.218222 CAT-based likelihood -1242.978035, best rearrangement setting 5
Inference[3]: Time 0.282072 CAT-based likelihood -1243.159855, best rearrangement setting 5
Inference[4]: Time 0.208645 CAT-based likelihood -1261.321621, best rearrangement setting 5


Conducting final model optimizations on all 5 trees under GAMMA-based models ....

Inference[0] final GAMMA-based Likelihood: -1388.324037 tree written to file /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpsanvl1cp/RAxML_result.q2.RUN.0
Inference[1] final GAMMA-based Likelihood: -1392.813982 tree written to file /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpsanvl1cp/RAxML_result.q2.RUN.1
Inference[2] final GAMMA-based Likelihood: -1388.073642 tree written to file /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpsanvl1cp/RAxML_result.q2.RUN.2
Inference[3] final GAMMA-based Likelihood: -1387.945266 tree written to file /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpsanvl1cp/RAxML_result.q2.RUN.3
Inference[4] final GAMMA-based Likelihood: -1387.557031 tree written to file /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpsanvl1cp/RAxML_result.q2.RUN.4


Starting final GAMMA-based thorough Optimization on tree 4 likelihood -1387.557031 .... 

Final GAMMA-based Score of best tree -1387.385075

Program execution info written to /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpsanvl1cp/RAxML_info.q2
Best-scoring ML tree written to: /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpsanvl1cp/RAxML_bestTree.q2

Overall execution time: 1.474086 secs or 0.000409 hours or 0.000017 days

Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command: raxmlHPC -m GTRCAT -p 1723 -N 5 -s /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/qiime2/jgc/data/d34420d9-336e-4d9e-aee8-99e64b3315f0/data/aligned-dna-sequences.fasta -w /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpsanvl1cp -n q2

Saved Phylogeny[Unrooted] to: raxml-cat-searches-tree.qza

Output artifacts:

raxml-cat-searches-tree.qza: view | download

raxml-rapid-bootstrap ¶

In phylogenetics, it is good practice to check how well the splits / bipartitions in your phylogeny are supported. Often one is interested in which clades are robustly separated from other clades in the phylogeny. One way, of doing this is via bootstrapping (See the Bootstrapping section of the first introductory link above). In QIIME 2, we’ve provided access to the RAxML rapid bootstrap feature. The only difference between this command and the previous are the additional flags --p-bootstrap-replicates and --p-rapid-bootstrap-seed. It is quite common to perform anywhere from 100 - 1000 bootstrap replicates. The --p-rapid-bootstrap-seed works very much like the --p-seed argument from above except that it allows anyone to reproduce the bootstrapping process and the associated supports for your splits.

As per the RAxML online documentation and the RAxML manual, the rapid bootstrapping command that we will execute below will do the following:

Bootstrap the input alignment 100 times and perform a Maximum Likelihood (ML) search on each.
Find best scoring ML tree through multiple independent searches using the original input alignment. The number of independent searches is determined by the number of bootstrap replicates set in the 1st step. That is, your search becomes more thorough with increasing bootstrap replicates. The ML optimization of RAxML uses every 5th bootstrap tree as the starting tree for an ML search on the original alignment.
Map the bipartitions (bootstrap supports, 1st step) onto the best scoring ML tree (2nd step).

qiime phylogeny raxml-rapid-bootstrap \
  --i-alignment masked-aligned-rep-seqs.qza \
  --p-seed 1723 \
  --p-rapid-bootstrap-seed 9384 \
  --p-bootstrap-replicates 100 \
  --p-substitution-model GTRCAT \
  --o-tree raxml-cat-bootstrap-tree.qza \
  --verbose

stdout:

Warning, you specified a working directory via "-w"
Keep in mind that RAxML only accepts absolute path names, not relative ones!

RAxML can't, parse the alignment file as phylip file 
it will now try to parse it as FASTA file



Using BFGS method to optimize GTR rate parameters, to disable this specify "--no-bfgs" 



This is RAxML version 8.2.12 released by Alexandros Stamatakis on May 2018.

With greatly appreciated code contributions by:
Andre Aberer      (HITS)
Simon Berger      (HITS)
Alexey Kozlov     (HITS)
Kassian Kobert    (HITS)
David Dao         (KIT and HITS)
Sarah Lutteropp   (KIT and HITS)
Nick Pattengale   (Sandia)
Wayne Pfeiffer    (SDSC)
Akifumi S. Tanabe (NRIFS)
Charlie Taylor    (UF)


Alignment has 157 distinct alignment patterns

Proportion of gaps and completely undetermined characters in this alignment: 39.77%

RAxML rapid bootstrapping and subsequent ML search

Using 1 distinct models/data partitions with joint branch length optimization



Executing 100 rapid bootstrap inferences and thereafter a thorough ML search 

All free model parameters will be estimated by RAxML
ML estimate of 25 per site rate categories

Likelihood of final tree will be evaluated and optimized under GAMMA

GAMMA Model parameters will be estimated up to an accuracy of 0.1000000000 Log Likelihood units

Partition: 0
Alignment Patterns: 157
Name: No Name Provided
DataType: DNA
Substitution Matrix: GTR




RAxML was called as follows:

raxmlHPC -f a -m GTRCAT -p 1723 -x 9384 -N 100 -s /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/qiime2/jgc/data/d34420d9-336e-4d9e-aee8-99e64b3315f0/data/aligned-dna-sequences.fasta -w /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpt__b6x2l -n q2bootstrap 



Time for BS model parameter optimization 0.021413
Bootstrap[0]: Time 0.071715 seconds, bootstrap likelihood -1199.758796, best rearrangement setting 12
Bootstrap[1]: Time 0.048405 seconds, bootstrap likelihood -1344.229251, best rearrangement setting 6
Bootstrap[2]: Time 0.044703 seconds, bootstrap likelihood -1295.343000, best rearrangement setting 8
Bootstrap[3]: Time 0.039735 seconds, bootstrap likelihood -1273.768320, best rearrangement setting 8
Bootstrap[4]: Time 0.046322 seconds, bootstrap likelihood -1253.402952, best rearrangement setting 6
Bootstrap[5]: Time 0.048485 seconds, bootstrap likelihood -1260.866113, best rearrangement setting 10
Bootstrap[6]: Time 0.047969 seconds, bootstrap likelihood -1293.636299, best rearrangement setting 14
Bootstrap[7]: Time 0.043383 seconds, bootstrap likelihood -1227.178693, best rearrangement setting 6
Bootstrap[8]: Time 0.047335 seconds, bootstrap likelihood -1321.820787, best rearrangement setting 13
Bootstrap[9]: Time 0.050986 seconds, bootstrap likelihood -1147.233446, best rearrangement setting 6
Bootstrap[10]: Time 0.036663 seconds, bootstrap likelihood -1220.766493, best rearrangement setting 13
Bootstrap[11]: Time 0.051767 seconds, bootstrap likelihood -1200.006355, best rearrangement setting 8
Bootstrap[12]: Time 0.055301 seconds, bootstrap likelihood -1346.392834, best rearrangement setting 14
Bootstrap[13]: Time 0.045147 seconds, bootstrap likelihood -1301.111096, best rearrangement setting 14
Bootstrap[14]: Time 0.047859 seconds, bootstrap likelihood -1262.253559, best rearrangement setting 11
Bootstrap[15]: Time 0.047531 seconds, bootstrap likelihood -1215.017551, best rearrangement setting 14
Bootstrap[16]: Time 0.044559 seconds, bootstrap likelihood -1238.832009, best rearrangement setting 7
Bootstrap[17]: Time 0.041015 seconds, bootstrap likelihood -1393.989732, best rearrangement setting 12
Bootstrap[18]: Time 0.043311 seconds, bootstrap likelihood -1173.921002, best rearrangement setting 15
Bootstrap[19]: Time 0.044978 seconds, bootstrap likelihood -1185.726976, best rearrangement setting 11
Bootstrap[20]: Time 0.041261 seconds, bootstrap likelihood -1158.491940, best rearrangement setting 6
Bootstrap[21]: Time 0.040119 seconds, bootstrap likelihood -1154.664272, best rearrangement setting 11
Bootstrap[22]: Time 0.044658 seconds, bootstrap likelihood -1244.159837, best rearrangement setting 10
Bootstrap[23]: Time 0.052462 seconds, bootstrap likelihood -1211.171036, best rearrangement setting 15
Bootstrap[24]: Time 0.044355 seconds, bootstrap likelihood -1261.440677, best rearrangement setting 12
Bootstrap[25]: Time 0.045268 seconds, bootstrap likelihood -1331.836715, best rearrangement setting 15
Bootstrap[26]: Time 0.046062 seconds, bootstrap likelihood -1129.144509, best rearrangement setting 5
Bootstrap[27]: Time 0.056580 seconds, bootstrap likelihood -1226.624056, best rearrangement setting 7
Bootstrap[28]: Time 0.056509 seconds, bootstrap likelihood -1221.046176, best rearrangement setting 12
Bootstrap[29]: Time 0.037257 seconds, bootstrap likelihood -1211.791204, best rearrangement setting 14
Bootstrap[30]: Time 0.047623 seconds, bootstrap likelihood -1389.442380, best rearrangement setting 5
Bootstrap[31]: Time 0.048621 seconds, bootstrap likelihood -1303.638592, best rearrangement setting 12
Bootstrap[32]: Time 0.052421 seconds, bootstrap likelihood -1172.859456, best rearrangement setting 12
Bootstrap[33]: Time 0.045644 seconds, bootstrap likelihood -1244.617135, best rearrangement setting 9
Bootstrap[34]: Time 0.043302 seconds, bootstrap likelihood -1211.871717, best rearrangement setting 15
Bootstrap[35]: Time 0.051914 seconds, bootstrap likelihood -1299.862912, best rearrangement setting 5
Bootstrap[36]: Time 0.040983 seconds, bootstrap likelihood -1141.967505, best rearrangement setting 5
Bootstrap[37]: Time 0.050897 seconds, bootstrap likelihood -1283.923198, best rearrangement setting 12
Bootstrap[38]: Time 0.040831 seconds, bootstrap likelihood -1304.250946, best rearrangement setting 5
Bootstrap[39]: Time 0.038789 seconds, bootstrap likelihood -1407.084376, best rearrangement setting 15
Bootstrap[40]: Time 0.046169 seconds, bootstrap likelihood -1277.946299, best rearrangement setting 13
Bootstrap[41]: Time 0.045286 seconds, bootstrap likelihood -1279.006200, best rearrangement setting 7
Bootstrap[42]: Time 0.044050 seconds, bootstrap likelihood -1160.274606, best rearrangement setting 6
Bootstrap[43]: Time 0.054604 seconds, bootstrap likelihood -1216.079259, best rearrangement setting 14
Bootstrap[44]: Time 0.041630 seconds, bootstrap likelihood -1382.278311, best rearrangement setting 8
Bootstrap[45]: Time 0.047468 seconds, bootstrap likelihood -1099.004439, best rearrangement setting 11
Bootstrap[46]: Time 0.038304 seconds, bootstrap likelihood -1296.527478, best rearrangement setting 8
Bootstrap[47]: Time 0.055747 seconds, bootstrap likelihood -1291.322658, best rearrangement setting 9
Bootstrap[48]: Time 0.036485 seconds, bootstrap likelihood -1161.908080, best rearrangement setting 6
Bootstrap[49]: Time 0.050040 seconds, bootstrap likelihood -1257.348428, best rearrangement setting 13
Bootstrap[50]: Time 0.057814 seconds, bootstrap likelihood -1309.422533, best rearrangement setting 13
Bootstrap[51]: Time 0.041945 seconds, bootstrap likelihood -1197.633097, best rearrangement setting 11
Bootstrap[52]: Time 0.047570 seconds, bootstrap likelihood -1347.123005, best rearrangement setting 8
Bootstrap[53]: Time 0.042366 seconds, bootstrap likelihood -1234.934890, best rearrangement setting 14
Bootstrap[54]: Time 0.048535 seconds, bootstrap likelihood -1227.092434, best rearrangement setting 6
Bootstrap[55]: Time 0.050268 seconds, bootstrap likelihood -1280.635747, best rearrangement setting 7
Bootstrap[56]: Time 0.041855 seconds, bootstrap likelihood -1225.911449, best rearrangement setting 6
Bootstrap[57]: Time 0.039120 seconds, bootstrap likelihood -1236.213347, best rearrangement setting 11
Bootstrap[58]: Time 0.056092 seconds, bootstrap likelihood -1393.245723, best rearrangement setting 14
Bootstrap[59]: Time 0.043312 seconds, bootstrap likelihood -1212.039371, best rearrangement setting 6
Bootstrap[60]: Time 0.039390 seconds, bootstrap likelihood -1248.692011, best rearrangement setting 10
Bootstrap[61]: Time 0.046376 seconds, bootstrap likelihood -1172.820979, best rearrangement setting 13
Bootstrap[62]: Time 0.053783 seconds, bootstrap likelihood -1126.745788, best rearrangement setting 14
Bootstrap[63]: Time 0.042842 seconds, bootstrap likelihood -1267.434444, best rearrangement setting 12
Bootstrap[64]: Time 0.041208 seconds, bootstrap likelihood -1340.680748, best rearrangement setting 5
Bootstrap[65]: Time 0.040630 seconds, bootstrap likelihood -1072.671059, best rearrangement setting 5
Bootstrap[66]: Time 0.049943 seconds, bootstrap likelihood -1234.294838, best rearrangement setting 8
Bootstrap[67]: Time 0.049007 seconds, bootstrap likelihood -1109.249439, best rearrangement setting 15
Bootstrap[68]: Time 0.038192 seconds, bootstrap likelihood -1314.493588, best rearrangement setting 8
Bootstrap[69]: Time 0.039451 seconds, bootstrap likelihood -1173.850035, best rearrangement setting 13
Bootstrap[70]: Time 0.042647 seconds, bootstrap likelihood -1231.066465, best rearrangement setting 10
Bootstrap[71]: Time 0.041697 seconds, bootstrap likelihood -1146.861379, best rearrangement setting 9
Bootstrap[72]: Time 0.035867 seconds, bootstrap likelihood -1148.753369, best rearrangement setting 8
Bootstrap[73]: Time 0.041821 seconds, bootstrap likelihood -1333.374056, best rearrangement setting 9
Bootstrap[74]: Time 0.037090 seconds, bootstrap likelihood -1259.382378, best rearrangement setting 5
Bootstrap[75]: Time 0.040414 seconds, bootstrap likelihood -1319.944496, best rearrangement setting 6
Bootstrap[76]: Time 0.046140 seconds, bootstrap likelihood -1309.042165, best rearrangement setting 14
Bootstrap[77]: Time 0.055568 seconds, bootstrap likelihood -1232.061289, best rearrangement setting 8
Bootstrap[78]: Time 0.045582 seconds, bootstrap likelihood -1261.333984, best rearrangement setting 9
Bootstrap[79]: Time 0.047099 seconds, bootstrap likelihood -1194.644341, best rearrangement setting 13
Bootstrap[80]: Time 0.041761 seconds, bootstrap likelihood -1214.037389, best rearrangement setting 9
Bootstrap[81]: Time 0.045652 seconds, bootstrap likelihood -1224.527657, best rearrangement setting 8
Bootstrap[82]: Time 0.052080 seconds, bootstrap likelihood -1241.464826, best rearrangement setting 11
Bootstrap[83]: Time 0.039323 seconds, bootstrap likelihood -1230.730558, best rearrangement setting 6
Bootstrap[84]: Time 0.042321 seconds, bootstrap likelihood -1219.034592, best rearrangement setting 10
Bootstrap[85]: Time 0.044859 seconds, bootstrap likelihood -1280.071994, best rearrangement setting 8
Bootstrap[86]: Time 0.038904 seconds, bootstrap likelihood -1444.747777, best rearrangement setting 9
Bootstrap[87]: Time 0.038686 seconds, bootstrap likelihood -1245.890035, best rearrangement setting 14
Bootstrap[88]: Time 0.044546 seconds, bootstrap likelihood -1287.832766, best rearrangement setting 7
Bootstrap[89]: Time 0.041072 seconds, bootstrap likelihood -1325.245976, best rearrangement setting 5
Bootstrap[90]: Time 0.047555 seconds, bootstrap likelihood -1227.883697, best rearrangement setting 5
Bootstrap[91]: Time 0.045679 seconds, bootstrap likelihood -1273.489392, best rearrangement setting 8
Bootstrap[92]: Time 0.018010 seconds, bootstrap likelihood -1234.725870, best rearrangement setting 7
Bootstrap[93]: Time 0.049324 seconds, bootstrap likelihood -1235.733064, best rearrangement setting 11
Bootstrap[94]: Time 0.040422 seconds, bootstrap likelihood -1204.319488, best rearrangement setting 15
Bootstrap[95]: Time 0.039192 seconds, bootstrap likelihood -1183.328582, best rearrangement setting 11
Bootstrap[96]: Time 0.045023 seconds, bootstrap likelihood -1196.298898, best rearrangement setting 13
Bootstrap[97]: Time 0.048966 seconds, bootstrap likelihood -1339.251746, best rearrangement setting 12
Bootstrap[98]: Time 0.017703 seconds, bootstrap likelihood -1404.363552, best rearrangement setting 7
Bootstrap[99]: Time 0.023584 seconds, bootstrap likelihood -1270.157811, best rearrangement setting 7


Overall Time for 100 Rapid Bootstraps 4.484976 seconds
Average Time per Rapid Bootstrap 0.044850 seconds

Starting ML Search ...

Fast ML optimization finished

Fast ML search Time: 1.812424 seconds

Slow ML Search 0 Likelihood: -1387.994678
Slow ML Search 1 Likelihood: -1387.994678
Slow ML Search 2 Likelihood: -1387.994676
Slow ML Search 3 Likelihood: -1387.994650
Slow ML Search 4 Likelihood: -1387.994685
Slow ML Search 5 Likelihood: -1388.092954
Slow ML Search 6 Likelihood: -1388.182551
Slow ML Search 7 Likelihood: -1388.182563
Slow ML Search 8 Likelihood: -1388.182547
Slow ML Search 9 Likelihood: -1387.994723
Slow ML optimization finished

Slow ML search Time: 0.892149 seconds
Thorough ML search Time: 0.231714 seconds

Final ML Optimization Likelihood: -1387.204993

Model Information:

Model Parameters of Partition 0, Name: No Name Provided, Type of Data: DNA
alpha: 1.227800
Tree-Length: 7.823400
rate A <-> C: 0.332564
rate A <-> G: 2.312784
rate A <-> T: 2.215466
rate C <-> G: 1.243321
rate C <-> T: 3.278770
rate G <-> T: 1.000000

freq pi(A): 0.243216
freq pi(C): 0.181967
freq pi(G): 0.319196
freq pi(T): 0.255621


ML search took 2.938256 secs or 0.000816 hours

Combined Bootstrap and ML search took 7.423289 secs or 0.002062 hours

Drawing Bootstrap Support Values on best-scoring ML tree ...



Found 1 tree in File /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpt__b6x2l/RAxML_bestTree.q2bootstrap



Found 1 tree in File /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpt__b6x2l/RAxML_bestTree.q2bootstrap

Program execution info written to /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpt__b6x2l/RAxML_info.q2bootstrap
All 100 bootstrapped trees written to: /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpt__b6x2l/RAxML_bootstrap.q2bootstrap

Best-scoring ML tree written to: /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpt__b6x2l/RAxML_bestTree.q2bootstrap

Best-scoring ML tree with support values written to: /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpt__b6x2l/RAxML_bipartitions.q2bootstrap

Best-scoring ML tree with support values as branch labels written to: /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpt__b6x2l/RAxML_bipartitionsBranchLabels.q2bootstrap

Overall execution time for full ML analysis: 7.427777 secs or 0.002063 hours or 0.000086 days

Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command: raxmlHPC -f a -m GTRCAT -p 1723 -x 9384 -N 100 -s /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/qiime2/jgc/data/d34420d9-336e-4d9e-aee8-99e64b3315f0/data/aligned-dna-sequences.fasta -w /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpt__b6x2l -n q2bootstrap

Saved Phylogeny[Unrooted] to: raxml-cat-bootstrap-tree.qza

Output artifacts:

raxml-cat-bootstrap-tree.qza: view | download

Tip

Optimizing RAxML Run Time. You may gave noticed that we haven’t added the flag --p-raxml-version to the RAxML methods. This parameter provides a means to access versions of RAxML that have optimized vector instructions for various modern x86 processor architectures. Paraphrased from the RAxML manual and help documentation: Firstly, most recent processors will support SSE3 vector instructions (i.e. will likely support the faster AVX2 vector instructions). Secondly, these instructions will substantially accelerate the likelihood and parsimony computations. In general, SSE3 versions will run approximately 40% faster than the standard version. The AVX2 version will run 10-30% faster than the SSE3 version. Additionally, keep in mind that using more cores / threads will not necessarily decrease run time. The RAxML manual suggests using 1 core per ~500 DNA alignment patterns. Alignment pattern information is usually visible on screen, when the --verbose option is used. Additionally, try using a rate category (CAT model; via --p-substitution-model), which results in equally good trees as the GAMMA models and is approximately 4 times faster. See the CAT paper. The CAT approximation is also Ideal for alignments containing 10,000 or more taxa, and is very much similar the CAT-like model of FastTree2.

iqtree ¶

Similar to the raxml and raxml-rapid-bootstrap methods above, we provide similar functionality for IQ-TREE: iqtree and iqtree-ultrafast-bootstrap. IQ-TREE is unique compared to the fastree and raxml options, in that it provides access to 286 models of nucleotide substitution! IQ-TREE can also determine which of these models best fits your dataset prior to constructing your tree via its built-in ModelFinder algorithm. This is the default in QIIME 2, but do not worry, you can set any one of the 286 models of nucleotide substitution via the --p-substitution-model flag, e.g. you can set the model as HKY+I+G instead of the default MFP (a basic short-hand for: “build a phylogeny after determining the best fit model as determined by ModelFinder”). Keep in mind the additional computational time required for model testing via ModelFinder.

The simplest way to run the iqtree command with default settings and automatic model selection (MFP) is like so:

qiime phylogeny iqtree \
  --i-alignment masked-aligned-rep-seqs.qza \
  --o-tree iqt-tree.qza \
  --verbose

stdout:

IQ-TREE multicore version 2.3.6 for MacOS Intel 64-bit built Aug  4 2024
Developed by Bui Quang Minh, Nguyen Lam Tung, Olga Chernomor, Heiko Schmidt,
Dominik Schrempf, Michael Woodhams, Ly Trong Nhan, Thomas Wong

Host:    88.local (SSE4.2, 18 GB RAM)
Command: iqtree -st DNA --runs 1 -s /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/qiime2/jgc/data/d34420d9-336e-4d9e-aee8-99e64b3315f0/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmptbdxg8mw/q2iqtree -nt 1
Seed:    346196 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Thu Apr 24 17:23:36 2025
Kernel:  SSE2 - 1 threads (12 CPU cores detected)

HINT: Use -nt option to specify number of threads because your CPU has 12 cores!
HINT: -nt AUTO will automatically determine the best number of threads to use.

Reading alignment file /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/qiime2/jgc/data/d34420d9-336e-4d9e-aee8-99e64b3315f0/data/aligned-dna-sequences.fasta ... Fasta format detected
Reading fasta file: done in 0.00030899 secs using 31.72% CPU
Alignment most likely contains DNA/RNA sequences
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
                                          Gap/Ambiguity  Composition  p-value
Analyzing sequences: done in 1.00136e-05 secs using 79.89% CPU
   1  e84fcf85a6a4065231dcf343bb862f1cb32abae6   40.65%    passed     90.91%
   2  5525fb6dab7b6577960147574465990c6df070ad   42.99%    passed     99.80%
   3  eb3564a35320b53cef22a77288838c7446357327   42.99%    passed     25.49%
   4  418f1d469f08c99976b313028cf6d3f18f61dd55   43.93%    passed     71.86%
   5  2e3b2c075901640c4de739473f9246385430b1ed   31.31%    passed     90.76%
   6  0469f8d819bd45c7638d1c8b0895270a05f34267   38.79%    passed     92.82%
   7  d162ed685007f5adede58f14aece31dfa1b60c18   40.65%    passed     97.17%
   8  1d45b2bce36cd995c5dcb755babf512e612ce8b9   41.59%    passed     39.04%
   9  5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8   31.31%    passed     87.21%
  10  206656bec2abdbc4aee37a661ef5f4a62b5dd6ae   42.99%    passed     85.00%
  11  606c23e79bb730ad74e3c6efd72004c36674c17a   47.20%    passed     87.78%
  12  682e91d7e510ab134d0625234ad224f647c14eb0   41.59%    passed     31.01%
  13  6a36152105590b1eb095b9503e8f1f226fc73e43   39.25%    passed     86.29%
  14  6ca685c39a33bfbcb3123129e7af88d573df7d6f   42.06%    failed      0.02%
  15  8a1c44eb462ed58b21f3fdd72dd22bb657db2980   31.78%    passed     54.40%
  16  9b220cae8d375ea38b8b481cb95949cda8722fcb   36.92%    passed     88.78%
  17  aa4698d2e2b1fa71d08e2934a923aad7374a18f6   37.85%    passed     90.52%
  18  b31aa3f04bc9d5e2498d45cf1983dfaf09faa258   31.78%    passed     72.69%
  19  d44b129a6181f052198bda3813f0802a91612441   41.59%    passed     41.69%
  20  ed1acad8a98e8579a44370733533ad7d3fed8006   48.13%    passed     58.15%
****  TOTAL                                      39.77%  1 sequences failed composition chi2 test (p-value<5%; df=3)


Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.000 seconds
Perform fast likelihood tree search using GTR+I+G model...
Estimate model parameters (epsilon = 5.000)
Perform nearest neighbor interchange...
Estimate model parameters (epsilon = 1.000)
1. Initial log-likelihood: -1396.575
2. Current log-likelihood: -1395.213
Optimal log-likelihood: -1394.464
Rate parameters:  A-C: 0.21819  A-G: 2.03593  A-T: 1.93394  C-G: 1.05109  C-T: 2.56337  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.033
Gamma shape alpha: 1.322
Parameters optimization took 2 rounds (0.007 sec)
Time for fast ML tree search: 0.032 seconds

NOTE: ModelFinder requires 1 MB RAM!
ModelFinder will test up to 484 DNA models (sample size: 214 epsilon: 0.100) ...
 No. Model         -LnL         df  AIC          AICc         BIC
  1  GTR+F         1411.054     45  2912.108     2936.751     3063.577
  2  GTR+F+I       1409.135     46  2910.270     2936.162     3065.105
  3  GTR+F+G4      1392.990     46  2877.979     2903.872     3032.814
  4  GTR+F+I+G4    1393.280     47  2880.560     2907.741     3038.761
  5  GTR+F+R2      1387.683     47  2869.367     2896.547     3027.567
  6  GTR+F+R3      1387.783     49  2873.566     2903.444     3038.499
 14  GTR+F+I+R2    1387.806     48  2871.612     2900.121     3033.179
 15  GTR+F+I+R3    1387.792     50  2875.584     2906.873     3043.883
 25  SYM+G4        1393.507     43  2873.014     2895.273     3017.751
 27  SYM+R2        1389.903     44  2867.807     2891.239     3015.910
 36  SYM+I+R2      1389.979     45  2869.959     2894.602     3021.428
 47  TVM+F+G4      1393.475     45  2876.951     2901.594     3028.420
 49  TVM+F+R2      1388.449     46  2868.898     2894.790     3023.733
 58  TVM+F+I+R2    1388.463     47  2870.925     2898.106     3029.126
 69  TVMe+G4       1393.626     42  2871.251     2892.374     3012.622
 71  TVMe+R2       1389.912     43  2865.823     2888.082     3010.560
 80  TVMe+I+R2     1389.955     44  2867.910     2891.342     3016.013
 91  TIM3+F+G4     1397.018     44  2882.036     2905.468     3030.139
 93  TIM3+F+R2     1391.446     45  2872.893     2897.535     3024.362
102  TIM3+F+I+R2   1391.495     46  2874.989     2900.882     3029.824
113  TIM3e+G4      1396.975     41  2875.949     2895.972     3013.954
115  TIM3e+R2      1393.203     42  2870.405     2891.528     3011.776
124  TIM3e+I+R2    1393.216     43  2872.431     2894.690     3017.168
135  TIM2+F+G4     1401.477     44  2890.953     2914.385     3039.056
137  TIM2+F+R2     1395.788     45  2881.575     2906.218     3033.044
146  TIM2+F+I+R2   1395.770     46  2883.540     2909.432     3038.375
157  TIM2e+G4      1406.341     41  2894.682     2914.705     3032.687
159  TIM2e+R2      1402.277     42  2888.553     2909.676     3029.924
168  TIM2e+I+R2    1402.296     43  2890.592     2912.851     3035.329
179  TIM+F+G4      1397.935     44  2883.870     2907.302     3031.973
181  TIM+F+R2      1392.172     45  2874.343     2898.986     3025.812
190  TIM+F+I+R2    1392.182     46  2876.364     2902.256     3031.199
201  TIMe+G4       1403.752     41  2889.505     2909.528     3027.510
203  TIMe+R2       1399.391     42  2882.783     2903.905     3024.154
212  TIMe+I+R2     1399.400     43  2884.799     2907.058     3029.536
223  TPM3u+F+G4    1397.356     43  2880.712     2902.971     3025.449
225  TPM3u+F+R2    1392.248     44  2872.495     2895.927     3020.598
234  TPM3u+F+I+R2  1392.253     45  2874.505     2899.148     3025.974
245  TPM3+G4       1397.121     40  2874.241     2893.201     3008.880
247  TPM3+R2       1393.229     41  2868.459     2888.482     3006.464
256  TPM3+I+R2     1393.237     42  2870.473     2891.596     3011.844
267  TPM2u+F+G4    1401.943     43  2889.887     2912.146     3034.624
269  TPM2u+F+R2    1396.528     44  2881.057     2904.489     3029.160
278  TPM2u+F+I+R2  1396.518     45  2883.036     2907.679     3034.505
289  TPM2+G4       1406.528     40  2893.056     2912.016     3027.696
291  TPM2+R2       1402.307     41  2886.613     2906.636     3024.618
300  TPM2+I+R2     1402.317     42  2888.633     2909.756     3030.004
311  K3Pu+F+G4     1398.533     43  2883.065     2905.324     3027.802
313  K3Pu+F+R2     1393.073     44  2874.146     2897.578     3022.249
322  K3Pu+F+I+R2   1393.047     45  2876.095     2900.738     3027.564
333  K3P+G4        1403.893     40  2887.786     2906.745     3022.425
335  K3P+R2        1399.412     41  2880.824     2900.848     3018.829
344  K3P+I+R2      1399.421     42  2882.841     2903.964     3024.212
355  TN+F+G4       1401.522     43  2889.044     2911.303     3033.781
357  TN+F+R2       1395.980     44  2879.961     2903.393     3028.064
366  TN+F+I+R2     1395.968     45  2881.937     2906.580     3033.406
377  TNe+G4        1406.408     40  2892.816     2911.775     3027.455
379  TNe+R2        1402.302     41  2886.605     2906.628     3024.610
388  TNe+I+R2      1402.317     42  2888.635     2909.758     3030.006
399  HKY+F+G4      1402.004     42  2888.008     2909.131     3029.379
401  HKY+F+R2      1396.737     43  2879.474     2901.732     3024.211
410  HKY+F+I+R2    1396.725     44  2881.451     2904.883     3029.554
421  K2P+G4        1406.585     39  2891.169     2909.100     3022.442
423  K2P+R2        1402.339     40  2884.678     2903.638     3019.317
432  K2P+I+R2      1402.348     41  2886.697     2906.720     3024.702
443  F81+F+G4      1410.210     41  2902.420     2922.444     3040.425
445  F81+F+R2      1405.831     42  2895.663     2916.786     3037.034
454  F81+F+I+R2    1405.837     43  2897.674     2919.933     3042.411
465  JC+G4         1414.850     38  2905.700     2922.637     3033.607
467  JC+R2         1411.456     39  2900.912     2918.843     3032.185
476  JC+I+R2       1411.464     40  2902.928     2921.888     3037.567
Akaike Information Criterion:           TVMe+R2
Corrected Akaike Information Criterion: TVMe+R2
Bayesian Information Criterion:         TPM3+R2
Best-fit model: TPM3+R2 chosen according to BIC

All model information printed to /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmptbdxg8mw/q2iqtree.model.gz
CPU time for ModelFinder: 0.542 seconds (0h:0m:0s)
Wall-clock time for ModelFinder: 0.545 seconds (0h:0m:0s)

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.100)
1. Initial log-likelihood: -1411.467
2. Current log-likelihood: -1394.204
3. Current log-likelihood: -1393.350
Optimal log-likelihood: -1393.276
Rate parameters:  A-C: 0.31514  A-G: 1.34673  A-T: 1.00000  C-G: 0.31514  C-T: 1.34673  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.693,0.361) (0.307,2.440)
Parameters optimization took 3 rounds (0.008 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.000485897 secs using 96.52% CPU
Computing ML distances took 0.000519 sec (of wall-clock time) 0.000495 sec (of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Setting up auxiliary I and S matrices: done in 2.59876e-05 secs using 134.7% CPU
Computing RapidNJ tree took 0.000090 sec (of wall-clock time) 0.000132 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1394.353
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.037 second
Computing log-likelihood of 98 initial trees ... 0.041 seconds
Current best score: -1393.276

Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1392.102
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 2: -1385.319
Iteration 10 / LogL: -1385.350 / Time: 0h:0m:0s
Iteration 20 / LogL: -1385.351 / Time: 0h:0m:0s
Finish initializing candidate tree set (4)
Current best tree score: -1385.319 / CPU time: 0.214
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
UPDATE BEST LOG-LIKELIHOOD: -1385.317
UPDATE BEST LOG-LIKELIHOOD: -1385.316
Iteration 30 / LogL: -1385.847 / Time: 0h:0m:0s (0h:0m:0s left)
Iteration 40 / LogL: -1386.370 / Time: 0h:0m:0s (0h:0m:0s left)
Iteration 50 / LogL: -1385.703 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 60 / LogL: -1385.343 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 70 / LogL: -1385.856 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 80 / LogL: -1385.568 / Time: 0h:0m:1s (0h:0m:0s left)
UPDATE BEST LOG-LIKELIHOOD: -1385.316
Iteration 90 / LogL: -1395.219 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 100 / LogL: -1385.703 / Time: 0h:0m:1s (0h:0m:0s left)
TREE SEARCH COMPLETED AFTER 103 ITERATIONS / Time: 0h:0m:1s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1385.316
Optimal log-likelihood: -1385.309
Rate parameters:  A-C: 0.39435  A-G: 1.57063  A-T: 1.00000  C-G: 0.39435  C-T: 1.57063  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.718,0.396) (0.282,2.537)
Parameters optimization took 1 rounds (0.002 sec)
BEST SCORE FOUND : -1385.309
Total tree length: 6.935

Total number of iterations: 103
CPU time used for tree search: 1.066 sec (0h:0m:1s)
Wall-clock time used for tree search: 0.873 sec (0h:0m:0s)
Total CPU time used: 1.629 sec (0h:0m:1s)
Total wall-clock time used: 1.437 sec (0h:0m:1s)

Analysis results written to: 
  IQ-TREE report:                /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmptbdxg8mw/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmptbdxg8mw/q2iqtree.treefile
  Likelihood distances:          /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmptbdxg8mw/q2iqtree.mldist
  Screen log file:               /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmptbdxg8mw/q2iqtree.log

Date and Time: Thu Apr 24 17:23:37 2025
n cores 1
Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command: iqtree -st DNA --runs 1 -s /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/qiime2/jgc/data/d34420d9-336e-4d9e-aee8-99e64b3315f0/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmptbdxg8mw/q2iqtree -nt 1

Saved Phylogeny[Unrooted] to: iqt-tree.qza

Output artifacts:

iqt-tree.qza: view | download

Specifying a substitution model ¶

We can also set a substitution model of our choosing. You may have noticed while watching the onscreen output of the previous command that the best fitting model selected by ModelFinder is noted. For the sake of argument, let’s say the best selected model was shown as GTR+F+I+G4. The F is only a notation to let us know that if a given model supports unequal base frequencies, then the empirical base frequencies will be used by default. Using empirical base frequencies (F), rather than estimating them, greatly reduces computational time. The iqtree plugin will not accept F within the model notation supplied at the command line, as this will always be implied automatically for the appropriate model. Also, the iqtree plugin only accepts G not G4 to be specified within the model notation. The 4 is simply another explicit notation to remind us that four rate categories are being assumed by default. The notation approach used by the plugin simply helps to retain simplicity and familiarity when supplying model notations on the command line. So, in brief, we only have to type GTR+I+G as our input model:

qiime phylogeny iqtree \
  --i-alignment masked-aligned-rep-seqs.qza \
  --p-substitution-model 'GTR+I+G' \
  --o-tree iqt-gtrig-tree.qza \
  --verbose

stdout:

IQ-TREE multicore version 2.3.6 for MacOS Intel 64-bit built Aug  4 2024
Developed by Bui Quang Minh, Nguyen Lam Tung, Olga Chernomor, Heiko Schmidt,
Dominik Schrempf, Michael Woodhams, Ly Trong Nhan, Thomas Wong

Host:    88.local (SSE4.2, 18 GB RAM)
Command: iqtree -st DNA --runs 1 -s /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/qiime2/jgc/data/d34420d9-336e-4d9e-aee8-99e64b3315f0/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpb_3um885/q2iqtree -nt 1
Seed:    378176 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Thu Apr 24 17:23:43 2025
Kernel:  SSE2 - 1 threads (12 CPU cores detected)

HINT: Use -nt option to specify number of threads because your CPU has 12 cores!
HINT: -nt AUTO will automatically determine the best number of threads to use.

Reading alignment file /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/qiime2/jgc/data/d34420d9-336e-4d9e-aee8-99e64b3315f0/data/aligned-dna-sequences.fasta ... Fasta format detected
Reading fasta file: done in 9.10759e-05 secs using 85.64% CPU
Alignment most likely contains DNA/RNA sequences
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
                                          Gap/Ambiguity  Composition  p-value
Analyzing sequences: done in 8.10623e-06 secs using 74.02% CPU
   1  e84fcf85a6a4065231dcf343bb862f1cb32abae6   40.65%    passed     90.91%
   2  5525fb6dab7b6577960147574465990c6df070ad   42.99%    passed     99.80%
   3  eb3564a35320b53cef22a77288838c7446357327   42.99%    passed     25.49%
   4  418f1d469f08c99976b313028cf6d3f18f61dd55   43.93%    passed     71.86%
   5  2e3b2c075901640c4de739473f9246385430b1ed   31.31%    passed     90.76%
   6  0469f8d819bd45c7638d1c8b0895270a05f34267   38.79%    passed     92.82%
   7  d162ed685007f5adede58f14aece31dfa1b60c18   40.65%    passed     97.17%
   8  1d45b2bce36cd995c5dcb755babf512e612ce8b9   41.59%    passed     39.04%
   9  5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8   31.31%    passed     87.21%
  10  206656bec2abdbc4aee37a661ef5f4a62b5dd6ae   42.99%    passed     85.00%
  11  606c23e79bb730ad74e3c6efd72004c36674c17a   47.20%    passed     87.78%
  12  682e91d7e510ab134d0625234ad224f647c14eb0   41.59%    passed     31.01%
  13  6a36152105590b1eb095b9503e8f1f226fc73e43   39.25%    passed     86.29%
  14  6ca685c39a33bfbcb3123129e7af88d573df7d6f   42.06%    failed      0.02%
  15  8a1c44eb462ed58b21f3fdd72dd22bb657db2980   31.78%    passed     54.40%
  16  9b220cae8d375ea38b8b481cb95949cda8722fcb   36.92%    passed     88.78%
  17  aa4698d2e2b1fa71d08e2934a923aad7374a18f6   37.85%    passed     90.52%
  18  b31aa3f04bc9d5e2498d45cf1983dfaf09faa258   31.78%    passed     72.69%
  19  d44b129a6181f052198bda3813f0802a91612441   41.59%    passed     41.69%
  20  ed1acad8a98e8579a44370733533ad7d3fed8006   48.13%    passed     58.15%
****  TOTAL                                      39.77%  1 sequences failed composition chi2 test (p-value<5%; df=3)

Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.000 seconds

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.100)
Thoroughly optimizing +I+G parameters from 10 start values...
Init pinv, alpha: 0.000, 1.000 / Estimate: 0.000, 1.245 / LogL: -1394.426
Init pinv, alpha: 0.040, 1.000 / Estimate: 0.008, 1.308 / LogL: -1394.715
Init pinv, alpha: 0.080, 1.000 / Estimate: 0.009, 1.319 / LogL: -1394.789
Init pinv, alpha: 0.120, 1.000 / Estimate: 0.009, 1.316 / LogL: -1394.785
Init pinv, alpha: 0.160, 1.000 / Estimate: 0.008, 1.311 / LogL: -1394.749
Init pinv, alpha: 0.200, 1.000 / Estimate: 0.009, 1.314 / LogL: -1394.777
Init pinv, alpha: 0.240, 1.000 / Estimate: 0.008, 1.309 / LogL: -1394.722
Init pinv, alpha: 0.280, 1.000 / Estimate: 0.008, 1.310 / LogL: -1394.735
Init pinv, alpha: 0.320, 1.000 / Estimate: 0.008, 1.312 / LogL: -1394.747
Init pinv, alpha: 0.360, 1.000 / Estimate: 0.009, 1.313 / LogL: -1394.756
Optimal pinv,alpha: 0.000, 1.245 / LogL: -1394.426

Parameters optimization took 0.271 sec
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.000690937 secs using 98.42% CPU
Computing ML distances took 0.000726 sec (of wall-clock time) 0.000707 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 2.81334e-05 secs using 131.5% CPU
Computing RapidNJ tree took 0.000096 sec (of wall-clock time) 0.000141 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1392.974
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.038 second
Computing log-likelihood of 98 initial trees ... 0.060 seconds
Current best score: -1392.974

Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1387.274
Iteration 10 / LogL: -1387.731 / Time: 0h:0m:0s
Iteration 20 / LogL: -1387.285 / Time: 0h:0m:0s
Finish initializing candidate tree set (2)
Current best tree score: -1387.274 / CPU time: 0.318
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
UPDATE BEST LOG-LIKELIHOOD: -1387.274
Iteration 30 / LogL: -1387.498 / Time: 0h:0m:0s (0h:0m:1s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.269
Iteration 40 / LogL: -1387.469 / Time: 0h:0m:0s (0h:0m:1s left)
Iteration 50 / LogL: -1387.281 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 60 / LogL: -1387.310 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 70 / LogL: -1387.522 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 80 / LogL: -1387.358 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 90 / LogL: -1398.185 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 100 / LogL: -1387.370 / Time: 0h:0m:1s (0h:0m:0s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.268
TREE SEARCH COMPLETED AFTER 102 ITERATIONS / Time: 0h:0m:1s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1387.268
Optimal log-likelihood: -1387.258
Rate parameters:  A-C: 0.32282  A-G: 2.22227  A-T: 2.10408  C-G: 1.15333  C-T: 3.22824  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.000
Gamma shape alpha: 1.318
Parameters optimization took 1 rounds (0.002 sec)
BEST SCORE FOUND : -1387.258
Total tree length: 6.801

Total number of iterations: 102
CPU time used for tree search: 1.780 sec (0h:0m:1s)
Wall-clock time used for tree search: 1.590 sec (0h:0m:1s)
Total CPU time used: 2.063 sec (0h:0m:2s)
Total wall-clock time used: 1.872 sec (0h:0m:1s)

Analysis results written to: 
  IQ-TREE report:                /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpb_3um885/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpb_3um885/q2iqtree.treefile
  Likelihood distances:          /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpb_3um885/q2iqtree.mldist
  Screen log file:               /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpb_3um885/q2iqtree.log

Date and Time: Thu Apr 24 17:23:45 2025
n cores 1
Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command: iqtree -st DNA --runs 1 -s /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/qiime2/jgc/data/d34420d9-336e-4d9e-aee8-99e64b3315f0/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpb_3um885/q2iqtree -nt 1

Saved Phylogeny[Unrooted] to: iqt-gtrig-tree.qza

Output artifacts:

iqt-gtrig-tree.qza: view | download

Let’s rerun the command above and add the --p-fast option. This option, only compatible with the iqtree method, resembles the fast search performed by fasttree. 🏎️ Secondly, let’s also perform multiple tree searches and keep the best of those trees (as we did earlier with the raxml --p-n-searches ... command):

qiime phylogeny iqtree \
  --i-alignment masked-aligned-rep-seqs.qza \
  --p-substitution-model 'GTR+I+G' \
  --p-fast \
  --p-n-runs 10 \
  --o-tree iqt-gtrig-fast-ms-tree.qza \
  --verbose

stdout:

IQ-TREE multicore version 2.3.6 for MacOS Intel 64-bit built Aug  4 2024
Developed by Bui Quang Minh, Nguyen Lam Tung, Olga Chernomor, Heiko Schmidt,
Dominik Schrempf, Michael Woodhams, Ly Trong Nhan, Thomas Wong

Host:    88.local (SSE4.2, 18 GB RAM)
Command: iqtree -st DNA --runs 10 -s /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/qiime2/jgc/data/d34420d9-336e-4d9e-aee8-99e64b3315f0/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpxir93wpu/q2iqtree -nt 1 -fast
Seed:    793549 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Thu Apr 24 17:23:50 2025
Kernel:  SSE2 - 1 threads (12 CPU cores detected)

HINT: Use -nt option to specify number of threads because your CPU has 12 cores!
HINT: -nt AUTO will automatically determine the best number of threads to use.

Reading alignment file /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/qiime2/jgc/data/d34420d9-336e-4d9e-aee8-99e64b3315f0/data/aligned-dna-sequences.fasta ... Fasta format detected
Reading fasta file: done in 8.98838e-05 secs using 83.44% CPU
Alignment most likely contains DNA/RNA sequences
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
Analyzing sequences: done in 8.10623e-06 secs using 74.02% CPU

---> START RUN NUMBER 1 (seed: 793549)

Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.00 seconds

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.50)
1. Initial log-likelihood: -1493.26
2. Current log-likelihood: -1403.08
3. Current log-likelihood: -1398.35
4. Current log-likelihood: -1396.98
5. Current log-likelihood: -1396.26
Optimal log-likelihood: -1395.75
Rate parameters:  A-C: 0.24339  A-G: 2.10097  A-T: 1.98596  C-G: 1.09180  C-T: 2.82193  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.355
Parameters optimization took 5 rounds (0.021 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.000701904 secs using 96.59% CPU
Computing ML distances took 0.000735 sec (of wall-clock time) 0.000703 sec (of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Setting up auxiliary I and S matrices: done in 2.69413e-05 secs using 126.2% CPU
Computing RapidNJ tree took 0.000097 sec (of wall-clock time) 0.000138 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1394.173
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1387.961
Finish initializing candidate tree set (4)
Current best tree score: -1387.961 / CPU time: 0.044
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1387.961
2. Current log-likelihood: -1387.803
3. Current log-likelihood: -1387.684
4. Current log-likelihood: -1387.593
5. Current log-likelihood: -1387.523
6. Current log-likelihood: -1387.468
Optimal log-likelihood: -1387.424
Rate parameters:  A-C: 0.33414  A-G: 2.26635  A-T: 2.14117  C-G: 1.17550  C-T: 3.28158  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.004
Gamma shape alpha: 1.353
Parameters optimization took 6 rounds (0.012 sec)
BEST SCORE FOUND : -1387.424
Total tree length: 6.743

Total number of iterations: 2
CPU time used for tree search: 0.086 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.044 sec (0h:0m:0s)
Total CPU time used: 0.147 sec (0h:0m:0s)
Total wall-clock time used: 0.091 sec (0h:0m:0s)

---> START RUN NUMBER 2 (seed: 794549)

Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.000 seconds

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1495.571
2. Current log-likelihood: -1402.008
3. Current log-likelihood: -1396.794
4. Current log-likelihood: -1395.393
5. Current log-likelihood: -1394.655
Optimal log-likelihood: -1394.081
Rate parameters:  A-C: 0.27755  A-G: 2.37595  A-T: 2.10647  C-G: 1.20302  C-T: 3.28731  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.386
Parameters optimization took 5 rounds (0.021 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.00069809 secs using 196.4% CPU
Computing ML distances took 0.000725 sec (of wall-clock time) 0.001414 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 1.78814e-05 secs using 95.07% CPU
Computing RapidNJ tree took 0.000068 sec (of wall-clock time) 0.000094 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1393.809
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.217
BETTER TREE FOUND at iteration 2: -1388.188
Finish initializing candidate tree set (4)
Current best tree score: -1388.188 / CPU time: 0.031
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1388.188
2. Current log-likelihood: -1387.973
3. Current log-likelihood: -1387.830
4. Current log-likelihood: -1387.725
5. Current log-likelihood: -1387.645
6. Current log-likelihood: -1387.584
Optimal log-likelihood: -1387.534
Rate parameters:  A-C: 0.36987  A-G: 2.31020  A-T: 2.11745  C-G: 1.22270  C-T: 3.27880  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.006
Gamma shape alpha: 1.332
Parameters optimization took 6 rounds (0.014 sec)
BEST SCORE FOUND : -1387.534
Total tree length: 7.502

Total number of iterations: 2
CPU time used for tree search: 0.061 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.031 sec (0h:0m:0s)
Total CPU time used: 0.304 sec (0h:0m:0s)
Total wall-clock time used: 0.170 sec (0h:0m:0s)

---> START RUN NUMBER 3 (seed: 795549)

Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.000 seconds

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1491.633
2. Current log-likelihood: -1402.007
3. Current log-likelihood: -1396.792
4. Current log-likelihood: -1395.393
5. Current log-likelihood: -1394.654
Optimal log-likelihood: -1394.081
Rate parameters:  A-C: 0.28077  A-G: 2.37447  A-T: 2.10134  C-G: 1.20130  C-T: 3.28121  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.386
Parameters optimization took 5 rounds (0.022 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.000696182 secs using 196.8% CPU
Computing ML distances took 0.000721 sec (of wall-clock time) 0.001410 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 1.50204e-05 secs using 79.89% CPU
Computing RapidNJ tree took 0.000058 sec (of wall-clock time) 0.000071 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1393.810
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.217
BETTER TREE FOUND at iteration 2: -1388.188
Finish initializing candidate tree set (4)
Current best tree score: -1388.188 / CPU time: 0.031
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1388.188
2. Current log-likelihood: -1387.973
3. Current log-likelihood: -1387.830
4. Current log-likelihood: -1387.725
5. Current log-likelihood: -1387.645
6. Current log-likelihood: -1387.584
Optimal log-likelihood: -1387.534
Rate parameters:  A-C: 0.36985  A-G: 2.31003  A-T: 2.11728  C-G: 1.22260  C-T: 3.27851  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.006
Gamma shape alpha: 1.332
Parameters optimization took 6 rounds (0.014 sec)
BEST SCORE FOUND : -1387.534
Total tree length: 7.502

Total number of iterations: 2
CPU time used for tree search: 0.062 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.031 sec (0h:0m:0s)
Total CPU time used: 0.461 sec (0h:0m:0s)
Total wall-clock time used: 0.250 sec (0h:0m:0s)

---> START RUN NUMBER 4 (seed: 796549)

Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.000 seconds

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1492.199
2. Current log-likelihood: -1404.591
3. Current log-likelihood: -1399.228
4. Current log-likelihood: -1397.831
5. Current log-likelihood: -1397.074
Optimal log-likelihood: -1396.495
Rate parameters:  A-C: 0.24620  A-G: 2.08306  A-T: 1.99580  C-G: 1.06240  C-T: 2.85598  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.432
Parameters optimization took 5 rounds (0.023 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.00068903 secs using 197.8% CPU
Computing ML distances took 0.000715 sec (of wall-clock time) 0.001408 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 1.78814e-05 secs using 78.29% CPU
Computing RapidNJ tree took 0.000066 sec (of wall-clock time) 0.000080 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1393.972
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.188
UPDATE BEST LOG-LIKELIHOOD: -1388.187
Finish initializing candidate tree set (3)
Current best tree score: -1388.187 / CPU time: 0.029
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1388.187
2. Current log-likelihood: -1387.966
3. Current log-likelihood: -1387.806
4. Current log-likelihood: -1387.687
5. Current log-likelihood: -1387.596
6. Current log-likelihood: -1387.525
7. Current log-likelihood: -1387.471
Optimal log-likelihood: -1387.426
Rate parameters:  A-C: 0.33228  A-G: 2.23741  A-T: 2.11202  C-G: 1.16006  C-T: 3.23503  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.004
Gamma shape alpha: 1.356
Parameters optimization took 7 rounds (0.015 sec)
BEST SCORE FOUND : -1387.426
Total tree length: 6.737

Total number of iterations: 2
CPU time used for tree search: 0.058 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.029 sec (0h:0m:0s)
Total CPU time used: 0.618 sec (0h:0m:0s)
Total wall-clock time used: 0.329 sec (0h:0m:0s)

---> START RUN NUMBER 5 (seed: 797549)

Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.000 seconds

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1495.571
2. Current log-likelihood: -1402.008
3. Current log-likelihood: -1396.794
4. Current log-likelihood: -1395.393
5. Current log-likelihood: -1394.655
Optimal log-likelihood: -1394.081
Rate parameters:  A-C: 0.27755  A-G: 2.37595  A-T: 2.10647  C-G: 1.20302  C-T: 3.28732  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.386
Parameters optimization took 5 rounds (0.022 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.000695944 secs using 198% CPU
Computing ML distances took 0.000721 sec (of wall-clock time) 0.001415 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 2.88486e-05 secs using 145.6% CPU
Computing RapidNJ tree took 0.000095 sec (of wall-clock time) 0.000140 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1393.809
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.217
BETTER TREE FOUND at iteration 2: -1388.188
Finish initializing candidate tree set (4)
Current best tree score: -1388.188 / CPU time: 0.031
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1388.188
2. Current log-likelihood: -1387.973
3. Current log-likelihood: -1387.830
4. Current log-likelihood: -1387.725
5. Current log-likelihood: -1387.645
6. Current log-likelihood: -1387.584
Optimal log-likelihood: -1387.534
Rate parameters:  A-C: 0.36987  A-G: 2.31020  A-T: 2.11745  C-G: 1.22270  C-T: 3.27880  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.006
Gamma shape alpha: 1.332
Parameters optimization took 6 rounds (0.014 sec)
BEST SCORE FOUND : -1387.534
Total tree length: 7.502

Total number of iterations: 2
CPU time used for tree search: 0.062 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.031 sec (0h:0m:0s)
Total CPU time used: 0.776 sec (0h:0m:0s)
Total wall-clock time used: 0.409 sec (0h:0m:0s)

---> START RUN NUMBER 6 (seed: 798549)

Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.000 seconds

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1492.097
2. Current log-likelihood: -1401.816
3. Current log-likelihood: -1396.523
4. Current log-likelihood: -1395.122
5. Current log-likelihood: -1394.389
Optimal log-likelihood: -1393.818
Rate parameters:  A-C: 0.27163  A-G: 2.41073  A-T: 2.17144  C-G: 1.24911  C-T: 3.27679  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.416
Parameters optimization took 5 rounds (0.023 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.00069499 secs using 198.4% CPU
Computing ML distances took 0.000726 sec (of wall-clock time) 0.001429 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 2.40803e-05 secs using 87.21% CPU
Computing RapidNJ tree took 0.000083 sec (of wall-clock time) 0.000118 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1393.794
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.214
Finish initializing candidate tree set (3)
Current best tree score: -1388.214 / CPU time: 0.024
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1388.214
2. Current log-likelihood: -1388.015
3. Current log-likelihood: -1387.868
4. Current log-likelihood: -1387.760
5. Current log-likelihood: -1387.676
6. Current log-likelihood: -1387.611
7. Current log-likelihood: -1387.560
Optimal log-likelihood: -1387.519
Rate parameters:  A-C: 0.35522  A-G: 2.35151  A-T: 2.13874  C-G: 1.20261  C-T: 3.36909  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.004
Gamma shape alpha: 1.362
Parameters optimization took 7 rounds (0.014 sec)
BEST SCORE FOUND : -1387.519
Total tree length: 6.815

Total number of iterations: 2
CPU time used for tree search: 0.047 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.024 sec (0h:0m:0s)
Total CPU time used: 0.920 sec (0h:0m:0s)
Total wall-clock time used: 0.483 sec (0h:0m:0s)

---> START RUN NUMBER 7 (seed: 799549)

Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.000 seconds

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1493.259
2. Current log-likelihood: -1403.078
3. Current log-likelihood: -1398.354
4. Current log-likelihood: -1396.979
5. Current log-likelihood: -1396.262
Optimal log-likelihood: -1395.753
Rate parameters:  A-C: 0.24339  A-G: 2.10097  A-T: 1.98595  C-G: 1.09180  C-T: 2.82193  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.355
Parameters optimization took 5 rounds (0.022 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.000694036 secs using 198.5% CPU
Computing ML distances took 0.000721 sec (of wall-clock time) 0.001414 sec (of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Setting up auxiliary I and S matrices: done in 2.71797e-05 secs using 154.5% CPU
Computing RapidNJ tree took 0.000089 sec (of wall-clock time) 0.000138 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1394.173
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1387.961
Finish initializing candidate tree set (4)
Current best tree score: -1387.961 / CPU time: 0.042
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1387.961
2. Current log-likelihood: -1387.803
3. Current log-likelihood: -1387.684
4. Current log-likelihood: -1387.593
5. Current log-likelihood: -1387.523
6. Current log-likelihood: -1387.468
Optimal log-likelihood: -1387.424
Rate parameters:  A-C: 0.33414  A-G: 2.26635  A-T: 2.14117  C-G: 1.17550  C-T: 3.28158  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.004
Gamma shape alpha: 1.353
Parameters optimization took 6 rounds (0.012 sec)
BEST SCORE FOUND : -1387.424
Total tree length: 6.743

Total number of iterations: 2
CPU time used for tree search: 0.084 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.042 sec (0h:0m:0s)
Total CPU time used: 1.096 sec (0h:0m:1s)
Total wall-clock time used: 0.572 sec (0h:0m:0s)

---> START RUN NUMBER 8 (seed: 800549)

Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.000 seconds

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1495.571
2. Current log-likelihood: -1402.008
3. Current log-likelihood: -1396.794
4. Current log-likelihood: -1395.393
5. Current log-likelihood: -1394.655
Optimal log-likelihood: -1394.081
Rate parameters:  A-C: 0.27755  A-G: 2.37595  A-T: 2.10647  C-G: 1.20302  C-T: 3.28731  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.386
Parameters optimization took 5 rounds (0.021 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.000697136 secs using 198.7% CPU
Computing ML distances took 0.000724 sec (of wall-clock time) 0.001426 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 1.5974e-05 secs using 75.12% CPU
Computing RapidNJ tree took 0.000064 sec (of wall-clock time) 0.000081 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1393.809
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.217
BETTER TREE FOUND at iteration 2: -1388.188
Finish initializing candidate tree set (4)
Current best tree score: -1388.188 / CPU time: 0.030
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1388.188
2. Current log-likelihood: -1387.973
3. Current log-likelihood: -1387.830
4. Current log-likelihood: -1387.725
5. Current log-likelihood: -1387.645
6. Current log-likelihood: -1387.584
Optimal log-likelihood: -1387.534
Rate parameters:  A-C: 0.36987  A-G: 2.31020  A-T: 2.11745  C-G: 1.22270  C-T: 3.27880  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.006
Gamma shape alpha: 1.332
Parameters optimization took 6 rounds (0.014 sec)
BEST SCORE FOUND : -1387.534
Total tree length: 7.502

Total number of iterations: 2
CPU time used for tree search: 0.060 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.030 sec (0h:0m:0s)
Total CPU time used: 1.252 sec (0h:0m:1s)
Total wall-clock time used: 0.652 sec (0h:0m:0s)

---> START RUN NUMBER 9 (seed: 801549)

Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.000 seconds

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1493.167
2. Current log-likelihood: -1403.041
3. Current log-likelihood: -1398.313
4. Current log-likelihood: -1396.957
5. Current log-likelihood: -1396.228
Optimal log-likelihood: -1395.709
Rate parameters:  A-C: 0.23146  A-G: 2.06957  A-T: 1.96268  C-G: 1.07937  C-T: 2.84174  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.322
Parameters optimization took 5 rounds (0.021 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.000703096 secs using 198.3% CPU
Computing ML distances took 0.000728 sec (of wall-clock time) 0.001435 sec (of CPU time)
WARNING: Some pairwise ML distances are too long (saturated)
Setting up auxiliary I and S matrices: done in 2.5034e-05 secs using 163.8% CPU
Computing RapidNJ tree took 0.000092 sec (of wall-clock time) 0.000140 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1394.184
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1387.955
Finish initializing candidate tree set (4)
Current best tree score: -1387.955 / CPU time: 0.025
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1387.955
2. Current log-likelihood: -1387.798
3. Current log-likelihood: -1387.680
4. Current log-likelihood: -1387.590
5. Current log-likelihood: -1387.521
6. Current log-likelihood: -1387.467
Optimal log-likelihood: -1387.423
Rate parameters:  A-C: 0.33566  A-G: 2.27095  A-T: 2.14605  C-G: 1.17829  C-T: 3.29012  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.004
Gamma shape alpha: 1.352
Parameters optimization took 6 rounds (0.012 sec)
BEST SCORE FOUND : -1387.423
Total tree length: 6.744

Total number of iterations: 2
CPU time used for tree search: 0.050 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.025 sec (0h:0m:0s)
Total CPU time used: 1.394 sec (0h:0m:1s)
Total wall-clock time used: 0.723 sec (0h:0m:0s)

---> START RUN NUMBER 10 (seed: 802549)

Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.000 seconds

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.500)
1. Initial log-likelihood: -1492.097
2. Current log-likelihood: -1401.816
3. Current log-likelihood: -1396.523
4. Current log-likelihood: -1395.122
5. Current log-likelihood: -1394.389
Optimal log-likelihood: -1393.818
Rate parameters:  A-C: 0.27163  A-G: 2.41073  A-T: 2.17144  C-G: 1.24911  C-T: 3.27679  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.027
Gamma shape alpha: 1.416
Parameters optimization took 5 rounds (0.022 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.000689983 secs using 197.8% CPU
Computing ML distances took 0.000716 sec (of wall-clock time) 0.001409 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 2.00272e-05 secs using 84.88% CPU
Computing RapidNJ tree took 0.000078 sec (of wall-clock time) 0.000096 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1393.794
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------

Do NNI search on 2 best initial trees
Estimate model parameters (epsilon = 0.500)
BETTER TREE FOUND at iteration 1: -1388.214
Finish initializing candidate tree set (3)
Current best tree score: -1388.214 / CPU time: 0.024
Number of iterations: 2
TREE SEARCH COMPLETED AFTER 2 ITERATIONS / Time: 0h:0m:0s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.050)
1. Initial log-likelihood: -1388.214
2. Current log-likelihood: -1388.015
3. Current log-likelihood: -1387.868
4. Current log-likelihood: -1387.760
5. Current log-likelihood: -1387.676
6. Current log-likelihood: -1387.611
7. Current log-likelihood: -1387.560
Optimal log-likelihood: -1387.519
Rate parameters:  A-C: 0.35522  A-G: 2.35151  A-T: 2.13874  C-G: 1.20261  C-T: 3.36909  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.004
Gamma shape alpha: 1.362
Parameters optimization took 7 rounds (0.014 sec)
BEST SCORE FOUND : -1387.519
Total tree length: 6.815

Total number of iterations: 2
CPU time used for tree search: 0.047 sec (0h:0m:0s)
Wall-clock time used for tree search: 0.024 sec (0h:0m:0s)
Total CPU time used: 1.538 sec (0h:0m:1s)
Total wall-clock time used: 0.796 sec (0h:0m:0s)

---> SUMMARIZE RESULTS FROM 10 RUNS

Run 9 gave best log-likelihood: -1387.423
Total CPU time for 10 runs: 1.547 seconds.
Total wall-clock time for 10 runs: 0.801 seconds.


Analysis results written to: 
  IQ-TREE report:                /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpxir93wpu/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpxir93wpu/q2iqtree.treefile
  Trees from independent runs:   /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpxir93wpu/q2iqtree.runtrees
  Likelihood distances:          /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpxir93wpu/q2iqtree.mldist
  Screen log file:               /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpxir93wpu/q2iqtree.log

Date and Time: Thu Apr 24 17:23:51 2025
n cores 1
Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command: iqtree -st DNA --runs 10 -s /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/qiime2/jgc/data/d34420d9-336e-4d9e-aee8-99e64b3315f0/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpxir93wpu/q2iqtree -nt 1 -fast

Saved Phylogeny[Unrooted] to: iqt-gtrig-fast-ms-tree.qza

Output artifacts:

iqt-gtrig-fast-ms-tree.qza: view | download

Single branch tests ¶

IQ-TREE provides access to a few single branch testing methods

SH-aLRT via --p-alrt [INT >= 1000]
aBayes via --p-abayes [TRUE | FALSE]
local bootstrap test via --p-lbp [INT >= 1000]

Single branch tests are commonly used as an alternative to the bootstrapping approach we’ve discussed above, as they are substantially faster and often recommended when constructing large phylogenies (e.g. >10,000 taxa). All three of these methods can be applied simultaneously and viewed within iTOL as separate bootstrap support values. These values are always in listed in the following order of alrt / lbp / abayes. We’ll go ahead and apply all of the branch tests in our next command, while specifying the same substitution model as above. Feel free to combine this with the --p-fast option. 😉

qiime phylogeny iqtree \
  --i-alignment masked-aligned-rep-seqs.qza \
  --p-alrt 1000 \
  --p-abayes \
  --p-lbp 1000 \
  --p-substitution-model 'GTR+I+G' \
  --o-tree iqt-sbt-tree.qza \
  --verbose

stdout:

IQ-TREE multicore version 2.3.6 for MacOS Intel 64-bit built Aug  4 2024
Developed by Bui Quang Minh, Nguyen Lam Tung, Olga Chernomor, Heiko Schmidt,
Dominik Schrempf, Michael Woodhams, Ly Trong Nhan, Thomas Wong

Host:    88.local (SSE4.2, 18 GB RAM)
Command: iqtree -st DNA --runs 1 -s /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/qiime2/jgc/data/d34420d9-336e-4d9e-aee8-99e64b3315f0/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpfrpo28h5/q2iqtree -nt 1 -alrt 1000 -abayes -lbp 1000
Seed:    158418 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Thu Apr 24 17:23:57 2025
Kernel:  SSE2 - 1 threads (12 CPU cores detected)

HINT: Use -nt option to specify number of threads because your CPU has 12 cores!
HINT: -nt AUTO will automatically determine the best number of threads to use.

Reading alignment file /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/qiime2/jgc/data/d34420d9-336e-4d9e-aee8-99e64b3315f0/data/aligned-dna-sequences.fasta ... Fasta format detected
Reading fasta file: done in 9.10759e-05 secs using 84.54% CPU
Alignment most likely contains DNA/RNA sequences
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
                                          Gap/Ambiguity  Composition  p-value
Analyzing sequences: done in 7.86781e-06 secs using 76.26% CPU
   1  e84fcf85a6a4065231dcf343bb862f1cb32abae6   40.65%    passed     90.91%
   2  5525fb6dab7b6577960147574465990c6df070ad   42.99%    passed     99.80%
   3  eb3564a35320b53cef22a77288838c7446357327   42.99%    passed     25.49%
   4  418f1d469f08c99976b313028cf6d3f18f61dd55   43.93%    passed     71.86%
   5  2e3b2c075901640c4de739473f9246385430b1ed   31.31%    passed     90.76%
   6  0469f8d819bd45c7638d1c8b0895270a05f34267   38.79%    passed     92.82%
   7  d162ed685007f5adede58f14aece31dfa1b60c18   40.65%    passed     97.17%
   8  1d45b2bce36cd995c5dcb755babf512e612ce8b9   41.59%    passed     39.04%
   9  5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8   31.31%    passed     87.21%
  10  206656bec2abdbc4aee37a661ef5f4a62b5dd6ae   42.99%    passed     85.00%
  11  606c23e79bb730ad74e3c6efd72004c36674c17a   47.20%    passed     87.78%
  12  682e91d7e510ab134d0625234ad224f647c14eb0   41.59%    passed     31.01%
  13  6a36152105590b1eb095b9503e8f1f226fc73e43   39.25%    passed     86.29%
  14  6ca685c39a33bfbcb3123129e7af88d573df7d6f   42.06%    failed      0.02%
  15  8a1c44eb462ed58b21f3fdd72dd22bb657db2980   31.78%    passed     54.40%
  16  9b220cae8d375ea38b8b481cb95949cda8722fcb   36.92%    passed     88.78%
  17  aa4698d2e2b1fa71d08e2934a923aad7374a18f6   37.85%    passed     90.52%
  18  b31aa3f04bc9d5e2498d45cf1983dfaf09faa258   31.78%    passed     72.69%
  19  d44b129a6181f052198bda3813f0802a91612441   41.59%    passed     41.69%
  20  ed1acad8a98e8579a44370733533ad7d3fed8006   48.13%    passed     58.15%
****  TOTAL                                      39.77%  1 sequences failed composition chi2 test (p-value<5%; df=3)

Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.000 seconds

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.100)
Thoroughly optimizing +I+G parameters from 10 start values...
Init pinv, alpha: 0.000, 1.000 / Estimate: 0.000, 1.239 / LogL: -1394.543
Init pinv, alpha: 0.040, 1.000 / Estimate: 0.010, 1.342 / LogL: -1394.886
Init pinv, alpha: 0.080, 1.000 / Estimate: 0.010, 1.353 / LogL: -1394.887
Init pinv, alpha: 0.120, 1.000 / Estimate: 0.009, 1.352 / LogL: -1394.871
Init pinv, alpha: 0.160, 1.000 / Estimate: 0.009, 1.348 / LogL: -1394.836
Init pinv, alpha: 0.200, 1.000 / Estimate: 0.009, 1.351 / LogL: -1394.862
Init pinv, alpha: 0.240, 1.000 / Estimate: 0.010, 1.352 / LogL: -1394.884
Init pinv, alpha: 0.280, 1.000 / Estimate: 0.008, 1.346 / LogL: -1394.826
Init pinv, alpha: 0.320, 1.000 / Estimate: 0.009, 1.347 / LogL: -1394.838
Init pinv, alpha: 0.360, 1.000 / Estimate: 0.009, 1.348 / LogL: -1394.841
Optimal pinv,alpha: 0.000, 1.239 / LogL: -1394.543

Parameters optimization took 0.269 sec
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.000710011 secs using 96.76% CPU
Computing ML distances took 0.000747 sec (of wall-clock time) 0.000714 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 2.5034e-05 secs using 103.9% CPU
Computing RapidNJ tree took 0.000088 sec (of wall-clock time) 0.000120 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1392.870
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.038 second
Computing log-likelihood of 98 initial trees ... 0.059 seconds
Current best score: -1392.870

Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1387.265
Iteration 10 / LogL: -1387.282 / Time: 0h:0m:0s
Iteration 20 / LogL: -1387.282 / Time: 0h:0m:0s
Finish initializing candidate tree set (1)
Current best tree score: -1387.265 / CPU time: 0.322
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Iteration 30 / LogL: -1387.285 / Time: 0h:0m:0s (0h:0m:1s left)
Iteration 40 / LogL: -1387.348 / Time: 0h:0m:0s (0h:0m:1s left)
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 44: -1387.168
Iteration 50 / LogL: -1387.349 / Time: 0h:0m:1s (0h:0m:2s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.168
Iteration 60 / LogL: -1387.629 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 70 / LogL: -1387.190 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 80 / LogL: -1387.336 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 90 / LogL: -1387.336 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 100 / LogL: -1387.183 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 110 / LogL: -1387.183 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 120 / LogL: -1387.384 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 130 / LogL: -1406.295 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 140 / LogL: -1395.178 / Time: 0h:0m:2s (0h:0m:0s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.168
TREE SEARCH COMPLETED AFTER 145 ITERATIONS / Time: 0h:0m:2s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1387.168
Optimal log-likelihood: -1387.167
Rate parameters:  A-C: 0.34634  A-G: 2.32101  A-T: 2.14181  C-G: 1.23358  C-T: 3.21640  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.000
Gamma shape alpha: 1.284
Parameters optimization took 1 rounds (0.002 sec)
BEST SCORE FOUND : -1387.167

Testing tree branches by SH-like aLRT with 1000 replicates...
Testing tree branches by local-BP test with 1000 replicates...
Testing tree branches by aBayes parametric test...
0.034 sec.
Total tree length: 7.607

Total number of iterations: 145
CPU time used for tree search: 2.468 sec (0h:0m:2s)
Wall-clock time used for tree search: 2.277 sec (0h:0m:2s)
Total CPU time used: 2.784 sec (0h:0m:2s)
Total wall-clock time used: 2.593 sec (0h:0m:2s)

Analysis results written to: 
  IQ-TREE report:                /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpfrpo28h5/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpfrpo28h5/q2iqtree.treefile
  Likelihood distances:          /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpfrpo28h5/q2iqtree.mldist
  Screen log file:               /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpfrpo28h5/q2iqtree.log

Date and Time: Thu Apr 24 17:23:59 2025
n cores 1
Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command: iqtree -st DNA --runs 1 -s /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/qiime2/jgc/data/d34420d9-336e-4d9e-aee8-99e64b3315f0/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpfrpo28h5/q2iqtree -nt 1 -alrt 1000 -abayes -lbp 1000

Saved Phylogeny[Unrooted] to: iqt-sbt-tree.qza

Output artifacts:

iqt-sbt-tree.qza: view | download

Tip

IQ-TREE search settings. There are quite a few adjustable parameters available for iqtree that can be modified improve searches through “tree space” and prevent the search algorithms from getting stuck in local optima. One particular best practice to aid in this regard, is to adjust the following parameters: --p-perturb-nni-strength and --p-stop-iter (each respectively maps to the -pers and -nstop flags of iqtree ). In brief, the larger the value for NNI (nearest-neighbor interchange) perturbation, the larger the jumps in “tree space”. This value should be set high enough to allow the search algorithm to avoid being trapped in local optima, but not to high that the search is haphazardly jumping around “tree space”. That is, like Goldilocks and the three 🐻s you need to find a setting that is “just right”, or at least within a set of reasonable bounds. One way of assessing this, is to do a few short trial runs using the --verbose flag. If you see that the likelihood values are jumping around to much, then lowering the value for --p-perturb-nni-strength may be warranted. As for the stopping criteria, i.e. --p-stop-iter, the higher this value, the more thorough your search in “tree space”. Be aware, increasing this value may also increase the run time. That is, the search will continue until it has sampled a number of trees, say 100 (default), without finding a better scoring tree. If a better tree is found, then the counter resets, and the search continues. These two parameters deserve special consideration when a given data set contains many short sequences, quite common for microbiome survey data. We can modify our original command to include these extra parameters with the recommended modifications for short sequences, i.e. a lower value for perturbation strength (shorter reads do not contain as much phylogenetic information, thus we should limit how far we jump around in “tree space”) and a larger number of stop iterations. See the IQ-TREE command reference for more details about default parameter settings. Finally, we’ll let iqtree perform the model testing, and automatically determine the optimal number of CPU cores to use.

qiime phylogeny iqtree \
  --i-alignment masked-aligned-rep-seqs.qza \
  --p-perturb-nni-strength 0.2 \
  --p-stop-iter 200 \
  --p-n-cores 1 \
  --o-tree iqt-nnisi-fast-tree.qza \
  --verbose

stdout:

IQ-TREE multicore version 2.3.6 for MacOS Intel 64-bit built Aug  4 2024
Developed by Bui Quang Minh, Nguyen Lam Tung, Olga Chernomor, Heiko Schmidt,
Dominik Schrempf, Michael Woodhams, Ly Trong Nhan, Thomas Wong

Host:    88.local (SSE4.2, 18 GB RAM)
Command: iqtree -st DNA --runs 1 -s /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/qiime2/jgc/data/d34420d9-336e-4d9e-aee8-99e64b3315f0/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmptma6fdg8/q2iqtree -nt 1 -nstop 200 -pers 0.200000
Seed:    321431 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Thu Apr 24 17:24:05 2025
Kernel:  SSE2 - 1 threads (12 CPU cores detected)

HINT: Use -nt option to specify number of threads because your CPU has 12 cores!
HINT: -nt AUTO will automatically determine the best number of threads to use.

Reading alignment file /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/qiime2/jgc/data/d34420d9-336e-4d9e-aee8-99e64b3315f0/data/aligned-dna-sequences.fasta ... Fasta format detected
Reading fasta file: done in 8.89301e-05 secs using 84.34% CPU
Alignment most likely contains DNA/RNA sequences
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
                                          Gap/Ambiguity  Composition  p-value
Analyzing sequences: done in 8.10623e-06 secs using 86.35% CPU
   1  e84fcf85a6a4065231dcf343bb862f1cb32abae6   40.65%    passed     90.91%
   2  5525fb6dab7b6577960147574465990c6df070ad   42.99%    passed     99.80%
   3  eb3564a35320b53cef22a77288838c7446357327   42.99%    passed     25.49%
   4  418f1d469f08c99976b313028cf6d3f18f61dd55   43.93%    passed     71.86%
   5  2e3b2c075901640c4de739473f9246385430b1ed   31.31%    passed     90.76%
   6  0469f8d819bd45c7638d1c8b0895270a05f34267   38.79%    passed     92.82%
   7  d162ed685007f5adede58f14aece31dfa1b60c18   40.65%    passed     97.17%
   8  1d45b2bce36cd995c5dcb755babf512e612ce8b9   41.59%    passed     39.04%
   9  5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8   31.31%    passed     87.21%
  10  206656bec2abdbc4aee37a661ef5f4a62b5dd6ae   42.99%    passed     85.00%
  11  606c23e79bb730ad74e3c6efd72004c36674c17a   47.20%    passed     87.78%
  12  682e91d7e510ab134d0625234ad224f647c14eb0   41.59%    passed     31.01%
  13  6a36152105590b1eb095b9503e8f1f226fc73e43   39.25%    passed     86.29%
  14  6ca685c39a33bfbcb3123129e7af88d573df7d6f   42.06%    failed      0.02%
  15  8a1c44eb462ed58b21f3fdd72dd22bb657db2980   31.78%    passed     54.40%
  16  9b220cae8d375ea38b8b481cb95949cda8722fcb   36.92%    passed     88.78%
  17  aa4698d2e2b1fa71d08e2934a923aad7374a18f6   37.85%    passed     90.52%
  18  b31aa3f04bc9d5e2498d45cf1983dfaf09faa258   31.78%    passed     72.69%
  19  d44b129a6181f052198bda3813f0802a91612441   41.59%    passed     41.69%
  20  ed1acad8a98e8579a44370733533ad7d3fed8006   48.13%    passed     58.15%
****  TOTAL                                      39.77%  1 sequences failed composition chi2 test (p-value<5%; df=3)


Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.000 seconds
Perform fast likelihood tree search using GTR+I+G model...
Estimate model parameters (epsilon = 5.000)
Perform nearest neighbor interchange...
Estimate model parameters (epsilon = 1.000)
1. Initial log-likelihood: -1391.281
2. Current log-likelihood: -1389.723
Optimal log-likelihood: -1388.882
Rate parameters:  A-C: 0.33811  A-G: 2.30746  A-T: 2.15809  C-G: 1.19223  C-T: 3.30165  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.033
Gamma shape alpha: 1.423
Parameters optimization took 2 rounds (0.008 sec)
Time for fast ML tree search: 0.033 seconds

NOTE: ModelFinder requires 1 MB RAM!
ModelFinder will test up to 484 DNA models (sample size: 214 epsilon: 0.100) ...
 No. Model         -LnL         df  AIC          AICc         BIC
  1  GTR+F         1402.497     45  2894.994     2919.637     3046.463
  2  GTR+F+I       1401.403     46  2894.806     2920.698     3049.641
  3  GTR+F+G4      1387.278     46  2866.556     2892.448     3021.391
  4  GTR+F+I+G4    1387.559     47  2869.117     2896.298     3027.318
  5  GTR+F+R2      1380.618     47  2855.236     2882.416     3013.437
  6  GTR+F+R3      1380.657     49  2859.313     2889.191     3024.246
 14  GTR+F+I+R2    1380.757     48  2857.515     2886.024     3019.081
 15  GTR+F+I+R3    1380.673     50  2861.347     2892.635     3029.645
 25  SYM+G4        1387.153     43  2860.305     2882.564     3005.042
 27  SYM+R2        1382.245     44  2852.489     2875.921     3000.592
 36  SYM+I+R2      1382.314     45  2854.629     2879.271     3006.098
 47  TVM+F+G4      1388.427     45  2866.854     2891.497     3018.323
 49  TVM+F+R2      1382.419     46  2856.838     2882.730     3011.673
 58  TVM+F+I+R2    1382.432     47  2858.863     2886.044     3017.064
 69  TVMe+G4       1387.149     42  2858.299     2879.421     2999.670
 71  TVMe+R2       1382.302     43  2850.603     2872.862     2995.340
 80  TVMe+I+R2     1382.335     44  2852.670     2876.102     3000.773
 91  TIM3+F+G4     1391.457     44  2870.914     2894.346     3019.017
 93  TIM3+F+R2     1384.431     45  2858.863     2883.506     3010.332
102  TIM3+F+I+R2   1384.453     46  2860.907     2886.799     3015.741
113  TIM3e+G4      1390.546     41  2863.091     2883.115     3001.096
115  TIM3e+R2      1385.234     42  2854.468     2875.591     2995.839
124  TIM3e+I+R2    1385.247     43  2856.495     2878.754     3001.232
135  TIM2+F+G4     1394.185     44  2876.371     2899.803     3024.474
137  TIM2+F+R2     1386.231     45  2862.463     2887.106     3013.932
146  TIM2+F+I+R2   1386.236     46  2864.471     2890.363     3019.306
157  TIM2e+G4      1397.735     41  2877.470     2897.493     3015.475
159  TIM2e+R2      1391.104     42  2866.209     2887.332     3007.580
168  TIM2e+I+R2    1391.105     43  2868.210     2890.469     3012.947
179  TIM+F+G4      1390.790     44  2869.581     2893.013     3017.684
181  TIM+F+R2      1383.201     45  2856.402     2881.044     3007.871
190  TIM+F+I+R2    1383.202     46  2858.404     2884.296     3013.239
201  TIMe+G4       1394.786     41  2871.571     2891.595     3009.576
203  TIMe+R2       1388.281     42  2860.561     2881.684     3001.932
212  TIMe+I+R2     1388.283     43  2862.565     2884.824     3007.302
223  TPM3u+F+G4    1392.566     43  2871.132     2893.391     3015.869
225  TPM3u+F+R2    1386.354     44  2860.708     2884.140     3008.811
234  TPM3u+F+I+R2  1386.357     45  2862.714     2887.357     3014.183
245  TPM3+G4       1390.565     40  2861.131     2880.090     2995.770
247  TPM3+R2       1385.310     41  2852.620     2872.644     2990.625
256  TPM3+I+R2     1385.313     42  2854.626     2875.749     2995.997
267  TPM2u+F+G4    1395.285     43  2876.569     2898.828     3021.306
269  TPM2u+F+R2    1388.120     44  2864.240     2887.672     3012.343
278  TPM2u+F+I+R2  1388.115     45  2866.230     2890.873     3017.699
289  TPM2+G4       1397.759     40  2875.518     2894.478     3010.158
291  TPM2+R2       1391.180     41  2864.359     2884.382     3002.364
300  TPM2+I+R2     1391.190     42  2866.380     2887.503     3007.751
311  K3Pu+F+G4     1392.049     43  2870.097     2892.356     3014.834
313  K3Pu+F+R2     1385.122     44  2858.244     2881.676     3006.347
322  K3Pu+F+I+R2   1385.122     45  2860.243     2884.886     3011.712
333  K3P+G4        1394.805     40  2869.610     2888.569     3004.249
335  K3P+R2        1388.356     41  2858.712     2878.735     2996.717
344  K3P+I+R2      1388.358     42  2860.717     2881.840     3002.088
355  TN+F+G4       1394.624     43  2875.247     2897.506     3019.984
357  TN+F+R2       1386.817     44  2861.634     2885.066     3009.737
366  TN+F+I+R2     1386.827     45  2863.655     2888.298     3015.124
377  TNe+G4        1397.746     40  2875.492     2894.452     3010.131
379  TNe+R2        1391.114     41  2864.229     2884.252     3002.234
388  TNe+I+R2      1391.120     42  2866.240     2887.363     3007.611
399  HKY+F+G4      1395.737     42  2875.475     2896.597     3016.846
401  HKY+F+R2      1388.679     43  2863.359     2885.617     3008.096
410  HKY+F+I+R2    1388.678     44  2865.356     2888.788     3013.459
421  K2P+G4        1397.765     39  2873.530     2891.461     3004.803
423  K2P+R2        1391.194     40  2862.387     2881.347     2997.026
432  K2P+I+R2      1391.197     41  2864.395     2884.418     3002.400
443  F81+F+G4      1406.462     41  2894.923     2914.946     3032.928
445  F81+F+R2      1400.568     42  2885.136     2906.259     3026.507
454  F81+F+I+R2    1400.569     43  2887.138     2909.397     3031.875
465  JC+G4         1408.458     38  2892.916     2909.853     3020.823
467  JC+R2         1403.008     39  2884.016     2901.947     3015.289
476  JC+I+R2       1403.015     40  2886.030     2904.990     3020.669
Akaike Information Criterion:           TVMe+R2
Corrected Akaike Information Criterion: TPM3+R2
Bayesian Information Criterion:         TPM3+R2
Best-fit model: TPM3+R2 chosen according to BIC

All model information printed to /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmptma6fdg8/q2iqtree.model.gz
CPU time for ModelFinder: 0.522 seconds (0h:0m:0s)
Wall-clock time for ModelFinder: 0.523 seconds (0h:0m:0s)

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.100)
1. Initial log-likelihood: -1403.013
2. Current log-likelihood: -1386.048
3. Current log-likelihood: -1385.319
Optimal log-likelihood: -1385.306
Rate parameters:  A-C: 0.39721  A-G: 1.56937  A-T: 1.00000  C-G: 0.39721  C-T: 1.56937  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.728,0.408) (0.272,2.583)
Parameters optimization took 3 rounds (0.007 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.00048089 secs using 97.32% CPU
Computing ML distances took 0.000515 sec (of wall-clock time) 0.000495 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 2.90871e-05 secs using 134.1% CPU
Computing RapidNJ tree took 0.000097 sec (of wall-clock time) 0.000141 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1393.877
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.037 second
Computing log-likelihood of 95 initial trees ... 0.039 seconds
Current best score: -1385.306

Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1385.304
Iteration 10 / LogL: -1385.339 / Time: 0h:0m:0s
Iteration 20 / LogL: -1385.331 / Time: 0h:0m:0s
Finish initializing candidate tree set (1)
Current best tree score: -1385.304 / CPU time: 0.201
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
UPDATE BEST LOG-LIKELIHOOD: -1385.304
UPDATE BEST LOG-LIKELIHOOD: -1385.304
Iteration 30 / LogL: -1385.305 / Time: 0h:0m:0s (0h:0m:1s left)
Iteration 40 / LogL: -1385.304 / Time: 0h:0m:0s (0h:0m:1s left)
Iteration 50 / LogL: -1385.715 / Time: 0h:0m:0s (0h:0m:1s left)
Iteration 60 / LogL: -1385.319 / Time: 0h:0m:0s (0h:0m:0s left)
Iteration 70 / LogL: -1385.320 / Time: 0h:0m:0s (0h:0m:0s left)
Iteration 80 / LogL: -1385.311 / Time: 0h:0m:1s (0h:0m:0s left)
UPDATE BEST LOG-LIKELIHOOD: -1385.304
Iteration 90 / LogL: -1385.305 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 100 / LogL: -1385.312 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 110 / LogL: -1385.310 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 120 / LogL: -1385.305 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 130 / LogL: -1385.307 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 140 / LogL: -1385.307 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 150 / LogL: -1385.304 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 160 / LogL: -1385.844 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 170 / LogL: -1385.306 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 180 / LogL: -1385.307 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 190 / LogL: -1385.373 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 200 / LogL: -1385.305 / Time: 0h:0m:1s (0h:0m:0s left)
TREE SEARCH COMPLETED AFTER 202 ITERATIONS / Time: 0h:0m:1s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1385.304
Optimal log-likelihood: -1385.304
Rate parameters:  A-C: 0.39753  A-G: 1.57422  A-T: 1.00000  C-G: 0.39753  C-T: 1.57422  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.725,0.404) (0.275,2.575)
Parameters optimization took 1 rounds (0.002 sec)
BEST SCORE FOUND : -1385.304
Total tree length: 6.837

Total number of iterations: 202
CPU time used for tree search: 1.356 sec (0h:0m:1s)
Wall-clock time used for tree search: 1.164 sec (0h:0m:1s)
Total CPU time used: 1.897 sec (0h:0m:1s)
Total wall-clock time used: 1.705 sec (0h:0m:1s)

Analysis results written to: 
  IQ-TREE report:                /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmptma6fdg8/q2iqtree.iqtree
  Maximum-likelihood tree:       /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmptma6fdg8/q2iqtree.treefile
  Likelihood distances:          /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmptma6fdg8/q2iqtree.mldist
  Screen log file:               /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmptma6fdg8/q2iqtree.log

Date and Time: Thu Apr 24 17:24:07 2025
n cores 1
Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command: iqtree -st DNA --runs 1 -s /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/qiime2/jgc/data/d34420d9-336e-4d9e-aee8-99e64b3315f0/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmptma6fdg8/q2iqtree -nt 1 -nstop 200 -pers 0.200000

Saved Phylogeny[Unrooted] to: iqt-nnisi-fast-tree.qza

Output artifacts:

iqt-nnisi-fast-tree.qza: view | download

iqtree-ultrafast-bootstrap ¶

As per our discussion in the raxml-rapid-bootstrap section above, we can also use IQ-TREE to evaluate how well our splits / bipartitions are supported within our phylogeny via the ultrafast bootstrap algorithm. Below, we’ll apply the plugin’s ultrafast bootstrap command: automatic model selection (MFP), perform 1000 bootstrap replicates (minimum required), set the same generally suggested parameters for constructing a phylogeny from short sequences, and automatically determine the optimal number of CPU cores to use:

qiime phylogeny iqtree-ultrafast-bootstrap \
  --i-alignment masked-aligned-rep-seqs.qza \
  --p-perturb-nni-strength 0.2 \
  --p-stop-iter 200 \
  --p-n-cores 1 \
  --o-tree iqt-nnisi-bootstrap-tree.qza \
  --verbose

stdout:

IQ-TREE multicore version 2.3.6 for MacOS Intel 64-bit built Aug  4 2024
Developed by Bui Quang Minh, Nguyen Lam Tung, Olga Chernomor, Heiko Schmidt,
Dominik Schrempf, Michael Woodhams, Ly Trong Nhan, Thomas Wong

Host:    88.local (SSE4.2, 18 GB RAM)
Command: iqtree -bb 1000 -st DNA --runs 1 -s /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/qiime2/jgc/data/d34420d9-336e-4d9e-aee8-99e64b3315f0/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpktr39d3q/q2iqtreeufboot -nt 1 -nstop 200 -pers 0.200000
Seed:    20894 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Thu Apr 24 17:24:13 2025
Kernel:  SSE2 - 1 threads (12 CPU cores detected)

HINT: Use -nt option to specify number of threads because your CPU has 12 cores!
HINT: -nt AUTO will automatically determine the best number of threads to use.

Reading alignment file /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/qiime2/jgc/data/d34420d9-336e-4d9e-aee8-99e64b3315f0/data/aligned-dna-sequences.fasta ... Fasta format detected
Reading fasta file: done in 8.89301e-05 secs using 84.34% CPU
Alignment most likely contains DNA/RNA sequences
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
                                          Gap/Ambiguity  Composition  p-value
Analyzing sequences: done in 9.05991e-06 secs using 88.3% CPU
   1  e84fcf85a6a4065231dcf343bb862f1cb32abae6   40.65%    passed     90.91%
   2  5525fb6dab7b6577960147574465990c6df070ad   42.99%    passed     99.80%
   3  eb3564a35320b53cef22a77288838c7446357327   42.99%    passed     25.49%
   4  418f1d469f08c99976b313028cf6d3f18f61dd55   43.93%    passed     71.86%
   5  2e3b2c075901640c4de739473f9246385430b1ed   31.31%    passed     90.76%
   6  0469f8d819bd45c7638d1c8b0895270a05f34267   38.79%    passed     92.82%
   7  d162ed685007f5adede58f14aece31dfa1b60c18   40.65%    passed     97.17%
   8  1d45b2bce36cd995c5dcb755babf512e612ce8b9   41.59%    passed     39.04%
   9  5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8   31.31%    passed     87.21%
  10  206656bec2abdbc4aee37a661ef5f4a62b5dd6ae   42.99%    passed     85.00%
  11  606c23e79bb730ad74e3c6efd72004c36674c17a   47.20%    passed     87.78%
  12  682e91d7e510ab134d0625234ad224f647c14eb0   41.59%    passed     31.01%
  13  6a36152105590b1eb095b9503e8f1f226fc73e43   39.25%    passed     86.29%
  14  6ca685c39a33bfbcb3123129e7af88d573df7d6f   42.06%    failed      0.02%
  15  8a1c44eb462ed58b21f3fdd72dd22bb657db2980   31.78%    passed     54.40%
  16  9b220cae8d375ea38b8b481cb95949cda8722fcb   36.92%    passed     88.78%
  17  aa4698d2e2b1fa71d08e2934a923aad7374a18f6   37.85%    passed     90.52%
  18  b31aa3f04bc9d5e2498d45cf1983dfaf09faa258   31.78%    passed     72.69%
  19  d44b129a6181f052198bda3813f0802a91612441   41.59%    passed     41.69%
  20  ed1acad8a98e8579a44370733533ad7d3fed8006   48.13%    passed     58.15%
****  TOTAL                                      39.77%  1 sequences failed composition chi2 test (p-value<5%; df=3)


Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.000 seconds
Perform fast likelihood tree search using GTR+I+G model...
Estimate model parameters (epsilon = 5.000)
Perform nearest neighbor interchange...
Estimate model parameters (epsilon = 1.000)
1. Initial log-likelihood: -1389.605
Optimal log-likelihood: -1388.793
Rate parameters:  A-C: 0.37543  A-G: 2.37167  A-T: 2.15334  C-G: 1.24271  C-T: 3.32365  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.034
Gamma shape alpha: 1.400
Parameters optimization took 1 rounds (0.003 sec)
Time for fast ML tree search: 0.036 seconds

NOTE: ModelFinder requires 1 MB RAM!
ModelFinder will test up to 484 DNA models (sample size: 214 epsilon: 0.100) ...
 No. Model         -LnL         df  AIC          AICc         BIC
  1  GTR+F         1402.600     45  2895.200     2919.843     3046.669
  2  GTR+F+I       1401.121     46  2894.242     2920.135     3049.077
  3  GTR+F+G4      1387.369     46  2866.737     2892.629     3021.572
  4  GTR+F+I+G4    1387.734     47  2869.468     2896.648     3027.669
  5  GTR+F+R2      1382.380     47  2858.759     2885.940     3016.960
+R3 reinitialized from +R2 with factor 0.500
+R3 reinitialized from +R2 with factor 0.250
  6  GTR+F+R3      1382.454     49  2862.909     2892.787     3027.842
 14  GTR+F+I+R2    1382.411     48  2860.821     2889.331     3022.388
 15  GTR+F+I+R3    1382.464     50  2864.928     2896.216     3033.227
 25  SYM+G4        1387.163     43  2860.326     2882.585     3005.063
 27  SYM+R2        1383.105     44  2854.209     2877.641     3002.312
 36  SYM+I+R2      1383.186     45  2856.372     2881.015     3007.841
 47  TVM+F+G4      1388.360     45  2866.721     2891.364     3018.190
 49  TVM+F+R2      1383.725     46  2859.451     2885.343     3014.286
 58  TVM+F+I+R2    1383.717     47  2861.433     2888.614     3019.634
 69  TVMe+G4       1387.152     42  2858.304     2879.427     2999.675
 71  TVMe+R2       1383.090     43  2852.179     2874.438     2996.916
 80  TVMe+I+R2     1383.142     44  2854.285     2877.717     3002.388
 91  TIM3+F+G4     1391.376     44  2870.752     2894.184     3018.855
 93  TIM3+F+R2     1385.912     45  2861.823     2886.466     3013.292
102  TIM3+F+I+R2   1385.947     46  2863.895     2889.787     3018.730
113  TIM3e+G4      1390.370     41  2862.741     2882.764     3000.746
115  TIM3e+R2      1385.927     42  2855.854     2876.977     2997.225
124  TIM3e+I+R2    1385.955     43  2857.911     2880.170     3002.648
135  TIM2+F+G4     1393.632     44  2875.264     2898.696     3023.367
137  TIM2+F+R2     1387.689     45  2865.378     2890.021     3016.847
146  TIM2+F+I+R2   1387.679     46  2867.359     2893.251     3022.194
157  TIM2e+G4      1396.798     41  2875.596     2895.619     3013.601
159  TIM2e+R2      1391.568     42  2867.135     2888.258     3008.506
168  TIM2e+I+R2    1391.562     43  2869.123     2891.382     3013.860
179  TIM+F+G4      1390.337     44  2868.673     2892.105     3016.776
181  TIM+F+R2      1384.915     45  2859.831     2884.474     3011.300
190  TIM+F+I+R2    1384.886     46  2861.772     2887.664     3016.607
201  TIMe+G4       1394.028     41  2870.057     2890.080     3008.062
203  TIMe+R2       1388.990     42  2861.980     2883.103     3003.351
212  TIMe+I+R2     1388.990     43  2863.980     2886.239     3008.717
223  TPM3u+F+G4    1392.293     43  2870.585     2892.844     3015.322
225  TPM3u+F+R2    1387.325     44  2862.650     2886.082     3010.753
234  TPM3u+F+I+R2  1387.333     45  2864.665     2889.308     3016.134
245  TPM3+G4       1390.386     40  2860.772     2879.731     2995.411
247  TPM3+R2       1385.935     41  2853.869     2873.893     2991.874
256  TPM3+I+R2     1385.953     42  2855.905     2877.028     2997.276
267  TPM2u+F+G4    1394.529     43  2875.058     2897.316     3019.795
269  TPM2u+F+R2    1389.057     44  2866.115     2889.547     3014.218
278  TPM2u+F+I+R2  1389.038     45  2868.077     2892.719     3019.545
289  TPM2+G4       1396.829     40  2873.658     2892.617     3008.297
291  TPM2+R2       1391.574     41  2865.147     2885.171     3003.152
300  TPM2+I+R2     1391.570     42  2867.139     2888.262     3008.510
311  K3Pu+F+G4     1391.377     43  2868.753     2891.012     3013.490
313  K3Pu+F+R2     1386.370     44  2860.739     2884.171     3008.842
322  K3Pu+F+I+R2   1386.340     45  2862.680     2887.323     3014.149
333  K3P+G4        1394.023     40  2868.047     2887.006     3002.686
335  K3P+R2        1389.000     41  2859.999     2880.022     2998.004
344  K3P+I+R2      1389.006     42  2862.011     2883.134     3003.382
355  TN+F+G4       1394.028     43  2874.056     2896.314     3018.793
357  TN+F+R2       1388.213     44  2864.425     2887.857     3012.528
366  TN+F+I+R2     1388.214     45  2866.428     2891.071     3017.897
377  TNe+G4        1396.818     40  2873.635     2892.595     3008.274
379  TNe+R2        1391.579     41  2865.158     2885.182     3003.163
388  TNe+I+R2      1391.584     42  2867.169     2888.291     3008.540
399  HKY+F+G4      1394.938     42  2873.876     2894.999     3015.247
401  HKY+F+R2      1389.592     43  2865.185     2887.444     3009.922
410  HKY+F+I+R2    1389.579     44  2867.157     2890.589     3015.260
421  K2P+G4        1396.828     39  2871.656     2889.587     3002.929
423  K2P+R2        1391.583     40  2863.165     2882.125     2997.804
432  K2P+I+R2      1391.585     41  2865.170     2885.193     3003.175
443  F81+F+G4      1405.730     41  2893.461     2913.484     3031.466
445  F81+F+R2      1400.797     42  2885.594     2906.717     3026.965
454  F81+F+I+R2    1400.790     43  2887.581     2909.839     3032.318
465  JC+G4         1407.635     38  2891.270     2908.207     3019.177
467  JC+R2         1402.843     39  2883.685     2901.616     3014.958
476  JC+I+R2       1402.837     40  2885.674     2904.634     3020.313
Akaike Information Criterion:           TVMe+R2
Corrected Akaike Information Criterion: TPM3+R2
Bayesian Information Criterion:         TPM3+R2
Best-fit model: TPM3+R2 chosen according to BIC

All model information printed to /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpktr39d3q/q2iqtreeufboot.model.gz
CPU time for ModelFinder: 0.523 seconds (0h:0m:0s)
Wall-clock time for ModelFinder: 0.525 seconds (0h:0m:0s)
Generating 1000 samples for ultrafast bootstrap (seed: 20894)...

NOTE: 0 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.100)
1. Initial log-likelihood: -1402.843
2. Current log-likelihood: -1386.465
3. Current log-likelihood: -1385.950
Optimal log-likelihood: -1385.940
Rate parameters:  A-C: 0.41103  A-G: 1.56375  A-T: 1.00000  C-G: 0.41103  C-T: 1.56375  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.722,0.414) (0.278,2.520)
Parameters optimization took 3 rounds (0.007 sec)
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.000473022 secs using 97.04% CPU
Computing ML distances took 0.000508 sec (of wall-clock time) 0.000486 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 3.09944e-05 secs using 154.9% CPU
Computing RapidNJ tree took 0.000095 sec (of wall-clock time) 0.000142 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1393.853
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.037 second
Computing log-likelihood of 97 initial trees ... 0.041 seconds
Current best score: -1385.940

Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1385.887
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 2: -1385.308
Iteration 10 / LogL: -1385.341 / Time: 0h:0m:0s
Iteration 20 / LogL: -1385.333 / Time: 0h:0m:0s
Finish initializing candidate tree set (2)
Current best tree score: -1385.308 / CPU time: 0.280
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
UPDATE BEST LOG-LIKELIHOOD: -1385.308
Iteration 30 / LogL: -1385.315 / Time: 0h:0m:0s (0h:0m:2s left)
UPDATE BEST LOG-LIKELIHOOD: -1385.308
Iteration 40 / LogL: -1385.929 / Time: 0h:0m:1s (0h:0m:2s left)
Iteration 50 / LogL: -1385.845 / Time: 0h:0m:1s (0h:0m:1s left)
Log-likelihood cutoff on original alignment: -1415.738
UPDATE BEST LOG-LIKELIHOOD: -1385.308
Iteration 60 / LogL: -1385.908 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 70 / LogL: -1385.657 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 80 / LogL: -1386.436 / Time: 0h:0m:1s (0h:0m:1s left)
Iteration 90 / LogL: -1385.831 / Time: 0h:0m:1s (0h:0m:1s left)
UPDATE BEST LOG-LIKELIHOOD: -1385.308
Iteration 100 / LogL: -1385.552 / Time: 0h:0m:1s (0h:0m:1s left)
Log-likelihood cutoff on original alignment: -1416.342
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.987
NOTE: UFBoot does not converge, continue at least 100 more iterations
Iteration 110 / LogL: -1385.636 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 120 / LogL: -1385.504 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 130 / LogL: -1385.310 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 140 / LogL: -1385.515 / Time: 0h:0m:1s (0h:0m:0s left)
Iteration 150 / LogL: -1385.518 / Time: 0h:0m:2s (0h:0m:0s left)
Log-likelihood cutoff on original alignment: -1416.342
Iteration 160 / LogL: -1385.312 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 170 / LogL: -1385.310 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 180 / LogL: -1385.835 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 190 / LogL: -1385.308 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 200 / LogL: -1385.318 / Time: 0h:0m:2s (0h:0m:0s left)
Log-likelihood cutoff on original alignment: -1417.215
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.994
TREE SEARCH COMPLETED AFTER 203 ITERATIONS / Time: 0h:0m:2s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1385.308
Optimal log-likelihood: -1385.305
Rate parameters:  A-C: 0.39510  A-G: 1.56732  A-T: 1.00000  C-G: 0.39510  C-T: 1.56732  G-T: 1.00000
Base frequencies:  A: 0.250  C: 0.250  G: 0.250  T: 0.250
Site proportion and rates:  (0.722,0.403) (0.278,2.550)
Parameters optimization took 1 rounds (0.002 sec)
BEST SCORE FOUND : -1385.305
Creating bootstrap support values...
Split supports printed to NEXUS file /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpktr39d3q/q2iqtreeufboot.splits.nex
Total tree length: 6.837

Total number of iterations: 203
CPU time used for tree search: 2.226 sec (0h:0m:2s)
Wall-clock time used for tree search: 2.039 sec (0h:0m:2s)
Total CPU time used: 2.796 sec (0h:0m:2s)
Total wall-clock time used: 2.611 sec (0h:0m:2s)

Computing bootstrap consensus tree...
Reading input file /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpktr39d3q/q2iqtreeufboot.splits.nex...
20 taxa and 141 splits.
Consensus tree written to /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpktr39d3q/q2iqtreeufboot.contree
Reading input trees file /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpktr39d3q/q2iqtreeufboot.contree
Log-likelihood of consensus tree: -1385.939

Analysis results written to: 
  IQ-TREE report:                /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpktr39d3q/q2iqtreeufboot.iqtree
  Maximum-likelihood tree:       /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpktr39d3q/q2iqtreeufboot.treefile
  Likelihood distances:          /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpktr39d3q/q2iqtreeufboot.mldist

Ultrafast bootstrap approximation results written to:
  Split support values:          /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpktr39d3q/q2iqtreeufboot.splits.nex
  Consensus tree:                /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpktr39d3q/q2iqtreeufboot.contree
  Screen log file:               /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpktr39d3q/q2iqtreeufboot.log

Date and Time: Thu Apr 24 17:24:15 2025
Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command: iqtree -bb 1000 -st DNA --runs 1 -s /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/qiime2/jgc/data/d34420d9-336e-4d9e-aee8-99e64b3315f0/data/aligned-dna-sequences.fasta -m MFP -pre /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmpktr39d3q/q2iqtreeufboot -nt 1 -nstop 200 -pers 0.200000

Saved Phylogeny[Unrooted] to: iqt-nnisi-bootstrap-tree.qza

Output artifacts:

iqt-nnisi-bootstrap-tree.qza: view | download

Perform single branch tests alongside ufboot ¶

We can also apply single branch test methods concurrently with ultrafast bootstrapping. The support values will always be represented in the following order: alrt / lbp / abayes / ufboot. Again, these values can be seen as separately listed bootstrap values in iTOL. We’ll also specify a model as we did earlier.

qiime phylogeny iqtree-ultrafast-bootstrap \
  --i-alignment masked-aligned-rep-seqs.qza \
  --p-perturb-nni-strength 0.2 \
  --p-stop-iter 200 \
  --p-n-cores 1 \
  --p-alrt 1000 \
  --p-abayes \
  --p-lbp 1000 \
  --p-substitution-model 'GTR+I+G' \
  --o-tree iqt-nnisi-bootstrap-sbt-gtrig-tree.qza \
  --verbose

stdout:

IQ-TREE multicore version 2.3.6 for MacOS Intel 64-bit built Aug  4 2024
Developed by Bui Quang Minh, Nguyen Lam Tung, Olga Chernomor, Heiko Schmidt,
Dominik Schrempf, Michael Woodhams, Ly Trong Nhan, Thomas Wong

Host:    88.local (SSE4.2, 18 GB RAM)
Command: iqtree -bb 1000 -st DNA --runs 1 -s /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/qiime2/jgc/data/d34420d9-336e-4d9e-aee8-99e64b3315f0/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmp9ar7hqqp/q2iqtreeufboot -nt 1 -alrt 1000 -abayes -lbp 1000 -nstop 200 -pers 0.200000
Seed:    380584 (Using SPRNG - Scalable Parallel Random Number Generator)
Time:    Thu Apr 24 17:24:21 2025
Kernel:  SSE2 - 1 threads (12 CPU cores detected)

HINT: Use -nt option to specify number of threads because your CPU has 12 cores!
HINT: -nt AUTO will automatically determine the best number of threads to use.

Reading alignment file /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/qiime2/jgc/data/d34420d9-336e-4d9e-aee8-99e64b3315f0/data/aligned-dna-sequences.fasta ... Fasta format detected
Reading fasta file: done in 8.79765e-05 secs using 85.25% CPU
Alignment most likely contains DNA/RNA sequences
Alignment has 20 sequences with 214 columns, 157 distinct patterns
104 parsimony-informative, 33 singleton sites, 77 constant sites
                                          Gap/Ambiguity  Composition  p-value
Analyzing sequences: done in 8.10623e-06 secs using 86.35% CPU
   1  e84fcf85a6a4065231dcf343bb862f1cb32abae6   40.65%    passed     90.91%
   2  5525fb6dab7b6577960147574465990c6df070ad   42.99%    passed     99.80%
   3  eb3564a35320b53cef22a77288838c7446357327   42.99%    passed     25.49%
   4  418f1d469f08c99976b313028cf6d3f18f61dd55   43.93%    passed     71.86%
   5  2e3b2c075901640c4de739473f9246385430b1ed   31.31%    passed     90.76%
   6  0469f8d819bd45c7638d1c8b0895270a05f34267   38.79%    passed     92.82%
   7  d162ed685007f5adede58f14aece31dfa1b60c18   40.65%    passed     97.17%
   8  1d45b2bce36cd995c5dcb755babf512e612ce8b9   41.59%    passed     39.04%
   9  5aba6bd9debc23ded7041ffdcfe5d68a427e8ce8   31.31%    passed     87.21%
  10  206656bec2abdbc4aee37a661ef5f4a62b5dd6ae   42.99%    passed     85.00%
  11  606c23e79bb730ad74e3c6efd72004c36674c17a   47.20%    passed     87.78%
  12  682e91d7e510ab134d0625234ad224f647c14eb0   41.59%    passed     31.01%
  13  6a36152105590b1eb095b9503e8f1f226fc73e43   39.25%    passed     86.29%
  14  6ca685c39a33bfbcb3123129e7af88d573df7d6f   42.06%    failed      0.02%
  15  8a1c44eb462ed58b21f3fdd72dd22bb657db2980   31.78%    passed     54.40%
  16  9b220cae8d375ea38b8b481cb95949cda8722fcb   36.92%    passed     88.78%
  17  aa4698d2e2b1fa71d08e2934a923aad7374a18f6   37.85%    passed     90.52%
  18  b31aa3f04bc9d5e2498d45cf1983dfaf09faa258   31.78%    passed     72.69%
  19  d44b129a6181f052198bda3813f0802a91612441   41.59%    passed     41.69%
  20  ed1acad8a98e8579a44370733533ad7d3fed8006   48.13%    passed     58.15%
****  TOTAL                                      39.77%  1 sequences failed composition chi2 test (p-value<5%; df=3)

Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.000 seconds
Generating 1000 samples for ultrafast bootstrap (seed: 380584)...

NOTE: 1 MB RAM (0 GB) is required!
Estimate model parameters (epsilon = 0.100)
Thoroughly optimizing +I+G parameters from 10 start values...
Init pinv, alpha: 0.000, 1.000 / Estimate: 0.000, 1.242 / LogL: -1394.542
Init pinv, alpha: 0.040, 1.000 / Estimate: 0.010, 1.345 / LogL: -1394.882
Init pinv, alpha: 0.080, 1.000 / Estimate: 0.010, 1.351 / LogL: -1394.883
Init pinv, alpha: 0.120, 1.000 / Estimate: 0.009, 1.355 / LogL: -1394.867
Init pinv, alpha: 0.160, 1.000 / Estimate: 0.009, 1.351 / LogL: -1394.832
Init pinv, alpha: 0.200, 1.000 / Estimate: 0.009, 1.354 / LogL: -1394.859
Init pinv, alpha: 0.240, 1.000 / Estimate: 0.010, 1.355 / LogL: -1394.881
Init pinv, alpha: 0.280, 1.000 / Estimate: 0.008, 1.349 / LogL: -1394.822
Init pinv, alpha: 0.320, 1.000 / Estimate: 0.009, 1.348 / LogL: -1394.833
Init pinv, alpha: 0.360, 1.000 / Estimate: 0.009, 1.347 / LogL: -1394.843
Optimal pinv,alpha: 0.000, 1.242 / LogL: -1394.542

Parameters optimization took 0.261 sec
Wrote distance file to... 
Computing ML distances based on estimated model parameters...
Calculating distance matrix: done in 0.00069809 secs using 98.41% CPU
Computing ML distances took 0.000735 sec (of wall-clock time) 0.000714 sec (of CPU time)
Setting up auxiliary I and S matrices: done in 3.09944e-05 secs using 129.1% CPU
Computing RapidNJ tree took 0.000100 sec (of wall-clock time) 0.000142 sec (of CPU time)
Log-likelihood of RapidNJ tree: -1392.914
--------------------------------------------------------------------
|             INITIALIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Generating 98 parsimony trees... 0.038 second
Computing log-likelihood of 97 initial trees ... 0.058 seconds
Current best score: -1392.914

Do NNI search on 20 best initial trees
Estimate model parameters (epsilon = 0.100)
BETTER TREE FOUND at iteration 1: -1387.266
Iteration 10 / LogL: -1387.271 / Time: 0h:0m:0s
Iteration 20 / LogL: -1387.282 / Time: 0h:0m:0s
Finish initializing candidate tree set (1)
Current best tree score: -1387.266 / CPU time: 0.407
Number of iterations: 20
--------------------------------------------------------------------
|               OPTIMIZING CANDIDATE TREE SET                      |
--------------------------------------------------------------------
Iteration 30 / LogL: -1387.483 / Time: 0h:0m:0s (0h:0m:4s left)
Iteration 40 / LogL: -1387.356 / Time: 0h:0m:0s (0h:0m:3s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.266
Iteration 50 / LogL: -1387.379 / Time: 0h:0m:1s (0h:0m:3s left)
Log-likelihood cutoff on original alignment: -1410.141
UPDATE BEST LOG-LIKELIHOOD: -1387.259
Iteration 60 / LogL: -1387.377 / Time: 0h:0m:1s (0h:0m:3s left)
Iteration 70 / LogL: -1387.279 / Time: 0h:0m:1s (0h:0m:2s left)
Iteration 80 / LogL: -1387.566 / Time: 0h:0m:1s (0h:0m:2s left)
Iteration 90 / LogL: -1387.349 / Time: 0h:0m:1s (0h:0m:2s left)
Iteration 100 / LogL: -1387.270 / Time: 0h:0m:1s (0h:0m:1s left)
Log-likelihood cutoff on original alignment: -1410.834
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.996
UPDATE BEST LOG-LIKELIHOOD: -1387.257
Iteration 110 / LogL: -1387.257 / Time: 0h:0m:2s (0h:0m:1s left)
Iteration 120 / LogL: -1387.393 / Time: 0h:0m:2s (0h:0m:1s left)
UPDATE BEST LOG-LIKELIHOOD: -1387.256
Iteration 130 / LogL: -1387.260 / Time: 0h:0m:2s (0h:0m:1s left)
Iteration 140 / LogL: -1396.823 / Time: 0h:0m:2s (0h:0m:1s left)
Iteration 150 / LogL: -1387.399 / Time: 0h:0m:2s (0h:0m:0s left)
Log-likelihood cutoff on original alignment: -1410.472
Iteration 160 / LogL: -1387.332 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 170 / LogL: -1387.270 / Time: 0h:0m:2s (0h:0m:0s left)
Iteration 180 / LogL: -1387.385 / Time: 0h:0m:3s (0h:0m:0s left)
Iteration 190 / LogL: -1387.261 / Time: 0h:0m:3s (0h:0m:0s left)
Iteration 200 / LogL: -1387.349 / Time: 0h:0m:3s (0h:0m:0s left)
Log-likelihood cutoff on original alignment: -1410.472
NOTE: Bootstrap correlation coefficient of split occurrence frequencies: 0.998
TREE SEARCH COMPLETED AFTER 202 ITERATIONS / Time: 0h:0m:3s

--------------------------------------------------------------------
|                    FINALIZING TREE SEARCH                        |
--------------------------------------------------------------------
Performs final model parameters optimization
Estimate model parameters (epsilon = 0.010)
1. Initial log-likelihood: -1387.256
Optimal log-likelihood: -1387.253
Rate parameters:  A-C: 0.32759  A-G: 2.24791  A-T: 2.12669  C-G: 1.16571  C-T: 3.26642  G-T: 1.00000
Base frequencies:  A: 0.243  C: 0.182  G: 0.319  T: 0.256
Proportion of invariable sites: 0.000
Gamma shape alpha: 1.320
Parameters optimization took 1 rounds (0.002 sec)
BEST SCORE FOUND : -1387.253

Testing tree branches by SH-like aLRT with 1000 replicates...
Testing tree branches by local-BP test with 1000 replicates...
Testing tree branches by aBayes parametric test...
0.033 sec.
Creating bootstrap support values...
Split supports printed to NEXUS file /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmp9ar7hqqp/q2iqtreeufboot.splits.nex
Total tree length: 6.740

Total number of iterations: 202
CPU time used for tree search: 3.317 sec (0h:0m:3s)
Wall-clock time used for tree search: 3.131 sec (0h:0m:3s)
Total CPU time used: 3.649 sec (0h:0m:3s)
Total wall-clock time used: 3.463 sec (0h:0m:3s)

Computing bootstrap consensus tree...
Reading input file /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmp9ar7hqqp/q2iqtreeufboot.splits.nex...
20 taxa and 161 splits.
Consensus tree written to /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmp9ar7hqqp/q2iqtreeufboot.contree
Reading input trees file /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmp9ar7hqqp/q2iqtreeufboot.contree
Log-likelihood of consensus tree: -1387.254

Analysis results written to: 
  IQ-TREE report:                /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmp9ar7hqqp/q2iqtreeufboot.iqtree
  Maximum-likelihood tree:       /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmp9ar7hqqp/q2iqtreeufboot.treefile
  Likelihood distances:          /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmp9ar7hqqp/q2iqtreeufboot.mldist

Ultrafast bootstrap approximation results written to:
  Split support values:          /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmp9ar7hqqp/q2iqtreeufboot.splits.nex
  Consensus tree:                /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmp9ar7hqqp/q2iqtreeufboot.contree
  Screen log file:               /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmp9ar7hqqp/q2iqtreeufboot.log

Date and Time: Thu Apr 24 17:24:24 2025
Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command: iqtree -bb 1000 -st DNA --runs 1 -s /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/qiime2/jgc/data/d34420d9-336e-4d9e-aee8-99e64b3315f0/data/aligned-dna-sequences.fasta -m GTR+I+G -pre /var/folders/1t/w4ys4pks4q5d5_kl7svl4t080000gn/T/tmp9ar7hqqp/q2iqtreeufboot -nt 1 -alrt 1000 -abayes -lbp 1000 -nstop 200 -pers 0.200000

Saved Phylogeny[Unrooted] to: iqt-nnisi-bootstrap-sbt-gtrig-tree.qza

Output artifacts:

iqt-nnisi-bootstrap-sbt-gtrig-tree.qza: view | download

Tip

If there is a need to reduce the impact of potential model violations that occur during a UFBoot search, and / or would simply like to be more rigorous, we can add the --p-bnni option to any of the iqtree-ultrafast-bootstrap commands above.

Root the phylogeny ¶

In order to make proper use of diversity metrics such as UniFrac, the phylogeny must be rooted. Typically an outgroup is chosen when rooting a tree. In general, phylogenetic inference tools using Maximum Likelihood often return an unrooted tree by default.

QIIME 2 provides a way to mid-point root our phylogeny. Other rooting options may be available in the future. For now, we’ll root our bootstrap tree from iqtree-ultrafast-bootstrap like so:

qiime phylogeny midpoint-root \
  --i-tree iqt-nnisi-bootstrap-sbt-gtrig-tree.qza \
  --o-rooted-tree iqt-nnisi-bootstrap-sbt-gtrig-tree-rooted.qza

Output artifacts:

iqt-nnisi-bootstrap-sbt-gtrig-tree-rooted.qza: view | download

Tip

iTOL viewing Reminder. We can view our tree and its associated alignment via iTOL. All you need to do is upload the iqt-nnisi-bootstrap-sbt-gtrig-tree-rooted.qza tree file. Display the tree in Normal mode. Then drag and drop the masked-aligned-rep-seqs.qza file onto the visualization. Now you can view the phylogeny alongside the alignment.

Pipelines ¶

Here we will outline the use of the phylogeny pipeline align-to-tree-mafft-fasttree

One advantage of pipelines is that they combine ordered sets of commonly used commands, into one condensed simple command. To keep these “convenience” pipelines easy to use, it is quite common to only expose a few options to the user. That is, most of the commands executed via pipelines are often configured to use default option settings. However, options that are deemed important enough for the user to consider setting, are made available. The options exposed via a given pipeline will largely depend upon what it is doing. Pipelines are also a great way for new users to get started, as it helps to lay a foundation of good practices in setting up standard operating procedures.

Rather than run one or more of the following QIIME 2 commands listed below:

qiime alignment mafft ...
qiime alignment mask ...
qiime phylogeny fasttree ...
qiime phylogeny midpoint-root ...

We can make use of the pipeline align-to-tree-mafft-fasttree to automate the above four steps in one go. Here is the description taken from the pipeline help doc:

This pipeline will start by creating a sequence alignment using MAFFT, after which any alignment columns that are phylogenetically uninformative or ambiguously aligned will be removed (masked). The resulting masked alignment will be used to infer a phylogenetic tree and then subsequently rooted at its midpoint. Output files from each step of the pipeline will be saved. This includes both the unmasked and masked MAFFT alignment from q2-alignment methods, and both the rooted and unrooted phylogenies from q2-phylogeny methods.

This can all be accomplished by simply running the following:

qiime phylogeny align-to-tree-mafft-fasttree \
  --i-sequences rep-seqs.qza \
  --output-dir mafft-fasttree-output

Output artifacts:

mafft-fasttree-output/masked_alignment.qza: view | download
mafft-fasttree-output/rooted_tree.qza: view | download
mafft-fasttree-output/alignment.qza: view | download
mafft-fasttree-output/tree.qza: view | download

Congratulations! You now know how to construct a phylogeny in QIIME 2!

Quick search